by Marie Dvorak Christ, Ph.D Michal Kliman, Ph.D. Mass Spec Lab

by
Marie Dvorak Christ, Ph.D
Michal Kliman, Ph.D.
Mass Spec Lab
For questions or info call or e-mail
949-872-2724 or [email protected]
www.massspeclab.com
Outline
 Biomedical Device
 Industry Overview
 Regulatory Aspects
 Extractables and Leachables (E&L) Analysis
 High Resolution Mass Spectrometry
 Instrumentation
 Time of Flight (TOF) Mass Spectrometers
 MS Data Overview
 Resolution and Accuracy
 Case Studies
 L&E Contaminant Analysis
 Surfactant Analysis
 Study of Polyurethane Acrylic Adhesive Hydrolytic Stability
Medical Devices
 Medical Device - any healthcare product that does not achieve its principal
intended purpose by chemical action or by being metabolized.
Biomedical Device- Regulatory Background
 Regulations, like drugs, defined in CFR Title 21
 Biomedical Device regulated by CDRH (Center for Device and Radiological





Health) Division of FDA
Three Biomedical Device Classes – Class I, Class II and Class III
Class I and II – 510K regulatory route (no clinical trial based on substantial
equivalence to predicate device)
Class III – IDE/PMA (involves clinical trial ; requires testing to demonstrate
safety and efficacy of new product)
Chemical Testing of Biomedical Devices required to demonstrate safety or
equivalence, i.e. “Extractables and Leachables” (E&L)
Objective of E&L Testing
 Identify and Quantitate “leachates” - chemical compounds that might leach
from the material under simulated physiological conditions
 Identify and Quantitate “extractables” chemical compounds that could
potentially migrate from the material under more extreme conditions of
temperature and solvent.
Leachable and Extractable Testing Aspects
 Leachables and Extractables (L&E) Test Guidance
 ISO 10993-12:2002 “Sample Preparation and Reference Materials”
 ISO 10993-18:2005 “Chemical Characterization of Materials”
 ISO 10993-13:2010 “Identification and Quantification
of Degradation Products from Polymeric Medical Devices
 Extraction Conditions
 Solvent
 Solvent:Mass ratio
 Time and Temperature
 Mode of Extraction
 Soxhlet
Extraction in swellable solvent (for crosslinked
 (elastomeric) materials such as butyl rubber, silicones, polyurethane)
 Repeated soak with heat and shaking
 Polymer Dissolution (for non-cross linked polymers)
ISO 10993 Guided Chemical Analysis –Identification and
Quantification
 Select analytical method justifying sensitivity and selectivity
 GC/MS and LC/UV and LC/MS required
 Typically require analytical method with ppm sensitivity for plastics monomers
and plastics additives.
 Semi-targeted screen – many possible analytes known (plastics monomers)
but many unknown (degradants and contaminants, polymerization
byproducts)
 If a polar analyte detected by UV (many chemicals are detectable at 1100ppm concentrations by UV), it must be chemically identified, preferably
source reported and amount quantified. The specificity of UV spectra is
not sufficient enough for chemical id.
 If a volatile analyte detected by GC/FID or GC/MS, it must be identified.
NIST library is a great aid to id but often analyte is not in the library.
Significant Challenges of
Leachable and Extractable Testing of Biomedical Device
 Device is a multiplicity of diverse materials






Different plastics
Adhesives
Metals
Lubricants
Manufacturing aids (surfactants)
Coatings
 Unknown Degradant Profile
 Process effects on diverse materials yielding unknown degradants
 Sterilization, molding, laser etching
 Device Aging Effects
 Shelf Life, hydrolytic aging, photoaging
 Raw material variability
 Each raw material source has potentially different impurity composition
 All plastics contain a significant proportion of processing additives
 E.g. antioxidants, heat stabilizers, UV absorbers, mold release agents and
dispersants
 Same plastic, e.g. polypropylene, but different additives package.

Not all polypropylenes are equivalent
UPLC Coupled to
G2-S (Waters) Q-Tof High Resolution Mass Spectrometer
ANALYTICAL FUNCTION: Chemical and Structural Identification of unknown
component analytes comprising a complex mixture.
UPLC Function : Separation of a complex liquid mixture into pure components
Mass Spectrometer Function: Determine the molecular mass of each pure component
Molecular Mass , if accurate enough, can be a powerful tool for chemical identification!
Definitions - Mass
Carbon 6C12 isotope = 12 Da = 12 g/mole
1 atomic mass unit (amu) = 1 Dalton =
1.660538921(73) × 10−27 kg
Cholesterol
Elemental composition: C27H46O
Theoretical exact monoisotopic mass (C12, H1, O16 ) =
386.35486 Da (uses isotope of maximum abundance)
Molecular weight (based on natural isotopic abundance =
386.65 g/mole
Nominal mass = 386 Da
Definitions – Mass to Charge (M/Z)
 Mass spectrometer does not detect mass but mass to charge (m/z) at various levels
of accuracy depending on instrument design
Cholesterol Singly Positively
Charged Ion
Singly Negatively Charged Ion
+
H+
386 + 1
1
= 387
2+
-
+
m/z =
Doubly Positively Charged Ion
+ 2H+
H+
m/z =
386 − 1
=
1
m/z =
385
386 + 2
2
= 194
Nominal Masses
m/z =
386.3549 + 1.0072
m/z =
= 387.3549
1
386.3549 − 1.0072
1
Accurate Masses
= 385.3477
m/z =
386.3549 + 2.0144
2
= 194.1847
Full Scan (Positive Mode) High Resolution MS Operation
Ion
UPLC
Charge
Ana
Ion
Mass Mass
m/z
TOF
Analyte of Mass 500 amu
CL+
+1(H+)
500
1001 1001 longest
+
+1(Na+)
500
523
523
+1(H+)
500
501
501
+2 (2H+)
500
502
251 shortest
++
Photodiode
Array UV
Detector
CL+
+
+
Relationship between
TOF (time of flight)
and m/z
m/z=k(TOF)2
++
CL+
+
CL+
++
++
CL+
+
20keV
impulse
++
•
Total Ion Chromatogram and Mass Spectra Output
Time of flight (TOF) data yields the mass of Detected Ion Data
•
•
spectrum, a plot of ion Intensity as a function
of TOF which is proportional to m/z.
Each push yields a mass spectrum
Many (20,000) mass spectra are added to yield a
Scan point intensity represents total ion count
Mass Spectra
Total Ion Chromatogram (TIC)
1
Intensity
Intensity
Total ion count
@ each
Scan point
1
2
Extracted m/z
ion count @
each
Scan point
Retention time (min)
Chromatographic
peaks not
resolved in TIC are
often resolvable in
XIC using TOF
mass selectivity
2
Intensity
Intensity
Extracted Ion Chromatogram (XIC)
Mass to charge
(m/z)
Intensity
Definition - Mass Resolution
m
 TOF 
z
Resolution 


whh  TOF 
whh
Mass to charge(m/z)
Detected mass
to charge (m/z)
2
MS
Instrument
Res
Whh @
m/z= 500
Tof
High 30,000
Res
0.0167
Quadrupole
Low
Res
0.5-1.0
5001000
Definition - Mass Accuracy
Mass Accuracy =
+
(m/z−theoretical m/z)
theoretical m/z
+ H+
Intensity
Theoretical
monoisotopic
mass to charge
Detected mass
to charge (m/z)
Theoretical m/z = 386.3549
Example detected m/z = 386.3599
Mass Accuracy =
Mass to charge (m/z)
(386.3599 − 386.3549)
= 1.29 x 10-5
386.3549
Mass Accuracy = 1.29 x 10-5 x 1,000,000 = 12.9 parts per million (ppm)
Plastics Additive UV Data
4-Allyloxy-2Hydroxybenzophenone
CAS 2549-87-3
Octabenzone
CAS 1843-05-6
3.0e-1
8.0e-2
2.5e-1
6.0e-2
287.7583
1.5e-1
AU
UV Spectrum
AU
2.0e-1
288.7583
4.0e-2
1.0e-1
2.0e-2
5.0e-2
nm
200
250
300
350
400
nm
200
450
250
300
350
400
450
1.8e+1
4.0e+1
1.6e+1
1.4e+1
3.0e+1
1.2e+1
AU
AU
Total photodiode
array UV
chromatogram
1.0e+1
2.0e+1
8.0
6.0
1.0e+1
4.0
Time
2.55
2.60
2.65
2.70
2.0
Time
3.55
3.60
3.65
3.70
Plastics Additive MS Data
Structural characterization is
limited with UV data. Two
related benzophenone
chromophores yield
equivalent UV spectra
(previous slide) but very
different mass spectra in this
slide
4-Allyloxy-2Hydroxybenzophenone
CAS 2549-87-3
%
Mass spectrum
C13H11N3O
monoisotopic
neutral mass
254.0943 Da
327.1960
C21H26O3
monoisotopic
neutral mass
326.1882 Da
%
255.1023
Octabenzone
CAS 1843-05-6
358.3680
200
250
300
350
400
450
m/z
500
0
100
405.3590
215.0708
150
200
250
300
350
400
450
m/z
500
%
Total ion
chromatogram
188.0530
150
%
0
100
15
Time
2.55
2.60
2.65
2.70
21
Time
3.55
3.60
3.65
3.70
MSMS High Resolution MS Operation
Ion
Charge
CL+
+
UPLC
++
Ana
Ion
Mass Mass
m/z
TOF
+1(H+)
500
1001 1001
+1(Na+)
500
523
523
+1(H+)
500
501
501
+2 (2H+)
500
502
251 shortest
+1(H+)
1200
1201 1201 longest
Analyte of Mass 500 amu
Analyte of Mass 1200 amu
Photodiode
Array UV
Detector
+
Filters specific Ions with EM Fields
1-2 amu narrow bandwidth filtering possible
Filter the 1201 ion only
++
CL+
CL+
++
+
Collision Induced Dissociation
(CID) – “Fragmentation”
Low Pressure Argon Gas Filled
Chamber
MSMS High Resolution MS Operation
Ion
Charge
CL+
+
UPLC
++
Ana
Ion
Mass Mass
m/z
TOF
+1(H+)
500
1001 1001
+1(Na+)
500
523
523
+1(H+)
500
501
501
+2 (2H+)
500
502
251 shortest
+1(H+)
1200
1201 1201 longest
Analyte of Mass 500 amu
Analyte of Mass 1200 amu
Photodiode
Array UV
Detector
F1
+
Filters specific Ions with EM Fields
1-2 amu narrow bandwidth filtering possible
Filter the 1201 ion only
++
F2
CL+
F3
CL+
++
F1 F2 F3 F4
F4
+
Collision Induced Dissociation
(CID) – “Fragmentation”
Low Pressure Argon Gas Filled
Chamber
Intensity
Mass spectrum
of fragments of
selected ion
(m/z)
Ion Fragmentation In Collision Cell
Ar +M+
eV
F+ + N + Ar
M+= Molecular Ion N= neutral fragment F+= ion
fragment M>F1>F2>F3>F4
Fragmentation – Collision Induced Dissociation (CID)
Site Specificity -Preferential fragmentation at polar bonds, clusters
Structural Information Encoded in Fragments
Searchable MSMS Fragmentation Libraries (Similar to EI GC/MS libraries, e.g. NIST) generated
with characteristic spectra. (Platform dependent) chemically identify fragments
Fragments can
fragment (F3->F4)
F3
F3
F2
F3
F4
F2
F4
F2
F3
F3
F1
F1
F1
N
N
N
F1
Triphenyl Phosphate High Energy MSMS of Dimer
MS/MS of 653 m/z
20 - 35 eV
Collision energy can be
used to break up clusters
(dimer) and simplify
mass spectrum
327.0801
%
[M+H]+
Triphenyl
Phosphate
Dimer absent in spectrum with collision energy on
0
100
MS Full Scan Low
Energy
200
300
400
500
327.0803
600
700
m/z
800
Dimer [2M+H]+
%
[M+H]+
653.1522
0
100
200
300
400
500
600
700
m/z
800
MSE High Resolution MS Operation
MSE Advantages
• In same time required for MS
alone, can acquire both MS and
MSMS scans for all ions in sample.
1. No quad selection
• MSMS might ultimately be
2. No collision energy
required for problem. But MSE is
3. Low energy mass spectrum
a great screening tool.
CL
+
+
++
+
+
CL+
++
++
Mass to charge (m/z)
In MSE mode collision cell energy is
rapidly alternating between on and off.
Two mass spectra are acquired;
1) low energy spectrum with molecular ions
2) High energy spectrum with fragments
+
1. No quad selection
2. Collision energy ON yields fragment
3. High energy mass spectrum with fragment
ions of all molecular ions
F1
+F2
CL
+
+
++
CL
+
+
++
F3
F1 F2 F3 F4
+++
F4
Intensity
+
++
CL
+
Intensity
CL
+
Mass to charge (m/z)
Two related benzophenone
chromophores yield
common fragments in red
due to their structural
similarity. But
fragmentation pathways are
so sensitive to structure that
distinguishing fragments
circled in green below are
also acquired
Plastics Additive MSE Data
4-Allyloxy-2Hydroxybenzophenone
CAS 2549-87-3
105.0340
137.0313
Octabenzone
CAS 1843-05-6
High
Energy
105.0340
%
%
High
Energy
215.0731
77.0388
0
50
75
100
137.0241
125
150
293.0481
175
200
225
250
275
300
325
m/z
350
0
50
75
100
125
150
175
200
225
250
275
300
255.1025
Low
Energy
100
125
150
175
200
339.0525
225
250
275
300
325
m/z
350
0
50
Low
Energy
C21H26O3
monoisotopic
neutral mass
326.1882 Da
%
139.0556 188.0548
75
m/z
350
327.1960
C13H11N3O
monoisotopic
neutral mass
254.0943 Da
%
0
50
325
75
100
125
150
284.2946
215.0709
137.0233
175
200
225
250
275
300
325
m/z
350
Plastics Additive MSE Data
C21H26O3
monoisotopic
neutral mass
326.1882 Da
Octabenzone
CAS 1843-05-6
Positive Mode MS
Negative Mode MS
137.0313
High
Energy
211.0416
105.0340
%
%
High
Energy
Fragments produced by positive
and negative ions can be
significantly different as shown. In
fact, positive mode and negative
mode spectra of Octabenzone
share no common fragments.
215.0731
169.0659
256.0260
0
m/z
100 120 140 160 180 200 220 240 260 280 300 320 340
Low
Energy
0
50
75
100
125
150
175
200
225
250
275
300
325
m/z
350
327.1960
325.1808
%
%
Low
Energy
211.0387 255.3548
0
m/z
100 120 140 160 180 200 220 240 260 280 300 320 340
0
50
75
100
125
150
284.2946
215.0709
137.0233
175
200
225
250
275
300
325
m/z
350
Theoretical Prediction of Plastic Additive Fragment Ions
in MassFrontier (Thermo) Software
Mass Frontier when used with
accurate mass data is an
effective tool in structural
interpretation of
fragmentation spectra
4-Allyloxy-2Hydroxybenzophenone
CAS 2549-87-3
Octabenzone
CAS 1843-05-6
O
HO
O
O
105.0340
OH
137.0313
OH
OH
%
O
%
HO
O
H2 O
105.0361
O
O
OH
215.0761
137.0241
293.0481
77.0388
0
50
75
100
125
150
175
200
225
250
275
300
325
m/z
350
0
50
75
100
125
150
175
200
225
250
275
300
325
m/z
350
Contaminant Structure Verification Using Mass Frontier Software (MFS)
 Mass Frontier (Thermo Scientific) Small Molecule Structural Elucidation Software
 MassFrontier is a predictive software that generates structures of expected
fragments from a given precursor ion
 Mass Frontier generates fragments using predictive algorithms based on
 rules of ionization, fragmentation and rearrangement
 MSMS Collision Induced Dissociation (CID) Fragmentation library based on
120,000 published small molecule fragmentation mechanisms
 The software predicts fragments as well as the pathways of their formation and so
yields precursor/fragment pairs.
Case Study I
E&L Study of Acrylic Intraocular Lens (IOL) Device
Chemical Identification of Low Level UV Absorber Contaminant in
Hydrophobic Acrylic Intraocular Lens (IOL) Extract
• IOL replaces crystalline lens
in cataract surgery
• Over 15 million implants
worldwide per year.
Hydrophobic Soft Acrylic IOL
Acrylic Chemistry
Lens Acrylic Components
Acrylic Monomers
Initiator (Azoisobutyronitrile)
Acrylic Crosslinker
Benzotriazole (UV absorber) monomer
H
O
Cl
N
N
N
R= short chain hydrocarbon or UV absorber
Elastomeric Polymer (acrylics,
polyurethane, EPDM rubber,
silicone, PVC)
Elastomer swells in a chemically
compatible solvent; it does not
dissolve
Acrylics swell in alcohols, e.g.
isopropanol (IPA)
UV Chromatogram of Lens E&L Extract
UV Absorber
Standard 5ppm
IPA E&L Extract
IPA Blank
330 nm UV Chromatograms
2.0e-2
0.0
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
-4.0e-3
-5.0e-3
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
-4.0e-3
Time
 UV absorber monomer
 Unknown residual analyte
elutes at 3.68 minutes
detected at RT 3.45 minutes
 330nm absorbance characteristic of UV absorber, so and
related. But
not UV absorber monomer (no coelution).
probably UV absorber
 Possible sources of ipa extractable UV absorber related analyte
A) generated from the monomer as a degradant in production or storage.
B) Introduced with UV absorber monomer (as a contaminant)
Contaminant Investigation – Analyte Source
 We identified an impurity in a high concentration UV absorber
standard that matched the device impurity in UPLC retention time, UV
response, and mass spectrum
Contaminant Chemical Characterization Using Accurate Mass
• Accurate Mass
was essential for
this structural
characterization
 Experimental accurate monoisotopic mass of UV absorber = 328.1204amu
of UV absorber impurity = 330.1021amu
 Software aided chemical formula determination
 Assumed element content C, O, H, N, Cl
 one chlorine containing chemical formula, C17H16ClN3O2 (monoisotopic mass
330.1009amu) matched the accurate experimental mass within 5 ppm
 Chemical deduction suggested the impurity structure with an aldehyde group replacing
the vinyl group
Theoretical = 330.1009amu
Experimental= 330.1021 amu
Mass accuracy =3.6ppm
=1.9793
+O
-CH2
UV Absorber Monomer
Contaminant
Comparison of Experimental MSE High Energy Spectral Fragments
and MFS Theoretical Predicted Fragments
100
A.
Cl
HO
N
119.0489
Cl
N
N
N
N
HO
N
%
HO
UV Absorber
H
272.0564
H2O
328.1160
274.0508
119.0868 147.1186
126.0089
0
100
120
140
175.1144
160
209.0784
198.9491 221.1139
180
200
220
286.0280
243.0501
240
260
280
330.1088
331.1131
314.0667
300
320
m/z
340
H
HO
100
246.0443
H2O
B.
Cl
Cl
N
N
N
HO
N
N
N
274.0373
O
Contaminant
%
330.0977
CO loss
loss of t-butyl
O
248.0410
183.0693
121.0639
121.1022
0
100
120
140
276.0355
CO loss
155.0739
160
249.0375
217.0295
180
200
220
302.1070 314.0667
240
260
280
300
320
m/z
340
• With the experimental
mass accuracy of 5 ppm
we were able to
unambiguously match up
theoretically predicted
fragment masses with
experimental data
Application of High Resolution MS to Surfactant Fingerprinting
 Surfactant Uses in Biomedical and Pharmaceutical Applications
 Formulation Solubilization or Compatibilization

OTC drugs, cosmetics, pharmaceutical formulations)
 Foam Control
 Drug Delivery Application
 Wipe down of clean room surfaces
 Final package (e.g. cap and closure) critical cleaning
 Process equipment cleaning
 Final cleaning of biomedical devices prior to final packaging
Non-ionic Surfactant Chemistry
 Non-ionic
 General Architecture
Linear Polymeric Surfactant Molecule
Hydrophobic
Tail
Hydrophilic
Head
R=cetyl, undecyl
PEG (polyethylene glycol)
( CH2CH2O )n
 High mass
 Liquids, paste, waxes
PPG (polypropylene oxide repeat unit)
PBO (polybutylene oxide)
•
•
•
TICs of Several Non-ionic Surfactants
Surfactants Often Coelute in LC due to chemical similarity (See Brijs below)
Polymeric chains are not chromatographically resolved. Broad chromatographic peaks encompass surfactant
molecules covering a range of molecular weights (polydisperse)
All surfactants shown not UV detectable at 20-200 ppm concentration
%
average Mn ~4,670
1
0.10
One Unsat bond
0.20
0.30
0.40
0.50
0.60
0.70
Brij S100
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2.60
2.70
%
avg. Mn ~1,150
2.80
2.90
3.00
3.10
3.20
3.30
3.40
3.50
3.60
3.70
3.80
3.90
3.00
3.10
3.20
3.30
3.40
3.50
3.60
3.70
3.80
3.90
2.90
3.00
3.10
3.20
3.30
3.40
3.50
3.60
3.70
3.80
3.90
2.90
3.00
3.10
3.20
3.30
3.40
3.50
3.60
3.70
3.80
3.90
2.90
3.00
3.10
3.20
3.30
3.40
3.50
3.60
3.70
3.80
3.90
Brij O20
1
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2.60
2.70
2.90
Brij 58
avg. Mn ~1,120
%
2.80
1
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
%
avg. Mn ~800
2.60
2.70
2.80
Valtron
1
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2.70
2.80
Micro 90
avg. Mn ~600
%
2.60
1
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2.60
2.70
2.80
PPG-PEG-PPG (Poloxamer)
%
avg. Mn ~2000
1
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
2.00
2.10
2.20
2.30
2.40
2.50
2.60
2.70
2.80
2.90
3.00
3.10
3.20
3.30
3.40
3.50
3.60
3.70
3.80
3.90
2.20
2.30
2.40
2.50
2.60
2.70
2.80
2.90
3.00
3.10
3.20
3.30
3.40
3.50
3.60
3.70
3.80
Time
3.90
Tween 80
avg. M.W. ~1310
%
1.90
1
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
Combined Mass Spectra of Surfactants
%
•
•
•
%
0
50
0
50
Higher Molecular Weight Surfactants Tend to Multiply Charge in ESI (see Brij S100)
Molecular weight distribution can be experimentally estimated from mass spectral data
Each surfactant displays a unique mass spectral fingerprint despite similarity in structure
Mixed
+1, +2, +3
and higher
charges
100
150
200
250
+4
300
350
400
450
500
550
600
650
700
750
800
850
900
+3
950
1000
1050
1100
1150
1200
+3
1250
1300
1350
1400
1450
1500
1550
1600
𝑚𝑎𝑠𝑠
= 1000 → 𝑚𝑎𝑠𝑠 = 2000𝑎𝑚𝑢
2
m/z
1650
1700
1750
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
PPG-PEG-PPG
+1
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
+1
Mixed
+1 and +2
charges
100
150
200
250
300
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
avg. Mn ~1,150 Brij O20 (C18 unsat)
m/z
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
+2
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
Brij 58 (C16)
avg. Mn ~1,120
+1
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
Tween 80
avg. M.W. ~1310
+1
%
1750
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
+1
100
150
200
250
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
avg. Mn ~800
%
Singly
charged
1800
2400
2450
Valtron
m/z
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
+1
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
Micro 90
avg. Mn ~600
%
0
50
1900
m/z
100
+2
0
50
1850
avg. Mn ~2000
%
%
0
50
1800
+2
+2
0
50
Brij S100 (C18 sat)
average Mn ~4,670
+2
0
50
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
High Resolution Essential for Identification of Multiply Charged
Species
• The carbon 13 isotope
+6
peaks of a molecule with
one positive charge are
spaced 1 Da. The spacing
is 0.5 Da for two charges
and 0.33 Da for three
charges.
1356.2906
1355.9536
Brij S100
1356.6245
0.33
2016.3560
1356.9546
2016.8531
0.5
%
%
+5
0
1356
%
+4
2017.3572
2015.3567
m/z
1357
2017.8481
+3
0
2015
2016
m/z
2018
2017
+2
0
400
m/z
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
+3
PPG-PEG-PPG
1650
1700
1750
1815.3431
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
2500
2550
2600
2650
2350
2400
2450
2500
2550
2600
2650
1816.3470
1
1817.3558
%
+2
%
1818.3522
0
1815
1816
1817
m/z
1819
1818
+1
0
400
m/z
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1650
1700
1750
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
Brij 58 vs Brij O20 Surfactant MS Peak Separation
• High resolution and accurate mass capability of TOF spectrometer
enables unequivocal identification of C2H2 chemical difference
between the two different Brij surfactants.
1122.8079
Brij O20
44.0262
Δ=C2H4O
=44.0272
=26.0113
avg. Mn ~1,150
%
HO(CH2CH2O)20C18H35
1166.8351
=26.0082
1099.7109
n
1143.7368
C18H35
1106.8162
0
1090
1100
1110
1187.7667
1150.8370
1120
1130
1140
1150
1194.8678
1160
1170
1180
1190
m/z
1200
1096.7966
Brij 58
=44.0303
HO(CH2CH2O)20C16H33
n
Δ=C2H2
1140.8269
=44.0237
avg. Mn ~1,120
1184.8506
%
26.0157
1117.7290
1161.7550
C16H33
1124.8235
0
1090
1100
1110
1120
1130
1168.8549
1140
1150
1160
1170
1180
1190
m/z
1200
Water Soluble Wax Analysis
• Hard wax is used to block
acrylic blanks during lathing
operation in contact lens or
IOL production.
• Hard wax is water soluble and
is removed from the lens after
lathing by soaking in water.
• Wax must be completely
removed for contact lens
comfort and IOL
biocompatibility.
Water Soluble Wax Analysis - Accurate Mass Extracted Ion
Chromatogram Improves Detection Limit
•
•
•
•
TOF is highly sensitive for high molecule weight waxy surfactant molecules
100ppb detection Limit achieved using accurate mass extracted ion chromatograms
Highly specific extraction of accurate masses of wax peaks is an excellent background noise filter.
Good example of how selectivity achieved with mass accuracy can enhance sensitivity
Wax Standard
Concentration
Accurate Mass (+/- 0.01 Da)
Extracted Ion Chromatogram
Areas
Total Ion Chromatogram
0
0
0.50
1.00
1.50
2.00
2.50
0.50
1.00
1.50
2.00
1.00
1.50
2.00
2.50
1.00
1.50
2.00
2.00
2.50
2.50
1.50
2.00
2.50
S/N:PtP=16.17
%
0
0.50
1.00
1.50
2.00
0.50
2.50
100
100
%
1.50
1.00
100
%
%
%
1.00
0.50
2.50
0
0.50
2.00
%
0.50
100
79
1.50
0
0
0.50
1.00
S/N:PtP=228.48
100
%
%
59
0.50
2.50
100
1 ppm
100
ppb
%
%
14
S/N:PtP=2691.57
100
%
10 ppm
%
100
Signal to Noise
Calculation
1.00
1.50
2.00
S/N:PtP=2.50
2.50
Adhesive Hydrolytic Stability
 Adhesives have widespread uses in biomedical devices.
 Adhesive bond must remain stable over time in physiological environment if a
device is to remain safe and effective.
 Adhesives are complex reactive mixtures whose chemical composition is often not
revealed by suppliers. Formulations are often trade secrets. Adhesive stability
requires early investigation.
 Investigated the chemical stability of photocured acrylic-based adhesive used in
adhesion two acrylic parts of long-term biomedical device implant.
 Exposed adhesive bond to Long term hydrolytic exposure. Analyzed hydrolysate.
 Accelerated aging in water at 85C for 2 months equivalent to five years real
time at physiological conditions (35C ) with 10grams water/gram material.
Adhesive Chemistry
Macromer + Acrylic Crosslinker
photoinitiator
Adhesive Network
Polyetherurethane Acrylic Macromer Architecture
Crosslinked Adhesive
PU
Polyurethane
PE
polybutoxyether
Polyetherurethane macromer
MW ?
Total Ion Chromatograms of 5 Year Equivalent Aged Hydrolysates
Normalized Ion Count
• The hydrolysate displayed
analyte peaks that were
related to the adhesive.
• “What is the solution
concentration of these
analytes?
• Is the analyte source a
residual fraction of the
adhesive or a degraded
fraction?
Time, Minutes
Total Ion Chromatograms – Macromer Std vs. Aged Hydrolysate
• There was no evidence that
the hydrolysate fraction
was a contaminant of the
macromer material.
• Both chromatograms share
a quartet repeat structure
that was found to be a
fingerprint of the
macromer.
Standard Solution
Normalized Ion Count
quartets
10 ppm
Time, Minutes
Combined Mass Spectra– Macromer Std vs. Aged Hydrolysate
72
Adhesive Macromer 1005.6
100
%
Normalized Ion Count
1006.6
730.5 766.5
767.0
694.4
802.5
803.0
658.4
413.3
0
200
300
400
500
469.3
100
1365.9
700
1294.9
1367.9 1440.0
923.1 934.5
800
1079.7 1151.7
900
1512.0
1582.1
1223.8 1295.9
1007.6
983.6
622.4
600
1078.7 1150.7
1510.0
1511.0
933.5
850.6
511.3 547.3
295.2
1438.0
1149.7 1221.8
1293.8
• No overlapping masses in
mass spectra either
411.3
1077.7
1000
1091.7 1163.7 1235.8 1307.9
1368.9 1441.0 1513.0
1100
1200
1300
1400
1500
1606.0 1678.1 1750.2
1608.0 1680.1
1600
1700
m/z
57D device hydrolysate
397.2
541.4
583.4
483.3
655.4
PE
polybutoxyether
369.2
%
613.4
271.2
343.2
329.2
425.3
727.5
699.4 741.4
257.2
255.2
0
200
• Polybutoxyether
repeat unit was
determined with
mass spectrometry.
Reverse engineering
possible
=72 Dalton
785.4
300
400
500
600
700
800
829.4
873.5 959.5
900
1000
Mass/Charge
1100
1200
1300
1400
1500
1600
1700
m/z
Total Ion Chromatograms – Adhesive Puck vs. Aged Hydrolysate
Normalized Ion Count
• Hydrolysis of cured
adhesive puck was
performed to investigate
kinetics of process
independent of other
device components.
• Puck was heated in
water at 100deg C for
100 hours.
• TIC of puck hydrolysate
was remarkably similar
to device hydrolysate.
• Puck peaks did not
increase with exposure
beyond 100 hours.
Time, Minutes
Mass Spectra -Adhesive Puck vs. Aged Hydrolysate
727.5
%
57D device hydrolysate
• Same masses aptly
highlights chemical
similarity of puck and
device hydrolysates
699.4
685.5
713.5
557.4
602.4
617.4
645.4 653.4
543.4
0
500
100
510
520
530
540
550
560
570
580
600
610
620
588.4
Adhesive puck
hydrolysate
543.4
590
677.5
631.4
630
640
650
670
714.5
680
690
700
710
557.4
730
742.5
740
750
760
770
780
790
m/z
800
780
790
m/z
800
727.5
639.4
603.4
714.5
686.4
645.4
589.4
544.4
509.3
0
500
720
729.5
713.5
685.4
631.4
741.5
687.5
663.5
660
728.5
705.5
686.5
588.4
%
Normalized Ion Count
100
510
529.4
522.3
520
530
545.4
540
550
611.4 617.4
558.4
571.3
560
570
625.4
590.4
580
590
600
610
620
728.5
653.4
655.4
630
640
650
660
699.5 705.5
670
Mass/Charge
680
690
700
741.4
742.4
715.5
687.5
663.5
710
720
730
740
750
771.5
760
770
Summary of Adhesive Case Study Results
 Macromer adhesive is not UV active and thus not detectable by
conventional UV analysis.
 Mass detection provided sensitive detection and mass spectral
data rich in chemical structure information.
 The adhesive related analyte concentration in the device
hydrolyate was estimated to be 45 ppm using the MS response of
adhesive macromer standard solution for quantification. Low
concentration equivalent to only 1% of the total macromer
applied in the adhesive joint
 Prolonged hydrolysis of the adhesive puck at 100C beyond 170
hours did not result in an increase in the adhesive analyte solution
concentration suggesting that hydrolysis was limited to a labile
macromer fraction.
Summary of Key Points
 Versatility of Waters G2-S QTOF as a highly effective Qual/Quan




Instrument
High Resolution Mass Spectrometry required for all three case
studies. No other method has both the sensitivity and selectivity
that was needed.
Fragment data acquired with QTOF MSMS is mandatory for
structural elucidation of small molecules.
The performance of Mass Frontier software in fragment prediction
was impressive. Its an important tool for successful small molecule
structural elucidation.
Mass Spec Lab’s fragment library built rapidly with MSE technology is
expected to be a superior tool for plastics and packaging additive
identification in E&L studies performed for pharma and biomedical
Acknowledgements
Richard Christ
Sandy Wong
Michelle Tran
Thermo Scientific & High Chem
Dr. Robert Mistrik
Waters Company
Bobbie Frye,
Keith Hermann,
Paul Salafia

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