Journal of Analytical Toxicology, Vol. 29, January/February 2005
Application of Accurate MassMeasurementto
Urine Drug Screening
Ilkka Ojanper~i1,*, Anna Pelander 1, Suvi Laks1, Merja Gergov 1, Erkki Vuori 1, and Matthias Witt 2
1Department of Forensic Medicine, P.O. Box 40, FIN-O0014, University of Helsinki, Helsinki, Finland and 2Bruker Daltonik
GmbH, Fahrenheitstrasse4, D-28359 Bremen, Germany
Abstract
Poor availability of reference standards for designer drugs,
metabolites, and new substancesprevents toxicology laboratories
from rapidly responding to the changing analytical challenges of
drug abuse. A novel screening approach comprising determination
of accurate masses of sample components and comparison of these
with databases of theoretical monoisotopic masses is described.
Using liquid chromatography-time-of-flight mass spectrometry
(LC-TOFMS), a routine mass search window of 20-30 ppm was
applied to urine samples. The ultimate reference technique, liquid
chromatography-Fourier transform mass spectrometry (LC-FTMS),
was capable of confirming the findings within a 3 ppm mass
accuracy. Using a target database of 7640 compounds, the number
of potential elemental formulas ranged from one to lhree with
LC-TOFMS, and it was always one with LC-FTMS. In contrast to
ordinary techniques requiring primary reference standards, the
formula-based databases can be updated instantly with fresh
numeric data from scientific literature and authority sources.
Introduction
Coupled chromatographic/mass spectrometric techniques
form the backbone of laboratory instrumentation in today's
well-equipped forensic and clinical toxicology laboratories. Although gas chromatography-mass spectrometry (GC-MS) is
currently the main tool used in drugs-of-abuse confirmation
analysis (1) and in comprehensive drug screening (2), liquid
chromatography-mass spectrometry (LC-MS) is quickly
gaining ground. LC-MS acquisitions have thus far been largely
directed towards single- or triple-quadrupole mass analyzers,
obviously because of their established position in quantitative
analysis. Recently, these techniques have also been successfully applied to comprehensive toxicological screening (3-5).
The utility of chromatographic techniques and the mentioned low-resolution MS techniques is limited by the lack of
ready access to the required reference standards. It often takes
months to acquire the pure drug metabolites, designer drugs,
or other rare compounds from manufacturers or commercial
* Authorto whomcorrespondenceshouldbe [email protected].
34
sources needed to obtain a reference spectrum for unequivocal
identification. Electronic spectrum libraries do not contain all
of the desired spectra. Production of generally usable LC-MS
libraries for screening is only in its early stages (6), partly due
to the use of different ionization techniques and to the lack of
harmonization of collision energies. Consequently, a demand
exists for a different type of approach: a high-accuracy, highresolution MS technique capable of producing conclusive
identification without the immediate need for primary
reference standards.
A few decades ago, GC coupled with high-resolution MS was
successfully applied to the analysis of biological fluids (7). This
technique was sporadically used in toxicological analysis (8),
but the complex instrumentation involving magnetic sector
MS and extensive data processing hindered routine use. Accurate mass determination with good resolution finally became
feasible with the development of orthogonal acceleration timeof-flight mass analyzers (TOFMS) (9), and modern instruments
can readily be combined with LC using electrospray ionization (10). Affordable benchtop LC-TOFMS instruments have
found widespread use in the analysis of small molecules, such
as in drug analysis (11) and in high throughput screening for
combinatorial chemistry libraries (12). Very recently, we
developedan LC-TOFMS method for toxicologicalscreening of
urine based essentially on accurate mass measurement and an
automated target database search with elemental formulas
(13,14).
The ultimate mass measurement technique is Fourier transform ion cyclotron resonance mass spectrometry (FTMS),
which provides very high mass accuracy and mass resolving
power (15,16), as well as high sensitivity in the attomole and
even in the zeptomole range (17). FTMS can be combined with
different external ion sources, for instance, electrospray (18)
and matrix-assisted laser desorption/ionization (MALDI)(19), or
with a combined electrospray/MALDl source (20) for metabolite
screening. The high mass accuracy of FTMS has been used for
several years for the accurate mass tag strategy in proteomics
to detect proteins using exact massesof protein tryptic digests
(21). These results have shown that very accurate masses can
dramatically improve confidence in identifying compounds
using a databasesearch.
Reproduction(photocopyin8) of editorial content of this journal is prohibitedwithout publisher's permission.
Journal of Analytical Toxicology, Vol. 29, January/February2005
This paper surveys the advantages of accurate mass measurement in toxicological drug screening by comparing the
results obtained with the authors' established LC-TOFMS
method (14) with those of the LC-FTMS. Findings of the
LC-TOFMSscreening for three urine samples were confirmed
by LC-FTMS, and the results were evaluated based on the applicable mass error tolerance and the corresponding lengths of
hit lists obtained with two different target drug libraries.
Experimental
Materials
Standard substances were kindly supplied by the manufacturers. All reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany), except for Jeffamine
D-230| (Fluka, Buchs, Switzerland) and !3-glucuronidase
(Roche, Mannheim, Germany). Acetonitrile and methanol were
high-performance liquid chromatography grade and purchased
from Rathburn (Walkerburn, U.K.). Isolute HCX-5 (100 rag)
mixed-mode solid-phase extraction (SPE) cartridges were
purchased from International Sorbent Technology (IST)
(Hengoed, U.K.).
Urine samples
Urine samples were collected at autopsies. They were chosen
for this study, after routine toxicological screening by various
immunoassays and by chromatographic and spectrometric
methods, to represent a wide range of commonly encountered
drugs.
Sample preparation
The sample preparation was carried out as follows (14): urine
samples (1 mL) were hydrolyzed with 13-glucuronidasefor 2 h
in a water bath at 56~ and 10 IJL of dibenzepin internal standard solution (10 lJg/mL in methanol) was added. The extraction was performed according to IST application note IST 1044
A (22) with minor modifications. The pH of the samples was adjusted between 5 and 7 by adding 2 mL of 0.1M pH 6 phosphate
buffer. The SPE cartridges were solvated and equilibrated with
2 mL of methanol, 2 mL of water, and 3 mL of 0.1M pH 6 phosphate buffer.After sample addition, the cartridges were rinsed
with I mL of 0.1M pH 6 phosphate buffer and dried under full
vacuum for 5 min. The cartridges were further rinsed with
1 mL of 1M acetic acid and again dried for 5 rain. The acidicneutral fraction was eluted with 3 mL of ethyl acetate/hexane
(25:75, v/v). The cartridges were dried for 2 min, rinsed with
3 mL of methanol, and dried again for 2 min. Basic drugs were
eluted with 3 mL of ethyl acetate/ammonium hydroxide (98:2,
v/v). After extraction, the eluates (acidic/neutral fraction and
basic fraction) were combined and evaporated to dryness at
40~ under nitrogen, and reconstituted in 150 IJL of acetonitrile/0.1% formic acid (1:9, v/v). For LC-FTMSanalysis, Sample
2 was further diluted to 1:20.
LC-TOFMS
The LC-TOFMSmethod has been described earlier in detail
(14). The LC was an Agilent (Waldbronn, Germany) 1100 series
system with a diode-array detector. Separation was performed
in gradient mode with a Phenomenex (Torrance, CA) Luna C18(2) 100 x 2-ram (3 pm) column and a 4 x 2-ram pre-column.
The column oven was kept at 40~ Mobile phase components
were 5mM ammonium acetate in 0.1% formic acid and
acetonitrile. The flow rate was 0.3 mL/min. The proportion of
acetonitrile was increased from 10% to 40% in 10 min, to 75%
in 13.5 min, to 80% in 16 rain, and held at 80% for 3 min. Posttime was 5 rain, and injection volume was 10 IJL.
The mass analyzer was an Applied Biosystems (Framingham,
MA) Mariner TOF MS equipped with a PE Sciex (Concord, ON,
Canada) TurboIon Spray source, and a 10-port switching valve.
The instrument was operated in the positive ion mode. The
eluent flow was carried to the ion source without splitting.
Spectrum acquisition time was 2 s, and a mass-to-charge ratio
range from 100 to 750 was recorded.
Daily instrument tuning and three-ion mass scale calibration
was carried out with 0.5 IJg/mLJeffamine D-230 solution in acetonitrile/0.1% formic acid (1:1) by infusion injection at a flow
rate of 50 IJL/min.The theoretical exact mass-to-charge ratios
of the calibration ions were 191.17544, 249.14731, and
317.25917, and a minimum resolution of 5000 was used in the
calibration. Automated post-run internal mass scale calibration
of individual samples was performed by injecting the calibration
solution at the beginning of each run 10 s after sample injection
via a 10-port switching valve equipped with a 20-1~Lloop, using
the same calibrator ions as in the instrument tune.
The database values of theoretical monoisotopic exact masses
of protonated compounds, based on elemental formulas, were
calculated with the Data Explorer software (Applied Biosysterns). The database also included the elemental formula, retention time if known, and a numerical code for each
compound connecting the metabolites together. MS data were
analyzed using an in-house macro program.
LC-FTMS
An Agilent 1100 series LC with a two-wavelength UVdetector
was used. Separation was performed in gradient mode with a
Bischoff(Loenberg,Germany) Prontosi1120-3-C18125 x 2-ram
(3 I~m) column without temperature controlling. The same
mobile phase components, flow rate, and gradient were used for
LC-FTMS as for LC-TOFMS.
The LC-FTMSmeasurements were carried out with an APEX
III FT-ICRMS (Bruker Daltonics, Billerica, MA) equipped with
a seven-tesla superconducting magnet (Bruker Biospin GmbH,
Karlsruhe, Germany), a cylindrical ICR cell ("infinity cell")
(23), and an Apollo electrospray source (Bruker Daltonics).
The instrument was operated in positive ion mode. The mobile
phase flow from the LC system to the MS was split 1:20. The
data acquisition time was 1 s for each spectrum scan, four
scans were averaged for one mass spectrum. Mass spectra were
acquired between m/z 130 and 3000. External calibration was
carried out with diester plasticizers of the ESI solvent using the
masses 207.15909, 229.14103, 279.15909, 301.14103,
315.25299, 337.23493, 391.28429, 413.26689, 447.34689, and
469.32883. For internal calibration, at least three of these
masses were used for a linear calibration. Data acquisition was
35
Journal of AnalyticalToxicology,Vol. 29, January/February2005
performed with 512 k data points, which resulted in a mass resolution between 50,000 and 100,000 in the mass range ofm/z
200 to m/z 400.
Results
The urine samples were first submitted to LC-TOFMS
screening and, after interpretation of results, the suggested
drug findings were confirmed with LC-FTMSby measuring the
accurate masses of the target drugs within the corresponding
narrow mass ranges. Table I lists the findings in each sample together with compounds' accurate masses measured by
LC-TOFMS and LC-FTMS. All LC-TOFMS findings could be
confirmed by LC-FTMS, except for amphetamine in Sample 1,
nicotine in Sample 2, and dibenzepin in Sample 2 (diluted 1:20
for LC-FTMS). The drug concentrations in urine varied from
0.01 to 31 mg/L, as previously measured by GC, thus representing a typical range encountered in forensic toxicology.The
relative differences in parts per million (AM/Mx 106,where AM
is the mass error) between theoretical masses and measured
masses by LC-TOFMS and LC-FTMS are shown in Figures 1
and 2, respectively.The performance of LC-TOFMSwas clearly
dependent on proper internal calibration,which had no marked
effect on LC-FTMS.
To determine the actual significance of mass accuracy in
comprehensive drug screening, two existing target drug
Table I. Drug Findings in Urine Samples with LC-TOFMS Screening and LC-FTMS Confirmation Together with the
Measured Masses
Compound
Name
MeasuredMass MeasuredMass MeasuredMass MeasuredMass
(M+H) FTMS
Molecular (M+H)TOFMS (M+H)TOFMS (M+H)FTMS
Mass(M+H) Ext.Calibration Int. Calibration Ext.Calibration Int. Calibration
Concentration
in Urine(mg/L)
Elemental
Formula
0.01
0.01
0.01
ND*
I
ND
ISTD
GsHnN2OCI
CIsHI.iN202CT
C16H13N2OC1
C16H13N202CI
CgH13N
CloH12N20
Q8H21N30
271.06327
287.05818
285.07892
301.07383
136.11208
177.10224
296.17574
271.08123
287.07264
285.09595
301.09263
136.12692
177.12000
296.1895
271.06638
287.05793
285.08121
301.07804
136.11244
177.10504
296.17487
271.06337
287,05829
285.07897
301.07388
ND
177.10255
296.17565
271.06341
287.05833
285.07902
301.07393
ND
177.10257
296.17567
4.1
ND
G8H18NSCI
C18H18NOSCI
316.09213
332.08704
316.09206
332.08669
C17H16NSCI
302.07648
QTHI6NOSCI 318.07139
C38H~sNO2SCl 348.08196
316.09114
332.0873/
332.08693t
302.07855
318.0702
348.08002
316.09203
332.08672
ND
ND
ND
316.10548
332.10210/
332.10204*
302.09250
318.08459
348.09515
302.07649
318.07145
348.08187
302.07658
318.07143
348.08187
2.5
ND
ND
ND
ND
16
ND
ND
ISTD
CloH~4N2
C~oHuN20
CIoH12N202
C14N2H2002
CI4H2oN203
Q7H2oN4S
Q6H18N4
S
C19H2oNO3F
GsH21N30
163.12298
177.10224
193.09715
249.15975
265.15467
313.14815
299.13250
330.15000
296.17574
163.13200
177.11182
193.11067
249.17279
265.16961
313.16134
299.14475
330.16409
296.19009
163.12249
177.10182
193.10013
249.16045
265.15679
313.14708
299.13088
330.14937
296.17649
ND
177.10257
193.09727
249.15978
265.15465
313.1475
299.13258
330.14994
ND
ND
177.10261
193.09731
249.15981
265.15472
313.14739
299,13252
330.14995
ND
0.4
ND
31
1.4
1.7
ND
1.6
ND
ISTD
C~7H18F3NO
C16H16NOF3
C16H21NO2
C18N2H2o
QoH14N2
CIoH12N20
C17H19N3
C~6H17N3
CIsH21N30
310.14"t33
296.12568
260.16451
265.16993
163.12298
177.10224
266.16517
252.14952
296.17574
310.15846
296.14347
260.18101
265.18512
163.13425
177.11405
266.18024
252.16398
296.19138
310.14291
296.12666
260.16643
265.17045
163.1221
177.10149
266.16555
252.14949
296.17619
310.14126
296.12575
260.16414
265.16962
163.12331
177.10243
266.16484
252.14961
296.17597
310.14129
296.12579
260.16405
265.16951
Sample 1
Nordiazepam
Oxazepam
Diazepam
Temazepam
Amphetamine
Cotinine
Dibenzepin
Sample2
Chlorprothixene
Chlorprothixenesulfoxide/
Hydroxychlorprothixene
Norchloprothixene
Norchlorprothixenesulfoxide
ChlorprothixeneN-oxide
sulfoxide
Nicotine
Cotinine
Hydroxycotinine
Pindolol
Hydroxypindolol
Olanzapine
Norolanzapine
Paroxetine
Dibenzepin
Sample3
Fluoxetine
Norfluoxetine
Propranolol
Mianserine
Nicotine
Cotinine
Mirtazapine
Normirtazapine
Dibenzepin
" Abbreviations:ND, not detectedand ISTD,internalstandard,
t Twopeaksdetected.
36
163.12337
177.10248
266.16474
252.14963
296.17578
Journal of Analytical Toxicology, Vol. 29, January/February 2005
databases of different sizes were selected for database search:
Database A is an in-house database containing the theoretical
masses of 637 drugs and metabolites (14), and Database B is a
larger commercial library of spectra developed for GC-MS and
consists of 7640 theoretical masses (24). Table II shows the
number of different elemental compositions and the number of
different compounds corresponding to the compositions.
low-mass ions in the storing unit (fnultipole) of the ion source,
but only a few toxicologically relevant drugs can be found in this
mass range. Here LC-FTMS was only applied to specific confirmation analyses; however, the technique is also suitable for
screening as there are no hardware limitations (25). The FTMS
technique is particularly we]l suited for metabolic profiling because of its high mass accuracy and an extremely high resolution of more than 100,000. For instance, compounds with the
same nominal mass but mass differences of only 0.005 Da,
which cannot be separated by LC, can simultaneously be sepaDiscussion
rated and detected by FTMS.
A key task is to define the mass error tolerance required for
LC-FTMS with accurate mass measurement was capable of
unambiguous identification of the elemental formula of a small
confirming drug findings from LC-TOFMS screening of urine
molecule (200-1000 Da). As shown in Table II, no differences
samples representing a wide range of concentrations. Internal
were present in the number of elemental formula candidates becalibration with LC-FTMS typically leads to mass accuracies
tween the mass windows used with Database A (637 combetter than 2 ppm. However, mass peaks with very high intenpounds). Using Database B (7640 compounds), the number of
sities can cause higher mass errors due to overloading of the ancandidates decreased drastically when changing from 30 ppm to
alyzer cell. This is why Sample 2 was diluted prior to analysis,
5 ppm, while virtually no difference was observed between
which in turn resulted in the internal standard dibenzepin
5 ppm and 3 ppm. Consequently, identification solely by elebeing undetectable.
mental formulas, that is, without retention times and metabolic
Compounds with masses below 200 Da could not be perfectly
pattern information, requires a better accuracy than that obanalyzed with LC-FTMS because of lower storage efficiencies of
tained by the present LC-TOFMS method. The 3 ppm accuracy
achieved with LC-FTMS seems to be sufficient
to elucidate the elemental formula even against
120
a relatively large target database, such as
105 - Database B. This finding is supported by a mea~-external calibration I
l " internal calibration
90
sured mass within 5 ppm generally being accepted as adequate to confirm the elemental
75
composition of an organic compound (26).
== 60
Theoretically, at a nominal parent mass of 500
oo
o
~
Da (C0_100H3_/400..4N0_4),there are five comr~
positions that have a neighboring candidate
30
fewer than 5 ppm away (27).
15
Table II indicates that as the database size increases several potential compounds may rep% 9
l
4'
0
m m
resent a single elemental formula. In these
-15
cases, additional information for differentia100
150
200
250
300
350
400
tion, such as the metabolic pattern, LC rem/z
tention time, or MS-MS fragmentation, is
Figure 1. Mass error of liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS)
required.
measurement.
In postmortem forensic toxicology, the benefit of broad-scale screening is self-evident, and
terms like "systematic toxicological analysis"
and "general unknown screening" have been
o external calibration
dedicated to procedures usually involving nunternal calibration
merous techniques (28,29). In the authors'
laboratory practice, only 20 drugs have been estimated to account for up to 80% of fatal poi9-= 0
sonings, and these cases can readily be solved
by, for example, a single efficient GC method
(30). Raising this percentage will rapidly in-2
crease the number of potential xenobiotics to
several hundred, addressing the need for the
100
150
200
250
300
350
400
same number of reference standards and a
multitude of parallel or complementary
Figure 2. Mass error of liquid chromatography-Fourier transform mass spectrometry (LC-FTMS)
analytical techniques. The present concept of
measurement.
qualitative analysis with accurate mass meao
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Journal of Analytical Toxicology, Vol. 29, January/February2005
surement meets the demands of systematic toxicological analysis in a very elegant way.
In emergency clinical toxicology situations, the need for comprehensive drug screening has been recognized in several
studies (31,32). Among surveys criticizing comprehensive drug
screens, one study stresses that management of overdose should
be based on clinical symptoms and signs with directed investigations only (33), and another admits that screens would still
enable optimal treatment in many cases (34). In addition to
acute poisonings, there are other important clinical and forensic
circumstances in which a comprehensive drug screen is indicated. These include treatment of drug abusers, monitoring of
compliance, psychiatric diagnostics, evaluation of ability to
work, child welfare, drug-facilitated sexual assault, occupational accidents, doping control, and driving under the influence. A limited screen based on only a few target analyses would
readily lead to erroneous actions in these instances.
Analogously to progress in characterization of libraries within
combinatorial chemistry, it can be anticipated that the present
approach, accurate mass measurements resulting in independence from reference substances, will open a new avenue for
toxicological screening. The instrumentation may involve a
particularly efficient LC-TOFMS method capable of 5-10 ppm
mass accuracy in biological material, or preferably LC-FTMS
with a mass accuracy to within 3 ppm. Disadvantages of the
latter are higher cost of purchase and maintenance, and more
demanding operation. A further logical step would be quantitative analysis of identified drugs using a chemiluminescence
nitrogen detector, which would produce an equimolar response
for all nitrogen-containing substances with a single secondary
reference standard (35).
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Manuscript received October 8, 2003;
revision received January 28, 2004.