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Abstracts 2014.indd - Eastern Analytical Symposium

2014 N
Eastern Analytical Symposium
& Exposition
CO 2
Abstract Book
O 17–19, 2014
Garden State Exhibit Center
Somerset, New Jersey
2014 EAS Abstracts
November 2014
2014 EAS Abstracts
This volume contains the final abstracts for the oral and poster presentations which take place
Monday, November 17, through Wednesday, November 19, 2014. Additional abstracts received
after this volume was finalized are provided in the Addendum to the Final Program. If an abstract
is not provided in this volume or the Addendum, then the presenting author did not supply an
abstract. For each abstract provided, a complete mailing address for the presenting author is
shown. Additional authors are indicated, however, their mailing addresses are not provided.
Schedule and meeting room information for the technical sessions, as well as information
concerning short courses, exhibitor workshops, and the exposition, are contained in the Final
Program Book.
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November 16 - 18, 2015
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March 1 – April 15, 2015
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2014 EAS Abstracts
November 2014
ples with DART. For samples that cannot be analyzed by DART, we can apply other
ambient ionization methods such as paper spray or several forms of inlet ionization
with minimal effort. Therefore, in the spirit of Father of American Microchemistry, I
look forward to celebrating Professor Gene Hall as he is awarded the 2014 Benedetti-Pichler Award!
Finger Prick Blood Analysis: The Omega-3 Index
Ken D. Stark, University of Waterloo, 200 University Ave. West,
Waterloo, ON N2L3G1 Canada
The fatty acid composition of blood can be used to determine dietary intake patterns, but also to indicate the potential risk of chronic disease. While over thirty
individual fatty acids can be determined, blood biomarkers of omega-3 polyunsaturated fatty status such as “the Omega-3 Index” have been developed to simplify
interpretation and to enable usage in clinical settings. However, these omega-3
blood biomarkers can still be influenced by sample collection and storage, chemical
preparation of specimens, gas chromatography methods, and data processing. The
interpretation of omega-3 biomarkers therefore requires consideration of the type of
blood sample analyzed (i.e., plasma, erythrocytes or whole blood), whether individuals are consuming their habitual intake or undergoing dietary modification, and other
biological factors known to effect fatty acid metabolism such as sex, age and genetics. Metabolically, measured fatty acid levels have been dictated by the metabolic
balance between synthesis and oxidation, but also by the selective incorporation
into complex lipid structures such as triacylglycerols and phospholipids. Differences
in the incorporation of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid
(22:6n-3) into blood with fish oil feeding and the use of omega-3 blood biomarkers
to confirm dietary adherence in clinical studies are examined, specifically. Insights
based on rodent models that examine the relationship between blood and tissue
levels including the expression of genes involved in fatty acid metabolism are also
presented. Future directions in lipid biomarkers, including the potential application
of lipidomic analytical techniques, are also discussed.
Chemical Microscopy for the 21st Century: Surface-Enhanced
Raman Microspectroscopy for the Ultrasensitive Detection of
Organic Colorants
Marco Leona, The Metropolitan Museum of Art, 1000 Fifth Ave., New
York, NY 10028
Surface-enhanced Raman scattering (SERS) is an ideal microchemical technique
for the analysis of dyes and organic pigments in cultural heritage material. Its sensitivity makes it possible to identify organic pigments in samples often smaller than
those required for polarized light microscopy. SERS is now moving beyond the classical microsampling and microanalytical approach, with the use of gels as sampling
aids for quasi-non-invasive analysis, and with inkjet system to precisely and reproducibly deliver silver colloid nanodroplets directly on the objects to be analyzed. Finally, the coupling of laser ablation with SERS microscopy is promising micrometer
scale spatial resolution in the analysis of complex samples.
From Archimedes Palimpsest to Water Analysis: My 35 Years of
Instrumental Analytical Chemistry at Rutgers
Gene S. Hall, Rutgers University, Chemistry Department, 610 Taylor
Rd., Piscataway, NJ 08854
This presentation focuses on my 35 years of analytical chemistry in the chemistry
department at Rutgers. I share with you some of the exciting research projects that I
have been involved with based on the use of several analytical instruments (Raman,
gas chromatography flame ionization detection mass spectrometry (GC-FID-MS),
MS-MS, energy dispersive X-ray fluorescence (EDXRF), Fourier transform infrared
(FTIR), high-performance liquid chromatography (HPLC), and thin-layer chromatography -FID) in my toolbox. Teaching has been an exciting method to pass on
my knowledge to up-and-coming students wishing to pursue a career in analytical
chemistry. My research focus is on environmental, biological, and forensic problems. Inductively coupled plasma (ICP) -MS is used for tap water analysis to determine sources of Pb by measuring its stable isotopes. ICP-MS was used to monitor
blood-lead concentrations from a gunshot victim. Lead isotope ratios from sequential blood samples were compared to ratios from Pb bullet fragments revealed interesting Pb exchange. Recently, we have used shotgun lipidomics to characterize designer omega-3 dietary supplements used to provide docosahexaenoic acid (DHA)
to Alzheimer’s patients. Forensic science is an exciting venture in our laboratory
especially for student participation. In this regard, I share with you our micro EDXRF
method to recover text for the first time on pages from the Archimedes Palimpsest. Keeping with the theme of micro analytical chemistry, results using Raman
microscopy, micro attenuated total reflectance (ATR)-FTIR, and micro EDXRF for
non-destructive analyses of analyzing postage stamps, jewelry (counterfeit Rolex
watches), counterfeit US and British banknotes, Coach handbags, mouse embryos,
and krill are also presented. Use of chemometrics with digital spectral libraries and
database mining software assisted in data interpretation.
Forensic DNA Analysis: The Gold Standard for Criminal
Investigation of Biological Evidence
Lawrence Kobilinsky, John Jay College of Criminal Justice, 524 West
59th St., New York, NY 10019
Criminalists are charged with the analysis of biological and other physical evidence
found at a crime scene in order to identify the chemical nature of the item (identification) and its source (individualization). Biological evidence can usually be identified using chemical and serological methods. However, determining the origin of
such evidence poses a greater challenge. There has been an evolution of techniques over the past several decades all of which attempt to determine genetic
information for the source. This paper presents an overview of methods which have
been employed to identify and individualize biological evidence. Methods have become more sensitive and more specific with the newer methods taking advantage
of polymerase chain reaction amplification of DNA extracted from evidence and
more sophisticated instrumentation and software to determine which alleles (genes)
are present. Currently, forensic scientists can perform PCR-STR DNA analysis to
routinely obtain genetic profiles of individuals at 24 loci using GlobalFiler (Applied
Biosystems) or PowerPlex Fusion (Promega) commercial kits. The introduction of
Next Generation Sequencing is revolutionizing the process of individualization even
with exceedingly small evidentiary specimens. Although the NGS systems are not
as yet validated for forensic casework, there is more interest in adopting this new
technology. It has the capability of performing autosomal s Short tandem repeat hort
tandem repeat (STR) analysis, X STR and Y STR analysis, and mitochondrial DNA
analysis simultaneously from the same evidentiary item. Testing will soon become
even more cost effective, more rapid, and more informative with respect to individualizing the specimen at hand.
Vibrational Optical Activity: An Established Tool for Research and
Routine Analytical Applications
Laurence A. Nafie, Syracuse University, Department of Chemistry,
Syracuse, NY 13244
Both infrared (IR) and Raman forms of vibrational optical activity (VOA) were discovered experimentally nearly 40 years ago. [1] The IR form is vibrational circular dichroism (VCD) and the Raman form is Raman optical activity (ROA). For the first 20
years of VOA, VCD and ROA passed through a period of gradual growth spearheaded by a few academic laboratories. Then in mid-1990s two developments occurred
for VCD, followed a few years later for ROA. These were the commercial availability
of instrumentation for measurements and software for calculations. The comparison
of measured and calculated VOA allows the determination of the absolute structure
and conformation of chiral molecules. In the past two decades VOA has undergone
a period of rapid growth and maturation. Academic labs now use VOA primarily to
determine the conformation of biological molecules, including the use of VCD to
probe the supramolecular chiral structure of amyloid fibrils. Pharmaceutical companies use VOA to determine the absolute configuration of new chiral drug substances
and to probe the higher order structure of biopharmaceutical molecules. This presentation shows how hundreds of scientists across a wide range of disciplines now
use VOA on a regular basis to determine absolute molecular structure and solve
Doing a Lot with a Little: Ambient Ionization and Microchemistry
Robert B. Cody, Jeol USA Inc., 11 Dearborn Rd., Peabody, MA 10960
When Gene Hall invited me to speak at the symposium honoring him with the American Microchemical Society’s 2014 Benedetti-Pichler Award, my first thought was
that ambient ionization has little to do with microchemistry. Reading about the “Father of American Microchemistry,” Anton Alexander Benedetti-Pichler, I realized that
the two have much in common! “Spur-of-the-moment improvisations” are a familiar
situation and “uncomplicated solutions” are the goal in our lab. Although mass spectrometry has excellent detection limits, a practical factor in handling small samples
is the preparation steps that precede mass spectrometric analysis. Open-air “ambient” ion sources like direct analysis in real-time (DART) present an opportunity
for chemistry on a small scale. Each day in our laboratory, we encounter new and
completely different analytical challenges. We treat the gap between the ion source
and the mass spectrometer as a small chemistry laboratory. Samples are often analyzed on the sealed end of a melting point tube, or positioned in the gas stream
with vacuum tweezers. Microextractions, in-situ derivatization of small samples, and
micro-scale reactions such as chelation and hydrogen–deuterium exchange are
among the many “tricks” that enhance our ability to analyze a wide range of sam-
2014 EAS Abstracts
November 2014
stereochemical problems of practical importance.
Application of UPLC-High Resolution MS in Drug Product
Comparability Studies
Pilsoo Kang, Genzyme, a Sanofi Company, 45 New York Ave.,
Framingham, MA 01701, Jianmei Kochling, Yimin Hua, Robert W.
A comparability study is conducted in order to evaluate the impact of the changes
to drug product for every biological manufacturing change, whether caused by site,
scale, or process. The comparability study will involve not only demonstration of
analytical equivalence for protein structures, but also verify that the products have
similar quality attributes and equivalent functionality. The analytical demonstration
in similarity commonly involves the use of forced degradation methods, by which
structure quality attributes as well as product degradation pathways are compared
using analytical methods. In this talk, comparability studies utilizing the state-or-art
technology with ultra-performance liquid chromatography (UPLC)-high resolution
mass spectrometry (MS) technique are presented.
[1] Laurence A. Nafie, Vibrational Optical Activity: Principles and Applications, John
Wiley & Sons, Ltd., Chichester, 2011.
Isao Noda, University of Delaware, 201 DuPont Hall, Newark, DE 19716
Two-dimensional (2-D) correlation spectroscopy has gained considerable popularity
over the last 25 years in not only academic but also industrial laboratories as generally applicable and versatile technique to analyze spectral data. The technique is
readily adaptable to many different fields, including materials design and development. In 2-D correlation spectroscopy, a set of spectra defined by two independent
variables, e.g., IR wavenumbers, is generated by applying a correlation analysis
to the changes in spectral signal intensities induced by an external perturbation,
such as mechanical deformation and change in temperature. Notable features of
2-D correlation spectra are: simplification of complex spectra consisting of many
overlapped peaks, enhancement of spectral resolution by spreading peaks over
the second dimension, and establishment of unambiguous assignment through
correlation of bands selectively coupled by various interaction mechanisms. 2-D
correlation spectroscopy has played a key role in the materials development effort
in my laboratory. An illustrative example of the successful industrial applications of
2-D correlation spectroscopy will be discussed, focused especially on the design
and development of bio-based biodegradable plastic material with excellent physical properties, which is now in commercial production.
Quality-by-Design Method Development of the 2nd Generation
Peptide Map Assays for Therapeutic Proteins
Yimin Hua, Genzyme, a Sanofi Company, 68 New York Ave.,
Framingham, MA 01701, Francisca Gbormittah, Alekhya Pochiraju,
Qian Guan, Robert W. Donaldson, Jianmei Kochling
Analytical method development process has evolved along with industry’s significant understanding of the “quality-by-design (QbD)” concept. Quality-by-design
approach analytical methods development relies upfront understanding of targeted
method attributes and acceptance criteria, process and product knowledge, regulatory requirements and the incorporation of the modern technology. In this presentation, the QbD method development process of peptide map assays utilizing UPLC
and mass spectrometry is presented. A platform approach applying for multiple therapeutic proteins is discussed and case studies are provided.
Electrospun PHA (polyhydroxyalkanoate) Fibers: Influence of
Processing on Structure
Bruce Chase, University of Delaware, Department of Materials Science,
210 Dupont Hall, Newark, DE 19716, Liang Gong, Isao Noda, John
Over the past decade, the production of polymeric fibers by electrospinning has
become a major focus for a large number of research groups. The ability to produce sub-micron diameter fibers, often in structures and morphologies different
from those found in conventional fiber spinning is potentially a powerful tool which
one can use to modify bulk properties for use in a variety of applications. Polyhydroxyalkanoates (PHAs), a class of biodegradable and biocompatible aliphatic
polyesters synthesized by a variety of bacteria as intracellular carbon and energy
storage materials, have properties which make them attractive for environmental,
electrical, pharmaceutical, and biomedical applications. Poly [(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHB-HHx) nanofibers have been electrospun and
collected on aluminum foil with an air-gap or on a rotary disk with a sharp edge.
The collection method has a significant effect on the morphology, crystal structure
and chain conformation found in the resultant fibers as demonstrated using scanning electron microscope (SEM), wide-angle X-ray diffraction (WAXD) and Fourier
transform infrared (FTIR). In addition, for the first time in the copolymer PHB-HHx,
we have succeeded in generating the strain-induced metastable ОІ-form crystalline
structure, with the chains extended and adopting a planar zig-zag conformation.
This crystalline structure was observed in macroscopically aligned electrospun
PHB-HHx fibers collected across the air-gap or on the sharp edge of the rotary disk.
Withdrawn by the author.
The Influence of Biocompatible Coating Thickness of Non-porous
Polymer Beads on Protein Recoveries and Resolution
Michael Lu, BioChrom Labs, 1719 S. 13th Street, PO Box 2404, Terre
Haute, IN 47802
The thickness of biocompatible coating was determined by molecular weight,
viscosity, and chemical composition of coating reagents. It can be changed. The
thickness of biocompatible coating of non-porous polymer beads affected protein
recoveries. The low coating thickness of hydrophilic beads had poor recoveries in
comparison with optimized and high coated thickness of hydrophilic beads. The
derivation of hydrophilic beads with hydrophobic groups such as C3 showed the
effects of coating thickness on the recoveries and resolution of proteins on hydrophobic interaction media. The low coating thickness of media, C3 NP10 showed
poor recoveries and long retention time. The optimized coating thickness of media
showed good recoveries, retention and resolution. The high coating thickness of
media showed good recoveries, but short retention. The further derivation of C3
NP10 with oxidizing agents showed the effects of coating thickness on the recoveries and resolution of proteins on weak cation exchange media. The low coating
thickness of media CM NP10 showed good resolution, longer retention time and
poor recoveries. The optimized coating thickness of media showed good resolution
and recoveries. The high coating thickness of media showed poor resolution, but
good recoveries. In Summary, the low coating thickness of polymer beads showed
poor recoveries. High coating thickness of polymer beads showed short retention
and poor resolution. The optimized coating thickness of polymer beads shows good
recoveries and resolution. The article shows the comparison of chromatograms of
the different biocompatible coating thickness of polymer beads on protein recoveries and resolution.
UV Resonance Raman Spectroscopic Studies of Protein Structure
and Dynamics
Sanford (Sandy) A. Asher, University of Pittsburgh, Chevron Science
Center, Rm. 701, Pittsburgh, PA 15260
UV Raman excitation into the ~200 nm peptide bond electronic transitions enhance
peptide bond amide vibrations of the backbone. A particular band (the amide III3)
reports on the Ramachandran psi angle and peptide bond hydrogen bonding. This
band is Raman scattered independently by each peptide bond with insignificant
coupling between adjacent peptide bonds. Isotope editing of a peptide bond (by
replacing the Calpha- H with Calpha- D) allows us to determine the frequency of individual peptide bonds within a peptide or protein to yield their psi angles. Consideration
of the Boltzmann equilibria allows us to determine the psi angle Gibbs free energy
landscape along the psi (un)folding coordinate that connects secondary structure
conformations. The psi angle coordinate is the most important reaction coordinate
necessary to understand mechanism(s) of protein folding. We examine the details
of peptide folding conformation dynamics with laser T-jumps where the water temperature is elevated by an 1.9 ВµM IR nsec laser pulse and we monitor the ~200 nm
UV Raman spectrum as a function of time. These spectra show the time evolution of
conformation. We discuss the role of salts on stabilizing conformations in solution.
Development, Validation and Comparison of a Competitive Ligand
Binding and a Functional Cell-Based Assay for Neutralizing
Antibody (NAb) Detection
Weifeng Xu, Bristol-Myers Squibb, Route 206 & Province Line Rd.,
Lawrenceville, NJ 08550, Michael Sank, Jennifer Cummings, Xuefeng
Li, Robert Dodge, Renuka Pillutla, Binodh DeSilva
Two assays were developed and validated to detect NAb against Bristol-Myers Squibb post-marketing mono-clonal therapeutics in clinical samples: an ELISA-based competitive ligand-binding assay (CLB) and a cell-based functional
assay. Both assays had minimal drug interference at the high concentration of
drug expected in clinical samples. In the competitive ligand binding assay, drug
competed with Ruthenium-labeled ligand for binding to plate-coated drug target.
Presence of NAb blocked binding of drug to its target thus increased signal. For the
functional bioassay, Jurkat cells overexpressing cell-surface restricted drug target
were incubated with ligand-expressing Raji cells. Without drug, drug target engaged
2014 EAS Abstracts
November 2014
with ligand and inhibited luciferase-reporter expression while presence of the drug
blocked this interaction and led to increased reporter expression. Presence of NAb
blocked drug and facilitated the interaction between drug and its ligand and decreased Luciferase-reporter expression. Since high drug concentration are present
in most of the clinical samples, a bead extraction method followed by acid dissociation was developed to remove both drug and serum factors. However, unlike CLB
assay, cells used in the functional bioassay were very sensitive to a broad range
of acid buffer commonly used for this purpose. Much more efforts were focused on
improving the extraction efficiency and buffer compatibility for the cell assay.
tion spectroscopy and inductively-coupled plasma spectroscopy with various modes
of detection) permit the quantification of a broad range of elements at low levels with
excellent precision and accuracy. USP has updated its heavy metals testing to procedures that are element specific and performance based. General chapters <232>
Elemental Impurities – Limits and <233> Elemental Impurities – Procedures have
been official since February 1, 2013 but will have a delayed implementation date
of December 1, 2015. This presentation discusses these general chapters, their
implementation and future directions based on the ICH Q3D Expert Working Group.
Simple Magnification, Optical Phase Contrast Microscopy, and
Desktop SEM-EDS: Characterizing a Cleaning Agent
John Scott, New York Conservation Foundation, 261 Fifth Ave., Rm.
2000, New York, NY 10016
No abstract submitted by the author.
Withdrawn by the author.
USP <233> Analysis of DMSO Soluble Drug Substances and
Excipients Using NexION ICP-MS
Jonathan L. Sims, PerkinElmer, Chalfont Rd., Seer Green, HP9 2FX
United Kingdom, Fadi Abou-Shakra
Compliance with the requirements of United States Pharmacopeia (USP) <232>
that “Elemental impurity levels present in drug substances and excipients must be
known, documented and made available on request” is demonstrated through the
application of NexION inductively coupled plasma mass spectrometry (ICP-MS) following the procedure presented in USP <233> for samples prepared directly as
organic solutions. A range of common excipients and drug substances that are used
in the formulation of readily available high dose oral medicines have been studied
and the levels of the 15 target elements discussed.
SEM-EDS and Multi-Instrumental Analysis: Studying the Corrosion
of Ag 999 and Ag 925 Inside and Outside Vitrines
Paula Homem, Faculdade de Letras da Universidade do Porto, Via
PanorГўmica s/n, Porto, 4
​ 150-564 Portugal
A study about fine silver and sterling silver tarnishing has been conducted in uncontrolled NW Portuguese exhibition contexts, not confined to vitrines. Outdoor/
indoor/inside vitrines atmospheric deposition, temperature and relative humidity
(RH) were studied. Ag 999 and Ag 925 samplers were exposed at 45В° angle and
used to model artifacts’ reactions, in response to seasonal conditions and annual
exposure. A multi-instrumental analysis approach was adopted to study interactions
effects, considering scanning electron microscopy coupled with X-ray microanalysis
(SEM-EDS), X-ray photoelectron spectroscopy (XPS), linear sweep voltammetry
(LSV) and colorimetry. The corrosion process proved to be complex and sensitive to
seasonal conditions, tending to slow down with time. Ag 925 is always more reactive
than Ag 999. Globally, the corrosion products detected were: Ag2O, Ag2O2, AgCl2ВЇ,
AgCl, AgHS, Ag2S, Cu2O, Cu(OH)2, CuO, CuCl, CuCl2ВЇ, CuCl2 and 3Cu(OH)2.
CuCl2. Formation of oxides and hydroxides correlated with relative absence of chloride ions inside vitrines and on down-facing surfaces, and was favored by relatively
high O3 in spring and summer and by relatively high NO2 and RH in autumn and
winter. Relatively high presence of chloride ions outside vitrines and on upward-facing surfaces, correlated with formation of chlorides and hydroxichlorides. Formation
of silver sulfides, detected only after a year-long exposure and mainly on Ag 999,
correlated with relative absence of chloride ions inside vitrines and on down-facing
surfaces. Surprisingly, their presence was only detected in association with particles
of sodium and ammonium sulfate. SEM-EDS contributions to the results are presented and discussed.
Determination of Elemental Impurities: A Practical Approach from
a Contract Lab
Samina Hussain, Exova, 9240 Santa Fe Springs Rd., Santa Fe Springs,
CA 90670
There are several challenges a contract lab faces with the determination of elemental impurities in finished drug products, active pharmaceutical ingredients (APIs),
and excipients. Due to the expense and complexity of inductively coupled plasma
instrumentation, a contract lab is often contacted by manufacturers to implement
compliance with the new elemental impurities chapters, such as United States Pharmacopeia (USP) <232>, USP <233>, EP 5.20, and International Conference on
Harmonization (ICH) Q3D. A common issue arising during the method development
and validation process concerns setting appropriate specifications for a particular
material. An evaluation of the specifications and how they relate to each and every
drug product, raw material, and the manufacturing process as a whole is required.
Regulatory compliance in different regions of the world may require a risk assessment involving different elements. Mastering sample preparation and analysis methods for a wide range of materials and specifications requires experience in these
sensitive techniques. Contamination controls at ultra trace levels is also a challenge.
An understanding of potential interferences associated with the instrumentation and
specific analytical approach is essential. Finally, there is a concern about analytical
capacity of existing contract labs for scheduling the numerous method validations
required once the chapters have been finalized. This presentation addresses these
Accessible Microanalysis in Authentication and Attribution: A Case
Soraya AlcalГЎ, The Hispanic Society of America, 613 W 155th St., New
York, NY 10032, HГЁlГ©ne Fontoira, Thiago Assan Piwowazrczyk, Marcelo
Myada RedГ­golo
Small to medium collections often lack ready access to advanced analytical expertise and services to augment curatorial and connoisseurship inputs in authentication
of works of art. However, with careful organization a lot may be accomplished using
basic photon microscopy, sometimes augmented by readily accessible methods of
elemental and molecular analysis. In this presentation we show how this is possible, by using basic materials analytical microscopical techniques to study a painting
from the collection of Fordham University, NY, NY. We wished to help determine
the period of the painting, which seems to be High Baroque or neo-Baroque Italian.
Our painting evokes the European Counter-Reformation, especially certain Italian
masters, but it has also been credibly suggested that this work is from Mexico. If the
provenance is Mexican, the painting could still be Italian, since Mexicans imported
much Italian art starting in the colonial period before 1821, and throughout the 19th
century. Moreover, the Academy of Fine Arts of San Carlos, established at Mexico City in the late 18th century, sent Mexican artists to Rome throughout the 19th
century, and their works reflect Italian influence. Our aim was to test attributed provenience and period, possibly involving painting techniques much associated with
Mexican artists, and to note alterations attributable to aging or to prior treatment.
Use of EDXRF for the Determination of Elemental Impurities in
Pharmaceutical Materials
Nancy Lewen, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903
United State Pharmacopeia (USP) and International Conference on Harmonization
(ICH) requirements for elemental impurity monitoring in pharmaceutical products
are changing and moving towards performance based requirements, not partial to
specific techniques. Although USP <233>, which describes the methods for elemental impurity testing, still focuses on inductively coupled plasma (ICP) and ICP-mass
spectrometry for historical reasons, other techniques are now allowed. Due to typical ICP challenges like sample digestion, waste streams and operation costs, companies seek for more simple, fast and cost effective means to perform these analyses. This study focuses on X-ray fluorescence (XRF), which is now a compendial
technique, using a PANalytical Epsilon 5 Energy Dispersive (ED) XRF spectrometer
as an alternative to ICP for determination of elemental impurities in pharmaceutical
products in a non-destructive manner.
Scientific Analysis of Pietro da Cortona’s The Triumph of David
Kristen E. Watts, Villanova University, 800 E. Lancaster Ave., Villanova,
PA 19085, Amanda Norbutus, Anthony Lagalante, Kristin deGhetaldi
A two-year restoration campaign at Villanova University has begun on a 17th century masterpiece attributed to baroque master painter Pietro da Cortona. The massive
painting, colloquially known as The Triumph of David (144”×228”), has been located
in a library reading room since its donation to Villanova University from Italy after
sustaining damages during the Battle of Nemi in World War II. The painting requires
a varnish replacement, canvas mends, and corrections to prior, less-scrupulous restoration campaigns. In order to provide the best conservation and treatment plan for
An Update on General Chapters <232> and <233> and
Implementation Timelines
Kahkashan Zaidi, United States Pharmacopeia, 12601 Twinbrook
Parkway, Rockville, MD 20879
Heavy metals tests have been available in the United States Pharmacopeia and
The National Formulary (USP-NF) for many years. The screening tests in <231> are
non-element-specific limit tests. Sample preparation and instrumental technology
available today (e.g., closed-vessel microwave digestion followed by atomic absorp-
2014 EAS Abstracts
November 2014
the painting, it is important to employ a multitude of analytical techniques to obtain
the most complete understanding of the painting’s provenance, history, and place in
Pietro’s oeuvre. Through scientific research, we can obtain a better understanding
of the materials and methods utilized in the painting’s original construction and subsequent restoration campaigns. This presentation summarizes our findings using a
host of traditional analytical methods to image and characterize the pigments and
binders present in the original and restoration layers of the painting, including: fluorescence microscopy, infrared microscopy, portable X-ray fluorescence, and scanning electron microscopy paired with energy dispersive X-ray spectroscopy. New
research in our laboratory is aimed at employing desorption electrospray ionization
mass spectrometry (DESI-MS) to image binding materials present in paint layer
cross-sections. DESI-MS is capable of 100 micron image resolution and can readily
distinguish between areas of oil paint versus areas of more modern materials based
upon the stratigraphy of characteristic molecular ions.
ples were correctly predicted as human in the species specific model. An external
validation was performed using hair and fiber samples, which were not used in the
training set. Overall, this method is able to quantitatively identify a sample of hair as
being of human origin with a high degree of confidence, using a non-destructive and
rapid approach, without the need for sample preparation or specialization, proving
its significance to the field of forensic science. The differentiation of animal species
based on the hair analysis is discussed.
Quantitation of Dyes in Forensic Fibers Using UHPLC-TOF MS and
Building a Dye Database with AxION EC ID Software
Joanne Mather, PerkinElmer, 940 Winter St., Waltham, MA 02451,
Sharanya Reddy, Bonnie Marmor
Fibers originating from textiles in carpet, furniture, and clothing are often used
as evidence at crime scenes. Fiber analysis is traditionally performed using various non-destructive techniques including optical microscopy, visible and infrared
micro-spectrophotometry. Chromatography techniques such as poor resolving
thin-layer chromatography or higher resolution high-performance liquid chromatography (HPLC) with UV or diode array detection (DAD) can also be used for fiber
identification. In this study, we show analysis of dyes extracted from fibers using ultra HPLC coupled to time-of-flight mass spectrometry (TOF- MS), providing a more
specific and sensitive technique. TOF-MS provides a higher degree of discrimination between closely resembling dyes with similar functional groups of detection
compared to UV or DAD detection using accurate mass measurements and isotope
ratio confirmations. We also provide a method to build a database for forensic dyes
using accurate mass and isotope profile information acquired from the time-of-flight
mass spectrometer along with the proprietary AxIONВ® EC IDв„ў software.
Synchrotron X-Ray Microtomography, Confocal Laser Microscopy
and SEM Imaging Applied to Characterization of Fungal
Biodeterioration of Paper in the Context of Cultural Heritage
Hanna M. Szczepanowska, Smithsonian Institution, 4210 Silver Hill Rd.,
Room E2101, Suitland, MD 20746, Diwaker Jha, Thomas G. Mathia
Biodeterioration of paper induced by fungi is one of the most complex phenomena
due to involvement of living organisms thriving on heterogeneous substrate such
as paper. Both materials, paper and fungi, are complex structurally and chemically,
making their characterization very challenging. Black stains produced by Dematiaceous (black fungi) and Chaetomiaceae family fungi were analyzed. Their spatial
and temporal distribution on the paper surface and in the matrix were investigated with Confocal Laser Scanning Microscopy (CLSM) and X-ray Microtomography
(XВµT) complementing scanning electron microscopy (SEM-VP) and transmitted
light microscopy (TLM) imaging. The XВµT data was obtained on ID 19 beamline at
the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The 3D
modeling of fungi in the paper matrix and statistical analysis of cells-size revealed
presence of yeast , information which could not be obtained solely from the surface
or microscopic analysis. This new analytical multi-scale approach combining several techniques elucidates dynamic interactions of fungi with paper, deposition of biological cells in the paper matrix and patterns of fungal growth on the surface of paper
providing base for designing preservation strategies for cultural heritage collections.
Characterization of the Binding Medium Used in Roman Encaustic
Paintings on Wall and Wood
Ruben M. Savizky, The Cooper Union, 41 Cooper Square, New York,
NY 10003, John L. Bove, Pedro Cuni, Jorge Cuni, Brielle Eisen
The characterization by means of attenuated total reflectance Fourier transform
infrared spectroscopy and gas chromatography-mass spectrometry of the binding
medium present in eight samples of Roman wall paintings coming from three archaeological sites in Spain and a sample of a Roman-Egyptian mummy portrait
on wood showed strong evidence that the medium in all the studied samples was
composed of beeswax and soap. These results suggest for the first time that Roman
artists used in wall and easel paintings a water soluble encaustic paint of beeswax
and soap. Experimental studies with a wax-and-soap technique showed that this
painting technique allows reproduction of the physical characteristics of many Roman-Egyptian encaustic mummy portraits with greater accuracy than the hot wax
encaustic paint and the alkali-treated encaustic paint often considered to be the
painting techniques used in these portraits. Wax-and-soap encaustic also showed
greater accuracy in reproducing the physical characteristics of Roman wall paintings
than the fresco painting technique, generally thought to be the technique used to
execute such paintings. This study suggests that wax-and-soap encaustic could be
a common painting technique among Roman artists, and its composition could correspond to a lost ancient encaustic formulation searched for the last five centuries
by many artists and researchers dissatisfied with the former reconstructions of the
ancient encaustic painting technique.
Investigation of Body Products on Worn Clothing Substrate Found
at a Fire Scene Conflicting with Ignitable Liquid Residue
Identification by GC-MS
Gina M. Guerrera, University of New Haven, 300 Boston Post Rd., West
Haven, CT 06516, Brooke W. Kammrath, Erika Chen, Michael Valetutti
The question of whether cosmetic and medicinal body product deposits on clothing
as well as the composition of clothing are being mistaken for ignitable liquids is
of considerable importance for criminalists in forensic science laboratories. Body
products and oil secretions can have similar chemical profiles to ignitable liquid
residues (ILR) as a result of comparable chemical compounds that are found in both
sources. This research investigates the substrate contribution of worn and unworn
clothing due to possible body oil, lotions, perfume/cologne, medicinal creams as
well as other body products. Clothing samples of varying fiber content were collected from three individuals having applied a body product provided for them by
the researcher before wearing the clothing item. Passive headspace by activated
charcoal strips (ACS) and desorption with carbon disulfide (CS2) was the method
of choice for sample preparation. The samples were then analyzed by gas chromatography-mass spectrometry with a quadrupole mass analyzer, and evaluated as
to whether the residues could be mis-identified as belonging to a class of ignitable
liquid residues. This research has important implications for criminal justice because
the identification of an ILR on clothing could result in a person being charged with an
arson crime, thus it is critical to be able to differentiate substrates from ILRs.
Withdrawn by the author.
Differentiating Authentic from Counterfeit Drugs by Raman
Fran Adar, Horiba Scientific, 3880 Park Ave., Edison, NJ 08820, Pauline
Leary, Thomas A. Kubic
The introduction of counterfeit drugs into the United States marketplace is important
for two reasons. These products appear without the oversight of regulatory agencies such as the Food and Drug Administration who can confirm the safety and
efficacy of the products. In addition, their sale deprives the legitimate corporations
(both innovator and generic companies) from benefiting from the results of the research and development that have been spent to produce these products. Raman
microscopy provides at least two means of screening these products. On the one
hand, the tablet coating can be removed enabling the acquisition of a Raman map
from which the components’ identities and distributions can be measured. On the
other hand, spectra of the packaging and the printing colorants can be measured for
comparison of suspected counterfeits to the authentic products. Examples of both
are shown in this talk.
Differentiation of Human, Animal, and Synthetic Hair by ATR FTIR
Jeremy M. Manheim, University at Albany, 1400 Washington Ave.,
Albany, NY 12222, Kyle Doty, Greg Mclaughlin, Igor K. Lednev
Hair and synthetic fibers are a common form of trace evidence found at crime
scenes. Currently, the methodology of microscopic examination of potential hair evidence is lacking statistical probability and is inherently subjective. Here attenuated
total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy along with
chemometric analysis was used to identify and differentiate human hair from a synthetic fiber and animal hair with a specific confidence and solely from its spectrum.
Two partial least squares-discriminant analysis (PLS-DA) models were constructed:
one to differentiate natural hair fibers from synthetic fibers (binary) and the second
discriminating human hair from animal hair (species specific). Both models were
successful in internally differentiating the classes examined; synthetic hair was
completely separated from actual hair in the binary approach and all human sam-
Withdrawn by the author.
2014 EAS Abstracts
November 2014
easily-interpretable spectrum with the increased volume sampling in NIR and its
sensitivity to physiochemical industrial processing parameters. Hyperspectral imaging has clear advantages, and has been brought to a number of types of instrumentation. We anticipate providing example data from chemical and biological material
analysis, such as precision agriculture, threat detection, contamination control, forensics, anti-counterfeiting, point-of-care medical diagnostics, and numerous other
in-field applications.
Fit-for-Purpose Miniature NIR Spectroscopy for Solid Dosage
Continuous Manufacturing
Krizia Karry, Rutgers University, 98 Brett Rd., Piscataway, NJ 08854
Near-infrared (NIR) spectrometers have long been the process analytical technology (PAT) tools of choice for pharmaceutical and industrial scientists seeking to gain
both physical and chemical information from a sample. More recently, the development of fast and inexpensive miniature NIR instruments have made possible the
real-time monitoring of individual unit operations within a process. For continuous
manufacturing operations this means that, NIR-based multivariate models can be
used as supervisory tools for closed loop control and eventually real-time release
of the drug product. In this presentation we discuss different case studies for which
the implementation of ultraportable spectrometers of low wavelength ranges (950
– 1650 nm) aided in formulation and process trouble-shooting and ultimately, how
these were used as PAT tools for the continuous manufacturing of tablets and orodispersible polymer films.
Off-Line and In-Line Monitoring of Mulling Processes
Savitha S. Panikar, Rutgers University, 19 Paulus Blvd., New Brunswick,
NJ 08901, Suyang Wu, Benjamin J. Glasser, Rohit Ramachandran
The characterization of mulling/extrusion of alumina, for the purposes of making
catalyst supports, is investigated. Specifically: 1) the optimal instrument set-up of
a near-infrared (NIR) spectrometer and, 2) monitoring and quantifying critical quality attributes (CQAs) of a continuous mulling process is reported. For this, off-line
and in-line calibration standards of alumina/nitric acid granules with known water
concentrations were prepared. An optimized unit operation was set up to produce
extrudable granules. Subsequently, the relationship between sample-detector distance and prediction accuracy was investigated for a JDSU microNIR spectrometer.
It was found that sample-detector distance was optimal around 15mm at 15mm of
sample thickness and 25 of scan number. Additionally, set-ups that could be used
for in-line monitoring have been investigated including monitoring from the side and/
or bottom surface through glass or plastic. Next, after ensuring adequate method
accuracy, NIR prediction models were built for the in-line measurement of critical
quality attributes: granule water content and agglomerate size.
The Next Generation of Long Wavelength Handheld Raman
Spectrometers for Raw Material Inspection and Beyond
Claire Dentinger, Rigaku Raman Technologies, 14 New England
Executive Park, Suite 102, Burlington, MA 01803, Mark Mabry, Claude
Handheld Raman analyzers are very well suited for material identification outside
the analytical laboratory and they are becoming ever more accepted and used for
portable measurements in such diverse areas as the pharmaceutical industry, safety and security professionals and academic institutions. We have recently designed
a smaller and higher performance handheld spectrometer with a 1064 nm excitation
wavelength. The use of 1064 nm excitation reduces fluorescence for most materials. This allows the 1064 nm excitation Raman to measure many materials that
cannot be measured at lower excitation wavelength as well those materials which
can. Using a handheld Raman, incoming raw material inspection can be done in a
warehouse or loading dock not just in analytical laboratory. This can reduce material
hold times and analysis costs. Using 1064 nm excitation allows a wider range of
materials to be investigated. For example polysorbates are often high purity and
used in sterile applications, this combined with the fact that they will degrade upon
exposure to oxygen makes analyzing these materials in the original amber bottles highly advantageous. We investigate how these Raman measurements in the
sealed containers compare at different excitation wavelength and for different polysorbates. Other incoming raw materials and pharmaceutical products are discussed
in terms of analysis with different Raman wavelengths and specificity of Raman to
identify closely related materials.
Achieving Greater Process Understanding through On-Line
Reaction Monitoring
Michael L. Hall, SABIC, 1 Noryl Ave., Selkirk, NY 12158, Nancy L.
Jestel, Eylem Tarkin-Tas, Mark Denniston, Carolyn Degonzague
It has been well demonstrated across different industrial environments that on-line
process analytical technology for reaction monitoring can provide chemists and engineers greater understanding of chemical processes. By simultaneously collecting
data from multiple sensors, quick transitions can be detected that likely would be
missed by off-line techniques due to delays associated with sampling and analysis.
During this talk, examples are presented where lab scale oxidative coupling polymerizations were monitored real-time by Fourier transform infrared (FTIR), near-infrared (NIR) and ultraviolet-visible (UV-VIS) spectroscopies. In the NIR spectra, the
signal reduction of hydroxyl overtones associated with phenolic monomers can be
observed while bands associated with oligomers and polymers were more easily
detected by FTIR. UV-VIS spectroscopy was employed to gain greater sensitivity
for the detection of low level reaction intermediates. These data sets were complemented by real-time temperature and agitation data streams. Chemometric decomposition and correlation techniques were employed to extract information from the
individual and combined data sets. The process understanding gained from these
models was leveraged to make more informed decisions during lab scale process
Mixture Analysis Using Handheld Raman Spectrometer
Dawn Yang, B&W Tek, 19 Shea Way, Newark, DE 19713
Over the past few years, Raman technology has been increasingly applied in fields
that require equipment that is portable, gives quick results, and is easy to use. While
the data (spectra) collected using Raman on pure chemical compounds are known
for their distinct peaks and current software with advanced algorithms allow for great
selectivity, attempting to define materials of impure compounds and mixtures requires a mixture analysis function with new and effective algorithms. As most materials found in the field are mixtures, a handheld Raman spectrometer that is easy to
carry around and is simple to use is ideal for onsite applications when equipped with
mixture analysis capabilities. In this study, mixture analysis on a handheld Raman
spectrometer is reviewed. Results for different types of mixtures are discussed.
In-Line PAT Method Development for Flowing Pharmaceutical
Blend Homogeneity Monitoring
Fan Zhang-Plasket, Merck, Sumneytown Pike and Broad St., West
Point, PA 19486, John P. Higgins, Cat MacConnell, Mano Ramasamy,
Robert F. Meyer, Charles E. Miller, Jennifer Pai
In-line, real-time measurement of flowing pharmaceutical blend homogeneity is a
key element for demonstrating the feasibility of continuous blending technology. The
same measurement can be used for the quantitative blend homogeneity in a feed
frame during tablet compression to detect powder segregation. Process analytical
technology (PAT) analysis could potentially be used for process control and/or real-time release. The development of quantitative multivariate models using in-line
near-infrared and Raman data is presented. The performance of the models on a
continuous blending process and during a tablet compression process along with
long term model robustness is discussed.
Miniaturized Multi-Spec (UV-NIR, Raman and Hyperspectral)
Instruments in Practical In-situ Analysis
Fredrick Haibach, BaySpec, 1101 McKay Dr., San Jose, MA 95131
Using “orthogonal” techniques that provide additional, and complementary data,
provides additional confidence in analytical models. The advantage can be powerful
if the data arises from the same source and the combination of instrumentation is
sufficiently reliable. Chromatography has become an extremely useful and powerful
tool due to the use of hyphenated techniques that use this “orthogonal information”
principle. BaySpec is creating advances in device miniaturization and integration,
efficiently combining ultraviolet near-infrared (UV-NIR), Raman and hyperspectral
imaging into reliable, and affordable, instrumentation that we call “Multi-Spec.”
The hyphenated methods have been described before; these instruments provide
a straightforward path to measurements or integration into a predefined sampling
setup. Improved reliability and stability arises from the use of volume phase gratings
(VPG), solid-state light sources and detectors. We report on a variety of BaySpec’s
newly developed miniaturized systems including the world’s first multi-excitation
wavelength miniature Raman spectral engines (Raman-Raman), Amphi-specв„ў
(NIR-Raman), and recently, handheld hyperspectral imagers (spatial-NIR). Advantages and specific, real-world examples of the different techniques are presented.
Raman-Raman instruments allow the efficient investigation of samples that exhibit
fluorescence, resonance Raman signatures, absorbance, or low Raman scattering cross-sections. NIR-Raman systems combine the specificity of Raman and its
NIR Analyses in the Field; How to get better Answers
Franklin E. Barton, Light Light Solutions Instruments, 165 Sunnybrooke
Dr., Athens, GA 30605, James A. de Haseth
For over a half century near-infrared (NIR) has been used to make analyses of agricultural commodities and crops easier, quicker and provide improved products to
all. In this half century the instrumentation has become easier to use, less expensive
and more reliable. The software has evolved from “homegrown” programs to very
powerful, user friendly chemometric packages. We have become very comfortable
in our heated, air conditioned, well-lighted laboratories and that is a good result of
years of research effort. However, there are issues which remain to be resolved.
First, there is the issue of sample preparation. This issue has been with us from the
beginning and there have been great improvements in instrument sampling geometries and ways to make the analysis with minimal sample preparation. Even so there
2014 EAS Abstracts
November 2014
is always the risk of some change in the sample that is correlated to the sample’s
modification more than to its composition. Second, the delay in analysis caused by
transport from the source, storage at the laboratory and subsequent preparation
can affect the results and therefore the accuracy of NIR methods. In recent years
we have spent our efforts to move NIR analyses from the laboratory to the manufacturing plant and the field. This presents a new and different set of challenges
and is illustrated by the applications we have developed. These challenges and
their solutions have resulted in new instrumentation that is flexible, lightweight, and
robust. It has required new approaches to software and the hardware, particularly
to scan the sample.
cal project is used to demonstrate how significant the method robustness impacts
the success of a product commercialization. The analytical method for the determination of assay and impurities of an oral solid dosage form was developed and
proven to be robust via successful validation and technology transfers. However,
when used to analyze samples from the manufacturing process transfer batches,
low potency results were observed. It is critical to understand whether it is due
to method robustness or product manufacturing issues which warrant further process optimization to ensure the success of commercialization. A comprehensive
investigation on the analytical method looking into many relevant parameters, such
as diluent composition and sample preparation procedure was conducted. It was
concluded that the method is sufficiently robust to consistently generate accurate
and precise results. With the demonstrated proof of confidence in the method, the
consistent analytical results allowed for the discovery of the low potency root cause.
The manufacturing process was subsequently optimized and the success of product
commercialization was ensured.
High-Throughput Virtual Slit Technology: Benefits for Polymer
Yusuf Bismilla, Tornado Spectral Systems, 555 Richmond St. West, PO
Box 218, Toronto, ON, M5V 3B1 Canada, Jeffrey T. Meade, Bradford
B. Behr, Andrew T. Cenko, Brandon DesRoches, Jared Slaa, Arsen R.
High-throughput virtual slit (HTVS) technology is a revolutionary new design paradigm for dispersive spectrometers that enables slit-like spectral resolution without
the associated throughput loss, significantly enhancing performance for any given
system size. This makes the HTVS particularly useful for real-time monitoring applications where speed and sensitivity are both required. Raman chemical identification is a highly sensitive method of determining the chemical makeup of samples as
well as inferring information on structural properties of the chemicals themselves.
On a manufacturing line, fast product verification leads to increased quality and lower costs—HTVS technology allows for significantly decreased measurement time,
providing substantial productivity increases. Raman measurements are traditionally signal limited due to the low Raman signal intensity. Slit-based spectrometer
designs must balance throughput and resolution requirements, severely impacting
system sensitivity when high spectral resolution is required. HTVS technology improves measurements by mitigating the standard tradeoffs between spectrometer
throughput and resolution. HTVS technology allows for size and weight reductions
while maintaining performance. This enables significant enhancements where small
and/or lightweight systems are required. HTVS spectrometers have the capability
to open entirely new regimes to spectroscopic work. We will present an in-depth
application case study showing the benefits of HTVS to polymer extrusion. Real
time measurements of the polymer melt in an extruder provide valuable information
on melt properties, both chemical and mechanical, and allow for quantitative and
qualitative analysis.
Incorporation of Benchtop NMR Spectroscopy into Undergraduate
Laboratories: An Active-Learning Approach
Susanne Riegel, Nanalysis, Bay 4, 4500 5 St., NE, Calgary, AB, T2E
7C3 Canada
Nuclear magnetic resonance (NMR) Spectroscopy is one of the most widely used
characterization techniques in chemistry. Despite pedagogical shifts towards active-learning and guided-inquiry approaches, incorporation of NMR spectrometers
directly into undergraduate curriculum has remained largely limited due to mitigating
factors of size, cost and availability of high-field spectrometers. As a result, students
do not gain hands-on access to this instrumentation, particularly in the beginning
states of their programs. An emergence of a new class of benchtop NMR spectrometers (42 – 60 MHz) that are affordable, portable and do not require weekly
upkeep or maintenance can facilitate the introduction of this technique at all stages
of chemical education. They also offer sufficient resolution and sensitivity for structure elucidation, reaction monitoring, and basic quantitation. Herein, we describe
methods for the incorporation of the NMReady benchtop spectrometer into common undergraduate laboratory experiments and illustrate how students can learn
the proper technique to use an NMR spectrometer, prepare samples, monitor and
characterize reaction mixtures. In combination with modern, easy-to-use interfaces
and network accessibility, these spectrometers are perfect for teaching and training
students use in the elementary chemistry laboratory.
Analytical Pipetting of Serum and Serum-Like Liquids
John T. Bradshaw, Artel Inc., 25 Bradley Dr., Westbrook, ME 04092,
Richard H. Curtis, Rachel Parshley
Many core types of chemical analyses are based upon analytical techniques involving accurate delivery of liquid components. A common tool developed to deliver
these liquid components is the handheld micropipette, as well as automated versions of the same. Handheld and automated pipettors have become commonplace
tools, especially used in many biological and pharmaceutical laboratories. While
these tools are familiar to many, their performance differences when pipetting different types of solutions are often over-looked and neglected aspects that relate
directly to the accuracy and reproducibility of pipetting performance. For example,
it is commonly known that water pipettes differently than serum. This presentation
addresses the difference in performance of a handheld micropipette when dispensing water versus various types of animal and human serum. These differences are
quantifiable and can be accounted for through careful experimentation and attention
to physical pipetting details. Details on achieving ideal performance when pipetting
serum are also discussed.
Analytical Testing for the Cannabis Industry: A New Era Presents
New Opportunities
Christopher J. Hudalla, ProVerde Laboratories, 420 Fortune Blvd.,
Milford, MA 01757, Nathan M. LaCroix
The cannabis industry has been thriving for many years, with the use of cannabis
tracing back thousands of years. However, the illicit status of the herb throughout
much of the world has stifled commercialization and research, forcing most activities
underground, often times with high risk and minimal accountability. As a result of
this, technological advances in science and analytical instrumentation have found
little to no application to this diverse field. Recent advances throughout the world in
legislation, regulation and public acceptance have opened the door for legitimacy
of this industry. This provides the new opportunity for the use of the latest advances
in scientific instrumentation and methodologies to be applied to different aspects of
this industry, including ensuring consumer safety, basic research, optimization of
cultivation practices, and the design and development of marijuana infused products (MIPs). Like most natural products, cannabis is comprised of an intrinsically
complex chemical profile. To further complicate our understanding of the complexity,
the high potential from contaminants due to human cultivation practices and from
microbiological contamination makes safety profiling critical for ensuring consumer
safety. Here we demonstrate the application of modern technology to the study of
cannabis, employing state-of-the-art chromatographic, mass spectrometric and microbiological methodologies to provide a better understanding of the plant constituents and the possible impurities or contaminants present. The high quality analytical
data generated leads to improved cultivation and manufacturing practices, and most
importantly, increased safety for patients and consumers.
S.M.A.R.T. Laboratory Practices
Julius Neil Buenconsejo, Keppel Seghers Engineering, 108 Pasir
Panjang Rd, Singapore 118535
In recent years, laboratories are facing immense pressure to continuously improve
quality while keeping up with increasing demand in efficiency, productivity and reliability of test results. From large testing facilities to small independent analytical
centers, the complex and intricate nature of the challenges to run optimum operations requires tailor-made solutions for various diversified and distinctive conflicting
needs to achieve maximum throughput. S.M.A.R.T. (S – Specific, M – Measurable,
A – Attainable, R – Relevant, T – Time-based) laboratory practices are simple but
practical approach in improving laboratory operations utilizing invaluable tools and
work-related enhancement programs. Proper application and execution not only
reduce waste and save energy, but also shorten turnaround times (TAT) through
efficient allocation of staff man-hours and resources - ensuring quick delivery of
analytical results and timely reporting of relevant information in accordance with
quality standards and regulatory compliance. For new and existing laboratories,
implementation of such programs drives higher cost-efficiency, improves cost-effectiveness and develops sustainable environment for the next generation of laboratory
Impact of Method Robustness to the Success of Product
Commercialization - Lesson Learned
Xin Bu, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ
08903, John Castoro, Joel Young
The robustness of an analytical method becomes increasingly important when a
new pharmaceutical entity transitions from the development stage to the commercialization stage. Whether a manufacturing process transfer from development to
commercial is deemed successful is heavily dependent on the analytical results,
thus the ability of the method to consistently deliver accurate and precise results
is of significant importance. In this presentation, an example from a pharmaceuti-
2014 EAS Abstracts
November 2014
decade sample requirements have decreased even further with the advent of low
volume (1.7 mm) cryo-probes. The sensitivity limits of these systems have been well
parameterized with samples made from serial dilutions of known compounds. While
these are important experiments, the practical aspects of creating usable samples
at these levels from biological isolates are daunting. Concomitant with these developments in the field of NMR, drug metabolism scientists have honed several in
vitro methods of incubation that can produce metabolites that reflect those found in
animal models. These methods include isolated cell lines, sub-cellular organelles or
recombinant enzyme systems. Together, these disparate developments have created a powerful tool for the structural elucidation and quantitation of metabolites much
earlier in the drug discovery/development paradigm. Using the in-vitro methods discussed above in conjunction with liquid chromatographic isolation and cryogenic
NMR probes, samples in the 10 nmole range can be routinely produced of sufficient
quality to allow full 1H and 13C characterization. In situations where full 13C characterization is not necessary the amount of material required may be decreased to less
than 5 nmole. Once characterized, these samples are of sufficient quantity that they
may be subsequently quantitated and used as reference standards in a variety of
discovery efforts. In this presentation several examples are discussed that highlight
the details and limitations of this process.
Taking the Pain Out of Chromatographic Peak Integration
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, Shaun Quinn
Peak detection and integration are fundamental tasks in chromatography, most
often done using chromatography software. Enabling software to detect and integrate the peaks as desired (or as required by laboratory rules) is challenging and
time-consuming. Common challenges in peak detection include distinguishing
peaks from noise, correctly identifying the underlying baseline, maintaining correct
peak and baseline detection throughout a sequence of chromatograms, and correctly handling rider peaks and other unresolved peaks. Ideally, these challenges
should be addressed using detection parameters so that the same treatment can
be applied across multiple chromatograms automatically, thereby minimizing variations introduced by different operators. However, finding parameter combinations
that produce the desired results has often been a tedious process, causing many
chromatographers to give up and resort to manual integration, which is subjective
and labor-intensive. In this presentation we look at a new peak detection algorithm
that addresses these issues by separating peak detection from baseline determination. It uses second-derivative signal analysis, providing reliable and consistent
peak detection even if the underlying baseline shape changes. An easy to use interface that guides the user through the correct set-up of peak detection parameters
is also shown. Examples of how the tools quickly solve problems with real-world
chromatograms are given.
Innovative Approach to Helium Carrier Gas Conservation in
Analytical Gas Chromatography
Lori Dolata, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale,
CA 94085, Edward B. McCauley, Massimo Santoro, Paolo Magni,
Alexander N. Semyonov
The global helium shortage and price increase cause more and more analytical laboratories employing gas chromatography (GC) to re-assess their consumption patterns of this non-renewable noble carrier gas. The existing solutions to this problem
include changing analytical column, migrating to another carrier gas (e.g., hydrogen) or passively reducing the helium consumption by reducing its usage during analytical runs or switching the GC or GC-mass spectrometry over to nitrogen during
the longer idling periods. In all these cases a considerable amount of time is spent
developing new methods or wasting daily the time needed for the instruments to
come back to normal operations. A new, innovative approach to the conservation of
helium carrier gas will be presented, comprised of separation of the flows inside the
standard split/splitless injector. While preserving the analytical GC column flow with
helium, the septum purge and the split flows are maintained with another inert gas
like nitrogen - even during the analytical run. On average, the helium consumption
is reduced such that a standard cylinder of compressed helium gas can last 3-6
years vs. 3-6 months without and changes in the analytical methods or conditions.
This new patented technology for saving Helium is presented, along with the data
validating its use; the additional practical benefits introduced to the quality of the
chromatographic data are also discussed.
Pulsed Field Gradient Nuclear Magnetic Resonance (PFGNMR)
Studies of Nanoparticle Surfaces and their Interactions
Kebede Beshah, The Dow Chemical Company, 400 Arcola Rd.,
Collegeville, PA 19426, Aslin Izmitli, Antony Van Dyk
Hydrophobically modified ethoxylated urethane resins (HEUR), telechelic polymers,
with molecular weights greater than 30 KDa, have been used to control viscosity of
nanoparticle dispersions. Extensive work has been done and published over the
past three decades on the associative nature of these amphiphilic HEUR polymers
in water. A molecular level study that shows the interaction between the HEUR polymer and the nanoparticles was conspicuously missing. The major reason for this
is the inability to detect the critical low level hydrophobic end group and urethane
linker signals in an overwhelming matrix of aqueous phase materials of the dispersions. We applied diffusion filtered one-dimensional (1-D) and two-dimensional
(2-D) nuclear magnetic resonance (NMR) experiments that enabled the elimination
of unwanted signals based on their higher diffusion coefficients and/or dynamics of
the various species to detect the desired HEUR signals without any perturbation of
the system. With these pulsed field gradient (PFG) NMR techniques, we are able
to probe HEUR polymer-nanoparticle interactions as a function of HEUR end group
and nanoparticle surface hydrophobicity. The method is used to obtain adsorption
isotherms of the composite in-situ providing unperturbed dynamic equilibrium systems compared to the widely used centrifugation method. Our observation has led
to a new model of HEUR-nanoparticle interaction that sheds light on the elusive
mechanism of high shear viscosity (10,000 s-1) of HEUR-latex composites. The details of the diffusion and dipolar filter 1-D and 2-D NMR techniques and implications
of these molecular level interactions to shear thinning rheology of HEUR-nanoparticle composites are discussed.
High-Throughput Screening of Solid Pharmaceuticals with 35Cl
Solid-State NMR
Robert W. Schurko, University of Windsor, 401 Sunset Ave., Windsor,
ON, N9B3P4 Canada, Michael J. Jaroszewicz, Anthony R. Sandre,
Andrew M. Namespetra, Kristopher J. Harris, Adonis Lupulescu, Lucio
Structural screening of solid active pharmaceutical ingredients (APIs) is a priority in
the pharmaceutical industry, and a major problem currently confronting solid-state
chemists. More than 80% of APIs are manufactured, stored and/or ingested as solids; therefore, they must undergo a rigorous quality assurance process to monitor their identities, structures and purities. Since hydrochloride (HCl) salts of APIs
constitute more than 50% of known pharmaceutical solids, 35Cl solid-state nuclear
magnetic resonance (SSNMR) is an appealing choice for their characterization.
35Cl is a quadrupolar nucleus (spin = 3/2), with NMR powder patterns influenced by
both chemical shift (CS) and quadrupolar interactions (QI). Though the patterns are
quite broad, and in some cases challenging to acquire, they are rich with structural
information, and provide a unique fingerprint for each API, as well as associated
polymorphs, solvates, hydrates, disproportionation forms and impurities. In this lecture the latest advances from my research group in the development of 35Cl SSNMR as a technique for high-throughput screening of APIs are presented. Discussion
includes: 1) the utilization of 35Cl SSNMR and plane-wave DFT calculations for
fingerprinting, identification and discovery of solid phases of APIs; 2) the application
of two powerful pulse sequences, WURST-CPMG and BRAIN-CP, which are used
for the rapid acquisition of broad 35Cl powder patterns associated with APIs; and
3) the use of powerful spectral editing techniques, including two-dimensional relaxation-assisted separation (2-D RAS) methods which can be used to differentiate
broad overlapping patterns and aid in making spectral assignments.
Automated, Rapid, Reliable Determination of Dissolved Gases in
Water by Static Headspace Gas Chromatography
Lori Dolata, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, Andrea Caruso, Richard Jack, Massimo Santoro
Hydraulic fracturing, the fracturing of a rock by a pressurized liquid is a well stimulation technique in which typically water is mixed with sand and chemicals, and
the mixture is injected at high pressure into a wellbore to create small (< 1mm)
fractures in order to maximize fluid removal and well productivity. While this oncevery diffused technique makes accessible big amounts of formerly non-accessible
hydrocarbons, the dissolved gases have become a controversial environmental and
health matter with some countries completely banning the practice. Public outcry
over preservation of water quality has led the U.S. EPA and other state agencies to
investigate the impact of hydraulic fracturing on the quality of environmental waters.
Some procedures for testing waters for dissolved gases through static headspace
sampling exist already, like RSK 175 standard operating procedure, but since the
target compounds are light hydrocarbons from methane through propane, are extremely volatile, a closed sampling system is required. A high- throughput test method using robust, automated and relatively inexpensive instrumentation like static
headspace and gas chromatography with flame ionization detection is used in this
paper, and data is shown for the quantitative determination of dissolved gases in
ground, waste and drinking waters.
Assessing Qualitative and Quantitative NMR Limits in a Drug
Metabolism Environment
Gregory Walker, Pfizer, Eastern Point Rd., Groton, CT 06340, Raman
Sharma, Shuai Wang
With the introduction of cryo-probes the minimum sample requirements for the
acquisition of interpretable and quantifiable one- and two-dimensional (1-D/2-D)
nuclear magnetic resonance NMR data has decreased significantly. Over the last
2014 EAS Abstracts
November 2014
Strategies to Enhance the Sensitivity and Reduce the Ambiguity of
NMR Spectra of Complex Biological Assemblies
Galia Debelouchina, Princeton University, Department of Chemistry,
Princeton, NJ 08540
Multidimensional nuclear magnetic resonance (NMR) spectroscopy is capable of
providing atomic-level structural information of complex and heterogeneous biological systems. As the size and the complexity of these systems increases, however,
the interpretation of NMR spectra becomes more challenging due to poor sensitivity
and ambiguity in the assignment of the observed interactions. Dynamic nuclear polarization, a method that significantly improves the sensitivity of magic angle spinning NMR experiments, can be used to generate spectra with greatly improved signal-to-noise ratios and unprecedented savings in data acquisition times. Advanced
isotopic labeling strategies such as site-specific, sparse and segmental labeling,
on the other hand, can be used to simplify the informational content of the spectra
and facilitate the interpretation of structural constraints. This talk focuses on the
combined use of dynamic nuclear polarization and specific labeling strategies used
to elucidate the higher order structural organization of complex protein assemblies
such as amyloid fibrils.
Automated Kinetic Forced Degradation Development and
Lina Liu, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065, Timothy
Rhodes, Roy Helmy, Keri Joy McNamara, Donna Carroll
Forced degradation studies are an important element in assessing a compound’s
drug-like properties and understanding the chemical liabilities that will need to be
addressed in preclinical development. Forced degradation studies help in early development to devise and to validate stability-indicating analytical methods. Kinetic
stability data allow for evaluation of degradation rates and can provide an understanding of the sensitivity to different degradation pathways and can even help to
distinguish between primary and secondary degradation products. Finally, this data
can help in devising a strategy to minimize excipient induced degradation and can
help to differentiate between excipient derived adducts and drug substance degradation products. Forced degradation studies are conducted multiple times over the
life of a drug development to serve a various purposes and, most often, the work
spans across multiple groups. In order to harmonize the procedures of forced degradation, an automated method for forced degradation was developed, utilizing the
CTC LEAP PAL workstation automation system. The automated forced degradation
approach significantly reduces the amount of manual labor used to perform the tests
and harmonizes the operational procedures of forced degradation. The automated
forced degradation system is user-friendly and is intended to be used as a “walk-up
system” that is able to prepare forced degradation and linearity samples, perform
on-line high-performance liquid chromatography analysis including sampling at multiple time points, and generate reports automatically using Empower customized
report template. The details of the system are discussed along with a number of
case studies demonstrating its use.
Understanding the Behavior of Different Tablet Disintegrants Using
High-Resolution FLASH Magnetic Resonance Imaging
Anuji Abraham, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, Dolapo Olusanmi, David Good, Denette Murphy, Daniel
McNamara, Venkatramana Rao, Andrew J. Ilott, Alexej Jerschow
Understanding the behavior of tablet disintegrants is valuable in the development
of pharmaceutical solid dosage formulations. High-resolution real-time magnetic
resonance imaging (MRI) allows viewing the full cross section of the tablet disintegration process. The fast low angle shot (FLASH) sequence has been used to
understand the behavior and mechanism of disintegration. Furthermore, FLASH sequence minimizes experimental time allowing high resolution images to be collected
in < 10s. In the current work, FLASH MRI methods have been used to understand
tablet disintegration mechanisms resolving swelling, gelling and erosion processes.
The study was done on a variety of disintegrants, such as sodium starch glycolate,
crospovidone and croscarmellose sodium. By studying different grades of these
disintegrants a correlation could be established between their disintegrating mechanism and rates with their chemical compositions, cross linking, hydrophilic nature
and ionic strengths. Quantification of the disintegration rates was possible using a
bespoke voxel analysis. Videos based on the MRI results were also produced helping to distinguish between different disintegration mechanisms. Overall, FLASH MRI
methods have been implemented and shown to be a useful tool for understanding
fast tablet disintegration giving valuable insights into the development of pharmaceutical formulations.
Resolving the Isolation Challenges of Color Impurities in NOXAFIL
Weidong Tong, Merck, 126 E. Lincoln Ave., Rahway, NJ 07065
NOXAFIL, a marketed posaconazole IV injection formulation, is proven to help prevent invasive fungal infections from Aspergillus (mold) and Candida (yeast). Both
fungi are relatively harmless in healthy people but can cause serious illness – and
even death – for individuals who have suppressed or severely weakened immune
systems. During stabillity study of the drug formulation solutions, a few low level
(0.02 area %) degradation impurities were observed. Initial attempts to purify and
identfy the impurities using liquid chromatography tandem mass spectrometry (LCMS-MS), or extractions for impurities enrichment, however, proved to be unsuccessful. Instead, by using Amberchrom CG161C resin treatment and eluting with
water and organic solvents with adjusted pH, we were able to successfully remove
sulfobutyl-ОІ-cyclodextrin, and enriched impurities to about 0.1 to 3% in a few fractions. Following that, prep high-performance liquid chromatography was employed
to successfully separate and purify the impurities.
Cleaning Verification: TOC or HPLC?
Elizabeth C. Moroney, Bristol-Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08903, Mariann Neverovitch
Validated cleaning verification methods are required by federal and international
regulatory agencies to comply with good manufacturing practice standards. Cleaning verification ensures the safety and purity of the product, satisfies regulatory
requirements and assures internal control over processes. Cleaning verification
methods are typically developed for each pharmaceutical compound individually,
as well as for detergents. Traditionally, high-performance liquid chromatography
(HPLC) has been the method of choice for analyzing cleaning verification samples
due to the widespread availability of instrumentation and the ability to quantify low
levels of product. Recently, total organic carbon (TOC) analysis has become more
prevalent owing to the simplicity of use and acceptance of the method by regulatory authorities. In this presentation, I summarize and compare the techniques
and explore a case study that highlights the challenges encountered during method
Analytical Challenges Encountered in Compatibility Testing of
Parenteral Dosage Formulations Used in Preclinical Studies
Lara D. Penn, Merck, 770 Sumneytown Pike, West Point, PA 19486,
Samantha Devine, Oksana Leidy, Michelle Mathe, Julie Novak,
Elizabeth Sander
Medical devices used for the storage and/or delivery of parenteral dosage
formulations to preclinical species involve plastic, rubber, glass, and stainless
steel. It is important to evaluate the compatibility of parenteral dosage formulations
with materials used for dose administration and storage. Ideally, an important
performance characteristic of the materials used in medical applications is chemical
inertness, and material compatibility is critical prior to conducting any preclinical
toxicology assessment. There exists a high level of concern for parenteral dosage
formulations that are solutions and suspensions since there is a higher risk for
interaction between storage and/or delivery component(s) and parenteral dosage
formulations. The three parameters typically evaluated in compatibility studies
are drug adsorption, device induced active degradation, and the appearance of
leachables and/or extractables. Analytical challenges encountered during the
evaluation of the compatibility of administration sets and storage containers with
preclinical formulations are discussed.
Justification of Applying Acceptable Residual Limit (ARL) Based
on Toxicological Data and Visual Inspection
Mariann Neverovitch, Bristol Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08903
Acceptable residual limit (ARL) value is based on subject exposure limit (SEL),
minimum batch size, maximum daily dose and total surface area. For total organic
carbon (TOC) analysis, the ARL value is corrected for percentage of carbon in the
molecule, and measured in parts per million units (ppm). Based on this calculation
and the molecular formula of a given compound, the value of ARL may vary from
very low (<0.1ppm C) to very high (>200 ppm C). If the ARL is lower than 1 ppm C,
the TOC technique will not be applicable due to background contribution from the
blank. If the ARL is too high, then the method limit should be evaluated and an alert
limit should be introduced. Historical cleaning validation data indicates that the vast
majority of drug product residue is visible at a level of 1 ppm C. The system suitability solution (KHP) for TOC analysis is prepared at 5 ppm C. Therefore it is scientifically justifiable that the value of the alert limit should be between 1 and 5 ppm C.
Migration of Ink Components into Transdermal Patch
Kenneth Wong, SGS Life Science Services, 75 Passaic Ave., Fairfield,
NJ 07004, Richard Bunnell, Sushmeet Singh, Gayatri Trivedi, Terry
Panetta, Xinjie Song
Food and drug packaging is printed with colorful labels; however, there is limited
knowledge about the potential migration of printed ink components into the product.
With no global legislation available and ill-defined terminology in the scientific community, formulating ink used in packaging becomes a challenge for suppliers and
users who are concerned about potential migration of harmful components. Possible sources of migratable compounds in inks and coatings include UV photo-initiators, mineral oils, and resins. No single analytical method can detect all components
in the ink due to the complexity of the composition and solubility of individual components. Hence, the aim of this study is to apply the most effective methods using
2014 EAS Abstracts
November 2014
minimal extraction combined with instrumental analysis to determine the amount of
ink components migrating into the contact adhesive layer of a transdermal patch
with printing directly on it. The instrumental analytical tests include gas chromatography flame ionization mass spectrometry (GC-FID-MS) for volatile compounds
(benzyl alcohol as ink reducer), ultra-performance liquid chromatography diode
array detection mass spectrometry (UPLC-DAD-MS) for non-volatile compounds
(polyamide, photo-initiators and their decomposition products) and inductively coupled plasma optical emission spectrometry (ICP-OES) for metal analysis (D&C Red
as color dye). The calibration curves showed linearity in the range of 0.1 - 5.0 Вµg/
mL for all three instrumental analyses, GC-FID-MS, UPLC-DAD-MS and ICP-OES
with r2 no less than 0.998. The limits of detection and the recoveries of individual ink
components from extraction samples were 89-102% and 0.2 Вµg/mL, respectively.
The proposed methods will be useful for the quantification of ink migration from the
transdermal patch.
requirements were easily met on the ultra high-performance liquid chromatography
(UHPLC) system similar to a standard HPLC system. This result means that this
system is also suitable for those who are running traditional USP methods on a
standard HPLC system and considering the adoption of a UHPLC system for high
speed analysis of USP methods in the future.
Quantitation and Characterization of Polysorbate in Biotherapeutic
Products Using Two-Dimensional HPLC
Kenichiro Tanaka, Shimadzu Scientific Instruments, 7102 Riverwood
Dr., Columbia, MD 21046, William Hedgepeth, Keiko Yamabe
Polysorbates are commonly used for biotherapeutic products to prevent aggregation and surface adsorption, as well as to increase the solubility of biotherapeutic compounds. A reliable method to quantitate and characterize polysorbates is
required to evaluate the quality and stability of biotherapeutic products. Several
methods for polysorbates analysis have been reported, but most of them require
time-consuming sample pretreatment such as derivatization and alkaline hydrolysis
because polysorbates do not have sufficient chromophores. Those methods also
require an additional step to remove biotherapeutic compounds. Here we report a
simple and reliable method for quantitation and characterization of polysorbates in
biotherapeutic products using two-dimensional high-performance liquid chromatography (HPLC).
Direct Determination of Native N-linked Glycans by HPLC with
Charged Aerosol Detection
David H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford,
MA 01824, Ian N. Acworth, Marc Plante, Matthew W. Neely
Glycoproteins of biological, diagnostic or therapeutic interest owe key aspects of
their normal function to the oligosaccharides attached to the protein backbone.
Changes in the number, type, composition or linkage pattern of these glycans may
serve as a biomarker of disease or influence the efficacy of a biotherapeutic product. For this reason, the ability to correctly identify and measure these glycans is
of scientific interest, and to do so reliably, quickly and inexpensively is of practical benefit. This work explores direct detection of native glycans as an alternative
to the common techniques for glycan analysis that rely on derivatization reactions
to render glycans detectable. The lack of a detectable chromophore in native glycans is overcome by using high-performance liquid chromatography (HPLC) with
charged aerosol detection, a detector that can quantitatively measure any non-volatile compound. N-linked glycans were released from proteins by PNGase-F. The
native glycans were separated by ultra HPLC on a column that employs both weak
anion exchange and hydrophilic interaction liquid chromatography (HILIC) separation mechanisms. The native glycans were detected directly without derivatization
by using charged aerosol detection. Sialylated glycans released from bovine fetuin
were analyzed by a fast separation according to charge as well as by a higher
resolution separation based on size, charge and polarity. Precision, detection limits
and dynamic range of quantitative measurements are presented. Typical figures of
merit include low-nanogram on-column sensitivity, over two orders of magnitude
of dynamic range, and peak area precision of approximately three percent relative
standard deviation.
High Sensitivity Profiling of Glycans Using High-Performance
Liquid Chromatography with Fluorescence Detection
Peter Ratsep, Shimadzu Scientific Instruments, 19 Schoolhouse Rd.,
Somerset, NJ 08873, Kenichiro Tanaka, William Hedgepeth
Glycans in antibody drugs may play roles in the antigenicity, pharmacokinetics and
high-order structural stability of drugs, which could adversely affect drug safety and
effectiveness. It is therefore necessary to investigate which glycans are present in
antibody pharmaceuticals. Non-uniformity of glycans due to the instability of culture
conditions used for antibody drugs is a concern which requires rigorous management of the production process. Here, using an amide column TSKgel Amide-80 and
a Prominence high-performance liquid chromatograph, we introduce an example of
the analysis of glycans. For detection, the high-sensitivity fluorescence detector RF20AXS was used. A mixture of three glycans was injected to the system and limit of
detection was found to be around 1 fmol.
A Two Level Limit Test for the Detection of Trace Hydrazine in a
Drug Substance by In-situ Derivatization Headspace GC-MS
Sarah G. Westerbuhr, Array BioPharma, 3200 Walnut St., Boulder, CO
80301, Peter A. Lobue, Philip N. Anderson
Hydrazine and its derivatives are common building blocks for the synthesis of drug
substances. However, hydrazines are known genotoxic compounds and must be
controlled in drug substances at very low levels. A method to detect trace hydrazine
in drug substance samples was developed and validated. The method is a two level
limit test for 1 and 8 ppm limits when the drug substance samples are prepared at
100 mg/mL. The method employs in-situ derivatization of hydrazine with acetone to
produce the acetone azine, which is then introduced via headspace sampling, chromatographed using gas chromatography, and detected using mass spectrometry in
single-ion monitoring mode. The limit of detection is 129 ppb. The two level limit test
approach allows for a distinction between samples with a relatively low concentration of hydrazine from higher concentration samples. However, because of the less
stringent validation requirements of a limit test, the method validation required only
minimal time and resource.
Identification of a Unique, Metformin Related, Impurity in a Drug
Product Stability Sample
Jonathan Marshall, Bristol-Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08903, Yande Huang, David Ayers, Michael Peddicord,
Scott Miller
Metformin is an oral antidiabetic drug which is prescribed primarily for the treatment
of type 2 diabetes. Metformin is also marketed in several fixed dose combination
products. A new impurity was recently observed during stability testing for a fixed
dose combination (FDC) drug product which contained Metformin as one of the
two active ingredients. Characterization of the impurity by liquid chromatography
(LC-MS) resulted in a structure proposal indicative of a reaction of Metformin with
formaldehyde. Formaldehyde is a known degradation product of polyethylene glycol
(PEG) which is used as a component in the coating of the FDC drug product. This
presentation will describe the LC-MS characterization performed on the impurity
as well as the details of its synthesis, purification, and nuclear magnetic resonance
characterization for absolute structural assignment.
Coupling of PXRD, ssNMR and HPLC for Impurity Identification in
a Batch of an Anti-Diabetic Agent
Roxana F. Schlam, Bristol-Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08903, Anuji Abraham, Ian Young, George Crull
Upon completion of the Bristol-Myers Squibb - A toxicology batch, a new peak was
observed in the powder X-ray diffraction (PXRD) pattern that did not correspond
to any allowed reflections of the selected crystalline form of the active pharmaceutical ingredient (API). The issue had to be resolved prior to the upcoming First
in Human study in order to facilitate API release for clinical use. A combination of
PXRD, solid-state nuclear magnetic resonance (SSNMR) and high-performance
liquid chromatography (HPLC) was used to identify the nature of the extra peak
observed in the tox batch. PXRD and ssNMR ruled out the possibility of amorphous
or other crystalline phases. Analysis of HPLC data identified very low levels of phosphine ligand utilized in the synthetic process which was not completely removed.
PXRD together with 13C and 31P ssNMR confirmed that the extra peak corresponds
to the phosphine ligand. This knowledge allowed Chemical Development to design
a strategy to control the level of the ligand, bellow the allowed safety limits, in future
clinical deliveries.
Ultra-High Speed USP Analysis within the New USP General
Chapter 621 Allowed Limits
Kenichiro Tanaka, Shimadzu Scientific Instruments, 7102 Riverwood
Dr., Columbia, MD 21046, William Hedgepeth
The United States Pharmacopeia (USP) defines allowed adjustments of high-performance liquid chromatography (HPLC) and gas chromatography (GC) parameters
in the general chapter <621> chromatography. If system suitability is met, method
parameters can be changed within the allowed limits without revalidation. The general chapter 621 was revised in the first supplement to USP37-NF32 published on
February 1, 2014 and will be official after August 1, 2014. The feature of the new
general chapter 621 is that a column packed with small particles can be used as
long as column length and particle ratio (L/dp) is kept constant between the designated and modified column. This enables the high speed analysis of USP methods
more than ever. In this poster, an adjustment of a USP method for speed is shown.
As a result, analysis time and solvent consumption were reduced to 1/10 and 1/15,
respectively, with the system suitability requirements met. Additionally, running a
USP method on the Nexera X2 UHPLC system is shown. The system suitability
2014 EAS Abstracts
November 2014
quantitative comparisons with powder patterns. However, the XRD peak positions
were generally consistent with calculated peaks for paclitaxel crystal forms found in
the Cambridge Structural Database. These collective results are consistent with up
to 30% of the active ingredient present as poorly soluble crystalline particulates in
some batches of AbraxaneВ®.
Effect of Organic Additives in Running Buffer on CapillaryElectrophoretic Analysis of Amino Acids
Jinmo Huang, The College of New Jersey, PO Box 7718, Ewing, NJ
08628, Margaret Chen, Kimberly Benitez
Organic additives have been used in running buffer to enhance the separation of
analytes in capillary electrophoresis. In this research, organic solvents including
acetonitrile, methanol, and ethanol are respectively added to phosphate running
buffer used in the capillary-electrophoretic analysis of some light-absorbing amino
acids. The effect of these organic additives on the separation and determination of
the amino acids are compared and discussed.
Comparison of the Performance and Spray Behavior of a New
Nasal Microemulsion to the Commercial Suspension
Tina Masiuk, Merck, 556 Morris Ave., Summit, NJ 07901, Elaine Pu,
Zhenyu Wang, Oscar Liu, Julianne Berry, Samir Shah
The mometasone furoate (MF) microemulsion nasal spray and mometasone furoate/azelastine (MF/AZ) microemulsion nasal spray were developed to treat allergic
rhinitis as lifecycle extensions to the Nasonex suspension. The aim of the microemulsions is to increase the solubility and reduce the dose strength of mometasone.
The new formulations solubilize drug(s) using surfactant and oil, and have a viscosity resembling water. These differences in viscosity, and surface active excipients
could change the spray behavior, which could affect nasal cavity deposition. Since
Nasonex has been shown to deposit a majority of its dose into the posterior nasal
cavity (Shah, 2011), this study was undertaken to compare its performance to the
microemulsions. Actuations from Nasonex (500 mcg/g MF), MF microemulsion (31
mcg/g) and MF/AZ microemulsion (31 mcg/g and 2.055 mcg/g) were characterized by plume geometry, spray pattern, droplet size distribution, and spray content
uniformity. Statistical analysis assessed the effect of formulation. Small differences
between microemulsions and suspension were observed and are likely due to the
differences in viscosity and excipient surface activity. While statistically significant,
it is uncertain if these in-vitro differences are practically significant, given studies
finding no changes in deposition due to small changes in in-vitro spray tests (Suman
2006). Therefore, deposition from these formulations will likely be similar. Since the
performance of the microemulsions and suspension are comparable, it is expected
that any efficacy differences may be attributed to the active ingredients and formulation, not to spray behavior. Therefore, the microemulsions have the performance
capability of Nasonex.
A New Ion Chromatography Method for the Trace Level Analysis of
Hydrazine in Povidone and Crospovidone
Lawrence J. Feeley Jr., Ashland Specialty Ingredients, 1005 US
Highway 202/206, Bridgewater, NJ 08807, James E. Brady
An accurate and precise ion chromatography (IC) method for the quantitative measurement of trace level residual hydrazine (N2H2) in Povidone and Copovidone
has been developed. The IC method has numerous advantages over the thin-layer
chromatography (TLC) monograph method, United States Pharmacopeia (USP) 37
including: (a) less cost per sample, (b) analysis speed and (c) increased sensitivity
and dynamic range. The IC method can monitor hydrazine levels over three orders
of magnitude from an MQL of 8 ppb to 8 ppm for a 6% sample solution and can be
extended with sample dilution. Sample preparation is fast and the method can be
automated. Finally, since there is no derivatization step, the analysis is direct. This
method is based on previous work by Jagota, et al, (Jour. Pharm Biomed Analysis
(1998) 16, 1083-7), in which N2H4 is oxidized to N2 and that reaction is directly
Examining the Thermal Stability of Compound A and Methacrylic
Copolymer Solid Dispersion formulation
Sanjaykumar Patel, Merck, 556 Morris Ave., Summit, NJ 07901, Pranav
Compound A is a weak base with steep pH dependent solubility. To enhance solubility at the intestinal pH a solid dispersion based formulation was made using hot-melt
extrusion (HME). In solid dispersion matrix active pharmaceutical ingredient (API)
was stabilized in methacrylic copolymer enteric polymer. To define the appropriate
HME processing temperature window, it is important to understand the thermal stability of methacrylic copolymer and compound A. For stability of methacrylic copolymer, a reverse-phase liquid chromatography charged aerosol detection ultraviolet
(RPLC-CAD/UV) analysis method was developed. The polymer was monitored using Corona detector and monomers are volatile hence monitored using UV detector.
Polymer cross linking can form a large molecular weight polymer while the backbone breakage could result in the formation of low molecular weight species. Either
of these impacts the molecular weight distribution of the polymer and its functionality and to confirm this, a size exclusion method with refractive index detector was
developed. API thermal stability was monitored using a stability indicating assay
method. Compound A and methacrylic copolymer extrudates were prepared with
temperature ranging between 145 В°C to 180 В°C on an 18 mm twin screw extruder.
RPLC-CAD/UV analysis method demonstrated that there was no change in polymer
assay over the range of processing temperature and this was further confirmed with
size exclusion chromatography. Therefore, methacrylic copolymer is chemically stable during hot-melt extrusion processing. API specific method exhibited increase in
level of Deg A as a function of processing temperature hence processing temperature need to be below 180 В°C. Overall processing temperature between 145 В°C to
170 В°C was recommended for compound A HME solid dispersion.
Using Skin-Pampatm for Transdermal Patch Testing
David A. Kwajewski, Pion Inc., 10 Cook St., Billerica, MA 01821, Balint
Using skin as absorption site presents interesting features that have facilitated the
progression of transdermal drug delivery in the past decades. Efforts in drug research have been devoted to find a quick and reproducible model for predicting the
skin penetration of molecules. The parallel artificial membrane permeability assay
(PAMPA) has been recently extended by this group for the prediction of transdermal
penetration (Skin PAMPATM)1. This commercially available system has been modified to make it suitable for transdermal patch testing. Four active pharmaceutical
ingredients (nicotine, fentanyl, ketoprofen and rivastigmin) have been investigated,
each applied in 1-3 marketed transdermal patches. The permeation vs. time profile
demonstrated linear release profile in every case, though the cumulative permeated
amount was about 30% higher than expected that can be caused by the edge effect reported by Hadgraft and co-workers2. In-vitro/in-vivo correlation of permeation
profiles were performed based on manufacturers’ data and resulted in acceptable
correlation. Skin PAMPA system appears to be a useful tool for transdermal patch
comparisons, though standard protocol needed to be modified. Results can be used
for patch comparison and for ranking; therefore Skin PAMPA can provide valuable
information for transdermal patch development.
Pharmaceutical Residual Solvent Analysis Using (FT-MRR)
Spectroscopy: Fourier Transform Molecular Rotational Resonance
Brent J. Harris, BrightSpec, 770 Harris St. #104B, Charlottesville, VA
22903, Robin L. Pulliam, Roger Reynolds, Justin L. Neill, Matthew T.
Muckle, Dave McDaniel, Brooks H. Pate
Residual solvent content is a critical quality attribute (CQA) in pharmaceutical manufacturing that has motivated the search for new process analytical technology (PAT). The
standard gas chromatography (GC) methods require hours of calibration and suitability
testing that are incompatible with process monitoring applications. Fourier transform
molecular rotational resonance (FT-MRR) offers a powerful alternative for fast, direct
analysis of volatiles (including water) in pharmaceutical drug development and process
monitoring applications. A single analyte can be detected at 100 ppm (m/m) levels in
as few as 10 seconds. The technology platform is inherently selective and adaptable to
enable monitoring of an extensive list of volatiles without chromatography or chemometrics. Detection targets can be re-assigned arbitrarily by selecting pre-defined measurement protocols that include a double resonance analysis for unambiguous identification
of analytes in the mixture. FT-MRR performance for residual solvent analysis is presented using the headspace of a custom mixture with a GC verified composition of common
solvents dissolved in water and dimethylacetamide (DMAC). The results summary includes cycle time, detection limits, linearity, and reproducibility. The broader implications
include applications of FT-MRR to in-line batch drying analysis, high throughput residual
solvent analysis, genotoxic impurity analysis, and chiral analysis.
Finding the Needle in the Haystack – Characterization of Trace
Crystallinity in a Commercial Formulation of Paclitaxel ProteinBound Particles by Raman Spectroscopy and Synchrotron X-Ray
Diffraction Enabled by Second Harmonic Generation Microscopy
Paul D. Schmitt, Purdue University, 560 Oval Dr., West Lafayette, IN
47907, Niraj S. Trasi, Shijie Zhang, Fengyuan Deng, Lynne S. Taylor,
Garth J. Simpson
Second harmonic generation (SHG) microscopy was used to rapidly identify regions of interest for localized confocal Raman spectroscopy and synchrotron X-ray
diffraction (XRD) measurements in order to quantify crystallinity within lyophilized
AbraxaneВ® powder (protein bound paclitaxel for injectable suspension). Water
insoluble noncentrosymmetric crystalline particles ranging from ~1 micrometer to
120 micrometers were identified by SHG, with wide variability in crystal size and
frequency observed between several lots of AbraxaneВ®. By targeting the Raman
and XRD analysis to these localized regions identified by SHG, the corresponding
reduction in background lowered the limits of detection for both Raman and XRD by
2-3 orders of magnitude relative to ensemble averaged measurements. Experimental Raman spectra of SHG active domains in AbraxaneВ® were in good agreement
with experimental spectra of pure crystalline paclitaxel. Peaks identified by synchrotron experiments exhibited significant preferred orientation effects complicating
2014 EAS Abstracts
November 2014
derivatization to be effective, it is important that both the derivatization procedure as
well as the chromatography be robust and reproducible. This is highlighted herein
via the automated pre-column derivatization and chromatographic separation of 17
primary/secondary amino acid hydrolysates. This approach also takes advantage of
in-line photodiode array (PDA) and fluorescence (FL) detection, as well as stressing
the importance of low peak dispersion. In addition, this application demonstrates
that, at times, running superficially porous particle columns under ultra high-performance LC conditions can be quite beneficial.
HPLC Separation Polar Compounds with a Focus on the Role
Stationary Phase
Matthew Przybyciel, ES Industries, 701 South Route 73, West Berlin,
NJ 08091
The chromatographic separation and the analysis of polar compounds using
high-performance liquid chromatography (HPLC) has always been a challenging.
It would seem that HPLC analysis would be well suited for the determination of the
polar compounds; however routine reverse-phase HPLC analysis has yielded poor
quality results for these types of compounds. The analysis of polar compounds via
routine HPLC analysis has been deficient in several main aspects including poor
retention, unacceptable low k’ values and poor peak shapes. One of few HPLC techniques capable of analyzing polar compounds has been traditional normal-phase
chromatography (polar stationary phase with a non-polar mobile phase). Unfortunately, normal phase chromatography suffers from several major deficiencies including poor retention time reproducibility, poor column to column reproducibility
and very low or no solubility of the target analytes. A number of HPLC stationary
phases have introduced to improve the analysis polar compounds. These HPLC
stationary phases include high-aqueous stable phases, hydrophilic interaction chromatography (HILIC) phases and polar embedded phases all of which utilize various
levels of water in the mobile phase. It is the focus of this presentation to compare
and contrast the HPLC stationary phases developed for the analysis of polar compounds. In addition, we introduce new stationary phases optimized for the separation of polar compounds. The ultimate goal of this work is to present a strategy for
the HPLC analysis of polar compounds and to provide the chromatographer with the
information necessary to match their polar compounds/samples with the appropriate
HPLC technique.
Determination of Zonyl FSA Surfactant in Anti-Bleed Additive
Using Ion-Pairing, Light Absorption Spectrophotometry, and HPLC
Peter Yeh, MacDermid, 245 Freight St., Waterbury, CT 06702, Shelby
Zonyl FSA surfactant is used in the anti-bleed additive to prevent or minimize bleeding or hazing in the deposition of fine-line metal traces. Analytical methods have
been developed for determining the Zonyl surfactant concentration in the anti-bleed
additive. In one of the analytical methods, methylene blue is added to react with
the Zonyl surfactant to form an ion-pair which, when extracted into chloroform, can
be determined spectrophotometrically. This analytical method is simple, fast, accurate, and sensitive, capable of detecting and determining low concentrations of
the surfactant in the process solution. Due to its unique light absorption behavior in
the UV region, Zonyl FSA surfactant in the additive can be determined spectrophotometrically with high accuracy and precision. Taking advantage of their different
chromatographic elution behavior, high-performance liquid chromatograph (HPLC)
analysis method has also been developed for determining the Zonyl FSA surfactant
and other active ingredient concentrations in the additive and process solutions.
The HPLC method utilizes a reversed-phase C18 column for separation in conjunction with an acidic mobile phase solution containing methanol for elution. The
separated and eluting components are detected using a spectrophotometric detector set at a selected wavelength. The same HPLC method can be used to detect
impurities present in the additive and process solutions for product quality control
and anti-bleed process control. In this paper, analytical methods developed in our
laboratory for determining Zonyl FSA surfactant in the anti-bleed additive and process solutions are described and the accuracy and precision of the analytical results
obtained will be discussed.
Characterization of CopaxoneВ® by Viscotek TDAmax
Revital Krispin, Teva Pharmaceutical, Hatruffa 12, Netanya, 4250483
Israel, Arthur Komlosh
CopaxoneВ® is an aqueous solution containing 20 mg/mL of the active ingredient
Glatiramer Acetate (GA) and 40 mg Mannitol. Glatiramer Acetate is a complex mixture of synthetic amino acid copolymer composed of a huge number of indiscriminate sequences of L-alanine, L-lysine, L-glutamate and L-tyrosine. This complex
mixture of linear polypeptides results in a varying chain sizes, therefore the molecular weight distribution of the GA components span over wide range of about 2,500
– 20,000 Daltons. Viscotek TDAmax is a dedicated multi-detector size exclusion
chromatography (SEC) system for polymers and macromolecules analysis. The Triple Detector Array (TDA) is comprised of three detectors that act simultaneously;
refractive index (RI), viscometer and light scattering detector (LSD) providing complimentary characteristic data on the sample being analyzed including molecular
weight and molecular size. In this study Viscotek TDAmax was used for characterization of CopaxoneВ® by certain molecular weight characteristics (such as Mn,
Mw, and Mz), Polydispersity, hydrodynamic radius and intrinsic viscosity properties.
These values are physical characteristics of the polypeptide chains and are derived
from their chemical structure. Using this technique CopaxoneВ® displayed a good
batch to batch consistency, whereas purported generic copies showed differences
within their characteristic parameters. The Viscotek TDAmax method demonstrated
to be specific and discriminatory, and thus suitable for the analysis of CopaxoneВ®.
Characterization of HILIC Columns with Zwitterionic Functional
Groups: Correlation between Retention, Selectivity, Stationary
Phase and Water Layer Thickness
David Lentz, EMD Millipore, 290 Concord Rd., Billerica, MA 01821,
Tobias Jonsson, Phuoc Dinh, Patrik Appelblad
Liquid chromatograph mass spectrometry (LC-MS) with hydrophilic interaction liquid chromatography (HILIC) separations facilitates identification and quantification
of hydrophilic species due to the orthogonality and enhanced sensitivity compared
to reversed phase. Zwitterionic columns, purposely having a 1:1 balanced ratio of
positive and negative charges, provide separation by HILIC partitioning plus weak
attraction and repulsion interactions. However, the limited information on stationary phases provided by several manufacturers might hamper the selection of best
column for a particular analysis situation. In this work we conducted several separations of hydrophilic model compounds with relevance to cell metabolism and signaling, including nucleotides, nucleosides, nucleobases and catecholamines. Results were correlated to measured characteristics of the stationary phases and their
ability to absorb water. Characterization of five different commercial silica-based
zwitterionic columns were carried out at typical HILIC conditions, more specifically in
eluents containing acetonitrile at 70-95% mixed with aqueous buffers of ammonium
acetate or formate at different pH. Columns were also assessed for background
in electrospray mass spectromentry in the range of 20-2000 m/z under these eluent conditions. Column materials were examined for stationary phase content and
surface area by elemental analysis and the BET-method, respectively. A subset of
materials was selected for further testing by determining the amount of bound water
using Karl Fischer titration.
Charged Aerosol Detection and Evaporative Light Scattering
Detection – Fundamental Differences Affecting Analytical
David H. Thomas, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford,
MA 01824, Ian N. Acworth, Marc Plante, Qi Zhang, Matthew W. Neely
At a fundamental level, both charged aerosol and evaporative light scattering detection share some similarities in that mobile phase exiting the column is first nebulized
and then dried to form analyte particles. However, the mechanism by which these
techniques measure analyte mass differ markedly and this has major impact on analytical performance. In charged aerosol detection, charged particles are measured
by an electrometer generating a signal that is proportional to particle size (ie., the
mass of analyte). For evaporative light scattering detection, signal is also proportional to particle size, but this relationship is much more complex, as the magnitude
of scattered light varies depending on particle size, resulting in sigmoidal response
curves. Unlike charged aerosol detection, evaporative light scattering detection
uses non-contiguous signal attenuation. As each attenuation setting has its own
unique sensitivity, response, calibration curve and dynamic range, samples may
have to be reanalyzed multiple times in order to quantify analytes occurring at different levels. In this poster the analytical performance of charged aerosol and light
scattering detection are evaluated and include: sensitivity, dynamic range, inter-analyte response, linearity, reproducibility and the effects of mobile phase flow rate.
Analysis of Phytosterols in Whole Blood by HPLC-ECD
Marc Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Bruce Bailey, Ian Acworth, Michael Hvizd
Phytosterols are a group of naturally occurring steroid alcohols found in plants.
They are key structural components of plant cell membranes, assuming the role
that cholesterol plays in mammalian cells. There is considerable interest in phytosterols as dietary supplements as they are reported to lower cholesterol levels and
also have a positive impact on cardiovascular diseases. However, recent research
suggests that phytosterol supplementation may aggravate atherosclerosis and lead
to aortic valve stenosis. Phytosterols are typically measured by gas chromatography. However, this approach is time-consuming since it requires saponification of
the sample, several extractions steps, and then derivatization. Presented here is
a simplified method using reversed-phase, high-performance liquid chromatogra-
Automated Pre-Column Derivatization and High Speed LC
Separation of Primary and Secondary Amino Acids
Wilhad M. Reuter, PerkinElmer, 710 Bridgeport Ave., Shelton, CT 06484
For any liquid chromatography (LC) application incorporating automated pre-column
2014 EAS Abstracts
November 2014
phy (HPLC) and electrochemical detection (ECD) using a boron doped diamond
electrode. Five standards, campesterol, cholesterol, stigmasterol, Гџ-sitosterol, and
stigmastanol were resolved in < 6 minutes using a solid core C8 phase. The limit of
detection (LOD) was ≤ 1 ng on column for all analytes. The method presented here
was evaluated by characterizing the phytosterol content of whole blood The HPLC
method with electrochemical detection is simple to implement, has good linearity
and sensitivity, and is capable of measuring numerous phytosterols in animal and
plant extracts. This approach can be used to examine product purity, supplement
content, and adulteration. The use of the BDD electrode extends the range of analyte to those whose structured are normally considered to be electrochemically inert
when using conventional carbon- or metal-based working electrodes.
The Use of Sequential Elution Liquid Chromatography of
Increasingly Complex Combinations of Compounds for Increased
Peak Capacity and Decreased Separation Disorder
Erin J. Ennis, Drexel University, 3141 Chestnut St., Philadelphia, PA
19104, Michael R. Fletcher, Donna M. Blackney, Catherine A. Kita, Joe
P. Foley
Sequential-elution liquid chromatography (SE-LC), through the use of multiple sequential elution modes, can separate analytes by class. By employing a pH gradient
followed by a solvent gradient, analytes composed of weak acids, weak bases, and
neutrals can be separated by class and also within each class (i.e. each analyte
from another). Previous work [1] shows a greater probability of successful separation utilizing SE-LC with increased peak capacity and decreased separation disorder over traditional liquid chromatography for the separation of a 12-component
sample. In this work, SE-LC experiments are extended to include additional elution
schemes, including serially coupled columns, as well as new and increased combinations of compounds. Experimental emphasis is on the optimization of chromatographic figures of merit and applications for future use are explored.
The Raptorв„ў ARC-18 Stationary Phase: Selectivity Accelerated
Ty W. Kahler, Restek Corporation, 110 Benner Cir., Bellefonte, PA
16823, Art Marigliano, Hanna Rutter
With Raptorв„ў LC columns, Restek chemists became the first to combine the speed
of superficially porous particles (SPP) with the resolution of highly selective USLCВ®
technology. This new breed of chromatographic column allows you to more easily
achieve peak separation and faster analysis times without expensive ultra high-performance liquid chromatography (UHPLC) instrumentation. The new Raptorв„ў ARC18 column was designed to stand up to even the harshest acidic mass spectrometry (MS) conditions. It utilizes a proprietary bonding procedure that arranges our
sterically protected ligand to resist acid hydrolysis and, therefore, also resist phase
degradation and bleed. Designed and intended specifically for use on liquid chromatography (LC) MS-MS systems, the Raptorв„ў ARC-18 column features a well-balanced retention profile without the drawbacks of using an ordinary C18 in the harsh,
acidic mobile phases needed for mass spectrometry. Even after extended use in
these low-pH (≤ 2.0) conditions, the sterically protected ARC-18 offers consistent
retention, peak shape, and response for charged bases, neutral acids, small polar
compounds, and more. The Raptorв„ў ARC-18 column has the performance needed
for critical multiclass workflows in any industry or lab.
[1] A. Socia and J. P. Foley, J. Chromatogr. A, 2014, 1324, 36-48.
Increasing Efficiency while Decreasing Solvent Usage: The
Development of a High-Throughput uHPLC Method for
Determination of the Aerodynamic Particle Size Distribution
(APSD) by Andersen Cascade Impaction (ACI) and Emitted Dose
Uniformity (EDU) for Dry Powder Inhalers (DPI)
Jagruti Patel, Merck, 556 Morris Ave., Summit, NJ 07901, Justin
As part of Merck’s commitment to the green chemistry, the Merck Research Labs
(MRL) has been exploring ways to reduce the formation of hazardous waste.
Chromatographic analysis generates a significant amount of solvent waste in
the analytical laboratories, providing great potential to decrease solvent usage
while increasing efficiency. The current example involves the implementation of a
high-throughput ultra high-performance liquid chromatography (UHPLC) method for
determination of aerodynamic particle size by Andersen cascade impaction (ACI) of
dry powder inhalers. The ACI test segregates particles based on their aerodynamic
size on a series of plates and provides insight into drug product aerodynamic properties. This information comes at the expense of a significant number of samples for
extraction and chromatographic analysis. A typical 5 inhaler ACI evaluation results
in 50 samples (10 / inhaler) providing a significant opportunity to increase efficiency
of analysis while reducing solvent usage. The resulting method was developed by
optimization of UHPLC parameters and sample preparations steps with the overall
goal of a one minute chromatographic run time. The newly developed one minute
UHPLC method has proven to be a significant improvement when compared to
historical methods which range from ~5-14 minutes. For a typical analysis set of
5 inhalers the UHPLC method uses only ~250 mL of solvent resulting in a 75-90%
reduction from typical HPLC methods for a total reduction of waste up to 1000-2750
mL. The efficiency gain results in a time savings of 8-24 hours per ACI set allowing
for a significant increase in analytical throughput.
The Raptorв„ў Biphenyl Stationary Phase: Selectivity Accelerated
Hanna Rutter, Restek Corporation, 110 Benner Cir., Bellefonte, PA
16823, Ty W. Kahler, Art Marigliano
With Raptorв„ў LC columns, Restek chemists became the first to combine the speed
of superficially porous particles (SPP) with the resolution of highly selective USLCВ®
technology. This new breed of chromatographic column allows you to more easily
achieve peak separation and faster analysis times without expensive ultra high-performance liquid chromatography (UHPLC) instrumentation. Our top priority when developing our new SPP line was to create a version of our innovative Biphenyl. The
Biphenyl ligand provides greater aromatic selectivity than commercially available phenyl-hexyl columns and a greater degree of dispersion than conventional phenyls. This
unique selectivity mechanism allows greater retention of dipolar compounds, conjugated compounds, and compounds containing strong electron withdrawing groups.
The industry-leading Biphenyl is Restek’s most popular LC stationary phase because
it is particularly adept at separating compounds that are hard to resolve or that elute
early on C18 and other phenyl chemistries. Increasing retention of early-eluting can
limit ionization suppression, and the heightened selectivity helps eliminate the need
for complex mobile phases that are not well-suited for mass spectrometry detection.
Use of Additives for Improving LC-MS Analysis
Shyam Verma, Supelco/Sigma-Aldrich, 595 North Harrison Rd.,
Bellefonte, PA 16823, Rudolf Kohling
Ultra high-performance liquid chromatography (UHPLC) using ultraviolet (UV) or
mass spectrometry (MS) detection is now a routine technique in most analytical laboratories. The development of instruments and LC columns in the recent years improved the performance of fast and highly resolved separations significantly. Fused
Core or the new Titan particles represent key developments that enable the full
performance of a UHPLC system with a maximum of sensitivity and chromatographic efficiency. Unfortunately these enhancements can be undermined by the use of
unsuitable solvents. The ion suppression effect is still a major issue of the most
common electrospray ionization (ESI) source and has not yet been addressed. The
results presented in this poster show the positive effect of a sample clean-up step
prior to LC-MS analysis. Complex sample matrices, such as food samples, often
cause ion suppression by co-eluting components, such as sugars or phospholipids.
These issues can be resolved by using high purity additives.
Withdrawn by the author.
A Novel Core-Shell HPLC Column with Unique Shape Selectivity
Ken Tseng, Nacalai USA, 10225 Barnes Canyon Rd., A103, San Diego,
CA 92121, Toshi Ono, Tsunehisa Hirose, Kazuhiro Kimata
A cholesterol-bonded high-performance liquid chromatography (HPLC) column
allows for separation of structurally similar compounds under the reversed-phase
condition. The column has similar hydrophobicity as a C18 column. However, the
relatively rigid structure of the cholesterol functional group gives it better shape selectivity than a typical C18 column. We have applied this phase to the core-shell
particle technology to further improve the column performance. Core-shell columns
have better performance and throughput than the fully-porous ones. We routinely
obtain the same separation performance on a 2.6- mm-particle core-shell column
at half the back pressure as a 1.7-mm fully porous column. Examples of method
development using this cholesterol-bonded core-shell column are shown with the
catechin mixture and other compounds. We have conducted experiments to show
the performance differences between fully porous 5-mm and 2.5-mm particle columns to the 2.6-mm core-shell.
Retention Characteristics of Fluorophenyl HPLC Stationary Phases
Brittany George, Ursinus College, Department Chemistry; 601 E. Main
St., Collegeville, PA 19426, Eric Williamsen
Fluorinated stationary phases are commercially available and are useful in separations of compounds of interest to the pharmaceutical as well as in other industries.
The retention characteristics of these phases however, are not well characterized,
and few comparisons between fluorinated and similar non-fluorinated phases have
been reported. Retention of approximately 40 analytes with phenyl, alcohol, nitro,
cyano, halogenated, and other functionality was measured on a fluorophenyl sta-
2014 EAS Abstracts
November 2014
tionary phase at temperatures ranging from 5.0 to 65.0 ЛљC and with various organic/
aqueous mobile phase compositions, ranging from 10 – 100% organic. Retention
measurements on a traditional, similar phenyl stationary phase have also been
made under the same conditions. By analyzing the retention data through van’t Hoff
and selectivity plots, and quantitative structure retention relationships, similarities
and differences in the retention characteristics of phenyl and fluorophenyl stationary
phases under various mobile phase and temperature conditions are described and
Selection of Mobile Phase Modifiers for High Efficiency HILIC
William L. Miles, Advanced Materials Technology, 3521 Silverside
Rd., Quillen Bldg., Ste. 1-K, Wilmington, DE 19810, Robert E. Moran,
Stephanie A. Schuster, Barry E. Boyes
In high-performance liquid chromatography (HPLC) separations, the use of mobile
phase modifiers (buffers, and other additives) has become common practice to
achieve better separations in both reverse-phase (RPLC) and hydrophilic interaction chromatography (HILIC). Trifluoroacetic acid (TFA) is widely used, but other
acidic additives such as formic acid (FA) and buffers like ammonium formate (AF)
have become more popular. The use of these and other additives were examined
for HILIC separations of small bases, acids, zwitterions, and peptides. Highly efficient superficially porous particles (Halo Fused-Core) bonded with the Penta-HILIC stationary phase was employed. Separations of small molecules and peptides
exhibited excellent peak symmetry, column efficiency, and load tolerance when
appropriate mobile phase additives were used. We show examples of how these
improvements can be enjoyed in liquid chromatography-mass spectroscopy (LCMS) applications with appropriate selection of volatile mobile phase modifiers.
Analysis of the Retention Characteristics of Fluorinated and NonFluorinated Octyl HPLC Stationary Phases
Peter Willard, Ursinus College, Department of Chemistry; 601 E. Main
St., Collegeville, PA 19426, Eric Williamsen
Although high-performance liquid chromatography (HPLC) is the one of the most
commonly used and successful separation techniques, work continues on completely understanding the processes that leads to retention. Fluorinated stationary
phases are commercially available and have been shown to be more useful than
traditional stationary phases in the separation of a number of compounds of pharmaceutical interest. To better understand the similarities and differences of retention
on fluorinated and similar hydrocarbon phases, retention of over 40 analytes with
phenyl, alcohol, nitro, cyano, halogenated, and other functionality have been measured on fluoro octyl and octyl stationary phases. Measurements have been made
at temperatures ranging from 5.0 to 65.0 ЛљC and with various organic/aqueous, mobile phase compositions. Because of the large number of variables, multivariate
analysis techniques, such as cluster analysis and principal component analysis,
have been used to emphasize distinctive patterns in the data. In order to better understand the underlying physical interactions that lead to retention, linear solvation
energy relationships are also used. The results of these analyses are presented
and discussed.
Integrating Real-World Chemical Separation Research Projects
into Analytical Chemistry Curriculum
Yuegang Zuo, University of Massachusetts-Dartmouth, 285 Old
Westport Rd., North Dartmouth, MA 02747
Analytical chemistry is increasingly becoming an integral part of all science and
engineering disciplines, and it plays a vital role in broader societal concerns. Thus,
today’s analytical chemistry education must be more relevant to modern analytical
laboratory practices. Although traditional undergraduate chemistry curricula provide
a solid foundation in the fundamental principles of analytical chemistry, they do not
formally value practical skills that enable students to adapt and be successful in
today’s rapidly changing and competitive analytical workplace. To bridge the current
gap between “real work” experiences and university training in analytical chemistry,
the author has integrated real world chemical separation research projects[1] into analytical chemistry curriculum to prepare our students academically for what the real
world wants from them. The integrated research projects challenged students to think
creatively and improved their skills in communication, teamwork, and problem-solving. The details of this innovative curriculum project are given at the presentation.
[1] Y. Zuo, 2014, High-Performance Liquid Chromatography (HPLC): Principles,
Procedures and Practices. Nova Science Publishers, Inc., New York.
The Concept of Standard Adsorption Isotherms: Comparison of
Excess Adsorption of Binary Aqueous Organic Mixtures on
Classical Packing Material Modified with Alkylated and Fluorinated
Margaret Figus, Seton Hall University, 400 South Orange Ave., South
Orange, NJ 07079, Yuri V. Kazakevich, Alexander Y. Fadeev
The absorbent surface area, bonded phase chemistry, and bonding density are the
major factors affecting the reverse-phase separation. The effect of bonded chain
chemistry, bonding density, chain length on adsorbent pore volume, and void volume
of high-performance liquid chromatography (HPLC) columns are described in this
work. A series of covalently attached monolayers of alkanes and perfluorinated-alkanes were prepared via solution phase reaction of CnH2n+1Si(CH3)2N(CH3)2
where n = 1, 4, 8, 18, and CnF2n+1C2H2Si(CH3)2N(CH3)2 where n = 1, 4, 6, 8 with
well characterized silica substrate adsorbent. The energies of adsorption, surface
area, and pore volumes of modified and unmodified silica were calculated using
nitrogen adsorption isotherm. Grafting density of bonded ligands was determined
from the weight percent of carbon and fluorine. The excess adsorption isotherms of
acetonitrile and methanol from water were measured on in-house packed columns.
Comparison of the excess adsorption isotherms measured on these columns and
expressed in surface specific form demonstrated significant similarity of the adsorption properties, which allows us to introduce the “standard adsorption isotherm” for
reversed-phase alkane type columns and suggests that adsorption depends on the
type of ligands and bonding density of the ligands.
Withdrawn by the author.
Quantitation of Creatinine and Albumin Biomarkers in Mouse Urine
by HPLC and Affinity Chromatography
Joe Shao, Bristol-Myers Squibb, 311 Pennington-Rocky Hill Rd.,
Pennington, NJ 08534, Yingru Zhang, Preeti Sejwal
Creatinine and albumin are two key renal biomarkers present in urine. A urinary
albumin/creatinine ratio (UACR) can be used to diagnose and monitor kidney disease. Clinically creatinine/albumin is tested using Jaffe’s colorimetric method, enzymatic or immunoassay method with low specificity. In this study, the optimized
reverse-phase high-performance liquid chromatography (HPLC) method for creatinine utilized a Zorbax Bonus-RP column and sodium phosphate buffer/methanol as
mobile phases. The method demonstrated no interference in urine samples from
six types of mouse models. The creatinine data obtained from the HPLC method
and enzymatic method were within 86-117% consistency. For albumin analysis,
affinity chromatography with commercial resin-bound single-domain mono-specific
antibody fragments coupled with MW cutoff membrane filtration was used for separation of albumin from the urine matrix. The sample was further quantified using
size exclusion chromatography with UV detection at 215 nm. Non-specific binding,
molecular cutoff membrane filter selection, pH/salt effect and in-house packing of
an affinity ligand column are discussed. The HPLC methods developed were able
to quantify creatinine and albumin with UV detection, and can be applied as the
reference or calibration methods for quantitation of UACR in urine.
Outperforming Sub-2-Вµm Totally Porous Particles using FusedCore Technology
Robert E. Moran, Advanced Materials Technology, 3521 Silverside Rd.,
Quillen Bldg., Ste. 1-K, Wilmington, DE 19810, Stephanie A. Schuster,
Barry E. Boyes, William L. Miles
Fused-core particles have shown distinct advantages over comparable totally
porous particles for separating molecules of all sizes and characteristics. The introduction of a new 2.0-Вµm particle size to the Fused-Core product line continues
to advance high speed, high efficiency separations into the future. These 2.0-Вµm
Fused-Core particles permit separation speeds and resolution improvements greater than sub-2-Вµm totally porous particles, but with about 20% lower column back
pressure. Based on chromatographic theory, the trend of using smaller particle sizes
has been accepted as a way to increase efficiency. Coupled with this trend, the
detrimental effects of extra column band broadening have become more significant.
This report describes the effect of these new particles on multiple factors of separation performance, including reduced plate height, column efficiency, and extra
column band broadening.
Simultaneous Analysis of APIs and their Counterions Using a
Mixed-Mode Column
Peter Ratsep, Shimadzu Scientific Instruments, 19 Schoolhouse Rd.,
Somerset, NJ 08873, Kenichiro Tanaka, William Hedgepeth
Approximately 50% of all drug molecules used in pharmaceutical products are
reported to be ionic compounds. Ion chromatography is generally appropriate to
analyze inorganic or organic ions, but not suitable for active pharmaceutical ingredients (APIs) analysis due to their hydrophobicity. On the other hand, reversed-phase
liquid chromatography (RPLC) is mainly applied for analysis of APIs, but cannot
retain commonly-used ions for drugs. Consequently, it is difficult to analyze APIs
and their counter-ions simultaneously. In this study, we evaluated the ReDual col-
2014 EAS Abstracts
November 2014
umn, our newly-developed mixed-mode column, for simultaneous analysis of APIs
and their counter-ions. Mixed-mode column usage has increased because of the
ability to analyze a wide range of compounds in a single run by multimode retention
mechanisms. The ReDual column was able to analyze APIs and their counter-ions
simultaneously. In addition, we investigated how retention behavior changes with
parameters such as pH, ion strength, and concentration of organic solvents in the
mobile phase.
High-Speed Amino Acid Analysis (AAA) on a New 2.7-Ојm
Superficially Porous High pH Stable Reversed-Phase (RP) Column
William J. Long, Agilent Technologies, 2850 Centerville Rd., Wilmington,
DE 19808, Anne E. Mack, Jason Link
Amino acid analysis (AAA) is commonly used food and fermentation testing as a tool
to determine the precise amino acid (AA) makeup of samples. In order to do this
in a timely fashion a short turnaround time is needed; to do it with limited sample
amounts, a highly sensitive method is needed. And to maximize laboratory productivity, an automated method is desirable. In many cases these samples contain
particulates which can clog many high resolution columns or necessitate additional sample clean-up, and the analysis is further complicated by the often rigorous
conditions used, resulting in poor column lifetimes. This work describes methodology using a high pH stable superficially porous column with a 2.7-Ојm engineered
particle size. These particles have 90% of the efficiency of sub two micron totally
porous columns at 50% of the pressure and offer longer lifetimes under the higher
pH conditions typically used in this analysis. Additionally since they incorporate a
2-Ојm frit, they are as resistant to clogging as 3.5 and 5-Ојm columns. In this work, an
automated pre-column OPA/FMOC amino acid method previously developed on 3.5
and 1.8-Ојm columns is expanded to include 2.7-Ојm superficially porous columns.
This column shows good lifetime and transferability to different column dimensions
which are shown in this work. Applications of this column to fermentation products
are also shown.
Fast GPC Analysis of Residual Lauryl Methacrylate in Polymer
Tian Lu, Ashland, 1005 US 202/206, Bridgewater, NJ 08807, Fan Wu
Lauryl methacrylate (LMA) is an important monomer in polymer chemistry. The long
alkyl chain makes it a key component for manipulating the hydrophobicity of polymers or copolymers. The determination of residual LMA in polymer products is required in product and application development. Both gas chromatography (GC) and
high-performance liquid chromatography (HPLC) methods have limitations for LMA
residual determination in a polymeric matrix. If the polymer is thermally labile, the
polymer will undergo degradation in the GC injector and column which can interfere
with quantitative LMA detection. Poor recovery is observed when HPLC is used as
the analytical approach. Therefore, a fast gel permeation chromatography (GPC)
method was developed for residual LMA analysis. LMA can be well separated from
other components and impurities and quantified directly from the polymeric matrix
on a fast GPC column platform.
Lead and Cadmium in Counterfeit Cigarettes: Implications for
Public Health and Research on the Illegal Cigarette Trade
Yi He, John Jay College, 524 W 59th St., New York, NY 10019, Klaus
von Lampe, Laura Wood, Marin Kurti
Lead (Pb) and cadmium (Cd) are two toxic heavy metals commonly found in regulated amounts in genuine cigarettes. However, information regarding their concentration in counterfeit cigarettes is limited. This study compares the concentration
of Pb and Cd in twenty-three samples of counterfeit cigarettes seized by federal,
state and local law enforcement agencies in the United States with that in genuine cigarettes using inductively coupled plasma-mass spectrometry (ICP-MS) after
microwave digestion. Significantly higher Pb and Cd concentrations were found in
the majority of counterfeit samples. The average Pb and Cd concentrations in counterfeits were 5.13 В± 0.17 mg/kg (n=23) and 5.13 В± 1.95 mg/kg (n=23), compared to
0.59 В± 0.08 mg/kg (n=9) and 1.08 В± 0.02 mg/kg (n=9) respectively in the genuine
samples. The counterfeit cigarette samples with the highest concentrations of Pb
and Cd originated from the People’s Republic of China. A sample originating from
Paraguay and classified as counterfeit had similar Pb and Cd content as genuine
brands. Analytical results suggest that if consumed prevalently, typically in an economically disadvantaged area, counterfeit cigarettes potentially pose a higher risk to
public health. In addition, the elemental distribution was related to the geographical
origin of the counterfeit products. This work provides important information regarding the illicit cigarette trade including the level of organization among counterfeiters,
who broker between availability of supplies (i.e., premium tobacco leaves, filter tips,
cigarette paper, etc.) and consumer demand for cheaper product that is assumed
to be genuine.
Use of New YMC-Meteoric Core Core-Shell and YMC-Triart Hybrid
Material for an Improved USP Chlorhexidine Gluconate Assay
Jeffrey A. Kakaley, YMC America, 941 Marcon Blvd., Allentown, PA
18109, Ernest J. Sobkow
United States Pharmacopeia (USP) methods are used widely in the pharmaceutical industry and are often not optimized for use with modern instrumentation and
packing materials. As it becomes increasingly important to boost throughput in the
modern laboratory, new instrumentation and smaller particle size packing materials
are allowing scientists to achieve these goals while also realizing cost savings in
terms of man-hours and solvent usage. In this poster we investigate the use of 3-Вµm
YMC-Triart C18 and 2.7-Вµm YMC-Meteoric Core C18 stationary phases for improving a USP assay method for chlorhexidine gluconate.
A Rapid Hydrophilic Interaction Liquid Chromatography (HILIC)
Method for Determination of Trace Nitrate and Nitrite in Snow and
Rain Samples
Xiaofei Lu, University of Massachusetts-Dartmouth, 285 Old Westport
Rd., Dartmouth, MA 02747, Yuegang Zuo
Nitrite and nitrate are two common ions in natural waters. It is of great significance
to determine the concentration of these ions because they can put potential risk to
human and environment health. A rapid, specific, and sensitive method for determining nitrite and nitrate in snow and drinking water has been developed based
on high-performance liquid chromatography. A hydrophilic amino column was used
to separate the nitrite and nitrate which were then detected by an ultraviolet-visible spectroscopy detector at wavelength of 215 nm. The mobile phase consists
of acetonitrile and acetate buffer solution. It was found that the retention times of
nitrite and nitrate increased with decrease of pH of mobile phase or decrease of
proportion of buffer solution in mobile phase. For the effect of acetate concentration,
the retention times of nitrite and nitrate increased with the increase of concentration
under low acetate concentrations range (below 2.5 mM), whereas in the high concentration range (2.5 mM to 10 mM), they decreased with increase of concentration.
The results may indicate an ion exchange mechanism to the separation of nitrite
and nitrate on amino column. When determining the nitrite and nitrate in snow and
rain water samples, the average recovery of the two compounds are 97.86% and
98.39%, respectively. Nitrate concentration varied from 0.072 to 1.39 mg/L in the
in snow or rain sample. The nitrite was quantified only in one snow sample with
concentration of 0.012 mg/L.
Blood Alcohol Determination Using Static Headspace Analysis
with Optimized Sample Throughput
Anne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014,
Mike Moses, Lindsey Pyron, Kelly Cravenor
There are several ways to determine the amount of alcohol that is in a person’s
system. The most common methods are breath analysis in the field and blood analysis in the lab. Blood alcohol determination in the laboratory is used predominantly
when a person refuses a breath test. In order to determine blood alcohol content,
a person’s blood has to be withdrawn as soon as possible after the occurrence.
Furthermore, the blood needs to be collected in duplicate in order to confirm the test
results. This poster will examine static headspace sampling of alcohol standards
using gas chromatography for separation and flame ionization detection for analysis. The linearity of the compounds of interest will be examined and compared using
a secondary column for confirmation. Additionally, as many forensic labs have an
excess of samples to examine, the use of software innovations will aid in optimizing
sample throughput.
Two-Dimensional GPC Analysis for Blends of Polymers
Yejia Li, Arkema, 900 First Ave., King of Prussia, PA 19406, Martin
Nosowitz, Mark Lavach, Joseph Mitchell, Robert Barsotti, Florence
Blending of different types of polymers offers a cost-effective way to develop high
performance materials. However when the polymers are miscible, determination
of each polymer’s molecular weight can be challenging. The two-dimensional gel
permeation chromatography (2D-GPC) method is a versatile technique for the separation and analysis of polymer blend’s molecular weight. Using liquid adsorption
chromatography (LAC) the blend is first separated by polarity. A second separation
using GPC provides molecular weight information for fractions eluted from the LAC
separation. Examples of analysis using the 2D-GPC system are discussed.
Application of Infrared Microscopy for the Chemical Analysis of
Security Documents
Ronald Rubinovitz, Thermo Fisher Scientific, 4410 Lottsford Vista Rd.,
Lanham, MD 20706, Monica Joshi
Government issued identity documents include credentials such as passports, driver’s licenses and travel visas. These documents generally incorporate various security features that are used to authenticate them. Many of these security features
can be verified visually or through the use of an alternate light source. Forensic
document examination includes the assessment of these security features and other characteristics of the identity documents. When a document is suspected of being
2014 EAS Abstracts
November 2014
fraudulent the document is subjected to more scrutiny of its physical characteristics
and the chemical analysis of inks or paper. Several recent high profile cases have
involved the use of counterfeit passports, visas and state issued ID’s. Document
examination and improving methods for document examination is essential to maintaining the integrity of homeland security and immigration services. In this poster we
explore the utility of Fourier transform infrared (FTIR) microscopy for the chemical
analysis of the inks and the substrates used in identity documents. Establishing reference collections and databases of inks and substrates used in security documents
is a crucial aspect of document examination. We present the chemical analysis
of representative documents in the categories of passports, visas and Pennsylvania driver’s licenses. We highlight the advantages of FTIR microspectroscopy in
conducting rapid, highly discriminatory and non-destructive chemical analysis of
inks and plastics used in security documents. The evaluation and optimization of
different spectral search methods to facilitate the use of a document component
database is also presented.
that, isotope labeling experiments such as deuterium and carbon 13 labeling were
used to confirm mass spectrometric fragmentation mechanisms that result in the
formation of some key fragment ions or to confirm the elemental composition of
these fragment ions. The gas chromatographic resolution is described for these
piperazines on a variety of stationary phases.
High-Throughput Methodology for Mechanical Testing of Polymer
Sara Reynaud, Arkema, 900 First Ave., King of Prussia, PA 19406, Jun
Fang, Liang Fang, Evan Crocker
The measurement of mechanical properties of thin polymer films under high speed
impact is often difficult. Standard weight dropping testing is performed at high strain
rate but it is not suitable for thin samples and does not provide information on
stress-strain. Other techniques such as tensile/flex or nanoidentation can only be
performed at low strain rate. A high-throughput methodology for mechanical testing
(HTMECH) of freestanding thin films under high speed impact is introduced in this
work. The main advantages of this technique are: high instrumental sensitivity; test
rapidity and simplicity of the data analysis. Toughness properties of polymers supplied by Arkema Inc. were measured and compared with dynamic mechanical analysis (DMA). Interesting correlations between HTMECH and DMA results were found.
In particular, highest impact performance was observed for lower Tg polymers.
The Use of Automated Direct Sample Analysis (DSA-TOF) for the
Rapid Screening of Illicit Street Drugs,
Bonnie Marmor, PerkinElmer, 940 Winter St., Waltham, MA 02451,
Joanne Mather, Sean Daugherty
Screening methods for drugs of abuse currently require long chromatographic
methods and sample preparation. These methods can be time-consuming and laborious. Due to the variety of the illicit and abused drugs available, it is important
to monitor the entire spectral range, which is easily performed using time-of-flight
mass spectrometry (TOF-MS). The ability to analyze samples rapidly is desirable,
as drug seizures may result in thousands of samples. A method was developed for
screening various classes of street seized illicit designer drugs, drugs of abuse and
abused human growth hormones in solid, liquids as well as in urine of possible illicit
drug offenders using the DSA-TOF. We present a fast screening and confirmation
method by AxIONВ® direct sample analysis with time-of-flight mass spectrometry
(DSAв„ў TOF-MS). Fast screening of seized samples in seconds, without sample
preparation, is easily performed using DSA-TOF-MS. We present a real life case
study for the rapid analysis of 369 unknown drugs from seized pills, vials, powders
and urine samples. The presence of the target analytes was confirmed using AxION
Soloв„ў software by exact monoisotopic masses of precursor ion and product ions.
Withdrawn by the author.
Method Implementation for THC, THC-OH, and THC-COOH Testing
and Application for Blood in Experimental Subjects
Alex J. Krotulski, The Forensic Sciences Mentoring Institute, 2300
Stratford Ave., Willow Grove, PA 19090, Kathryn Peavy, Ian WietechaReiman, Samiyah Cornwell
Cannabis sativa, or marijuana, is a plant containing over 70 psychoactive compounds. Delta-9-tetrahydrocannabinol (THC) is the primary psychoactive compound
and is metabolized to 11-hydroxy-delta-9-tetraydrocannbinol (THC-OH) and 9-carboxy-11-nor-delta-9-tetrahydrocannbinol (THC-COOH) during first pass metabolism
in the liver. This project aimed to develop and optimize a method for detecting and
quantifying low levels of THC, THC-OH, and THC-COOH in blood. After smoking,
THC levels quickly spike in blood and then fall rapidly due to metabolism. Blood
taken at varying times after inhalation will be analyzed so it is important to be able to
quantify high and low levels of THC and its metabolites. Solid-phase extraction was
used to separate out the analytes from blood. Once the analytes were extracted,
the samples were dried down and derivatized with BSTFA + 1% TMCS for analysis by gas chromatography-mass spectrometry (GC-MS). Calibration models were
created using analyte standards spiked into sheep blood. Correlation coefficients
for the calibrations curves produced were 0.995 for THC, 0.996 for THC-OH, and
0.999 for THC-COOH. Recovery of our analytes after solid-phase extraction was
lower than expected at 49% for THC, 41% for THC-OH, and 51% for THC-COOH.
Precision studies were also carried out and determined to be acceptable (<20%)
for all analytes at 5 ng/mL and 200 ng/mL. Once the method is validated, real-world
blood samples will be analyzed from known marijuana users. The results gathered
from the blood samples will be used to correlate blood concentrations with driving
records, as part of a larger driving under the influence of drugs study.
Lawsone’s Derivatives for Fingerprint Detection
Joo Meang, John Jay College, Science Department, 524 W 59th St.,
New York, NY 10019, Natalia Fernandez, Gloria Proni
Lawsone, 2-hydroxy-1,4-naphthoquinone, is a naturally occurring coloring agent. It
is a relatively new reagent for developing latent fingerprints compared to ninhydrin
and 1,8-diazofluoren (DFO) that are more routinely used for the same purpose.
Lawsone has the property of coloring and fluorescing in a single step, which is a
beneficiary property when used to develop fingerprints. In this research, derivatives of the lawsone compound with selected fluorophores are being analyzed for
the enhanced properties in development of fingerprints using mass spectrometry
and nuclear magnetic resonance spectrometry. This enhancement is expected to
be seen in both coloring and fluorescing properties. The synthesized compounds
were studied after individually adding several amino acids and treating fingerprints
using ultraviolet-visible spectroscopy and fluorescence spectroscopy to view and
compare their fluorescence properties. The furthermost objective of the research
is to find the best compound with upgraded properties of both fluorescence and
solubility for fingerprint detection.
Development of an Analytical Method for Nootropic “Smart” Drugs
in Biological Fluids
Mollie M. Mares, The Forensic Sciences Mentoring Institute, 2300
Stratford Ave., Willow Grove, PA 19090, Iryna Kurochka, Rini Gupta,
Dymere Taylor, Mandi Mohr, Barry Logan
Smart drugs, or nootropics, are stimulants that allegedly boost brain function and
cognition. The media attention on these drugs has increased within the last few
years. The drugs have developed an underground following and are commonly sold
on-line and in illicit supply chains. Most have not been approved or scheduled in the
United States, and are therefore of concern to regulators such as the Food and Drug
Administration (FDA) and Drug Enforcement Administration (DEA). There are ongoing investigations in the applications of smart drugs in the treatment of Alzheimer’s
disease, Huntington’s disease, and attention deficit hyperactivity disorder (ADHD).
The stimulant properties of the drugs have led to their use in academic doping and
as drugs of abuse. Some drugs are also prohibited by the World Anti-Doping Agency (WADA). The goal of this project was to develop a single analytical method for
screening, confirmation, and quantification of a series of the widely known smart
drugs in blood and urine using supported liquid extraction (SLE). Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry
(LC-MS) were investigated to determine the optimum approach for sensitivity and
ability to detect a broad range of compounds, specifically piracetam, pramiracetam,
aniracetam, modafinil, adrafinil, ciproxifan, and noopept. Due to the thermal instability of the compounds and the inability to produce single, stable chromatographable
analytes, LC-MS provided a superior means of analysis. All seven analytes were
Analytical and Synthetic Studies on Substituted 1-n-Pentyl-3Benzoylindoles: Isomeric Synthetic Cannabinoids
Karim M. Abdel-Hay, Auburn University, 3306 Walker Building HSOP,
Auburn, AL 36849, Jack DeRuiter, Forrest Smith, C. Randall Clark
Gas chromatography mass spectrometry (GC-MS), GC-time-of-flight (TOF)-MS and
GC-IRD techniques provide complimentary data in the differentiation of regioisomeric and isobaric piperazine drugs of abuse and controlled substances belonging
to synthetic cannabinoids. The mass spectra for regioisomeric and isobaric piperazines are almost identical with no marked differences that would allow for differentiation between these compounds. Perfluoroacylation of the secondary amine nitrogen
of these isomeric piperazines gave mass spectra with differences in the relative
abundance of some fragment ions but did not alter the fragmentation pathway to
provide unique ions for discrimination among these isomers. Gas chromatography
coupled with time-of-flight mass spectrometric detection provided a means of discrimination among the isobaric piperazines by confirming the elemental composition
of the major fragment ions in the mass spectra of these compounds. On the other
hand, GC-TOF-MS was not successful in differentiating between regioisomeric piperazines. The vapor phase infrared spectrum obtained by GC-IRD was successful
in differentiating among the regioisomeric and isobaric piperazines by the characteristic bands in the region 700 – 1700 cm-1. These techniques have been applied
to the analysis of the ring substituted methoxy-, methylenedioxy-, dimethoxy- and
bromodimethoxy-, in both the benzyl and benzoylpiperazine series. In addition to
2014 EAS Abstracts
November 2014
successfully extracted from blood, providing an effective method for the analysis of
nootropics in biological fluids.
reclaim derelict industrial land for house building is driving the need for air monitoring methods that can reliably accommodate very high concentration samples
in potentially aggressive matrices. Many techniques are available for the sampling
and measurement of volatile organic compounds in air/gas, but which are the best
to use? Several international standard methods have been introduced that dictate
the sampling and analytical methodology for the study of air toxics, with sampling
generally carried out using evacuated canisters or sorbent tubes. In response to the
global interest in this application area, a number of instrument developments have
been made to address this analysis. This work describes a cryogen-free, analytical
system designed to offer flexibility of sampling methodology, fully compliant with
United States Environmental Protection Agency Methods TO-15 (canisters) and TO17 (sorbent tubes).
Automating Liquid-Liquid Extractions Using a Bench-Top
Fredrick D. Foster, Gerstel, 701 Digital Dr., Suite J, Linthicum, MD
21090, Oscar G. Cabrices, Jacqueline A. Whitecavage, John R. Stuff,
Edward A. Pfannkoch
Liquid-liquid extractions have long been performed manually and are used to extract
and concentrate analytes from aqueous matrices. Inclusion of liquid-liquid extraction
in many official methods attests to the wide acceptance of the technique. Following
solvent extraction it is also common to include an evaporation and reconstitution
step to improve detection limits or exchange solvents for compatibility with subsequent chromatographic separations. Modern analytical labs are looking to automation to help reduce solvent usage and increase sample throughput while ensuring
the high quality of the resulting data. A single robotic X-Y-Z coordinate autosampler
commonly used for sample introduction in gas chromatography (GC) or high-performance liquid chromatography (HPLC) can be used to perform a wide variety of
sample preparation techniques using a single instrument and controlling software.
The sampler can be configured as part of a GC or LC system or can be configured
as a bench-top workstation and can also include a six position evaporation station.
In this report, the automation of liquid-liquid extractions by the robotic autosampler
is discussed. Examination of a new, automated vortexing option that allows samples
to be rapidly and effectively mixed using speeds of up to 3000rpm is described.
Automated liquid-liquid extractions methods for a variety of analytes from different
matrices are examined and resulting precision and accuracy data are provided.
Increasing Extraction Efficiency of Pesticides and Dioxins from
Wet Samples Using a Novel New Polymer during Accelerated
Solvent Extraction
Aaron Kettle, Thermo Fisher Scientific, 1214 Oakmead Parkway,
Sunnyvale, CA 94085, Pranathi Perati, Kannan Srinivasan, Rahamt
Accelerated solvent extraction (ASE) is a high-temperature and high-pressure extraction technique that is widely used for sample extractions in the environmental,
chemical and food analysis industries. Extraction efficiencies when extracting volatile or semi volatile analytes from wet solids are often low, as the analyte of interest may partition between the extracting solvent and the water phase. Traditional
pre or post extraction methods of heat evaporation cannot be used for volatile and
semi-volatile compounds. Drying techniques that involve mixing the wet samples
with an inorganic salt that has a high affinity for the aqueous phase are unsuitable
for in-cell extractions. Drying methods with inorganic salts suffer from the limitations
of clumping or precipitation making post extraction clean-up difficult. Off-line drying
methods like freeze drying are extremely tedious and time consuming. This study
presents the use of a novel new polymer designed to remove moisture from wet
samples like soil, tissue and food products and increase the extraction efficiency
of volatile and semi volatile compounds. The polymer has a high capacity for water
removal and does not suffer from some of the limitations of clumping or precipitation observed in some of the traditional drying methods. Data showing recoveries
of organochlorine pesticides, polychlorinated dibenzo-p-dioxins, polychlorinated
dibenzofurans, and polychlorinated biphenyls in different matrices are presented.
New Stationary Phases for Large Volume SPE
Pranathi R. Perati, Thermo Fisher Scientific, 1214 Oakmead Pkwy.,
Sunnyvale, CA 94085, Jing Hong, Rosanne Slingsby
Five new polymeric stationary phases were developed for large sample volume
solid-phase extraction (SPE). The phase chemistries include a hydrophilic reversed-phase material as well as mixed mode, strong and weak, anion and cation
exchange. Base particles are grafted with polymeric and monomeric moieties to
achieve a useful balance between reversed-phase and ion exchange capacities.
The hydrophilic reversed-phase material bears specific functionality to retain polyphenolic and azo-containing species through a charge transfer mechanism and
H-bonding. This allows the retention of such species as humic acids along with the
ability to elute other organic species using conventional reversed-phase techniques.
These new resins are formatted to be compatible with sample volumes larger than
20 mL in automated SPE instrumentation. Several applications are shown. The
targets include triclosan, a popular but very hydrophobic bactericide, anionic and
cationic surfactants, the weak base atenolol and others with specific advantage for
the phases. Recoveries greater than 95% for sample volumes of 20-200 mL at loading flow rates of 5-30 mL/min in different matrices ranging from tap water, surface
waters to the American Society for Testing and Materials formulation of wastewater
were observed. Comparisons illustrating the right choice of bed weight to minimize
cost are also presented.
Automated Solid-Phase Extraction of Organochlorine Pesticides
from Drinking Water
Aaron Kettle, Thermo Fisher Scientific,1214 Oakmead Parkway,
Sunnyvale, CA 94085, Rosanne Slingsby, Jing Hong, Pranathi Perati
Organochlorine pesticides are chlorinated hydrocarbon compounds that have a
history of wide spread use both in the United States and globally. Organochlorine
compounds degrade slowly and can bio-accumulate over time, with increasing concentrations in animals high in the food chain. Their ability to volatilize in warm regions allows them to spread over long distances, with measurable concentrations
detected near the Arctic Circle and in alpine areas where they have not been used.
Organochlorine pesticides have found their way into sediments and drinking water
supplies posing serious health risks. Organochlorines have a wide range of both
acute and chronic health effects, including cancer, neurological damage, and birth
defects. In response to growing health concerns, the United States and Europe has
banned several of these compounds such as DDT, dieldrin and chlordane. Others
still in use include lindane, endosulfan and methoxychlor. This study evaluated extraction recoveries of twenty five chlorinated pesticides from drinking water using
automated solid phase extraction (SPE). This study also compared recoveries of
the twenty five pesticides when using automated SPE to the traditional liquid-liquid phase extraction. The quantitative determinations of the twenty five chlorinated
pesticides were performed by gas chromatography followed by electron capture
Adsorption of Steroid Hormones on Filter Membranes during
Analytical Sample Preparation
Jingjing Xie, University of Massachusetts-Dartmouth, 285 Old Westport
Rd., North Dartmouth, MA 02747, Si Zhou, Yuegang Zuo
Determination of steroid hormones (SH) in aquatic environments has been a challenge for environmental scientists because of the extremely low analyte concentration levels and complex matrix composition. Consequently, it is necessary to
preconcentrate the selected analytes and clean-up the samples from interfering
components, especially in the presence of humic subtances or suspended particles
in waste and surface waters. In this study, the effects of filter materials on the concentration of SH were systematically examined with commonly used commercially
available filter membranes, including glass fiber, acetate cellulose, polycarbonate
and nylon membranes. The pH and electrolyte concentration of the sample solutions can also affect the SH adsorption on the filter membranes. Under all conditions
tested, nylon membrane has the highest adsorption efficiency for SH. Approximately
64% of 17О±-ethinylestradiol and 55% estrone was adsorbed on the nylon membrane
after 10 mL of 5.0 mg L-1 of these analytes were filtered through the membrane.
Abstracts 115 - 116
Development of Jatropha Oil Extraction from Biodiesel Feedstocks
Using Accelerated Solvent Extraction
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, Aaron Kettle, Linda Lopez
The determination of oil content in biodiesel feed stocks can be performed using
several methods, including mechanical press, solvent extraction, and nuclear magnetic resonance (NMR). For the feedstock quality control in terms of oil content, it
is important that the applied method is universally accepted so as to obtain results
that can be compared with those reported from alternate sources. The European
Norm (EN) has specified two methods for the determination of oil content in oil seed
crops: conventional Soxhlet extraction and NMR imaging. However, these methods
have several disadvantages. Both methods are time, labor, and cost intensive, and
both require highly skilled labor and a significant amount of sample. These methods
are also unfriendly to the environment. The Thermo Scientificв„ў Dionexв„ў Accelerated Solvent Extractor (ASEв„ў) has great potential to overcome these constraints.
New Advances in Environmental Air Monitoring
Charles Haws, Markes International, 11126-D Kenwood Rd., Cincinnati,
OH 45242, Pete Grosshans, Nicola Watson
As the harmful effects of low-level exposure to hazardous organic air pollutants
become more evident, there is constant pressure to improve the detection limits of
indoor and ambient air monitoring; for example, by collecting larger air volumes and
by optimizing the sensitivity of the analytical detector. However, at the other end of
the scale, rapid industrialization in the developing world and growing pressure to
2014 EAS Abstracts
November 2014
Furthermore, ASE also has a high potential for application to oil-content testing of
third generation biodiesel feed stocks (e.g., micro algae). In this poster we present
the extraction of oil from Jatropha seeds in compliance with accuracy and reproducibility requirements described in the EN method. Oil extraction using an ASE system
requires only one to one-and-a-half hours as compared to nine hours consumed by
the Soxhlet extraction. In this research, the effects of ASE system conditions on the
percentage of oil extracted, the optimum conditions for oilseed extraction, and the
minimum amount of oilseeds required per test are also investigated.
The linear Langmuir and Freundlich models were applied to describe equilibrium
isotherms and both models fitted well.[1-3]
[1] Yang G, Fena W, Lei C, Xiao W, Sun H. Study on solid phase extraction and
graphite furnace atomic absorption spectrometry for the determination of nickel,
silver, cobalt, copper, cadmium and lead with MCI GEL CHP 20Y as sorbent. J.
Hazard. Mater. 2009; 162:44-49.
[2] Malekpour A, Hajialigol S, Taher MA. Study on solid-phase extraction and flame
atomic absorption spectrometry for the selective determination of cadmium in
water and plant samples with modified clinoptilolite. J. Hazard. Mater. 2009; 172:
Accelerated Solvent Extraction for Alternative Fuel Research
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, Aaron Kettle
As global interest in alternate fuel sources increases, many laboratories are researching effective ways to test, develop, and produce fuel from renewable energy
sources. One of these is the production of alcohol from biomass, called bioalcohol.
Alcohol produced from biomass has several benefits of fossil fuels because it is produced from crops, a renewable energy source, and causes little environmental pollution when burned. As bioalcohol is produced from biomass via sugar fermentation,
it is necessary to research which types of plants produce the best yields of usable
sugars. Thermo Scientific Dionex ASE Accelerated Solvent Extractors provide a fast
and efficient way to extract various biomass samples for sugar analysis to determine
sample viability for alcohol production. The Dionex ASE 150 and ASE 350 Accelerated Solvent Extractors with the pH-hardened pathways provide additional benefits
to the method by allowing pre-hydrolyzed biomass samples or those with acidified
solvents to be extracted. This allows samples to be hydrolyzed in the extraction cell.
This poster describes methods for the extraction of sugars from biomass using acidified solvents (in-cell hydrolysis) and extracting pre-hydrolyzed samples.
[3] Eren E, Tabak A, Eren B. Performance of magnesium oxide-coated bentonite
in removal process of copper ions from aqueous solution. Desalination 2010;
257: 163–169.
New Sources for Ambient Desorption/Ionization Mass Spectrometry
Gary M. Hieftje, Indiana University, 800 East Kirkwood Ave.,
Bloomington, IN 47405, Jacob T. Shelley, Steven J. Ray, Kevin P.
Pfeuffer, Gregory T. Schilling
Ambient desorption/ionization mass spectrometry burst upon the analytical scene
roughly ten years ago in the form of two novel sources: desorption electrospray
ionization (DESI) and direct analysis in real time (DART). Since then, more than 40
additional sources for the same field have been introduced. Of these sources, more
than half have been based on atmospheric-pressure electrical discharges. In this
presentation, the features of some of these sources are examined, with particular
emphasis on the flowing atmospheric-pressure afterglow (FAPA) developed at Indiana University. The FAPA is an unusually simple source, operated in flowing helium
and powered by a dc supply. There are two basic FAPA designs, one that uses a
hole or capillary in the discharge chamber and the other that generates a toroidal
(halo-shaped) discharge in the open air. Both have advantages, limitations, and
optimal areas of application. These aspects are explored in detail and tales spun
to show how they relate to the many achievements of Professor Joseph Caruso.
Applying a New and Unique Microwave Sample Digestion
Technique towards the Determination of Metals Present in Wine,
Berry and Soil Samples
Reynhardt Klopper, Anton Paar, 10215 Timber Ridge Dr., Ashland, VA
The presence and concentration of several metals can have a large impact on the
quality and preservation of wines. Various metals can have a negative impact on
several properties of wines, which include color, clarity, flavor and oxidation stability.
Generally the source of these metals is environmental, i.e., soils, berries. Metals
present in wine can be determined by a number of analytical techniques, the most
common include AAS, inductively coupled plasma optical emission spectrometry
(ICP-OES) and ICP-mass spectrometry. Although liquid samples can be introduced
directly into the ICP instruments, certain wines which contain high amounts of solids
or sugars need to be digested or mineralized before introduction. Moreover, solid
sample matrices such as soils and berries also need to be completely digested to
prevent spectral interferences during the analysis step. In this study we illustrate
how new and unique microwave digestion technology allows for the efficient and
fast processing of several wine and beverage industry related sample types which
contain high amounts of sugar. The compact and lightweight Multiwave GO microwave system features the patent pending directed multimode cavity (DMC) technology, which enables efficient and focussed application of microwave energy to a very
compact cavity system, while allowing for the simultaneous processing of up to 12
samples in 18 minutes, yielding clear digestions of multi-gram samples.
The Liquid Sampling-Atmospheric Pressure Glow Discharge (LSAPGD) Microplasma: An Example of What I Learned from Joe
Caruso – It’s More Fun Living (a little) Outside of the Box
R. Kenneth Marcus, Clemson University, Department of Chemistry,
Clemson, SC 29634
While the field of analytical atomic spectrometry has a long rich history, no one can
say that it is a history of high evolution in terms of the base tools. Beyond the venerable inductively-coupled plasma (ICP), flame, and graphite furnace sources, the
vast majority of advances have occurred on the sample introduction and analyzer
components of the instruments. Sample introduction strategies in atomic spectrometry have centered on application-driven developments. For this, Joe Caruso looked
outside of the box and put the power of the ICP to work in the solving of real-world
problems dealing with enviro/bio/medicinal chemistries. It is this sort of pragmatism-driven research, driving hardware developments, where Joe has left his mark
and showed me a path through my career. He demonstrated that this approach is a
valuable way to attract high quality students of highly diverse interests and talents
(oh ya, and funding from diverse sources). In this presentation, I pay homage to Joe
Caruso in describing the developments of the liquid sampling-atmospheric pressure
glow discharge (LS-APGD) microplasma as a source for elemental, isotopic, and
molecular species analysis by optical emission and mass spectrometry. While the
Caruso group led the way in sample introduction for ICP- optical emission spectrometry mass spectrometry, the LS-APGD had as its genesis the desire to have
a chromatography-friendly source for those cases where the ICP was not needed.
Our latest results in the development and characterization of this microplasma will
point to new directions in its use, a little outside of the box.
SmartSampleв„ў Titration Weighing Technology
Matt Eby, Mettler Toledo, 1900 Polaris Pkwy, Columbus, OH 43240,
David Zavich
SmartSample technology eliminates transcription and ordering errors, while optimizing efficiency by wirelessly transferring sample information and weights to a titration
beaker to be automatically read during a titration on an InMotionв„ў Autosampler.
Trace Determination of Some Heavy Metals by Solid-Phase
Extraction and Atomic Absorption Spectrophotometry
Sevgi Kocaoba, Yildiz Technical University, Faculty of Art and Science,
Department of Chemistry, Davutpasa Д°stanbul, 34210 Turkey, Zeynep
Aslan, Gonul Celen
The presence and movement of potentially toxic metals such as Cd, Pb, Cu, Zn and
Ni in soils and aqueous systems have created concerns for environmental and human health. Heavy metals can be introduced into soils and aqueous environments
either naturally or as a result of human activities such as agricultural, industrial and
mining operations. The adsorption of Cd(II), Pb(II), Cr(III) and Mn(II) onto dolomite
and sepiolite was examined in aqueous solutions with respect to the pH, concentration, adsorbent dosage, contact time, eluent type and volume of the sample solution,
flow rate of solution on the retention of the metal ions have been studied. Also,
for investigation of exchange equilibria different amounts of dolomite and sepiolite
contacted with a fixed volume and concentration of a heavy metal bearing solutions.
Sorption data have been interpreted in terms of Langmuir and Freundlich equations.
Plasma Sources Pushing the Limits in Analytical Chemistry
David W. Koppenaal, Pacific Northwest National Laboratory, EMSL, PO
Box 999, Richland, WA 99352
Plasma sources continue to be the source of choice for most elemental analyses,
approximately 50 years following their introduction to the community during the
1960’s. The use of plasma sources as ionization sources, although more recent
(1980’s introduction), has revolutionized inorganic mass spectrometry. In this paper,
the use of both standard and non-standard plasma sources for elemental/isotopic
analyses is discussed, with emphasis on new approaches and techniques designed
to make such sources even more powerful and adaptable to a wide range of scientific problems. In our work we are using both inductively coupled plasma (ICP) and
glow-discharge (GD) plasma sources for environmental and biological applications.
In the latter category, and in recognition of this year’s Field’s award winner, we
are especially interested in the development of metallomic applications of these
2014 EAS Abstracts
November 2014
Office of Generic Drugs has received numerous questions related to this document.
Therefore, an addendum was published to clarify many of these requirements. This
session introduces the stability requirements and how it impacts the analytical development sector of generic industry.
Spectroelectrochemistry as a Strategy for Improving Sensor
William R. Heineman, University of Cincinnati, Department of Chemistry,
PO Box 210172, Cincinnati, OH 45221, Samuel A. Bryan
Spectroelectrochemistry improves selectivity for sensors by electrochemically
changing the optical signal associated with the analyte and thereby distinguishing it
from interfering species whose signal is constant. The sensor consists of an optically transparent electrode (OTE) coated with a thin polymer film that preconcentrates
the target analyte adjacent to the electrode for spectroelectrochemical detection.
The OTE serves as an optical waveguide for attenuated total reflectance (ATR)
spectroscopy, which detects the analyte that has partitioned into the polymer film by
UV-vis absorption spectroscopy. Alternatively, ATR can provide the excitation light
for fluorescence detection, which is generally more sensitive than absorption. The
analyte partitions from the sample into the film where it is electrochemically reduced
or oxidized at the OTE surface. The change in the spectral response associated
with the change in oxidation state of the analyte is used for quantification. Absorption sensors for metal ion complexes such as [Fe(CN)6]4- and [Ru(bpy)3]2+ and
fluorescence sensors for [Ru(bpy)3]2+ and the polycyclic aromatic hydrocarbon
1-hydroxypyrene have been developed. The sensor concept has been extended to
binding assays for a protein as demonstrated using avidin–biotin and 17β-estradiol–
anti-estradiol antibodies. The sensor has been demonstrated to measure analytes
in complex samples such as nuclear waste and natural water.
Advances on the Modernization of USP Monographs
Leonel Santos, United States Pharmacopeia, 12601 Twinbrook
Parkway, Rockville, MD 20878, Clydewyn M. Anthony
The modernization of monographs continues to be a key initiative at United States
Pharmacopeia (USP) after a year of formal implementation. The key benefit of the
modernization scheme is the evaluation of the identity, strength, and purity of other
genetic drugs. A summary of the past year’s accomplishments and specific examples of the influence of monograph modernization of generic drugs is presented.
Important issues and challenges affecting modernization of monographs as well as
future plans to modernize drug product monographs based on the over-the-counter
(OTC) family of monographs are presented and discussed.
Comparison of Dissolution Profiles Including Bioequivalence,
Method Transfer/Validation, and f2
Vivian Gray, V. A. Gray Consulting, 9 Yorkridge Trail, Hockessin, DE
In dissolution, one of the most important tasks is the comparison of profiles. Minimizing variability is paramount so that when comparing profiles significant differences
can be observed. In method validation, the comparison of profiles is expected with
intermediate precision, manual versus automated testing, and robustness determinations. The appropriate criteria for method validation will be discussed. Sources
of variability and failures during method transfer will be explored. The comparison
of dissolution profiles is also critical to determine bioequivalence, as in the case of
f2. The improper applications of the f2 criteria, some ideas to explain failures, and
possible remedies for these failures are presented.
From Solution Chemistry to Mass Spectrometry – My Fields of
Joseph A. Caruso, University of Cincinnati, PO 210172, Cincinnati, OH
Following an MSU Ph.D. with Alex Popov and a postdoc at Texas Austin with Joe Lagowski, my first years at Cincinnati continued the solution physical/analytical chemistry in non-aqueous solvents, but inevitably moved to atomic spectrometry. Our
first AA studies were on lead in evaporated milk – much to the consternation of the
“contented cow folks,” since the cans at that time were Pb soldered, which the milk
clearly showed. With inspiration from a Velmer Fassel lecture, we moved modestly
into a low-power microwave plasma, which required vapor generation for sample
introduction. The Varian mini-atomizer fit the bill and was heavily utilized, leading us
to a brief study with a PAR Vidicon system for multielement work; perhaps one of
the earliest studies of analytical plasma emission with an array detector. As a means
of sample vaporization at about the same time, we began hydride generation for
sample introduction and found good results for As and Se, especially. With better
power coupling methods, the TM010 cavity allowed us to use helium plasmas and
the ultimate departure into ICPMS and many speciation studies, such as, metal
species translocation in plants. Further, while the glow-discharge was primarily used
for solids analysis, we showed that these plasmas could be used for vapor sample
analysis. Ultimately, speciation studies were to move us to biological/clinical studies
of metals, metal species and metalloproteins. With the help of Agilent Technologies,
our work with proteomics became possible. This presentation tiptoes through my
fields of analytical.
3-D Digital Microscopy and Cultural Heritage: A Successful
Ilaria Cacciari, IFAC-CNR, Via Madonna del Piano 10, Sesto Fiorentino,
50019 Italy, Salvatore Siano
The need for observing the microscopic details on artwork surface often occurs
during the course of the same restoration work. Standard optical systems such as
eyepieces, videomicroscopes or stereomicroscopes represent powerful aids for the
restorer during the examination of the preservation status, the decision of the treatment to be applied and interpretation of the peculiarity of the decoration process.
In recent years there has been an increasing use of digital videomicroscopes due
to their low cost and high convenience, but where the observation of the depth is
an important element in the assessment, they cannot fully replace the stereomicroscope. The “feeling” of three-dimensionality provided by the latter can allow to
decode textural aspects of interest, but in turn is limited if quantitative data on the
depth of the surface is needed. In this case, a versatile solution is represented by
the three-dimensional (3-D) digital microscopes. Since 2007 we are directly involved
in the development of devices for 3-D reconstruction of surfaces, and now we improve, promote and accelerate the technology transfer to increase the access to this
technology. Here, after an introduction on the main features of the 3-D digital microscopes we have developed, a review of several case studies is presented. These
are representative of microrelief problems which can arise in practice in cultural heritage domain. In particular we report the micromorphological studies of the punchworks on medieval painting, the granulation motives decorating jewels, the minting
toolmarks on Roman coins, and the test of laser cleaning on marble artifacts.
Regulatory Perspectives on Good Analytical Practices
Linda Ng, United States Food and Drug Administration, WO 51-4324,
10903 New Hampshire Blvd, Silver Springs, MD 20993
The selection of analytical procedures, method validation or verification, and instrument qualification are critical backbones for reliable analytical data to support
product quality in pharmaceuticals approved by the United States Food and Drug
Administration (US FDA). The current regulatory issues on analytical procedures
especially for the generic industry with the related requirements for method validation and instrument qualification will be shared. Discussion may include International Conference on Harmonization (ICH) Q2, Q3, Q6 and Q7; USP <621>, <1225>,
<1226>, <1010>, <1058>, <1086>; and related FDA guidances. The definition of
terms, tools, responsibilities of the respective parties, and current practices for
selecting scientifically appropriate analytical methods will be covered. In addition,
recommendations on information that should be included in the chemistry, manufacturing, and controls portions of drug applications to optimize the efficiency of
the review process, and data stored in the laboratory for the investigative team are
shared. Discussions also include Field Alert Reports, 483 observations and warning
letters attributed to the laboratories generating these data. These observations can
be avoided by an understanding of CGMP and the Agency’s expectations.
Pairing 3-D Laser Scanning Confocal Microscopy with Reflectance
Transformation Imaging to Gain Insight into the Techniques Used
to Create Relief and Contour Lines on Greek Red-Figure Vases
Philip A. Klausmeyer, Worcester Art Museum, 55 Salisbury St.,
Worcester, MA 01609, Paula Artal-Isbrand
This presentation offers an overview of research published in Studies in Conservation (Oct 2013, vol. 58:4 pp 338-359) in which the relief and contour lines on a
group of ancient Greek red-figure vases and vase fragments in the collection of the
Harvard Art Museums and the Worcester Art Museum were examined using two
surface examination methods – reflectance transformation imaging and three-dimensional (3-D) laser scanning confocal microscopy. These methods helped resolve longstanding debate among art historians, archaeologists, and scholars in the
field, regarding the tools, techniques, and production sequence used by Greek vase
painters to decorate vessels. This research also incorporated fabricated mock-ups
to gain a better understanding of the ancient technology and ultimately established
that two distinct types of relief lines exist on these ancient vases: the �laid line’ and
the �pulled line’. This study also determined the sequence of application between relief and contour lines. Based on observations made during this research, the authors
propose a likely evolution for the techniques used to produce decorative features
throughout the period of red-figure vase production. The role of 3D laser scanning
Impact of New Stability Guidance on Generic Drug Development
Kim Huynh-Ba, Pharmalytik Consulting and Training, 32 Forest Ridge,
Newark, DE 19711.
Stability is defined as a critical quality attribute to determine an expiry and storage
condition for pharmaceutical products. The new abbreviated new drug application
(ANDA) stability guideline was issued in September 2012 established the regulatory
requirements for submitting an Abbreviated NDA based on International Conference
on Harmonization (ICH) requirements; however, over the past couple of years, The
2014 EAS Abstracts
November 2014
confocal microscopy in this research is presented along with a brief introduction to
how the technique differs from better-known types of confocal microscopy.
modify, the functional properties that drive the quality or performance of numerous
industrial products. In this presentation I reflect on a variety of developments, applications, perspectives and insights that describes not just how we measure chemical
images but just as importantly why.
Optical Coherence Tomography for the Non-Invasive 3-D Volume
Imaging of Cultural Heritage
Haida Liang, Nottingham Trent University, School of Science &
Technology, Nottingham, NG11 8NS United Kingdom
Optical coherence tomography (OCT) is a non-invasive, non-contact imaging
technique based on a fast scanning Michelson interferometer capable of imaging
surface and subsurface structure in three-dimensional (3-D). In the last 10 years,
optical coherence tomography has been successfully applied to art conservation, art
history and archaeology. OCT has the potential to become a routine non-invasive
tool in museums allowing cross-section imaging anywhere on an intact object where
there are no other methods of obtaining subsurface information. While current OCTs
have shown potential in this field, they are optimized for biomedical applications.
Some major limitations are: 1) lower depth resolution compared to conventional
microscopic examination of sample cross-sections; 2) limited probing depth through
highly scattering material such as paint. Increasing the depth resolution involves
broad band laser sources and increasing the probing depth needs sources at longer
wavelength than conventionally used in biomedical imaging. To achieve this, we
have recently built two new OCT systems so that when used in conjunction, they
would aim to match the information from conventional invasive microscopic examination of sample cross-sections: 1) a long wavelength (2000nm) OCT optimized for
deeper penetration suitable for imaging cultural heritage objects consisting of highly
scattering materials; 2) an ultra-high depth resolution OCT at a central wavelength
of 800nm (axial resolution ~1.2 microns) targeted towards applications where the
highest resolution is required but the material is relatively transparent, such as imaging of multiple thin varnish layers on painting surfaces and gel layers on degraded
Historical Development of Mid-Infrared Spectroscopy
John Reffner, John Jay College, Sciences Department, 524 West 59th
St., New York, NY 10019
When William Weber Coblentz began his graduate studies at Cornell University
in 1900, infrared spectroscopy was in a primitive state. He built and calibrated his
own infrared spectrometer. Collecting infrared spectra with his spectrometer was tedious. Manual alignment of the spectrometer required skill. Tabulating wavelengths
and recording the radiometer’s response was laborious. Coblentz plotted all his
infrared spectra point by point. By 1905, he had collected several hundred spectra.
His pioneering research was published by the Carnegie Institution of Washington
D.C. in a book titled: Investigations of Infrared Spectra. His 40-year career at the
National Bureau of Standards (now NIST) is the cornerstone for the science of infrared spectroscopy and radiometry. Until the late 1930’s, infrared spectroscopy was
an academic curiosity. Analytical chemists took little note of its utility. As World War
II approached, the US government recognized its need to develop synthetic rubber.
On June 28, 1940, President Franklin D. Roosevelt designated rubber as a strategic
and critical material. Scientists from academia and industry joined forces to produce
synthetic rubber. Mid-infrared analysis of polymers and the raw materials needed to
make synthetic rubber was a pivotal part of this program. After the war, the sale of
commercial infrared spectrometers soared. By the mid-1960’s, the market matured
and other instrumental methods became serious competition. Beginning in the late
70’s, the development of Fourier transform IR spectrometers resurrected infrared
spectroscopy. The higher through-put, noise reduction, wavelength stability and
computer integration revived infrared analysis.
Pump-Probe Microscopy Enables Noninvasive, Three-Dimensional
Imaging of Artwork
Warren S. Warren, Duke University, Department of Chemistry, Durham,
NC 27708, Tana Villafana, Martin Fischer
The layering structure of a painting contains a wealth of information about the artist’s
choice of materials and working methods, but currently, no 3-D noninvasive method
exists to replace the taking of small paint samples in the study of the stratigraphy.
Here, we adapt femtosecond pump-probe imaging, previously shown in tissue, to
the case of the color palette in paintings, where chromophores have much greater
variety. We show that combining the contrasts of multispectral and multidelay pumpprobe spectroscopy permits nondestructive 3-D imaging of paintings with molecular
and structural contrast, even for pigments with linear absorption spectra that are
broad and relatively featureless. We show virtual crosssectioning capabilities in
mockup paintings, with pigment separation and nondestructive imaging on an intact
14th century painting (The Crucifixion by Puccio Capanna)[1]. Our approach makes
it possible to extract microscopic information for a broad range of applications to
cultural heritage. Additional applications to paper, pottery, and photodegradation
analysis are presented.
Historical Development of Raman Imaging - the Impact of the
Instrumentational Developments on the Quality of the Results
Fran Adar, Horiba Scientific, 3880 Park Ave., Edison, NJ 08820
After calculations showed that the loss of signal produced by small sample volumes
would be more than compensated by highly effective sampling optics, the Raman
microscope was conceptualized as a molecular analogue of the electron microscope, and then prototyped and commercialized, in northern France in the early
1980s. Originally the microscope was mounted on a scanning double monochromator which detected one wavelength at a time. As the instrumentation evolved, it
became faster, more sensitive and less subject to fluorescence interference. Consequently many new materials could be studied, and the number of successful applications exploded. Each new success promoted new instrumentation innovations.
In addition, the software became an integral part of the instrumentation, simplifying data acquisition, as well as data treatment and interpretation. This talk reviews
these developments, indicating the synergism between the evolution of the instrumentation and software, with the expansion of the applications. In particular, with the
maturation of the technology, the implementation of Raman imaging which was key
in the original concept of the early 1970’s, is now a practical reality.
Terahertz Spectroscopy: Past, Present and a Very Bright Future
Richard A. McKay, Advantest America, 508 Carnegie Center, Suite 102,
Princeton, NJ 08540
The terahertz (THz) spectrometer has, only recently, entered the realm of robust
and user-friendly laboratory tools. This mode of vibrational spectroscopy extends
the reach of the spectroscopist further down the electromagnetic frequency spectrum and is becoming an important complement to laboratory mainstays such as
near-infrared and Raman spectroscopy. There has been a lag in implementation
of this spectroscopic method due to the so-called �terahertz gap’, a technological
limitation in our ability to reliably and efficiently generate and detect THz waves.
Recent improvements in this field have made this technology more accessible. This
talk explores the history of terahertz spectroscopy, its current status as a growing
field, and it’s very promising future.
[1] T. E. Villafana, W. P. Brown, J. K Delaney, M. Palmer, W. S. Warren, and M. C.
Fischer, Femtosecond Pump-Probe Microscopy Generates Virtual Cross-Sections in Historic Artwork, Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):1708-13.
doi: 10.1073/pnas.1317230111
Diffuse Reflections on Chemical Imaging
Neil Lewis, Malvern Instruments, 7221 Lee Deforest Dr., Columbia, MD
The maturation of chemical (spectroscopic) imaging as a valuable problem solving
tool in the life sciences, pharmaceutical and polymer industries is well documented. In particular near-infrared (NIR) chemical imaging has proven to be one of the
most rugged and versatile implementation when industrial, routine or even process
analytical measurements are desired. This is due in part to the fundamental robust
measurement characteristics that it inherits from conventional or single-point NIR
spectroscopy. Commercial near-infrared imaging platforms are particularly flexible
with respect to the range of sample sizes that can be evaluated, addressing microscopic and remote sensing (satellite) applications and are in some cases capable
of generating chemical images in real-time. This flexibility enables NIR chemical
imaging to interrogate the characteristics of many different types of spatially and
chemically complex manufactured or naturally occurring materials delivering information not readily available using more traditional analytical methods such as
high-performance liquid chromatography (HPLC) or �point average’ spectroscopic
measurements. That is, information about not only what or how much of a particular component or chemical species is in a sample, but where it is, and how it is
distributed. This insight into the structure/function relationship of complex materials
ultimately enables an understanding of the factors that can influence, or be used to
Paper on Glass: A Practical Guide to the Implementation of an
Electronic Lab Notebook
James Exarchakis, Colgate-Palmolive Company, 909 River Rd.,
Piscataway, NJ 08855
Electronic laboratory notebooks (eLN) have proliferated throughout modern laboratories. This presentation describes the implementation of eLN within a multi-disciplinary analytical department. The transition from traditional notebooks to an
electronic system was facilitated by a paper on glass philosophy. Well trained
power-users helped define the eLN processes in terms that the end-users already
practiced eliminating the need to change behavior and practices of record keeping.
An emphasis on end-user return on investment of their time and on consistency in
formatting contributed to a speedy adoption. Additional experiences with instrument
integration, internal audits, and the relationships the IT department are discussed.
2014 EAS Abstracts
November 2014
lum to a research-rich, research supportive curriculum, and provide some practical
advice and examples of how to do this.
Implementing a Process Intelligence System for Pharmaceutical
Edward Keefe, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, Megerle Scherholz
For drug formulation understanding the process is paramount. For most formulators
retrieving all the data necessary to do any type of analysis is often a laborious endeavor. The data, which includes process parameters, analytical results and batch
genealogy, is often dispersed across different electronic systems, exists in paper
records, and resides with multiple individuals. Once the data collection is complete
the formulator faces another challenge. Since data capture is not standardized the
data must be normalized before any analysis can be conducted. In this type of
environment where process intelligence can have a beneficial impact on the drug
development process the knowledge must be extracted in a reasonable time frame.
This presentation discusses the implementation of a process intelligence system
along with the benefits, challenges, and lessons learned in two distinct areas of
pharmaceutical development: oral solid dosage formulation and parenteral formulation. It focuses primarily on three tools: the electronic lab notebook (Symyx ELN),
lab information management system (LabWare LIMS) and process intelligence software (Discoverant). It also presents actual examples showing some of the benefits
that can be derived from a process intelligence system.
Integration of Research into the Curriculum Using XRD and NMR
Structural Analysis
Benny Chan, The College of New Jersey, PO Box 7718, Ewing, NJ
08628, Abby R. O’Connor
The College of New Jersey has recently received two National Science Foundation
- Major Research Instrumentation grants to perform structural studies using single
crystal X-Ray diffraction (XRD) and nuclear magnetic resonance (NMR) spectroscopy. The inorganic faculty has integrated instruments into our curriculum through
research projects in our courses required of chemistry majors. Students have developed organometallic projects and studied the metallation of a variety of potential
ligands. Students either purchase the ligands or perform ligand design synthesis
strategies. Ligands chosen are studied through NMR and XRD. The metallation
studies are first performed via NMR and then crystals of the complex are studied.
The two faculties are particularly interested in these projects as they are novel research area and are soon entering the publication stage after only two cycles of
our courses.
Electronic Orchestration of Knowledge Creation and Management
Joel Young, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ
08903, Ronald Behling, Larry Fulton
Due to the disconnected and un-integrated nature of the various operational software solutions across the pharmaceutical development workspace (e.g., chemical
development, formulation development, analytical), it is extremely challenging to
manage, aggregate and connect the diverse, but related, knowledge inputs leading to tremendous inefficiency and rework. The ability to transform these collective
data inputs to knowledge is limited, sporadic, and inconsistent across platforms and
applications. A solution to orchestrate the creation of pharmaceutical development
knowledge (with a focus on analytical development) is discussed.
Involving Undergraduate Students in Collaborative Research: The
Union College Aerogel Team
Mary K. Carroll, Union College, Department of Chemistry, 807 Union St.,
Schenectady, NY 12308
The Aerogel Team is a cross-disciplinary research group established at Union College by the presenter, an analytical chemist, and Prof. Ann Anderson, a mechanical
engineer. Founded in 1795, Union College was the first college to be chartered in
New York State, to create a bachelor’s degree in science and mathematics (1822),
and to establish an engineering degree program within the context of the liberal
arts (1845). An analytical chemistry laboratory course was first offered at Union in
1857. Since 2001-02, more than 90 undergraduate students have participated in
aerogel research at Union, working alongside faculty mentors, using state-of-the-art
instrumentation, presenting and, in some cases, publishing their results. The Union
College Aerogel Team has received considerable internal and external (ACS PRF,
NSF MRI, NSF RUI, NSF I-Corps) support. Undergraduate students are involved in
all aspects of the research: they learn about the statistical design of experiments,
employ cutting edge instrumentation and data acquisition systems, perform experiments and reduce and interpret the results. In addition to fundamental studies of
aerogel materials, the team investigates applications of aerogels in areas as diverse
as chemical sensing, catalysis, drag reduction and drug delivery. Being part of a
cross-disciplinary team provides students with a valuable opportunity to learn how
to work beyond the confines of their own core disciplines. This approach engages
students, excites them to construct their own investigations, gives them practical
experience, and prepares them well for postgraduate study and careers in science,
technology, engineering, and mathematics fields.
The Role of the BIOVIA (Accelrys) Laboratory Execution System
(LES) and Inventory Management System (IM) in a CRO cGMP
Kathy Shanks, PPD, 8551 Research Way, Suite 90, Middleton, WI
To improve data quality and security, gain efficiencies, decrease review and turnaround time, and to eliminate manual data entry into analytical reports, the PPD
LLC cGMP Laboratory implemented a laboratory execution system. In addition, the
laboratory deployed a system for the control and maintenance of consumables. A
synopsis of the implementation of the BIOVIA (Accelrys) LES in conjunction with
Inventory Manager is discussed including lessons learned, best practices, benefits
derived, and the plan going forward.
Structure/Function Studies of Obscurin Ig58/59
Nathan T. Wright, James Madison University, 901 Carrier Dr.,
Harrisonburg, VA 22807
The giant protein obscurin (~800-900 kDa) plays a role in cytoskeletal organization and myofibrillogenesis. A mutation in the 58th Ig domain of this highly modular
protein causes hypertrophic cardiomyopathy in humans, possibly through ablation
of obscurin binding to its molecular target titin. Here, we analyze this obscurin-titin
interaction in more detail. We present the high resolution structure of Ig58, Ig59, and
a model of Ig58/59 together. While both obscurin domains are necessary to bind to
titin, the domains do not interact extensively with each other, and in fact exist in a
semi-extended conformation. Further analysis shows that the disease-causing mutation is on the surface of the Ig58 domain, and disrupts an extensive electrostatic
surface of the molecule.
Incorporating Research Method Development into the Analytical
Kimberley Frederick, Skidmore College, 815 N Broadway, Department
of Chemistry, Saratoga Springs, NY 12866, Maryuri Roca, Lisa Quimby
Many types of chemical research involve the process of analytical method development making it the most transferrable part of an analytical laboratory course. Unfortunately, traditional labs in analytical chemistry focus on giving students a broad survey of wet and instrumental techniques. We have chosen to replace the second half
of the lab experience with a research-based method development project. Students
work in teams with a faculty member or community member to develop, optimize
and validate an analytical method that supports a research project. Teams have developed preparative separation methods for an organic research lab, optimized and
validated detection methods for a geosciences lab group and devised and optimized
a detection reaction for a microfluidic assay. Because the results are of interest to
the research group or community member that the students are partnering with, students are more engaged, willing to work many additional hours and more invested
in the quality of their results. Students have been shown to be able to transfer the
skills learned in this course to new situations as well. As a bonus, their results have
also made important contributions to research projects and they have been listed as
co-authors on papers resulting from their project.
Liquid State, High-Resolution 14N-NMR of Symmetrically
Substituted Nitrogen-Containing Compounds
Benjamin Liu, Princeton University, 4589 Frist Center, Princeton, NJ
08540, Istvan Pelczer
Nuclear magnetic resonance spectroscopy (NMR) is one of the most powerful tools
available to chemists today. Conventional high-resolution liquid state NMR experiments, involving 1H, 13C, 19F, 31P and 15N routinely are used for structure determination, kinetic studies and quantitative analysis. However, one nucleus that has not
been studied in much detail is 14N, the primary isotope of nitrogen (99.7% natural
abundance). Nitrogen-14 is a nucleus has a nuclear spin of 1, meaning it has a
quadrupole moment. This quadrupole moment makes studying the nucleus more
difficult as severe line broadening often is observed. However, if the nitrogen atom
has symmetric substitutions, this can effectively collapse the quadrupole moment
and the peak narrows. Conventionally, the substances that have been looked at
have mostly been tetrahedral ammonium salts that can completely collapse this
Integrating Teaching and Research: Strategies and Practices for
Building a Research-Supportive Curriculum
Roger S. Rowlett, Colgate University, Department of Chemistry, 13 Oak
Dr., Hamilton, NY 13346
Undergraduate research is a high-impact educational practice that should be a high
priority for incorporation into science curricula. Developing a research-rich, research
supportive curriculum is an excellent way to accomplish this goal. This presentation
addresses the challenges of transitioning from a traditional content-based curricu-
2014 EAS Abstracts
November 2014
quadrupole moment. However, through an assay of common functional groups containing nitrogen we have identified a surprisingly large family of compounds suitable for high-resolution 14N–NMR. We have found that nitrogen atoms of both linear
and planar symmetry can also be visible in the NMR spectra with little broadening,
greatly expanding the substances available for this type of spectroscopy. Since this
nucleus is highly selective for molecules with appropriate symmetry, 14N-NMR potentially has a great number of applications. Complex or biological mixtures can
easily be analyzed with no background, since only the symmetrically substituted
nitrogen-containing compounds will appear in these spectra. We are in the process
screening a large variety of nitrogen-containing substances to exploit this opportunity.
columns representing the range of possibilities from the column database. We compare results from different columns to assess the feasibility of this approach.
Ultrahigh Performance Supercritical Fluid Chromatography of
Lipophilic Compounds with Emphasis on Synthetic and
Commercial Biodiesel
Larry T. Taylor, Virginia Tech, Department of Chemistry, 117 Surge
Building, Blacksburg, VA 24061, Mehdi Ashraf-Khorassani, Kenneth J.
Fountain, Giorgis Isaac
Ultra high-performance supercritical fluid chromatography (UHPSFC) in combination with sub-2 micron particles and either diode array UV, evaporative light scattering or mass spectrometric detection has been shown to be a valuable technique for
the determination of acylglycerol composition in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an unendcapped single C18 column (3.0 x 150
mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10
minutes served greatly as an improvement on first generation SFC instrumentation.
Separation of residual tri-, di-, mono-acylglycerols, glycerol itself, fatty acid ethyl esters, and free fatty acids at 0.2% (w/w) was easily obtained. The methodology was
applied to an in house synthetic biodiesel (i.e., fatty acid ethyl esters) derived from
tobacco seed oil both before and after purification by column chromatography on
bare silica. The established UHPSFC mass spectrometry (MS) approach has potential application in lipidomics and food testing as a complementary method alongside
high-performance liquid chromatography-MS and gas chromatography-MS, as the
former can separate both polar and nonpolar lipids to improve both detection limits
and peak shape.
Noncanonical Polyubiquitin Chains: Insights from Structural and
Dynamical Studies,
Carlos CastaГ±eda, Syracuse University, Chemistry Department,
Syracuse, NY 13244, Susan Krueger, Ashton Cropp, David Fushman
Polyubiquitination, an important post-translational modification of proteins, signals
for a wide variety of cellular events including proteasomal degradation, DNA repair,
cell cycle regulation, etc. The molecular basis of the diversity of polyubiquitin signaling lies in the different structural and dynamical properties of polyubiquitin chains,
imparted by the different possible covalent linkages between the C-terminus of one
ubiquitin (Ub) and the Оµ-NH2 of a lysine side chain (K6, K11, K27, K29, K33, K48
or K63) or О±-NH2 of M1 on a second Ub. While the canonical K48-linked and K63linked chains have been well characterized, little is known about the structural and
functional properties of so-called noncanonical polyUb chains (K6, K11, K27, K29,
K33) largely due to a lack of linkage-specific Ub-conjugating enzymes for these
polyUb chains. Recent studies suggest these chains are implicated in many nonproteolytic functions, including innate immune system activation and mRNA stability.
To study these chains biochemically and biophysically, we devised a chemical assembly strategy to make polyubiquitin chains composed of every lysine linkage, of
specific length, and with selective isotopic labeling. Solution nuclear magnetic resonance measurements, including chemical shift perturbations, 15N relaxation measurements and residual dipolar couplings, in conjunction with small angle neutron
scattering (SANS) measurements, have enabled us to describe the structural and
dynamical properties of free polyubiquitin chains of every lysine linkage for the first
time. These studies provide a foundation for future work in elucidating the molecular
recognition properties of these chains.
Thinking Outside the Valve: Pressure Control and Other Important
Spectrometry (SFC-MS)
J. David Pinkston, Kellogg Company, WK Kellogg Institute, 2 East
Hamblin Ave., Battle Creek, MI 49017
SFC bears many similarities to high-performance liquid chromatography (HPLC),
but there are important differences. One of these is the importance of controlling
mobile phase pressure at the column outlet to maintain a single chromatographic
mobile phase and to control the strength of the mobile phase. This presentation
describes novel approaches for pressure control and interfacing in SFC-MS which
eliminate some of the disadvantages of a traditional mechanical valve and of flow
splitting. I also highlight differences with a comparison of the applicability of SFCMS to that of LC-MS for a wide range of pharmaceutical compounds. Finally, nature
and function of mobile phase additives in HPLC and SFC can be quite different. For
example, HPLC has traditionally been the only technique routinely used for peptide
and protein separations, but I show that large, hydrophilic peptides can be eluted in
SFC-MS when the proper mobile phase additives are used. These additives are not
usually needed in HPLC.
Using Complexity to Make Separations Simpler
Thomas L. Chester, University of Cincinnati, Department of Chemistry,
PO Box 210172, Cincinnati, OH 45221
Chromatography is becoming more complex as the ranges of parameters are
pushed beyond conventional boundaries. This is not to be feared because increasing complexity provides new opportunities for creating rapid separations that more
fully satisfy the underlying analysis needs of resolution and speed. However, knowledge and expertise are required to get the most benefit. Short courses and on-line
training resources have rarely met the needs of developing frontier-pushing knowledge in the workplace. The next level of complexity in supercritical fluid chromatography (SFC) is to link reversed-phase liquid chromatography, hydrophilic interaction
liquid chromatography, and SFC into a seamless, unified behavior. This seemingly
impossible task is theoretically possible and has been demonstrated piecewise.
The key to accomplishing continuity is to develop understanding of phase behavior
and the formation and role of excesses of mobile phase components on stationary
phase surfaces. The ability to control surface excesses and to predict how solute retention in SFC is affected by parameter changes is a largely unexplored opportunity
that will lead to easily adjustable selectivity among dissimilar solutes. This flexibility,
combined with accurate models to quickly sort through complexity, will provide the
simplest and fastest separations within the allowed ranges of parameter values.
Spherical Nanoparticle Supported Lipid Bilayers for the Study of
Membrane Proteins
Fang Tian, Pennsylvania State University, Biochemistry and Molecular
Biology, Penn State Medical School, Hershey, PA 17033, Richard L. Gill Jr.
We are developing spherical nanoparticle supported lipid bilayers (SSLBs) to model various degrees of membrane curvature and study membrane proteins. SSLBs
were first introduced in 1990, and preserve many characteristics of cell membranes
such as lateral fluidity, impermeability to ionic species, and flexibility along the fatty
acyl chain. SSLBs are easy to prepare with high reproducibility and stability. Moreover, unlike the commonly used liposomes SSLBs can be homogeneously prepared
with well-defined sizes. In our preliminary study, we found that spectra from SSLBs
and liposomes are nearly identical, validating the use of SSLBs for the structural
study by solid-state nuclear magnetic resonance. Furthermore, spectra of SSLB
samples display higher resolution than those of liposome preparations.
Liquid Chromatographic Estimates of Octanol - Water Partitioning:
Is there a Rational Stationary Phase Choice?
John G. Dorsey, Florida State University, Department of Chemistry,
Tallahassee, FL 32306
There is a 40 year history of modern liquid chromatography being used for estimating octanol – water partition coefficients and hydrophobicity. Even with the many
hundreds of papers that have appeared, there is not a consensus of how best to
make these measurements. Mobile phase composition and additives have been
extensively studied, approaches to measure or estimate k’w, the retention factor in
100% water are many, but there are very few studies of the effect of the choice of
stationary phase on these estimates. Snyder developed the hydrophobic subtraction model for characterizing reversed phase stationary phases, and this has proven
a useful tool in picking alternative columns for methods development. We are now
asking if this model might provide a rational approach for choosing an appropriate
stationary phase for octanol – water measurements. We are investigating a diverse
set of compounds of varying structure, size and functional group on a series of
Probing Dynamic Protein-Glycosaminoglycan Interactions Using
Paramagnetic Ligands
Xu Wang, Arizona State University, Physical Sciences, Bldg. D 102,
Tempe, AZ 85287
Glycosaminoglycans (GAG) are large, sulfated, linear polysaccharides ubiquitous
to all mammalian cells. Their polyanionic nature sets them apart from other glycans,
and allows GAGs to act as receptors for many classes of signaling molecules. However, studying GAGs’ interactions with proteins has been challenging for nuclear
magnetic resonance (NMR) because non-labile protons needed by intermolecular
Nuclear Overhauser Enhancement Spectroscopy (NOESY) experiments are rarely
found at the intermolecular interfaces of these complexes. To improve the sensitivity
of detection, we have developed methods to functionalize GAG ligands specifically
with paramagnetic tags. Investigations of several GAG-binding proteins showed that
not only can the ligands indicate the location of the GAG-binding epitopes, but they
can also reveal orientations of the bound ligands on the protein, and, because of the
2014 EAS Abstracts
November 2014
sensitivity of NMR signals to paramagnetic relaxation enhancement effects, uncover
existence of binding conformation heterogeneity as well as secondary binding sites.
The application of this technology to the investigation of GAGs’ interactions with
the pro-inflammatory chemokine CCL5/RANTES is discussed in detail. In particular,
we were able to confirm that CCL5 possessed more than one major GAG binding
epitope, and its interactions with GAGs are exquisitely sensitive to the sulfation
patterns of the GAG ligands. Use of these ligands in the investigations of other
GAG-binding systems is also discussed.
lyzing individual aerosols in real-time that we have applied to the analysis of inhaled pharmaceuticals. A SPAMS draws individual particles directly from the aerosol phase at atmospheric pressure and directs them across the paths of two laser
beams. The first is a continuous wave laser with a square profile and duration of a
particle’s light scattering as it crosses it is related to the aerodynamic diameter of
the particle. As the particle passes out of the square laser and into the path of the
second laser, that second laser is pulsed, ionizing material from the particle. The
ionization event takes place at the center of the source region of a dual polarity
time-of-flight mass spectrometer and positive and negative ion mass spectra are
acquired from the particle. In this manner, the aerodynamic diameter and chemical
composition are determined for each individual particle that the SPAMS analyzes.
Up to 250 particles per second ranging from 0.1 to more than 8-Вµm in aerodynamic
diameter may be analyzed. Results from three series of experiments on inhalational
pharmaceuticals are reported. In the first, different formulations of individual and
combination commercial products were analyzed and the internal versus external
mixing states determined at the single particle level. In another, analytical consistency of the SPAMS was demonstrated over weeks in measuring a commercial formulation of indacaterol. A third series of experiments demonstrated the quantitation
of nebulized ciprofloxacin.
NMR Studies of Bacterial Masonry that Provide Protective Barriers
Charles V. Rice, University of Oklahoma, Department of Chemistry and
Biochemistry, Stephenson Life Sciences Research Center, Norman, OK
Although a fundamental aspect of bacterial physiology; little is known about the organization of peptidoglycan and teichoic acid in the cell wall of Gram positive bacteria. Both components influence lysozyme function, repulsion of antibiotics, and metal binding. In bacterial spores, the coat proteins and peptidoglycan cortex provide a
means of survival against conditions that normally kill vegetative bacteria. Peptidoglycan is a network of alternating N-acetylglucosamine (NAG) and N-acetylmuramic
acid (NAM) groups modified with a peptide. The peptides form crosslinks between
glycan chains, creating the cell wall framework. Interlaced within the peptidoglycan
are the teichoic acid molecules, phosphodiester polymers decorated with D-Alanine
and NAG. With nuclear magnetic resonance (NMR) spectroscopy, we are able to
identify how teichoic acid interacts with the peptidoglycan. Studies of metal absorption reveal that strong binding helps metals stabilize the peptidoglycan structure.
However, teichoic acid has a weaker interaction with metals, allowing diffusion into
the bacterial cytoplasm. Peptidoglycan also forms a protective shield around bacterial spores and additional NMR studies reveal that this shield affects the penetration
of sterile water into the bacterial spore and may explain resistance to oxidative attack by hydrogen peroxide.
Using Particle and Polymer Properties to Tailor Aerosol
Christopher Kuehl, University of Kansas, 2030 Becker Dr., Lawrence,
KS 66047, Nashwa El-Gendy, Laird Forrest, Cory Berkland
The prevalence of asthma has increased steadily in the United States from 20 million to 30 million people over the past decade and is associated with over $56 billion
dollars in healthcare costs annually. Asthma is expected to increase worldwide from
300 million to 400 million patients by 2025. To help treat asthma and its growing
prevalence, there has been a push for longer duration treatments by utilizing chemical approaches: by chemically modifying therapies such as beta-2 agonists or by
using combined therapeutics such as beta-2 agonists and corticosteroids to extend duration from 6 to 24 hours recently. This work looks to increase duration and
efficacy by employing a physical approach compared to a chemical method. The
first approach uses agglomerated nanoparticles called NanoClusters to improve
deposition into the peripheral lung and enhance dissolution of corticosteroids. The
other technique is to employ a polymer, hyaluronic acid (HA), as a delivery vehicle specifically for biomolecules. By choosing the appropriate molecular weight of
HA, different body compartments can potentially be selectively accessed from the
delivery vehicle into the lungs. The drainage kinetics and distribution from delivery
to the lungs (based on HA molecular weight) can enhance the persistence in the
lungs, thereby increasing the therapeutic efficacy of the therapeutic. By utilizing
physical as opposed to purely chemical methods, better aerosol formulations can be
achieved with enhanced duration for inflammatory respiratory diseases.
Challenges and Triumphs in Glycoprotein NMR
Megan Macnaughtan, Louisiana State University, 437 Choppin Hall,
Baton Rouge, LA 70803
Glycosylation is one of the most common post-translational modifications of human
proteins, but its effect on the proteins’ biophysical properties is poorly understood.
Almost all human proteins are modified with a form of glycosylation. Small O-linked
glycans, like elongated O-fucose and intracellular O-GlcNAc, have been shown to
directly regulate enzyme function, but the molecular mechanism is unclear. Nuclear
magnetic resonance (NMR) spectroscopy is recognized tool to study proteins, but
it is also an effective method to study glycans. One limitation of studying glycoproteins with NMR is the production of isotopically labeled sample. Some proteins can
be modified in-vitro with glycosyltransferases, after metabolic labeling in E. coli,
to produce isotopically labeled glycoprotein. Notch1 epidermal growth factor-like
(EGF) repeats can be modified with O-fucose, and even elongated to the tetrasaccharide form, with glycosyltransferases in-vitro. Wh ile some EGF repeats do
not show structural changes upon O-fucosylation, EGF27 of Notch1, a key player in the activation of Notch signaling, shows significant changes in structure. A
second strategy to produce glycoproteins for NMR analysis is to co-express the
glycosyltransferase with the substrate protein in E. coli. This method provides the
advantage of isotopically labeling the protein and the glycans. We have strived to
produce a co-expression system to produce O-GlcNAc-modified proteins using human O-GlcNAc transferase. With the discovery that E. coli NagZ sabotages the
in-vivo production of O-GlcNAc-modified proteins, we have engineered a functional,
co-expression system and produced O-GlcNAc-modified human cAMP responsive
element-binding protein (CREB1) and human Abelson tyrosine-kinase 2 (ABL2).
Using Spray Pattern Area as a Surrogate for SAC in Optimizing
pMDI Test Methods
Zachary Pitluk, Proveris Scientific, 290 Donald Lynch Blvd., Ste. 100,
Marlborough, MA 01752, Dino Farina
No abstract submitted by the author.
Near-Real Time Detection of Counterfeit Pharmaceuticals
William E. Mayo, Veracity Networks Inc., 745 North Dr., Melbourne, FL
X-ray diffraction (XRD) is an accepted characterization method (e.g., United States
Pharmacopeia <941>) that can be used to detect counterfeit pharmaceuticals.
However, the method is limited to laboratory environments, is relatively slow (15
– 60 min) and is a destructive ex-situ technique. Hence, it can only be used as a
screening tool that is unsuitable for field use or 100% floor inspection. To overcome
these obstacles, a novel technique has been developed based on energy dispersive X-ray diffraction (ED-XRD), which can detect sub potency, contamination and
counterfeit formulations with high speed (<1 min), high detection rate (>99%) and
low false alarm rate (<1%). The instrument is a table top unit with low power and no
external cooling requirements. Moreover, nondestructive testing is performed in-situ
and can inspect pharmaceuticals inside bottles and blister packs, with a maximum
throughput of 250,000 bottles per year per machine. The ED-XRD method relies on
scattering of incident X-ray beams by the crystalline and amorphous components of
the tablet/capsule, which produces a molecular fingerprint that is unique to that material. A test sample is then determined to be legitimate or counterfeit by comparing
its diffraction pattern against the reference standard in the material database. High
speed is achieved by use of an energy dispersive detector that collects the entire
diffraction pattern simultaneously, which eliminates the sample movements required
in conventional XRD analyses. The system can be used by virtually anyone and
minimally adds to the wholesale average cost (WAC), thus providing an incentive to
screen non-salable returns and overstock acquisitions.
Solid-State Characterization Techniques for Inhalation Product
Matthew Lamm, Merck, 556 Morris Ave., Summit, NJ 07901
In this presentation, solid-state characterization methods specific for inhaled drug
product active pharmaceutical ingredient phase screening and selection as well as
formulation development are presented. Case studies and retrospectives are used
to highlight the importance of specific phase attributes with respect to millability, particle size stability in the presence of moisture, and phase stability during drug product processing and storage. The utility of solid-state characterization techniques
such as X-ray diffraction, solid-state nuclear magnetic resonance, Fourier transform-Raman spectroscopy, electron microscopy, thermal analysis, laser diffraction
and dynamic vapor sorption is discussed.
Real-Time Inhalation Analytics Using Single Particle Aerosol Mass
Spectrometry (SPAMS)
David P. Fergenson, Livermore Instruments, 2038 Livingston St., Suite
B, Oakland, CA 94606, Maria Balaxi, David Kohler, Anna Susz, Bradley
D. Morrical
Single particle aerosol mass spectrometry (SPAMS) is a general method of ana-
2014 EAS Abstracts
November 2014
XRF profile of the different paints and help identify the pigments and fillers in them.
The different paints where then mapped to help visualize the artist strategy for laying
in the composition through several application techniques, including brushwork, oil
paint dragged directly from the tube or applied to his hands and pressed on the
canvas and industrial house paints dripped or poured across the surface.
Analytical Testing of Counterfeit Drugs in the Field Using
Vibrational Spectroscopy and GC-MS
Pauline Leary, Graduate Center, City University of New York, 1934 Bulls
Head Rd., Stanfordville, NY 12581, John A. Reffner
The counterfeit-drug problem is complex and integrates matters of science, law,
criminal justice, public health, and public policy. Proposed solutions require an integrative approach. Forensic science, local laws and law enforcement, drug-development regulations, international law, public treaties and policies, funding of drug
development, and intellectual property (IP) rights and enforcement all need to be
considered if an appropriate solution is to be presented. The impact of these goods
is devastating; ranging from localized fatalities resulting from small-scale product
adulteration, to large-scale public-health crises due to the development of drug resistance and fatal diseases caused by substandard medications. These goods present a significant regional and global problem and are a serious threat to public health
and safety. The nature of these goods as well as the internet and other factors that
have streamlined global trade are rendering current practices ineffective. The field
of forensic science struggles to establish analytical methods to identity composition
and provenance of counterfeit goods. No method has been shown to be universally
applied to achieve this goal. The authors present results of research performed on
counterfeits to identify and source these drugs in the field. Methods based upon
infrared spectroscopy, Raman spectroscopy and gas chromatography-mass spectrometry (GC-MS) are presented. Method limitations as well as discrimination potential are reviewed. In addition, challenges to admissibility in court are discussed.
Multiphoton Microscopy: An Efficient and Promising Tool for InSitu Characterization of Historical Artifacts
GaГ«l Latour, Imagery and Modelisation in Neurobiology et Cancerology
(IMNC), Batiment 440, Orsay, 91405 France, Laurianne Robinet, JeanPhilippe Echard, Marie Didier, Marie-Claire Schanne-Klein
Characterization of coatings (stratigraphy, composition) is of the utmost importance
for the understanding and conservation of historical artifacts. In the past few years,
optical coherence tomography (OCT) has become a well-established technique for
in-situ three-dimensional (3-D) imaging with a micrometer-scale resolution. However, discrimination of the various components is strongly limited with OCT. Multiphoton microscopy (MPM), also called non-linear optical microscopy, appears as a
promising alternative imaging technique for investigating cultural heritage artifacts.
This technique performs non-invasive 3-D imaging with micrometer-scale resolution
based on an intrinsic optical sectioning. A key advantage of MPM is its multimodal
capability with different modes of contrasts that are directly linked to the structural
and chemical nature of the materials. Two-photon excited fluorescence (2PEF) is
emitted by a wide range of materials (fluorophores) in historical artifacts with specific
absorption and emission spectra. As an example, we used spectral discrimination of
2PEF signals to separate gelatin-based film from sandarac film and to perform 3-D
imaging of cochineal lake pigments. Second harmonic generation (SHG) signals
are specific for non-centrosymmetric structures, with no counterpart in usual (linear)
optical techniques. In historical artifacts, we observed this signal in plaster particles
composed of bassanite crystals, crystalline cellulose and fibrillar collagen. We studied the potential of this imaging technique for the investigation of varnishes, musical
instruments and parchments. Our results demonstrate that multimodal MPM is an
efficient and promising technique for 3-D in-situ investigation of historical artifacts
and woods.
Paper Test Cards for Presumptive Identification of Falsified and
Very Low Quality Pharmaceuticals
Marya Leiberman, University of Notre Dame, Department of Chemistry
and Biochemistry, 271 Stepan, South Bend, IN 46556
Finding falsified or very low quality medications in low resource settings is a very
different task for the analytical chemist than finding them in a developed-world setting. Among the constraints: very limited access to instrumental analysis; lack of
funding, difficulty in obtaining reagents, solvents, supplies, and lab space; unreliable
electricity, water, and mail service, shortage of trained technicians and chemists.
We developed a paper test card that can be used to quickly screen pharmaceutical
dosage forms. The test card combines a dozen chemical color reactions and can be
evaluated by comparison to stock images or via an image analysis program. In ten
minutes, a user can detect the presence of unauthorized fillers or substitute active
pharmaceutical ingredients (APIs), and with a little more effort can spot formulations
in which the concentration of API is below 50%. We are working with pharmacists
and regulatory agencies in Kenya to improve supply chain testing and follow up on
adverse medical outcomes. I show how the cards work, then focus on recent results
from genuine samples of falsified medications and from anti-infective and antimalarial drugs collected in Kenya and South East Asia.
Advances in the Imaging and Microscopy of Semiconductor
Pigments: From Laser-Based Time-Resolved Luminescence
Imaging to Micro X-Ray Fluorescence Analysis Using Synchrotron
Austin Nevin, Institute of Nanotechnologies and Photonics, Department
of Physics, Piazza Leonardo da Vinci 32, Politecnico di Milano, Milano,
20133 Italy, Sara Bellei, Anna Cesaratto, Valentina Capogrosso,
Gianluca Valentini, Daniela Comelli, Caroline Tokarski, HervГ© Vezin
Semi-conductor pigments are found in paintings and include oxides and sulphides
of zinc and cadmium-based sulphoselenides. While today these pigments are stable and very pure, due to advances in synthetic processes and purification during
production, in the past impurities in pigments may have a significant impact on the
long-term stability of historical paint. The analysis of historical and modern Zn- and
Cd- based semiconductor pigments is presented using a variety of non-destructive
techniques to probe their chemical and physical properties with an aim to define
useful markers for their detection in paintings and other indicators for their degradation. Micro-photoluminescence imaging and spectroscopy using laser-based
analysis are presented for the detection of emissions and lifetimes from the bandgap and trap states; differences in signals are ascribed to defects and impurities in
the pigments. Complementary analysis of pigments using alternative approaches
provides data regarding the chemical nature of the impurities and their distribution
in micro samples. High-resolution X-ray spectroscopy (using both micro-X-ray fluorescence mapping and X-ray absorption spectroscopy), and electron paramagnetic
resonance (EPR) demonstrate the presence of various transition metal impurities,
some of which were intentionally added to pigments during production to improve
. Finally, examples of the application of luminescence imaging to paintings by Van
Gogh and other 19th C. artists are shown.
Analysis of Counterfeit Pharmaceutical Products Using Visible and
Vibrational Spectroscopies Inside and Outside of the Laboratory
Adam Lanzarotta, United States Food and Drug Administration,
Forensic Chemistry Center, 6751 Steger Dr., Cincinnati, OH 45237,
Nicola Ranieri
Counterfeit pharmaceuticals pose a serious health problem worldwide, and the
United States Food and Drug Administration’s Forensic Chemistry Center (FCC)
has been involved in their detection and characterization for several years. FCC
analysts employ handheld devices for field examinations and utilize state-of-the-art
bench-top instrumentation in the laboratory for confirmatory analyses. This presentation discusses some recent advances by the FCC for the screening and analysis
of counterfeit pharmaceuticals including the use of handheld alternate light sources,
infrared spectrometers and Raman spectrometers as well as the use of bench-top
alternate light sources, macroscopic infrared spectrometers, infrared spectroscopic
imaging microscopes and Raman microspectrometers.
Jackson Pollock’s Number 1A (1948): Using Spatially Resolved
XRF and Multivariate Analysis to Characterize the Artist Palette and
Ana Martins, Museum of Modern Art, 11 W53rd St., New York, NY
10019, Jim Coddington, Donald Dahlberg, Geert van der Snickt, Birgit
van Driel, Joris Dik
Analysis of historical paintings using non-destructive spatially resolved X-Ray fluorescence (XRF) microscopy has brought a tremendous insight into the materials and
methods used by artists. The volume of information to interpret, however, can be as
overwhelming as the amount of data that is gathered for a single object. Multivariate
image analysis (MIA) can assist in the interpretation by extracting the meaningful
information contained in the XRF hyper-spectra and by presenting this information
in two dimensional maps. This presentation shows how multivariate image analysis
methods were used to simultaneously characterize and map the different paints
used by Jackson Pollock in Number 1A (1948). Principal component analysis (PCA)
and multivariate curve resolution (MCR) were used to elucidate the characteristic
Laser Ablation Surface-Enhanced Raman Microspectroscopy
Pablo Londero, The Metropolitan The Metropolitan Museum of Art, 1000
Fifth Ave., New York, NY 10028, Marco Leona, Anna Cesaratto, John
R. Lombardi
Surface-enhanced Raman spectroscopy (SERS) has become a powerful tool for
the identification of organic colorants in cultural heritage studies, particularly when
only trace amounts of sample are available. However, it has been limited by the
protocols and instrumentation with which it is applied. These generally require water
solubility and, for the best spatial resolution, mechanical removal of a micro-sample
typically >20 Вµm. We have developed a new approach based on laser ablation in
the desorption regime, in which a microscopic region is visualized, selected, and
vaporized directly onto a SERS-active active substrate which one can immediately
excite with a Raman laser to detect a spectroscopic signature. High quality spectroscopic signatures are obtained with up to 4-5 Вµm resolution and ~100 attomole
2014 EAS Abstracts
November 2014
1:1 SNR sensitivity, though 10-100 picomoles is more likely for most samples. This
provides comparable performance to some mass spectrometry measurements and
opens the door to a number of new SERS-based studies. Measurements of insoluble pigments and the stratigraphy of thin paint layers have been performed, on both
reference samples and genuine works of art.
functionalized carboxylate deposition (FCD), was used to rapidly deposit a self-assembled monolayer of targeted alpha-carbon modified carboxylic acid containing
small molecules to porous silica solid supports. The FCD process rapidly (15-30
min) produces novel stationary phases, including a stationary phase coined Halophase. Halophase is created by depositing haloacetic acids via FCD and has shown
tunable retention properties based on their electronegativity and substitution (i.e.,
mono, di, or tri).
Method Development with Zirconia Based Stationary Phases
Clayton McNeff, ZirChrom Separations, 617 Pierce St., Anoka, MN
55303, Daniel Nowlan, Kelly S. Johnson, Richard A. Henry
The surface chemistry of zirconia-based reversed-phase high-performance liquid
chromatography (RP-HPLC) stationary phases differs considerably from the bulk of
silica and polymeric HPLC phases. These differences impart zirconia-based phases
with both extraordinary chemical stability (pH 1-14 and up to 200 В°C) and unique
chromatographic selectivity. However, these differences must be taken into account
in method development in order to achieve optimal results. This presentation will
cover the appropriate selection of pH, buffer type and organic modifier type when
using a zirconia-based phases as well as the use of elevated temperature. Carefully
selected applications highlighting zirconia-based stationary phase method development techniques are presented.
The Use of Elevated Temperature in HPLC Biomolecule Separation
to Shorten Analysis Time and Improve Peak Shape with Wide-Pore,
Core-Type Silica Columns
Stacy Squillario, Sigma-Aldrich/Supelco, 595 North Harrison Rd.,
Bellefonte, PA 16823, Roy Eksteen, Hillel Brandes
Temperature elevation has proven to be beneficial for decreasing the analysis time
and improving the peak shape of small molecule separations by high-performance
liquid chromatography (HPLC). Antia and Horvath demonstrated in 1988 that significant advantages are realized when columns containing superficially porous silica
particles rather than fully porous particles are utilized in these elevated temperature
separations. In this talk, we extend this concept to the analysis of biomolecules by
HPLC, demonstrating a similar trend. Briefly, several globular proteins and various
monoclonal antibodies were studied using commercial HPLC columns with large
pore diameters suitable for reversed-phase chromatography of biomolecules. The
operating capability of columns packed with either superficially or fully porous silica
particles was examined for stability at temperatures up to 90 В°C for a prolonged
period of time. In general, wide pore columns packed with superficially porous silica
particles were found to provide higher peak heights, narrower peak widths, and
in some cases larger peak areas under conditions of elevated temperature than
columns packed with fully porous particles. Larger peak areas were due to greater
biomolecule recovery on columns containing the superficially porous silica particles.
These results show the importance of temperature as well as particle morphology in
biomolecule separation. This data benefits analysts who are tasked to find optimum
conditions for the separation of proteins or peptides during drug development or
biomarker research.
High-Pressure Ion Chromatography: Following the Lead of Ultra
High-Pressure Liquid Chromatography
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, Frank Hoefler, Maria Rey
Typical ion chromatography systems and analytical columns in standard 4 mm and
microbore 2 mm formats operate below a maximum pressure of 3000 psi using well
established reagent-free ion chromatography (RFIC) system techniques. However, when using smaller particle-size analytical column, a higher-pressure system
may be required. Capillary high-pressure ion chromatography (HPIC) systems can
operate continuously at up to 5000 psi without eluent generation. The modified
system used here extends the high system backpressure capabilities to the entire
flow rate range of the system. Running any columns at increased flow rates may
cause a decrease in peak efficiencies. However, the superior chromatographic fidelity achieved using the new 4-Ојm columns, minimizes such losses, to provide
better separation of ions in complex matrices and can provide faster analysis times
and higher throughput. Higher peak efficiencies can be achieved, even when running at faster flow rates, providing shorter analysis times and increased productivity.
This poster demonstrates the use of smaller-particle-size (4-Ојm) analytical columns
combined with high-pressure ion chromatography to achieve efficient separations,
fast analyses, and high-throughput.
Easy Method Transfer and Scalability with Superficially Porous
Anne E. Mack, Agilent Technologies, 2850 Centerville Rd., Wilmington,
DE 19808, William J. Long, Wu Chen, Xiaoli Wang, Jason Link, Maureen
Superficially porous particles make transferring and scaling existing liquid chromatography (LC) analyses flexible and easy. Compared to similarly sized totally porous
particles, superficially porous particles have a shorter mass transfer distance and
substantially narrower particle size distribution, resulting in more efficient analyses
and possible time savings. In many cases methods using totally porous particle
columns can be directly transferred to superficially porous particle columns without
adjustment. This is particularly true when similar or identical bonding chemistries
are available on both types of particles. Method scalability is also facilitated by the
increase in choices of particle sizes. Together, these qualities allow for quick and
reliable method scaling or transfer, ranging from method development for preparative laboratories to methods for high throughput analyses, while also catering to the
use of a wide range of instruments and pressure limitations. In this presentation, we
share various practical examples of successful method transfer and scaling using
superficially porous particles with both isocratic and gradient elution.
Achieving Maximum Kinetic Performance from UHPLC Columns
Richard A. Henry, Sigma-Aldrich/Supelco, 595 North Harrison Rd.,
Bellefonte, PA 16823, William H. Campbell, David S. Bell
During the past decade, developments in high-performance liquid chromatography
(HPLC) columns confirmed predictions of Knox and Saleem in 1969 that smaller
particles and much flatter van Deemter (h- ОЅ) curves would have a dramatic impact
on HPLC speed and resolution, if suitable instruments could be developed. Instruments have made necessary advances in pressure and extra-column dispersion,
and porous and core-type silica ultra-HPLC columns in the 2-Ојm range perform well
with small, nonpolar test solutes at high velocities, but it is not that simple. Modern
UHPLC columns still show larger C terms that reduce separation performance for
high molecular weight, complex molecules at high mobile phase velocities. In 1988,
Horvath and Neue described fundamental equations behind this mass transfer limitation. The magnitude of this undesired resistance to mass transfer will be documented for different polar solutes, columns and operating conditions. Techniques
are demonstrated for plotting plate height, efficiency and resolution as a function
of flow velocity to define practical operating limits for high speed separation of real
sample mixtures on modern columns. Certain limitations could be reduced or even
eliminated by learning more about the nature of separation processes (diffusion,
partition and adsorption) that occur inside pores of silica particles. The full benefits
of modern, small-particle columns and instruments cannot be realized until flat van
Deemter curves are achieved with samples containing larger, polar and ionic solutes
on silica-based columns in reversed-phase, hydrophilic interaction liquid chromatography and other operating modes. Several areas for possible improvement in
columns and instruments are described.
Monolithic Silica’s for “Dirty Sample” HPLC Analysis with Less
Sample Preparation
Egidijus Machtejevas, EMD Millipore, Frankfurter 250, Darmstadt,
64293 Germany
In contrast to conventional particle-packed columns, monolithic silica columns are
made of a continuous piece of high purity porous silica. High permeability and
porosity of the silica skeleton and the resulting low backpressure allow for more
flexible flow rates compared to particulate columns. Monolith columns are just as
versatile and, in addition, provide several advantages over packed columns especially for “dirty” sample analysis. Especially the much higher tolerance for sample
matrix makes monolith columns the preferred choice for “dirty” sample analysis.
This presentation contains several methods, with workflows; including calibration
curves, recovery calculations, and method robustness overview for “heavy” samples
such us beer, milk, sun lotions, lipsticks, etc., with minimal sample preparation. For
example 2700 analysis of Bisphenol A in baby milk formulation (total volume of
injected sample was 27 ml + 65 l of mobile phase) were performed, and the results
demonstrated the excellent long-term stability and method robustness obtained with
monolithic silica columns.
Halophase: A Novel Stationary Phase with Tunable Retention
Properties through Functionalized Carboxylate Deposition of
Halogenated Small Molecules
Brian A. Logue, South Dakota State University, Box 2202, Brookings,
SD 57002, Michael W. Stutelberg, Venkataiah Mallam, Robert P. Oda,
Raj K. Bhandari
The current state-of-the-art liquid chromatography stationary phase materials are
produced by bonding chloro- or methoxysilanes to surface hydroxyls of highly
porous silica solid supports. A promising gas-phase deposition technique, coined
2014 EAS Abstracts
November 2014
with high fidelity spectroscopy. By combining spatial and spectral (color) information, and the use of statistical processing methods, the isolation of objects with very
similar color or objects within complex backgrounds can be achieved. ChemImage
and its customers have used hyperspectral imaging in many forensic applications.
This presentation gives a background of hyperspectral imaging and its application to
forensic investigations. Applications shall include question documents, finger prints,
biological fluids, gunshot residue and bruise detection.
Discriminating Power of Fiber Associations in Forensic
Examination Practice
Edward G. Bartick, George Washington University, Department of
Forensic Sciences, 2100 Foxhall Rd. NW, Washington DC 20007,
Stephen L. Morgan, Kevin Roberts
The objective of this work was to evaluate the statistical significance of measurement variance of like and unlike fibers using multivariate statistical methods, and
to determine the discriminating power for the comparison of sets of red cotton and
acrylic fibers was determined. Using visual light microscopy, visible microspectrophotometry (MSP) and Fourier transform infrared spectroscopy, data was collected
on fibers. Studies were done on 21 red cotton and 21 red acrylic fibers using multivariate analysis. The absorption spectra of visible microspectrophotometry scans
on each fiber were compared by using principal component (PCA) and linear discriminant analysis (LDA) to determine how well fiber color matched on similarly dyed
fibers. The discriminating power (DP) for the classification performance achieved
was calculated as the ratio of the number of discriminated pairs of fibers divided by
the total number of pairs of fibers under examination. The discriminating power was
calculated for the red cotton fibers based on the discrimination achieved by PCA
and LDA of the MSP data. On the 21 red acrylic fibers, the discrimination of the dyes
was also done by MVA. The IR spectra were discriminated by spectrometer search
software and the fiber diameters and cross-sectional shapes were discriminated by
visual light microscopy. The final discriminating power based on all the parameters
measured was then calculated.
Fate and Effects of Engineered Nanomaterials in Agricultural
Jason White, Connecticut Agricultural Experiment Station, 123
Huntington St., New Haven, CT 06504, Joseph Hawthorne, Roberto De
la Torre-Roche
Although engineered nanomaterial (NM) use has increased, the fate and effects of
these substances in the environment is poorly understood. This lack of understanding is of particular concern in agriculture, where food chain contamination may occur. The accumulation and transfer of NM from soil through terrestrial food chains is
being measured. Bulk or nanoparticle (NP) cerium oxide (CeO2) was added to soil
at 0 or 1000 mg/kg and zucchini were planted. After 28 d, the plant Ce content was
determined by inductively coupled plasma mass spectrometry (ICP- MS). Leaves
from each treatment were fed to crickets for 14-d; crickets were harvested and fed
to wolf spiders for 7 days. The cricket and spider Ce content was measured. CeO2
exposure had no effect on total biomass but NP CeO2 significantly suppressed
reproductive tissue production. The Ce content of zucchini was significantly greater
when exposure was in the NP form. The flowers, leaves, stems, and roots of bulk
CeO2-exposed zucchini was 93.3, 707, 331, and 119,000 ng/g, respectively; levels
in NP-exposed plants were 153, 1510, 479 and 567,000 ng/g, respectively. Crickets
fed bulk and NP CeO2-exposed leaves contained 13.5 and 19.7 ng/g, respectively.
Feces from bulk and NP-fed crickets contained Ce at 393 and 1010 ng/g, respectively. Spiders that consumed crickets from bulk treatments contained non-quantifiable Ce amounts but NP-fed spiders contained Ce at 4.9 ng/g. These findings show
that NP CeO2 accumulates in zucchini at levels significantly greater than equivalent
bulk materials and that this greater NP intake may result in trophic transfer and food
chain contamination.
Raman Microspectroscopy and Advanced Statistics for the
Analysis of Biological Stains and Gunshot Residue
Igor K. Lednev, University at Albany-SUNY, 1400 Washington Ave.,
Albany, NY 12222, Greg McLaughlin, Kyle C. Doty, Justin Bueno
The identification of traces of body fluids discovered at a crime scene is a major part of forensic investigation today. The main problem with current tests is the
destruction of the sample. The forensic community is in great need of a reliable,
non-destructive, on-field method for identification of all common body fluids. We
report here on the latest development of a new method for identification of body fluid
traces using Raman spectroscopy combined with advanced statistics. Multidimensional Raman spectroscopic signatures of dry traces of sweat, vaginal fluid, semen,
saliva, and blood were developed. The ability of identification of contaminated and/
or mixed samples, differentiating menstrual and peripheral blood as well as determine species (human vs. animal) based on dry traces of blood is discussed. The
development of a novel and alternative method for detection, identification and discrimination of gunshot residue (GSR) is also discussed. We implemented advanced
statistics for differentiating experimental spectra collected from non-equivalent GSR
samples. Specifically, the differentiation of GSR particles originating from two different caliber ammunitions was achieved with high confidence. Automated Raman
microspectroscopic mapping was utilized for GSR detection and identification on
an adhesive tape used for GSR collection. Our preliminary results indicate that this
new technique is more comprehensive than all currently accepted methods of GSR
Locating Metal Oxide Nanoparticle Transformation in Plants Using
Synchrotron X-Rays
Jorge Gardea-Torresdey, University of Texas-El Paso, Department of
Chemistry, El Paso, TX 79968, Jose Hernadez-Viezcas, Hiram CastilloMichel
With the increased use of engineered nanomaterials such as ZnO and CeO2
nanoparticles (NPs), these materials will inevitably be released into the environment, with unknown consequences. In addition, the potential storage of these NPs
or their biotransformed products in edible/reproductive organs of crop plants can
cause them to enter into the food chain, and the next plant generation. Few reports
thus far have addressed the entire life cycle of plants grown in NP-contaminated
soil. Soybean (Glycine max) seeds were germinated and grown to full maturity in
organic farm soil amended with either ZnO NPs at 500 mg/kg or CeO2 NPs at
1000 mg/kg. At harvest, synchrotron micro X-ray fluorescence (Ој-XRF) and micro
X-ray absorption near edge spectroscopy (Ој-XANES) analyses were performed on
soybean tissues, including pods, to determine the forms of Ce and Zn in NP-treated
plants. The X-ray absorption spectroscopy studies showed no presence of ZnO NPs
within tissues. However, Вµ-XANES data showed O-bound Zn, in a form resembling
Zn-citrate, which could be an important Zn complex in the soybean grains. On the
other hand, the synchrotron Ој-XANES results showed that Ce remained mostly as
CeO2 NPs within the plant. The data also showed that a small percentage of Ce(IV),
the oxidation state of Ce in CeO2 NPs, was biotransformed to Ce(III). To the authors’
knowledge, this is the first report on the presence of CeO2 and Zn compounds in
the reproductive/edible portion of the soybean plant grown in farm soil with CeO2
and ZnO NPs.
Chemometrics in Action: Deployment of Handheld Spectroscopic
Devices for First Responders
Suzanne Schreyer, Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury,
MA 01876, Lin Zhang, Michael Hargreaves
In recent years, there is an increasing trend of moving analytical chemistry testing from laboratory environments to field based material identification. The use of
analyzers in the field has characteristics which clearly distinguishes itself from the
use in a traditional laboratory setting. Portable instruments in the field need to be
able to give rapid, consistent results over a range of conditions – from testing in a
warehouse to high stress situations involving emergency personnel and unknown
conditions. This puts additional requirement on the software and underlying data
treatment. For field based applications, merely presenting a spectrum is not enough.
Rather the instrument is required to present a clear and unambiguous result that
can be quickly interpreted by the user. This puts greater emphasis on analytical
method development and is a big hurdle for wide adoption of new analyzers. Intelligent chemometrics algorithms must be used in these cases to give users clear
answers for their intended use in the field. In this presentation, an overview is given
on recent developments of chemometrics algorithms for handheld analyzers based
on Raman, infrared and near-infrared spectroscopy. Specific examples are given of
field based applications for safety and security applications, narcotic detection, and
forensic analysis.
Determination of Carbon Nanotube Fate and Exposure Using a
Microwave-Induced Heating Method
Jacyln CaГ±as-Carrell, Texas Tech University, PO Box 41163, Lubbock,
TX 79409, Amanda Parra, Sabrina Deleon, Fahmida Irin, Mohammad
Saed, Micah Green
Carbon nanotubes (CNTs) are currently one of the most widely used nanomaterials. However, there are currently limited analytical methods to quantify carbon
nanotubes. A novel technique utilizing microwave-induced heating was developed
to determine carbon nanotube concentrations in biological samples. The new
method has been used to determine multi-walled carbon nanotube (MWNT) and
single walled carbon nanotube (SWNT) uptake and bioaccumulation in terrestrial and aquatic organisms. Various forms of carbon nanotubes (functionalized and
non-functionalized MWNTs and SWNTs) were taken up into corn exposed to various
concentrations in soil for 40 d. Translocation of nanotubes to the above ground
portion of the plant (leaves and stem) was very low. The new method has also been
Hyperspectral Imaging and its Applications in Forensics Science
Jeffrey Beckstead, ChemImage Corporation, 7301 Penn Ave.,
Pittsburgh, PA 15208, Carley Chwal
Hyperspectral imaging has been shown to improve the detection of challenging
samples within many forensic applications such as question document, fingerprint
and biological fluids. Hyperspectral imaging combines high fidelity spatial imaging
2014 EAS Abstracts
November 2014
used to evaluate the potential for bioaccumulation of CNTs in earthworms. Carbon
nanotubes were also taken up into earthworms, but did not bioaccumulate with a
low bioaccumulation factor of 0.015 В± 0.004. Finally, the new method has been used
to investigate uptake of nanotubes in aquatic organisms such as Daphnia and fish.
Considering the lack of detection and quantification methods for carbon nanotubes
in biological samples, this new method is not only novel but will be useful in the risk
assessment of carbon nanotubes.
tron spectroscopy and Fourier transform infrared) demonstrate that benzoquinone
has reacted with the surface. Density functional calculations are consistent with a
photoexcited benzoquinone species reacting with the surface.
Electron Beam Induced Reactions of Adsorbed ПЂ-Allyl Ruthenium
Tricarbonyl Bromide: Towards Design of Electron Beam Induced
Deposition (EBID)-Specific Precursors
Julie A. Spencer, Johns Hopkins University, Department of Chemistry,
Baltimore, MD 21218, Rachel Thorman, Michael Barclay, Howard
Fairbrother, Joseph A. Brannaka, Lisa McElwee-White, Oddur IngГіlfsson
Electron beam induced deposition (EBID) is a resistless process with a great deal
of promise in depositing well-defined, metal-containing nanostructures. To date,
EBID of nanostructures has used precursors designed for thermal processes, such
as chemical vapor deposition (CVD); this underscores the need to design precursors specifically for EBID. This surface science study focuses on elucidating the
reaction mechanism involved in EBID of ПЂ-allyl ruthenium tricarbonyl bromide
(ПЂ-C3H5Ru(CO)3Br), an organometallic precursor synthesized specifically to test
its suitability as an EBID precursor. Experiments studied the effects of 500 eV incident electrons on nanometer scale films of ПЂ-C3H5Ru(CO)3Br under ultrahigh
vacuum conditions using X-ray photoelectron spectrometry complemented by mass
spectrometry. Preliminary results indicated that the initial reaction step involved
electron stimulated desorption of one to three carbonyl ligands from the parent
ПЂ-C3H5Ru(CO)3Br molecules, while continued electron processing or annealing
both resulted in almost full loss of Br from the thin film. A parallel gas phase single
electron impact study on ПЂ-C3H5Ru(CO)3Br evaluated the dissociative ionization
and dissociative electron attachment mechanisms potentially involved in EBID. This
parallel study indicated that loss of a single CO ligand is most prevalent, with minimal loss of Br, in agreement with surface science experiments; conversely, the gas
phase study showed significant loss of the ПЂ-allyl group, which was not observed
in surface experiments. Insights from ПЂ-C3H5Ru(CO)3Br studies are compared
with results from other organometallic precursors (e.g. cis-dicarbonyldichloro platinum(II), cis-PtCl2(CO)2) and discussed with regards to increased understanding of
EBID processes and potential precursor design implications.
Identification and Avoidance of Potential Artifacts and
Misinterpretations in Nanomaterial Ecotoxicity Measurements
Elijah Petersen, National Institute of Standards and Technology, 100
Bureau Dr., Gaithersburg, MD 20899,Vincent Hackley, Teresa Kirschling,
Theodore Henry, Jian Zhao, Baoshan Xing, Robert MacCuspie, Marina
Dobrovolskaia, Jason White
Novel physicochemistries of engineered nanomaterials (ENMs) offer considerable
commercial potential for new products and processes, but also the possibility of
unforeseen and negative consequences upon ENM release into the environment.
Investigations of ENM ecotoxicity have revealed that the unique properties of ENMs
and a lack of appropriate test methods can lead to results that are inaccurate or
not reproducible. The occurrence of spurious results or misinterpretations of results
from ENM toxicity tests that are unique to investigations of ENMs (as opposed to
traditional toxicants) have been reported, but have not yet been systemically reviewed. Our objective in this talk is to highlight artifacts and misinterpretations that
can occur at each step of ecotoxicity testing: procurement or synthesis of the ENMs
and assessment of potential toxic impurities such as metals or endotoxins, ENM
storage, dispersion of the ENMs in the test medium, direct interference with assay
reagents and unacknowledged indirect effects such as nutrient depletion during the
assay, and assessment of the ENM biodistribution in organisms. We recommend
thorough characterization of initial ENMs including measurement of impurities, implementation of steps to minimize changes to the ENMs during storage, inclusion
of a set of experimental controls (e.g., to assess impacts of nutrient depletion, ENM
specific effects, impurities in ENM formulation, desorbed surface coatings, the dispersion process, and direct interference of ENM with toxicity assays), and use of
orthogonal measurement methods when available to assess ENMs fate and distribution in organisms.
Formation of High-Coverage Nanoparticle Monolayers through
Click Chemistry
Mackenzie G. Williams, University of Delaware, 111 Lammot DuPont
Laboratory, Newark, DE 19716, Yue Liu, Timothy Miller, Andrew
Gold and silicon surface modification was accomplished using click chemistry. Modified nanoparticles were attached to this functionalized surface through a 1,3-dipolar cycloaddition click reaction between terminal alkyne and azide groups. Silica
nanoparticles were functionalized with azide and alkyne groups using 2-azidoethanol and 4-pentyn-1-ol respectively. In a separate set of experiments, magnetic iron
(III) oxide nanoparticles were functionalized with alkyne groups using 10-undecynylphosphonic acid and 5-hexynoic acid. The chemistry of nanoparticle deposition
was verified through scanning electron microscopy (SEM), single-point attenuated
total reflectance infrared spectroscopy (ATR-IR), X-ray photoelectron spectroscopy
(XPS), and atomic force microscopy (AFM) studies. The quality of the monolayers
formed and the coverage obtained were interrogated by SEM and AFM. The successful application of the click reaction was verified by the presence of the triazole
ring and disappearance of azide and alkyne groups in ATR-IR and XPS results and
compared to density functional theory (DFT) calculations. Continuing work will focus
on the formation of a complete monolayer and the development of pre-defined multilayers controlled by click chemistry.
Silicon Surface Functionalization to Control the Oxidation State of
Copper during Metalorganic Chemical Vapor Deposition of Cu(I)
and Cu(II) Precursors
Yichen Duan, University of Delaware, 114 Lammot DuPont Laboratory,
Newark, DE 19716, Andrew Teplyakov
This work focuses on the surface-limited deposition reactions leading to the formation of copper-containing nanoparticles on functionalized Si(111) surface. The
study compares the previous work on the deposition of Cu(I) precursors and a newly developed deposition of solid-state Cu(acac)2 compound. H-terminated Si(111)
surface is prepared by modified RCA method. Then the sample is loaded into a high
vacuum chamber with the base pressure 1.0 x 10-6 torr. The precursor, Cu(acac)2
powder, is placed in a metal-organic solid state doser which is directly attached to
the high vacuum chamber. The precursor is sublimed by heating and dosed into the
chamber where it reacts with the H-terminated Si(111) surface. A reduction reaction from Cu(II) to Cu(I) is confirmed by X-ray photoelectron spectroscopy analysis.
Atomic force microscopy images reveal the morphology of the nanoparticles formed
on the surface following deposition. A reaction pathway is proposed based on the
densityfunctional-theory calculations to explain the mechanism of this surface-limited reduction reaction and to compare with the deposition of Cu(I) precursor, Cu(hfac)VTMS.
Correlation of Carbon Growth on Ru-Capped Multilayer Mirrors
Irradiated by Extreme-Ultraviolet Light and Electrons
Michael S. Barclay, Johns Hopkins University, 3400 North Charles
St., Baltimore, MD 21218, Nadir S. Faradzhev, Howard Fairbrother,
Shannon B. Hill, Thomas B. Lucatorto
Extreme-ultraviolet lithography (EUVL) is the next likely step in improving chip production for the semiconductor industry. Using a shorter (13.5 nm) wavelength of
light, manufacturers can mass-produce chips with even smaller nanoscale features
than possible with current lithographic techniques. Due to the nature of 13.5 nm
light, EUVL must be performed in a vacuum chamber. This exposes the highly sensitive optics in the EUVL tool to contamination by outgas products from the photoresists used during the lithographic process. To protect the delicate optics, industry
has established a resist-outgas testing protocol to determine a resist’s efficacy before introducing it to the EUVL tool. Unfortunately, a key component of this protocol
is the use of a dedicated, bright, EUV source. To mitigate the large capital investment necessary for such a source, an electron beam is often used as an analog for
the EUV source. To this end, we have measured rates of carbon growth on samples
of Ru-capped multilayer optics exposed to both electron and EUV irradiation in the
presence of admitted hydrocarbon vapors of two model species: benzene and tetradecane. We find that the carbon growth rate for both exposure methods have
Mechanism of Electrical Passivation of Silicon Surfaces with
Meixi Chen, University of Delaware, 201 Dupont Hall, Newark, DE
19716, Nicole A. Kotulak, Nikolas J. Schreiber, Robert L. Opila
Quinhydrone in methanol is known to interact with hydrogen terminated Si surfaces
in such a way that this interaction greatly diminishes excited electron recombination
at the surface. In this work quinhydrone has been separated into its constituent
parts, p-benzoquinone and hydroquinone, and each was dissolved in methanol in
order to test the electrical passivation of the silicon surfaces. P-benzoquinone is
the active component; however, hydroquinone solutions improve in performance
when in contact with the substrates for a longer duration. The effect of light was also
examined. Substrates passivated with benzoquinone in full ambient light conditions
displayed the highest effective carrier lifetimes. The passivation effect of solutions
exposed to light during preparation, but with measurements taken in the dark, were
compared with that of substrates passivated using a process completely performed
in the dark. The passivation effect from these procedures is much less effective than
full light passivation. It is confirmed that the presence of light facilitates the passivation. The presence of a free proton in the solution (i.e., the hydroxyl proton) is also
found to play an important role in passivation. Surface analyses (X-ray photoelec-
2014 EAS Abstracts
November 2014
sub-linear pressure dependence at low irradiance which transitions to linear scaling
at higher irradiance. The growth rates at which this transition occurs, however, are
different for EUV and e-beam irradiation.
Development of SERS Methods for Rapid Detection of MultiPesticides in Food
Lili He, University of Massachusetts-Amherst, 102 Holdsworth Way,
Amherst, MA 01003, Hua Zhang
Pesticide residues in food are under strict regulations due to their potential toxicity
to environmental and human health. Due to the large variety of insecticides, herbicides, and fungicides, it is challenging and time consuming for factory, governmental
agency or test labs to test every possible pesticide residue in food. There is an
increasing need for rapid detection of pesticides in food, as traditional high-performance liquid chromatography (HPLC) and gas chromatography (GC) methods
are usually time consuming. Here I demonstrate the superior capacity of surface
enhanced Raman spectroscopy (SERS) for rapid detection of pesticides on apples,
lettuce and in apple juices; this research is funded by the United States Department
of Agriculture (USDA). SERS is a combination technique of Raman spectroscopy and nano-techniques. The use of nanostructures like silver dendrites increases the Raman signals by more than a million times. Contaminants can be either
swabbed from apple surfaces or captured in food slurries with or without selective
agent (i.e., aptamer), then rapidly measured using a Raman instrument. Our results
show SERS can detect multiple pesticides in different food products and at parts
per million (ppm) level. All detections can be done within 30 minutes including the
sample preparation. A portable Raman instrument can be used to detect pesticides
on site. In summary, the use of SERS shows great success in rapid detection of
pesticides in food.
Controlled Synthesis and Optical Studies of ZnO Nanostructures
Zhengxin Li, University of Delaware, Department of Chemistry and
Biochemistry, 105 Lammot DuPont Laboratory, Newark, DE 19711, Jolie
Blake, Lars Gundlach
In this work, various ZnO nanostructures were synthesized via chemical vapor deposition, and hydrothermal method. The morphology change of ZnO nanostructures
was investigated by changing growth parameters. Scanning electron microscope,
X-ray diffraction, and fluorescence measurements are compared for differently prepared ZnO nanostructures. Ultrafast time-resolved luminescence measurements
can reveal preparation dependence of electronic parameters. Controllable synthesis
of ZnO nanoparticles has great potential in design of desirable ZnO nanostructures
for nanoscale applications such as solars cells, photochemical analysis, and laser
Detection of Olive Oil Adulteration Using Benchtop NMR
Susanne Riegel, Nanalysis, Bay 4, 4500 5 St. NE, Calgary, AB, T2E
7C3 Canada
Olive oil adulteration remains of considerable concern on a global scale. Methods
and techniques for rapid, easy identification of adulterants in olive oil have generated considerable interest. Typical techniques rely on hyphenated chromatography-mass spectrometry (MS) systems (typically liquid chromatography-MS or gas
chromatography-MS). These methods can be time consuming, requiring extensive
sample preparation and data interpretation; and, depending upon the scope of the
method being used, it can be possible to miss certain contaminants if, for example,
they have different retention times or potential dangerous volatiles. We address the
efficacy of a robust, easy-to-use 60 MHz nuclear magnetic resonance spectroscopy
for rapid sample preparation and data acquisition.
Very Large Range Pesticide Screening in Food Using GC Triple
Quadrupole MS
Lori Dolata, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, Massimo Santoro
Greater than before ease of access to high selectivity gas chromatography mass
spectrometry (GC-MS) is allowing more rapid and generic sample preparation in
pesticide testing, is permitting consolidation of multiple analyte lists and matrices
into one analytical qorkflow. While GC-MS-MS is well suited to multi-residue analysis in a diverse range of matrices, an increased number of targeted compounds
also amplify the complexity of method optimization and analytical performance may
become problematic. Furthermore, there is sometimes a desire to look beyond targeted lists for other potentially harmful food contaminants. Presented is the use
of smart instrument control and data processing software applied to GC-MS-MS
analysis of >600 pesticides in matrix to mitigate analytical performance degradation
through MS duty cycle optimization. Also discussed is combining this optimized targeted quantitation with general unknown analysis through fullscan-MRM.
Conditions Using Accelerated Solvent Extraction (ASE) and LCMS-MS
Changling Qiu, South Dakota State University, Chemistry and
Biochemistry Department, Avera Bldg.131, Brookings, SD 57007,
Douglas Raynie
Perfluorooctanoic acid (PFOA) is used as a polymerization aid in the production of
fluoropolymers. These polymers provide oil and water repellency as well as stain resistance, which make them as ideal coating materials for non-stick cookware. PFOA
is bio-accumulative and potentially harmful to humans. PFOA is not supposed to be
found in the final products of non-stick cookware after processing. This study presents a method to determine the potential leaching of PFOA from the cookware under
simulated cooking conditions. PFOA cookware was extracted with ethanol/water
mixture using accelerated solvent extraction (ASE), and the extraction parameters
such as pressure, temperature, and time were optimized. The resulting extracts
were analyzed by liquid chromatography tandem mass spectrometry (LC-MS-MS).
Analysis of Beer, Wine and their Agricultural Constituents (Hops,
Grapes, Grains) for Pesticides Using QuEChERS Extraction and
High-Throughput Sample Preparation
Patricia L. Atkins, SPEX CertiPrep, 203 Norcross Rd., Metuchen, NJ
There are hundreds of commercial pesticides in use in industrial and private agriculture. The concern over human pesticide exposure over the past few decades has
led to the monitoring of these pesticides. Increasing concern over the health effects
of residual pesticides on fruits and vegetables has led to increased testing of these
products to determine the levels of pesticides on produce when it goes to market. In
this study, commercial red wine and beer samples were examined for their pesticide
concentrations. In addition to the examination of the finished alcoholic beverage,
the constituent agriculture products of wine and beer production: grains, malts hops
and wine grapes; were also examined to determine the levels of pesticides found
in those products. The sample preparation and extraction process efficiency and
recovery were examined by processing samples using manual versus high-throughput techniques. The QuEChERS (quick, easy, cheap, effective, rugged, and safe)
method was used to process a greater number of samples in a shorter period of time
than other extraction methods.
Determination of Perfluorooctanoic Acid (PFOA) from the Surface
of Cookware under Simulated Cooking HPLC Analysis of a
Commercial Whirlpool Hopping Process in the Beer Brewing
Jessica S. Henry, University of New Hampshire, 23 Academic Way,
Durham, NH 03824, Kelsey A. Packard, Elizabeth A. Brady, Sterling A.
Tomellini, Austin D. Gregoire
Hops are a key ingredient in the brewing industry due to the bittering and aromatic
qualities they contribute to the final product. In their raw form, hops contain little
bitterness; however, adding hops to the wort kettle and boiling the hops in the wort
will cause an isomerization of insoluble О±-acids to soluble iso-О±-acids, which are the
main component of the bitter flavor. An addition of hops can also occur during the
whirlpool, when no boiling occurs. This late addition of hops is believed to increase
the aroma of the final product, yet little is known about the efficiency of the isomerization during this process. This study aimed to investigate the effects of whirlpool
hopping on the iso-О±-acids in wort. A series of brews were conducted with different
kettle hopping methods to substantiate previous work. These test brews assisted
in removing assumptions that brewers use when talking about hop utilization that
were used in past reports. The test brews where also hopped and fermented as
the standard operating procedure brews at the brewery and then put in front of
professional sensory panels for taste evaluation. The analysis utilized a modified
high-performance liquid chromatography (HPLC) method to monitor the concentration of iso-О±-acids over time in wort samples collected from an industrial brewhouse.
Results show the increase of the iso-О±-acids concentration over time.
Vacuum Ultraviolet Detection for the Identification and
Quantification of Pesticides by Gas Chromatography
Hui Fan, University of Texas-Arlington, Department of Chemistry and
Biochemistry, 700 Planetarium Pl., Arlington, TX 76019, Doug D. Carlton
Jr., Ian Sawicki, Kevin A. Schug, Phillip Walsh, Dale Harrison
Pesticide analysis by gas chromatography (GC) is a tedious process due the activity, thermal instability, and the prevalence of isomeric analyte species. Identification
of these analytes, typically by mass spectrometry, also becomes taxing, since the
ionization routes for many of the compounds result in low analyte response and
similar spectra used for database identification. The vacuum ultraviolet detector
which we have recently introduced is capable of measuring gas phase absorbance
spectra within the wavelength range of 115-185 nm (VUV region) and up to 240
nm. Within this region, all species absorb energy and possess a unique spectral
response, even for ionizably labile pesticides which limit mass spectrometry. Specifically, chlorinated pesticides have sections of fine structure absorbance throughout
2014 EAS Abstracts
November 2014
this wavelength range, further aiding to identification. The mass spectral responses
for captafol, folpet, and methidathion have reported low spectral database matching
capabilities for qualitative identification. The VUV absorbances of these pesticides,
along with various others that are isomers, structurally similar, or tend to coelute,
are very unique, allowing for confident identification and spectral deconvolution if
analyte or matrix coelution occurs. The ability to deconvolute is demonstrated by
quantifying captan under fast GC conditions used to reduce thermal decomposition
on column and the coeluting pair of fenthion and chlorpyrifos.
sions containing Compound X and a stabilizing excipient; the same spiked mixtures
were analyzed by XRD and FT-Raman. Results indicate that both techniques can be
exploited for identification and quantification of crystalline content in initially amorphous samples. An XRD method alone may be sufficient for routine characterization of these dispersions, especially when measurement parameters are optimized.
However, XRD in conjunction with FT-Raman can provide more definitive evidence
of crystallinity. FT-Raman also offers information about the state of the drug in the
formulation matrix.
Development of a Sensitive LC-UV Method to Determine Two Trace
Aldehydes in Maltitol
George Wang, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, Zhen Wu, Holly M. Shackman, Mark S. Bolgar, David K. Lloyd
A high-performance liquid chromatography (HPLC) method for the determination of
glyceraldehyde (GA) and pyruvic aldehyde (PA) in maltitol, based on derivatization
with the 2, 4-di-nitrophenylhydrazine (DNPH), is presented. Maltitol is a commonly
used excipient in drug formulations. It may contain trace levels of GA and PA, which
can react with primary or secondary amines in drug actives to generate impurities. In Baraclude oral solution, maltitol is present at 380 mg/mL while the primary
amine-containing active ingredient is only 0.05 mg/mL. Both high maltitol concentration and low drug loading pose challenges for the method sensitivity. The derivatization of GA and PA by DNPH facilitated the detection with UV absorbance. As
a bi-molecular reaction, the derivatization rate is proportional to the concentrations
of both aldehydes and DNPH. Since the aldehydes are at trace level, to allow the
reaction to proceed quickly, the concentration of DNPH needs to be relatively high. It
was found that some GA-DNPH converted to PA-DNPH when stored for an extended period. A mechanism based on enolate tautomerization is proposed for this conversion. To prevent maltitol from forming a gel, the content of acetonitrile should be
kept low (<25%) in the sample diluent. Finally, the method sensitivity was enhanced
by optimizing HPLC conditions including detection wavelength, injection volume and
maltitol concentration. The finalized method has easy sample preparation and is
suitable for use in a quality control environment. The method was validated with a
quantitation limit of 0.01 ug/mL (~25 ppb relative to maltitol).[1]
Nanoparticle Synthesis Using Aerosol Reactions
Ho Yeon Yoo, Stanley Bruckenstein Chemical Consulting, 115 Foxpoint
W, Williamsville, NY 14221, Stanley Bruckenstein
Nanoparticles were synthesized by merging two aerosols containing the different
constituents of the nanoparticles. The feasibility of this concept for three classes
of reactions in aerosols was demonstrated using the chemiluminescent reaction
between Luminol and Potassium Ferricyanide, the pH sensitive fluorescence of
Umbelliferone and the precipitation of silver chromate by reaction of silver nitrate
with potassium chromate. We postulate that using oppositely charged aerosols are
more efficient synthetically and will produce a narrower distribution of nanoparticle
sizes than using uncharged aerosols. We also describe how to construct a desk top
apparatus to carry out the synthesis of nanoparticles.
Withdrawn by the author.
Moisture Determination in Rubber Stoppers Using Relative
Humidity Sensing Technology; A Karl Fischer Titration Alternative
Garrett Rowe, Arizona Instrument, 3375 N. Delaware St., Chandler, AZ
85225, Christopher Altamirano
The use of rubber stoppers in the pharmaceutical industry has an important purpose
of keeping the active ingredients within drugs protected from the external environment. Keeping the contents within a vial dry (or at least within a controlled humidity
range) is essential for long term storage and drug efficacy. Although rubber stoppers
provide an airtight seal to a vial, the rubber material itself is inherently hygroscopic
and prone to moisture absorption. Therefore, knowing the baseline moisture level
of certain rubber stoppers during drug processing and packaging has become part
of the quality assurance monitoring of many pharmaceutical companies. Moisture
analysis of rubber stoppers is traditionally performed using a Karl Fischer (KF) titration method, which tend to be lengthy and consume expensive wet chemical
reagents. Using a moisture specific relative humidity (RH) sensor to detect inherent moisture within rubber stopper provides fast and accurate results that can be
used without the use of wet chemical reagents. The % moisture of various rubbers
stoppers analyzed by both the RH and KF methods statistically correlate between
various data sets. Moreover, using the RH sensor method proved to yield quicker
test times with a smaller standard deviation when compared to the traditional KF
[1] C. Pathirana et al, Tetrahedron Letters, 54 (2013) 132–133.
Particulate Contamination Control - Current Technology versus
Oliver Valet, rap.ID Inc., Princeton Corporate Plaza, 11 Deer Park Dr.,
Ste. 201, Monmouth Junction, NJ 08852
The thorough root cause investigation of particle contamination in pharmaceutical
production requires reliable chemical identification techniques. Particle identification
techniques including: scanning electron microscopy / energy dispersive spectrometer, Fourier transform infrared (FTIR) or Raman microscopy are necessary to unveil
a particles chemical nature. Traditional laboratory methods use different equipment
for each technique, meaning micro-objects must be transferred between methods.
It is difficult and time consuming to continuously locate small particles on micrometer scales, and errors can occur. Besides manual obstacles, every instrument has
its own software and chemometric system, producing independent data-sets. The
pharmaceutical industry works in a regulated environment, and 21 CFR Part 11
compliance is stringent regarding the collection of raw data. State of the art equipment combines multiple techniques in one system and produces traceable, reliable,
and compliant data. This data enables chemical characterization, and matches micro-particle materials with materials used in manufacturing. The rap.ID SPE unit
eliminates need for sample transfer between machines. Furthermore, analysis is
completely automated and user friendly, thus there is no need for a spectroscopic
expert; results are compared to a built-in matrix database, and corresponding reports generated. Once the database establishes a match for the samples spectral
fingerprints, differentiation between samples can be made and their origins determined. Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS)
techniques are vital for the accurate identification of microscopic materials. Raman
provides information regarding molecular structure, while LIBS yields elemental
composition. Thus, their combination provides a unique tool for identifying unknown
particles such as foreign particulate matter.
Detection and Quantification of As, Cd, Hg, and Pb in API and FDP
Using Procedure 2 of Updated USP <233>
Michael Murphy, Intertek Pharmaceutical Services, PO Box 470,
Whitehouse, NJ 08807, Philip Costello, Diana Buro
The United States Pharmacopeia (USP) has replaced the non-specific colorimetric test with two new chapters <232> (Limits) and <233> (Procedures). The limits
used to assess active pharmaceutical ingredients (API) and formulated drug products (FDP) will be updated this year pending the adoption of guidance International
Conference on Harmonization (ICH) Q3D. The limits between the USP and ICH
documents will not be perfectly aligned and hence the limits chosen for testing will
depend on the intended market of the API or FDP. A simplified testing scheme will
allow rapid transfer of methodology between different APIs and FDPs reducing/eliminating the time required to develop adequate sample preparation/analytical conditions. The Milestone Ultrawave microwave digestion system and an Agilent 7700X
ICP-MS can be used to develop a general methodology for sample preparation
and analysis. Data presented for multiple sample types demonstrates how this type
of procedure can streamline method development/validation for the required USP
elements at the new monograph limits.
Determination of Crystallinity in Amorphous Solid Dispersions
Cynthia S. Randall, Particle Sciences, 3894 Courtney St., Bethlehem,
PA 18017, Ajoy Koomer, Rabi Bello
Solid dispersions are an attractive option for formulating poor water soluble drugs.
But the inherent physical instability of amorphous materials is a major concern, and
methods are needed to detect the onset of crystallization. X-ray diffraction (XRD)
and Fourier transform (FT)-Raman were evaluated for this purpose in analysis of
Compound X solid dispersions. The basic approach involved spiking pure amorphous material with known amounts of the crystalline drug form. Spiking was done
with the amorphous drug substance alone, as well as with amorphous solid disper-
Impact of Croscarmellose Sodium on Recovery of Active from
Formulated Capsules
Songling Yu, Celgene Corporation, 86 Morris Ave., Summit, NJ 07901,
Xiaolu Liao, Mauro Solorzano, Naijun Wu
A new formulation with 0.3 mg and 1 mg capsule strengths was developed for a
weakly basic developmental compound. During the development of assay and related impurity methods by high-performance liquid chromatography (HPLC), low
drug recoveries were observed. However, no significant impurities or degradation
products were found in the sample. In this study, the effects of formulation excipient,
sample diluent pH, diluent composition, and sonication time on assay recovery were
2014 EAS Abstracts
November 2014
investigated. The results showed that the low recovery was related to the use of 4%
w/w Croscarmellose sodium disintegrant. The drug recovery was improved significantly by using low pH diluents and less impacted by acetonitrile percentage of the
diluents or sample sonication time. The data suggests that the low recovery could
be attributed to interactions between the amino group of drug and the carboxylic
group of Croscarmellose sodium. The interaction between drug and Croscarmellose
sodium is pH dependent and can be minimized using acidic diluents. Accordingly,
assay and related impurity methods were optimized and a recovery of 100.3% was
achieved using an ACN:0.1% HCl, 20:80 (v/v) diluent at pH ~2.
Determination of the Enantiomeric Purity of Dextromethorphan via
Mark Canestrano, Anton-Paar USA, 10215 Timber Ridge Dr., Ashland,
VA 23005
Methorphan is a chiral drug that differs in its effects and pharmacology with respect
to its two enantiomers. The dextrorotatory dextromethorphan is applied as an antitussive drug in cough medicines, whereas the levorotatory enantiomer levomethorphan is a strong opioid analgesic that is listed as a schedule II drug in the United
States. As dextromethorphan is approved for use in over-the-counter drugs, accurate control of enantiomeric purity is highly essential. Enantiomeric purity is controlled by measurement of optical rotation, which is dependent on the wavelength.
The specific rotation of dextromethorphan is increased approximately 10 fold when
measuring in the UV range, compared to values obtained from measurements performed at the sodium D line that is typically used in pharmaceutical applications. As
the UV range wavelength results in an increased optical rotation that also correlates
with a higher sensitivity of the measurement, international pharmacopeias demand
the measurement of optical rotation in dextromethorphan solutions at 325 nm. To
determine optical rotation in dextromethorphan according to international pharmacopeias at 325 nm, Anton Paar developed the MCP 500/MW325 polarimeter.
Characterizing Ligand Binding to Serum Albumin Using Taylor
Dispersion Analysis
David Goodall, Paraytec, York House, Outgang Lane, Osbaldwick, York,
YO19 5UP United Kingdom, Thomas Allen, Alexander Chapman
Characterizing the interaction of small molecule ligands with selectors is important
for drug discovery in the pharmaceutical industry. For serum albumin, the binding
affinities for ligands are routinely screened in order to characterize plasma protein
binding, with the gold standard method equilibrium dialysis. The objective of this
work was to determine whether unbound fractions and binding affinities in the milliand micro-molar ranges can be measured by Taylor Dispersion Analysis (TDA). A
Viscosizer 200 (Malvern Instruments) was used for TDA, with wavelengths for study
214, 254 and 280 nm. Human serum albumin (HSA) was the selector, with naproxen
and warfarin as ligands. The method uses less than 10 ВµL of material, and is shown
to be applicable in biorelevant media (40 mg/mL HSA and plasma). New TDA methodology is demonstrated showing how binding information can be extracted over a
range of 2 orders of magnitude variation of sample concentration in a single run for
injection of ligand as sample into a carrier containing a fixed concentration of HSA.
At low absorbance/low concentration the TDA profile is that of the bound ligand,
whereas at high absorbance / high concentration the profile is that of the free ligand.
Advantages relative to alternative techniques (e.g., surface plasmon resonance,
affinity chromatography) include having the protein in free solution, with no immobilization required which might change affinity and binding properties.
Determination of Carbohydrates in Various Matrices by Capillary
Terri T. Christison, Thermo Fisher Scientific, 1214 Oakmead Parkway,
Sunnyvale, CA 94088, Peter Bodsky, Carl Fisher, Hua Yang, Monika
Verma, Linda Lopez
Monosaccharide and disaccharide determinations are important to various industries from ensuring quality product to researching biological pathways and disease
states. Because carbohydrates are poor chromophores, chemical derivatization is
needed for absorption. However, derivatization is costly, labor-intensive and may
cause changes in molecular configuration. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is a proven sensitive
method to directly and selectively determine carbohydrates. In HPAE-PAD, carbohydrates are ionized in strong base and separated by anion-exchange chromatography. The carbohydrates are detected by PAD with a gold working electrode using
a four-potential waveform selective and sensitive for carbohydrates. This sensitivity
allows carbohydrate analysis down to pmole concentrations or when the samples
are limited. This sensitivity is moderated in beverage samples which contain g/L
concentrations by minimizing the flow path combined with moderate dilution. Here
we combine the advantages of a reagent-free ion chromatography (RFIC) system and a capillary format IC to determine monosaccharides and disaccharides
in various applications, from low concentrations in synthetic urine samples to high
concentrations in beverage samples. In an RFIC system, the hydroxide eluent is
electrolytically generated inline to deliver accurate and precise concentrations for
isocratic or gradient separations by only adding deionized water. Eluent generation
eliminates carbonate contamination and errors from manual preparation. A capillary
scale system with ВµL/min flow rates can run 24/7, always on and always ready for
samples. Eluent consumption and waste generation are reduced to 5.2 L/yr and
eluent generator cartridges can last up to 18 months.
Dual Wavelength Imaging of Whole Tablet Dissolution
Alexander Chapman, Paraytec, York House, Outgang Lane, Osbaldwick,
York, YO19 5UP United Kingdom, David Goodall
The aim of this study was to provide proof of concept for whole tablet imaging with
dual wavelength detection. The instrumentation used an ActiPix UV area imaging
module (Paraytec Ltd). A standard 1 cm fused silica spectrophotometer cuvette was
used as the dissolution vessel. The tablet was mounted on a fused silica tube, held
in the teflon cap. A miniature stir bar motor was positioned underneath the cuvette.
The light sources were 255 (UV) and 505 nm (Vis) light emitting diodes, with sequential on/off switching at frame rate 2 Hz. The sample tested was Sulazine EC,
an enteric-coated sulfasalazine delayed release tablet. Measurements were carried
out in 0.1 N HCl and in PBS. A narrow boundary layer develops in both the UV and
Vis, consistent with initial swelling of the polymer film coating the tablet. In PBS
there is a sharp increase in the gradient of absorbance with time at 42 minutes,
with UV-Vis absorbance ratio 10:1 mirroring the ratio of extinction coefficients of sulfasalazine at 255 and 505 nm and indicating the start of the main phase of release
of sulfasalazine. This timing agrees well with literature data (Bali et al, Int. J. Biol.
Pharm. Res. 2013, 4, 491-494). Spatially- and time-resolved images provide an immense amount of detail and prove the benefits of both visualization and quantitative
measurements of absorbance at the two wavelengths, one in the UV and one in
the Vis to characterize physical and chemical changes occurring during the tablet
dissolution process.
High-Throughput and Highly Reproducible Sub-4 min Separation
of Proteins and Antibodies Using Size Exclusion Chromatography
Justin Steve, Tosoh Bioscience, 3604 Horizon Dr., King of Prussia, PA
19406, Atis Chakrabarti
Aqueous size exclusion chromatography (SEC), popularly known as gel filtration
chromatography (GFC) is a powerful analytical tool in the separation of protein
species of differing size and their molar mass variants and impurities. Traditionally,
GFC columns with a dimension of 7.8 mm ID x 30 cm L are used for analytical
purpose. The longer column dimension lead to longer run times, sample dilution
as well as substantial solvent waste. Instead a 4.6 mm ID x 15 cm L column may
give high-throughput separation with shorter run time, high resolution and minimal
solvent waste using conventional high-peformance liquid chromatography (HPLC).
Here we show the use of a 4.6 mm ID x 15 cm L TSKgel SuperSW mAb HTP SEC
column for the highly reproducible separation of proteins and antibodies in less than
3.5 minutes by using a moderate flow rate of 0.75 mL/min. Even at the slower flow
rate of 0.35 mL/min, the overall run time was less than 7 minutes without considerable decrease in resolution. Here we report the lifetime study and performance of
the TSKgel SuperSW mAb HTP column in the separation of mAb using fast assay
conditions. The retention time and other peak parameters were reproducible with a
low percentage relative standard deviation (%RSD). This study illustrates adequate
stability of the TSKgel SuperSW mAb HTP column for high speed, sub-4 minute
separations of monoclonal antibodies.
Development of a 2-Butanol Background Electrolyte for NonAqueous Chiral Capillary Electrophoresis
Erin J. Ennis, Drexel University, 3141 Chestnut St., Philadelphia, PA
19104, Joe P. Foley
Non-aqueous capillary electrophoresis (NACE), an alternative to aqueous electrokinetic chromatography, is a technique that utilizes organic modifiers to enhance
the separation of charged hydrophobic compounds. NACE can be applied to the
separation of enantiomers by utilizing chiral additives or an enantiopure solvent,
although the latter has received little attention. The separation of charged enantiomers will be examined for various preparations of non-aqueous enantiopure solvent to optimize experimental methods. While previous work examined 2-butanol
as a potential NACE background electrolyte at both low (mainly aqueous) and high
(mainly non-aqueous) concentrations, this work examines a novel and completely
non-aqueous background electrolyte comprised of various concentrations of 2-butanol and acetonitrile for the separation of charged pharmaceutical enantiomers.
Experimental emphasis is on the optimization of resolution, migration time, and enantioselectivity. Chromatographic figures of merit are examined and applications are
explored for future work.
2014 EAS Abstracts
November 2014
Glycerophospholipids Analysis by Comprehensive TwoDimensional Liquid Chromatography
William Hedgepeth, Shimadzu Scientific Instruments, 7100 Riverwood
Dr., Columbia, MD 21046, Kenichiro Tanaka, Tetsuo Iida, Yoshiyuki
Watabe, Junichi Masuda, Yoshihiro Hayakawa
Comprehensive two-dimensional liquid chromatography (LCxLC) is a powerful tool
for the analysis of complex samples including pharmaceutical, biological, and natural products. It can provide higher resolution that traditional LC (1-D/LC) can ever
achieve because it combines two separation modes in a single analysis. Recently,
the use of ultra high-performance liquid chromatography (UHPLC) has been successfully employed to remarkably decrease analysis time in the second dimension
of comprehensive LC, and it is becoming a popular way to speed up 2-D/LC separations. In this poster, we present glycerophospholipids (GPLs) analysis using our
newly-developed comprehensive two-dimensional liquid chromatograph system.
GPLs are the major component of biological membranes. They can not only act as
a barrier from the external environment, but can also play a key role in a variety of
biological processes including membrane trafficking and signal transduction. Thus,
analysis of GPLs is one of the most important studies in the metabolomics field.
Although reversed-phase (RP) HPLC coupled with electrospray ionization tandem
mass spectrometry (ESI-MS-MS) is an effective strategy for lipidomics, there is still
room for further improvement of the analytical methods. One drawback to performing determination of GPLs is ion suppression by co-eluting compounds. To obtain
reliable results, complete separation of target GPLs by comprehensive two-dimensional liquid chromatography with ESI-MS-MS is an effective strategy.
Environmentally Friendly HPLC Methods for the Determination of
Renal Function Biomarkers, Creatinine and Uric Acid, in Human
Si Zhou, University of Massachusetts Dartmouth, 285 Old Westport
Rd., North Dartmouth, MA 02747, Zhuo Zhu, Ruiting Zuo, Xiaofei Lu,
Yuegang Zuo
Two environmentally-friendly high-performance liquid chromatography (HPLC)
methods for simultaneous determination of creatinine and uric acid in human urine
samples have been developed. Human urine samples were pretreated by dilution,
protein precipitation, centrifugation and filtration, followed by reversed-phase (RP)
or hydrophilic HPLC separations. The developed methods provide simple, rapid
separation and sensitive detection for the interest species using a C18 or an Agilen
Zorbax SB-CN column (4.6 x 150 mm) in 6 minutes with UV detection at 205 nm
by using an isocratic elution containing highly aqueous mobile phase at pH 4.75.[1]
Quantification was carried out by relating the peak areas of identified compounds
to that of the cimetidine as an internal standard. The detection limits for creatinine
and uric acid were 0.04 Вµg/mL and 0.06 Вµg/mL, respectively. The recoveries of the
standards added to urine samples were 91.6% - 97.7% for creatinine and 89.7% 100.9% for uric acid and the relative standard deviation for both analytes was less
than 6.0%. For the RP-HPLC, a simple retention recovery procedure is required
after each run. These methods are suitable for the estimation of creatinine and uric
acid in human urine and other bio-fluid samples.
[1] Y. Zuo, 2014, High-Performance Liquid Chromatography (HPLC): Principles,
Procedures and Practices. Nova Science Publishers, Inc., New York.
Development of Stability Indicating HPLC Assay and Degradation
Product Methods for a Peptide Drug Product
Kaimeng Zhou, Merck, 556 Morris Ave., Summit, NJ 07901, Jing Zhang,
Zheng Zhao, Wendy Zhong, Claudia Neri, Yun Mao
Peptide separation is very challenging especially for peptide drug product containing impurities and degradants whose structures are highly similar to their parent
peptide. To guide peptide formulation optimization, good stability-indicating methods which provide baseline separation of peptide and its impurities and degradants
is essential. This presentation focuses on the development of reversed-phase
high-performance liquid chromatography (HPLC) methods for peptide A impurities
and degradants. Our analytical strategy to develop good stability-indicating methods
involves multiple HPLC modes to achieve the separations of complex peptide impurities and degradation products for the peptide stability characterization. A number
of conventional and new columns including C18 and phenyl-hexyl stationary phases
were screened. To optimize resolution, several mobile phases containing different
organic modifiers and salts were examined. A broad range of separation parameters
were evaluated. It was found that column temperature and gradient have strong
impact to the critical pair resolution and need to be carefully tuned and controlled.
The optimum conditions for assaying Peptide A and its impurities were obtained with
a mobile phase containing acetonitrile with perchlorate (NaClO4) at acidic pH as an
ion-pairing reagent. A mobile phase containing trifluoroacetic acid (TFA) was found
to provide better resolution for the peptide degradation products. Liquid chromatography tandem mass spectrometry was utilized to identify the degradants and verify
the separation. This degradant method has been successfully validated and applied
in stability monitoring of clinical formulations.
2,4-Bis(dimethylbenzyl)phenol and Bisphenol A in Blue Crab and Prawn
Zhuo Zhu, University of Massachusetts-Dartmouth, 285 Old Westport
Rd., North Dartmouth, MA 02747, Mohammed Alshanqiti, Joseph
Michael, Sarah Pereira, Yuegang Zuo
Bisphenol A (BPA), 4-cumylphenol (4-CP) and 2,4-bis-(dimethylbenzyl)phenol (2,4DCP) are all high production volume chemicals and widely used in plastic and other
consumer products. During the past two decades, BPA has attracted a great deal
of scientific and public attention due to its presence in the environment and estrogenic property. Although 4-CP and 2,4-DCP are much more estrogenic and toxic
than BPA, little information is available about their occurrence and fate in the environment. In this study, a rapid, selective, accurate and reliable analytical method
was developed for the simultaneous determination of 4-CP, 2,4-DCP and BPA in
blue crab and prawn Macrobrachium rosenbergii. The method comprises an ultrasound-accelerated extraction followed by capillary gas chromatographic (GC) separation. The detection limits range from 1.50 to 36.4 ng kgв€’1 for the three alkylphenols. The calibration curves are linear over the concentration range tested with the
coefficients of determination, R2, greater than 0.994. The developed method was
successfully applied to the simultaneous determination of 4-CP, 2,4-DCP and BPA
in prawn samples. The peak identification was confirmed using GC mass spectrometry. Bisphenol A, 2,4-bis-(dimethylbenzyl)phenol and 4-cumylphenol were found in
blue crab and prawn samples in the concentration ranges of 0.67–5.51, 0.36–1.61,
and 0.00–1.96 ng g−1 (wet weight), respectively. All relative standard deviations are
less than 4.8%. At these environmentally relevant concentration levels, 4-CP, 2,4DCP and BPA may affect the reproduction and development of aquatic organisms,
including negative influence on crustaceans’ larval survival, molting, metamorphosis
and shell hardening. This is the first study reported on the occurrence of 4-CP, 2,4DCP and BPA in blue crab and prawn M. rosenbergii.
Carbohydrate Analysis in Beverages and Food Using Pulsed
Amperometric and Charged Aerosol Detection
Qi Zhang, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Marc Plante, Bruce Bailey, David Thomas, Ian Acworth, Michael
Carbohydrates are important food components affecting taste and nutrition. The
determination of the types and levels of carbohydrates in foods is important for
energy evaluation, nutritional labeling, quality control and for identifying possible
product adulteration. Separation and detection of carbohydrates can be challenging.
Simple carbohydrates are highly polar, uncharged, and as they lack a chromophore
cannot be measured directly by UV absorbance detectors. High-performance liquid chromatography (HPLC) with various detection techniques has been used for
carbohydrate analysis, such as infrared, mass spectrometry, UV or fluorescence
following derivatization. Although methods utilize derivatization improve the chromatographic resolution and detector sensitivity, they can lead to increased assay
variability. This poster presents two approaches for carbohydrate analysis that solve
the challenges for separation and detection of carbohydrates without requirement
for laborious derivatization: high performance anion exchange chromatography with
pulsed amperometric detection, hydrophilic interaction liquid chromatography and
charged aerosol detection. The electrochemical detector now with pulsed amperometric detection capabilities when coupled with a gold working electrode provides
high sensitivity and selectivity for the measurement of carbohydrates in complex
food sample matrices. The charged aerosol detector is a mass-sensitive detector
that can measure all non-volatile, and many semi-volatile compounds in a sample,
The Building Blocks of Life: Comparing Methods for Amino Acid
William Hedgepeth, Shimadzu Scientific Instruments, 7100 Riverwood
Dr., Columbia, MD 21046, Kenichiro Tanaka, Steve Wishnies
Amino acids are the building blocks of proteins and contain an amine and carboxylic acid functional group. High-performance liquid chromatography (HPLC) is the
most popular technique for analyzing amino acids, and their usage has increased
as supplements in the health food industry. Traditionally, amino acid analysis has
been run with cation exchange chromatography with post-column derivatization.
Pre-column derivatization with higher speed reversed-phase ultra-high pressure
liquid chromatograhy has become more popular to shorten analysis times. Additionally, analysis time can be further shortened with automation and the use of a mass
spectrometry detector. This poster discusses the various methods used to analyze
amino acids by LC and the benefits and disadvantages of each method.
2014 EAS Abstracts
November 2014
typically with low nanogram sensitivity. Application examples for various juice samples, milk and other dairy products, syrup and honey are discussed.
exist in their mean concentrations over the three years studied. Signals for as of yet
unidentified compounds also displayed seasonal and yearly trends. These results
are presented along with a discussion of the challenges of identifying the selected
phenols in the maple sap matrix.
Targeted Research of Peptides by LCxLC-MS-SQD
Joanne Mather, PerkinElmer, 940 Winter St., Waltham, MA 02451,
Noelle Elliott, Sean Daugherty, Sabine Heinisch
One-dimensional liquid chromatography coupled to mass spectrometry detection
(LC-MS) is a widely used analytical method which can be applied to a broad variety
of analysis fields such as pharmaceuticals, biological compounds as well as environmental samples. However, the complexity of such samples causes two relevant
issues in LC-MS which can be overcome with comprehensive two-dimensional liquid chromatography coupled to MS (LCxLC-MS): 1) The maximum peak capacity
that can be achieved in 1D-LC for peptide analysis, taking into account the current
equipment constraints, is about 1000-1500 at the expense of very long gradient
times and very long columns. Conversely, the theoretical peak capacity in LCxLC
is the product of peak capacities in each dimension and hence could lead, for instance, to 2500 considering a peak capacity as low as 50 in each dimension. 2) In
LC-MS, poorly separated compounds reach together the mass spectrometer source
and as a result are subjected to an ion suppression phenomenon which may compromise their detection. The increase in total peak capacity in LCxLC, and hence the
better separation of compounds before entering the ionization source is expected to
reduce ion suppression effects. This study shows the great benefit of LCxLC-MS for
peptide analysis via reverse-phase (RPLCxRPLC) separations of protein digests.
These separations led to much higher peak capacities than those expected in LCMS. A significant reduction of ion suppression effects is brought out. Moreover,
these results are associated to a huge decrease in analysis time. Our results point
out the right adequacy between a simple quadruple mass spectrometer coupled to
LCxLC, and the targeted research of peptides in complex protein tryptic digests.
Rapid Identification and Determination of Polyphenols in Herbal
Plants by UPLC/MS/MS
Saliha Esin Г‡elik, Istanbul University, Faculty of Engineering,
Department of Chemistry, 34320 AvcД±lar, Istanbul, Turkey 34320, Burcu
BekdeЕџer, AyЕџe Nur Tufan, Mustafa Г–zyГјrek, Kubilay GГјГ§lГј, ReЕџat Apak
This study reports the characterization of polyphenolic compounds in pennyroyal
(Mentha pulegium), marjoram (Origanum marjoram) and lavender (Lavandula vera)
extracts by using ultra performance liquid chromatography (UPLC) coupled with
tandem mass spectrometry (MS-MS) in negative mode of electron spray ionization (ESI). 9 phenolic acids (gallic acid, protocatechuic acid, vanillic acid, caffeic
acid, ferulic acid, sinapic acid, syringic acid, p-coumaric acid, rosmarinic acid) and
9 flavonoids (catechin, rutin, hesperidin, morin, fisetin, quercetin, naringenin, kaempferol and apigenin) were monitored within 12 minute gradient elution program.
The developed method was validated with good precision (RSD % 0.54- 2.72 for
intra-day, 1.71-4.64 for inter-day), reproducibility (REC% 95.1-104.6) and linearity
(r=0.9989-0.9999). Polyphenols were quantitatively analyzed in the plant extracts.
Total antioxidant capacity and total phenolic content of samples were measured by
using CUPRAC (cupric reducing antioxidant capacity) and Folin assays. Microwave
assisted extraction (MAE) technique was used to extract phenolic antioxidants in
herbal plants. The extracts exhibited high antioxidant capacity (ranged from 0.33
mmol T/g to 0.70 mmol T/g). The proposed method was found to be easy, fast,
reproducible and convenient for quantitative analysis of polyphenols.
Determination of Corilagin by Hybrid Solid-Phase Extraction (SPE)
Ultra-Performance Liquid Chromatography Coupled with Triple
Quadrupole Mass Spectrometry in Rat Plasma and its Application
to Pharmacokinetic and Bioavailability Studies
Atul S. Rathore, Poona College of Pharmacy, Bharati Vidyapeeth
University, Erandawane, Paud Rd., Pune 411038, India,
Sathiyanarayanan Lohidasan, Kakasaheb R. Mahadik
A sensitive, specific and rapid hybrid SPE ultra performance liquid chromatography
tandem mass spectrometry (UPLC–MS-MS) method has been established to study
pharmacokinetic properties of corilagin. Corilagin is an ellagitannin, was determined
in rat plasma with ellagic acid as internal standard. Chromatographic separation
was performed on a Waters ACQUITY UPLC BEH C18 column (50 mm Г— 2.1 mm,
1.7-µm) with gradient elution system at a flow rate of 0.3 mL/min. The mobile phase
was composed of 0.1% formic acid water and 0.1% formic acid acetonitrile solution.
Analysis was performed under a triple-quadruple tandem mass spectrometer with
an electrospray ionization (ESI) source via the multiple reaction monitoring (MRM)
mode to determine corilagin at [M-H]- m/z 633.0 в†’ m/z 301.0 and that of IS at
[M-H]- m/z 300.9 в†’ m/z 145.0 within 3 min. The assay method exhibited good
separation of corilagin from the interference of endogenous substances. The lower
limit of quantification (LLOQ) was 20 ng/mL, with a good linearity within the concentration range of 20–1000 ng/mL (r = 0.9993). Intra-day and inter-day precision RSD
was less than 7.83%; intra-day and inter-day accuracy was 90.26% and 102.12%
respectively. The extraction recoveries of corilagin was over 90% in rat plasma, and
the extraction RSD was less than 2.06%. The established UPLC–ESI-MS-MS method is rapid and sensitive, which has been successfully applied to a pharmacokinetic
study of corilagin in rats for the п¬Ѓrst time following oral (10 mg/kg) and intravenous
(1 mg/kg) administration.
Benefits of Using Wide Pore Superficially Porous Particles for
Biomolecule Separations
Wu Chen, Agilent Technologies, 2850 Centerville Rd., Wilmington, DE
19808, Anne Mack, Jim Martosella, Kunqiang Jiang
Superficially porous particles with pore size ranging from 90Г… to 120Г… have been
a great success for fast separation of small molecules over totally porous particles
in the recent years. However, superficially porous particles with wide pore size of
≥300Å provide even better benefits over totally porous particles for separation of
large biomolecules such as proteins and monoclonal antibodies (mAbs) because
the analyte diffusion length inside a particle plays a more important role for fast
separation in large molecule separation than in small molecule separation. Wide
pore superficially porous particles in 5 Вµm size have been used for protein separations for a decade.[1] And more recently, wide pore superficially porous particles in
smaller size have been developed to meet the need of continuing interest in larger
therapeutic molecules by biopharmaceutical companies.[2] This report includes fast
separations of intact protein mixtures, as well as examples of very high-resolution
separations of larger monoclonal antibodies and associated variants by using wide
pore superficially porous particles, and their advantages over wide pore totally porous particles.
[1] J. Chromatogr. Sci.; 2008; 46(3); 261-8; Ricker R. D., Woodward C. B. 3rd, Forrer
K., Permar B. J., Chen W.; Options for rapid analysis of peptides and proteins,
using wide-pore, superficially porous, high-performance liquid chromatography
particles with unique bonded-phase ligands.
[2] J. Chromatogr. A; 2013; 1315; 118-26; Schuster S. A., Wagner B. M., Boyes B.
E., Kirkland J. J.; Optimized superficially porous particles for protein separations.
Rapid Quantitative Determination for Comparative Analysis of
Biologically Active Piperamides, Phenolics, Flavonoids and
Terpenoid in Fruits and Leaves of Ten Piper Species using UPLCESI-MS-MS
Preeti Chandra, Central Drug Research Institute, Sector 10, Jankipuram
Extension, Sitapur Rd., Lucknow 226031 India, Renu Pandey, Brijesh
Piper species are of high commercial and economic importance have been used
extensively in traditional medicines. Piperamides are the main bioactive compounds
in the species other than, phenolics, flavonoids and terpenoid. To determine the
distribution and content of these compounds in different plant parts (fruits & leaves)
of ten Piper species, a reliable method has been established using ultra high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem
mass spectrometry (UPLC-ESI-MS/MS). Thirteen constituents, namely piperamides
(piperine, piperlongumine and piperlonguminine), phenolics (caffeic acid, ferulic
acid, protocatechuic acid, rosamarinic acid and vanillic acid), flavonoids (quercetin,
kaempferol, apigenin and luteolin) and terpenoid (ursolic acid) were simultaneously
determined in 8 min. The analysis was accomplished on an Acquity UPLC BEH C18
(2.1 mm Г— 50 mm, 1.7-Вµm) column using gradient elution with 0.1% (v/v) formic
acid water and acetonitrile. Piperamides were detected in positive mode while phe-
Analysis of Phenolic Compounds in New Hampshire Sugar Maple
Sap by LC-MS
Elizabeth Brady, University of New Hampshire, 23 Academic Way,
Durham, NH 03824, Walter C. Shortle, Sterling Tomellini, Martha
Carlson, Barrett N. Rock
A liquid chromatography-mass spectrometry (LC-MS) method has been developed
for the analysis of selected phenolic compounds in sugar maple sap. The method
employs a direct injection analysis in order to avoid expensive and time consuming sample preparation steps. Interest in phenolic compounds has grown in recent
years as research has demonstrated their potential antioxidant, antiradical and antimutagenic capabilities. While many studies have focused on these compounds in
relation to human health, relatively less is focused on the relationship of phenolic
compounds to the health of the plant in which they are produced. The LC-MS method developed in this study provides the capability to detect selected phenolic compounds in sugar maple sap samples provided by volunteers throughout the state of
New Hampshire using only a syringe filter for sample preparation. Quantitative data
from the 2011, 2012 and 2013 tapping seasons were analyzed for trends and variability. Preliminary observations show that a wide range of variability are possible
for these compounds over individual tapping seasons and significant differences
2014 EAS Abstracts
November 2014
nolics, flavonoids and terpenoid were detected in negative electrospray ionization
tandem mass spectrometry operating in multiple-reaction monitoring (MRM) mode
with continuous polarity switching. A good linear regression relationship for each
analyte was obtained over the range from 1-250 ng/ml. The intra- and inter-day
assay precision ranged from 0.63 to 3.37% and 0.91 to 4.09% respectively. The
recovery relative standard deviation (RSD) measured at three concentration levels
varied from 0.78-2.78%. The method sensitivity expressed as limit of quantitation
was typically 0.06-3.88 ng/ml. The present study can provide necessary information
for the rational utilization and quality control of Piper species.
Investigation of Tumor Differentiation Factor (TDF)-Induced Cell
Differentiation Using Mass Spectrometry Based Proteomics
Devika Channaveerappa, Clarkson University, 8 Clarkson Ave.,
Potsdam, NY 13699
Tumor differentiation factor (TDF) is a protein, produced by the pituitary gland and
is secreted into the bloodstream. TDF and TDF derivatives induce differentiation of
breast and prostate cells, but not of other cells. However, the mechanism of action of
TDF to induce differentiation is unknown. Therefore, one option to identify the members of the TDF pathway is to investigate the proteomes of the TDF-treated and
–untreated human breast and prostate cells. In our preliminary studies, we observed
substantial differences in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein patterns of untreated cells and cancer cells treated
with TDF analogs. Hence, the current study is focused on the proteomic analysis
of TDF-untreated and TDF treated MCF7 and DU145 human breast and prostate
cancer cells, on the functional investigation of TDF as well as on the localization
of TDF. These experiments will help elucidate the mechanism through which TDF
induces cell differentiation. To carry out these investigations, MCF-7 human breast
and DU-145 human prostate cancer cells in culture were grown to confluence under
standard cell culture conditions and transferred to six-well plates in a serum-free
medium. The cells were treated with various concentrations of TDF analogs (0 to
10 Вµg), lysed and the proteins were separated by SDS-PAGE for qualitative inspection and direct comparison of the samples. The gel pieces were digested and the
different peptide mixtures were analyzed using a nanoAcquity ultra-performance
liquid chromatography coupled with a quadrupole time-of-flight micro mass spectrometer followed by data analysis. Further validation and functional studies will
also be performed.
Analysis of Emulsifiers in Foods by High Pressure Liquid
Chromatography and Charged Aerosol Detection
Marc Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Bruce Bailey, Ian Acworth, Qi Zhang
Emulsifiers are used to maintain a uniform suspension of immiscible materials.
These compounds are typically surfactants, and can be designed for use in specific applications and products. Acylglycerols are used in food products containing
oil and water (e.g. margarine, mayonnaise); lecithin is commonly found in chocolate and spray oils; acid esters of monoglycerides are used for dough conditioners;
and hydroxypropylmethyl cellulose (HPMC) is use to thicken dairy products and
help improve flavor characteristics. HPMC is also an important emulsifier used in
the pharmaceutical industry. The analysis of emulsifiers is becoming increasingly
important, for product quality, consistency and stability properties. High-pressure
liquid chromatography is one of the more prevalent methods for analyzing these
compounds. However, the majority of analytes do not contain suitable chromophore
characteristics for UV detection, which then requires the use of a universal detector,
such as evaporative light scattering, refractive index, or charged aerosol detection.
The charged aerosol detector was used in the analyses of two emulsifiers (HPMC
and lecithin) that were extracted from food products. The charged aerosol detector,
a sensitive mass-based detector, is ideally suited for the direct measurement of
emulsifiers, as they are non-volatile and non-chromophoric compounds. It offers
excellent sensitivity (down to low nanogram amounts on column), a dynamic range
of over four orders of magnitude, and similar inter-analyte response independent of
chemical structure.
Reverse-Phase UPLC Assay for Purity and Concentration
Determination during Admixtrue Compatibility Study of a
Therapeutic Protein Program
Shenjiang Yu, Merck, 2000 Galloping Hill Rd., Kenilworth, NJ 07033,
Shuai Shi, Joseph Rizzo, Venus Hashemi, Chakravarthy Narasimhan,
Valenthyn Antochshuk, Daisy Richardson, Mohammed Shameem
A pharmaceutical admixture consists of a drug product mixed with a suitable diluent in an intended dosing/delivery device for the purpose of parenteral infusion to
the patient. Regulatory agencies have specific requirements for the demonstration
of the compatibility of the drug product with reconstitution diluents, because many
of the therapeutic proteins are dosed intravenously in the form of admixtures. A
reverse-phase liquid chromatography (RPLC) method has been developed to monitor the concentration and purity during the admixture study. A ultra-performance
liquid chromatography (UPLC) system and sub-2-Вµm column have been utilized to
obtain improved separation. An appropriate column pore size has chosen for the
therapeutic protein. Protein loss through the dosing device and infusion lines at low
concentration level has been discovered. A preventive action has been taken by
conditioning the infusion lines by protein sample. In addition, some new impurities
during the admixture have been detected by the RPLC method. Some preliminary
investigation about the impurities has been reported.
Analysis of Phospholipids in Natural Samples by Normal Phase
HPLC and Corona Charged Aerosol Detection
Marc Plante, Thermo Fisher Scientific, 22 Alpha Rd., Chelmsford, MA
01824, Bruce Bailey, Ian Acworth, Qi Zhang
Phospholipids are a broad class of lipids that can be divided into glycerophospholipids (GPLs) and sphingolipids. Both groups show great structural diversity. Phospholipids are amphiphilic molecules, having a hydrophilic head group, and a lipophilic
fatty acid tail. Several families of GPLs exist biologically, differing in the type of polar
head group present, and include: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol
(PI), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine
(DPPC), and lysophophatidylcholine (LPC). Each compound contains many species resulting from differences in their fatty acid composition. For example, PC may
contain several different fatty ligands, which will result in multiple peaks by reversed
phase chromatography. Differences in polar moieties were used to separate these
analytes by normal phase liquid chromatography and provided superior quantitative
data with less effort. Analysis of phospholipids in red palm oil and krill oil are provided. The Corona Veo charged aerosol detector, a sensitive mass-based detector, is
ideally suited for the direct measurement of phospholipids, as they are non-volatile
and non-chromophoric compounds. It offers excellent sensitivity (down to low nanogram amounts on column), a dynamic range of over 4 orders of magnitude, and
similar inter-analyte response independent of chemical structure. The developed
method is based on an original publication by Rombaut, R., et al., (J. Dairy Sci.,
2005, 88, 482), that enables the direct measurement of a number of GPL and sphingolipid species, each as near-single peaks.
The Use of Diamond-Based Core-Shell HPLC Particle in the
Analysis of Proteins
David S. Jensen, Diamond Analytics, 1260 S 1600 W., Orem, UT 84660,
Janusz Zukowski, Andrew E. Dadson
Diamond core-shell particles are composed of a solid, spherical carbon core and a
functionalized porous nano-diamond shell. The composite material utilizes nano-diamond to create a permeable, but extremely stable shell that can tolerate high temperatures and extreme pH separation conditions. Nano-diamonds also have a much
higher thermal conductivity when compared to silica. This innovative material offers
a new standard in liquid chromatography performance, providing a powerful tool for
difficult drug and protein separations when compared to more traditional columns.
Herein, a variety of protein applications are demonstrated under conditions that will
normally stress and degrade a traditional high-performance liquid chromatography
(HPLC) column. Resolution of oxidized, native and reduced forms of Interferon variants is shown which were separated under highly acidic conditions. Additionally, a
systematic approach is discussed on how to use this diamond-based HPLC material
for protein separations.
Proteomic Investigation of Saliva from People with Smith-LemliOpitz Syndrome (SLOS) and Controls
Emmalyn J. Dupree, Clarkson University, 49 Market St., Apt. 3, Potsdam,
NY 13699, Megan M. Borland, Costel C. Darie, Alisa G. Woods, Kelly
L. Wormwood
Saliva samples were analyzed from people with Smith-Lemli-Opitz Syndrome
(SLOS) and controls using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in-gel trypsin digestion, and nanoliquid chromatography-tandem
mass spectrometry (nanoLC-MS/MS) to investigate the differences between the
proteomes of people with SLOS and controls. The pending results will hopefully
provide more information about the protein changes that occur in SLOS, potentially
leading to identification of treatment targets. These results may also shed some light
on the possible link between SLOS and Autism Spectrum Disorder (ASD), as ASD
is almost always co-morbid with SLOS. This could possibly lead to better diagnosis,
treatment and understanding of ASD as well.
Accelerated Solvent Extraction and In-Cell Sample Clean-Up of
Marine Sediment for the Analysis of Persistent Organic Pollutants
Emma L. Gatley, University of Connecticut, 3107 Horsebarn Hill
Rd. U-4210, Storrs, CT 06269, Emmanuel O. Omari, Aliaksandr
V. Yeudakimau, James D. Stuart, Christopher R. Perkins, Molly B.
Williams, Anthony A. Provatas
Contamination of sediment is an important factor when evaluating the overall quality of water since it holds a crucial role in ecosystem and human health. Continuous pollution from non-point source run-off, petroleum production and transport,
EAS is pleased to announce that we have partnered
with Pharma Webinars to offer new low-cost
webinars to all analytical scientists
launching in 2015
Overview of Protein Analysis: Analytical Testing and
Robert Johnson, Consultant
ICP and Elemental Impurities Testing in
Pharmaceutical Analysis
Stay tuned to; more details to follow
Follow us for more info:
2015 Eastern Analytical
Symposium & Exposition
п‚Ѓ Three-day technical program
п‚Ѓ State-of-the-art exposition featuring analytical equipment and services
п‚Ѓ Extensive selection of short courses and professional development workshops
п‚Ѓ Employment bureau, and more
Garden State Exhibit Center | Somerset, NJ
November 16–18, 2015
Call for Papers
March 1–June 15, 2015
Abstracts received from June 16–Sept 30, 2015 will be reviewed for quality to be included in the
poster session. You will be notified via email when/if the abstract is placed.
EAS seeks contributed abstracts in
these and other analytical fields:
п‚Ѓ Bioanalysis
п‚Ѓ Microscopy
п‚Ѓ Capillary Electrophoresis
п‚Ѓ Nanoparticles
п‚Ѓ Chemometrics
п‚Ѓ Near-Infrared (NIR) Spectroscopy
п‚Ѓ Conservation Science
п‚Ѓ NMR Spectroscopy
п‚Ѓ Environmental Analysis
п‚Ѓ Pharmaceutical Analysis
п‚Ѓ ESR Spectroscopy
п‚Ѓ Process Analytical Science
п‚Ѓ Food Analysis
п‚Ѓ Protein Analysis
п‚Ѓ Forensic Analysis
п‚Ѓ Quality-by-design
п‚Ѓ Gas Chromatography
п‚Ѓ Quality/Regulatory/Compliance
п‚Ѓ Raman Spectroscopy
п‚Ѓ Sample Preparation
п‚Ѓ Immunochemistry
п‚Ѓ Science Education
п‚Ѓ Industrial Hygiene
п‚Ѓ Sensors
п‚Ѓ Ion Chromatography
п‚Ѓ Size Exclusion Chromatography
п‚Ѓ IR Spectroscopy
п‚Ѓ Solid State Analysis
п‚Ѓ Laboratory Automation
п‚Ѓ Space Analytics
п‚Ѓ Laboratory Management
п‚Ѓ Supercritical Fluid Chromatography
п‚Ѓ Laboratory Miniaturization
п‚Ѓ Surface Science
п‚Ѓ Ligand Binding Assays
п‚Ѓ Very High-Pressure LC/
Ultra High-Pressure LC
п‚Ѓ Microchemistry
п‚Ѓ Vibrational Spectroscopy
Call for Proposals
Please help EAS serve you better. If you have suggestions for a short course offering, or would
like to teach a short course, please contact us at [email protected] If you have suggestions for
program, or would like to organize a session, please contact us at [email protected]
2014 EAS Abstracts
November 2014
incomplete combustion of fossil fuels, industrial operations, agricultural spraying,
and improper disposal of industrial materials all contribute to the concentration of
polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and
pesticides in sediment. Elevated levels of PAHs within the environment are considered hazardous to human health, with exposure proven to have carcinogenic and
mutagenic effects. Similarly, PCBs and pesticides also are carcinogenic while contributing to reproductive complications and nervous system damage respectively.
Subsequently, various methods have been developed to detect PAHs, PCBs, and
pesticides in sediment with the majority involving extensive clean-up steps such
as gel permeation chromatography (GPC) and solid-phase extraction. The objective of this project was to analyze for Environmental Protection Agency’s 16 priority
PAHs, select PCBs and pesticides in marine sediment utilizing accelerated solvent
extraction and in-cell alumina clean-up for rapid sample preparation. This was followed by gas chromatography tandem mass spectrometry (GC-MS-MS) analysis
in a “soft” Electron ionization setting of 40 eV instead of the conventional 70 eV to
increase analyte sensitivity. To expedite the evaporation process, a Genevac EZ-2
evaporator was utilized. This validated methodology is presented for 16 PAHs, 22
PCBs and 19 pesticides of interest.
Recognition of N-Oxides and Quaternary Ammonium Compounds
by Chloride Ion Attachment LC-MS
Shirley A. Rodriguez, Boehringer Ingelheim, 900 Ridgebury Rd., PO
Box 368, Ridgefield, CT 06877, Keith B. McKellop, Scott Pennino,
Daniel L. Norwood
Gas phase chloride ion attachment negative ion atmospheric-pressure chemical
ionization (APCI) liquid chromatography mass spectrometry (LC-MS) has been applied to the analysis of drug molecules, such as nitroglycerin, which are difficult to
ionize by more conventional LC-MS methods. [M+Cl]- ions are formed by non-covalent gas phase interactions of analyte molecules with Cl- derived from the addition
of chlorinated reagents (e.g., chloroacetonitrile) to the high-performance (HPLC)
mobile phase. At the EAS 2013 we presented a comparison of two approaches
(APCI and electrospray ionization) to evaluate the selectivity of chloride attachment
ionization to a diverse group of compounds including hydroxylated (sugars), phenols, nitro, N-oxides and sulfur containing, and aliphatic vs. non-aliphatic structures
using standard LC-MS platforms and ionization conditions. From these studies we
observed that N-oxides showed an interesting pattern of attachment ions, in cases
where the attachment worked, of [M+Cl]- and [M+Cl+HCl]-. N-oxides can be genotoxic impurities in pharmaceuticals and identification by MS can be inconclusive and
many times requires confirmation by other techniques, such as, LC-nuclear magnetic resonance. Therefore, the work has continued on the recognition of N-oxides
and quarternary ammonium compounds by chloride ion attachment LC-MS and is
presented here.
Characterization of the Points and Extent of Glycosylation in
Steviol Glycosides
Li Chen, International Flavors & Fragrances, 1515 State Highway 36,
Union Beach, NJ 07735, Richard Hiserodt
Steviol glycosides are a sweet principal of Stevia rebaudiana Berotoni.They are up
to 450 times sweeter than sucrose. Steviol can be glycosylated at the C19-carboxyl
group or the C13-hydroxyl group. The position and extent of glycosylation will affect
the sweetness and mouth feel attributes of the steviol glycosides. The aim of this
study is to evaluate the extent to which mass spectrometry can be used to predict
the position and extent of glycosylation. To accomplish this, the product ion spectra of fourteen steviol glycosides were acquired using a Linear Trap Quadropole
Orbitrap Discovery high resolution, accurate mass-mass spectrometer. The data
from this study showed that using both full scan and product ion spectra (MS2), the
position and extent of glycosylation can be predicted for these compounds.
Analysis of Drugs of Abuse by LC-TOF MS: A Unique Quantitation
Viewer for Streamlining Workflows
Leslie Sullivan, PerkinElmer, 740 Bridgeport Ave., Shelton, CT 06484,
George Perkins, John Moncur, Scott Campbell, Stephen Roberts, Bill
Workplace drug screening has been in existence in the United States since the
1980’s. Employers commonly use drug screening to test prospective employees for
the existence of alcohol and illegal drugs. It is also common during an accident at
the job site. Most illegal substances remain in the body for at least 24 hours following use. The Substance Abuse and Mental Health Services Administration (SAMHSA) panel (formally referred to as the NIDA panel) classes are chosen to represent
the most commonly abused drugs in the general public, this list includes cocaine,
marijuana, amphetamines, opiates and phencyclidines. Liquid chromatography tandem mass spectrometry (LC-MS-MS) is often used to quantitate drugs of abuse in
biological fluids due to its sensitivity and selectivity. However, triple quadrupole techniques can have an undesirable high cost and lack the ability to easily identify new
or unknown compounds. This becomes significant with the constant development of
drugs, making it harder to identify and quantify them as they appear in the samples.
We present an alternative technique to quantitate drugs of abuse in urine utilizing a
rapid dilute and shoot with LC separation method in combination with time-of-flight
mass spectrometry. We use a unique quantitation viewer to rapidly review the data
for integrity and final results, thus streamlining workflows.
Evaluation of Low- and High-Resolution Mass Spectrometry
Techniques for Metabolite Profiling and Identification
Khushbu G. Gajjar, Kean University, 1000 Morris Ave., Union, NJ 07083,
Dil Ramanathan
Preclinical species drug metabolism studies are integral part of drug discovery
process to understand and predict the metabolism of drugs in humans. Loratadine
(LOR) is a second-generation histamine H1 receptor antagonist drug which is used
in the treatment of allergic symptoms. In the present study, LOR and its primary metabolites such as desloratadine (DL), hydroxy desloratadine (OH-DL) and glucuronide metabolites of LOR and DL in plasma and urine of male and female rats treated
with a single oral 8 mg/kg dose of LOR were analyzed by a low resolution linear trap
quadropole (LTQ) and a high resolution Orbitrap mass spectrometers with a run
time of 3 min. The main purpose of this analysis is to compare the two types of mass
spectrometers to understand which type of mass spectrometer should be used to
accurately and precisely identify and characterize drugs and their metabolites. Plasma and urine from rats were collected at different time intervals over 48 hours to
identify and profile circulating and excretory metabolites. The pharmacokinetics of
LOR and DL were also studied.
High-Throughput UPLC-MS-MS Method Development for Phenolic
Acids (Chlorogenic Acids) Determination in American Cranberry
Yifei Wang, Rutgers University, RM 379, Foran Hall, 59 Dudley Rd., New
Brunswick, NJ 08901, Nicholi Vorsa, Ajay P. Singh
Phenolic acids are the class of compounds abundant in Rubiaceae family and other plant species including Vaccinium species. Based on biosynthetic pathway the
accumulation of phenolic acids may vary in the different organs of the plant e.g.
seeds, stem, leaf, fruit epidermis and fruit flesh. Chlorogenic acids (CGA) are a
family of esters formed between quinic acid and certain trans cinnamic acids such
as caffeic acid, ferulic acid, p-coumaric acid and their dimers. As per literature the
chlorogenic acids absorbed in humans through dietary digestion, and have been reported to possess anti-oxidant, anti-hypertensive activities as important implications
to human health. A numerous diversity of structures exist in the CGA family, varying
by the identity, number and position of their acyl residues, identification of the various species has been difficult and problematic due to their structural similarities.
A high-throughput ultra-performance liquid chromatography coupled with tandem
mass spectrometry (UPLC-MS-MS) method has been developed to identify 10 individual CGA compounds simultaneously, including 3 caffeoylquinic acids (CQA), 3 dicaffeoylquinic acids (diCQA), 1 feruloylquinic acid (FQA) and 3 caffeoylferuloylquinic acids (CFQA). The method was successfully applied to compare different varietal
cranberry organ extractions for their CGA composition. Six CGAs were successfully
identified, including 3/4/5-CQA, 5-FQA, 3,5-diCQA and 4,5-diCQA. Quantitation of
characterized CGAs in cranberry organs was determined by multiple reaction monitoring (MRM) mode in precursor/product ion pair of m/z 353.2/191.5 for 3/4/5-CQA,
m/z 366.7/191.0 for 5-FQA and m/z 515.3/353.1 for 3,5-diCQA and 4,5-diCQA.
Gas-Phase Fragmentation of Metal-Cationized Acid Salts
Robert D. Hale, Stevens Institute of Technology, Department of
Chemistry, Hoboken, NJ 07030, Athula B. Attygalle, Carl S. Weisbecker
Very few detailed investigations have been undertaken on the mass spectrometric fragmentation of cationic adducts of salts of small acidic molecules. Generally,
collision-induced mass spectra of small cationized molecules are considered to be
rather uninformative, because most spectra show only a single product-ion peak
for the ejected metal cation. Nonetheless, in our exploration of oxalate and carbonate salts, we found that these ions fragment in an unusual manner, generating
product ions that retain all three metal atoms of the precursor ion. Upon collision induced dissociation (CID), the [Na3C2O4]+ (m/z 157); [39K3C2O4]+ (m/z 205); [7Li3C2O4]+ (m/z 109); and [Na3CO3]+ (m/z 129) ions ejected a 44-Da fragment even
at a collision energy as low as 2 eV. DFT calculations predicted that the product
ions, [Na3C2O4]+ and [Na3CO3]+ are planar in geometry , and that the ejection of
CO2 requires less energy than an ejection of a Na+. When the sodium adducts from
the labeled compounds [Na2C218O4] (m/z 165) and [Na2C18O3] (m/z 135) were
subjected to CID, the spectra showed a peak at m/z 117 and m/z 87, respectively, which confirmed that the ejected fragment was CO2. Furthermore, product-ion
spectra for [Na2C2O4•Na+], [39K2C2O4•39K+] and [Na2CO3•Na+] recorded at
higher collision energies showed an additional peak for an even-electron ion formed
by a loss of an M• from the m/z 113 ion to form the M3O+ ion, which violates the
even-electron rule.
2014 EAS Abstracts
November 2014
Screening and Sequencing of Gangliosides from Human
Glioblastoma Multiforme by High-Performance Mass Spectrometry
Loredana M. Lupu, Clarkson University, Department of Chemistry and
Biomolecular Science 5810, Potsdam, NY 13699, Megan Borland
Glioblastoma multiforme represents one of the most common types of brain cancer known to man. It is a grade IV primary neoplasm of the central nervous system, usually located in the cerebral cortex. Following drastic surgical resection and
chemotherapy the prognosis is very poor. At the present time one strategy for the
investigation of tumor treatments is to target invading tumor cells by using specific ligands. Gangliosides, syalilated glycosphingolipids represent a class of such
ligands; therefore characterization of their expression and structure in gliblastoma
tumor is of a high clinical importance. In the present work, a systematic profiling of
ganglioside composition followed by structural analysis of individual species in a
glioblastoma specimen vs. normal brain tissue was carried out using the advanced
mass spectrometric (MS) method based on chip-nano electrospray ionization quadrupole time-of-flight (QTOF) MS. It was discovered that the total number of distinct
individual gangliosides is actually much larger than reported before. Tandem MS
provided for the first time the complete and accurate set of structural elements for
glioma-associated GD2. The results represent a comprehensive evaluation of the
ganglioside heterogeneity and structural elucidation of several species considered
as human glioma-associated structures.
An Investigation of Biodiesel Stability: A Study of the Behavior of
Antioxidants in Soybean and Canola Derived Biodiesel
Casey Camire, University of Connecticut, 100 Oak St., Meriden, CT
06450, James D. Stuart, Anthony A. Provatas
Biodiesel is a renewable fuel that is synthesized via the transesterification of the
triglycerides found in vegetable oil, used cooking oil, or animal fats. Unlike ethanol
fuels, which often can take the place of important food supplies, biodiesel precursors are usually made from waste products. Biodiesel can be used on its own as a
replacement for regular, petroleum-based diesel, but is more commonly used as a
blend with the usual petroleum-based diesel in order to reduce fossil fuel consumption and greenhouse gas emissions. However, biodiesel can be prone to undergo
degradation thru a series of radical reactions of oxygen and other species involving
the conjugated double bonds of the long chain fatty acids and resulting in the formation of shorter fatty acids, carboxylic acids and networked polymers. In order to
minimize these effects, antioxidants have been added to both prevent oxygen from
interacting with the biodiesel and to inhibit the free-radical degradation of the ester
tails. A standardized test has been developed which provides a measure of oxidative stability of biodiesel. A modification of this method can be used to determine the
kinetics of antioxidants added to biodiesel. This project seeks to use that method
to assess the kinetics of commonly used antioxidants in canola and soy derived
Identification of the O-Glycoforms Expressed in the Urine of a
Patient Suffering from Schindler Disease Using Chip-Based
Nanoelectrospray Tandem Mass Spectrometry
Adrian C. Robu, Clarkson University, Department of Chemistry and
Biomolecular Science, 8 Clarkson Ave., Box 5810, Potsdam, NY 13699,
Megan Borland, Costel C. Darie, Mirela Sarbu, Aurel Vlaicu, Jasna
Peter-Katalinic, Alina D. Zamfir
Schindler disease is a rare, however severe condition, recently recognized as an
autosomal recessive disorder, defined as a О±-N-acetylgalactosaminidase (О±-NAGA)
deficiency. Deficient activity of the lysosomal enzyme О±-NAGA leads to an abnormal accumulation of sialylated and asialo-glycopeptides and oligosaccharides with
О±-N-acetylgalactosaminyl (GalNAc) residues, causing a 100 times higher concentration of O-glycans in patient urine than in healthy controls. Screening, structural
characterization and complete identification of O-glycosylated amino acids and peptides extracted from patient urine using tools of high sensitivity, reproducibility and
accuracy is nowadays of major clinical importance. In this study an integrative mass
spectrometry (MS) approach based on fully automated chip-nanoelectrospray quadrupole time-of-flight mass spectrometer was optimized and applied for the discovery
and structural characterization of O-glycopeptides in a fraction from the urine of a
patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides extracted and purified from an age matched healthy subject served as control. Structures with relevant biological significance, previously not described, such as O-fucosylated tetrasaccharides and chains up to pentadecamers O-linked to threonine,
serine or serine-proline were identified in the pathological urine and characterized
by tandem MS (MS/MS). 49 glycoforms were discovered in the investigated urine
fraction from Schindler disease vs. only 14 in control urine. A number of species with
long chain glycans were not previously reported as associated to this condition. All
glycopeptides were detected in only 1 min analys is time, with a sensitivity situated
in the low picomole range.
Developing a Sensitive and Robust Method for Container Closure
Integrity (CCI) Testing
Casey A. Tyrrel-Pawlowic, Bristol-Myers Squibb, One Squibb Dr., New
Brunswick, NJ 08903, Nikunj Vasoya, Chris Knutsen, Jayshree Patel,
Steve Klohr, Antonio Fernandez
Container closure integrity testing (CCIT) is performed to measure the ability and
quality of a container closure system to provide protection and maintain efficacy and
sterility of a sterile drug product. Methylene blue dye has typically been used for dye
ingress testing of syringes and vials, but we found it unsuitable when used for sensitive detection of breaches in drug product filled containers. Changing regulatory
expectations necessitated the development of CCIT methods for stability studies, so
sensitive detection of breaches in the presence of drug product was required. Here
we describe the use of fluorescein dye to enable development of robust, sensitive,
and quality control friendly CCIT methods suitable for long-term stability (LTS) and
market-life stability (MLS) studies.
Characterizing Nanoparticles in Cosmetic Formulations to
Quantitate and Compare the Contribution of Absorbance from
Large Particle and Nanoparticle Scattering
Chris Lynch, PerkinElmer, 940 Winter St., Waltham, MA 02451, Jeffrey
Nanoparticles have been growing in use in a large number of application areas.
Cosmetic products such as sun screens are one major application area. Nanoparticles are commonly used to enhance the protection from harmful ultraviolet (UV)
radiation by scattering it away from the skin. Characterization and quantitation of
nanoparticles used in creams and lotions is a key element in understanding how
homogenously materials are distributed and the overall effectiveness of these
nanoparticles. The unique use of UV-visible spectroscopy coupled with a 150 mm
integrating sphere and center mount sample holder provides important information
to quantitate and compare the contributions of absorbance, large particle scattering,
and nanoparticle scattering.
Air Sampling of Fragrance Compounds Using the GERSTEL GSSFP Automated Sampler
John Stuff, Gerstel, 701 Digital Dr., Linthicum, MD 21090, Jacqueline
Air fresheners are commonly used in households to mask unpleasant odors. Many
types of fragrances are commercially available. The fragrance can be a complex mix
of many components. The effectiveness of the freshener to mask the off odors over
time can be directly related to the air concentration of the fragrance components.
Therefore, an important aspect in product development is airborne concentration
of the fragrance components. This study describes the use of the GERSTEL GSSFP sampler for the collection of fragrance compounds in air. The GSS-FP is a 28
position active sorbent tube gas/air sampling system designed to collect sample
onto sorbent tubes. The sampler can collect multiple samples on a pre-programmed
schedule or on demand from the user interface. Air fresheners were dispersed in a
room and samples collected using the GSS-FP. The sorbent tubes were analyzed
by thermal desorption gas chromatography mass spectrometry. Several fragrance
components contained in the dispersed products were monitored and quantified
over time. The study also shows validation of an air sampling method for the compounds. This type of experiment can be used to show the rate at which compounds
decay in the room air as a function of time as well as identifying which compounds
are important contributors to the perceived odor in the room.
Authentication of Extra Virgin Olive Oil Using Fluorescence and
Fourier Transform Infrared Spectroscopy
Fred Behringer, Surveillant LLC, 81 Halls Rd., Ste. B, Old Lyme, CT
06371, Emily Paine
Extra virgin olive oil (EVOO) is considered susceptible to economic adulteration.
Rapid and sensitive screening methods would be useful to aid in the identification of
impure products. Multivariate models based on fluorescence and Fourier transform
infrared (FTIR) spectra of EVOOs were developed and evaluated for the ability to
detect blends with various non-EVOOs. Spectra obtained from 43 brands of EVOO
purchased from retail outlets over a 14 month period were used to build models that
factored in the normal variation of EVOO. Canola, corn, grape, hazelnut, peanut,
rice, soybean, sunflower, and safflower oils were blended with EVOO to assess
limits of detection. At least three brands of each type of non-EVOO were evaluated
and the brand representing the mean was chosen for blending. The non-EVOOs
were detected from a low of 5% for rice bran oil to 20% for sunflower oil. The other
oils were detected at either 10% or 15%. The model based on fluorescence resulted
in lower detection limits than the FTIR model for a majority of the adulterants. However, the FTIR model provided lower detection limits for soybean and sunflower oils.
The results suggest that fluorescence and FTIR used together can provide a rapid
method for detecting adulteration of EVOO with other types of oils.
2014 EAS Abstracts
November 2014
through multiple different chelating agents as samples interact differently with each
chelating structures. In the current protocol the sample is simply mixed with the
chelates and signal is measured within five minutes. Until now the technology platform has been shown to be powerful in a number of food applications. Here olive oil
adulteration (spiked) with canola oil down to 1% is measured with a large dynamic
range as the signal-to-background ratio of assay was more than 500. Identification
of vegetable oils is also presented.
Analysis of Cocoa Content in Chocolate Using Tribo-Rheometry
and its Correlation to Mouthfeel
Alina Higham-Latshaw, TA Instruments, 159 Lukens Dr., New Castle,
DE 19720
A large amount of resources in the food science industry are directed to understanding properties of food products such as texture and mouthfeel before, during,
and after the consumption of food and beverages. These properties play a large
role in consumer selection and acceptability, directly impacting the success of new
formulations. Unfortunately, many sensory properties of food are difficult to characterize. Numerous studies have been shown to correlate some properties, such as
firmness, fattiness, and creaminess with bulk rheological properties and recently,
studies have begun to focus on thin-film tribological properties. Tribology is defined
as the study of friction, wear, and lubrication between two interacting substrates
in relative motion. Within the mouth alone, the number of interacting substrates is
quite plentiful: teeth-teeth, tongue-teeth, tongue-palette, tongue-food, just to name
a few. It is believed that tribology can be used to analyze friction properties of substrates within the mouth and food surfaces to correlate with consumer perception
and mouthfeel. In this poster, the tribo-rheometry accessory in conjunction with a
rotational rheometer are described and the tribological properties of commercially-available chocolates. We demonstrate how differences in chocolate composition,
such as fat and cocoa content, affect the properties and behavior of chocolate thin
films between two sliding surfaces. The coefficient of friction as a function of sliding
velocity and load force is given for the chocolate samples.
Forensic Genetic Analyses of Mitochondrial DNA Haplogroups and
Control Region Variation in the Korean Population
Wook Kim, Dankook University, Department of Biology Science, 1st
Science Hall #406-1, Cheonan, South Korea 330-714, Ki Cheol Kim,
Seung Beom Hong
We analyzed the distribution of mitochondrial DNA (mtDNA) haplogroups and
control region sequence variation using a 20-plex SNaPshot assay/sequencing to
evaluate the possible genetic structure and differentiation as well as forensic purpose from 704 unrelated males residing in six major provinces in Korea. The most
common mtDNA haplogroups were found to be D4 and B4, followed by A, D4a,
and M7, which are prevalent in South/Northeast Asian populations. Based on the
result of control region variation, a total of 558 different haplotypes characterized by
271 polymorphic sites were identified, of these 471 haplotypes were unique. The
gene diversity and random match probability were 0.9989 and 0.0025, respectively.
According to the pairwise comparison of the 704 control region sequences, a mean
number of pairwise differences between individuals found to be 13.47 В± 6.06. Pairwise FST genetic distances revealed population homogeneity of six Korean provinces on a peninsula level, except samples from Jeju Island. In contrast, statistically
significant distances were observed between Asian populations (p < 0.001). The
present data may help not only in personal identification but also in determining
maternal lineages for forensic purpose. These data will be available on the EMPOP
database via accession number EMP00661.
Analysis of Lead in Commercial Wine Samples by Graphite Furnace
Atomic Absorption Spectroscopy
Samuel M. Abegaz, Columbus State University, 4225 University Ave.,
Columbus, GA 31907, Matthew Mireles, Kydric Smith
The purpose of this study was to investigate the level of lead in red and wine samples produced in different parts of the world. Forty five wine samples (twenty five red
and twenty white) were obtained from local wine stores and treated before analysis.
The thermal behavior of lead during pyrolysis and atomization stages was studied
with and without matrix modifier. The analytical procedure was validated using standard reference material of water origin. The concentrations of lead ranged from 12.4
– 35.0 µg/L. These concentrations were well below the maximum permissible levels
set by the International Organization of Vine and Wine. The precision was better
than 5% for 95% of the samples.
Using Pure Shift HSQC to Characterize Microgram Quantities of
Yong Liu, Merck, 126 E. Scott Ave., Rahway, NJ 07065, Mitchell
D. Green, Rosemary Marques, Tony Pereira, Roy Helmy, Robert T.
Williamson, Wolfgang Bermel, Gary E. Martin
Difficulties in isolating samples from complex biological matrices and sensitivity limitations have long stymied the utilization of heteronuclear two-dimensional (2-D) nuclear magnetic resonance (NMR) for the characterization of drug metabolites. Small
diameter cryogenic NMR probes have largely ameliorated sensitivity limitations and
the recently reported pure shift heteronuclear single quantum coherence (HSQC)
2-D NMR pulse sequence offers a further and marked improvement in both resolution and sensitivity. Using a 7.4 Вµg sample of the commercially available metabolite
3-hydroxy carbamazepine dissolved in 30 ВµL of deuterated solvent and a 600 MHz
NMR equipped with a 1.7 mm cryogenic NMR probe, it was possible to acquire
high signal-to-noise pure shift HSQC data in just over 30 minutes. A conventional
HSQC spectrum acquired with identical parameters had approximately half the signal-to-noise of the pure shift HSQC spectrum. Collapsing the vicinal homonuclear
couplings in the pure shift HSQC spectrum also significantly improves resolution.
A practical, real world application of the technique is illustrated with the chromatographically isolated metabolite 3-hydroxy amiodarone from incubation with CYP2J2
recombinant enzyme. High quality pure shift HSQC data were recorded in slightly
over 14 h for a 3 Вµg sample of the metabolite.
The Determination of Preservatives in Cosmetic Products by LCMS-MS
Emily A. Myers, Cedar Crest College, 100 College Dr., Allentown, PA
18104, Thomas A. Brettell, Thomas H. Pritchett
Preservatives are natural or synthetic ingredients that are commonly added to products in order to prevent spoilage, including but not limited to microbial growth or
undesirable chemical changes, ultimately extending the products shelf life. Without
the addition of preservatives cosmetic products can easily become contaminated
leading to product degradation increasing the risk of irritation or infection. A liquid
chromatography tandem mass spectrometry (LC-MS-MS) method has been developed which identifies and quantifies multiple preservatives in cosmetic products.
LC-MS-MS data was acquired using an ABI Sciex 3200 QTRAP triple quadrupole
mass spectrometer interfaced with a Shimadzu LC system. The instrument utilized
electrospray ionization (ESI) and all samples were run in positive-ion mode monitoring. Chromatography was performed on a 5.0 cm x 3.0 mm x 2.7-Вµm RaptorВ®
biphenyl capillary column (RestekВ®). The strong mobile phase used was 0.1% formic acid in 2-propanol and the weak mobile phase used was 0.1% formic acid in
HPLC grade methanol. Various cosmetic product samples were prepared by adding
approximately 100 mg to 5 mL of methanol:acetonitrile (1:1) and sonicated for 10
minutes. After sonication the solution was placed into centrifuge tubes and centrifuged for 5 minutes at 3000 rpm. After centrifugation the supernatant was carefully
removed using disposable pipettes and filtered using a 0.2-Вµm Millipore filter. Approximately 1 mL of the supernatant was added to a vial along with 60 ВµL of the
internal standard (BHA). Lastly, 2.0 ВµL of sample was injected onto the LC column.
The Power of the Combination of HILIC-MS-MS and 31P-NMR
Techniques for the Characterization of Phospholipid Compositions
in Different Lecithin Products
Ying Yang, International Flavors and Fragrances, 1515 Highway 36,
Union Beach, NJ 07735, Richard Hiserodt, Jing Li
The chemical composition of phospholipids strongly influences the physicochemical
properties and performance of different types of lecithin. Consequently, a rapid and
reliable method to characterize phospholipid composition is necessary for many
applications. However, due to the complex nature and poor solubility of these phospholipids in water, it is not practical to use the most common analytical approach of
reverse-phase high-performance liquid chromatography tandem mass spectrometry
(HPLC-MS-MS). In the present study, an hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization (ESI)-MS-MS method was employed for the
separation and identification of the chemical composition of different phospholipid
classes and molecular species by ion trap MS-MS technique. SIEVE TM was used
for post-acquisition data analysis in order to rapidly achieve a comprehensive structural characterization for key components in a variety of lecithin products. Quantitative results for each different phospholipid class were obtained using 31P-nuclear
magnetic resonance (NMR). The results from the present study revealed the different strengths of these two techniques and demonstrated the power of the combination of HILIC-MS-MS and NMR for a rapid and comprehensive characterization
of phospholipids.
Rapid and Inexpensive Method for Olive Oil Adulteration and
Harri Harma, University of Turku, Tykistokatu 6A 5th Floor, Turku, 20300
Olive oil is the most adulterated food product. Recent reports show that fraudulent
activities are obvious as olive oil is sold at a higher price than other vegetable oils.
To preserve its quality and authenticity new analytic methods are required. We have
developed a chemical detection fingerprinting technique to detect subtle changes
in the content of vegetable oils. The core of the technique bases on time resolved
luminescence (TRL), label chemistries and a fingerprint that provides a unique
sample-specific signature of virtually any liquid or liquidized sample. TRL chelating
structures are highly unstable, and the sample interacts non-specifically with the
chelating structures modulating the TRL luminescence signal. The array is created
2014 EAS Abstracts
November 2014
poster we investigate the use of YMC-Triart C18 1.9Вµm hybrid and YMC Meteoric
Core C18 2.7Вµm core-shell materials for improving peptide mapping methods.
Ultrasensitive Carbohydrate-Peptide Microarray for Diagnosing
IgE Mediated Peanut Allergy Using Surface Plasmon Resonance
Amit A. Joshi, University of Connecticut, 55 North Eagleville Rd., Storrs,
CT 06269, Mark W. Peczuh, Challa V. Kumar, James F. Rusling
Quantification of allergen specific IgEs specific to individual peptide and carbohydrate epitopes is a promising strategy to reliably predict severity of allergic response.
Here the first prototype microarray for IgE detection using peptide and carbohydrate
epitopes is shown. A surface plasmon resonance imaging (SPRi) microarray is spotted with a 28-mer peptide and a ОІ-xylosyl glycoside (BXG). Both are known to be
antigenic epitopes from the Arachis hypogaea h2 (Ara h2) peanut glycoprotein. IgEs
from human serum are precaptured onto to magnetic beads, loaded with polyclonal
anti-IgE antibodies, and injected into the SPRi microarray to identify IgEs in dilute
serum. As low as 0.1 attomoles of IgEs are detected in 45 min assay. Amounts of
IgEs binding to the Ara-h2 peptide and BXG are identified by SPRi in patient samples and compared to standard immunoCAP assay values.
Development of a Quantitative Method for Synthetic Cannabinoid
Metabolites in Urine Using Liquid Chromatography Tandem Mass
Spectrometry (LC-MS-MS)
Craig T. Leopold, The Forensic Sciences Mentoring Institute, 2300
Stratford Ave., Willow Grove, PA 19090, Alonzo Elias, Christina
Bangura, Monica Marcuse
Over the past few years, synthetic cannabinoids have become increasingly popular in an effort to achieve a “legal” high. At the federal level in the United States,
most are quickly regulated as Schedule I substances, so “manufacturers” produce
compounds that are structurally different, but still give similar effects to achieve that
high. JWH-018, UR-144, ADB-PINACA, AKB-48, PB-22, and 5F-PB-22 have been
scheduled by the Drug Enforcement Agency (DEA); while AB-PINACA, ADBICA,
and BB-22 are currently not scheduled but have recently been identified as components in synthetic cannabis blends in Japan and the United States. An updated
method was developed to extract, identify, and quantify the N-pentanoic acid metabolites and the 3-carboxyindole metabolites of the compounds listed above from
urine, using liquid-liquid extraction (LLE) followed by liquid chromatography tandem
mass spectrometry. Samples were made strongly acidic using HCl and extracted
into chloroform, isopropyl alcohol, and n-heptane (50:17:33) and the LC-MS-MS
was operated in positive ionization mode. The method achieved baseline separation
for all analytes and identification was based on their multiple reaction monitoring
(MRM) transitions. Calibration models were produced in the range of 1-100 ng/mL
for JWH-018, UR-144, AKB-48, PB-22, and 5F-PB-22 metabolites; 1-50 ng/mL for
ADBICA, AB-PINACA, and ADB-PINACA metabolites; and 5-100 ng/mL for BB-22
metabolite (R2 > 0.98). The limits of detection were ≤ 2 ng/mL and limits of quantitation were ≤ 5 ng/mL. Currently, the method is being validated for use as quantitative
assay in the field of forensic toxicology as it incorporates several scheduled compounds and recently emerging synthetic cannabinoids.
Versatile Low-Cost Electrochemical Microfluidic Immunosensors
for Cancer Diagnostics
Brunah A. Otieno, University of Connecticut, 55 North Eagleville Rd.,
Storrs, CT 06269, Colleen E. Krause, Gregory W. Bishop, James F.
Rapid, accurate and sensitive detection of multiple biomarker proteins holds significant promise for early diagnosis of cancer and personalized therapy guidance.
Here we describe a simple, low-cost, modular microfluidic system for on-line capture
and detection of cancer protein biomarkers. The system features a small chamber
for on-line protein capture from serum by magnetic beads labeled with many copies of analyte-specific antibodies and signal-transducing enzyme labels, positioned
upstream of a detection chamber housing a nanostructured 8-electrode sensor array. Microfluidic chambers are made by templating polydimethylsiloxane (PDMS)
channels on machined aluminum molds and mounting on hard flat poly(methyl
methacrylate) (PMMA) plates equipped with inlet and outlet panels. The chambers
are interfaced with a sample injector, syringe pump and switching valves to deliver
sample and reagents. Gold immunosensors fabricated by ink-jet printing of 4 nm
alkylthio l gold nanoparticles ($0.2) or commercial screen printed carbon sensors
coated with 5 nm glutathione-gold nanoparticles($6) are fitted into the microfluidic
detection chamber to achieve high sensitivity. Ultralow detection limit in the low fM
range was achieved for multiplexed detection of four oral cancer biomarker proteins
from as little as 5 ВµL sample within 30 minutes. The incorporation of electrochemical
immunoassays for protein biomarkers in a microfluidic platform with nanostructured
sensors thus provides a rapid, sensitive and effective tool for cancer diagnostics.
Metabolic Profile Determination of Novel Psychoactive Substances
by Use of Human Liver Microsomes
Sarah E. Wolf, The Forensics Mentoring Institute, 2300 Stratford Ave.,
Willow Grove, PA 19090, Noah Gubernick, Timothy Martin, Grace Pak
Novel psychoactive substances have been increasingly abused, particularly within
the electronic dance music (EDM) scene. With the continued popularity of “designer
drugs,” the market is constantly changing, requiring continued research, including
the metabolic pathways of these newly emerging drugs. The aim of this research
was to develop and optimize a method for in-vitro metabolism using human liver microsomes (HLM). In-vitro metabolism using pooled HLMs was first optimized
by incubating the microsomes with diazepam, and monitoring for the presence of
nordiazepam and temazepam. Analysis occurred using liquid chromatography triple quadrupole tandem mass spectrometry. The incubation mixtures contained a
phosphate buffer (pH= 7.4), magnesium chloride, nicotinamide adenine dinucleotide phosphate, reduced (NADPH), diazepam, and HLM, and were incubated at 37
В°C. After optimizing the time of incubation and the amounts of NADPH, HLM, and
substrate, the following values of each were used: incubations were incubated for
two hours with 25 ВµL of microsomes (20 mg/ml), 50 ВµL NADPH (10 mM), and 5000
ng substrate with a final volume of 600 ВµL. Once it was established that the microsomal incubation mixture was effectively producing metabolites as seen in-vivo,
this optimized method was used to produce the metabolites of the drugs of interest,
including О±-PVP. For this, analysis occurred by liquid chromatography quadrupole
time-of-flight mass spectrometry to produce exact mass data.
A Novel Device for Plasma Micro-Sampling Technique Developed
for Bioanalysis
Ji Zhang, Takeda Pharmaceutical International, 40 Landsdowne St.,
Cambridge, MA 02139, David Lok, Jesse Gray, Kelly Connolly, Steve
Micro-sampling technique was widely used in the past five years and its benefits
have been recognized throughout the pharmaceutical industry. Traditionally, plasma
is gold standard matrix of choice. But it is a technical challenge to accurately and
precisely sample plasma directly from derived plasma layer at micro volume level.
Here, we report a novel device for isolating plasma from red blood cells at micro
volume level (as low as 20 mL) which allows direct pipetting plasma from generated
plasma layer. All experiments were performed API 5500 Qtrap mass spectrometer
interfaced with a Shimazu high-performance liquid chromatography (HPLC). Twenty
or 40 ВµL mouse blood was pipetted into the novel device developed in-house for
plasma micro-sampling followed by 6 min centrifugation at 14000 rpm. Pipette 5
or 10 ВµL plasma from top layer derived plasma for Bioanalysis. Roughly 10 or 20
ВµL plasma can be isolated from 20 or 40 ВµL blood in the devices after centrifugation. Five or 10 ВµL plasma can be easily pipetted out. Side-by-side comparison of
this new plasma micro-sampling and conventional plasma collection method using
in-house discovery compounds has demonstrated that the mean percentage difference between two methods is less than 15%. The precision (%CV) falls within
В±15%. Animal studies have also been performed to show good agreement between
the data obtained from conventional plasma collection and that from micro-sampling
method using this new device. The device is novel, simple to use and cost-effective.
LC-MS-MS Method Development and Analysis of Sofosbuvir
Metabolites Using PGC (Porous Graphitic Carbon) Column:
Determination of the Metabolites in Human Liver and Lung Cell
Prabhakar G. Reddy, Emory Institute for Drug Development, 954
Gatewood Rd. NE, Atlanta, GA 30329, Taylor J. Evers, Adhitya R.
Katkam, Gregory Bluemling, Michael G. Natchus, Damien Kuiper,
Manohar Saindane, Shuli Mao, Mark Lockwood, Michael Hager, Richard
F. Arrendale
A sensitive and selective liquid chromatography tandem mass spectrometry (LCMS-MS) method was developed for Sofosbuvir metabolites (monophosphate, triphosphate, intermediate metabolite, and nucleoside), and the method was used to
determine these metabolites in human liver (Huh7) and lung (A549) cell lines. The
method utilized a porous graphitic carbon (PGC) stationary phase, Hypercarb (Thermo), under conditions suitable for liquid chromatography mass spectrometry (LCMS). The separation of the Sofosbuvir metabolites was optimized using gradient
elution with acetonitrile and ammonium bicarbonate buffer. The pH of the ammonium bicarbonate buffer solution proved to be critical for selectivity and retention of the
mono and triphosphate metabolites of Sofosbuvir. Baseline separation of the monophosphate, triphosphate, intermediate metabolite, and the nucleoside was achieved
Use of New 1.9-Вµm YMC-Triart C18 and 2.7-Вµm YMC-Meteoric Core
C18 Bio Stationary Phases for Fast Peptide Mapping of Monoclonal
Jeffrey A. Kakaley, YMC America, 941 Marcon Blvd., Allentown, PA
18109, Ernest J. Sobkow
Peptide mapping methods have historically been lengthy, time-consuming methods requiring large amounts of solvent. Modern instrumentation and smaller particle
size stationary phases now allow for these methods to be scaled down; improving
throughput, saving solvent, and increasing scientists’ overall productivity. In this
2014 EAS Abstracts
November 2014
in less than 10 minutes. An internal standard spiking technique was used to produce
a quantifiable linear range for these metabolites from 1 ng/ml to 1000 ng/ml when
using the appropriate blank cell extract for preparation of calibration curves. Sofosbuvir was incubated with cells from human liver (Huh7) and human lung (A549) cell
lines for up to 24-hours, and the formation of the Sofosbuvir metabolites were monitored and quantitated. ATP levels were also monitored to assess the reproducibility
of the cell incubation procedure. This presentation outlines the method development
and application of this methodology to these cellular metabolism studies.
Finding Order Amid Chaos: In-Vitro Challenges Working with
Boronic Acids
Teresa Mulder, Genentech, 1 DNA Way, S. San Francisco, CA 94618,
Xiaorong Liang, Chenghong Zhang, Hoa Le, Matt Baumgardner, Quynh
Ho, Savita Ubhayakar, Yuan Chen, S. Cyrus Khojasteh, Cornelis E.C.A.
Hop, Peter Fan
As bacteria are becoming increasingly resistant to antibiotics, novel therapies are
becoming necessary. Compound A is a boron-containing compound we are assessing for this purpose. Its boronic acid component is similar to that of Bortezomib, the dipeptidyl boronic acid proteasome inhibitor used to treat myeloma. Both
compounds undergo rapid deboronation in the conditions typical of in-vitro DMPK
screening assays such as protein binding and liver microsome stability. The mechanism of deboronation may be the same in both compounds, although the abundant
metabolite/degradant of Compound A is different from that of Bortezomib. This may
be due to an amine offering intra-molecular stabilization of the abundant metabolite
in compound A. With similar mechanisms causing instability, we were able to use
published Bortezomib conditions to accelerate our discovery of in-vitro methods.
Comparing metabolite percentages from microsomes incubated with and without
nicotinamide adenine dinucleotide phosphate (NADPH) allowed us to determine
the NADPH-dependent component we were interested in. For our plasma protein
binding assay, Compound A was more stable in freshly prepared plasma than the
frozen plasma that was provided to us. These in vitro methods can be used to test
the properties of similar antibiotic candidates in the future, and to guide the development of further in vitro tests as they become necessary.[1]
Supported Liquid Extraction as a Bioanalytical Sample Preparation
Matthew Cleeve, Kinesis Ltd., 9 Orion Court, Ambuscade Rd., St Neots,
PE19 8YX United Kingdom
An increasingly diverse range of drug candidates present challenges in bioanalysis.
The selected sample preparation method should be simple, efficient and optimized
for high sample throughput. One of the key challenges is to remove interferences
present in biological fluid samples, commonly plasma. Protein precipitation, liquid–liquid extraction (LLE) and solid-phase extraction (SPE) are commonly used
techniques to extract drug molecules. It is clear traditional techniques are not always suited to removing protein and phospholipid interferences, with phospholipids
significantly affecting the reliability of results. Removing phospholipids and proteins
is key to the effectiveness of the sample prep procedure. Recently, diatomaceous
earth has gained popularity in bioanalytical sample preparation. Its porous channel
structure provides excellent dispersive properties, trapping large molecules including phospholipids. When the plasma sample passes through the material, it penetrates the pores, forming an aqueous film on the surface. When a water-immiscible
solvent is passed through LLE occurs. This is referred to as supported liquid extraction (SLE). Using this approach, analytes will be eluted while lipids are retained.
It is believed the acidic property of the hydroxyl groups and metal centers on the
substrate contribute to the retention of phospholipids via the interaction with its chlorine and phosphate groups. In this study, we have compared LLE, PPT, SPE and
SLE methods to extract dexamethasone in plasma prior to liquid chromatography
tandem mass spectrometry (LC-MS-MS). Spiked plasma samples containing 10ng/
ml, 100ng/ml, and 1000ng/ml dexamethasone were used in each case. The results
presented will show the responses from SLE are between four and six times higher
than the other techniques.
[1] S Wu, et al. J. Pharm. Sci. (2000) 758-765.
Aggregation Behavior of Ionic Liquids in Low Polarity Solvents
Elise Cade, The College at Brockport, 41 Stuart Rd., Churchville, NY
14428, Markus Hoffmann
Ionic liquids are salts that are liquid below 100 oC. Normally, salts do not dissolve in
solvents of low polarity. However, many ionic liquids are very soluble or completely
miscible in low polarity solvents and their physical chemistry is not well understood.
The goal of our ongoing research is to elucidate the speciation (freely dissolved
ions, ion pairs and aggregates) present for ionic liquids dissolved in solvents of
low polarity. We present experimental results for concentration and temperature
dependent self-diffusion coefficients measured by nuclear magnetic resonance
spectroscopy in conjunction with viscosity measurements to determine the average
hydrodynamic radii of the present species. Five systems were investigated: [C6mim]
[NTf2] dichloromethane , tetrahydrofuran , and chlorobenzene , as well as [C4mim]
[NTf2] and [C2mim][NTf2] in dichloromethane.
Extraction of Indomethacin and Ibuprofen from Small Volume
Biological Fluid Samples
Matthew Cleeve, Kinesis Ltd., 9 Orion Ct., Ambuscade Rd., St Neots,
PE19 8YX United Kingdom
Developments in liquid chromatography tandem mass spectrometry (LC-MS-MS)
detection levels have seen biological fluid sample volumes reduced to such an extent that conventional solid-phase extraction (SPE) formats are not always suitable,
or provide considerable analyte dilution and extended evaporation times. When extracting from sample volumes of less than 100 Вµl, it is important elution volumes and
the sample prep format are fully compatible with the original sample volumes being
processed. The TELOS MicroPlate is a modular design, allowing for flexibility in
sample numbers. Full or partially populated plates can be processed using vacuum
or positive pressure. In addition, the well outlet design ensures good collection plate
penetration, removing any possibility of well to well cross contamination. This poster
summarizes a solid-phase extraction method for two drugs from human plasma, Indomethacin and Ibuprofen using TELOS neo PRP, a water-wettable polymer-based
SPE sorbent. Analyte concentrations range from 5 to 50ng/ml. The data highlights
the minimum elution volumes that can be achieved from the 5mg sorbent mass,
providing high reproducible recoveries and RSDs. Excellent linearity was achieved,
along with LODs of 0.5 and 1ng/ml for Indomethacin and Ibuprofen respectively.
Linked Scanning of He and Compensation Voltage to Improve the
Resolving Power of Differential Ion Mobility Separations
Rachel Harris, University North Carolina-Chapel Hill, Caudill
Laboratories Rm. 326, 131 South Rd., Chapel Hill, NC 2514, Brandon
Santiago, Samantha Isenberg, Gary Glish
One of the benefits of gas phase separations such as differential ion mobility spectrometry (DIMS) is the ability to improve signal-to-background in mass spectrometry-based analyses. Helium can be added to the DIMS carrier gas to improve the
resolving power of the separations. However, the addition of He also leads to a loss
of ion transmission, especially for ions of higher differential ion mobility. In an effort
to improve both the resolving power and ion transmission of DIMS separations, the
“linked scan” was developed, which varies the DIMS compensation voltage and
carrier gas He content simultaneously in a fixed relationship. Throughout the linked
scan, as the compensation voltage increases, the He content of the carrier gas
is decreased. Experiments were performed using dispersion fields of 24, 26, 28,
30, 32, and 34 kV/cm. The compounds analyzed include Agilent ESI tuning mix
and ubiquitin. Linked scans were performed by varying the carrier gas He content
during the CV scan. CV scans with a constant helium composition were run with
He compositions ranging from 0% to 60% He in 5% increments. Resolving powers
of peaks from linked scans are compared to those from static scans at the nearest
He composition. For both analytes, peak widths are reduced and resolving power
increased when using linked scans. Generally, higher dispersion fields (ED = 32 kV/
cm) and larger He ranges give larger increases in resolving power. The average
percent increase in resolving power across all experiments is 37В±18% for tuning mix
and 51%В±17% for ubiquitin.
Development of an Ultra-Sensitive Affinity Capture Method for the
Quantification of Microdosed Dog Toxicology Samples
Lorell N. Discenza, Bristol-Myers Squibb, Route 206 & Province Line
Rd., Princeton, NJ 08543, John Mehl, Georgia Cornelius, Charu
Chaudhry, Randy Talbott, Jinping Gan, Celia D’Arienzo, Timothy Olah
Because a millamolecule of interest was not well tolerated in dogs at 0.01 mg/kg, a
new study was designed and dosed at 0.0001, 0.0003, and 0.001 mg/kg to evaluate
toxicity, tolerability and to provide guidance for future toxicology studies. In order to
measure the low concentration levels expected from this study, an affinity capture
sample preparation method was developed. With this improved methodology, we
were able to lower the lower limit of quantitation (LLOQ) ~ 100 fold down to 0.076
nM. Further reduction of the LLOQ was achieved by analysis on a recently acquired
Waters Xevo TQ-S with ionKey technology. How this strategy was implemented and
future applications is discussed.
2014 EAS Abstracts
November 2014
Photochemical and Thermal Control over Electrolessly GoldPlated Film Structure on Thin Silicon Nitride to Target Sensing
Caitlin M. Masterson, University of Rhode Island, Department of
Chemistry, 51 Lower College Rd., Kingston, RI 02881, Brian D. Velleco,
Buddini Iroshika Karawdeniya, Julie C. Whelan, Y.M. Nuwan D.Y.
Bandara, Jason R. Dwyer
Thin silicon nitride films are a common building block of nanofabricated devices.
They offer favorable physical properties, such as the ability to form free-standing <100nm-thick films, but their complex surface chemistry presents challenges.
We developed a process to electrolessly plate thin gold films onto silicon nitride
membranes, and into through-membrane pores, so that we could use thiol-based
chemistry to more readily modify the surface chemistry. We discovered that the gold
film grain structure allowed us to perform surface-enhanced Raman spectroscopy
(SERS). Thermal annealing of the films dramatically changed the gold film structure,
as revealed by both the SERS signal and field-emission scanning electron microscopy (FESEM). Mean grain radii, for example, could be readily increased by tens
of nanometers by post-deposition annealing under suitable conditions. We exerted
control over the film spatial distribution across longer length scales by preceding the
plating with a photochemical surface protection route that allowed us to generate
patterned gold films. We will describe the various gold film plating approaches and
present applications.
Using Nuclear Magnetic Resonance (NMR) Techniques to Study
Small (d = 2-3 nm) Gold-Transition Metal Nanoparticle Alloys
Lauren Marbella, University of Pittsburgh, Chevron Science Center, 219
Parkman Ave., Pittsburgh, PA 15227, Christopher M. Andolina, Jill E.
Small gold-transition metal nanoparticle alloys (diameter = 2 – 3 nm) display physical behaviors distinct from either their bulk or molecular counterparts, and are desirable for applications ranging from heterogenous catalysis to therapeutics. However,
nanoscale alloyed architectures are difficult to synthesize because metal mixing
behavior exhibits length scale dependence. We hypothesize that alloy formation
at the nanometer scale can be manipulated by controlling surface chemistry. Using
classical colloidal methods, we have been able to synthesize a range of gold-transition metal nanoparticle alloys with fully tunable compositions, regardless of bulk
miscibilities. With a unique combination of NMR-based molecular characterization
techniques and traditional materials characterization strategies, we are able to provide crucial insight into particle formation, as well as directly correlate particle composition with particle physical properties.
Toxicity of Silver Nanoparticles in the Environment
Maral P.S. Mousavi, University of Minnesota, Department of Chemistry,
207 Pleasant St. South East, Minneapolis, MN 55455, Ian L. Gunsolus,
Carlos E. PГ©rez De JesГєs, Christy L. Haynes, Philippe BГјhlmann
Silver nanoparticles, Ag NPs, are widely used in many consumer products. As a
result, they can leach into the environment during product usage, raising concern
about their toxicity in the ecosystem. Natural organic matter, (NOM), which is abundant in water supplies, soil, and sediments, can interact with Ag NPs and influence
their environmental persistence. Moreover NOM can bind with Ag+ that is released
from Ag NPs resulting in formation of silver-species with altered bioavailability and
toxicity. Using fluorous-phase Ag+ ion-selective electrodes (ISEs), we studied Ag+
binding to NOM and showed that Ag+ binding to NOM is favored by a higher pH.
Moreover, effect of NOM chemical composition on kinetics and extent of binding
was investigated. It was demonstrated that NOM samples with a low sulfur and
nitrogen content have weak Ag+ binding, while stronger binding was observed for
Pony Lake NOM, which has higher amounts of these elements. Fast kinetics of
Ag+ and NOM binding was confirmed. Using fluorous-phase Ag+ ISEs it was shown
that presence of NOM does not alter the concentration of Ag+ released from Ag
NPs. NOM samples with high sulfur and nitrogen content were shown to reduce
silver toxicity to the bacterium Shewanella oneidensis. More generally, this work has
shown that fluorous-phase Ag+ ISEs are effective tools for studying Ag NP toxicity
in the environment.
Quantification of Local Bonding Environments in Cesium Silicate
Glasses Using Si-29 Magic–Angle Flipping NMR
Pyae Phyo, Berea College, Chemistry Program, CPO 1119, Berea,
KY 40404, Jay H. Baltisberger, Kevin Sanders, Eric Keeler, Philip J.
Cesium silicate glasses were studied using Si-29 magic-angle flipping Nuclear Magnetic Resonance (NMR) spectroscopy. The chemical shift anisotropy parameters
were extracted for each glass with a variety of Q(4) and Q(3) sites. The Q(4) site
increases in width as the amount of cesium is increased. This is consistent with an
increasingly disordered environment around these sites as the neighboring sites
have more non-bridging oxygen atoms. Two distinct Q(3) sites are seen with very
different anisotropies at the low cesium content and become more similar as cesium
is added. Previous work has shown that larger anisotropies correspond to short
non-bridging Si–O bond distances for Q(3) sites. This leads to a general interpretation that the large anisotropy site arises from an increasing number of coordinating
cesium cations near that site. The other Q(3) site with increasing anisotropy with
increasing cesium content must arise from either a smaller number of coordinating
cations or a reduced local microscopic density to increase the Si-O bond lengths as
cesium is added. This model indicates a microscopically phase separated mixture
of isolated cesium cations scattered throughout the silica matrix, as well as some
channels filled with clustered cesium cations. No macroscopic evidence for phase
separation is seen in these glasses, however the degree of homogeneity of the melt
is difficult to quantify given that samples were quenched from 1200 ЛљC. Smaller cations such as sodium, rubidium, and potassium have been studied in silicate glasses
with no evidence of multiple Q(3) sites.
Enzyme Modulator Screening Using Droplet Samples and SubSecond Microchip Electrophoresis
Erik D. Guetschow, University of Michigan, Department of Chemistry,
Ann Arbor, MI 48109, Robert T. Kennedy
Traditional high-throughput screening (HTS) utilizes multiwell plate based assays
and fluorescence detection to perform tens of thousands of assays per day. While
this method has been successful, fluorogenic reagents or coupled reactions are
required and multi-analyte detection is difficult. We have developed a novel method
to couple multiwell plate-based assays to microchip electrophoresis (MCE) using
droplet microfluidics. Samples contained in multiwell plates are reformatted in to
nanoliter volume plugs segmented by an oil carrier phase. Droplet streams are coupled to a hybrid polydimethylsiloxane-glass microfluidic device capable of selectively extracting the aqueous samples from the droplet stream and rapidly analyzing
droplet contents by MCE. To validate this new method, a test library containing 140
small molecules was screened for inhibitors or protein kinase A (PKA). Each sample
was reformatted as two droplets allowing at least 6 MCE injections per sample.
Sub-second separation of three analytes – rhodamine, substrate peptide, and product peptide – was achieved in 5 mm using an applied electric field of 2000 V/cm with
an efficiency of 16,000 plates/second. Using 700 injections total, 96 samples were
analyzed in 12 minutes (0.13 Hz) and several small molecule inhibitors of PKA were
identified and verified through dose-response experiments. While this method has
been applied to screening applications, the device layout and operation is amenable
to many applications including coupling two-dimensional separations and chemical
sensing. This work was supported by funding from the National Institutes of Health.
Mass Spectrometry Analysis of NXS/T Glycosylation Sites in
Recombinant Glycoproteins
Armand G. Ngounou Wetie, Clarkson University, 8 Clarkson Ave.,
Potsdam, NY, 13699, Izabela Sokolowska, Urmi Roy, Alisa G. Woods,
Costel C. Darie
To fulfill the United States Food and Drug Administration’s requirements, therapeutic
proteins have to be fully characterized with regard to their size, sequence and their
post-translational modifications (PTMs). One of the most important PTMs that play
a role in the solubility, stability and function of therapeutic proteins is glycosylation.
In this study, we investigated N-glycosylation at NXS/T sequons of a fusion protein using a mass spectrometry-based proteomic strategy to determine if the introduction of new N-glycosylation sites into a chimeric protein would influence known
N-glycosylation sites. Therefore, a chimeric protein IgG-Fc-ZP3E7, composed of
the Fc part of IgG1 heavy chain and of exon 7 of the zona pellucida (ZP) protein,
was over-expressed and the protein product purified. As a control, IgG-HC (Fc part
of IgG1 heavy chain) was used. Samples were treated with and without PNGase F
and analyzed by SDS-PAGE, Western blotting, trypsin-AspN double digest followed
by nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using
data dependent analysis (DDA) and information dependent analysis (IDA or DDA
with inclusion list). Our results show that addition of a new potential NXS/T glycosylation site within a fusion or chimeric protein could prevent the glycosylation of
original NXS/T glycosylation sites.
Hard-Modelling - From Titrations to Solving Climate Tribulations
Graeme Puxty, Commonwealth Scientific and Industrial Research
Organization, PO Box 330, Newcastle, NSW 2300 Australia
In the chemometrics/chemistry world hard-modelling is the fitting of models that
represent some sort of chemical phenomena to measurements of that phenomena.
A simple example is the fitting of a straight line to spectrophotometric measurements of absorbance at a characteristic wavelength as a function of concentration
of the absorbing chemical species. In its most complex it is the fitting of a chemical
model incorporating reaction kinetics and equilbria (between species and phases),
thermodynamics, and physical processes to multidimensional measurements of dif-
2014 EAS Abstracts
November 2014
ferent type. This presentation covers a range of examples where a combination of
intelligent measurement combined with appropriate model selection, development
and fitting has led to understanding, insights and developments of chemistry and
chemical processes. From the fitting of a pH curve to determine the pKa of an acid,
to multidimensional potentiometric and spectrophotometric titrations and stoppedflow kinetics to elucidate the equilibrium constants, rate constants and thermodynamics of complex multi-step reactions. Finally, how this has developed into new
and improved absorbents and processes for the capture of carbon dioxide from gas
streams. Developments that are now leaving the lab and moving to real-world large
scale testing.
States. The advent of tandem mass spectrometry (MS-MS) coupled with “soft” ionization techniques significantly increased the number of disease states amenable to
detection by DBS analysis. This presentation describes the advent of DBE analysis
with tandem mass spectrometry, and traces its history from the earliest applications
to modern mass newborn screening, toxicokinetics, pharmacokinetics, and therapeutic drug monitoring.
Dried Blood Spots - From Newborn Screening to Forensic Science
Donald Chace, Pediatrix Medical Group, 1301 Concord Terrace,
Sunrise, FL 33323
The most frequent application of dried blood spot analysis is its clinical use as a
blood collection matrix for metabolic screening of infants for genetic disease screening (newborn screening) and forensic pathology (postmortem screening). The dried
blood spot is collected between 24-48 hours after birth and tested for more than 40
conditions using a variety of technologies such as immunoassays, fluorescence assays, electrophoresis and tandem mass spectrometry. Metabolic diseases screened
by tandem mass spectrometry analysis of primarily amino acids and acylcarnitines
which account for more than half of all disorders screened. This comprehensive
analysis of more than 50 metabolites is achieved by a single sampling (a punch) of
the dried blood spot and in an analysis that takes approximately two minutes per
sample. The forensic pathology analysis uses primarily tandem mass spectrometry
for primarily acylcarnitines in dried blood samples which are collected at autopsy
(the metabolic autopsy) from unexplained infant or child deaths or in medico-legal
cases. The dried blood spot is unique because it requires small volumes of blood
and can be easily shipped to a laboratory for analysis. These advantages coupled
with mass spectrometry analysis are continuing to lead to new applications in areas
such as premature infant metabolism and nutrition.
Hybrid Hard and Soft-Modeling and Local Rank Analysis: Two
Relevant Ideas to Describe and Explore Chemical Systems
Anna de Juan, University of Barcelona, Chemometrics Group,
Department of Analytical Chemistry, Diagonal, 645, Barcelona, 08028
No abstract submitted by the author.
Calculation of Feasible Bands for Visualizing the Constraint Effects
in Model-Free Methods
Hamid Abdollahi, Institute for Advanced Studies in Basic Sciences,
Department of Chemistry, PO Box 4, Zanjan, 5195-1159 Iran
The curve resolution (CR) methods magnetize substantial research efforts aimed
to discover knowledge of multicomponent systems. The soft methods establish a
specified part in CR methods, as they allow going into insight the data without prior
knowledge of the studied system. Using soft methods, the outcome of the analysis
of one system is not usually the true one due to rotational ambiguity. The rotational
ambiguity leads to a range of possible solutions that fulfill constraints and represent
the measured data correctly. Consequently, taking into consideration of all feasible
solutions can provide useful information about the system and the process under
study when there is no unique solution for the system. Additionally, the calculation of
feasible bands is dramatically advantageous not only for quantitative and qualitative
application of the resolved profiles but also for visualizing and deep understanding the effects of constraints on results of using soft-modeling methods. Different
methods have been proposed in the literature to calculate the feasible solutions.
Analytical methods for calculating the feasible solutions in two and three-component
systems developed as Lwton-Sylvester and Borgen-Rajko plots. Then the exact and
explicit calculations of range of feasible solutions were approximated by numerical methods as grid search procedures up to four component systems. The micro
structure of chemical data can be visualized by showing the feasible solution regions in abstract spaces under considered constraints. The effects of non-negativity,
equality, selectivity-zero regions and hard modeling constraints on micro structure
of chemical data have been investigated basically and some new conclusions have
been obtained from interpretation of observations.
Recent Progress in Implementing Dried Blood Spot Technology in
Drug Development
Qin Ji, Bristol-Myers Squibb, Rte. 206 & Province Line Rd., Princeton,
NJ 08540
No abstract submitted by the author.
Automated Bioanalysis of Dried Blood Spots and Dried Plasma
Spots Coupled with High Resolution Mass Spectrometry and
Selected Reaction Monitoring LC-MS Technologies
Jack Henion, Quintiles Bioanalytical and ADME Labs, 19 Brown Rd.,
Ithaca, NY 14850, Robert Sturm, Regina Oliveira
The pharmaceutical industry has recently shown interest in dried blood spot (DBS)
techniques which has expanded to other important biological matrices including
plasma (DPS), cerebrospinal fluid, saliva, bile, etc. DBS and DPS techniques represent micro sampling techniques which are also of increasing interest when limited
quantities of biological samples are available. If large numbers of DBS cards require
liquid chromatography mass spectrometry (LC-MS) analysis which is beginning to
occur in certain drug discovery programs, it is very helpful for automation which can
preclude tedious off-line manual punching and extraction of these micro samples.
Challenges from micro samples include the need for high sensitivity, but modern
LC-MS techniques can meet these needs. In contrast, the use of “DBS techniques”
has raised new issues including the effects of hematocrit on the distribution of whole
blood on the cellulose substrate. New strategies are being explored to deal with
these issues. For our DBS bioanalytical studies commercially available cards were
employed coupled with commercially available automated DBS extraction robotic
equipment coupled with high resolution accurate mass using a quadrupole timeof-flight MS. For our DPS studies we have produced a home-made laminated DPS
card device that is well-suited for micro sampling low volumes of whole blood (10to
25 ВµL) to produce dried plasma without the need for centrifugation. These devices
have undergone accuracy and precision LC-MS experiments employing the prodrug, guanfacine and its stable isotope internal standard. A comparison of automated DBS and DPS analysis with the title techniques is presented.
Chemometrics: The Balance Between �Chemistry’ and �Metrics’
Marcel Maeder, University of New Castle, Department of Chemistry,
University Dr., Newcastle, 02308 Australia
Chemometrics is a term that does not have a clear definition; it is rather a substantial collection of computational methods that are used by chemists for the analysis
of usually large data sets. Selecting the �optimal’ method, the one that delivers the
maximum amount of useful information is clearly an important but also challenging
task. One way of bringing together and differentiating between the diverse methods
of chemometrics is to investigate the nature and amount of prior information used
for the analysis. Specifically, we will consider the amount of chemical insight that is
used as the backbone for the analysis of the process under investigation. There is
little doubt that the more such prior information is available and efficiently used, the
more robust the analysis and also, at least potentially, the more useful the extracted
information. Conversely, it is not immediately clear how such prior information can
be incorporated into the different algorithms. It is interesting to investigate the history of chemometrics, e.g., by compiling the chapter headings of the bi-annual reviews
on Chemometrics in Analytical Chemistry: the trend is clearly in the direction of less
rather than more chemical content in the analyses. The presentation argues that this
is a �sub-optimal’ development.
Enhanced-Fluidity Liquid Chromatography (EFLC) for Highly Polar
Biologically Relevant Compounds
Susan Olesik, Ohio State University, Department of Chemistry and
Biochemistry, 100 West 18th Ave., Columbus, OH 43016, Martin Beres,
Rafael Bennett
Enhanced-fluidity liquids (EFLs) are organic solvents or organic/aqueous solvents
mixed with high proportions of liquefied gases. These solvents share the positive
attributes of supercritical fluids (fast diffusion rates and low viscosities) and the positive attributes of common liquids (high solvent strength). These liquids when used in
liquid chromatography provide high efficiency and decreased separation time. Our
group previously illustrated that EFLC when combined with hydrophilic interaction
liquid chromatography (HILIC) stationary phases is quite effective for the separation
of highly polar nucleotides and nucleosides without the need for gradient conditions.
This talk presents data illustrating the separation of even more polar biologically
History of DBS and its Development from Clinical to Pharmaceutical
Daniel L. Norwood, Boehringer Ingelheim, 900 Ridgebury Rd., PO Box
368, Ridgefield, CT 06877
Dried blood spots (DBS) on filter paper have been employed since the early 1960s
for the collection, storage and transport of small (< 100 ВµL) samples for the purpose
of newborn screening. Originally, newborns were screened for phenylketonuria by
measurement of phenylalanine using a bacterial inhibition assay. Advances in technology over the last two decades of the 20th century led to a significant increase in
the number of disease conditions assessed in newborns by DBS analysis, as well as
virtually universal application of DBS analysis for newborn screening in the United
2014 EAS Abstracts
November 2014
relevant compounds using HILIC and ion-exchange stationary phases. The range of
operating conditions that are important in EFLC-HILIC or EFLC-IE is discussed and
a deep characterization of the retention mechanism for various operating conditions
is included.
LC-QTOF, and RapidFire-MS-MS. The effectiveness of these different screening
methods was compared; 75 of the 104 urine samples collected (72%) were positive
for drugs of interest by one or more screening techniques, with good agreement
between the various methods. This presentation describes the results from four
complementary approaches in screening these samples and evaluates ease of use,
breadth of analyte detection, sensitivity and ability to detect unknown compounds.
Recent Advances in Supercritical Fluid Chromatography
Jennifer Van Anda, Agilent Technologies, 330 Locust Grove Rd., West
Chester, PA 19382, Rick Wikfors
The key market for the application of supercritical fluid chromatography (SFC) has
been in the pharmaceutical industry, primarily in chiral separations. Many recent
improvements have enabled the use of SFC in a variety of other application areas
previously deemed unavailable. Previously, major vendors had limited the supported mass spectrometers to single quadrupole systems. Recently, the coupling and
support of triple quadrupole and time of flight spectrometers have become available. These additional detectors will allow increased sensitivity in application areas
such as drug residue analyses and with additional discrimination power, applications such as lipids and metabolomics. A recently introduced flow splitter contains
componentry for enhancing robustness and allowing either split or direct interfaces
to various mass spectrometers. When using the flow splitter, the split flow across a
gradient is more constant, assuring greater accuracy in peak quantitation after the
splitter. The use of a solventless injection mode has been developed with minimal
additional hardware. Solventless injection enables very large volumes, comparable
to a high-performance liquid chromatography (HPLC) injection, to be injected. Additionally, the solventless injection allows for previously incompatible sample solvent
injections including an aqueous injection. Examples of applications utilizing the solventless injection mode, high discrimination mass spectrometry outside of the chiral
arena are shown.
Screening Novel Psychoactive Substances (NPS) by High
Resolution Accurate Mass Spectrometry (HRAMS)
Jared Castellani, South Carolina Law Enforcement Division, 4400 Broad
River Rd., Columbia, SC 29212, Alex L. Magitti III, Barry K. Logan,
Francis X. Diamond, Matthew M. McMullin
In the realm of forensic toxicology, recent interest has developed over the use of
HRAMS for drug screening casework due to its superior specificity and sensitivity to
historical screening methods. Time-of-flight (TOF) identifies compounds based on
a peak’s retention time and accurate mass relative to the expected retention time
(when available) and each compound’s exact mass based on its molecular formula
and constant calibration. The issue with this type of testing on complex forensic
specimens is that artifacts, minor metabolites and isomers can create false positives
that lead to unnecessary confirmatory testing and additional manual evaluation/manipulation to characterize potential candidate compounds. This study demonstrates
techniques that have the ability to fragment the molecular ion, allowing for identification of fragment ions to enhance specificity. Using an Agilent 1290/6530 high-performance liquid chromatography (HPLC)-quadropoleTOF mass spectrometer, parent and respective fragment masses can be monitored to identify positive findings.
Three fragmentation modes were evaluated using LC-QTOF including: quadrupole-TOF liquid chromatography tandem mass spectrometry (QTOF LC-MS-MS),
LC-all product ions-MS with collision induced dissociation in the source (CIDS) and
LC-all product ions-MS with collision induced dissociation in collision cell (CIDCC).
Three fragmentation modes were evaluated by screening patient whole blood and
spiked synthetic blood for NPS. Using an Agilent developed database containing
MS-MS spectra for 140 target compounds, the three modes were evaluated. Assay
performance was comparable across all three methods at elevated drug concentrations for the spiked blood samples, but at lower concentrations both CIDS and
CIDCC performed superior to QTOF with respect to spiked drug identification.
Effect of Particle Size on the Speed and Resolution of Chiral
Separations Using Supercritical Fluid Chromatography
Mirlinda Biba, Merck, PO Box 2000, MS: RY818-B218, Rahway, NJ
07065, Erik L. Regalado, Christopher J. Welch, Naijun Wu
Fast chiral supercritical fluid chromatography (SFC) separations have become
important due to the increasing use of high-throughput experimentation (HTE) in
organic synthesis. These HTE experiments can generate hundreds of samples for
chiral analysis that need to be assayed in a short time. In general, chiral SFC can
provide much faster analysis times compared to liquid chromatography (LC). Additionally, columns packed with smaller particles can provide faster and more efficient
separations. In this study, the effect of the particle size on the speed and resolution
of chiral separations by SFC was evaluated. The performance of Chiralcel OD columns packed with either 5-Вµm or 3-Вµm particles were compared using van Deemter
or other kinetic plots. The benefits of using smaller particle columns for chiral SFC
analysis are illustrated.
Determination of 22 Antiepileptic Drugs in Post-Mortem Blood,
Serum and Plasma Using LC-MS-MS with Focus on their Rule in
Forensic Cases
Karen Scott, Arcadia University, 450 S. Easton Rd., Glenside, PA 19038,
Shaza Deeb
In the last decade, there has been a growth in reports of antiepileptic drugs (AEDs)
being misused both on their own and in combination with other drugs of abuse in
a variety of toxicological case types such as drug abuse, suicide, overdose and
drug facilitated crime. To date, there are no methods for the simultaneous analysis
of the most commonly encountered AEDs in post mortem whole blood and clinical
plasma/serum samples. A simple, accurate and cost effective liquid chromatography
tandem mass spectrometric (LC-MS-MS) method was developed and validated for
the quantification of carbamazepine and its metabolite carbamazepine-10,11-epoxide, eslicarbazepine acetate, oxcarbazepine and S-licarbazepine as a metabolite,
gabapentin, lacosamide, lamotrigine, levetiracetam, pregabalin, phenobarbital, phenytoin and its metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin, retigabine (ezogabine) and its metabolite N-acetyl retigabine, rufinamide, stiripentol, topiramate,
tiagabine, valproic acid, vigabatrin and zonisamide in post-mortem whole blood,
serum and plasma which is suitable for routine forensic toxicological analysis and
therapeutic drug monitoring. Drugs were extracted from biological matrix using a
simple effective liquid liquid extraction. All AEDs were detected and quantified within
17 minutes without endogenous interferences. The correlation coefficient (R2) was
greater than 0.994 for all drugs with accuracy ranging from 90 to 113% and precision
<13% for all analytes. The recovery ranged from 70% to 98%. No carryover was
observed in a blank control injected after the highest standard and the matrix effect
was acceptable and ranged from 90% to 120%. The method has been successfully
verified using authentic samples from clinical, postmortem and prison case samples.
The Power of Ultra Performance Convergence Chromatography:
Theoretical Considerations and some Examples of Use
Francois J. Huby, The Dow Chemical Company, 400 Arcola Rd.,
Collegeville, PA 19426, Robert M. Campbell
Supercritical carbon dioxide (CO2) enhanced chromatography (supercritical fluid
chromatography and ultra performance convergence chromatography) is complementary to gas chromatography (GC) since it can separate higher boiling compounds and to high-performance liquid chromatography (HPLC) since it can separate and detect closely related compounds which may co-elute or be hard to detect
by liquid chromatography. New instrumentation has allowed implementing various
CO2 mediated separation techniques with greater efficiency and ease of use than
earlier versions. Theoretical evidence and examples of use supporting these statements are presented.
Comparison of GCMS, EIA, and LC-QTOF Screening Methods for
Novel Psychoactive Substances in Urine Samples
Jill Yeakel, Lehigh Valley Toxicology, 3864 Courtney St., Ste. 150,
Bethlehem, PA 18017, Mandi Mohr, Barry Logan
Designer drugs, such as synthetic cathinones and substituted phenethylamines
have been introduced into the drug user community over the last 5 years. US authorities have moved to control dangerous compounds as they are discovered, and
in response, illicit manufacturers make structural modifications to the drugs in an effort to create legal yet potent stimulant and hallucinogens. The ever-changing drug
market has presented a unique problem for medical professionals and toxicologists
in identifying the compounds responsible for intoxication or adverse medical effects.
Attendees at electronic dance music (EDM) festivals have been documented as
having high rates of use of novel psychoactive substances (NPS). We obtained
biological samples including urine from anonymous volunteer attendees at a large
EDM festival in the US in early 2014 to determine the patterns of use of known
NPS as well as to identify new and emerging compounds. The urine samples were
screened via a battery of tests including immunoassay (EIA), full scan GC-MS,
Development of a Pyrrolidinophenone Panel in Blood and Detection
in Toxicology Case Work
Donna Papsun, NMS Labs, 3701 Welsh Rd., Willow Grove, PA 19090,
Barry K. Logan, Robert Middleberg, Carol Beck
Pyrrolidinophenones are an emerging group of compounds characterized as novel
psychoactive substances (NPS) as part of the designer drug movement. Routine
forensic toxicology assays may not be detecting these compounds, consequently underestimating their use. For this reason, it was necessary to develop screen
and confirmation tests for blood to identify compounds in this group, which included alpha PVP, naphyrone, pyrovalerone, alpha PBP, alpha PPP, MePPP, MDPPP,
MOPPP, MPBP, and MPHP. Traditional screening tools use gas chromatography-mass spectrometry (GC-MS) library matching; however, validated reporting lim-
2014 EAS Abstracts
November 2014
its for this group of pyrrolidinophenones varied from 5 to 80 ng/mL, which poses a
significant problem when trying to identify small amounts of drugs in biological specimens. A second screening technique, Liquid Chromatography Time of Flight Mass
Spectrometry (LC-TOF-MS), demonstrated 100% sensitivity and specificity for all
compounds at a reporting limit of 10 ng/mL. A quantitative method was developed
using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry
(HPLC-ESI-MS-MS) operated in electrospray positive ionization multiple reaction
monitoring mode. Calibration was satisfactory for all 10 analytes from 5-500 ng/mL.
Accuracy data were within the acceptance interval of +/- 15% of the nominal values
for all drugs. Within-day (repeatability) and intermediate precision data were within
20% relative standard deviation (RSD). After analysis of de-identified patient cases,
positives were seen for alpha PVP and alpha PBP. These findings suggest that a
combination of LC-TOF-MS and LC-MS-MS provides two valuable tools for the sensitive and specific analytical identification and measurement of pyrrolidinophenones
in forensic casework.
Tip-Enhanced Infrared Nanospectroscopy via Molecular Expansion
Force Detection
Mikhail A. Belkin, University of Texas-Austin, Department of Electrical
and Computer Engineering, Austin, TX 78759, Feng Lu, Mingzhou Jin
Mid-infrared photoexpansion nanospectroscopy, or atomic force microscopy infrared (AFM-IR), is a powerful technique for nanoscale chemical analysis in which
light absorption is detected by measuring associated local sample thermal expansion with an atomic force microscope (AFM). Originally developed by Alexandre
Dazzi and co-workers, this approach required high-power laser pulses to induce
significant sample heating and samples of ~100 nm or more in thickness. Recently,
we demonstrated that the sensitivity of this technique may be improved to take
nanoscale spectra of samples as thin as molecular monolayers with better than
30 nm spatial resolution. This was achieved by sending low-power laser pulses at
a repetition frequency that is tuned in resonance with the mechanical vibrational
frequency of the AFM cantilever and by employing tip-enhancement of the optical
field below a sharp gold-coated AFM tip. I describe the details of our approach and
present the latest results, including nano scale spectra of samples in aqueous environment. This project was supported by the Welch Foundation grant F-1705 and
STTR program from the DOE.
How Photoacoustic and Nanomechanics Combine to Perform IR
Spectroscopy at the Nanoscale
Alexandre Dazzi, University of Paris-Sud, Laboratory of Physical
Chemistry, Batiment 349, Orsay 91405 France
Atomic force microscope-based infrared spectroscopy (AFM-IR) has been developed in recent years providing extremely high spatial resolution chemical characterization and imaging. The technique is based on the combination of a tunable
infrared laser with an atomic force microscope that can locally map and measure
thermal expansion of nanoscale regions of a sample resulting from the absorption
of infrared radiation. The results obtained these last years in microbiology and polymer sciences show how this approach is a powerful tool. The principle is based on
detecting the local thermo expansion of the sample, irradiated at the wavelength of
its absorption bands. This expansion is detected by the AFM tip in contact mode. As
the duration of expansion and relaxation of the sample is always shorter than the
response time of the cantilever in contact, the excitation transmitted to the cantilever acts as an acoustic shock, exciting oscillations at resonant frequencies of the
cantilever. The technique can create nanoscale IR absorption spectra by recording
the amplitude of these oscillations as a function of wavelength and chemical maps
by measuring the oscillation amplitude as a function of position. [1] Because the AFM
probe tip can map the thermal expansion on very fine length scales, the AFM-IR
technique provides a robust way to obtain interpretable IR absorption spectra at
spatial resolution scales well below the diffraction limit.
NanoIR to Investigate Parchment and its Degradation
Laurianne Robinet, Center of Research for Conservation, 36, rue
Geoffroy Saint Hilaire, Paris, 75005 France, GaГ«l Latour, Ariane
Deniset-Besseau, Alexandre Dazzi, Marie-Claire Schanne-Klein
The characterization of cultural heritage materials is essential to understand the
artefact technology and its alteration, so to develop suitable restoration treatment or
conservation conditions. These analyses are often complex due to the heterogeneity of the material at different length scales and its composite nature originating from
the manufacturing or the degradation. Additionally, artefacts are generally fragile or
rare thus sampling is often restricted or impossible. For these reasons non- or micro-invasive analytical approaches using sensitive techniques are being developed.
Parchment was the main writing material in the Middle Ages in Western Europe up
to the growth of paper production in the 14-15th centuries. Made from an untanned
animal skin it was preserved by liming, scraping and drying the skin under tension.
Parchment is very sensitive to water, which can cause denaturation of collagen, its
main constituent. This degradation, known as gelatinization, induces modifications
at all structural levels in the molecule, thus complementary analytical techniques are
required to better understand the involved mechanisms. Infrared spectroscopy is
used to examine parchment modifications at the molecular level; however the structural heterogeneity within a collagen fiber and the interference of the carbonate signal (from the liming process) in the spectra often renders the interpretation difficult
when working at the micro scale. To overcome this limitation, infrared analysis at the
nanoscale is crucial. This presentation presents the first results in parchment fibers
analysis at the nanoscale using atomic force microscopy infrared (AFM-IR) and discusses the potential of coupling nanoIR with other microscopy imaging techniques.
[1] Dazzi et al. J Appl. Phys. 107, 124519 2010
Extending AFM-Based Infrared Spectroscopy to a Wide Range of
Craig Prater, Anasys Instruments, 325 Chapala St., Santa Barbara, CA
Atomic force microscope-based infrared spectroscopy (AFM-IR) is a growing technique for nanoscale chemical characterization of materials. Initial AFM-IR instruments had two key limitations: 1) Samples had to be prepared as thin sections with a
very limited thickness range; and 2) the thin sample sections needed to be mounted
on an infrared transparent prism. Recent research has enabled instrumentation that
overcomes both of these limitations. Now samples can be measured on almost arbitrary substrates, opening up nanoscale chemical analysis to a much wider range
of samples, including many in-situ measurements. In addition, recent innovations
have enabled dramatic improvements in AFM-IR measurement sensitivity. Using
resonant enhanced AFM-IR, it is possible to perform spectroscopic analysis and
chemical imaging on films as thin as a single molecular monolayer. This presentation describes recent innovations in AFM-IR technology and applications in various
fields including energy, materials and life sciences.
Quantitating Protein Panels and Normalizing Hematocrit from
Dried Blood Spots (DBS) Using an Automated SISCAPAВ® Mass
Spectrometry Workflow
Morteza Razavi, SISCAPA Assay Technologies, Box 53309, Washington
DC 20009, Selena S. Larkin, Terry W. Pearson, Leigh Anderson
The use of dried blood spots (DBS) for bioanalysis has gained considerable momentum with reports demonstrating that both small molecules and proteins can be
reliably extracted and analyzed by mass spectrometry. However, lack of a standardized method to normalize for sample hematocrit and volume variations has slowed
the adoption of DBS in clinical settings. Moreover, standard separative means used
in mass spectrometry are either overly complicated and low-throughput or do not
provide enough sensitivity to measure low-abundance proteins. To address these
challenges, we devised an automated approach using a Bravo liquid handling robot (Agilent Technologies, CA) whereby proteotypic peptide targets are selectively enriched from digested human blood using high-affinity anti-peptide antibodies
and quantitated based on their relative ratio to internal stable isotope standards; an
approach known as SISCAPAВ®. The approach was complemented with a proprietary algorithm that normalizes sample-to-sample hematocrit and volume variations
based on measurement of high-abundance proteins of plasma and red blood cells.
Using this novel strategy, we successfully measured normalized levels of two 11plex protein panels, with abundance spanning 8 orders of magnitude, from a single
¼” diameter DBS punch. By monitoring longitudinal DBS specimens, we were able
to track the kinetics of changes in concentration of these proteins through conditions such as a common cold, pneumonia and kidney infection. We also observed
an interesting �protein baseline-concentration fingerprinting pattern’ unique to each
individual under study. These results bode well for personalizing medicine through
longitudinal collection of dried-blood-spots.
AFM-IR: Nanoscale IR Spectroscopy for the Materials and Life
Curtis Marcott, Light Light Solutions, 2558 High Hammock Rd., Johns
Island, SC 29455, Craig Prater, Qichi Hu, Michael Lo, Kevin Kjoller
Atomic force microscope-based infrared spectroscopy (AFM-IR) has been developed in recent years providing extremely high spatial resolution chemical characterization and imaging. The technique is based on the combination of a tunable infrared
laser with an atomic force microscope that can locally map and measure thermal
expansion of nanoscale regions of a sample resulting from the absorption of infrared
radiation. Because the AFM probe tip can map the thermal expansion on very fine
length scales, the AFM-IR technique provides a robust way to obtain interpretable IR
absorption spectra at spatial resolution scales well below the diffraction limit. Several applications of AFM-IR spectroscopy and imaging to problems in the materials
and life sciences are presented.
2014 EAS Abstracts
November 2014
Identification of Volatile Organic Compounds in Beer by
TurboMatrix Headspace Trap System-Gas Chromatography-Mass
Hetal Rana, Kean University, 1000 Morris Ave., Union, NJ 07083, Dil
Beer is the most popular drink around the world, after water and tea. It has become a standard and most consumed alcoholic beverage. Beer is made by the
saccharification of starch and fermentation of sugar, a process called brewing. The
saccharification enzymes, required for fermentation process, are extracted from
malted cereal grains like wheat and barley. Female flowers called hops are used as
flavoring agents, which also give bitterness to the beer and act as a natural preservative. Beer is a complex mixture of various compounds such as alcohols, sugars,
esters, proteins, ketones and acids. In this study, a rapid and sensitive method is
developed to identify and quantify many of these compounds using a TurboMatrix
headspace trap (TMHST) system connected to gas chromatography-mass spectrometry (GC-MS). The selectivity and inertness of the stationery phase, the chromatography column (60 m x 0.25 mm x 1.0 Вµm Elite-5MS), and the gaseous mobile
phase give precise retention times to separate the peaks. In the MS, each peak will
be further separated according to their mass-to-charge ratio and then identified and
characterized. The method developed is used to create a library of volatile organic
compounds present in beer. In the results all beers contain 3-methylbutanol and all
hops contain Limonene which is responsible for citrus aroma. The 3-methylbutanol
is one of the fusel oils or fusel alcohols associated with fermentation of beer.
Validated Determination of Dapagliflozin and Qualitative
Determination of Its Glucuronide Metabolite in Human Dried Blood
Spot: Multiple-Site Pediatric Clinical Experience
Xiaohui Xu, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08540,
Jane Liu, Jim Shen, Melanie Pe Benito, Marsha Epstein, Guowen Liu,
David Boulton, Pathanjali Kadiyala, Mark E. Arnold, Qin Ji, Michael
Dry blood spot (DBS) technology has garnered acceptability as a microsampling
technology to support drug discovery and development process due to the ease
of sample storage and shipment. Bristol-Myers Squibb has extensively applied this
technique in toxicokinetic studies and started to extend the application into pediatric studies. DBS sample collection, including collections from both venous and
finger-stick draws, were incorporated in a pediatric study protocol for dapagliflozin
(DAPA, Farxigaв„ў). Plasma sample collection was also made in parallel and compared against DBS data to ascertain the fitness of the DBS as a sampling scheme.
We have developed a DBS liquid chromatography tandem mass spectrometry (LCMS-MS) assay to simultaneously quantify DAPA while qualify the major glucuronide
metabolite (GLU) in dry blood spots with a volume 15-ВµL of blood collected on Whatman FTA DMPK-C cards. Each analysis utilizes a circular punch size of 6mm in diameters. Analytes were eluted from the cards with 75:20:5 water / 1.0 M ammonium
acetate / formic acid, v/v/v which was cleaned up via solid-phase extraction and
analyzed under negative ion electrospray LC-MS-MS detection in multiple reaction
monitoring. A lower limit of quantitation of 1 ng/mL was achieved for DAPA and 2.0
ng/mL for GLU, which were suitable for the pediatric study. The between-run %CV
from assay qualification batches was ≤ 8.60 and the within-run %CV was ≤ 8.03.
The mean % deviation from nominal concentration was within В±8.61. The concentration data from DBS venous and finger stick draws, and plasma draw are comparable
and we have demonstrated the utility of DBS in this pediatric clinical study.
Assuring Water Purity by Monitoring Water Contaminants at
Ultratrace Levels
Satinder (Sut) Ahuja, Ahuja Consulting, 1061 Rutledge Court, Calabash,
NC 28467
After air, water is the most essential material for human survival. Without water,
life as we know it in this world would not be possible. Even though our earth is
composed largely of water, fresh water comprises only 3% of the total water available to us. Of that, only 0.06% is easily accessible. An estimated 1.2 billion people
drink unclean water today. Drinking water comes mainly from the following sources:
rivers, lakes, wells, and natural springs. These sources are exposed to a variety
of conditions that can contaminate the water. The failure of safety measures relating to production, utilization, and disposal of a large number of inorganic/organic
compounds (from arsenic to zinc) can cause contamination of our water supplies.
Whereas zinc amounts is in small desirable, arsenic is harmful over 10 parts per
billion (ppb). The most notable case is the massive arsenic contamination of groundwater in Bangladesh. Arsenic contamination of water has also been observed in the
US, Australia, Asia, Europe, and South America. Inorganic arsenic above 10 ppb
level can increase the risk of lung, skin, bladder, liver, kidney, and prostate cancer.
This lecture discusses the impact of such effects on the basis of studies in Bangladesh where 60 million people are at risk. Analytical chemists can play a leading
role in the prevention of a number of illnesses by monitoring our water supplies by
ultra-trace analysis at or below ppb level, using various sophisticated techniques.
Advantages and Challenges of Applying LC-MS-MS to Regulated
Protein Bioanalysis
Hao Jiang, Bristol-Myers Squibb, Route 206 & Province Line Rd.,
Princeton, NJ 08543, Jianing Zeng, Craig A. Titsch, Linlin Luo,
Dharmesh D. Desai, Anne F. Aubry, Binodh S. DeSilva, Mark E. Arnold
Liquid chromatography-mass spectrometry (LC-MS) has been recently applied to
protein bioanalysis due to its ease of measuring multiple analytes, unique selectivity,
and significant flexibility. In this presentation, the authors discuss the advantages
and challenges of applying LC-MS in regulated protein bioanalysis, followed by
elaborating the strategy of supporting protein bioanalysis using either LC-MS or ligand-binding assays (LBA). A recent case study comparing the two assay formats in
the bioanalysis of monoclonal antibody (mAb) drugs is presented. In this regulated
toxicokinetic study, two co-administered mAb drugs in monkey serum were simultaneously quantitated with LC-MS by monitoring a pair of tryptic peptides (quantitation
peptide and confirmatory peptide) derived from different regions of each mAb, after
a simple and efficient protein pellet digestion. Stable isotopically labeled peptides
with flanking amino acids on C- and N-terminals were used as internal standards to
minimize the variability during sample processing and detection. In the assay, the
confirmatory peptides played a critical role in confirming quantitation accuracy and
the integrity of the drugs in the study samples. Meanwhile, two separate LBAs were
applied for quantitation of each mAb for the same set of samples. The equivalency of the data between LC-MS and LBA was demonstrated from cross-validations
using quality control samples and incurred study samples. The comparable bioanalytical concentrations, toxicokinetic parameters, and statistical correlations from the
both assay formats confirm that LC-MS is a reliable and complementary approach
for mAb bioanalysis.
Recent Developments in Stationary Phases for Ion Chromatography
Christopher A. Pohl, Thermo Fisher Scientific, 445 Lakeside Dr.,
Sunnyvale, CA 94085
Although ion chromatography is a relatively mature science, research into the development of new stationary phases for ion chromatography has continued at an active
pace for the more than 35 years since the original introduction of ion chromatography as an analytical technique. In this work we review the latest developments in
new ion exchange phases developed specifically for ion chromatography. We cover
a variety of different stationary phase architectures and include examples illustrating
their application to water quality analytical challenges.
Improved LC-MS Method Development for Pain Management
Thomas A. Russell, Shimadzu Scientific Instruments, 7102 Riverwood
Dr., Columbia, MD 21046
In order to maximize turnaround time in a pain management lab setting there has
been a focus on combining multiple drug panels in a short analysis time. This may
present challenges when considering isobaric pairs and fast polarity switching.
This study was undertaken to demonstrate the ability to quickly develop a rugged
method using ultra-performance liquid chromatography tandem mass spectrometry
(UHPLC-MS-MS). Method development software is used to efficiently determine the
best mobile phase and column combination for one method. This assay meets requirements for sensitivity and quantitation for all classes of drugs of abuse: barbiturates, THC metabolites, amphetamines, benzodiazepines, cocaine and metabolites,
opiates and opioids. The focus is on selectivity of all compounds, especially isobaric
pairs, while optimizing the shortest runtime possible.
Novel Electrochemical and Reflectance Photometric Techniques
for the Measurement of Trace Arsenic in Water
Abul Hussam, George Mason University, 4400 University Dr., Fairfax,
VA 22030, Douglas Mays, Jinsoo Hong, Joan Rozario
The measurement of trace arsenic in the environment at a low cost is a continuing
analytical challenge. Here, we explore some promising analytical techniques costing less than $10,000 and field deployable for large scale measurement efficiency.
Recent advances in electrochemical techniques using B-doped diamond electrode,
Au-NP modified electrode, Au-micro electrode in flow cell, and electrochemical detection of gas phase arsine appears promising. In a novel reflectance photometric
technique the formation of Agx-AsHy (x=1,2,3 y= 2,1,0) (s) complex by the reaction
of Ag+ and AsH3(g) was monitored as a function of time. The concentration of trace
arsenic was determined by a kinetic method for faster analysis.
2014 EAS Abstracts
November 2014
rators: R. A. Bartynski, E. Garfunkel, T. Gustafsson, H.D. Lee, D. Mastrogiovanni,
V. Podzorov, L. S. Wielunski, G. Liu, J. Williams, S. Dhar, P. Cohen, E. Conrad, Yi
Xu, P. Batson
Pharmaceuticals in Environment and Water Quality: The Role of
Analytical Chemistry
Hua Yao, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ
Thanks to advances in analytical chemistry, trace levels of active pharmaceutical
ingredients (APIs) have been discovered in the environment, especially in water
systems, around the globe. Pharmaceuticals in the Environment (PIE) raises public
interest and concerns among regulatory, scientific, environmental and media stakeholders. Although environmental risk assessment on APIs is now required by regulatory agencies such as the Food and Drug Administration and European Medicines
Agency prior to new drug approval, a framework is still lacking to evaluate and
monitor the potential impact on the environment once a new drug is launched. Only
a small percentage of the total of over 3000 APIs have been studied for their occurrence and fate in environment. Models have been proposed to estimate the concentrations of APIs in aquatic environment. At Bristol-Myers Squibb collective efforts
have been initiated to advance the understanding of its drug products’ impact on
the environment. Analytical chemistry plays an important role by providing analytical
tools to monitor the occurrence of APIs post drug approval. Quantitative analytical
methods for twelve APIs have been developed using solid-phase extraction and liquid chromatography with electrospray ionization source tandem mass spectrometry.
Multiple APIs are analyzed simultaneously. Stable isotopically labeled analogs or
structure analogs have been used as internal standards to improve method performance. Lower limits of quantification as low as parts per trillion are achieved.
Structural Motifs in Carbon Nanoparticle Films: Nanoparticle
Functionalization and Nanorystallization
Janice Reutt-Robey, University of Maryland, Department of Chemistry
and Biochemistry, College Park, MD 20742
We explore structural motifs that can be achieved in monolayer films of C60 and
C70, and their phenyl-butyric-acid methyl ester derivatives, at neat and chemically-modified Au (111) and Ag (111) substrates. On metallic substrates, we generate
glassy films of C60-PCBM and C70-PCBM (nanoparticle) monolayers through a
microaerosol deposition method. We then reveal how the ordering of the films is
frustrated by nanoparticle-nanoparticle interactions through UHV-STM measurements. Transition to crystalline (hcp) fullerene arrangements proves to be highly
activated and dependent on local PCBM density. Anisotropic PCBM-PCBM interactions account for the observed packing arrangements in PCBM films, which include
disordered, dimer-row phases, and hcp phases, and barriers for nanocrystallization.
Additionally, we present comparative studies of nanostructure evolution in binary
C60-ZnPc and C60-PCBM-ZnPC films. In the first case, we identify and explore
the formation mechanisms of a serpentine C60 chain phase structure that arises at
the ZnPc/Ag(111) interface. In the case of functionalized carbon nanoparticles, we
explore the vertical phase separation that results in the formation of PCBM/ZnPc/
Au(111) stacked film structures. We show how molecular Electrostatics – including
contributions from the interface dipole and from the intrinsic molecular dipoles –
provide key driving forces that lead to remarkable - and predictable - pattern formations. Implications for functionalized carbon nanoparticles in organic electronic applications are discussed. This research is supported by the NSF-Chemistry (MSN)
through Award # CHE1310380.
Withdrawn by the author.
Analysis of High and Low Dielectric Constant Oxides for
Microelectronics Applications
Robert Opila, University of Delaware, 201 DuPont Hall, Newark, DE
19716, Jonathon Church, James Krajewski, Dan Yang, Venkateswara
Pallem, Conan Weiland, David Gidley
Electronic materials are becoming compositionally and structurally more complicated. Thus, characterization of these materials is becoming more and more of a
challenge. We discuss two examples from a collaboration between Air Liquide and
the University of Delaware. In the first study, atomic layer deposition (ALD) of ZrO2
and Nb2O5 thin films has been studied using high-energy synchrotron-based X-ray
photoemission spectroscopy. Given the relatively high thickness of the layers (tens
of nanometers), the X-rays are able to effectively probe the interface of ZrO2 and
Nb2O5 layers with their respective substrates. Binding energy shifts associated with
the chemical environment at the interface of up to 1.4 eV and 1.1 eV for the ZrO2
and Nb2O5 films respectively are shown. These energy shifts suggest significant
charge transfer at the interface between the high-k oxide and Si. In addition, the
composition and chemistry of the films as a function of thickness has been determined non-destructively by converting a series of spectra taken with different incident X-ray energies (and simultaneously, different photoelectron sample depths) to
depth profiles using Bayesian analyses. In the second example, the characterization
of porosity of porous low-k dielectric films is a challenge in part due to the limited
sampling volume. The application of three non-destructive porosimetry techniques:
positron annihilation lifetime spectroscopy, ellipsometric porosimetry, and X-ray reflectivity to characterize porous low-k thin films, are examined. The advantages and
limitations of each technique, especially the sensitivity to pore interconnectivity and
its relationship to the mechanical properties, are discussed.
Understanding the Electron Stimulated Surface Reactions of
Organometallic Complexes to Enable Design of Precursors for
Electron Beam Induced Deposition
Howard Fairbrother, Johns Hopkins University, Department of
Chemistry, 3400 North Charles St., Baltimore, MD 21218, Julie Spencer,
Mike Barclay, Samantha Rosenberg, Lisa McElwee-White
Standard practice in electron beam induced deposition (EBID) is to use precursors
designed for thermal processes, such as chemical vapor deposition (CVD). However, organometallic precursors that yield pure metal deposits in CVD often create
EBID deposits with high levels of organic contamination. This contamination negatively impacts the deposit’s properties (e.g., increasing resistivity, decreasing catalytic activity) and severely limits the range of potential applications for metal-containing EBID nanostructures. To provide the information needed for the rational design
of precursors specifically for EBID, we have employed an ultra-high vacuum (UHV)
surface science approach to elucidate the elementary reactions of organometallic
precursors during EBID. Our results have shown that the initial electron induced
depositions of surface bound organometallic precursors proceed through desorption
of one or more of the ligands present in the parent compound. In specific cases this
deposition step has been shown to proceed via dissociative electron attachment
(DEA), involving low energy secondary electrons generated by the interaction of the
primary beam with the substrate. However, electron beam processing of the surface
bound species produced in the initial deposition event usually causes decomposition of the residual ligands, creating non-volatile fragments. This process is believed
to be responsible for the bulk of the organic contaminants typically observed in
EBID nanostructures. Our results also suggest that simple ligands architectures that
involve a combination of CO and halogen atoms as the exclusive ligands may yield
deposits with significantly higher metal contents compared to the values that can
currently be attained with CVD precursors.
Interface Analysis of Nano-scale Materials
Leonard Feldman, Rutgers Institute for Advanced Materials, Devices
and Nanotechnology, 607 Taylor Rd., Piscataway, NJ 08854
Modern material research has provided the means of creating structures controlled
at the atomic scale. Examples include the formation of hetero-structures grown with
atomic precision, monolayer (graphene and graphene-like) films, nanostructures
with designed electronic properties, shaped nano-plasmonic materials and new organic structures employing the richness of organic chemistry. The current forefront
of such nano-materials research includes the creation and control of new materials
for energy, bio/medical and electronics applications. The performance of these diverse materials systems is invariably determined by their fabrication and their interfacial structure. Interfaces are the critical component and least understood aspect
of such materials-based structures. Interfacial analysis is described in the context
of a number of projects underway at the Rutgers Institute for Advanced Materials,
Devices and Nanotechnology. These include: 1) self-assembled monolayers on organic single crystals resulting in enhanced surface mobility; 2) interfacial analysis of
complex oxide hetero-structures to elucidate the enhanced two-dimensional electron mobility; 3) characterization of the semiconductor-dielectric interface in SiC/
SiO2 for energy efficient power transmission. We describe two new analytical tools:
the Zeiss-Orion 0.25nm He ion beam and the Rutgers-NION Scanning Transmission Electron Microscope with ~ 12 meV electron energy loss resolution. Collabo-
Reducing Solvent Consumption in High-Performance Liquid
Chromatography during Column Re-Equilibration after Gradient
Michael R. Fletcher, Drexel University, 3141 Chestnut St., Philadelphia,
PA 19104, Erin J. Ennis, Donna M. Blackney, Catherine A. Kita, Joe P.
In traditional high-performance liquid chromatography (HPLC), column re-equilibration between runs is necessary to prepare a column for subsequent experiments
when using gradient elution. This process replaces the final mobile phase in the column, interstitially (between the particles), intra-partically (within the pores), and interfacially with the initial mobile phase. Column re-equilibration, which is presumably
limited by the rate of diffusion within the pores, traditionally consumes a large quantity of mobile phase due to the high flow rates commonly employed. In the interest
of green chemistry, an alternative method of column re-equilibration after gradient
elution is presented. This work aims to minimize solvent consumption by substantially reducing the flow rate during column re-equilibration, thus allowing more time
2014 EAS Abstracts
November 2014
for new mobile phase to diffuse into all regions of the column while consuming less
solvent. A variety of column sizes and composition are studied to optimize equilibration time, flow rate, and solvent consumption.
Determination of Common Counter and Impurity Anions in
Pharmaceutical Using a High-Pressure Modular Capillary Ion
Chromatography System (HPIC) with Suppressed Conductivity
(CD) and Charge (QD) Detection
Hua Yang, Thermo Fisher Scientific, 1214 Oakmead Pkwy., Sunnyvale,
CA 94085, Linda Lopez
Ion analysis is important for pharmaceutical industry because many active pharmaceutical ingredients (APIs) exist in their salt form. Pharmaceutical products are
strictly regulated by the United State Food and Drug Administration (FDA) and other regulatory agencies, and must be tested for composition to verify their identity,
strength, quality and purity. Recently, identification and quantification of ions in early
stage drug development has gained increasing attention, because the APIs maybe
contaminated with different counter ions from synthesis steps, and because selecting the counter ion to enhance APIs’ solubility and stability is becoming a key
step in formulation development. Since many of the ions are non-chromophoric, Ion
chromatography (IC) with suppressed conductivity detector (CD) is an established
method used for ion determination. The recently introduced high-pressure capable
capillary IC systems (HPIC) combined with 4-Вµm particle ion-exchange columns
have improved separation efficiency and significantly reduced the eluent consumption to 5.2 L/year which saves money and time. Use of a newly invented charge
detector (QD) combined with CD permits peak purity assessment to further simplify
identification and confirmation of ions. This study demonstrates the identification
and quantification of 22 commonly found anions in pharmaceuticals in a single run
using a high-pressure capillary IC system (HPIC) with 4-µm particle ion –exchange
column, and CD-QD dual detectors.
The Importance of Tolerance Limits in UHPLC Method Modelling
for Robustness Evaluation
Imre L. Molnár, Molnár-Institute, Schneeglöckchenstr.47, Berlin, 10407
Germany, Hans-JГјrgen Rieger
From over 100,000 diseases only 20,000 can be treated with drugs, as the development costs of the drugs are too high to cover the investments because the numbers
of patients are too small. This can only be changed, if ultr high-performance liquid
chromatography (UHPLC) method development becomes faster and more efficient
and allows to simplify also the complexity of regulatory interactions. By modelling
the separation, the development process, which is based on solid science, can be
dramatically shortened. Multifactorial influences can be modeled in seconds and the
best separation with the best column can be selected in a revolutionary way, much
faster than in the past. Tolerance limits will be varied and the separation quality
will be unically visualized. The presentation shows how to develop UHPLC methods with high efficiency and excellent robustness, based on modern computational
technologies and reliable chromatographic rules. Case studies support the presentation in an effective way demonstrating a breakthrough in modern UHPLC method
The Use of Eluent Generation in Ion Chromatography
Peter Bodsky, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale, CA
94085, John Madden
In ion analysis, mobile phases and regenerant solutions are typically prepared using
acidic (ie: HCl) or basic (ie: NaOH) stock solutions. Manual preparation of these
reagents requires the need to handle acids or bases and thus a skilled chemist.
Manual preparation is also labor intensive and can often lead to errors if the reagents used to make the solutions and the inevitable dilutions are not made precisely. Reagent-free ion chromatography (RFIC) use electrolytic technologies to
generate eluents from deionized water. Using RFIC, laboratories spend less time
on equilibration, calibration, method verification, troubleshooting, and consistency
checks because the systems minimize unintentional variations in the preparation of
eluents and regenerants. In this presentation, we review how RFIC or eluent generation works and provide examples demonstrating that it can be superior to manually
prepared mobile phases and regenerats.
Recent Compendial Activity for Dissolution Testing when Gelatin
Crosslinking is an Issue
Gregory P. Martin, Complectors Consulting, 804 Bauman Cir., Pottstown,
PA 19465, Vivian A. Gray
Crosslinking of gelatin in pharmaceutical dosage forms is known to cause dissolution slowing, and the USP allows use of enzymes to mitigate this. There are many
issues with the two tier testing allowed in United States Pharmacopeia (USP) General Chapter Dissolution <711> where enzyme is added to media when dissolution
failures are observed on stability with capsules shell/gelatin products. New proposed revisions to Dissolution General Chapter <711> and new General Chapter
on Capsules <1094> are discussed, including when enzyme use is appropriate,
strategies when enzyme activity is adversely affected by the dissolution medium
and consideration of use of additional enzymes in the pH region where pepsin and
pancreatin have little enzyme activity.
What Performance can we Expect from Microfluidic LC-MS
Martin Gilar, Waters, 34 Maple St., Milford, MA 01757, Thomas S.
McDonald, Gregory Roman, Jay S. Johnson, James P. Murphy
When dealing with sample limited applications or when striving for lower limits of
detection, microfluidic liquid chromatography (LC) is a promising alternative to conventional scale chromatography. In this presentation, we discuss the implications of
dispersion in microfluidic chromatography, both pre-column (sample injection, sample trapping) and post-column (connection to the detector), and illustrate how these
affect the results of an analysis. We illustrate how the holistic design of a microfluidic
LC-mass spectrometry (MS) system can minimize many of these difficulties, providing the analytical scientist with robust methods and a high level confidence in data
quality. Theoretically predicted and experimental results are shown to demonstrate
the performance of a well optimized microfluidic LC system. We discuss the performance of the recently introduced microfluidic systems (0.15 mm ID) and compare its
performance to industry standard 2.1-mm I.D. Ultra-performance LC columns. We
show that microfluidic LC does not have to be difficult to use, and ease of use does
not have to be traded for an increased sensitivity.
Characterizing the Dynamic Formation of Disease Protein
Aggregates Using Dark-State Exchange Saturation Transfer (DEST)
Nicolas L. Fawzi, Brown University, Department of Molecular
Pharmacology, Physiology, and Biotechnology, 70 Ship St., Rm. 119,
Providence, RI 02903
A common feature of numerous neurodegenerative diseases is the formation of
neurotoxic inclusions of proteins. However, the structures along the protein assembly pathway, made up of large, disordered, and transient aggregates, are often
invisible to traditional atomic resolution techniques. Here we describe techniques
in solution nuclear magnetic resonance (NMR) spectroscopy to characterize the
aggregation intermediates of proteins associated with neurodegenerative diseases.
Alzheimer’s disease is characterized by the accumulation of amyloid β peptides.
Recently, the 43-amino-acid peptide AОІ43 has garnered attention as a highly aggregation-prone, neurotoxic species shown to deposit sooner and with higher likelihood than its shorter variants. We present the biophysical characterization of AОІ43
structure and dynamics in the monomeric and protofibrillar states to determine if
and why this peptide has enhanced aggregation propensity. Slower dynamics and
chemical shift differences are present across residues 30-43 in the monomeric
state of AОІ43 as compared to AОІ42. Dark-state exchange saturation transfer NMR
demonstrates that, compared to AОІ42, AОІ43 is less often directly-bound to the surface of protofibrils, but the C-terminus directly binds more frequently. Dynamics in
protofibril-bound states are slowed at the C-terminus. Our results suggest that faster
aggregation of AОІ43 is due to slowed dynamics and the formation of aggregate-favoring structure in the C-terminal region in both the monomeric and protofibril-bound
states of AОІ43. Recent extensions of the DEST method to methyl sidechains in
AОІ and application to aggregation of prion-like domains of RNA-binding proteins in
amytrophic lateral sclerosis are also reviewed.
Chromatographic Separation of Complex Mixtures of Closely
Related Species
Erik L. Regalado, Merck, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ
07065, Christopher J. Welch
In this presentation we describe our recent investigations into the separation of
complex mixtures of constitutional isomers, stereoisomers and other closely-related
species using a variety of single- and multi-dimensional chromatography approaches. Strategies and approaches for application of chiral or achiral stationary phases,
mass spectrometry deconvolution and the use of tandem supercritical fluid chromatography, offline two-dimensional (2-D) liquid chromatography and comprehensive
2-D liquid chromatography are discussed, and several examples of the practical
use of multi-dimensional chromatography methods for resolution of multicomponent
mixtures are presented.
2014 EAS Abstracts
November 2014
ylated at multiple serine residues in-vivo. A growing body of evidence supports the
hypothesis that phosphorylation modulates the binding of transcription factors that
are required for mRNA biogenesis, yet little is known about how phosphorylation
affects CTD structure or dynamics. Here we present the combined application of
C-direct detect NMR and mass spectrometry to address the hypothesis that serine
phosphorylation fundamentally alters the structure and dynamics of the CTD.
Structure, Dynamics, and Function of Opa60: A Neisserial Гџ-Barrel
Membrane Protein that Mediates Host Phagocytosis
Linda Columbus, University of Virginia, Department of Chemistry,
McCormick Rd., PO Box 400319, Charlottesville, VA 22904, Alison K.
Criss, Peter M. Kasson
The family of Opa proteins from Neisseria gonorrhoeae and N. meningitidis are
eight-stranded ОІ-barrel proteins that induce phagocytosis of the bacterium by engaging three different host receptors: carcinoembryonic antigen cellular adhesion
molecules (CEACAM), heparansulfate proteoglycans (HSPG), or integrins via
HSPG and fibronectin or vitronectin. The receptor engaged depends on the sequence of two hypervariable (HV) regions in two extracellular loops, which differ between isolates. There are hundreds of HV sequences identified; however, multiple
sequence alignment of the HV loops does not reveal specificity motifs among the
family of Opa proteins due to the extreme variability in the amino acid sequences.
Towards understanding the molecular recognition required to gain entry into human
cells, the structure of Opa60, which binds CEACAM1, 3, 5 and 6 was determined.
Structure determination of membrane proteins is challenging and even more so
for proteins that have large portions of both soluble and membrane-embedded regions. Thus, Opa proteins presented some methodological obstacles in both solution nuclear magnetic resonance (NMR) assignments and structure calculation and
refinement. Strategies for the assignment of the protein included trypsin cleavage,
peptide synthesis, and assignment at various temperatures. To overcome structure refinement obstacles, a hybrid method that used the restraints determined with
NMR spectroscopy in detergent micelles in conjunction with molecular dynamics
(MD) simulations in a lipid bilayer. The resulting Opa60 structure, coupled with
in-vivo and in-vitro Opa reconstituted liposome assays, has facilitated investigations
of the molecular determinants of Opa-receptor interactions.
Studying Networks of Weak Protein Interactions by NMR
Assen Marintchev, Boston University School of Medicine, Department of
Physiology and Biophysics, Boston, MA 02118, Nabanita Nag, Jielin Yu,
Devika Nadkarni, Katherine A. Edmonds, Boriana Marintcheva
Macromolecular complexes are built from networks of interactions, many of which
are weak, allowing dynamic remodeling. Such weak, and often transient, interactions are notoriously difficult to study because they usually cannot be observed in
isolation, when the respective proteins are not part of the large complex. Nuclear magnetic resonance (NMR) is uniquely suited to provide structural information
about such weak interactions because binding is observed at equilibrium, at high
protein concentrations. Furthermore, transient interactions are readily observed,
including those involving intrinsically disordered peptides. The translation initiation
complexes represent an example of macromolecular complexes, whose assembly,
remodeling and regulation involve a number of weak interactions. Two eukaryotic
translation initiation factors (eIFs), eIF1A and eIF5B are at the center of an intricate
network of protein-protein and protein-RNA interactions. We used a combination of
NMR, site-directed mutagenesis, and deletion analysis to identify both intra- and
intermolecular interactions involving eIF1A and eIF5B and to characterize the respective binding interfaces. Further, we analyze these interfaces in the context of
the recently determined positions of these proteins in ribosomal complexes. Some
of these interactions appear to be enabled by ribosome binding, while others are
hindered. For example, ribosome binding is expected to disrupt an intramolecular
interaction interface within eIF1A, exposing a binding site for eIF5B. Taken together,
the observed overlaps in intra- and intermolecular contact surfaces offer a mechanism for coordination of the interactions of eIF1A and eIF5B with each other, the
ribosome and with other eIFs.
Characterization of the Active Site of Vanadium Chloro-peroxidase
by 51V Solid-State NMR Spectroscopy
Rupal Gupta, University of Delaware, Department of Chemistry, Newark,
DE 19716, Guangjin Hou, Tatyana Polenova
Vanadium-dependent haloperoxidases (VHPOs) are enzymes found in seaweeds,
fungi, and lichens that catalyze a two-electron oxidation of halides. Understanding
the electronic structure and coordination geometry of the vanadium site remains
a challenge because vanadium is in a “spectroscopically silent” diamagnetic V(V)
state throughout the catalytic cycle, precluding its characterization by EPR and UVvis. We have previously demonstrated that 51V Magic Angle Spinning (MAS) NMR
spectroscopy provides a detailed glimpse of the geometry and electronic structure
at the vanadium site in VHPOs.[1-2] 51V MAS NMR spectra are dominated by the
quadrupolar and chemical shift anisotropy interactions, which are highly sensitive
probes of the active site environment and report on protonation state and coordination geometry. In this work, we present a 51V MAS NMR investigation of the resting
state of vanadium chloroperoxidase (VCPO), a 67-kDa member of this family, and of
its mutant (P395D/L241V/T343A) showing 100-fold increased brominating activity,
as a function of pH. We demonstrate that, with fast MAS (40 kHz), the required sample amounts and the experiment time are drastically reduced, permitting acquisition
of high-quality spectra. This work is supported by the US-Israel Binational Science
Foundation (Grant 2011077).
My Contributions to NIR Spectroscopy
Mark Westerhaus, FOSS, 2379 Corinna Ct., State College, PA 16803
Since 1978, I have worked with near-infrared (NIR) spectra developing new ways
to make NIR predictions more reliable and accurate. Instrument standardization removed some of the differences between instruments. Repeatability files made calibrations less sensitive to variation not included in the calibration dataset. Modified
partial least squares (PLS) improved the calibration accuracy for some constituents.
Local calibrations provided accurate predictions when the calibration set was too
variable for linear models to handle. Global H and Neighborhood H values provided
confidence that a new spectrum looked like the calibration spectra. Good product
definition detected non-conforming spectra in a process environment. Many recommendations were also developed over the years. The history and current status of
these developments are described.
Classification and Interpretation of NIR Data: Recursive Weighted
Partial Least Squares Discriminant Analysis (rPLS-DA)
SГёren B. Engelsen, University of Copenhagen, Rolighedsvej 30,
Frederiksberg, 1958 Denmark
Near-infrared (NIR) spectra contain holographic information about the samples being investigated. The same information is repeated and the spectral variables are
thus highly correlated (collinear data structure). For this type of data, multivariate
projection methods are very suitable and indeed the combination of multivariate
data analysis and NIR spectroscopy is the working principle in high throughput quality control (QC) and process analytical technology (PAT). Despite the high redundancy in NIR spectra, the multivariate methods can often be improved by variable
selection such as for example interval partial least squares (iPLS). The primary
reason for the improvements is the reduced number of interferences in the reduced
set of variables. Perhaps even more important than improved and parsimonious
models is the improved interpretation. This paper introduces the recently developed
recursive weighted PLS algorithm (rPLS) which is related to jack-knifing, but instead
of iteratively eliminating variables, rPLS iteratively uses the regression coefficients
to magnify important variables and thus down-weight less important variables. The
rPLS model has the advantage that it will converge to a limited number of variables
(good for interpretation) but it will exhibit optimal performance before normally including co-linear neighbor variables. This talk demonstrates the performance for
regression (rPLS) and classification (rPLS-DA) on selected NIR data sets.
[1] N. Pooransingh-Margolis, R. Renirie, Z. Hasan, R. Wever, A. J. Vega, and T.
Polenova, J. Am. Chem. Soc., 2006, 128, 5190-5208.
[2] T. Polenova, N. Pooransingh-Margolis, D. Rehder, R. Renirie, and R. Wever,
In “Vanadium, The Versatile Metal”, Eds. Ken Kustin, Debbie Crans, and Jose
Costa-Pessoa, Oxford University Press, ACS Publications, 2007, pp. 178-203.
C-Detect NMR and Mass Spectrometry Applied to Study the
Effects of Post-Translational Modifications on Disordered Proteins
Eric B. Gibbs, The Pennsylvania State University, 104 Chemistry
Building, University Park, PA 16802, Debashish Sahu, Scott A. Showalter
Recent advances in spectrometer and cryogenic probe technology have resulted in
the re-emergence of 13C-direct detection spectroscopy as a tool for the study of proteins in solution. For intrinsically disordered proteins (IDPs), a highly dynamic class
of biomolecules that lack temporally stable secondary/tertiary structure, 13C-direct
detect NMR has become indispensable for the characterization of structural and
dynamic properties at atomic resolution. Our biomolecular “tool-kit” built from 13C′15
N and 13C′-13Cα heteronuclear correlation spectroscopy, has provided enhanced
peak dispersion and line width narrowing. This along with the use of multi-dimensional spectra and non-uniform sampling has greatly improved the spectral resolution achievable for these systems while also reducing acquisition times. With
these techniques we can now address fundamental questions about IDP structure
function relationships. For example, we are interested in whether post-translational
modifications like serine phosphorylation, enact long range structural changes or
merely influence structure locally. The carboxy-terminal domain of RNA polymerase
II (CTD), an essential regulator of Eukaryotic gene expression, becomes phosphor13
2014 EAS Abstracts
November 2014
40) fibrils. Remarkably, AОІ(1-40) is not compatible with the structure of AОІ(1-42)
fibril as seeding the AОІ(1-42) fibrils did not promote conversion of monomeric AОІ(140) into fibrils via cross-replication. The results provide crucial structural insight into
how Aβ(1-42)-selective amyloid propagation is introduced in early-stage Alzheimer’s without involving Aβ(1-40). In the second topic, we present characterization of
graphene-based materials derived from graphene/graphite oxide by various SSNMR methods. We examine structural changes including graphene-edge modifications through reduction of graphite/graphene oxide by hydrazine (H2-N-N-H2).[1] We
present the structural features of other reduced graphene/graphite oxide systems
obtained via milder modifications by azide (N3-)[2] or time-controlled thermal reduction.[3] Other topics on recent development of solid-state NMR methods are covered.
Two Major Advanced Algorithms in NIRS: Cloning Instruments and
Local Calibration
Pierre Dardenne, Walloon Agricultural Research Centre, Chaussee de
Namur, Gembloux, 5030 Belgium
This lecture presents an overview of two major algorithms implemented into the
package called Winisi: clone and local. Already in the middle of the 80’s, Prof.
John Shenk and Mark Westerhaus proposed in their package these methods to
increase the performances of the NIR instruments networks. Cloning (matching or
standardization) consists of correcting the spectra from “slave” instruments to make
them as close as possible to the spectra of a “master” instrument. The method has
been applied successfully for 30 years inside numerous networks. Nowadays the
standardization between instruments of the same brands or series is adjusted by
the manufacturers and the need of correction becomes less important. Anyway, the
method remains a very practical tool to transfer data bases between instruments of
different brands (i.e., dispersive to Fourier transform or reverse). The second algorithm concerns the concept of “local” calibration. It is well known that including large
variations in a common data set improves the robustness but can affect negatively
the prediction performances. To analyze one sample, the “Local” method selects N
close samples from a big data set based on the highest correlations between the
spectrum to be processed and the library. Trials with real large data sets show that
“Local” outperforms the others methods as classical partial least squares, artificial
neural network and even support vector machines. Mark Westerhaus is the author
of the computer codes to run these algorithms included in the Foss WinISI package
and his contributions are recognized as major impacts to the development of the
NIR technology.
[1] Park, S. et al. Nature Communications 2012, 3, 638.
[2] Eigler, S. et al. Nanoscale 2013, 5, 12136.
[3] Wen, Y. Nature Communications 2014, 5, 5033.
Structural and Dynamic Studies of Protein Assemblies by SolidState NMR Spectroscopy
Christopher P. Jaroniec, Ohio State University, 100 West 18th Ave.,
Columbus, OH 43221
In this talk I discuss our recent structural and dynamic studies of large protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance (NMR)
techniques. The main focus of the talk is on our progress toward providing an atomic
level understanding of the phenomena of amyloid strains and cross-seeding barriers
for Y145Stop prion protein variants. Our recent results related to the characterization of flexible histone N-terminal tail domains in the context of large nucleosome
arrays under experimental conditions corresponding to extended, folded and highly
condensed chromatin are also presented.
Application of Non-Linear Chemometric Models to NIR Data
Lars NГёrgaard, FOSS, Foss AllГ© 1, HillerГёd, DK-3400, Denmark, Morten
With Pedersen, Pedro HГёjen-SГёrensen
Linear partial least squares regression modeling is extensively used for near-infrared (NIR) spectroscopic calibration modeling despite the fact that many NIR
spectroscopic data sets often exhibit different levels of non-linearities between the
spectra and the property of interest. In this paper the background for the application of artificial neural networks (ANN) from an industrial research and development
perspective is provided including examples of the benefit by this approach. The talk
also touches upon alternative machine learning methods like the Gaussian process
regression that has recently been introduced to the chemometric society as an alternative to both linear and non-linear modeling methods like partial least squares
and ANN.
Solid-State NMR of Protein Assemblies: New Methods and New
Robert Tycko, National Institutes of Health, Bldg. 5, Rm. 112, Bethesda,
MD 20892, Jun-Xia Lu, Marvin Bayro, Alexey Potapov, Eric Moore,
Dylan Murray, Kent Thurber, Wai-Ming Yau
Our lab works on solid-state nuclear magnetic resonance (NMR) methods and on
the application of these methods to structural problems in biological systems. We
are particularly interested in non-crystalline protein assemblies, including amyloid
fibrils and viral capsids, where solid-state NMR can provide truly unique information.
In the area of Alzheimer’s disease (AD), we have recently reported the first detailed
structural studies of amyloid-beta fibrils that develop by spontaneous self-assembly
in brain tissue of AD patients, including the first detailed molecular structural model
(Lu et al., Cell 2013; PDB 2M4J). I discuss this work and describe ongoing efforts
to determine whether patient-to-patient variations in fibril structures, as revealed by
solid-state NMR, correlate with variations in clinical history and neuropathology. In
the area of AIDS research, we are investigating the molecular structures of tubular
and non-tubular assemblies formed by the HIV-1 capsid (CA) protein. I describe how
solid-state NMR data can be used to identify structurally ordered, disordered, and
dynamic segments of CA within CA tubes and to characterize conformational changes that accompany CA self-assembly. I also describe methodological developments
that enable progress in these areas, including the use of ultra-low-temperature dynamic nuclear polarization in studies of transient intermediates in the amyloid-beta
self-assembly process.
Solid-State NMR of Viral Fusion Proteins
David Weliky, Michigan State University, Department of Chemistry, 578
S. Shaw Lane, East Lansing, MI 48824
Solid-state nuclear magnetic resonance (SSNMR) has been applied to probe the
structures and membrane locations of domains of the HIV gp41 and the influenza virus HA2 membrane fusion proteins. Advantages of SSNMR include: 1) use of membrane bilayers without detergent and with lipid and cholesterol composition similar
to that of host cells; and 2) preparation of samples under conditions very similar to
those used for functional vesicle fusion assays. The N-terminal ~25-residue HA2
fusion peptide (IFP) domain has helix-turn-helix structure and in detergent there are
reports of: 1) an open interhelical topology with a hydrophobic interhelical pocket
and inverted V membrane insertion; and 2) a closed topology with tightly-packed
antiparallel helices and membrane contact with a hydrophobic protein surface. SSNMR measurements in membranes lacking cholesterol show that the closed structure
is predominant. Both the IFP and the corresponding N-terminal gp41 fusion peptide
(HFP) have predominant oligomeric beta structure in membranes with 30 mole%
cholesterol which is typical for viral and host cell membranes. SSNMR measurements for HFP show that there is antiparallel arrangement of adjacent stands with
a broad distribution of antiparallel registries. The central regions of the antiparallel
sheets are deeply inserted in the membrane and contact the methyl termini of the
lipid acyl chains and cholesterol. This antiparallel distribution is also observed for a
much larger segment of gp41 that includes the fusion peptide. A very different registry distribution is detected for the non-functional V2E mutant and suggests that the
hydrophobicity of the antiparallel sheet is correlated to membrane fusion.
Finding Homes for all of the Orphan Assays
Brian Buckley, Environmental and Occupational Health Sciences
Institute (EOHSI), Rutgers University, 170 Frelinghuysen Rd.,
Piscataway, NJ 08854, Andreia Valente, Ill Yang, Min Liu, Kyle Buckley,
Frances Acevedo Mariani, Stephanie Marco, Elizabeth McCandlish
Just as there are orphan drugs looking for homes to be either tested or manufactured, there are orphan assays looking for separation techniques that have not been
made available because there is not enough interest to make them commercially
viable. Most of the separation applications; solid phases, clean-ups, etc., are driven
by the large commercial needs of the various industries. There are a handful of solid
phases for high-performance liquid chromatography and essentially two phases for
ion chromatography offered commercially with the belief that all your needs can be
met with these products and the right gradient. When an orphan application comes
around, e.g., potentially toxic small molecule adducts to chemotherapeutic agents
or pesticides separated from small quantities of brain, there is no application guide
to visit and no column recommended by a manufacturer. The chromatographer is
left to their own ingenuity, trying to make the column fit the application. This presentation focuses on the many challenges faced by methods development analysts,
challenged by assays that others have not explored. This is because the application
is new or there is not enough interest in supporting the columns to make the separation commercially viable. In addition to the aforementioned examples other sepa-
Structural Examination of Amyloid Proteins and Graphene-Based
Nanomaterials by Solid-State NMR
Yoshitaka Ishii, University of Illinois-Chicago, 845 W Taylor St.,
Department of Chemistry, SES Rm. 4500, Chicago, IL 60607
In this study, we report two separate topics on solid-state nuclear magnetic resonance (SSNMR) methodologies and its applications to amyloid proteins and
graphene-based carbon nano-materials. In the first topic, we discuss structural features of amyloid fibrils and spherical intermediates of 42-residue Alzheimer’s amyloid-β (Aβ(1-42)). In particular, we present the first high-resolution structure model
of AОІ(1-42) amyloid fibril. The model based on SSNMR data displays structural features, which are critically different from reported structures of less pathogenic AОІ(1-
2014 EAS Abstracts
November 2014
ration challenges are discussed including bile acids without buffer and microbiome
profiling of isoprostanes.
surfaces is demonstrated, and its potential use with field portable and clinical mass
spectrometers is discussed along with mechanistic insights.
Agroecosystem Analysis Using Chromatographic Methods
Jeffrey S. Buyer, United States Department of Agriculture, Bldg. 001,
Rm. 124, BARC-West, Beltsville, MD 20705
The agroecosystem is a complex community of organisms in a heterogeneous environment which is altered periodically by agricultural management. Soil microbial
communities, which carry out biochemical functions essential to ecosystem sustainability, are the most diverse and complex biological assemblages known. These
communities are poorly understood, but chemical, biochemical, and genomic methods are revealing their structure and function. In this talk we discuss three chromatographic methods used to analyze different components of the agroecosystem
that are relevant to microbial community structure and function. First, phospholipid
fatty acid analysis by gas chromatography (GC) and GC-mass spectrometry (MS)
is used to measure soil microbial biomass and broad-scale microbial community
structure. Second, the chemical composition of soil organic matter is analyzed by
pyrolysis-GC-MS. Third, microbial metabolites extracted from agricultural samples
are analyzed by derivatization and GC-MS. These analyses illustrate just a few of
the many chromatographic methods useful in agricultural and ecosystem analysis.
[1] Trimpin, S.; Inutan, E. D.; Herath, T. N.; McEwen, C. N. Mol. Cell. Proteomics
2010, 9, 362.
[2] McEwen, C. N.; Pagnotti, V. S.; Inutan, E. D.; Trimpin, S. Anal. Chem. 2010, 82,
[3] Trimpin, S.; Inutan, E. D. J. Am. Soc. Mass Spectrom. 2013, 24, 722.
From Laser Ablation and Electrospray Fundamentals to Ambient
Ionization by LAESI
Akos Vertes, George Washington University, Department of Chemistry,
Washington DC 20052, Tarek R. Mansour, Sylwia A. Stopka, Hang Li,
Bindesh Shrestha
Mass spectrometry inherently relies on converting neutral molecules in the sample
into ions. In recent years, there has been a proliferation of ionization techniques to
expand the variety of sample types and sampling conditions to include, for example:
single cells and ambient ionization, respectively. Many of the new ionization techniques rely on laser-matter interactions and electrosprays produced from liquids.
Both of these phenomena have been extensively studied from the perspective of
fundamental processes under idealized conditions. This presentation describes the
path from the fundamental understanding of mid-infrared laser ablation of samples
with high water content to the microsampling of biological cells and tissues, from describing the nanophotonic interactions between a laser pulse and silicon nanopost
arrays (NAPA) to ultratrace analysis of adsorbates, and from the disintegration of
an electrified liquid meniscus at the end of an electrified capillary to the electrospray
ionization of a laser ablation plume. Recent applications of the high-throughput ambient ionization method, laser ablation electrospray ionization (LAESI) are demonstrated to biological tissue imaging and to metabolic profiling of lipid production in
microalgae. The combination of LAESI-mass spectrometry with stable isotope labeling pulse-chase experiments sheds a new light on the dynamics of the metabolic
network in these microorganisms. The ultratrace analysis capability of NAPA opens
the door for the metabolic analysis of single human cells.
Deciphering 4-Billion Year Old Meteorite Chemistry through Liquid
Chromatography-Mass Spectrometry
Michael P. Callahan, National Aeronautics and Space Administration
Goddard Space Flight Center, 8800 Greenbelt Rd., Greenbelt, MD
Laboratory analyses of meteorites can provide a window into the extraterrestrial
organic chemistry that took place during the formation of the Solar System. The
detection of numerous biologically-relevant organic compounds in carbonaceous
chondrites, a class of chondritic meteorites, has led to the assertion that meteorites
delivered important organic compounds for the origin of life on early Earth. I present
recent investigations of amino acids and nucleobases in meteorites and discuss
potential formation pathways for these compounds, their significance for the origin
of life, the challenges associated with analyzing meteorites, and the application of
emerging analytical technologies (such as liquid chromatography coupled to Orbitrap mass spectrometry) in the field of meteoritics.
Mass Spectrometry: A Tool for Studying Metabolomics
Ann M. O’Brien, DuPont, 200 Powder Mill Rd., Wilmington, DE 19803
Metabolomics is the systematic study of the unique chemical fingerprints that result from specific cellular processes. Metabolomics studies can be targeted and
non-targeted. In a targeted approach the metabolites are known and experiments
are conducted to measure the flux of the metabolites as a function of changing cellular processes. In an untargeted approach the metabolites are unknown and must be
identified. Mass spectrometry is a valuable tool for characterizing small molecules
with high sensitivity. However, because metabolites vary greatly in chemical nature
a full metabolomics profile cannot be accomplished by applying only one analytical
technique alone. This presentation aims to discuss development, validation and implementation of a suite of mass spectrometric and separation methods in order to
have a comprehensive strategy to characterize the cellular metabolites. Additionally,
this presentation addresses the importance of robust, validated sample preparation
protocols in support of proper interpretation of data in the context of the biological
Utilizing a Semi-Automated Sample Preparation Workflow to
Achieve Accurate, Precise, Rugged and Robust Analytical Assay
Methods for Pharmaceutical Solid Oral Dosage Forms
Khanh Ha, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ
Sample extraction of pharmaceutical solid oral dosage forms is a critical step of any
assay method. An accurate, precise, and robust data set typically include a well
prepare standard solution and a sample preparation that has a robust extraction
process. The overall sample preparation process can be challenging and very time
consuming. Traditionally, manual weighing, agitation, such as shaking and/or sonication are used to deliver a known accurate amount of material, and facilitate dispersion / dissolution of the solid dosage form over a given period of time. In addition,
a well-developed sample preparation method needs to be rugged and robust with
short sample preparation times to ensure accurate and precise method execution
by analysts with varying analytical experiences at different facilities over the life
of the pharmaceutical product. This presentation discusses the evaluation of an
automated weighting system, and novel sample extraction apparatus that employs
homogenizing multiple samples of solid oral dosage forms in parallel that significantly reduces the sample extraction time and increases sample throughput. This
semi-automated sample workflow achieves faster sample turnaround time, resources saving while ensuring extraction ruggedness and robustness for the analysis of
pharmaceutical solid dosage forms.
Biological Applications of FTICR Imaging Mass Spectrometry:
Overcoming the Analytical Challenges of Direct Tissue Analysis
Jeffrey Spraggins, Vanderbilt University, Rm. 9160, 465 21st Ave. South,
Nashville, TN 37232, Raf Van de Plas, Richard Caprioli
While imaging mass spectrometry (MS) development has traditionally focused on
spatial resolution and throughput, mass resolution and accuracy are becoming important new focal points. Driving factors include the need to identify spatially resolved ions directly from tissue and the difficulties introduced by nominally isobaric
species. Low-resolution instrumentation can produce ion images that are in fact
merged images of several species that fall under a given peak. Not only can this
make ion identification extremely difficult, in many cases biologically relevant information is lost. Matrix-assisted laser desporption/ionization Fourier transform ion
cyclotron resonance (MALDI FTICR) provides the unique performance capabilities
to overcome these challenges while maintaining high-throughput, sensitivity and
dynamic range. In this work, a series of case studies that demonstrate the analytical
power of MALDI FTICR imaging mass spectrometry (IMS) for lipid, peptide and protein analysis are highlighted. All imaging experiments were performed using a 15T
solariX MALDI FTICR MS (Bruker Daltonics, Billerica, MA) equipped with an Apollo
II dual ion source and 355 nm solid-state laser. Images were acquired with spatial
resolutions ranging from 10-150 Вµm and resolving powers up to 1,000,000. Initial
results have demonstrated the necessity for high performance molecular imaging
to overcome the challenges of biological complexity and structural identification.
Matrix-Assisted Ionization: A Simple Low Energy Ionization
Approach for Volatile and Nonvolatile Compounds
Charles N. McEwen, University of the Sciences, 600 South 43rd St.,
Philadelphia, PA 19104, Khoa Hoang, Milan Pophristic
The initial finding that laser ablation of a small molecule matrix at atmospheric pressure (AP) produces ESI-like charge states of incorporated analyte[1] was later shown
not to require the laser.[2] Ionization occurs simply by passing the matrix:analyte
sample through a heated inlet tube linking AP and the vacuum of a mass analyzer.
Trimpin, et al.[3] later demonstrated that by selecting the proper matrix, ionization
occurs spontaneously when the matrix:analyte sample is simply exposed to the
vacuum inherent with any mass spectrometer, even without added heat. Thus, matrix:analyte preparations, identical to those used in matrix-assisted laser desporption-ionization, can be introduced to a mass spectrometer through any AP inlet to
produce ions regardless of analyte volatility. An ion source is not necessary. In this
presentation, the utility of this simple and highly sensitive matrix assisted ionization method for high throughput analysis of peptides, proteins, small molecules and
2014 EAS Abstracts
November 2014
with a large number of bacterial and yeast species (e.g., >20) has not been published. Current clinical identification systems; e.g., matrix-assisted laser desporption-ionization time-of-flight (MALDI-TOF) mass spectrometry, have the capability
to correctly identify over 180 species. As such it has been difficult to increase interest in commercialization of Raman bacterial identification systems. To this end, we
have recently completed a study using multiple strains from 40 clinically relevant
microorganisms to evaluate the ability of Raman to correctly identify the each of the
species. Raman results are compared to correct identification of the same strains
that have been evaluated using a MALDI-TOF mass spectrometer with a commercially available database. This talk reviews clinical microbiology work flow, sample
preparation for either Raman or MALDI-TOF analysis and the comparative results of
the two methods as well as the clinical significance of the findings.
Quantitative Analysis of Moisture in Intact Lyophilized Protein
Samples by NIRS
Suzanne Schreyer, Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury,
MA 01876, Michelle Pressler
Lyophilization is a common process for the storage of biopharmaceuticals yet the
challenge remains on how to analyze the moisture content once the vials of protein
are sealed. Near-infrared spectroscopy (NIRS) has been used effectively and reliably to measure moisture in a wide variety of pharmaceutical samples due to the
large overtone band for water in NIR, and relatively high levels of accuracy for resulting predictive models. In this study NIRS, using a portable instrument, is used to
generate a predictive model for the quantitative determination of moisture in protein
samples. The use of a portable instrument is also evaluated and compared with a
lab based NIR instrument.
Terahertz Spectral Profiling and Imaging of Human Skin for early
Detection of Cancer
Aisha Masud, Applied Research & Photonics, 345 East 37th St., Ste.
317, New York, NY 10016, Babar Rao, Aunik Rahman, Anis Rahman
Terahertz scanning reflectometry was used to investigate both the surface and the
sub-surface of human skin biopsy. The non-ionizing nature of T-ray eliminates radiation damage or perturbation of sensitive tissues while able to probe disease conditions in the deeper layers leading to an effective early diagnostic tool. In this study,
a terahertz technique has been developed that is comprised of terahertz scanning
reflectometry, terahertz time-domain spectroscopy and terahertz imaging (all instruments from Applied Research & Photonics, Harrisburg, PA 17111) for detection of
cancerous skin with basal cell carcinoma (BCC) in comparison to benign skin samples. Two groups of samples were studied; the first group samples are benign skin
and the second group samples are biopsy from cancerous area. Thickness profile
exhibits significant differences in profiles of the respective skin samples both in their
layer structure and also in their total reflected intensities; thus indicating presence
and lack of cellular order for respective specimens. Similarly, terahertz spectra acquired in transmission exhibit quantifiable differences for both groups. More interestingly, terahertz image of the benign skin shows regular cell patterns while the
image of a sample with BCC exhibit no clear cell pattern. The lack of clear cell order
in the skin, thus, may be used as an indication of cancerous area and this finding
may be used as an early diagnosis tool. It is notable that this is the first of such a
concerted observation of benign versus BCC skins from three different experiments.
The results are consistent from individual experiments and collectively provide a
conclusive means of early diagnosis of skin cancer detection.
Noninvasive In-Vivo Glucose Sensing on Human Subjects Using
Mid-Infrared Light
Sabbir Liakat, Princeton University, F310 G4 EQUAD, Olden St.,
Princeton, NJ 08544, Kevin A. Bors, Callie M. Woods, Claire F. Gmachl
The realization of a clinically accurate, noninvasive, in vivo glucose sensor would
improve the quality of life of the more than 343 million diagnosed diabetics in the
world because it would eliminate the necessity of drawing blood to monitor blood
glucose levels. We have made significant progress towards such a sensor by utilizing fundamental vibrational resonances of the glucose molecule between 10001200 cm-1, which yield stronger absorption cross sections than overtone absorption
or Raman scattering. Previously, we showed promising initial results of clinically
accurate glucose concentration prediction using human in-vivo mid-infrared spectra.
Here we report recent improvements to our prediction results for human subjects
utilizing an integrating sphere to enhance collection efficiency, as well as derivative
spectroscopy for analysis. In-vivo spectra of backscattered light from human forearms was recorded between 1020 and 1220 cm-1 (a prominent glucose absorption
feature centers around 1080 cm-1) after propagating through an integrating sphere,
which improved collection efficiency by allowing the collection of all backscattered
angles. This reduced noise present in consecutive human spectra by 5%. Spectral
datasets were created for multiple subjects, with expected concentration quantified
using a commercially available glucose monitor. A small subset of spectra was set
aside for calibration, and PLSR predicted concentrations of the rest. By performing
regression on first derivative spectra, clinically accurate predictions were achieved
approximately 80% of the time, a net 6% improvement from our previously presented results. We continue to work on improving our optical setup and data processing
to achieve greater accuracy.
Microscopy’s Importance in Cases I Have Worked
Peter DeForest, Forensic Consultant, 16 Bryant St., Hartsdale, NY
A revolution has taken place in the field of analytical chemistry over the last 70 years,
made up of sweeping, profound changes. Physical methods have replaced chemical ones for the vast majority of chemical analyses conducted. This is especially
true for analyses conducted in high-volume testing facilities. In these high-volume
facilities, scientists have been replaced by technicians, as large quantities of fundamental knowledge of chemistry and chemical reactions is set aside and forgotten.
This presentation will not argue for a return to the “good old days” in such modern
laboratories; automated physical methods are ideal for routine high-volume applications. The difficulty arises when laboratories encounter non-routine problems. Problems in the forensic science laboratory provide an example. Even forensic science
laboratories deal with sets of routine problems, but non-routine problems, intellectually challenging and difficult to solve, frequently arise in complex cases. For this
reason, microscopy and chemical microscopy remain exceedingly powerful tools in
the forensic science laboratory. Illustrative case examples are discussed.
Surface Enhanced Raman Scattering Characterization of one
Changchu Ma, University of Massachusetts-Amherst, 100 Holdsworth
Way, Chenoweth Lab 247, Amherst, MA 01003, Hang Xiao, Lili He
Polymethoxylflavones (PMFs), a unique class of flavonoids mainly found in citrus
fruits. Our previous study using surface enhanced Raman spectroscopy (SERS)
found that the presence and numbers of hydroxyl played an important role on the
interaction with sliver (Ag) dendrites, the SERS substrate. The objective of this study
is to further characterize PMFs, and investigate the role of hydroxyl position using
SERS. Serial concentrations of 3’-demethylnobiletin (3DMN), 4’-demethylnobiletin
(4DMN), and 5-demethylnobiletin (5DMN) in methanol were incubated with Ag dendrites. Below saturation, 5DMN had the highest peak intensity, while 3DMN had the
lowest peak intensity. The high-pressuer liquid chromatography (HPLC) analysis
revealed that 36.13% В±1.06% 5DMN, following 18.40% В± 3.31% 4DMN and 9.66%
В± 0.94% 3DMN were bound to Ag. This result indicates that higher peak intensity may due to higher binding affinity of the molecule onto Ag dendrites. Different
binding affinity also influenced the saturated concentration. Meanwhile, the overall
spatial conformation of PMFs bound on Ag surface was determined by the position
of hydroxyl. 3DMN and 4DMN tended to form relative vertical conformation on Ag.
The lower binding of 3DMN on Ag surface was further interfered by the nearby
methyl. 5DMN tended to lay on the Ag surface and occupied larger space resulting
in low saturated concentration. The carboxyl nearby could enhance binding affinity by chelating Ag. This study reports how the position of hydroxyl in these one
hydroxyl-PMFs is crucial for the interaction with Ag dendrites, and provides value
information for further application of the SERS technique for molecular characterization of flavonoids.
Microscopy of Tattoo Inks
Michelle Miranda, SUNY- Farmingdale, 2350 Broad Hollow Rd.,
Farmingdale, NY 11735
No abstract submitted by the author.
Microscopy of Shotgun Buffers (Not your Common pH)
Peter Diaczuk, John Jay College, Department of Sciences, 524 W 59th
St., New York, NY 10019, Jack Hietpas
A multi-projectile shotgun shell is more complex and consists of more components
compared to a standard piece of handgun or rifle ammunition. Besides the shell,
primer, and propellant, an unfired shotgun shell is available in a multitude of payload
options, consisting of a single projectile or a variety of pellet or “shot” sizes. Some
shotgun ammunition manufacturers use an additive, called “buffer,” in their products, which are small polymer granules added to shotgun shells to cushion larger
sized pellets, named buckshot, and some smaller pellets, such as turkey shot. The
comingling of softer polymer buffer with harder metal pellets provides a cushioning
effect that reduces deformation of the pellet’s spherical shape. When a buffered
shotgun shell is fired, numerous polymer granules are discharged with the pellets
and wad, which imbeds in close range wounds or clothing and becomes scattered
Bacterial Identification Using Raman Spectroscopy: How does the
Technique Perform with many Species of Bacteria?
Bradford G. Clay, bioMГ©rieux, Inc., 595 Anglum Rd., Hazelwood, MO
63042, Pierre Mahe, Maud Arsac, Brenna Moss, Olivia Jobes, Chris
Huff, Virginia D. Shortridge, Ron Robinson, Sonia Chatellier, Alex van
Raman spectroscopy has been discussed by many researchers as a potential,
next-generation technology for bacterial identification. To date, an in-depth study
2014 EAS Abstracts
November 2014
about downrange of the muzzle for several yards. Being able to associate a sample
of buffer to a specific ammunition manufacturer offers useful information to shooting
scene reconstructions when spent shells are not present. Several brands of ammunition were obtained, representative shotgun shells were dissected and buffers were
examined microscopically in pre- and post-discharged instances. Comparison of
post-discharged to pre-discharged buffer samples revealed no significant changes
in buffer size or shape. However, stereomicroscopy showed morphological differences in unfired buffer used by different manufacturers. Using particle micrometry
and infrared spectroscopy, it was possible to determine and differentiate among
different buffers used in different brands of shotgun shells.
between the trans and the gauche forms and 168 cm-1 (2.01 kJ/mol) for the cis form
which is a transition state.
Comparison of Target Detection and Maximum Signal Factors
Neal B. Gallagher, Eigenvector Research, 160 Gobblers Knob Ln.,
Manson, WA 98801
The mathematics behind target detection and maximum signal factors (MSF) can
be shown to be identical. The difference in the approaches is that target detection
is a supervised pattern recognition technique while MSF is an exploratory analysis
method. Target detection typically employs known, well-characterized, library spectra for detection and classification. Unfortunately, due to measurement related differences between the library and the measured data there may be some mismatch
between the two resulting in a compromise in detection performance. Additionally,
only the selected target is considered in the detection task. On the other hand, MSF
can be used with current data to explore anomalies where the observed anomaly
is used as the measurement relevant target. Recent advances in MSF algorithms
have improved detection performance, reduced memory and time requirements,
and reduced the number of scores images that to be inspected during exploratory
analysis. However, the detected anomaly must be compared to library spectra for
classification. As a result, it should be seen that target detection and MSF form a
complimentary approach to detection and classification. Examples of the two approaches are shown with hyperspectral images.
Microscopy of Dust Samples
Skip Palenik, Micro Trace, 790 Fletcher Dr., Ste. 106, Elgin, IL 60123
No abstract submitted by the author.
Enhancement of Curcumin Fluorescence by Ascorbic Acid in
Bicontinuous Microemulsion
Maurice O. Iwunze, Morgan State University, 1700 E. Coldspring Lane,
Baltimore, MD 21251
Bicontinuous microemulsion is a thermodynamically stable and isotropic solvent
system that has been found to solubilize both hydrophobic and hydrophilic solutes
in a given reaction. In this work bicontinuous microemulsion made up of a four component system (water:oil:surfactant and co-surfactant (amyl pentane)) in the ratio of
0.35:0.12:0.18:0.35 w/w is used to solubilize curcumin that is known to be of very
low solubility in water. However, it was found to be very soluble in the prepared
bicontinuous microemulsion. The oil used in the preparation is tetradecane and the
surfactant is cetyltrimethylammonium bromide (CTAB). The observed fluorescence
of this compound was enhanced by introduction of ascorbic acid to the solution.
The association constant, Ka, that resulted between curcumin and the ascorbic acid
was determined using the Benesi-Hildebrand type of equation. The value obtained
is 6.19 x 106 with the free energy of association, ∆Ga, of -23.92 kJ/mole. The implication of this observation is discussed with regards the synergism of the two compounds for combating the reactive oxygen species, ROS that is very prevalent in
any given metabolic system.
Polymer Gel Permeation Chromatography with Luminescence
Martin Nosowitz, Arkema, 900 First Ave., King of Prussia, PA 19406
Gel permeation chromatography (GPC) is a workhorse tool in the polymer analysis
laboratory. Determinations of polymer molar mass distributions are commonly made
using GPC with differential RI or evaporative light scattering detectors. Various configurations of angle-dependent light scattering detectors (often in conjunction with
a viscometer) are used to obtain additional qualitative information such as polymer
branching. By exploiting the ability of GPC to separate molecules by molar mass
and then applying more specific detection such as UV-Vis absorbance and luminescence, compositional information may be extracted from samples. Some polymers
exhibit characteristic luminescence derived from their backbone or pendant groups.
The presence (and sometimes identity) of additives such as pigments and anti-oxidants can be confirmed. It is sometimes possible to elucidate end groups or defects
within the polymer structure, given the high sensitivity of luminescence detection.
Examples are presented.
An Investigation of Solution Phase Copper ESR Spectra as a
Means to Study Speciation by Ligands in Aqueous Solution,
Including those of Geochemical Significance
Christopher J. Bender, Fordham University, 441 E Fordham Rd., Bronx,
NY 10458
The electron spin resonance (ESR) spectrum of the copper (II) ion in aqueous solution appears as one of three forms that are indicative of the degree of sequestration
by anionic ligands. A survey of Cu(II) ESR spectra in aqueous solutions containing
inorganic acids, mono- and di-basic organic acids, amines, amino acids and amides was conducted on samples prepared according to complexometric titrimetric
procedures described by Schwarzenbach. A consistency of behavior is observed
regarding the transition of ESR lineshape among three forms that differ with respect to effective g-value and resolution of the copper hyperfine lines. The degree
of copper sequestration is accompanied by an improved resolution of the copper
hyperfine structure and a shift of the effective g-value towards the pure spin g-value. Thus one may investigate copper speciation in mixed ligand solutions by using
ESR spectroscopy to identify and make quantitative determinations of magnetically
distinct species. In cases of two different ionizable functional groups on the ligand,
one may detect and resolve the spectra of complexes formed by these independent
ionic species. For example, among the aliphatic alpha-amino acids, first indication
of copper coordination appears at approximately pH 2.5; a second complex makes
its appearance in the form of a second ESR line with g-value closer to 2 as the pH
is raised past 5. Subtractive methods of data analysis permit one to recover the
spectrum of each copper species along the ligand concentration or pH coordinate.
Fast GC-TOF-MS for High-Throughput Screening of Environmental
Pete Grosshans, Markes International, 11126-D Kenwood Rd.,
Cincinnati, OH 45242, Nicola Watson, Charles Haws, Laura McGregor,
Nick Bukowski
The introduction of recent legislation, such as the EU Environmental Liabilities Directive 2004/35/EC, has encouraged the development of precise and robust analytical
systems for identifying pollutants. However, the sheer number of toxic compounds
which may require monitoring at any given time makes this a challenging prospect.
Conventional quadrupole gas chromatography mass spectroscopy (GC-MS) methods often employ selective ion monitoring (SIM) for trace-level detection of target
compounds. However, this protocol prevents retrospective searching of data for the
latest contaminants of emerging concern. The use of time-of-flight mass spectrometry (TOF MS) can overcome this issue by providing highly sensitive detection whilst
acquiring full range mass spectra, to allow both target and unknown identification in
a single, rapid analysis. Nevertheless, trace-level identification may become complicated for compounds which exhibit weak molecular ions and extreme fragmentation,
e.g., the �drin’-type pesticides, such as dieldrin and endrin. Select-eV ion source
technology aims to combat this problem by allowing both hard and soft electron ionisation with no inherent loss in sensitivity. Select-eV provides enhanced molecular
ions whilst retaining structurally-significant fragment ions, delivering both confident
compound identification and lower limits of detection. This work demonstrates the
use of fast GC-TOFMS with Select-eV ionisation for accurate identification of a suite
of ultra-trace level environmental contaminants.
Raman, Infrared and Microwave Spectra, r0 Structural Parameters,
and Conformational Stability of Isopropylisocyanate
Bhushan S. Deodhar, University of Missouri-Kansas City, 5100 Rockhill
Rd., Kansas City, MO 64110, James R. Durig
The microwave spectrum of isopropylisocyanate, (CH3)2CHNCO, has been investigated from 11,000 to 21,000 MHz and 18 transitions for the more stable trans
conformer were assigned and A=6693.23(15), B=2263.10(3), C=1960.05(2) MHz
were obtained. By utilizing these rotational constants along with ab initio MP2(full)/6-311+G(d,p) predicted structural values, adjusted r0 parameters have been obtained for the trans conformer along with estimated values for the gauche conformer. Variable temperature Raman and infrared spectra of xenon solutions have been
recorded and a О”H value of 115В±11 cm-1 (1.38В±0.13 kJ/mol) has been determined.
The conformational stabilities have been predicted from ab initio calculations utilizing several different basis sets up to cc-PVQZ for MP2(full) and 6-311+G(3df,3pd)
for density functional theory calculations by the B3LYP method. From the MP2(full)/
cc-PVQZ calculations an energy difference of 87 cm-1 (1.04 kJ/mol) is predicted
Using Solid-Phase Extraction to Concentrate Human Hormones in
Drinking Water and High-Performance Liquid Chromatography to
Analyze the Recovery
Carl A. Fisher, Thermo Fisher Scientific, 1214 Oakmead Pkwy.,
Sunnyvale, CA 94085, Hua Yang, Pranathi Perati
The presence of hormones in drinking water is a human health concern with several being routinely monitored as part of the United States Environmental Protection Agency (EPA) Unregulated Contaminant Monitoring Rule 3 (UCMR3). Various
forms of estrogen are prescribed as a hormonal contraceptive device, for estrogen
deficiency syndromes, and to counter the negative effects associated with the natural decline in estrogen levels, such as accelerated bone loss, in postmenopausal
2014 EAS Abstracts
November 2014
women. Due to the widespread use of hormone pharmaceuticals, these often end
up in the sewage system as a result of excretion and disposal of unwanted quantities. Additionally, hormones from livestock waste can find its way into drinking water
sources. There is evidence that hormones may not be effectively removed during
wastewater treatment, and as a result, significant amounts of these hormones may
be present in drinking water sources. To monitor the levels of the most common hormones, EPA method 539 was developed, which describes a procedure for quantifying hormones in drinking water using solid phase extraction (SPE) followed by liquid
chromatography electrospray ionization tandem mass spectrometry. The present
work describes the use of high surface area solid phase extraction cartridges to
extract seven sex hormones from drinking water using a SPE instrument. Extracts
were subsequently quantified by high performance liquid chromatography to determine the recovery.
ENMs will inevitably lead to their release into the environment, which raises concern
about their potential adverse effects on the ecosystems and, subsequently, human
health. This talk goes over the theory of single particle inductively coupled plasma
mass spectrometry (SP-ICP-MS), the importance of data acquisition speed, and
describes its use in analyzing metal-based Nanoparticles. The single particle analysis technique allows for the differentiation between ionic and particulate signals,
quantitation of each fraction, the measurement of particle concentration (particles
/ mL), determination of particle size and size distribution. In addition, it allows the
user to explore particle agglomeration. SP-ICP-MS is a key analytical instrument in
assessing the fate, behavior and distribution of (ENMs) in several types of matrices
(environment, food, etc.). It is an essential technique used in evaluating ENMs bioavailability and bioaccumulation in the biota, and improving bio-labeling capabilities
as well as furthering advancements in the field of nanomedicine.
Low Level Chrysotile in Soil: A Method Assessment
Frank Ehrenfeld, International Asbestos Testing Laboratories, 9000
Commerce Pkwy., Mount Laurel, NJ 08054, Raymond Kennedy, Kristen
Goedde, Thomas Barkley
The detection of soil surface asbestos contamination presents particular analytical
challenges. This is especially true when the analyte is present at extremely low levels. Recently, a new test method for determination of asbestos in soil was approved
by the American Society for Testing and Materials (ASTM) Subcommittee D22.07 on
sampling and analysis of Asbestos (ASTM D7521-13 Standard Test Method for the
Determination of Asbestos in Soil). In this study, commercially available topsoil was
used as a blank matrix material. A chrysotile standard reference material was then
added to produce known percent laboratory spikes. Multiple spikes were formulated to contain both 0.1% and 0.01% chrysotile by weight. These spikes were then
subjected to the test method. The findings are very encouraging for the applicability
of this test method.
Quad vs. Magnet, Battle of the ICPMS Instruments for Superiority
of Cr Speciation in NJ Drinking Water Samples
Elizabeth McCandlish, Rutgers University, 170 Frelinghuysen Rd.,
Piscataway, NJ 08854, Sanjeev Rai, Brian Buckley, Lee Lippincott
Disinfection of raw water to be used for drinking, generally employs chemical oxidation. An Ion chromatography- inductively coupled plasma mass spectrometry (ICICPMS) was used to quantify Cr3+ and Cr6+, to measure Cr3+ oxidation to Cr6+
during disinfection. Measurements of NJ drinking water both before and after the
disinfection process demonstrated the vast of total chromium found in both raw and
finished water were far below (0.02-0.1 ppb) the federal drinking water standard.
To measure Cr concentrations at these low levels, significant signal processing of
raw data was required. Isotopic ratios of signals of 8.9 to 1 of 52Cr to 53Cr isotopes
were used to validate Cr signal vs. isotopic interferences and background signal.
Until now this very labor intensive method of post-data processing was the only
means for achieving the required sensitivity, even with a great deal of uncertainty.
To facilitate Cr measurement for future studies a magnetic sector ICPMS was employed to measure concentrations of both species at much lower levels removing
the isobaric interferences of a quadrupole instrument. This presentation focuses on
the study results from the drinking water study that demonstrated the need for greater resolution and sensitivity. In comparison, results using the Nu instrument’s HR
magnetic sector ICPMS for quantitation of Cr are presented. The greater sensitivity
and resolution were able to overcome many of the difficulties encountered in the
original study, opening up the possibility for future studies where these limitations
currently restrict progress.
Direct Mercury Analysis of Gaseous Samples Using Sorbent Tubes
Sumedh P. Phatak, Milestone Inc., 25 Controls Dr., Shelton, CT 06484
Direct mercury analysis (DMA) is a technique that is ubiquitous in analytical labs
within the environmental industry for analyzing mercury in solid and liquid samples. However, environmental labs are also required to test for mercury in gaseous
samples quite frequently. In addition to continuous emission testing and long term
testing for relative accuracy test audits (RATA), regulations in the environmental
industry have also driven the need for mercury gas testing and analysis. Most conventional direct mercury analysis systems do not offer the flexibility of analyzing
mercury in gases along with solid and liquid samples, which affects throughput,
productivity and costs. To address the need for a highly flexible system, we have
studied a configuration with sorbent tubes connected to a mass flow controller for
trapping mercury gases and subsequent release and analysis on a DMA-80 Dual
Cell (DL – 0.003 ng) or Tri Cell (DL – 0.001 ng) Mercury Analysis System, offering
the capability to directly analyze gases along with solid and liquid matrices without
sample preparation. The sorbent tubes contain a special mercury adsorbing material, which traps the mercury from the gases as they flow through the tubes. These
mercury containing sorbent tubes can be directly placed on the auto-sampler tray
and be directly analyzed along with the regular solid or liquid samples. We present
data on the flexibility of analyzing gas samples along with routine operation of mixed
Polyol Induced Extraction (PIE) of Water from Organic Solvents
Mithilesh Deshpande, Seton Hall University, Department of Chemistry
and Biochemistry, 400 South Orange Ave., South Orange, NJ 07079
Acetonitrile (ACN) is used in huge quantities for reverse-phase high-performance
liquid chromatography (RP-HPLC) experiments. For most part, this results in waste
that is only contaminated with large amounts of water (H2O), buffer and trace organic impurities. It would seem worthwhile to purify the ACN from H2O using a mass
separating agent (MSA). Our investigation revealed polyols as a new class of MSAs.
It was observed that when a polyol such as glycerol or sorbitol or erythritol or xylitol
or Isomalt or maltitol was added to a mixture of ACN-H2O, the ACN separates out of
mixture to form a new immiscible upper phase. We term the process: Polyol-induced
extraction (PIE). The study revealed that glycerol could initiate phase separation of
1:1 (v/v) ratio of ACN-H2O mixture at 20 ВєC with a glycerol concentration of 17.5 wt/
wt %. The results demonstrate that glycerol can improve purity of ACN up to 96%
at lower temperatures. We show that polyols as separating agents are equivalent
or better than MSAs such as sugars and salts. Our thermodynamic investigation
based on determination of change in free energy (∆ G) and change in enthalpy (∆ H)
revealed that glycerol and sorbitol induced phase separation is driven by decrease
in free energy of process and is primarily an exothermic process. Our study also
demonstrated that solvents in all of the major categories, non-polar, polar aprotic
and polar protic can be purified by the PIE process.
Novel QCell Technology for Inference Removal in ICP-MS Combining Low Mass Filtration with Kinetic Energy Discrimination
Fergus Keenan, Thermo Fisher Scientific, 1228 Titan Way, Sunnyvale,
CA 94085, Craig Seeley
Polyatomic interferences are one of the fundamental challenges in inductively
coupled plasma mass spectrometry (ICP-MS). They can be suppressed using a
collision/reaction cell with a neutral gas like Helium combined with kinetic energy
discrimination (KED) or with a reactive gas like Hydrogen or Ammonia. The iCAP Q
ICP-MS features the new QCell which combines KED with mass filtration of interference precursors. The QCell uses an innovative flatopole design that not only utilizes
KED with low mass cutoff but also achieves best in class sensitivity and lowest
background in a simple, automated process. The QCell is described along with data
from environmental, industrial and food safety applications.
Biphasic Dissolution Studies of Felodopine: Understanding the
Effect of Amorphous Solubility on the Rate of Partition
Jon Mole, Sirius Analytical, 100 Cummings Ctr., Beverly, MA 01915, Karl
Box, John Comer, Rebeca Ruiz, Robert Taylor, Lynne S. Taylor
A biphasic dissolution assay has been developed to measure the partition rate of
active pharmaceutical ingredients (APIs) from an aqueous compartment into an
immiscible organic phase to mimic absorption through biological membranes. A
stock solution of felodopine, was injected into a chamber containing 45mL of phosphate buffer adjusted to pH 6.8 and 20 mL of nonanol which formed an immiscible
layer above the aqueous compartment to represent a biological sink. A fibre-optic
probe was immersed in both the aqueous compartment and organic layer, in order
to collect UV spectra in both phases. The partition rate increased proportionally
with concentration up to 10 ug/mL. Above this concentration, the rate continued to
increase, but the trend was no longer proportional to concentration. The solution in
the aqueous compartment was found to be clear at concentrations of 6ug/mL and
Single Particle ICP-MS (SP-ICP-MS) a New Analytical Technique for
Counting and Sizing Metal Based Nanomaterials: Theory and
Chady Stephan, PerkinElmer, 25 Nuttall, Brampton, ON, L6S4V7
Engineered nanomaterials (ENMs) are synthesized by a manufacturing process that
produces and controls ENMs to have at least one dimension in the range of 1 to
100nm in size. ENMs often possess different properties than their bulk counterparts of the same composition, making them of great interest to a broad spectrum
of industrial and commercial applications. The widespread use and application of
2014 EAS Abstracts
November 2014
below, but turbid at 10 ug/mL and above. Trend lines were drawn through the data
with R2 values >0.98. The concentration at which the trend lines intercepted was
close to the reported amorphous solubility. At concentrations below the amorphous
solubility of felodopine, the partition rate was directly proportional to concentration.
However, above the amorphous solubility, the concentration in solution was expected to remain constant as the precipitation/dissolution kinetics of the amorphous
form, were expected to be faster than the partition rate. The observed increase in
partition rate above the amorphous solubility was thought to be due to a dissolution
event at the interface.
errors. Therefore a prominent goal in the scientific community is to identify whether
differences in data are genuine or the result of non-product quality factors causing
variability in ACI measurements.[2] For this study, several factors related to analyst
error and test design were identified. Next, a design of experiments approach was
used to randomize experiments and determine which effects caused statistically
significant (p<0.05) changes to the data. Significant effects were then modeled using PCA where each error defined a discrete class and each size fractionated group
represented a distinct variable. Two components were sufficient to describe 80%
the total variance and separate each class. The model was further successfully
validated with three data sets. This research therefore establishes that PCA may aid
in the investigation of atypical ACI data.
Dissolution Performance of Inhaled Product APIs in Simulated
Lung Fluid Using Novel Low Volume Assay
Jon Mole, Sirius Analytical,100 Cummings Ctr., Beverly, MA 01915,
Rebeca Ruiz, Karl Box, John Comer, Robert Taylor, Antonio LlinГ s,
Johan Solandt, Ulrika Tehler
The aim of this study was to measure the dissolution characteristics of active pharmaceutical ingredients (APIs) contained in inhalation products, using novel low volume assays. Samples were compared in a standard phosphate buffer and in a simulated lung fluid (SLF) media. The dissolution performance of six APIs (budesonide,
indacaterol, pranlukast, rofleponide, salmeterol and zafirlukast) was measured at
37 В°C using two novel technologies. In the first technique, the SiriusT3 was used
to run powder dissolution assays in 2mL volumes with quantitation using in-situ
UV probes. The second technique used a surface dissolution imaging system (Sirius SDI) allowing direct measurement and visualization of dissolution events at the
surface of the API in real-time. Dissolution measurements were compared using
standard phosphate buffer and a simulated lung fluid media containing 1% sodium
dodecyl sulphate (SDS). Powder dissolution results in phosphate buffer ranged between 1% released for pranlukast up to 10% for zafirlukast. There was a marked increase in dissolution performance in SLF medium for zafirlukast (up to 20% release)
and for salmeterol (up from 12% release in phosphate buffer to 18% in SLF). However, for some APIs there was no little or no increase in dissolution performance.
The increase in dissolution in SLF could be attributed to increased wettability in the
presence of a surfactant in combination with surface active properties of the API
itself. Direct UV imaging of the surface confirmed the increased accessibility of the
media at the interface when surfactant was present.
[1] Mitchell, J. and Nagel, M (2004). Particle size analysis of aerosols from medical
inhalers. KONA. 22: 32-65.
[2] Glaab, V., Goodey, A., Lyapustina, S., and Mitchell, J. (2011). Efficient data analysis for MDIs and DPIs: failure mode effect analysis. Respiratory Drug Delivery
Europe. 225-236.
New Dispersive Technology (Grinder) Sample Preparation
Improvements for Disintegration and Dissolution of a Challenging
Extended Release Solid Oral Dosage Form
Peter I. Tattersal, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, Nalini Anand, Marlon Rutherford, Shan Xiao, Khanh Ha, John
A risk assessment was performed on an extended release high-performance liquid
chromatography (HPLC) assay method following data trending concerns with imprecision and accuracy (~3% relative standard deviation with low bias (97%) along with
a number of object-oriented analysis investigations) in support of manufacturing.
This presented an opportunity to evaluate a new dispersive technology that uses a
grinding / blending action for the sample extraction process to efficiently disintegrate
tablets in a fraction of the current method time. The approach was optimized through
extensive studies on three different formulations / packages (hundreds of assay
values) to statistically compare and ensure more consistent / accurate / precise
data could be routinely obtained. A robustness study on key sample preparation
variables (n=4) was performed as a design of experiment (multi-factorial, 20 experiments) based on the optimized conditions. The development studies performed
clearly demonstrated that the new sample preparation conditions are effective and
Characteristics of Felodipine: HPMCAS Solid Dispersions:
Dissolution Performance and Particle Size Effects
Jon Mole, Sirius Analytical, 100 Cummings Ctr., Beverly, MA 01915, Karl
Box, John Comer, Robert Taylor, Rebeca Ruiz, Brian Stockton, George
Butcher, Darren Matthews
To assess the dissolution performance of felodipine: hypromellose acetate succinate (HPMCAS) solid dispersions prepared using different spray drying conditions
and to characterize the particle properties using laser obscuration time (LOT) and
digital image analysis. Nine solid dispersions of felodipine in HPMCAS were prepared using different spray drying conditions. Outlet temperature, nozzle pressure
and feed concentration were varied. The dissolution profiles of were determined in
20mL volumes of phosphate buffer at pH 7.0, using a SiriusT3 autotitrator with in-situ UV-spectroscopy. The particle size distribution of each dispersion was measured
using the Sirius Insight using combined techniques of LOT and image analysis. The
dispersing medium was isooctane. The dissolution rate from all nine solid dispersions was greater than that of pure felodopine. The total dissolved mass from the
dispersions ranged between 7 and 15 Вµg/mL in a 60 minute time frame, whereas
pure felodopine achieved less than 1 Вµg/mL in the same period. The LOT and image
analysis showed that the batches ranged in particle size distribution from a lowest
d50 value of 17-Вµm up to the largest d50 value of 76-Вµm. Dissolution performance
was found to correlate directly with particle size, where the greatest dissolution enhancement occurred for the two dispersions with the smallest d50 values. Dissolution performance could be measured in situ from low volume assays using sparing
amounts of material and was found to correlate directly with dispersion particle size.
LOT and digital image capture enabled fast characterization of the particle size distribution and analysis of particle shape, with minimal application.
NMR Spectroscopy as a Tool in PAT: An Essential Tool in Chemical
Process Understanding
Brian L. Marquez, Bruker Corporation, 15 Fortune Dr., Billerica, MA
01821, Kim Colson, Anna Codina
Process analytical technology (PAT) has become an increasingly more powerful
as a tool for the understanding and monitoring of chemical processes. Traditionally
these tools vary from pH meters to infrared spectroscopy. However, in recent years,
nuclear magnetic resonance (NMR) spectroscopy has become a very powerful tool
in the area of process understanding, owing to its capabilities to discern different
chemical moieties via unique chemical shift properties (multinuclear) and inherent
quantitative capabilities. In this presentation, we will demonstrate the use of a novel
flow cell, powerful acquisition and processing software that allows one to monitor
chemical reaction in real time providing a tremendous amount of kinetic, quantitative
mass balance and mechanistic understanding of example synthetic steps.
Optimized Karl Fischer Titration of Lyophilized Substances
Matt Eby, Mettler Toledo, 1900 Polaris Pkwy, Columbus, OH 43240,
David Zavich
The new LabXВ® Balance Software method optimizes external extraction of lyophilized, especially when the sample weight is unknown, with a completely guided workflow utilizing an Excellence Analytical balance and Compact Karl Fischer
Continuous Improvement in Productivity of Pharmaceutical
Development through Enhanced Usage of UV Fiber Optic
Lili Lo, Bristol Myers Squibb, One Squibb Dr., New Brunswick, NJ
08903, Xujin Lu
Ultraviolet fiber optic dissolution has been accepted in the pharmaceutical industry
since it was endorsed by United States Pharmacopeia in monograph <1092> in
2006. Unlike conventional dissolution testing performed manually or automatically,
which requires sample withdrawal during dissolution and HPLC analysis and data
processing after the run, the UV fiber optic dissolution system performs in-situ detection in the vessel without sample removal and collection, nor post dissolution
detection. It saves the costs of sampling, filtration and collection of consumables. It
also saves time and labor in set up and data processing of the off-line detection sys-
A PCA Model of Atypical Andersen Cascade Data
Lauren Seabrooks, Merck, 181 Passaic Ave., Summit, NJ 07901,
Jennifer Wylie, Justin Pennington
This research details a novel application of principal component analysis (PCA) to
aid in the root-cause determination of atypical Andersen cascade impaction (ACI)
data. Andersen cascade impaction is a technique used to measure the aerodynamic
particle size distribution of medical inhalers an important indication of drug deposition in the lung. It is one of several methods which provide fractionation of particles
based on the differences in inertia. A particle passes through numerous stages each
with decreasing nozzle apertures, increasing linear velocity, and either remains
entrained in the laminar flow paths or breaks through the stream and impacts on
a collection surface located beneath each stage. [1] Although information rich this
technique is labor intensive, involving many steps, and ultimately susceptible to
2014 EAS Abstracts
November 2014
tem. Although the benefits of using UV fiber optic dissolution are obvious, the rate
of adoption has been modest. HPLC is currently still the primary dissolution finish
in some working environments. The resistance to change is in large part because
analysts are not comfortable to change working habits and adapt to a new application. In an environment of continuous improvement within the industry, a proposal
to enhance the use of UV fiber optic dissolution has been executed in our department. In this presentation, we demonstrate the significant savings and productivity
improvement achieved by expanded use of UV fiber optic dissolution, with a case
study of how the cost and savings were generated and evaluated.
less hydrophilic polymer. It was hypothesized that the addition of certain classes of
excipients into the dispersion would help to improve hydrophilicity and/or promote
disintegration thus maintaining faster dissolution rate at higher drug loads. The following classes of additives were evaluated: highly soluble excipients, effervescent
salts, superdisintegrants, surfactants, and milling aids. Dispersions were manufactured using a small scale hot melt extruder. Dissolution was performed to evaluate
the effect on API dissolution rate and nanoparticle formation. The majority of the
excipients evaluated did not increase the dissolution rate of the API in high drug
loaded extrudates and in some cases even impeded the dissolution. The additives
also did not increase nanoparticle formation during dissolution. Only extrudates containing high levels of surfactant (10%) provided similar dissolution performance to
the lower drug loaded extrudate. However, this level of surfactant is not a viable
commercial formulation due to compromised physical stability. Therefore it was concluded that the addition of excipients intra-extrudate is not a feasible formulation
solution for increasing the dissolution rate of high-drug loaded dispersions.
Turbidity Measurement of Infusion Solutions
Alex White, Anton Paar USA, 10215 Timber Ridge Dr., Ashland, VA
Turbidity is a parameter that cannot be overlooked when performing quality control
checks on infusion solutions for clinical use. Haze or particles in these solutions
may result in life-threatening complications. Undesirable reactions may occur when
preparing intravenous drugs and solutions. Incompatibility reactions are those which
occur inside a fluid container or infusion line and are often visible. These reactions
can be either physical or chemical. Physical reactions are those when drugs react
and either phase separate or precipitate. Such reactions can increase or decrease
drug effectiveness, but can also produce completely different interactions in the
body that can often be toxic. Precipitations based on drug incompatibilities are responsible for the most common particle formation seen in infusions. Such reactions
can be detected and monitored through turbidity measurement. Chemical reactions
that may occur, such as oxidation, reduction, hydrolysis, or decomposition, often result in changes in turbidity. Chemical degradation decreases the amount and/or effectiveness of the active agent and can also form toxic by-products. Turbidity should
be measured during production as well as for final quality control checks. Turbidity
measurement can also provide critical stability and shelf-life determinations.
Application of Multivariate Curve Resolution Approach to Complex
Thermal Desorption Data
Jing Zhao, University of Delaware, Department of Chemistry and
Biochemistry,102 Brown Laboratory, Newark, DE 19716, Jia-Ming Lin,
Andrew V. Teplyakov, Juan Carlos F. Rodriguez-Reyes
The qualitative and quantitative analysis for temperature-programmed desorption
(TPD) experiments in surface and catalysis chemistry is often difficult due to overlapping mass spectral cracking patterns of the compounds produced and complex
reaction channels that are difficult to decipher. Empirical methods to analyze TPD
results always need a priori knowledge and initial assumptions specific to systems investigated, thus the input of an operator onto the outcome of process of
analysis cannot be efficiently eliminated. Multivariate curve resolution (MCR) is a
mathematical approach to analyze spectral data in the form of matrix calculation. In
this way, spectra of different m/z (mass-to-charge ratio) traces changing with temperature can be translated into significant cracking patterns of several desorbates
with variations and correlations. This provides a straightforward comparison to the
known cracking pattern of every component to understand the reactions followed by
desorption. For better understanding of thermal process by MCR approach, three
reaction processes were used to demonstrate: 1) the cryogenic desorption of vinyltrimethylsilane from silicon, an introductory system where the known multilayer and
monolayer components are resolved. 2) The thermal reaction of tetrakis(dimethylamino)titanium (Ti[N(CH3)2]4) on a Si(100) surface, where the products of surface
decomposition are identified and an estimation of the surface composition following
thermal reaction is afforded. 3) The thermal reaction of ethylchloride-d5 (C2D5Cl)
on a Si(100) surface, where the mechanism is not apparent but the products of
thermal reaction can be identified, is followed. The original TPD data and results
achieved following MCR process are discussed.
Material Identification with Advanced Handheld 1064 nm Raman
Claire Dentinger, Rigaku Raman Technologies, 14 New England
Executive Park, Ste. 102, Burlington, MA 01803, Mark Mabry, Jen Lynch
Portable and handheld Raman spectrometers, especially those with long wavelength excitation lasers, are becoming smaller, lighter, more rugged and easier to
use without compromising quality. Long wavelength excitation Raman spectrometers expand the range of different materials that can be identified compared to
Raman spectrometers using shorter excitation wavelengths. This combined with
their small size, ease of use and high spectral quality makes them ideal for material identification outside the laboratory. Advancements in optical components and
electronics have enabled this improvement in long wavelength handheld Raman
spectrometers. Applications which benefit from high quality 1064 nm handheld Raman spectrometers are presented in the areas of pharmaceutical raw material identification (RMID), finial product verification, biopharmaceuticals, cosmetics, dietary
supplements and anti-counterfeiting.
Evaluation of a Low-Cost Mass Spectrometer: Waters QDa
Michael W. Dong, Genentech, 1 DNA Way, S. San Francisco, CA 94080,
Christine C. Gu
A low-cost, compact single-quadrupole mass spectrometer was evaluated for pharmaceutical analysis in supporting method development of stability indicating assays and cleaning verification testing. The advantages found were: easy to use and
maintain, sensitive, and excellent software integration of UV and mass spectrometry (MS) data in Acquity ultra high-performance liquid chromatography (UHPLC)
Empower systems with automated annotation and reporting functions. Some of
limitations found were: not very “portable,” electrospray ionization only, no adjustment needed or possible, and “not yet integrated with other vendors’ HPLCs.” The
poster includes a description of the system, schematics and empower instrumental
method. Case studies shown include: a stability-indicating assay with a peak table
containing UV and MS data plus system suitability values for each peak; and a
generic cleaning verification method of 12 new chemical entities with automated
annotations of UV and MS spectra indexed to each peak and performance data of
the UV and MS channels.
In-Situ Monitoring of Form Change as a Function of Relative
Humidity in the Solid-State by VTI-Raman
Candice Choi, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08904, Duohai Pan, Denette Murphy, Anisha Patel, Roxana Schlam,
Shawn Yin, Sruthi Janakiraman
Understanding changes that occur as a function of water activity has become an
important aspect of physical characterization of the active pharmaceutical ingredient
(API) in all stages of pharmaceutical product development due to the recent increase in number of compounds forming both stoichiometric and nonstoichiometric
solvates and/or hydrates. Currently, moisture sorption isotherms are acquired to
better understand the relationship between water activity and moisture content at a
given temperature for an API form/material in the solid state. However, the additional complexity of API form/phase change that can occur during a sorption analysis
can make the interpretation of the moisture sorption isotherm difficult. Integration of
moisture sorption isotherm and Raman spectroscopy provides better understanding
of these changes that may be occurring to the API as a function of equilibrated water
content and/or %RH in the solid-state. This poster illustrates, via three examples,
applications of VTI/Raman in the pharmaceutical field.
Performance Comparison of Gel-IEF and cIEF with a Recombinant
Model Protein
Leu-Fen Lin, SGS Life Science Services, 75 Passaic Ave., Fairfield, NJ
07004, Jeffrey Hulmes
In this study, we compare the performance of traditional gel-based isoelectric focusing (gel-IEF) and capillary-based IEF (cIEF) methods for the detection of impurities
and variants of a model protein based upon differences in net charge. The protein
in the current study is a proprietary recombinant enzyme of MW ~140 kDa. In traditional gel-IEF analysis, the protein has a major (>90% of total) species having a
pI of approximately 5.0 and additional bands at pI’s slightly higher (2-3% of total)
and lower (6-7% of total). For gel IEF, Novex pH 3-7, precast IEF Minigel (Life Technologies) was employed with ServaВ® IEF pH 3-10 protein markers and quantified
with densitometer. For cIEF, a Beckman Coulter PA 800 plus system was employed
with the vendor’s synthetic peptide marker pI 4.1-10.0 and 280 nm detection. In this
An Attempt to Increase Hot Melt Extrusion Drug Loading without
Compromising Dissolution
Julie M. Novak, Merck, 770 Sumneytown Pike, West Point, PA 19486,
Karen Pica, Melanie J. Marota, Pavithra Sundararajan, Mary Ann
Johnson, Wei Xu, Paul A. Harmon
High drug loaded amorphous solid dispersions (>40%) are desirable in formulation
of poorly potent active pharmaceutical ingredients (APIs) requiring higher doses and
in formulation of combination products in order to minimize image size. High drug
loaded dispersions have been found to have significantly reduced dissolution rate
compared to lower drug loads due to the presence of more hydrophobic drug and
2014 EAS Abstracts
November 2014
study, we investigated if any differences exist in the pI assignment for the major
species between the two methods. Inherent differences between the two methods
include pI markers to generate standard curves (native proteins [gel-IEF] versus
synthetic peptides [cIEF]), and the standard curve of pI versus mobility (sigmoidol
[gel-IEF] and linear [cIEF]. Additionally, we explored if lyophilization of the liquid protein followed by reconstitution would affect the IEF profile with both methods. The
data shows that more species from the sample were clearly resolved with the cIEF
method while retaining similar quantitation and precise pI measurement.
home to disease sites where proteolytic cleavage of the peptide substrate by thrombin releases the reporter, which subsequently filters into the urine. For detection, we
employ a digital immunoassay with single-molecule resolution. Liberated reporters
are captured by microbeads coated with specific capture antibodies and labeled
with streptavidin-ОІ-galactosidase. The labeled immunocomplexes are localized into
individual microwells and sealed with fluorogenic substrate, generating high local
concentrations of fluorescent product. The number of fluorescent wells relative to
entrapped beads correlates directly to bulk solution concentration. Conventional
ELISA detects these reporters at low picomolar concentrations, but our assay extends into the low femtomolar range, increasing sensitivity by 1000x. This assay
discriminates healthy mice from those with thromboembolic disease using doses as
low as 3.9 ng, corresponding to a human dose of approximately 8.7 Ојg.
The Development and Validation of a LC-MS-MS Method for the
Determination of CEP-32496 in Human Plasma
Charisse Green, Teva Pharmaceutical, 145 Brandywine Pkwy, West
Chester, PA 19380, Edward Hellriegel
B-Raf is an isoform of the RAF serine/threonine kinases family. Mutations in the
B-Raf gene are found in approximately 7% of all cancers evaluated. While several mutated forms of B-Raf have been identified, the V600E mutation found in the
majority of cases (approximately 80%) is a B-Raf-kinase-activating mutation with
approximately 500-fold higher activity than wild-type B-Raf. CEP-32496 is a potent orally active B-Raf kinase inhibitor that is currently under clinical development
by Teva Branded Pharmaceutical Products R&D, Inc., for the treatment of various
cancers. A liquid chromatography tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the rapid determination of CEP-32496 in
human plasma. The method was validated over a dynamic range of 1.00-1000 ng/
mL. The method utilizes a protein precipitation plate (Biotage) to extract CEP-32496
from 50 ОјL of human plasma. 13C6-CEP-32496 was used as an internal standard.
Chromatographic separation was achieved using a Phenomenex Synergi Fusionв„ў
RP 30.0 mm x 2.00 mm, 4 Ојm 80Г… column with isocratic elution followed by tandem
mass spectrometric detection. The run time was 2 minutes. The intra-and inter-day
accuracy (%bias) of this method at the quality control (QC) levels tested was <15%
(20% at the lower limit of quantitation) of the nominal concentrations. The intra- and
inter-day precision (%CV) of this method at the QC levels tested was within 15%
(20% at the limit of quantitation). The validated method is currently in use to quantify the plasma concentrations of CEP-32496 in patients with advanced carcinoma.
Determination of Latanoprost and Latanoprost Acid by UPLC-MSMS
Hemantkumar Naik, Millennium Research Laboratories, 160 New
Boston St., Woburn, MA 01801, Monish Chaddha, Teresa Pekol, Ram
Latanoprost, marketed as a generic and under the brand name Xalatan (Pfizer), is
a prostaglandin analog prescribed for reducing intraocular pressure in patients with
or at risk for glaucoma or ocular hypertension. The marketed formulations are ophthalmic solutions (0.005% or 50 Вµg/mL), and typical dosing is by the application of
drops to the eyes, once daily. For many patients, chronic use of the drug is required
to control eye pressure, prompting the investigation of controlled/extended release
formulations and implants to reduce dosing frequency. In-vitro and in-vivo release
testing of latanoprost from these formulations and implants over time requires an
analytical method with both specificity and a low limit of detection. Additionally, because latanoprost is an isopropyl ester pro-drug that is quickly hydrolyzed by esterases in the cornea to the biologically active latanoprost acid, both the pro-drug and
the active drug must be monitored. In this work, a ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) method for the determination
of latanoprost and latanoprost acid was developed and qualified. The LC-MS-MS
system comprised a Waters Acquity UPLC with an AB Sciex API 5000 mass spectrometer. Deuterated (d4) internal standards were included for both latanoprost and
latanoprost acid. The total method cycle time was less than 5 minutes. Method development rationale and the results from qualification assessments (stability, specificity, calibration curve, lower limit of quantitation, accuracy, precision, and matrix
effect) will be presented as well as the application of the method to samples from
in-vitro release tests.
Application of Micro Flow Image (MFI), ARCHIMEDES, and Quartz
Crystal Microbalance with Dissipation (QCM-D) on the Particle
Characterization for a Therapeutic Protein in Formulation
Songyan Zheng, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, Aastha Puri, Vishal Nashine, Jinjiang Li, Monica Adams
During product storage and transportation, proteins can aggregate to form particles either under the influence of shear force or due to interaction with silicon oil
in prefilled syringes. This study aims to characterize the particulate matters in the
solutions of a therapeutic protein in prefilled syringes after shaking using two analytical methods-micro flow imaging (MFI) and resonant mass measurement (ARCHIMIDES). Specifically, the quantity of silicon oil particles leached from the syringe
was correlated with formation of proteinaceous particles. In addition, the inhibitive
effect of polysorbate 80 (PS-80) on particle formation was evaluated. The results
from both MFI and ARCHIMIDES indicated that PS-80 can prevent the formation of
proteinaceoues particles and the leaching of silicon oil particles from the syringes.
Mechanistically, as shown by the QCM-D results, the presence of PS-80 inhibited
the adsorption of proteins on the silicone oil through a competitive adsorption. The
results suggest that the contact of the protein with hydrophobic silicon caused the
formation of proteinaceous particles, and the preferred adsorption of PS-80 on silicon oil likely prevented the interaction of protein and silicon particles. Overall, this
study also demonstrated the capabilities and utilities of MFI, ARCHIMEDES, and
QCM-D for the particle analysis respective to types and sizes.
Fluorescence-Based Sugar Sensor on a Gold Nanoparticle
Ishan V. Soni, Kutztown University, 15200 Kutztown Rd., Kutztown, PA
19530, Zachary M. Semian, Gary A. Baker, Thomas A. Betts
One of the driving forces behind the development of sugar-sensing platforms has
been the need to quantify blood glucose levels for the management of diabetes. As a
result a wide variety of spectrophotometric and electrochemical methods are geared
towards sugar quantitation. Our sugar sensor relies on boronic acid-capped gold
nanoparticles which have a fluorescent dye bound to the boronic acid sugar-binding
site. The addition of sugars can displace the fluorescent dye from the proximity of
the gold nanoparticle surface resulting in a change in fluorescence intensity.
Generic Ligand Binding Immunoassay Method for Rat IgG1-Based
Surrogate Bio-Pharmaceuticals
Wonmi K. Rooney, Bristol-Myers Squibb, Route 206 and Province Line
Rd., Lawrenceville, NJ 08543
In antibody drug discovery and development, use of surrogate antibodies, particularly rat IgG1-based antibodies, is an advantageous alternative to using humanized
antibody therapeutics in expensive non-human primates. As effective bioanalytical
support for pharmacokinetic assessment of surrogate antibodies, a generic assay
method using commercially available antibody reagents was made by design of
experiment on an ELISA platform and was assessed for accuracy and precision.
Quantitation of rat IgG1-based surrogate therapeutics in various commonly used
preclinical species was with a total error of <20%. The reliability of this method
was confirmed orthogonally by, side by side testing using a generic liquid chromatography mass spectrometry method. This easy to use method facilitates efficient
pharmacokinetic testing of surrogate antibody candidates.
Thromboembolic Disease Discrimination through an Ultrasensitive
Assay of Microdosed Synthetic Urinary Biomarker
Kevin C. Ngan, Tufts University, Department of Chemistry, 62 Talbot
Ave., Medford, MA 02155, Shonda T. Gaylord, Milena Dumont
Milutinovic, David R. Walt, Andrew D. Warren, Gabriel A. Kwong,
Sangeeta N. Bhatia
The discovery of clinically significant biomarkers has been limited, despite extensive
research. It is difficult to resolve endogenous molecules from complex biological
matrices, prevent rapid ex vivo degradation, and establish quantification criteria
robust enough to account for patient-to-patient variability. An alternative to endogenous biomarkers is synthetic exogenous agents. These agents can not only be customized to exploit specific physiological processes, but also continuously improved
upon and optimized through further testing. We describe an ultrasensitive assay
to detect thromboembolic disease using microdosed synthetic urinary biomarkers.
These synthetic biomarkers are composed of iron oxide nanoworm carriers bound
to thrombin-sensitive peptide substrates containing a fluorescein reporter and a biotin tag (nanoworm-peptide substrate-fluorescein-PEG-biotin) designed to facilitate
immunocomplex formation. After intravenous injection, the nanoworms passively
Proteomic Investigation of Saliva and Sera from Children with
Autism Spectrum Disorder (ASD) and Matched Controls
Kelly L. Wormwood, Clarkson University, Box 5810, 8 Clarkson Ave.,
Potsdam, NY 13699, Armand G. Ngounou Wetie, Izabela Sokolowska,
Katherine M. Beglinger, Alisa G. Woods, Costel C. Darie, Jeanne P.
Approximately 1/88 children are believed to be affected by Autism Spectrum Disorder (ASD). A more recent study even suggests numbers as high as 1/50. With
2014 EAS Abstracts
November 2014
the increased diagnosis of ASD, treatment and understanding of the disorder is
a pressing health concern. Protein biomarkers found for ASD may potentially be
used for ASD diasgnosis, subtyping, monitoring, treatment and identifying therapeutic targets and may also provide clues about the causes of ASD. Here, sera and
saliva samples from children with ASD and matched controls were analyzed using a
combination of gel electrophoresis (Tricine-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Blue Native PAGE), in gel digestion
or insolution digestion and nanoliquid chromatography-tandem mass spectrometry
(nanoLC-MS-MS) to investigate difference between the proteomes of ASD patients
and matched controls. These results will hopefully shed some light on the possible
causes of ASD. In the SDS-PAGE and Tricine-PAGE based experiments using sera,
increased levels of Apolipoproteins (Apos) ApoA1 and ApoA4, involved in cholesterol metabolism, and of serum paraoxanase/arylsterase 1, involved in preventing
oxidative damage, were discovered in the sera of children with ASD, compared with
their matched controls. These three proteins are parts of high density lipoproteins
(HDLs) and are presumed to interact with one another. This data suggests that there
may be a dysregulation of cholesterol metabolism in children with ASD. Additional
dysregulated proteins were also detected, suggesting a dysregulated immunological response and an enhanced response to environmental toxic factors. This investigation is currently ongoing.
strategy only included liquid-liquid extraction (LLE) methodology with three extraction solvents under three pH conditions and four sub-2-Вµm UHPLC columns for
method screening. UHPLC separation was optimized to separate metabolites from
analyte(s) using extracted incurred plasma samples with selected reaction monitoring (SRM) transitions of metabolites generated from AB Sciex IDAв„ў software
and ADME knowledge. This methodology has led to the development of a robust
assay for Compound X, a Bristol-Myers Squibb development compound with curve
range of 50-20000 ng/mL in rat plasma. Plasma samples were extracted into Methyl
tert-Butyl Ether (MTBE) by automated LLE in 96-well format. Chromatographic separation was achieved with a gradient elution on an ACQUITY Cortecsв„ў UPLC C18+
column (2.1 x 50 mm, 1.6-Вµm). Positive electrospray ionization for Compound X
and its isotopically-labeled internal standard was performed using selected reaction
monitoring (SRM) mode on an AB Sciex API4000 mass spectrometer. The intra-assay precision was within 3.9% CV, inter-assay precision was within 1.6% CV, and
the assay accuracy was within В±2.7% deviation (% Dev) of the nominal values in
rat plasma. An inconsistent internal standard response was overcome by resetting
the needle penetration in the autosampler, suggesting that a thorough examination
of the hardware settings during method development is essential because vendor-to-vendor variations in 96-well plates and other consumables are very common.
Electrochemical Sensor Using Redox Polymer as an Indicator
Zahilis Mazzochette, Rowan University, 201 Mullica Hill Rd., Glassboro,
NJ 08028, Amos Mugweru
DNA hybridization biosensors are important diagnosis of generic diseases including
environmental and food safety analysis. Electrochemistry has superior advantages
over other existing DNA hybridization detection methods; they provide rapid, easy to
use, and inexpensive detection. In this work, a simple electrochemical DNA hybridization sensor was assembled using osmium redox polymer as the electrochemical
indicator. First the 3’ thiolated single stranded DNA (ssDNA-SH) probe self-assembled on the surface of the gold electrode. Then the hybridization was carried out
by immersing the ssDNA-SH/Au system containing the complementary ssDNA
strand. The electrochemical behavior of osmium redox polymer on the ssDNA and
the hybridized double stranded DNA (dsDNA) was determined using cyclic voltammetry (CV) and impedance methods. Formation of the hybridized ds-DNA on the
gold electrode was indicated by a great increase in the peak currents observed on
the voltamograms in comparison with those obtained for the single-stranded DNA
modified electrode. These results demonstrate that, osmium redox polymer used in
this work can be an excellent hybridization indicator.
Human Breast Milk Proteomics for Cancer Biomarkers Discovery
Roshanak Aslebagh, Clarkson University, 8 Clarkson Ave., Chemistry
Department, Box 5810, Potsdam, NY 13699, Devika Channaveerappa,
Armand G. Ngounou Wetie, Costel C. Darie, Sallie S. Schneider, Susan
R. Sturgeon, Kathleen F. Arcaro
Early detection and treatment of breast cancer is a challenging subject of study
since breast cancer is a major cause of morbidity and mortality in the United States.
Mammography has greatly increased the early detection of breast cancer, but has
known limitations, especially in younger women with dense breasts. Breast milk
can be an appropriate option for identifying breast cancer biomarkers because it
provides access to breast tissue, in the form of exfoliated epithelial cells, that when
combined with mass spectrometry-based proteomics may offer a novel way to identify young women at increased risk of developing breast cancer. In this study, breast
milk samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained by Coomassie for qualitative analysis and direct
comparison of the samples. Then the gel lanes of cancerous and healthy sample
(as a control) were cut into several bands which were digested with trypsin and
then analyzed by nanoliquid chromatography-tandem mass spectrometry (nanoLCMS-MS) using a nanoAcquity ultra-pressure liquid chromatography coupled with
quadrupole time-of-flight (Q-TOF) micro mass spectrometer. Data processing and
analysis were performed using MassLynx (version 4.1), ProteinLynx Global Served
(PLGS 2.4), Mascot and Scaffold 4.0. In the Coomassie-stained SDS-PAGE, although the overall protein patterns of most of the major bands are similar, sample
specific-bands are also present which make the differences between cancerous and
healthy samples. These outcomes are currently being investigated.
Withdrawn by the author.
Unknown Identification via Vibrational Spectroscopy Coupling
with Databases and Analysis Algorithms
Dana Garcia, Arkema, 900 First Ave., King of Prussia, PA 19406, Farrel
Borden, Marie Scandone
Identification, classification, and interpretation of vibrational spectra present ongoing challenges. The objective of coupling vibrational spectroscopy with databases
and analysis algorithms is to minimize experimental work, analyze small/intractable
samples and maximize the use of data mining tools. The toolboxes currently available include large cheminformatics spectral and property databases and specialized
algorithms for queering optimization based on spectra and property. Additionally
chemometrics principal component analysis methods, mixture analysis and advanced visualization tools can be incorporated within the toolbox repertoire. In this
presentation we provide examples of solutions to multi-components identification
based solely on effective utilization of computational methods. The flowchart approach provides a logical protocol for efficient utilization of available tools.
The Separation and Identification of Omega-3 Fatty Acids in
Commercially Available Fish Oil Products Using Capillary
Pasquale W. Carione, Drexel University, 3141 Chestnut St., Philadelphia,
PA 19104, Donna M. Blackney, Joe P. Foley
Fish oil supplements are primarily composed of polyunsaturated omega-3 fatty acids that possess multiple double bonds starting at the third carbon from the methyl
group. The two main omega-3 fatty acids in fish oil supplements are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Capillary electrophoresis was
used to separate EPA and DHA from each other and from other components found
in commercially available oil capsules. The two fish oil supplement brands studied,
NatureMade and CVS, do not report the exact quantities of EPA and DHA present
in each tablet; only the total amount of EPA and DHA combined is reported. These
products also report an amount of “other” omega-3 fatty acids on the product label without reporting the names or individual amounts of each of these fatty acids.
Therefore, a quantitative study of the individual amounts of EPA and DHA, as well
as the “other” fatty acids was initiated.
Monmouth County Coastal Lakes: Analysis of Nutrients, Dissolved
Oxygen, and Salinity
Ellen Rubinstein, Monmouth University, 400 Cedar Ave., West Long
Branch, NJ 07746, Katlynn Muratore, Payal Patel
Due to concerns based upon 2012 studies, which found higher than minimum eutrophic level nitrate concentrations in several Monmouth County coastal la-kes, monitoring of these lakes has been continued and expanded. Potential negative effects
on the aquatic ecosystem include damage to fish populations and pollution of the
ocean, which is in close proximity to the eastern ends of these lakes. Dissolved oxygen and total salinity analysis were incorporated in 2013; our 2014 studies added
phosphate measurements. Narrow land masses separate these coastal lakes from
the ocean. In October of 2012, breaches occurred in several locations due to the
impact of Superstorm Sandy. This year, we returned to a few lakes not studied since
the summer of 2012, to examine recovery by comparing current nutrient and pH values in these lakes with pre-storm data. Samples were collected at the eastern ends
of the lakes, since the outflow will affect the ocean. Vernier sensors were used, in
conjunction with a LabQuest2, to measure dissolved oxygen, pH, and temperature
on-site. Vernier electrodes were used in the laboratory, with the LoggerPro comput-
Simplified Strategy to Develop a Robust UHPLC-MS-MS Assay with
Emphasis on Assay Performance and Metabolite Interference
Adela Buzescu, Bristol-Myers Squibb, Route 206 and Province Line
Rd., Princeton, NJ 08540, Naiyu Zheng, Jianing Zeng, Anne-Francoise
Aubry, Mark Arnold
In regulated liquid chromatography tandem mass spectrometry (LC-MS-MS) bioanalysis, the development of a robust assay is very desirable. A simplified method
screening strategy was utilized for ultra high-pressure (UHPLC-MS-MS) method
development to remove phospholipids, minimize metabolite interference and ensure assay’s ruggedness. As compared to a systematic screening method, this
2014 EAS Abstracts
November 2014
er program, to determine concentrations of nitrate and phosphate ions and salinity.
Contributions to the understanding of the health of Monmouth County lakes are
made via these analyses. Future studies may benefit from sample collection and
data analysis throughout the year, to examine possible changes in these parameters with temperature over time. Where continued high levels of nutrients or low
levels of dissolved oxygen are measured, efforts at remediation may be indicated.
solution to create PMMA films having various void volumes. A porous PMMA film
that originally contained 50% by weight PDLL had a 3.7 times larger QCM sensitivity for water vapor than a nonporous PMMA film that contained the same weight
of PMMA.
Factors that Contribute to the Air Pressure Dependence of Particle
Size Analysis by Laser Diffraction
Zheng (Eric) Li, Ashland Specialty Ingredients, 1005 Route 202/206,
Bridgewater, NJ 08807, William Thompson, Guanglou Cheng, Chi-San
Laser diffraction is currently the prevailing technology for particle size analyzing,
comparing to its wet measurement method, the dry measurement method is often
preferred for simpler sampling procedure and less variables introduced. In the dry
method set-up, particle breakage may occur due to the energy introduced by excessive air pressure during the sample dispersion process, results in the dependence
of particle size and distribution on the amount of air pressure applied. For a certain
sample, choosing the proper air pressure becomes the key to establish the standard
operating procedure (SOP) to determine the particle size accurately and precisely,
which is crucial for batch-to-batch quality control. Ideally, a proper pressure should
be sufficient to fully disperse the particles, without breaking them; however, this
proves to a difficult task because it largely depends on the property of the samples,
including the chemical nature, the size and the morphology, etc. More importantly,
it is found in this study that the difference in equipment design plays inevitable role
on this dependence particle size analysis on air pressure. The goal of this study is
to find the “true” particle size using laser diffraction technique. Materials of different
chemical nature, size and morphology were studied, and results by equipment from
different vendors (Horiba, Malvern, Sympatec, etc.) were compared and discussed
regarding to this issue.
Low Temperature 13C Nuclear Magnetic Resonance to Explore the
Formation and Structure of a Carbamoyllithium Anion Used in
Diastereoselective Synthesis of Alpha-Amino Amides
Scot Campbell, Boehringer Ingelheim Pharmaceuticals, 39 Briar Ridge
Rd., Danbury, CT 06810, Nina Gonnella, Jonathan Reeves, Keith
Carbamoyl anions, generated from N,N-disubstituted formamides and linear discriminant analysis, add with high diastereoselectivity to chiral N-sulfinyl aldimines
and ketimines to provide alpha-amino amides. The methodology enables direct
highly diastereoselective carbonyl introduction. This asymmetric addition reaction
is efficient and gives direct access to a wide range of different substituted amido
amides as well as to dipeptides. Low temperature 13C nuclear magnetic resonance
and density functional theory computational studies were carried out to investigate
the nature of the reactive carbene intermediate. The data support formation of a
lithium coordinated oxyanionic carbene structure.
Automated Online Desorption and Analysis of DNPH Derivatives of
Airborne Aldehydes and Ketones
Fredrick D. Foster, Gerstel, 701 Digital Dr., Ste. J, Linthicum, MD 21090,
John R. Stuff, Kurt C. Thaxton, Edward A. Pfannkoch
The analysis of airborne aldehydes and ketones first involves collection of the
analytes by passing air through a cartridge containing 2,4-dinitrophenylhydrazine
(DNPH). As the air passes through the cartridge, the analytes react with the DNPH
to form hydrazones which are immobilized on the cartridge. The cartridges are then
eluted with solvent and the DNPH derivatives can be analyzed using high-performance liquid chromatography (HPLC) with UV detection. Using the GERSTEL
MPS2-xt robotic sampler with a special tray to hold DNPH cartridges, the entire process of desorbing the analytes and injection of the samples into the LC-UV system
can be easily controlled. Automating the desorption of these cartridges can result in
significant improvement in accuracy and reproducibility as well as reducing potential
experimental errors by the operator. The intuitive software includes tools that allow
the desorption of a cartridge to take place during the chromatographic separation of
a previously injected sample to ensure maximum sample throughput. In this report,
the automation of the online desorption and analysis of airborne DNPH derivatives
by the robotic autosampler is discussed. Examination and calibration for a variety
of aldehyde- and ketone-DNPH derivatives is described. Finally, DNPH cartridges
collected from representative matrices are desorbed and analyzed online and the
resulting precision data are provided.
Accurate Determination of Moisture Content of Soft Contact
Lenses by Near-Infrared (NIR) Spectroscopy
Keith Freel, Metrohm USA, 6555 Pelican Creek Cir., Riverview, FL
33578, Hari Narayanan
Moisture content determination is a popular application of near-infrared (NIR)
spectroscopy due to the strong absorption bands of water near 1450 nm and 1940
nm. The delicate properties of soft contact lenses require a minimal-contact method for analysis to avoid damage to the lens itself. NIR spectroscopy provides a
quick and damage-free solution. Reference values used for calibration were based
on gravimetric analysis. To improve the accuracy water content of contact lenses
were determined using a KF titration with oven sampling. The water content of the
lens samples ranged from 24% to 58%. A quantitative model was developed using
chemometric software using the water absorption band at 1450 nm. A partial least
squares regression model was developed to correlate the spectra profile with embedded moisture percentage. A variety of reflectance and transmission sampling
presentations were tested and the accuracy and the precision of the sampling methods were compared. Details of correlation values and the standard error of calibration for each sample presentation are summarized.
Portable Raman Spectroscopy for Monitoring Polymorphic
Philip Zhou, B&W Tek, 19 Shea Way, Ste. 301, Newark, DE 19713
Portable Raman spectroscopy is used as process analytical technology (PAT)
tool for online continuous monitoring of chemical reactions as well as polymorph
transformations. Citric acid is studied here as a model system because citric acid
crystalline powder has two polymorph phases monohydrate and anhydrous, which
crystallize from water at different temperatures. With appropriate portable Raman
sampling methods, the crystalline forms Raman spectra can be measured and thus
by comparing these Raman spectra with standard signatures from pure known samples, the crystalline material formed can be qualified, and even quantified when a
chemometric model is well established. Various sampling methods are provided to
accord with the real situation showing the versatility of portable Raman for in-situ
Combined Headspace Karl Fischer and NIR for Quick, Safe, and
Reliable Method Development, Validation, and Routine Analysis as
Demonstrated with Lyophilized Products
Keith Freel, Metrohm USA, 6555 Pelican Creek Cir., Riverview, FL
33578, Hari Narayanan, Denise Root, George E. Porter III
Near-infrared (NIR) spectroscopy has been used for decades as a rapid and cost effective tool for determining the water content in numerous different products across
a wide range of industries. The most common challenge of NIR testing is creating a
new quantitation model when the matrix or packaging goes through a change. This
time-consuming process usually involves creating a number of samples, collecting
NIR spectra and then analyzing by Karl Fischer (KF) for water content. In this poster,
we show that by mounting a NIR probe on the turntable of a KF oven, we are able
to streamline this process. By integrating NIR and headspace KF, there is immense
time savings, higher productivity and more reliable results than the former method.
This efficiency and accuracy of this method was demonstrated with the quantitative
analysis of moisture in lyophilized products. NIR has no sample prep and is fully
compatible with glass vials making it the perfect candidate for rapid screening of
mass produced freeze dryer vials. The quantitative NIR model for lyophilized products was developed and validated based on the KF values. The error of prediction
was 0.3% within the range 0.9 – 5.3%. A comparison of volumetric vs. coulometric
titration as the primary method is compared, and method details and cost savings
are discussed.
Unique Porous QCM Sensor Coatings: A High Sensitivity Vapor
Sensor Based on Porous Poly(Methyl Methacrylate)
Ho Yeon Yoo, Stanley Bruckenstein Chemical Consulting, 115 Foxpoint
W, Williamsville, NY 14221, Stanley Bruckenstein
A unique way is described on how to create porous films on a solid substrate. This
method is advantageous particularly when a quartz crystal microbalance (QCM) is
the basis of a sensor. QCMs coated with films having large porosities produce more
sensitive sensors than those coated with nonporous films. Here, an initially nonporous Poly(methyl methacrylate) (PMMA) film was used to demonstrate our technique for the model system of water vapor analysis in flowing nitrogen gas. A film of
nonporous PMMA on a QCM is a sensor for water vapor in a gas phase. However,
a much more sensitive sensor was created by dip coating a 10 MHz AT cut quartz
QCM crystal into solutions containing mixtures of PMMA and PDLL(Poly(D,L-Lactide)), and then evaporating the solution on the QCM. This forms mixed polymer
films of varying PDLL content. The PDLL was then leached by exposure to a NaOH
2014 EAS Abstracts
November 2014
Three-Dimensional Solution Structure of Cyclic Antibiotic
Teicoplanin Aglycone Using NMR Distance and Dihedral Angle
Restraints in a DMSO Solvation Model
Nina C. Gonnella, Boehringer Ingelheim, 900 Ridgebury Rd., Ridgefield,
CT 06877, Nelu Grinberg, Shengli Ma, Mark Mcloughlin
Teicoplanin is a potent antibiotic existing as a mixture of several glycopeptide like
antibiotics which all share a common core system. [1] These compounds are members of the vancomycin group of glycopeptide antibiotics. A fused ring structure
forms the common core of the teicoplanin antibiotic family that is termed teicoplanin
aglycone. [2] This work describes the three-dimensional solution structure of teicoplanin aglycone in dimethylsulphoxide (DMSO) using nuclear magnetic resonance
(NMR) spectroscopy. The structure was derived from NMR inter proton distances and targeted dihedral angle restraints based on measured coupling constants.
Structures were generated using systematic searches of conformational space for
optimal satisfaction of distance and dihedral angle restraints. Comparison of the
NMR derived structure with the X-ray structure of a teicoplanin aglycone analog
revealed a common backbone conformation with small deviation of two aromatic
side chain substituents. The results revealed flexibility of the core aromatic side
chain consistent with the region of the molecule involved in protein binding. Overall
the results showed that a combination of NMR derived restraints and a hydrogen
bonded DMSO solvation model were needed to generate structures that satisfied
experimental data.
Comparison of FT-NIR and NMR Spectral Analysis
Herman He, Thermo Fisher Scientific, 4410 Lottsford Vista Rd.,
Lanham, MD 20706
Fourier transform near-infrared (FT-NIR) and nuclear magnetic resonance (NMR)
spectroscopy are the popular analytical tools for many chemists because of its
versatility in elucidating molecular structure, optimizing reaction dynamics, measuring reaction kinetics, monitoring reaction content and controlling product purity.
These two technologies are both nondestructive and complimentary to each other;
and provide a wealth of information on physical, chemical and structural aspects
of molecular systems. This poster presents spectral data of common liquids and
application in wine analysis using a Thermo Antaris FT-NIR analyzer and a Thermo
picoSpin Portable NMR spectrometer.
Stereochemical Analysis of Organophosphates
Kristi Tami, John Jay College, Science Department, 524 West 59th St.,
New York, NY 10019, Cristina Kinahan, Gloria Proni
Many commercially available organophosphorus (OP) insecticides are chiral, yet
they are sold in racemic mixtures. Since enantiomers possess different levels of
toxicity on the various pests they target, it is prudent to isolate each enantiomer
and test its relative toxicity in order to find out which enantiomer is most effective.
Then, only the more effective enantiomer should be used to eliminate the target
pests. High-performance liquid chromatography (HPLC) was used to separate the
enantiomers of acephate, methamidophos, and N-methyl methamidophos. The column used to separate the enantiomers was a ChiralcelВ® OJ-H 250 mm x 20 mm ID.
Three complimentary spectroscopic techniques were used to determine the absolute configuration of the enantiomers: Optical rotatory dispersion (ORD), electronic
circular dichroism (ECD), and vibrational circular dichroism (VCD). Three separate
techniques were used in order to ensure a higher degree of confidence. Time-dependent density functional theory (TD-DFT) was used to generate theoretical ORD,
ECD, and VCD data. Once the absolute configurations were established, biological
assays were performed in order to determine the relative toxicity of the enantiomers
compared to the racemic mixture.
[1] Hunt, A. H., et. al. J. Am. Chem. Soc. 1984, 106, 4891.
[2] Barna, J. C. J, et. al. J. Am. Chem. Soc. 1984, 106, 4895.
Withdrawn by the author.
Quantitative Trace Metals Analysis Using Hydrofluoric Acid
Jane Ramsey, E. I. du Pont de Nemours and Company, PO Box 80228,
Wilmington, DE 19880, Thomas Connell, Daniel Iversen, Robert
Lockerman, Michael Karney
Hydrofluroic acid (HF) is one of the most dangerous and corrosive acids found in today’s chemistry laboratories. Despite the hazards, HF is necessary for the complete
dissolution of many samples, most commonly those containing silicon, titanium, or
zirconium. Only experienced and trained laboratory personnel should attempt to use
HF in their protocols due to the severity of burns. HF will readily penetrate the skin
and react with calcium in the blood, leading to cardiac arrest. In previous efforts,
researchers from CEM and DuPont worked together to evaluate fluoride surrogates
other than HF to provide total dissolution of the sample matrix. Our team used titanium, silicon and zirconium dioxide as target analytes for this experiment. Ammonium
bifluoride (NH4HF2) and fluoroboric acid (HBF4) were chosen as fluoride sources
and validated against digestions with HF. The results indicated that the HF, HBF4,
and NH4HF2 preparations were statistically the same for the titanium and silicon
measurements. Zirconium was less promising as it did not go into solution with the
HBF4 and had lower than expected recoveries with the NH4HF2. It was determined
that further investigation was required. Our current efforts focus on providing optimized conditions for the dissolution of titanium and silicon dioxide as well as to
provide a better digestion for zirconium. These optimized methods are then applied
to reference materials containing trace amounts of our analytes of interest to provide
a general procedure for analysis using hydrofluoric acid alternatives.
Withdrawn by the author.
SERS Detection and Characterization of Gold Nanoparticles on
Spinach Leaves
Zhiyun Zhang, University of Massachusetts-Amherst, Department
of Food Science, 240 Chenoweth Laboratory, 100 Holdsworth Way,
Amherst, MA 01003, Lili He
The increased prevalence of engineered nanomaterials in environment and their
potential toxicity urge researchers to study whether those engineered nanomaterials
could possibly contaminate agricultural food products. However, many techniques
require invasive and complicated sample preparation procedures to detect and
characterize engineered nanomaterials. In this study, we aim to explore the feasibility of surface enhanced Raman spectroscopic (SERS) mapping to detect and characterize gold nanoparticles (GNPs), as the model engineered nanomaterials, on
spinach leaves. SERS is a technique that studies the enhanced Raman scattering
signals on noble metal nanostructures. We prepared various sizes of GNPs (15, 30,
50, 90, and 150 nm) and characterized their background and enhancement signals
on chlorophyll, the major pigment in spinach leaves. Results show SERS was able
to detect the background and the chlorophyll signals from the GNPs of 30 to 90 nm,
and the 50 nm GNPs produced the highest signal. Then, we deposited 10 Вµl (1010^5 mg/L) 50 nm GNPs onto the surface of spinach leaves, and detected and characterized these GNPs using optical light scattering and SERS mapping before and
after washing. The results showed that 1) GNPs were able to attach on the surface
of spinach leaves even after washing and their distribution were heterogeneous; 2)
characteristic peaks, like the chlorophyll peak, could be observed, which indicates
molecular interaction between GNPs and surface leaf cell bio-components. These
results demonstrated the feasibility of SERS for detecting and characterizing GNPs
contaminates on plant leaves.
NMR Analysis of Synthetic Polymers Utilized in Medical Devices
Robert A. Schiksnis, Johnson and Johnson Global Surgery Group, PO
Box 151, Rte. 22 West, Somerville, NJ 08876, Wendy Feng
Johnson & Johnson Global Surgery Group is a leading producer of surgical care
products. It manufactures various medical devices and biologics products such as
sutures, meshes, breast implants, hemostasis and antimicrobial devices, etc. These
diverse product lines involve wide ranges of materials made using a variety processes. Suitable analytical techniques and critical in-house expertise are required
to support new product development and product life cycle management. Synthetic polymers produced for use in our medical devices undergo extensive chemical
characterization. Solution nuclear magnetic resonance (NMR) spectroscopy is an
enabling technology utilized regularly to analyze a polymer’s chemical composition,
sequence distribution, and minor component profile. These parameters, amongst
other chemical and physical characteristics, are experimentally determined and
evaluated throughout the product development process. We also use NMR to detect, identify, and quantify new chemical species that could be produced by specific
polymer processing steps. An example is given describing the NMR characterization
of a polymeric device component that undergoes laser processing.
Wide Field Raman Chemical Imaging for Determination of Content
Uniformity in Pharmaceutical Blends
Lauren Seabrooks, Merck, 181 Passaic Ave., Summit, NJ 07901, Justin
Content uniformity is a critical quality attribute measured and monitored throughout
the development and commercial supply of pharmaceutical products. Herein we
report the successful utilization of wide field Raman chemical imaging to assess
content uniformity. This approach is non-destructive, obviates the need for sample preparation and is highly sensitive allowing sample sizes of less than 3 mg.
Furthermore, unlike previous reports, a direct sample to sample model approach
was leveraged through first scanning the samples for Raman spectra and then diluting to volume for quantitative analysis. Model blends included caffeine/lactose
and albuterol sulfate/lactose. Additionally, each blend included the following active
2014 EAS Abstracts
November 2014
to excipient ratios: 1:3; 1:1, and 3:1. Quantitative analysis was achieved using ultra
high-performance liquid chromatography-mass spectrometry (UHPLC-MS) as the
model comparison method to develop multivariate calibration based models from
Raman spectra. In each case a partial least squares (PLS) algorithm provided the
most robust and accurate model with r-squared values greater than 0.9 and rootmean-square error (RMSE) for calibration, cross-validation and prediction of approximately 0.1.
Comprehensive Ion Analysis of Various Water Matrices in the
Hydraulic Fracturing Process
Stuart J. Procter, Metrohm USA, 6555 Pelican Creek Cir., Riverview, FL
33578, Jay Gandhi, Anne Shearrow
Oil and natural gas are fossil fuels that are a valuable diminishing resource that
are utilized for energy and materials production. These naturally occurring products cannot be easily replenished, so to maintain a local and sustainable source,
fracking is becoming of increasing importance to the USA’s long term economic
stability. Fracking technology represents a doorway to vast untapped reserves, but
with it comes environmental concerns and analytical challenges that need to be addressed moving forward. Therefor analytical techniques for the long term monitoring
of ground and surface water is important for environmental conservation, as well
as the analysis off back waters and discharge water from the fracking process that
can have up to 30% sodium chloride as an ionic matrix. In this poster presentation,
chemical analysis methodologies using ion analytical instruments are discussed.
The new methods enable the simultaneous analysis of anions, cations and organic
acids in a 30% sodium chloride matrix that are of environmental interest.
Chemical Analysis and Characterization of Dried Figs and Dried
Jerzy Mierzwa, Tennessee State University, Chemistry Department,
3500 John A. Merritt Blvd., Nashville, TN 37209
Dried dates and figs are very popular dried fruits for snacking. The author of this
analytical study has carried out chemical analysis, characterization and a comparison of several samples of dried figs and dates (samples of different origins).
The concentration of twelve metals was determined in digested samples by atomic
absorption spectrometry (AAS) and inductively coupled plasma – optical emission
spectrometry (ICP-OES). Several organic compounds belonging to certain groups
(e.g. sugars, vitamins) were analyzed by HPLC (reverse-phase gradient chromatography, standard C18 column, and UV/Visible absorption detector) and some trace
level compounds were also analyzed by gas chromatography mass spectrometry
(with ion-trap mass spectrometer). Moreover, these samples were examined by
FTIR spectrometry and UV-Visible spectrophotometry. The results of this comparative analysis are summarized, and some basic analytical figures-of-merit are presented. A possibility of a reliable geographic fingerprinting of the samples is also
Developing Mobile LIBS Solutions for Real World Applications
Qun Li, B&W Tek, 19 Shea Way, Ste. 301, Newark, DE 19713, Katherine
Bakeev, Jing Li, Sean Wang
We present a new type of handheld laser-induced breakdown spectroscopy (LIBS)
spectrometer, NanoLIBS for developing mobile solutions for real world applications.
A micro diode-pumped passive Q-switched solid-state laser with high repetition rate
of well above 1kHz in comparison to 1-10Hz as used in a traditional LIBS instrument
is employed to produce a train of laser pulses. The focused laser spot is further fast
scanned over a pre-defined area, hence generating several hundreds of micro-plasmas per second at different locations. Synchronized miniature CCD array spectrometer modules collect and generate pre-conditioned LIBS spectra. The NanoLIBS is
measured at less than 1.8kg and operable for more than four hours with embedded
batteries. The limits of detection are in the range of 1 to 1,000ppm, which is element
and matrix dependent. The typical measurement time is within 1-2 second. The
handheld LIBS spectrometer has a Linux based operation system, NanoLIBS Operation System (NOS). NanoLIBS is an open platform for developing mobile LIBS
solutions. For a custom analyzer for certain specific applications, the user will: 1)
collect the LIBS data using NanoLIBS; 2) export the collected LIBS data to a PC; 3)
using the chemometrics software, BWIQ for model and calibration development; 4)
conversion under NOS with custom graphic user interface (GUI). Some examples
of successful application development are presented.
Spectral Studies of Dipicolinic Acid with Bovine Serum Albumin
Anne E. Okafor, St. John’s University, 8000 Utopia Pkwy., Queens, NY
11439, Enju Wang, Neil Jespersen
Dipicolinic acid (DPA) 2,6 pyridinedicarboxylic acid, is a common chemical compound of bacterial spores. Endorspores typically contain 2 to 15% of DPA by dry
weight. Endospores can germinate into active cell when the appropriate nutrients
and environmental conditions suitable for spore germination are present. The
presence of spores is sensitively detected through the concentration of DPA. In
this present work we investigate whether DPA will alter the conformation of bovine
serum albumin, (BSA). BSA is the most abundant protein in the plasma. It has a
strong affinity to reversibly bind to large number of endogenous and exogenous
substances. It has very many molecular functions. Serum albumins are special carriers that chaperones important nutrient not soluble in water to hungry cells. BSA is
widely used in the laboratory when a generic protein is needed. BSA contribute to
colloid osmotic blood pressure and are chiefly responsible for the maintenance of
blood pH. BSA has been used as a model protein to study protein aggregation and
for biotechnological folding. This present work seeks to investigate the interaction
of DPA and its binding mode in BSA. The study is carried out using fluorescence
spectroscopy, Fourier transform infrared, and ultraviolet-visible spectroscopies. This
study provides insight into the potential toxicity between DPA and the protein. Spectral changes of BSA with DPA are looked into. The shift of fluorescence wavelength
of BSA with DPA is seen as an interaction of the protein conformational changes.
Localized Surface Plasmon Resonance of Gold Nanoparticle
Random Arrays
Julie A. Jenkins, University of Connecticut, 55 North Eagleville Rd.,
Storrs, CT 06238, Sravan Thota, Xiangdong Tian, Xiaowen Zhao, Jing
Zhao, Yadong Zhou, Shengli Zou
When electromagnetic radiation interacts with a metal nanoparticle, it excites the
particle and causes the conduction electrons to oscillate collectively, known as localized surface plasmon resonance (LSPR). The shape and size of the nanoparticle,
as well as the local dielectric environment around the nanoparticle will affect the
LSPR peak. Therefore, molecular binding with metal nanoparticles can be used
as an optical sensing technique. In this work, a highly uniform, LSPR substrate
was produced based on the self-assembly of colloidal gold nanoparticles onto a
pretreated glass substrate. LSPR wavelength of the gold nanoparticle random array
blue-shifts and the LSPR band width narrows compared to that of the single gold
nanoparticle on glass substrate as well as the gold nanoparticles in solution, resulted from dipole interaction between the nanoparticles. It was also found that not
only is the LSPR wavelength sensitive to changes in the dielectric environment, but
the LSPR peak width as well. The LSPR substrates are reproducible, uniform and
robust with potential applications in LSPR sensing and imaging.
Chiral Analysis by Fourier Transform Rotational Spectroscopy
Brooks H. Pate, University of Virginia, McCormick Rd., Charlottesville,
VA 22904, Kevin K Lehmann, Simon Lobsiger, Cristobal Perez, Luca
A new spectroscopic method to measure the enantiomeric excess using molecular rotational spectroscopy was introduced by Patterson, Schnell, and Doyle in
2013 [Nature 497, 475–477 (2013)]. The technique uses the fact that enantiomers
can be distinguished by the way that the dipole moment projects onto the principal
axis system for molecular rotation. Three wave mixing experiments are performed
that generate a molecular signal proportional to the product of the dipole moment
components. The enantiomers differ in the sign of this product and the three wave
mixing signal measured using Fourier transform spectroscopy has different signal
phases for the enantiomers. A new method for generating the optimal signal using
time-separated microwave pulses is demonstrated in measurements of solketal.
The measurement uses high-resolution spectroscopy so that chiral analysis can
be performed directly in a complex sample matrix. High-resolution spectroscopy
also makes it possible to perform chiral analysis on the large number of stereosiomers that are possible for a molecule with multiple chiral centers without requiring
chromatographic separation. The technique has the potential determine the absolute configuration of the molecule by determining the absolute signal phase – an
experimental challenge that is currently being tackled – and using relatively simple
quantum chemistry calculations of the molecular dipole moment.
Determination of BPA in Aqueous Solution Using Fluorescence
Sarah J. Pereira, University of Massachusetts Dartmouth, 789 Wilbur
Ave., Swansea, MA 02777, Yuegang Zuo
Bisphenol A (BPA) is a compound widely used in plastics and other industrial consumer products. However, BPA has been found to show toxicity as well as endocrine
disrupting effects and so release of BPA into the aqueous environment has been of
great concern. Methods of detection of BPA in aqueous medium have, therefore, become of great importance. Current detection methods of BPA are slow, so a quicker
method using fluorescence to measure concentration of BPA is being developed. It
has been reported that BPA shows fluorescence in organic solvent but is too low to
be detected in aqueous solution. (Olmo, 2000). A method for using florescence to
measure BPA in aqueous solvent must then be developed. In this study the fluorescence of BPA in acetonitrile (ACN) and water were compared and solutions of BPA
made from solvents with varying percent of acetonitrile to water were measured to
determine the effect of water on the fluorescence of BPA in acetonitrile solution. It
was found that BPA does show fluorescence in water but is low, especially com-
2014 EAS Abstracts
November 2014
pared to ACN. The results also showed that fluorescence increases slightly from
pure ACN as water is added but decreases once the solvent is more than 20%
water. If fluorescence detection is to be used to detect BPA in real water samples
then the method must be able to distinguish between BPA and other compounds
with similar structure. To do this constant-wavelength synchronous fluorescence
spectroscopy was used.
Effect of Split Ratio on USEPA Method 8260 Compounds
Anne Jurek, EST Analytical, 503 Commercial Dr., Fairfield, OH 45014,
Mike Moses, Lindsey Pyron, Kelly Cravenor
United State Environmental Protection Agency (USEPA) Method 8260 involves
purging analytes out of a water matrix. During a split injection, the sample volatilizes
in the inlet and is swept by the carrier gas through the liner onto the gas chromatography (GC) column with a portion of the sample being split off and sent out the
split vent line. The amount of sample reaching the detector is dependent upon the
split flow rate. Thus, the higher the split rate, the smaller amount of sample on the
column. Furthermore, a higher split ratio minimizes column exposure to moisture.
During purge and trap sampling moisture control is crucial to decreasing split flow
and enhancing detection limits. This application explores the effect split ratios have
upon USEPA Method 8260 analytes.
Coupled Exciton and Charge-Transfer Resonances in the Raman
Enhancement of Phonon Modes of CdSe Quantum Dots (QDs)
Syed K. Islam, City College, City University of New York, 160 Convent
Ave., New York, NY 10031, John R. Lombardi, Mohammad A. Sohel
We report the observation of the enhancement of a (TO) phonon mode of e1 symmetry and a normally forbidden (SO) phonon mode of b1 symmetry in CdSe quantum dots (QDs) due to adsorption of 4-mercaptopyridine molecules. The former is
observed in 3, 4, and 5 nm diameter particles, while the latter is observed only in the
2 nm particles. Maximum enhancement of the phonon modes is obtained through
a coupling of the charge-transfer transition and the exciton transition which are in
resonance with the laser energy. Selection rules using Herzberg-Teller (vibronic)
coupling are invoked to explain the observed enhancements.
Synthesis, Spectroscopic and Electrochemical Characterization
and Catalytic Activity of Mn(III) Complexes of Tridentate Schiff
Base for the Epoxidation of Olefins
Rita N. Egekenze, Howard University, 525 College St. NW, Washington
DC 20059, Yilma Gultneh
The Mn(III) complexes of tridentate Schiff-base ligands of 2-aminoethyl-2-pyridine
with 5-methoxy-, 4,6- dimethoxy-, 5-bromo-, 3,5-dibromo– and nonsubstituted-salicylaldehyde (complexes 1, 2, 3, 4 and 5 respectively) were prepared and characterized by Fourier transform infrared, UV-Visible spectroscopy, liquid chromatography mass spectrometry (LC-MS) and thermal gravimetry.X-ray crystallographic
structures showed six-coordinate ML2 (L = tridentate Schiff base) complexes. In
their cyclic voltammetry in acetonitrile solution, the Mn(III) complexes of the Schiff
bases each showed one reversible redox process in the -1.5 to +1.5 V (vs Ag/AgCl)
window (Mn(III) ⇔ Mn(II)). The thermogravimetry show that the decomposition
takes place in three steps for all complexes. Evaluation of the catalytic activity of
the Mn (III) complexes for the epoxidation of olefins recorded higher yield than in
the absence of the complexes. UV-visible absorption studies of the reactions of
the complexes with aqueous H2O2 at room temperature and in dichloromethane
showed appearance of new peaks at 255 and [323-332] indicating the formation
Mn-peroxo and Mn-oxo complexes that are the oxygen atom transfer intermediates
leading to the epoxidation of the olefin. We report the crystal structures, LC-MS
result, cyclic voltammogram, UV-visible absorption spectra, 1HNMR and gas chromatography-MS results for the catalytic epoxidation experiment.
New Techniques for the Analysis of Volatiles and Gases: FT-MRR
Spectroscopy (Fourier Transform Molecular Rotational Resonance)
Justin L. Neill, BrightSpec, 770 Harris St., #104 B, Charlottesville, VA
22903, Robin L. Pulliam, Roger Reynolds, Matthew T. Muckle, Brent J.
Harris, Dave McDaniel, Brooks H. Pate
For gas mixture analysis, there is a need for new instruments that offer standalone
operation in combination with broad composition analysis capabilities. Infrared
based detectors come with constraints in selectivity and chemical coverage, and
gas chromatography techniques come with the need for a dedicated analyst. A new
commercial molecular spectroscopy technique, FT-MRR, is capable of direct, quantitative gas mixture analysis without chromatography. The FT-MRR spectral fingerprint is specific to the unique mass distribution of a molecule (including its isotopes)
and arises from the quantized rotational energy levels. It is also a high resolution
technique that offers superior chemical selectivity over infrared spectroscopy for
complex mixture analysis. Without the need for optical cavities, a broadband FTMRR instrument can monitor a wide range of volatile species, and is reprogrammable in order to incorporate new compounds. The detection limits for small, polar
molecules (e.g., hydrogen cyanide, ammonia, hydrogen sulfide, formaldehyde, water) in direct sampling are in the parts-per-billion (ppb), with parts-per-trillion (ppt)
attainable with a pre-concentration sampling system. FT-MRR performance for
quantitative analysis using calibration standards in nitrogen will be presented. Sampling methods for direct analysis as well as pre-concentration for trace monitoring of
volatile organic compounds in air are described.
GCxGC-TOFMS with Variable-Energy Electron Ionization for
Enhanced Screening of Pollutants in Complex Environmental
Pete Grosshans, Markes International, 11126-D Kenwood Rd.,
Cincinnati, OH 45242, Laura McGregor, Nick Bukowski, Nicola Watson,
Charles Haws
Comprehensive two-dimensional gas chromatography with time-of-flight mass
spectrometry (GCxGC-TOF MS) offers greatly enhanced peak capacity, through the
coupling of two columns of different selectivity, as well as highly sensitive detection
and confident mass spectral identification of trace-level analytes. Nevertheless, the
identification of individual compounds may be hindered by weak molecular ions or
when similar mass spectral characteristics are evident across entire chemical classes. Select-eV ion source technology aims to combat this problem by allowing both
hard and soft electron ionisation with no inherent loss in sensitivity. The availability
of full-fragmentation spectra for easy library matching is complemented by the ability
of Select-eV to enhance the heavier ions that are critical for confirming compound
identity and distinguishing between similar compounds. At low ionisation energies,
fragmentation is greatly suppressed, resulting in simplified spectra dominated by
structurally-significant ions. Furthermore, the fragment ions that ordinarily dominate
high matrix samples are greatly reduced, enhancing selectivity and further improving detection levels. This work shows the suitability of this novel analytical platform
for the analysis of both target pollutants and unknown chemicals within various environmental matrices.
Mid-Infrared Optical Coherence Tomography System Using a High
Power, Low Coherence Quantum Cascade Superluminescent
Deborah M. Varnell, Princeton University, Department of Electrical
Engineering, Equad, Olden St., Princeton, NJ 08544, Ahmed Musse,
Nyan Aung, Mei Zheng, Germano Penello, Claire Gmachl
Visible and near-infrared (IR) optical coherence tomography (OCT) imaging systems
use light reflected at different depths in a sample to provide high resolution, three-dimensional images and are used extensively in medicine, research and industry.
Using mid-IR light as an OCT source would decrease scattering and give stronger
reflections, and therefore better images, from materials with absorption fingerprints
in the mid-IR; however no mid-IR OCT systems are commercially available, most
likely due to the lack of good sources. Broadband lasers have previously been used
as a source but although they have high enough power to give long depth scans,
they have poor depth resolution due to the long coherence lengths. We demonstrate
progress in developing a mid-IR OCT system using a new high power (3 mW), low
coherence (55 Вµm) super-luminescent emitter to achieve both long depth range and
high depth resolution. A free space OCT system has been designed, assembled and
tested. This work was supported in part by MIRTHE (NSF-ERC).
Residual Solvent Analysis of N-Methylpyrrolidone Using
Headspace Sampling and NPD Detection
Amy F. Birch, Boehringer Ingelheim, 5175 Briar Ridge Rd., Danbury, CT
06810, Johanna Ubben
N-Methylpyrrolidone (NMP) is a class 2 solvent commonly used in pharmaceutical
synthesis, with an International Conference on Harmonization Q3C alert level of
530 ppm. The high boiling point (204 В°C) and low vapor pressure of this compound
allows only a small amount to be partitioned into the headspace for residual solvent analysis by headspace/gas chromatography (GC) analysis at 140 В°C sample
incubation temperature. The literature generally recommends that a liquid injection
technique be used to quantitate NMP for residual solvent analysis. However, liquid
injection of large concentrations of a drug substance can create analyte recovery
challenges due to liner contamination. Headspace sampling is preferential in providing a robust method. A nitrogen specific detector (NPD) can be used to quantitate
Optimizing Bio-Energy Production by Imaging Nutrient Exchange
in the Plant Root Rhizosphere Using Infrared Microspectroscopy
Tiffany Victor, Stony Brook University, 350 Circle Rd., Stony Brook, NY
Plant based biomass is an efficient renewable energy source in the United States,
but there is a limited understanding of the nutrient uptake processes that facilitate
its availability. By studying the nutrient distribution within the plant rhizosphere, qualitative and quantitative information can be gathered to spatially resolve nutrients
example phosphates and nitrates. This project uses an infrared microscopy method
that enables imaging of the nutrient distribution in the rhizosphere to be imaged both
in the presence and absence of growth promoting microbes in order to assess how
different combinations affect the production of biomass.
2014 EAS Abstracts
November 2014
NMP, using a headspace injection technique. The NPD detector is specific to nitrogen compounds and also is more sensitive than the flame ionization (FID) detector
in detecting nitrogen compounds by 1-2 orders of magnitude. Thus, the detector
provides an increase in sensitivity for NMP, while still using a headspace injection
technique to maintain a more robust analytical method. Validation data from a dual
detector (FID/NPD) method is presented here to support the use of an NPD/headspace method for the quantitation of NMP in residual solvent analysis.
logical functions. Gas chromatography – mass spectrometry is the gold standard for
analysis of FAs and FAMEs; however, the differentiation of many isomeric species
requires specialized columns, as the mass spectrometry detector does not provide
sufficient specificity. A vacuum ultraviolet (VUV) detector was recently developed
and tested in fatty acid methyl ester analysis in GC. Here we demonstrate the ability
of GC-VUV as a universal detector to classify fatty acids (e.g., unsaturate vs. saturate, mono-unsaturate vs poly-unsaturate), and differentiate cis/trans isomers (e.g.,
elaidic vs. oleic acid) based on their absorption in the 125 – 240 nm wavelength
range. The analysis of standards as well as real world samples, such as rapeseed
oil demonstrates that less effort is needed to chromatographically separate different
species of interest, due to the greater specificity of the VUV detector.
GCxGC Column Kits Offer Choice Plus Convenience
Len Sidisky, Supelco/Sigma-Aldrich, 595 North Harrison Rd., Bellefonte,
PA 16823, Gustavo Serrano, Dan Shollenberger, Jamie Desorcie, Greg
Baney, Michael Buchanan
Gas chromatography x gas chromatography (GCxGC) is one of the fastest growing
areas in analytical chemistry due to its ability to resolve a large number of compounds, even in the most complex samples. It employs two columns in series, separated by a modulator. One key to the successful operation of GCxGC is that the two
columns must have orthogonal selectivity, that is, they must utilize different retention
mechanisms. Column selection strategies to achieve orthogonal selectivity include:
1) Non-polar to polar strategy. Analytes are separated on a non-polar column in the
first dimension, and on a polar, highly polar, or extremely polar column in the second
dimension. 2) Polar to non-polar strategy. Analytes are separated on a polar, highly
polar, or extremely polar column in the first dimension, and on a non-polar column
in the second dimension. 3) High resolution polar to non-polar strategy. Analytes
are separated on a long (100 m) highly polar or extremely polar column in the first
dimension, and on a non-polar column in the second dimension. Details, benefits,
and GCxGC plots for each of these three column selection strategies are presented.
Fourier Transform Ion Cyclotron Resonance and Infrared
Multiphoton Dissociation Studies of Cadmium Sulfide Metal
Douglas P. Ridge, University of Delaware, Department of Chemistry and
Biochemistry, Newark, DE 19716, Kaitlin Papsin, Una Kim, Giel Berden,
Jos Oomens
Fourier transform ion cyclotron resonance mass spectrometry involves injecting
ions into an electromagnetic trap where they can be detected and mass analyzed
on the basis of their cyclotron frequency. The method facilitates manipulating the
trapped ions in various informative ways. The reactivity of trapped ions can be examined by exposing the trapped ions to reactive gases. Structure and spectroscopic
properties can be examined by subjecting the trapped ions to collisional or photon
induced decomposition. The application of these techniques to the study of cadmium sulfide metal clusters is described. These metal clusters are important in marine
environments both as potentially toxic pollutants and in relation to the exotic biology
found around hydrothermal vents in the ocean floor. Although cadmium sulfide is
insoluble in water metal sulfide clusters can be prepared by electrospraying soluble
metal salts such as cadmium acetate and allowing the resulting cadmium acetate
clusters to react with H2S in the ion trap. These results in clusters containing multiple Cd atoms and bisulfide and sulfide counter ions. The structure of these clusters
have been probed by subjecting them to collision induced decomposition, infrared
multiphoton dissociation, and density functional theory calculations. Among the conclusions is that when excited the clusters tend to lose preferentially metal atoms
forming a series of clusters containing hypervalent polysulfides. The energetics of
these processes and the consequences for marine environments are discussed.
Determination of Natural Gas Components in Drinking Water by
Gas Chromatography and Vacuum Ultraviolet Detection
Ling Bai, University of Texas-Arlington, 700 Planetarium Pl., Rm. 130,
Arlington, TX 76019, Hui Fan, Kevin A. Schug, Jonathan Smuts, Phillip
Walsh, Dale Harrison, Zacariah L. Hildenbrand
Natural gas is a complex mixture consisting primarily of low molecular weight hydrocarbons, inert gases, and other impurities including a variety of sulfur compounds.
Mixtures of fixed gases are typically difficult to speciate by modern capillary gas
chromatography (GC). A new gas chromatography detector based on vacuum
ultraviolet (VUV) spectroscopy (GC-VUV) simultaneously collects full scan (120
– 240 nm) VUV and UV absorption of eluting analytes. GC-VUV can be used to
provide universal detection in combination with high quality analyte-specific absorption spectra. The vacuum ultraviolet spectrum opens up a wide spectrum of
advantages, resolves co-eluting analytes and limits needs to achieve fully resolved
analytes chromatographically. Gas chromatography was performed on a Shimadzu GC-2010, and separations were performed with a HP-PLOT-Q BOND column.
Many sample mixtures of gases were tested, ranging from prepared gas mixtures, to
natural gas samples collected from a laboratory natural gas supply, as well as samples taken from private water wells in close proximity to unconventional natural gas
extraction. Presented will be the ability of VUV detection to analyze natural gas and
natural gas contamination samples, including the potential for direct quantification
based on absorption cross-section of the measured compounds.
Mass Spectrometry of Airborne Particulate Matter: How it Works
and What it Tells Us About the Air We Breathe
Murray Johnston, University of Delaware, Chemistry and Biochemistry
Department, Newark, DE 19716
Our research group has developed a variety of mass spectrometers for on-line
characterization of individual airborne particles. In this presentation, I highlight one
particular aspect of this work, particles smaller than 100 nm in diameter. Nanoparticles are ubiquitous in the atmosphere and are produced by a variety of natural and
anthropogenic processes. These particles represent a potential health hazard to
humans when inhaled, and influence climate by acting as seeds for cloud formation.
Determining the chemical composition of these particles aids identification of sources and assessment of environmental impacts. The results of several measurement
campaigns are summarized, which span both urban and remote locations. These
measurements give insight into the air we breathe and why it matters.
Analysis of a PIONA Sample Using Gas Chromatography and
Vacuum Ultraviolet Spectroscopy
Phillip Walsh, VUV Analytics, 16525 Ennis Trail, Austin, TX 78717,
Jonathan Smuts, Daniel Klopp, Dale Harrison
Analysis of a PIONA standard using gas chromatography coupled with a novel vacuum ultraviolet spectrometer detector are presented. The vacuum ultraviolet detector
generates a three-dimensional dataset consisting of time-resolved 125-240nm absorbance spectra. Generally, vacuum ultraviolet absorbance spectra exhibit a high
level of uniqueness, providing a selective component to gas chromatography data
allowing for deconvolution of co-eluting species. Even in cases where individual
species within a given class are harder to distinguish, vacuum ultraviolet absorbance is often able to distinguish between general classes of analytes. This combination lends itself particularly well to the PIONA analysis, and offers the possibility
of reducing the number of separation steps involved in typical hydrocarbon analysis
of fuels.
Mass Spectrometry to Support Industrial Fermentation
Barbara Larsen, DuPont, Experimental Station E228/120A, 200 Powder
Mill Rd., PO Box 8352, Wilmington, DE 19803
A variety of analytical methods are used to support strain development in metabolic
engineering of organisms for the production of industrial chemicals. Strain engineering requires changes to cell metabolism to direct the carbon flux to produce the
industrially valuable chemical. Mass spectrometric methods have been developed
to ascertain changes in the protein profile through the fermentation process. Proteomics to support the fermentation processes are discussed.
Mass Spectrometry Explores Communication in the Tumor
Catherine Fenselau, University of Maryland, Department of Chemistry
and Biochemistry, Bldg. 091, College Park, MD 20742, Suzanne
Ostrand-Rosenberg, Meghan Burke, Rebecca Rose, Lucia GeisAsteggiante
Communication between cells in the tumor microenvironment is conducted primarily
by chemical moieties that are secreted by cells or transported in exosomes shed
by cells. We have focused on the potential for communication by proteins carried
by exosomes. What proteins are present? How are they modified? What are their
biological activities? We have used tandem high resolution mass spectrometry to
answer the first two questions, combined with various chromatographies, bioinformatics and strategies for sample preparation of proteins from exosomes shed by
Gas Chromatography–Vacuum Ultraviolet Spectroscopy in Fatty
Acid Methyl Ester Analysis
Hui Fan, University of Texas-Arlington, 700 Planetarium Pl., Arlington,
TX 76019, Kevin A. Schug, Jonathan P. Smuts, Phillip Walsh, Dale
Fatty acid methyl esters (FAMEs) are common transformation products of fatty acids, present in biodiesel, various consumer products, and as a common derivatization product of fatty acids for gas chromatography (GC). The long chain components
of these compounds can contain variable degrees of saturation and unsaturation,
the differentiation of which can be critical for assessing health benefits or other bio-
2014 EAS Abstracts
November 2014
myeloid derived suppressor cells collected from tumor-bearing mice. This talk merges the analysis of exosome surface glycoproteins, exosome lysate by top down
and bottom up strategies, and ubiquitinated proteins captured by immunoaffinity, to
provide insight into the formation and some biological activities of these exosomes.
Advances in Applying Near Infrared Measurements to Cell Cultures
and Fermentation Processes
Gary Ritchie, Infratrac, Silver Spring Innovation Center, 8070 Georgia
Ave., Silver Spring, MD 20910
Near-infrared (NIR) spectroscopy has matured as a useful analytical tool for the
measurement and control of biopharmaceutical processes. At the present time, NIR
has both a scientific, risk-based regulatory footing and extensive empirical basis
for supporting the manufacture of biologic drug substances and drug products. Recent publications indicate a surge of interest in the use of this versatile tool for the
production of biologics; yet, very little has been said about how biopharmaceutical manufacturers go about adopting NIR for the production of lifesaving drugs.
Many factors must be considered before a NIR analyzer is put at, in or on-line for
any purpose, but especially in the case of living systems, these considerations are
many and extremely complex. This presentation briefly reviews the progress that
has been made over the last several years for the use of NIR in the production of
biologics and then further presents what should be considered if this tool will have
any future role to play in supporting biopharmaceutical manufacturing. Based on
recent developments in both the regulatory and industrial arenas, this presentation
provides examples that touch upon developments in regulations, equipment, facilities and techniques to offer a better understanding of the potential and limitation that
NIR spectroscopy may hold for the production of biologics in the twenty-first century.
The Incredible Shrinking NIR Instrument; and Why that is Great for
the Pharmaceutical Industry
Emil Ciurczak, Doramaxx Consulting, 77 Park Rd., Goldens Bridge, NY
The near-infrared (NIR) instruments available in 1980 were larger than counter-top
microwave cookers and had to be used in a laboratory setting. As the decades
advanced, not only did the instruments become more rugged, but the sizes also
began to shrink. Simultaneous advancements in computers and software allowed
spectra to be generated and information generated at an amazing speed. The first
qualitative software, for example, in 1984, took hours to generate a usable equation.
Today, far more complex equations, based on many hundreds of spectra, take seconds to generate and apply. The size of the hardware has also been shrinking (partially based on the influx of components from the telecom industry) steadily. By the
1990’s, very good instruments could be purchased with dimensions less than 1/5th
those of the 1980’s, equipped with internal power sources and WiFi abilities. This
has progressed logically until units are being developed that simply plug into “smart
phones,” using the software of the phone for calculations. Why is this so good? Not
only can process analytical technology/quality-by-design applications be fulfilled,
but field-ready units can be pressed into service to assist in the control of counterfeit
products. In short, size does matter; in this case, smaller is better.
Fast and Efficient Peptide Mapping of Monoclonal Antibodies
Using Superficially Porous Particles
Timothy Rice, Agilent Technologies, 2850 Centerville Rd., Wilmington,
DE 019808
Peptide mapping by reverse phase chromatography is the mainstay technique in bio
therapeutic analysis, delivering comprehensive characterization of biopharmaceutical products. When interfaced with a mass spectrometer, it can deliver the identification of proteins and their variants, determine post translational modifications and
locations, and confirm protein sequences. However, peptide mapping represents
a significant chromatographic challenge due to the inherent complexity of protein
digests. Developing robust and reliable peptide maps is a struggle because of low
sensitivity, poor peak shapes, and very long separation times. This talk focuses on
the AdvanceBio Peptide Mapping column from Agilent as means to overcome these
challenges and achieve rapid, highly sensitive, and reproducible peptide maps.
Validation and Transfer of NIR Methods for Real-Time Release
Testing for Continuous Manufacture
Jianfeng (Frank) Qi, Vertex Pharmaceuticals, 50 Northern Ave., Boston,
MA 02210, Kelly A. Swinney, Henrik T. Rasmussen, David E. Nadig
The validation of near-infrared (NIR) methods, or process analytical technology
(PAT) methods in general, can follow the general principles in International Conference on Harmonization Q2(R1) with some adaptations. For validating a NIR
final blend active pharmaceutical ingredient (API) content method, specificity, robustness, accuracy, precision, linearity, and range are assessed. Specificity and
robustness are satisfied by demonstrating that the NIR signal (following chemometric treatment) is attributable to the API content and is not impacted by likely interferences across the product design space. Accuracy is determined by comparing the
NIR results with results from the reference method (high-performance liquid chromatography (HPLC)). The precision of the PAT method is assessed using repeat
measurements of the same sample at low, middle, and high concentrations across
the design space (the range). To facilitate determining only the analytical error (vs.
the error associated with both the measurement and sample heterogeneity), NIR
precision measurements are made with the powder flow stopped to make sure the
same sample is being measured. Linearity is determined by comparing the PAT
results to the HPLC results across the range. Since chemometric model development and validation require a large number of samples to be analyzed to generate
reference values, a high-throughput HPLC method, optimized for accuracy, was
developed for this purpose. The NIR method is validated on a lab NIR instrument
in a PAT stack that is equivalent to the PAT system on the continuous manufacture
system (CM-PAT). The method is then transferred to the CM-PAT using a limited
method validation approach.
Chromatographic Analysis and Separation of Short RNA
Oligonucleotides with Novel Liquid Chromatography Methods
Mirlinda Biba, Merck, PO Box 2000, MS: RY818-B218, Rahway NJ
07065, Christopher J, Welch, Joe P. Foley
Synthetic oligonucleotides have become increasingly important as part of new developments in the use of antisense and small interfering ribonucleic acid (siRNA) as
potential therapies for the treatment of various different diseases. The development
of analytical methods for the sensitive and quantitative analysis and separation of
oligonucleotides is an essential part for the advancement of this research area.
A typical oligonucleotide therapeutic sample is a short RNA-based oligonucleotide
with about 21-mer length (~7-8 kDa), and with possible chemical modifications prepared by chemical synthesis using an automated synthesizer. Due to their relatively
large sizes compared to typical small-molecule compounds, oligonucleotides can
be difficult to analyze with traditional analytical methods. There are many different
analytical techniques reported for the analysis and separation of oligonucleotides,
including capillary gel electrophoresis (CGE), anion-exchange high-performance
liquid chromatography (AEX-HPLC), and ion-pair reversed-phase liquid chromatography (IP-RPLC). In these research studies, existing liquid chromatography methods for the separation of oligonucleotides were evaluated for improved separations.
New and novel approaches were also explored for more efficient analysis and separation of oligonucleotides.
Understanding Hot Melt Extrusion via NIRS
Brandye Smith-Goettler, Merck, 770 Sumneytown Pike, PO Box 4, West
Point, PA 19486
Process analytical technology (PAT) and hot melt extrusion (HME), commonplace
in the food and polymer industries, are increasingly being implemented in the pharmaceutical industry. Hot melt extrusion utilizes pharmaceutical grade polymer(s),
potentially high temperatures, and mechanical energy to convert drug substance
from a crystalline to an amorphous state. This processing platform is ideal for poorly soluble drug substances, maximizes production efficiency by combining several
pharmaceutical processing steps and enables continuous manufacturing. PAT, in
general, yields numerous manufacturing advantages but those specific to HME are
highlighted via discussion of in-line, transmission mode, Fourier transform near-infrared spectroscopy (FT-NIR) as applied to two Merck drug products. In both applications, NIR and partial least squares models were developed for real-time drug
loading (%) predictions. These predictions have been successfully used for process
development, process fault detection and real-time quality assurance via waste gate
automation control.
Case Study of Challenges in ADC Method Development
Girija Krishnamurthy, Bristol-Myers Squibb, 519 Route 173, Bloomsbury,
NJ 08804
No abstract submitted by the author.
iCE3: A Powerful Analytical Tool for Antibody Drug Conjugate
Jiaqi Wu, ProteinSimple, 27 Coronet Rd., Toronto, ON M8Z 2L8 Canada
Antibody-drug conjugates (ADCs) are a new class of therapeutic proteins. In an
ADC, a targeted monoclonal antibody (mAb) is covalently linked to a cytotoxic small
molecule. This allows for targeted drug delivery as the ADCs can selectively bind
and kill tumor cells that over express the target antigen while limiting toxicity to
normal tissues. Ion exchange chromatography is typically used to characterize the
charge heterogeneity of therapeutic mAbs, however analysis of ADCs is problematic due to the small molecule drug and linker that increase their hydrophobicity
compared to unconjugated mAbs. iCE3 is a powerful tool in the charge characterization of ADCs. iCE3 performs free solution information exchange forum (IEF) in
an open capillary column and the separation of the charge species is purely based
2014 EAS Abstracts
November 2014
on overall charge and isoelectric point differences. Because the iCE separation is
based on overall charge in free solution it can analyze a wide variety of samples
including ADCs. In addition, iCE has the advantages of high speed, high-throughput
and quantitation with a typical throughput for ADCs is 4-5 injections per hour with
the ability to run 100 analyses in a single batch. In this presentation, the principle of
the iCE3 is reviewed and its application to ADCs is discussed.
DNA extraction capabilities when applied towards a variety of DNA samples and
matrices. High extraction efficiencies were obtained for smaller single-stranded and
double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III)
MIL, while the dicationic 1,12-di (3-hexadecylbenzimidazolium) dodecane bis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III) MIL produced higher extraction
efficiencies for larger DNA molecules. The recovery of DNA from the MIL extraction
solvent and its implications in downstream analysis including polymerase chain reaction (PCR) is discussed.
Application of SPME in High-Throughput and In-Vivo Drug
Janusz Pawliszyn, University of Waterloo, 200 University Ave. W.,
Waterloo, ON N2L 3G1 Canada
In this presentation, an automated, high-throughput method based on thin-film solid-phase microextraction and liquid chromatography mass spectrometry is introduced for simultaneous quantitative analysis of 110 drugs chosen from ten classes
and varying in physical and chemical properties. The developed solid-phase microextraction (SPME) method was optimized in terms of sorbent selection, extraction
pH, ionic strength of the sample, washing solution, extraction and desorption times
for analysis of urine, blood and saliva samples. Chromatographic separation was
obtained in reversed-phase mode and detection was utilized with full scan orbitrap
or triple quadrupole mass spectrometer. The developed method was validated according to the Food and Drug Administration (FDA) criteria, taking into account minimum required performance level (MRPL) values provided by the World Anti-Doping
Agency (WADA). The developed assay offers fast and reliable multi residue analysis as an attractive alternative to the standard methods that are currently used in
anti-doping laboratories. We also introduce an in-vivo solid-phase microextraction
method, which combines sample preparation, metabolism quenching and extraction
as well as eliminates sample collection as an approach to monitor graft function at
different stages of the medical procedure related to organ transplantation. To ensure the best analytical performance of the method, various aspects of the protocol
were studied and optimized including selection of coating length, analyte coverage,
transportation and storage conditions with particular attention paid to stability of
the extracted compounds and convenience of the approach for clinical setup. The
applicability of the developed method for determination of metabolic profile of the
organs and for monitoring of drug metabolism was verified during lung and liver
transplantation in pig models.
Advances in Sample Preparation for Dietary Supplements and
Other Complex Matrices
Derick Lucas, Agilent Technologies, 2850 Centerville Rd., Wilmington,
DE 19808, Jerry Zweigenbaum, Bruce Richter, Irina Dioumaeva
Sample preparation is the essential first step of chemical analysis and is increasingly important with more chemically complex samples. Laboratories demand methods
that can deliver cleaner samples, at the appropriate concentrations, in less time,
with minimal cost and method development. The QuEChERS (quick, easy, cheap,
effective, rugged, and safe) method addresses many of these sample preparation
deficiencies; however, co-extracted matrix components in the form of lipids, sugars,
proteins, and pigments can greatly affect instrument cleanliness and analytical results. This work demonstrates the effectiveness QuEChERS provides for the preparation of dietary supplements and advances in selective lipid removal from fatty
sample extracts. Yohimbine and its numerous analogues were identified and quantified by liquid chromatography quadrupole time-of-flight and liquid chromatography
triple quadrupole; providing excellent sensitivity (limit of quantitation = 0.1 ng/g),
recovery (99% to 103%), and reproducibility (< 4% relative standard deviation). Results indicated larger than expected amounts of yohimbine in some supplements
while others showed undeclared alkaloids and evidence of synthetic adulteration
with exogenous Yohimbine. Additionally, advances in selective lipid removal from
lipid rich samples such as avocado, oils, and plasma are presented. These results
indicated that matrix co-extractives were dramatically reduced over conventional
cleanup methods without the loss of target analytes.
HPLC Method Development Strategies for Molecules with Multiple
Chiral Centers
Michael W. Dong, Genentech, 1 DNA Way, S. San Francisco, CA 94080,
Kelly Zhang, Meenakshi Goel, Nik P. Chetwyn
Development of new chemical entities (NCE) with high chemical and chiral purity is
a regulatory expectation in new drug development. Today, it is not unusual to have
complex molecules with three or four stereogenic centers which show high affinity
for disease targets. For the process development of these “multi-chiral” molecules,
numerous analytical methods capable of separating all stereoisomers (enantiomers
and diastereomers) must be developed rapidly to assess and control the stereochemistry of raw materials, intermediates and the final active pharmaceutical ingredient (API). Achiral reversed-phase (RP) methods, used to assess the overall
chemical purity assays, can typically be developed to monitor the diastereomeric
content in a single run. In many cases, these achiral methods often become the
primary quality control (QC) and stability-indicating purity assay methods. This paper describes the method development and QC strategies used for these complex
multi-chiral drug molecules, which include: 1) adoption of a 3-pronged high-performance liquid chromatography (HPLC) method development template approach; 2)
development of a single RP method using multi-segment gradients for determination
of overall purity and diastereomeric content; 3) SFC for rapid initial screening for chiral separations; 4) two-dimensional liquid chromatography for peak purity determination and achiral/chiral combinational assays. These strategies are described with
actual examples used during clinical development of several new drug candidates.
Investigations into Analytical Extractions Using Green Solvents
Douglas Raynie, South Dakota State University, Department of Chemistry and Biochemistry, Brookings, SD 57007
Extractions and related sample preparation procedures generally represent the largest generation of solvent waste in the analytical process. Over the past 15-20 years,
solvent minimization as a way of avoiding waste has made great strides through
the development of new techniques like supercritical fluid extraction, accelerated
solvent extraction, microwave-assisted extraction, solid-phase microextraction, and
single-drop extraction. But despite this reduction in solvent use, often the nature
of the extracting solvent is overlooked. In this presentation, a variety of solvents
and solvent alternatives are reviewed from the perspective of green chemistry. That
is, the environmental and health considerations are discussed in four classes of
solvent alternatives: 1) Conventional organic solvents. The American Chemical Society’s Green Chemistry Institute Pharmaceutical Roundtable, and member companies, has developed lists of solvent alternatives to conventional organic solvents.
For example, ethyl acetate is generally preferred over dichloromethane. These
solvent considerations are reviewed. 2) Supercritical fluids. As mentioned, supercritical fluids have become an established solvent alternative and are not thoroughly
discussed. 3) Ionic liquids and deep eutectic solvents. These novel solvents have
recently found their way into the analytical laboratory. However, because of viscosity
and other concerns, their use as traditional solvents is somewhat limited, though
utility as stationary phases or in unique experimental set-up is justified. 4) Green
solvents. Natural products, such as limonene, or non-peroxide forming ethers are
becoming more than a research novelty. As appropriate, these solvent classes are
compared for use in the analytical extraction process.
Strategies of Advanced LC-MS-MS Screening and Development for
Regulated Bioanalysis
Jian Wang, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543,
Ryan Gu, Eliza Fung, Ang Liu, John Lute, Mark Arnold, Anne Aubry
It is important to optimize/select a liquid chromatography tandem mass spectrometry (LC-MS-MS) bioanalytical method that provides good sensitivity, accuracy,
precision, ruggedness, and selectivity; the latter to avoid potential interference
from metabolites or endogenous components. With the advance of technology and
changing requirements from regulatory agencies in different regions, the strategy to
screen bioanalytical assay conditions is also constantly evolving. This presentation
discusses the current strategy of bioanalytical method screening and development
in regulated bioanalysis at Bristol-Myers Squibb regarding the optimization of mass
spectrometer, chromatography and extraction parameters. Comprehensive, but
also fit-for-purpose, method screening and development ensures the data quality
and meanwhile avoids unnecessary complexity in execution and data interpretation. Two different LC optimization procedures are applied in various applications:
1) using standards of parent and metabolites to screen mobile phases/columns and
optimize gradient followed by confirmation and further refining of the LC method
using incurred samples; 2) using pooled incurred samples from the start to screen
Selective Extraction of DNA Using Magnetic Ionic Liquids
Jared L. Anderson, University of Toledo, 2801 W. Bancroft St., Toledo,
OH 43606, Kevin D. Clark, Omprakash Nacham, Honglian Yu
DNA extraction represents a significant bottleneck in nucleic acid analysis. Traditionally, liquid-liquid extraction with phenol-chloroform was used for the purification
of DNA from biological samples. Several adaptations to this method involving the
addition of detergents to assist in the removal of proteins and polysaccharides have
been made. However, the dependence of these protocols on organic solvents and
often time-consuming centrifugation steps has resulted in the development of more
environmentally benign techniques that are capable of high-sample throughput.
This talk discusses the synthesis of hydrophobic magnetic ionic liquids (MILs) and
their implementation as solvents for the rapid and efficient extraction of DNA from
aqueous solution. The DNA-enriched MIL microdroplets were manipulated by application of a magnetic field. The three MILs examined in this study exhibited unique
2014 EAS Abstracts
November 2014
columns and mobile phases, and build a chromatography method. The strategy also
considers the impact of mobile phase on MS-MS fragmentation and optimization, as
well as including the rationale and procedure to maximize speed and resolution in
rapid gradient LC-MS-MS bioanalysis.
Extraction of Volatile Organics Using Carbonnanotubes
Immobilized Composite Membranes
Smruti Ragunath, New Jersey Institute of Technology, 2211 Woodbridge
Commons Way, Iselin, NJ 08830, Somenath Mitra
Extraction of volatile organics from air involves a broad range of techniques including the use of sorbents and cryogenics. Membrane extraction of volatile organics
provides a wide range of selectivity and enrichment factor which can be effectively
applied towards separation of organics from atmosphere. Carbon nanotubes (CNTs)
were immobilized to the existing membrane surface to study the effect of nanotubes
to enhance the separation process. Separations of organic species from atmosphere
were compared using composite member and carbon nanotubes immobilized membranes. The effect of temperature, flow rate and concentration in reference to the
flux attained were studied with both the membranes. A maximum enhancement of
94% was obtained for the separation of toluene using carbon nanotubes immobilized membrane, followed by 44% for ethanol and 22% for dichloromethane which
can be used in effective separation of organics from the atmosphere.
Selected Ion Flow Tube Mass Spectrometry (SIFT-MS): Real-time,
Sensitive Detection of Residual Solvents in Pharmaceuticals
Barry J. Prince, Syft Technologies, 3 Craft Place, Christchurch, 8024
New Zealand, Daniel B. Milligan, Vaughan S. Langford, Chuck Renner
Fast analysis, high selectivity, high sensitivity, and elimination of chromatographic
separation make selected ion flow tube mass spectrometry (SIFT-MS) an ideal technique for rapid screening for solvent residues in pharmaceuticals. In this presentation, the principles of SIFT-MS are presented together with data that demonstrate
very effective analysis, rapid of residual organic solvents. Traditional analytical techniques, such as gas chromatography mass spectrometry (GC-MS), usually take
15 to 45 minutes to analyze samples due to use of chromatography. They also
struggle to achieve good recoveries for very volatile compounds, including important
solvents used in the pharmaceutical industry. SIFT-MS is a direct mass spectrometric technique [1] that detects and quantifies volatile organic compounds (VOCs)
and inorganic gases within seconds to low part-per-trillion levels.[2] Direct sample
analysis is achieved without sample preparation, pre-concentration, or chromatography, because soft chemical ionization is utilized. High selectivity is maintained in
SIFT-MS through application of three reagent ions that can be switched in 10 milliseconds, markedly increasing the selectivity of SIFT-MS versus other direct mass
spectrometry techniques. Analytical results compare well with those obtained using
a regulatory GC-MS method at an accredited laboratory.[3]
The Introduction of PDMS-Overcoated Adsorbent Based Fiber
Len Sidisky, Supelco/Sigma-Aldrich, 595 North Harrison Rd., Bellefonte,
PA 16823, Robert Shirey, Yong Chen
A new line of fibers that contain an overcoating-layer of a proprietary polydimethylsiloxane (PDMS) over an adsorbent fiber coating has been developed. The primary
advantage of adsorbent based fiber coatings is that the porous materials retain analytes more tightly than absorbent type fiber coatings. This analyte retention advantage may become a disadvantage if the fiber has to be immersed in a complex
matrix sample. The properties of adsorbent coatings that retain analytes tightly also
may tightly retain nonvolatile components in the matrix. This can result in fouling of
the fiber coating and shorten the lifespan of the fiber. By applying a layer of PDMS
coating over the adsorbent coating, many of the large components in the matrix are
repelled, while the smaller analytes of interest can pass through the outer PDMS
layer and be retained by the adsorbent. This presentation compares the lifespan
of overcoated fibers to adsorbent fiber coatings without the PDMS overcoat layer
out of complex matrices such as fruit juices. The effect of the matrix on the lifespan of the coatings is demonstrated with pictures and data. The effectiveness of
the overcoated fibers from a variety of matrices is presented. Because the PDMS
overcoating slightly changes analyte uptake kinetics, data is shown that compare
the differences. Also, we share some tips on how to use the overcoated fibers more
[1] Smith, D., & Spanel, P. (2005). Mass Spec. Rev., 24, 661–700.
[2] Prince, B.J., Milligan, D.B., & McEwan, M.J. (2010). Rapid Commun. Mass
Spectrom., 24, 1763-1769.
[3] Langford, V.S., Graves, I., & McEwan, M.J. (2014) Rapid Commun. Mass Spectrom., 28, 10–18.
Real-Time Dissolution Behavior of Indomethacin in Spray Dried
Dispersion (SDD) in Biorelevant Media Using UV Surface Imaging
Steven Ponce, Bristol-Myers Squibb, One Squibb Dr., New Brunswick,
NJ 08903, Xujin Lu, Christopher Zordan
Drugs with low solubility and high permeability are classified as Class II in which
dissolution is the rate limiting step to drug absorption according with the Biopharmaceutics Classification System. In order to increase the oral bioavailability of insoluble
drugs several formulation strategies have been developed. One of these strategies
includes the utilization of spray dried dispersion (SDD). An SDD formulation consists of active pharmaceutical ingredient (API) in a polymer matrix. SDD are formed
when a crystalline or amorphous drug is co-dissolved in organic solution with a
polymer, sprayed into very fine droplets, and the droplets are rapidly stripped of
the solvent. Hydroxypropyl methylcellulose acetate succinate (HPMCAS) is one of
the polymers commonly used in formation of SDD. This work uses the UV surface
imaging system (ActiPix SDI300) to study the dissolution of SDD in traditional and
biorelevant media. Indomethacin co-spray dried with HPMCAS was used as a model drug. Samples with three different drug/polymer ratios were studied. Dissolution
rate was measured in a flow cell at a flow rate of 0.2 mL/min and UV detection at
280 nm. UV images were collected across the solid liquid boundary of the samples
to examine the drug diffusion. The swelling profiles from the matrix (HMPCAS) were
also generated to assess the polymer effect on the drug release. High spatially and
temporally resolved mapping of Indomethacin surface gradient concentration were
achieved from the UV images. Results obtained from the study of the model drug
are beneficial to SDD formulation development.
Comparison of Green Solvents during Chemical Extraction by
Diffusion Studies
Shanmugapriya Dharmarajan, South Dakota State University, 1424, 8th
St., Apt. F, Brookings, SD 57006, Douglas E. Raynie
Safety, health, and environmental concerns of conventional solvents such as hexane and related hydrocarbons stimulate the use of green solvents in the extraction
processes. Green solvents have been characterized for their low miscibility, high
boiling point, and easy biodegradability to nontoxic products under environmental
conditions, which makes them safer and less risky to human health and environment. Oils have been extracted from soybeans using green solvents such as d-limonene, 2-methyltetrahydrofuran, cyclopentyl methyl ether, ethyl lactate, tert-butyl
methyl ether, α–Pinene and deep eutectic solvents. The extractions are carried out
using accelerated solvent extraction. We study the extraction kinetics with these
green solvents and compare the experimental data to the hot-ball model. The performances of these green solvents compared to the conventional hazardous solvents in the extraction of soybean oil are validated.
Expanding Method Capabilities through Dynamic Sample
Keith Griswold, Pepsico, 100 East Stevens Ave., Valhalla, NY 10595
Dynamic sample preparation is an approach used to provide flexibility and expand
the capabilities of an existing validated method. Designing robust chromatographic
analysis methods that function across a diverse portfolio of products presents many
challenges. Avoiding method proliferation resulting from product development innovations and new ingredient technologies is among a few. Mode of detection and
conditions can be chosen to eliminate most identifiable interferences, but what measures can be taken to prevent an existing method from failing when faced with new
ingredients and formulations? Polymeric based mixed-mode solid phase extraction
has been investigated as a possible technique for “difficult” sample matrices. The
versatility available for exploiting analyte/matrix interactions allow for tunable selectivity, creating a great deal of method flexibility and, if required, enhanced sensitivity.
To address some of the issues commonly associated with solid-phase extraction, a
fully automated version of this process is currently being explored by PepsiCo R&D.
Understanding the Dynamic Process of Dissolution and Hydrolysis
of Aspirin by ATR-FTIR
Vrushali M. Bhawtankar, Seton Hall University, 400 South Orange Ave.,
South Orange, NJ 07001
Dissolution studies are critical tests for measuring the performance of a drug product. In the past few years, the importance of the dissolution test has increased.
Using in-situ attenuated total reflection Fourier transform infrared spectroscopy
(ATR-FTIR) spectroscopy we developed a methodology of analyzing and monitoring dissolutions of pharmaceutical active pharmaceutical ingredients (APIs). The
accuracy of this technique was found to be В± 3% relative to high-performance liquid
chromatography and Ultra-violet. In this presentation we discuss a dynamic analysis
of the dissolution and subsequent hydrolysis of aspirin by ATR-FTIR. This technique
allows real time analysis of the behavior of aspirin under simulated physiological
conditions (pH 1.2, 4.5, 6.8) as aspirin (1205 cm-1) and salicylic acid (1488 cm-1)
are detected as separate and distinct peaks in the FTIR. This technique suggests a
future potential for real-time studies of dissolution and hydrolysis of other pro-drugs.
2014 EAS Abstracts
November 2014
Extraction of Steroids from Water and Herbal Medicines Using
QuEChERS Sample Preparation
Michelle L. Schmidt, Seton Hall University, 400 South Orange Ave.,
South Orange, NJ 07079, Nicholas H. Snow
Quick, easy, cheap, effective, rugged, safe (QuEChERS) was an extraction technique originally developed for the extraction of veterinary drugs from animal tissue
by Anastassiades; however, its potential for the extraction of basic and polar compounds was quickly realized and it has since been primarily used for the extraction
of pesticides from food products. QuEChERS is a liquid-liquid microextraction combined with a dispersive solid phase extraction cleanup and has been applied to
a wide array of pesticides in a multitude of food matrices including fruits, vegetables, teas, fatty food products, etc. Since its inception, different methods have been
adapted to enable the analysis of not only basic pesticides, but acidic pesticides
as well by the addition of citrate buffer. The overall methodology used including
solvent, salts, buffers, and dispersive sorbents depends upon the target analyte(s).
This presentation discusses an overview of QuEChERS and its methodology as
it pertains to the extraction of steroids from water and herbal medicinal products.
Herbal medicinal products are frequently adulterated with steroids in order to speed
the healing process and thus it is necessary to develop an analytical method to detect the presence of these illegal adulterants. Optimization of the different parameters involved in the extraction process is discussed as well as the use of a depletion
study for the determination of partition coefficients for the steroids of interest.
Portable Raman Spectroscopy Opens the Door for Site Analysis
Katherine Bakeev, B&W Tek, 19 Shea Way, Newark, DE 19713, Dawn
Yang, Philip Zhou
The portability of Raman spectrometers broadens the reach of this tool for diverse
applications in analysis of materials from the ancient past, not to mention through
the modern era and even into the future. The ability to perform nondestructive analysis on archaeological samples without removing them from the site of discovery
helps to preserve the artifacts while giving us the information that we seek, showing
the flexibility of portable Raman. This flexibility can also prove useful in the fields
of minerals and mining, such as in the study of the nanomaterials such as carbon
Single Reaction Chamber Microwave Digestion: Sample Prep for
Better Trace Analysis
David Gunn, Milestone Inc., 25 Controls Dr., Shelton, CT 06484, Njies
The demand for a simplified approach for trace metals analysis is at an all-time
high with the development of new regulations, the complexity of new materials and
the lower limits of detection. Current sample preparation has challenges with the
wide variety of sample types, including United State Pharmacopeia (USP) pharmaceutical matrices, highly organic samples and difficult inorganic materials. Single
reaction chamber (SRC) microwave technology has been utilized in a cross-section
of these industries with significant improvements in temperature and pressure capability, digestion quality, and productivity. Trace metal analysis data following new
sample preparation techniques for the current USP 232/233 methods along with
highly organic samples are discussed.
Repair of CFRP Composite with Localized Incipient Thermal
Damage Guided by Handheld FTIR Inspection
Tucker Howie, University of Washington, Roberts Hall, Box 352120,
Seattle, WA 98195, Ashley Tracey, Brian Flinn, Paul Vahey, Paul Shelley
Incipient thermal damage (ITD) of carbon-fiber-reinforced polymer (CFRP) composites cannot be detected by common nondestructive evaluation (NDE) methods,
such as ultrasound or visual inspection. ITD can reduce matrix dominated mechanical properties such as short beam strength (SBS) of composites and thus requires
detection to insure the integrity of composite structures that may be subjected to
thermal exposures. Fourier transform infrared (FTIR) spectroscopy is an established laboratory technique that shows promise for NDE of ITD. The purpose of
this research was to determine the effectiveness of using a handheld ExoScanTM
FTIR for detecting localized heat damage in a panel. Calibration models were developed by using a partial least squares (PLS) to correlate SBS measurements
from coupons with a range of thermal exposures to FTIR spectra. The panels were
24-ply unidirectional 177 В°C cure carbon fiber reinforced epoxy laminates. Localized
heating was performed using a heat blanket coupled with multiple layers of insulation in the center of the panels. Cured panels were subjected to a 1 hour post-cure
exposure at peak temperatures of 227, 241, and 254 В°C. The FTIR was used to
perform ply-by-ply inspection during the scarfed repair process to remove remaining ITD material. From the inspection a three-dimensional map was generated to
evaluate progression of damage through the thickness direction of the panel. The
ExoScanTM FTIR was found capable of guiding a repair inspection for ITD; however, performance could be improved by obtaining a better match of surface finish
between the calibration standards and samples.
Improved Bonding of Composites, Polymers and Coatings by
Monitoring Surface Preparation with Handheld FTIR Spectroscopy
John Seelenbinder, Agilent Technologies, 14 Commerce Dr., Danbury
CT 06795
Surface preparations are key to both adhesive bonding and coating applications.
Without the proper preparation, bond strengths deteriorate and performance falls
below acceptable levels. Additionally, there are few ways of inspecting the bond
after it has cured. These factors lead to a situation where surface testing is require insuring that bonding surfaces have been prepared correctly. Infrared (IR)
spectroscopy has long been known to be an effective surface analysis technique.
Infrared is sensitive to many of the chemical changes which preparations impart to
the surface. Additionally, IR’s specificity allows for easy identification of organic materials, making it especially suitable for detection of surface contamination. Recent
advances in the miniaturization of Fourier transform infrared (FTIR) spectrometers
have produced a new range of handheld, non-destructive instruments. These instruments now allow qualification of surface preparations prior to bonding applications.
Handheld use allows these measurements to be truly non-destructive, allowing for
increased inspection of the actual surfaces being bonded. We’ll demonstrate methods developed to verify several surface preparations including plasma treatment
prior to bonding, evaluation of UV damage prior to coating and evaluation of surface
contamination by non-volatile residue. This talk features both new applications and
the latest advances in handheld FTIR technology.
What’s in your “Natural” Supplement? Application of NMR
Spectroscopy to Natural Products
Jimmy Yuk, Bruker BioSpin, 15 Fortune Dr., Billerica, MA 01821, Mark
Garvey, Christian Fischer, Kim Colson
Natural products such as herbal remedies have been increasing in sales with annual
revenue of 5.11 billion dollars in the United States alone. This has raised concerns
as surges in adulteration of natural products with other materials have been detected in the market. In some cases, this is due to accidental species misidentification,
but in other cases, non-disclosed materials were added to increase product efficacy.
This illegal practice can be detrimental to the natural product industry and extremely
dangerous to the health of the consumer. Due to the diversity and complexity of
chemical compounds within botanical materials, a demand exists for methods to
understand the composition of natural products for proper quality control and authentication. In this study, a nuclear magnetic resonance (NMR)-based screening
tool was applied to natural products. Targeted and non-targeted NMR screenings
for adulterated dietary supplements are described. This approach shows the simultaneous analysis of natural products which involves: 1) Identification of known
components, impurities and adulterants in reference spectrum database; 2) quantification using a calibrated NMR spectrometer; and 3) multivariate modeling for
product classification. With the increasing demand for robust high-throughput analytical methods, an automated NMR-based screening tool is a powerful approach
for validating the identity, purity, strength, and composition of many natural products.
Non-Contact Material Characterization Using Mid-Infrared Quantum
Cascade Laser Spectroscopy
Mark Norman, Block Engineering, 377 Simarano Dr., Ste. 130,
Marlborough, MA 01752, Anish Goyal
We review the applicability of mid-infrared (IR) quantum cascade laser (QCL) spectroscopy to characterize coatings on a variety of surfaces as well as bulk materials. This includes characterization of surface cleanliness prior to coating, chemical
composition identification, curing validation, adhesion verification, and coating thickness determination. Such testing has traditionally been performed using laboratory-based Fourier transform infrared (FTIR) spectrometers, but advances in laser
and opto-mechanical technologies are enabling the field testing of coatings using
non-contact and non-destructive methods in a hand-held manner. We discuss the
pros and cons of various measurement modes including specular reflectance, diffuse reflectance, and attenuated-total reflectance.
Enabling Strategies and Platforms for NMR Metabolite
Janet Caceres Cortes, Bristol-Myers Squibb, PO Box 4000, Princeton,
NJ 08540, Xiaohua Huang, Kim Johnson, Sarah Traeger, Haiying
Zhang, Xiaohong Liu, Yue-Zong Shu, Vikram Roongta, Michael Reily
Truly understanding the efficacy and safety profile of drugs and their metabolites
places a heavy emphasis on metabolite identification (ID) in the research and development process, so much so that over time, metabolite ID has garnered attention
at earlier stages within the drug discovery-development continuum. Liquid chromatography mass spectrometry (LC-MS) based approaches are the front-line analytical tool to analyze metabolites from biological matrices in an efficient time frame.
Nuclear magnetic resonance (NMR) spectroscopy has also become an important
technique in support of metabolite ID, particularly when challenges arise in precisely identifying the exact site and isomeric consequences of metabolic modifications
using LC-MS. In this presentation, we discuss two key challenges for metabolite
2014 EAS Abstracts
November 2014
identification by NMR. It is well known that low amounts of isolated desired metabolites as well as contaminants arising from incubation matrices, coeluting metabolites
and solvents are major impediments to NMR metabolite ID analyses. Strategies
that address the challenges of isolating sufficient amounts of metabolites with high
purity for speedy NMR analysis will be discussed. Another challenge for metabolite
ID is the issue of sensitivity since critical characterizations may need to be carried
out on high nanogram-low microgram material. Whereas the most common NMR
platform utilizes room temperature probes and sample volumes of 180 or 600 microliters, they are not well-suited for the analysis of metabolites in these low microgram quantities. This presentation provides selected examples of the benefit of
utilizing nitrogen cryocooled probe technology, as well as reducing sample volumes
to between 7 and 40 microliters in specially designed room temperature and helium
cryogenic NMR probes.
data processing, and data interpretation are also presented with specific examples
from real-world samples from my personal collection. Characterization of colonial
currency from New Jersey illustrates the power of micro multi-elemental analyses.
John Scott, New York Conservation Foundation, 261 Fifth Ave., Rm
2000, New York, NY 10016
No abstract submitted by the author.
Emerging Designer Drugs: The New Frontier for Microcrystalline
Thomas A. Brettell, Cedar Crest College, Forensic Science Program,
Department of Chemistry and Physical Sciences, 100 College Dr.,
Allentown, PA 18104
Microcrystalline testing is based on chemical-precipitation reactions that form crystals observed by microscopy. Most of the tests were developed in the late nineteenth century. Over the years, they have been modified to aid in the identification
of controlled substances and offer a low-cost alternative to GC/MS and FTIR. The
Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) has established criteria for their use in the identification of controlled substances. Additionally,
the American Society of Testing and Materials (ASTM) have established methods
for identifying cocaine, methamphetamine and phencyclidine using microcrystalline
testing. A wide variety of new psychotropic substances have emerged over the past
few years. These new emerging designer drugs are challenging traditional forensic
methodologies that have been in place for many years. Most of these new drugs
have never been seen in crime laboratories and hence forensic scientists are in
need of new identification methods. Microcrystalline testing can offer the drug analyst an alternative for identification of closely related structures of these substances.
The presentation will give an overview of these new emerging drugs and a summary
of the present state of microcrystalline testing in the crime laboratories. The talk will
highlight some recent developments and the opportunity for new research.
New Methods for the Simultaneous Measurement of Homo- and
Heteronuclear Coupling Constants
Josep Sauri, Merck, 126E. Lincoln Ave., Rahway, NJ 07065, Teodor
A long-range optimized P.E. heteronuclear single quantum multiple bond correlation
experiment, named P.E.HSQMBC, is proposed for the simultaneous measurement
of a complete set of homo- and heteronuclear coupling constants from a single
two-dimensional (2-D) cross-peak. The sign and the magnitude of proton–proton
coupling constants are measured along the direct dimension from the relative E.COSY-type multiplet pattern displacement generated thanks to the passive one-bond
coupling constant; whereas long-range proton–carbon coupling constants are independently determined in the detected dimension thanks to the In-phase-anti-phase
(IPAP) methodology. On the other hand, it will also be shown how using a 13C Boltzmann polarization instead of the regular insensitive nuclei enhanced by polarization
transfer (INEPT)-based 1H Boltzmann polarization in the original P.E.HSQC experiment, the C-HA cross-peak corresponding to a diastereotopic CHAHB methylene
spin system, which exhibits a characteristic 1:0:1 multiplet pattern along the indirect
dimension of a F1-coupled HSQC spectrum, makes visible the central lines of this
multiplet pattern. Therefore, a spin-state-selective method is proposed for the efficient measurement of both 1J(CHA) and 1J(CHB) along the indirect dimension of
a 2-D spectrum as well as to the magnitude and the sign of the 2J(HAHB) coupling
constant from the straightforward analysis of a single four-component E.COSY
cross-peak. The extraction of 1J(CH) values for CH and CH3 multiplicities is also
performed from the same spectrum. The success of the method is also illustrated
for the determination of residual dipolar 1D(CH) and 2D(HH) coupling constants in a
small molecule weakly aligned in a polymethyl-methacrylate swollen gel.
Microscopy and Microanalysis Applied to Pharmaceuticals
John A. Reffner, John Jay College, 524 West 59th St., New York, NY
Hal Ferrari was a member of the greatest generation a pharmacist and an analytical chemist with a devotion to family, baseball, EAS and microanalysis. He joined
Lederle Laboratories in the early 1950’s and spent 37 years there as an analytical
chemist. Hal was a cherish colleague and friend. When I first met Hal, he was a
senior research chemist at Lederle. Later, I joined American Cyanamid I had the
opportunity to work with Hal on various projects. We shared our interest in microanalysis and its application to pharmaceuticals. In Fritz Pregl’s acceptance of the
1923 Nobel Prize in Chemistry he concluded, “I therefore am very hopeful that in
the future quantitative organic microanalysis will find many more fields of application
and expansion, and that it will make possible much insight and discernment which
otherwise might not have been possible, especially following the magnificent recognition that has been awarded to it by the Swedish Academy of Sciences.” (From
Nobel Lectures, Chemistry 1922-1941, Elsevier Publishing Company, Amsterdam,
1966) Dr. Pregl’s vision inspired many scientists to follow his lead and it lead to
many advances in pharmaceutical science. Thinking small continues to produces
large changes in analytical chemistry.
Experiment Approaches to Probe Long-Range H-X Correlations in
2-D NMR Spectroscopy
Ronald Crouch, NMR Consultant, 1609 S. Del Norte Ave., Loveland, CO
80537, Craig Butts
An overview of both historical and current sequence techniques for efficient observation of long-range H-X coupling pathways is presented. Both heteronuclear multiple-quantum correlation (HMQC) and recent powerful heteronuclear single quantum
coherence (HSQC)-derived experimental approaches are discussed and compared.
The importance of phased spectra in F1 and in some instances both F1 and F2
when high-resolution is required is a key requirement for quality data. Strategies to
obtain high F1 resolution using both F1 band-selection and non-uniform-sampling
can afford a tremendous savings of time and some guidance to the use of these
strategies are discussed.
What can Non-Destructive Micro Multi-Elemental Analyses of
Counterfeit Banknotes Reveal?
Gene S. Hall, Rutgers University, Department of Chemistry, 610 Taylor
Rd., Piscataway, NJ 08854
Banknote counterfeiting in the United States has a long history starting during the
issues of Early American currency. This currency had security features such as mica
that the papermaker added to the paper pulp. During the Revolutionary War, the
British counterfeited this paper currency but did not use the same mica. Energy dispersive X-ray fluorescence (EDXRF) is a multi-elemental technique that is non-destructive and provides feature rich qualitative and quantitative information about the
bulk. However, if a micro X-ray beam (50-microns) is used to induce X-ray emission,
multi-elemental analysis can be used to analyze specific features such as individual
printed or written letters, embedded security features, and natural imperfections in
the banknote. Then, by operating the EDXRF spectrometer in the scanning mode,
elemental imaging can reveal mineral composition and formulations of the printing
or written ink. This memorial presentation will provide new insights into multi-elemental composition of counterfeit Confederate States of America (CSA) and obsolete banknotes. Also presented will be elemental imaging of contemporary United
States and British counterfeit banknotes to reveal the power of multi-elemental imaging to uncover printing secrets of the modern-day counterfeiter. Instrumentation,
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