Journal of Chromatography B, 713 (1998) 3–25

Review

Liquid chromatography–mass spectrometry in forensic and clinical

toxicology 1
Hans H. Maurer
Institute of Pharmacology and Toxicology, Department of Toxicology, University of Saarland, D-66421 Homburg ( Saar), Germany

Abstract

This paper reviews liquid chromatographic–mass spectrometric (LC–MS) procedures for the identification and / or
quantification of drugs of abuse, therapeutic drugs, poisons and / or their metabolites in biosamples (whole blood, plasma,
serum, urine, cerebrospinal fluid, vitreous humor, liver or hair) of humans or animals (cattle, dog, horse, mouse, pig or rat).
Papers published from 1995 to early 1997, which are relevant to clinical toxicology, forensic toxicology, doping control or
drug metabolism and pharmacokinetics, were taken into consideration. They cover the following analytes: amphetamines,
cocaine, lysergide (LSD), opiates, anabolics, antihypertensives, benzodiazepines, cardiac glycosides, corticosteroids,
immunosuppressants, neuroleptics, non-steroidal anti-inflammatory drugs (NSAID), opioids, quaternary amines, xanthins,
biogenic poisons such as aconitines, aflatoxins, amanitins and nicotine, and pesticides. LC–MS interface types, mass spectral
detection modes, sample preparation procedures and chromatographic systems applied in the reviewed papers are discussed.
Basic information about the biosample assayed, work-up, LC column, mobile phase, interface type, mass spectral detection
mode, and validation data of each procedure is summarized in tables. Examples of typical LC–MS applications are
presented.  1998 Elsevier Science B.V. All rights reserved.

Keywords: Reviews; Therapeutic drugs; Drugs of abuse; Poisons

Contents

1. Introduction ............................................................................................................................................................................ 4
1.1. LC–MS interfaces........................................................................................................................................................... 4
1.2. LC–MS detection modes ................................................................................................................................................. 5
1.3. Applicability of LC–MS in forensic and clinical toxicology ............................................................................................... 5
1.4. Choice of the references .................................................................................................................................................. 5
2. LC–MS determinations of drugs, poisons and / or their metabolites in biosamples ........................................................................ 5
2.1. Sample preparation ......................................................................................................................................................... 6
2.2. Liquid chromatographic systems ...................................................................................................................................... 6
2.3. LC–MS procedures......................................................................................................................................................... 15
2.3.1. Illicit drugs of abuse ............................................................................................................................................ 15
2.3.2. Therapeutic drugs relevant to forensic or clinical toxicology .................................................................................. 16
2.3.3. Poisons............................................................................................................................................................... 19
2.3.4. Applications in studies on pharmacokinetics or metabolism of future drugs ............................................................. 21

1
¨
Dedicated to Prof. Dr. Gottfried Blaschke, Munster, Germany, on the occasion of his 60th birthday.

0378-4347 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved.

PII: S0378-4347( 97 )00514-8
4 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

3. Conclusions and perspectives ................................................................................................................................................... 22

4. List of abbreviations ................................................................................................................................................................ 22
Acknowledgements ...................................................................................................................................................................... 23
References .................................................................................................................................................................................. 23

1. Introduction already with ES [2,8,9,11,32–52] or APCI [4,6,53–

69]. Modern (benchtop) apparatus supply both tech-
In forensic and clinical toxicology analytical meth- niques. They allow very sensitive, gentle and univer-
ods must provide high reliability and accuracy. The sal procedures.
combinations of mass spectrometry with suitable ES enables the determination of analytes of high
chromatographic procedures are the methods of molecular mass up to several hundred thousand units
choice, because they are very sensitive, precise, such as peptides or proteins [14,15,18,70,71], and / or
specific, universal and fast. Today, GC–MS is the very high polarity such as quaternary amines, sulfate
golden standard for detection and quantification of conjugates, nucleotides or phospholipids
drugs and poisons volatile under GC conditions ([1] [9,28,70,72–74]. A prerequisite is that the analyte
and other papers of this Special Volume), whereas must be ionizable in solution, so that the mobile
non-volatile compounds require LC–MS [2–18]. phase often contains a small amount of a volatile
While GC can easily be coupled with MS, LC can acid or base. If such additives impair the chromato-
only be coupled with MS via sophisticated inter- graphic separation, they can be added after the
faces. separation before the eluent enters the ES interface
[25,29,43].
APCI allows very sensitive determination of ana-
1.1. LC–MS interfaces lytes with moderate polarity and molecular mass.
Since ES and APCI are soft ionization techniques,
Since the 1970s, different LC–MS interface types, they usually produce quasi-molecular ions. If more
such as moving belt, direct liquid introduction, information on the chemical structure is needed,
continuous-flow or frit-terminated fast atom bom- fragmentation can be obtained either by collisionally
bardment (FAB), particle beam (PB), thermospray induced dissociation (CID) in the ion source or by
(TS), electrospray (ES) or atmospheric-pressure using tandem mass analysis (MS–MS) [16,17].
chemical ionization (APCI) were developed to re- However, interpretation of the resulting mass spectra
move the mobile phase and to ionize the analyte. The may be difficult, since on the one hand the frag-
technical principles, and the advantages and dis-
advantages of these interfaces were recently de-
scribed in the reviews of Gelpi [15], Careri et al. [16]
or Hoja et al. [17], so that this topic needs not be
repeated here. In Fig. 1 the relation of the molecular
mass range and the polarity of analytes that can be
analyzed by GC–MS and different LC–MS interface
techniques are sketched. As shown, LC–MS with ES
is the most universal of these analytical techniques.
In the last 2 years only few papers using PB
[10,19–21], FAB [3,5,12,22] or TS [7,23–31] have
appeared. These interfaces have several limitations,
such as less sensitivity or less universality. Today,
two relatively robust LC–MS interface types seem to
have become the golden standards of LC–MS, the Fig. 1. Relation of the molecular mass range and the polarity of
atmospheric-pressure ionization techniques, ES and analytes analyzable by GC–MS and different LC–MS interface
APCI. The majority of the papers reviewed here deal techniques.
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 5

mentation depends on several parameters (e.g. CID used for screening in order to differentiate between
voltage) and on the other hand adduct ions can be negative and presumptively positive samples. Posi-
formed, e.g. by ammonia or sodium present in the tive results must be confirmed by a second in-
eluent. dependent method that is at least as sensitive as the
screening test and that provides the highest level of
1.2. LC–MS detection modes confidence in the result. Some recent LC–MS papers
on drugs of abuse testing deal with confirmation of
Mass analysis of the substances ionized by the positive immunoassay results [23,24,33–
different interfaces is performed using one or two 36,41,53,55] or with the detection of drugs of abuse
mass analyzers (MS–MS), which consist of ion not detectable by immunoassay [25,26,37–40,53–
traps, sector field instruments or predominantly of 57]. Some authors describe procedures for identifica-
quadrupoles. They can operate in the full scan mode tion and / or quantification of therapeutic drugs rel-
or in the more sensitive selected-ion monitoring evant to analytical toxicology in human or animal
mode (SIM) detecting positive or negative ions. The samples [2–11,19–22,25–28,42–52,58–66,87],
MS–MS combination provides additional possibil- while others describe the detection of non-volatile
ities, because both analyzers can be operated in a poisons relevant to diagnosis of an acute or chronic
scan or in a selected-ion mode resulting in four intoxication [12–14,67,69]. Further papers deal with
combinations: parent-ion scanning (scan mode in the the quantification of (future) therapeutic drugs as part
first analyzer, SIM in the second), daughter-ion of pharmacokinetic or metabolic studies [9,23,29–
scanning (DIS; SIM in the first analyzer, scan mode 31,37,38,55,72,73,75–86,88–98]. Such papers are
in the second), constant neutral-loss scanning not reviewed in detail, but they are mentioned in
(CNLS; scan mode in both analyzers) or selected- Section 2.3.4. They were not omitted for two
reaction monitoring (SRM; SIM in both analyzers) reasons: on the one hand such therapeutic drugs may
[16,17]. DIS is preferable for the identification of also occur in future toxicological cases, and on the
drugs and / or their metabolites in complex matrices, other hand they may give hints for developing new
since separation is performed on the LC and in the procedures for similar compounds of toxicological
first mass analyzer, while structural information is interest.
obtained by fragmentation in the second analyzer.
SRM (sometimes named multiple-reaction monitor- 1.4. Choice of the references
ing, MRM) is the most powerful technique for
quantification of small amounts of analyte in com- The reviewed references were selected by on-line
plex matrices. Especially pharmaceutical companies searching the Medline database, the Chemical Ab-
are using this technique for series of quantifications stract Services ( CAS) and Current Contents. Since
during pharmacokinetic studies of new (polar and Hoja et al. [17] most recently published a review
low dosed) drugs [75–86]. considering papers until the beginning of 1995, the
period from January 1995 to April 1997 was taken
1.3. Applicability of LC–MS in forensic and into consideration. Only papers written in English
clinical toxicology were considered.

The choice of the method in analytical toxicology

depends on the problems which have to be solved. 2. LC–MS determinations of drugs, poisons
Usually, the compounds which have to be analyzed and / or their metabolites in biosamples
are unknown. Therefore, before quantification, the
drug or poison must first be identified. The analytical LC–MS procedures for the identification (ID)
strategy – especially in drugs of abuse testing – and / or quantification (QU) of drugs or poisons
includes a screening test and a confirmatory test. If relevant to forensic or clinical toxicology published
only a single drug or a single category of drugs must in the last 2 years are critically reviewed in this
be monitored, immunoassays, if available, can be chapter. Most of the papers concerning this topic
6 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

have been published in early 1997 workstation to perform the complete process. Three
[2,12,14,32,33,39,40,44,50,53,55,61,99]. This fact different columns were used in the column switching
indicates that LC–MS is a new but upcoming system: an IAE column packed with the bound
technique also in this field of scientific research and antibodies, a trapping column and an analytical
application. column. The work-up process consisted of the fol-
The principal information on each procedure is lowing steps: preconditioning of the IAE column,
summarized in Tables 1–3 to simplify the rapid loading of the urine sample, elution of the unbound
selection of a method suitable for an actual analytical matrix to waste, equilibration of the trapping and
problem. The information, whether a paper deals analytical column with mobile phase, elution of the
with a quantitative assay, can be taken from the bound analytes and trapping in the trapping column,
Validation column. Retention time and mass spectral back-flushing of the trapped analytes onto the ana-
data are not listed in the tables to save space. lytical column, separation and introduction into the
MS(–MS). The advantage of such a procedure is not
2.1. Sample preparation only the full automation but also the highly selective
and sensitive detection. For example, the LOD for
Suitable sample preparation is an important pre- the IAE-LC–LC–MS–MS detection of LSD in urine
requisite for chromatography of biosamples. It in- was 20-fold below that using off-line SPE and LC–
volves isolation after cleavage of conjugates by MS–MS [35]. However, a disadvantage of IAE is
enzymatic (EHY) or acid hydrolysis (if required), that overloading the antibodies with the analyte (e.g.
and / or derivatization of the drugs and their metabo- in overdose cases) leads to poor linearity. This is in
lites. Isolation was performed by liquid–liquid ex- accordance with our studies on IAE of amanitins
traction at a pH at which the analyte is uncharged [103,104].
(see LLE in Work-up column in Tables 1–3) or by A simpler alternative way (e.g. for determination
solid-phase extraction (see SPE) preceded or fol- of plasma levels) is the direct injection of the
lowed by clean-up steps. Afterwards, the extract is biosample onto a pre-column, washing the matrix
concentrated and finally dissolved in an appropriate from this column to waste and back-flushing the
volume of mobile phase. The pros and cons of both analyte onto the analytical column [64]. On-line
types of extraction are discussed in detail in the extraction was also described using two analytical
review of Franke and De Zeeuw in this Special columns (LC–LC–MS–MS) [27] or using an SPE
Volume [100]. column connected to the analytical column (SPE–
Most analytes do not require derivatization for LC LC–MS–MS) [19]. By applying such techniques, the
separation or MS detection. However, some analytes analysis cycle time can drastically be reduced (e.g.
such as amines show only poor chromatographic ,5 min), especially in combination with MS–MS
behaviour without derivatization. Therefore, Bogusz [64], in which the analyte can be separated further by
et al. [53] have derivatized their analytes (amphet- its mass in the first mass analyzer and specifically
amine derivatives) to improve the LC separation and detected in the last.
the MS or the diode array detection (DAD). How-
ever, if derivatization was necessary, the advantage 2.2. Liquid chromatographic systems
of such an LC–MS procedure seems to be ques-
tionable in comparison to GC–MS procedures as As shown in Tables 1–3, the most commonly used
reviewed by Kraemer and Maurer in this Special stationary phases were – as usual – reversed-phase
Volume [101]. C 18 columns. For enantioselective determination,
An excellent way to isolate analytes from biosam- normal-phase columns with alkane–alcohol mixtures
ples is immunoaffinity extraction (IAE), especially if as mobile phase were used [43,47]. Ammonium
coupled on-line with the LC–MS [34,35,61,102]. Cai acetate must be mixed into the non-aqueous eluent
and Henion [61], for example, have recently de- [47] or it must be introduced post-column to the
scribed an automated on-line clean-up and enrich- eluent [43] to improve the ionization in the MS and
ment procedure using a commercially available to reduce the risk of explosion in the heated nebul-
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 7

Table 1
LC–MS procedures for the identification and / or quantification of illicit drugs of abuse and / or their metabolites in biosamples
Compound Sample Work-up Stationary phase Mobile phase Interface Detection Validation data Ref.
(mixtures in v/v) mode

Amphetamines: S, U LLE, Superspher Select 50 mM ammonium APCI ID: scan REC: 70–75% [53]
Amphetamine (AM), phenylisocyanate Ba ECOcart formate (pH 3)– QU: SIM LOD: 5 ng/ml (AM,
methamphetamine (MA), derivatization (12533 mm I.D.) ACN (55:45) MDA); 1 ng/ml (others)
methylenedioxy-AM (MDA), LIN: 5–1000 ng/ml
methylenedioxy-ma (MDMA),
methylenedioxyethyl-AM
(MDE); ephedrine,
fenfluramine,
norfenfluramine,
phentermine,
phenylethylamine,
phenylpropanolamine,
propylhexedrine
Amphetamines: U SPE Ultron ES-PhCD 100 mM TS ID: scan, SIM; REC: 96.5–98.5% [24]
Amphetamine (AM), (15036 mm I.D., ammonium acetate QU: UV, 220 nm LOD scan : 10 ng/ml
methamphetamine (MA) 5 mm); chiral (pH 6)–MeOH– (AM); 20 ng/ml (MA)
separation ACN (60:30:10) LOD SIM : 0.5 ng/ml
(AM); 0.8 ng/ml (MA)
For HPLC–UV:
LIN: 0.5–10 ng/ml
Amphetamines and other U LLE L-column ODS gradient elution: TS ID: scan REC: 88–99% [23]
drugs of abuse: (15034.6 mm I.D.) 100 mM QU: SIM LOD scan : 50–400 ng/ml
Amphetamine (AM), ammonium acetate- LOD SIM : 2–40 ng/ml
methamphetamine (MA), -ACN (100:0– LIN:?–2000 ng/ml
(methyl)ephedrine; morphine1M (M3G, M6G), 60:40) (M3G, M6G)
cocaine1M LIN: 40–500 ng/ml
(others)
Cocaine: Blood spots Elution of BZE 2 Perkin-Elmer C 18 25 mM ammonium APCI MS-MS, SRM REC:? [54]
Cocain-M (BZE) from the spot by in series (3034.6 mm acetate in MeOH– LOD: 2 ng/ml
aqueous ammonium I.D., 3 mm) water (50:50) LIN: 4–166 ng/ml
acetate,
deproteination by
MeOH, evaporation
Cocaine and other drugs U LLE L-column ODS gradient elution: TS ID: scan REC: 88–99% [23]
of abuse: (15034.6 mm I.D.) 100 mM QU: SIM LOD scan : 50–400 ng/ml
Cocaine1M ammonium acetate- LOD SIM : 2–40 ng/ml
(benzoylecgonine, BZE); -ACN (100:0– LIN:?–2000 ng/ml
amphetamine (AM), 60:40) (M3G, M6G)
methamphetamine (MA), LIN: 40–500 ng/ml
(methyl)ephedrine, (others)
morphine1M (M3G, M6G)
Lysergide: U SPE Nucleosil C 18 2 mM ammonium ES SIM REC: 93%, 80% (M) [32]
LSD1M (nor-) (15031 mm I.D.) formate (pH 3)– LIN: 0.05–20 ng/ml
ACN (70:30) LOD: 0.025 ng/ml,
0.035 ng/ml (M)
LOQ: 0.1 ng/ml
(metabolite)
Lysergide: U SPE Hypersil C 18 100 mM ES SIM REC: ? [33]
LSD1M (nor-) (12533 mm I.D., 3 mm) ammonium acetate LIN: 0.5–10 ng/ml
(pH 8)–ACN
(75:25)

(Cont.)
8 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

Table 1. Continued
Compound Sample Work-up Stationary phase Mobile phase Interface Detection Validation data Ref.
(mixtures in v/v) mode

Lysergide: U SPE, IAE Hypersil C 18 100 mM ES SIM REC: 83–100% (SPE); [34]
LSD (12533 mm I.D., 3 mm) ammonium acetate 64–166%! (IAE)
(pH 8)-ACN LIN: 0.5–10 ng/ml
(75:25)
Lysergide: U IAE (on-line IAE column: antibodies ACN–MeOH– ES MS-MS, SRM REC: ? [35]
LSD1M extraction) bound to protein G HOAc–5 mM LOD: 0.0025 ng/ml
(2.133.3 mm I.D.) ammonium acetate
Trapping column: LC (30:30:0.1:39.9)
Packings C 18
(1530.5 mm I.D., 5 mm)
Analytical column: LC
Packings C 18
(15030.3 mm I.D., 3 mm)
Lysergide: U, human SPE Spherisorb ODS-2 Gradient elution ES MS-MS, REC: 45–74% (U) [36]
LSD1M liver microsomes (10031 mm I.D., 5 mm) from 44 to 100%B: CNLS LOD: 0.05 ng/ml
(A) ACN–MeOH–HOAc–5 mM (microsomes),
ammonium acetate SRM (U)
(10:10:0.1:79.9)
(B) ACN–MeOH–
HOAc–5 mM
ammonium acetate
(49.5:49.5:0.1:0.9)
Opiates: B, U, SPE Superspher Select B 50 mM ammonium APCI QU: SIM REC: 82–89% [55]
Morphine, morphine-3- cerebro-spinal (12533 mm I.D.) formate (pH 3)– LOD: 0.1–1 ng/ml
glucuronide (M3G) fluid, vitreous ACN (95:5) or LIN: 5–500 ng/ml
morphine-6-glucuronide (M6G), humor (90:10) for MAM
6-monoacetylmorphine
(MAM)
Opiates: S SPE Supelcosil ABZ Gradient elution: ES QU: SIM REC: 70% (M3G) [37]
Morphine1M (M3G, M6G) (25034.6 mm I.D., 5 mm) water-MeOH REC: 95% (others)
(85:15–40:60) LIN: 10/100/50–
1000 ng/ml
(morphine/M3G/M6G)
Opiates: S SPE YMC ODS-AL gradient elution: 3 ES QU: SIM REC:? [38]
Morphine1M (M3G, M6G) (10034.6 mm I.D.) mM formic acid in LIN: 0.84–17/5–
water–3 mM 500/2–100 ng/ml
formic acid in ACN (morphine/M3G/M6G)
(4:96–70:30)
Opiates and other drugs U LLE L-column ODS Gradient elution: 100 mM TS ID: scan REC: 88–99% [23]
of abuse: (15034.6 mm I.D.) ammonium acetate–ACN QU: SIM LOD scan : 50–400 ng/ml
Morphine1M (M3G, M6G, (100:0–60:40) LOD SIM : 2–40 ng/ml
MAM); amphetamine (AM), LIN:? –2000 ng/ml
methamphetamine (MA), (M3G, M6G)
(methyl)ephedrine, LIN: 40–500 ng/ml
cocaine1M (BZE) (others)

izer. Some authors recommended a short guard in the tables. It is incomprehensible, why referees
column with the same type of stationary phase and editors accept papers in which neither the length
[2,14,27,42,45,47,49,52,59,63,64,68]. Others used nor the diameter of the column were reported [4].
two analytical columns in series [29,54]. If the The mobile phases consisted of mixtures of vola-
column was heated, the column temperature is given tile buffers (e.g. ammonium acetate or formate) with
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 9

Table 2. LC–MS procedures for the identification and / or quantification of toxicologically relevant drugs and / or their metabolites in
biosamples
Compound Sample Work-up Stationary phase Mobile phase Interface Detection mode Validation data Ref.

Anabolic steroids: U EHY, SPE-LC Ultrasphere ODS 10 mM ammonium PB scan REC: .80% [19]
Excretion studies on (on-line extraction) (25032.1 mm I.D., acetate (pH 7.35)– LOD: th. conc.
testosterone, boldenolone, 5 mm) ACN (55:45)
methenolone,
methyltestosterone,
trenbolone; for further
10 steroids and 13 of their
metabolites only t R and
MS data are given
Anabolic steroids: U SPE? (not Hypersil BDS C 18 Gradient systems: ES MS-MS, SRM No data [9]
Sulfate and glucuronide described!) (15031 mm I.D.) or (A) 0.11% acetic acid– recorded in
conjugates of (epi)- Hypersil BDS C 18 7.5 mM ammonium urine samples
testosterone, (epi)- (25030.3 mm I.D., acetate in water; were given!
androsterone, 3 mm) (B) 0.11% acetic acid–
dehydroepiandrosterone, 7.5 mM ammonium
estrone, etiocholanolone acetate in MeOH or
ACN
Antihypertensive drugs: S LLE Puresil C 18 (15032.1 10 mM ammonium APCI MS-MS, SRM REC: ? [58]
Amlodipine mm I.D., 5 mm) acetate (pH 4)– LIN: 0.014–
MeOH (33:67) 7.2 ng/ml
Antihypertensive drugs: P LLE Chiralpak AD CSP n-Hexane–2-propanol– ES MS-MS, SRM The authors [43]
Doxazosin (10032.1 mm I.D., diethylamine state that
10 mm), 308C; chiral (70:30:0.1) validation is
separation in progress
Antihypertensive drugs: P LLE Chira OJ MOD 2 mM ammonium ES MS-MS, SRM REC: 90% [47]
Nimodipine (the method (25032 mm I.D., acetate in ethanol– LOD: 0.25 ng/ml
could be transfered to 8 mm) with a guard n-heptane (20:80) LIN: 0.25–
other dihydropyridines like column (1032 mm 75 ng/ml
felodipine, nisoldipine, I.D.), 358C; chiral LOQ: 0.5 ng/ml
nitrendipine) separation
Antihypertensive drugs: P (horse) LLE, SPE Betasil C 18 (10031 5 mM ammonium ES MS-MS, SRM For LLE extracts: [50]
Reserpine mm I.D., 5 mm) acetate–ACN (20:80) REC: 68–76%
For LLE extracts: pH 7.12 LOD: 0.01 ng/ml
For SPE extracts: pH 5.47 LIN: 0.01–5 ng/ml)
LOQ: 0.05 ng/ml
For SPE extracts:
REC: 44–58%
LOD: 0.01 ng/ml
LIN: 0.1–5 ng/ml
LOQ: 0.2 ng/ml
Antihypertensive drugs: P LLE Chiralpak AD CSP Ethanol–n-hexane–2- ES MS-MS, SRM The authors [43]
Sotalol, verapamil1M (10032.1 mm I.D., propanol–diethylamine state that
(nor-) 10 mm), 208C; (30:63:7:0.17), validation is
chiral separation addition of 800 ml/min in progress
of 25 mM ammonium
acetate–2-propanol
(25:75) post-column to
improve the ionization
and to reduce the risk
of explosion

(Cont.)
10 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

Table 2. Continued
Compound Sample Work-up Stationary phase Mobile phase Interface Detection mode Validation data Ref.

Benzodiazepines: S, U SPE LiChrospher 60-RP MeOH–water–ACN ES MS-MS, SRM REC: 91–110% (S), [41]
Diazepam, nordazepam, (10032 mm I.D., (1:1:1), pH 6 90–104% (U)
nitrazepam, flunitrazepam, 5 mm) LIN: 1–1000 ng/ml)
medazepam
Benzodiazepines: B SPE Nova-Pak C18 50 mM ammonium TS MS-MS, SRM REC: about [25]
Flunitrazepam1M (nor-), (15033.9 mm I.D., acetate–MeOH 50% LOD:
lormetazepam (for 4 mm) (40:60), addition of not reported
brotizolam, clonazepam, 800 ml/min of 50 mM for biosamples
diazepam, ketazolam, ammonium acetate
loprazolam, nitrazepam post-column to
and triazolam only t R and improve the ionization
on-column LOD data are
given)
Benzodiazepines: P (pig) SPE Hitachi C 18 (15034.6 100 mM ammonium APCI SIM REC: 93–98% [56]
Midazolam, (atipamezole, mm I.D., 5 mm) acetate–MeOH LIN: 0.5–
medetomidine) (35:65) 100 ng/ml)
LOQ: 1–2 ng/ml
Benzodiazepines: P, U LLE Nomura ODS-HG-5 Gradient: 50 mM APCI SIM REC: 77–84% (P), [57]
Triazolam1M (15034.6 mm I.D., ammonium acetate 79–85% (U)
5 mm) (pH 4)–MeOH (50:50– LIN: 0.02–300 ng/ml
0:100)
b2 -Agonists: P SPE Upchurch a-Chrom Gradient: 10 mM APCI MS, SIM; REC: 62–104% [59]
Clenbuterol, fenoterol, C 18 (25033 mm I.D., ammonium acetate MS-MS, SRM LOD: 2.5 ng/ml
metaproterenol, 5 mm) with a guard (pH 7)–water–ACN for salbutamol LIN: 2.5–50 ng/ml
salbutamol, terbutaline column (C 18 silica (12:11:77) to 25 mM
(532 mm I.D., ammonium acetate
10 mm)) (pH 3.5)–ACN
(35:65)
b2 -Agonists: U, liver EHY, SPE Upchurch a-Chrom Gradient: APCI MS-MS, SRM REC: 60–80% (U); [60]
Cimaterol, clenbuterol, (cattle) C 18 (25033 mm I.D., (A) 10 mM 45–55% (liver)
mabuterol, salbutamol, 5 mm) ammonium acetate LOD: 0.05 ng/ml (U);
terbutaline (pH 7)–water–ACN 0.05–0.1 ng/g
(12:11:77) to 25 mM (liver)
ammonium acetate LIN: 0.5–
(pH 3.5)–ACN 25 ng/g (liver)
(35:65)
b2 -Agonists: U (cattle) LC-LC (on-line 2 columns Microspher MeOH–water TS MS-MS, SRM REC: 102–107% [27]
Clenbuterol, salbutamol sample enrichment) C 18 (5035.6 mm I.D., containing 100 mM LOD: 0.05 ng/ml
3 mm) with a guard ammonium acetate, LIN: ?
column each 10 mM triethylamine LOQ: 0.1 ng/ml
(Chrompack (1033 and 170 mM formic
mm I.D.)) acid (30:70) for
clenbuterol, (18:82)
for salbutamol
b2 -Agonists: U (cattle) IAE (on-line extraction) IAE column: ACN–MeOH–0.1% aqueous acetic acid APCI MS-MS, SRM REC: 94–108% [61]
Clenbuterol, mabuterol, antibodies bound to (A) 2.5:2.5:95 LIN: 0.05–250 ng/ml
mapenterol, protein G (1032 mm (B) 47.5:47.5:5
methylclenbuterol, I.D.) ID: gradient 90%A–100B;
tolubuterol Trapping column: QU: 28%A, 72%B
pellicular C 18 (2031
mm I.D., 30–
40 mm)
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 11

Table 2. Continued
Compound Sample Work-up Stationary phase Mobile phase Interface Detection mode Validation data Ref.

Analytical column:
LC Packings C 18
(15030.3 mm I.D.,
3 mm)
Cardiac glycosides: P LLE Nova-Pak C 18 (15032 Gradient: 2 mM ES Scan, SIM REC: 67–90% [2]
Digoxin, digitoxin, a-, mm I.D., 4 mm) ammonium acetate LOD: 0.15–0.6 ng/ml
b-acetyldigitoxin, with a guard column (pH 3)–ACN (20:80–
lanatoside C, oleandrin (Optiguard C 18 (1531 70:30)
mm I.D., 5 mm))
Cephem antibiotics: S SPE Pre-column: For pre-column: FAB Scan, SIM REC: .60% [3]
24 Antibiotics Develosil PhA 10 mM acetic acid– LOD: th. conc.
(3030.5 mm I.D., glycerol (or
10 mm), analytical diethanolamine)
column: (99.5:0.5);
Develosil PhA water–MeOH–acetic
(15030.5 mm I.D., acid–glycerol
5 mm) (59:40:0.5:0.5) or
water–MeOH–acetic
acid–diethanolamine
(57:40:2.5:0.5)
Corticosteroids: U (cattle) EHY, SPE Spherisorb ODS2 100 mM ammonium APCI MS-MS, SRM REC: 80–86% [62]
Betamethasone, cortisone, (25034.6 mm I.D., acetate (pH 6.8)– LOD: 0.05 ng/ml
dexamethasone, 5 mm) ACN (60:40)
flumethasone, flunisolide,
hydrocortisone,
methylprednisolone,
prednisolone, prednisone
Corticosteroids: S, U (horse) LLE Lichrospher 100 C 18 Gradient: 10 mM ES MS-MS, SIM For serum: [52]
Triamcinolone (12534 mm I.D., ammonium acetate– REC: 83%
5 mm) with a guard MeOH (50:50–0:100) LOD: 0.1 ng/ml
column (C 18 (434 mm LIN: 0.3–12 ng/ml
I.D., 5 mm)) For urine:
REC: 81%
LOD: 0.1 ng/ml
LIN: 0.5–50 ng/ml
Immunosuppressants: B SPE Resolve C 18 (15033.9 MeOH–1% formic ES SIM (MS-MS REC: 85–91% [8]
Sirolimus1M mm I.D., 5 mm), acid (90:10) for identification LIN: 0.25–
408C of M) 250 ng/ml
Immunosuppressants: B, U SPE Spherical C 18 MeOH–water (90:10) PB SIM REC: 90% [10]
Tacrolimus1M (15033.9 mm I.D., LIN: 0.2–
5 mm) 100 ng/ml
Immunosuppressants: B SPE Brownlee C-4 40 mM ammonium ES MS-MS, SRM REC: 54.3% [11]
Tacrolimus Column (3032.1 mm acetate (pH 5.1)– LIN: 0.2–
I.D.) MeOH (20:80) 100 ng/ml
Neuroleptics P LLE Nucleosil C 18 2 mM ammonium ES SIM REC: 58%; [44]
( Butyrophenones): (15031 mm I.D.) formate (pH 3)– 70% (M)
Haloperidol1M (dihydro-) ACN (55:45) LOD: 0.075 ng/ml;
0.1 ng/ml (M)
LIN: 0.1–
50 ng/ml; 0.25–
50 ng/ml (M)

(Cont.)
12 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

Table 2. Continued
Compound Sample Work-up Stationary phase Mobile phase Interface Detection mode Validation data Ref.

Neuroleptics B SPE Nova-Pak C 18 Gradient: 50 mM TS MS-MS, SRM REC: 80–90% [26]

( Butyrophenones): (15033.9 mm I.D., ammonium acetate– LOD: 0.05 ng/ml
Haloperidol (for 4 mm) MeOH (35:65–25:75) LIN: 0.5–12.5 ng/ml
penfluridol, pimozide,
benperidol, droperidol and
pipamperone only t R and
on-column LOD data are
given)
Neuroleptics B SPE Nova-Pak C 18 50 mM ammonium TS MS-MS, SRM REC: 80–90% [25]
( Butyrophenones): (15033.9 mm I.D., acetate–MeOH LOD: not
Haloperidol (for 4 mm) (25:75), addition reported for
penfluridol, pimozide, of 800 ml/min of biosamples
benperidol, droperidol and 50 mM ammonium
pipamperone only t R and acetate post-column to
on-column LOD data are improve the ionization
given)
Neuroleptics B SPE Asahipak OPD-50 50 mM ammonium TS MS-MS, SRM REC: 80–90% [25]
( Phenothiazines): (12534 mm I.D., acetate–ACN (15:85), LOD: not
Chlorprothixene (for 5 mm) addition of 800 ml/min reported for
flupentixol, thiothixene of 50 mM ammonium biosamples
and clopenthixol only t R acetate post-column to
and on-column LOD data improve the ionization
are given)
Non-steroidal anti- U (horse) SPE Hypersil ODS For PB and UV: PB, ES scan REC: 88.5% [21]
inflammatory drugs (10032.1 mm I.D., 5 mm), gradient: hexane– LOD: 10 ng/ml (PB)
( NSAIDs): 458C 95% 2-propanol–water
Flunixin1M (hydroxy-); (98:2–0:100),
for a further 40 NSAIDs 400 ml/min For ES:
only t R and MS data are gradient: 1% aqueous
given acetic acid–ACN
(70:30–0:100)
Opioids: B SPE Nova-Pak C 18 50 mM ammonium TS MS-MS, SRM REC: 80–90% [25]
Dextromoramide (for (15033.9 mm I.D., acetate–MeOH LOD: not
dextropropoxyphene and 4 mm) (25:75), addition of reported for
methadone only t R and on- 800 ml/min of 50 mM biosamples
column LOD data are ammonium acetate
given) post-column to
improve the ionization
Opioids: B SPE Nova-Pak C 18 50 mM ammonium TS MS-MS, SRM REC: 80–90% [26]
Dextromoramide (for (15033.9 mm I.D., acetate–MeOH LOD: 0.05 ng/ml
dextropropoxyphene and 4 mm) (25:75) LIN: 0.5–12.5 ng/ml
methadone only t R and on-
column LOD data are
given)
Opioids: B SPE Nucleosil C 18 2 mM ammonium ES SIM REC: 56–60% [39]
Buprenorphine1M (nor-) (15031 mm I.D.) formate (pH 3)– LOD: 0.05 ng/ml
ACN (55:45) or LIN: 0.1–
for MAM (90:10) 100 ng/ml
Opioids: B, U, hair LLE Nova-Pak C 18 2 mM ammonium ES SIM REC: 87–94%; [40]
(15032 mm I.D., acetate (pH 3)–
4 mm) ACN (20:80)
Table 2. Continued H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 13

Compound Sample Work-up Stationary phase Mobile phase Interface Detection mode Validation data Ref.

Buprenorphine1M (nor-) 61–66% (M)

LOD blood :
0.1 ng/ml;
0.05 ng/ml (M)
LOD hair :
4 pg/mg; 2 pg/mg
(M)
Quarternary ammonium P LLE Hamilton PRP-1 50 mM ammonium TS MS-MS, SRM REC: 80% [28]
drugs: (resin) (15034.1 mm acetate–ACN (70:30) LIN: 1–
Oxyphenonium bromide I.D., 5 mm) 100 ng/ml
(antrenyl), (for
methylbenactyzine,
mepenzolate, pipenzolate,
clidinium, neostigminium,
valethamate, isopropamide
and propantheline only LC
and MS data are given)
Xanthins: P, U SPE Pre-column: For pre-column: 17 M FAB Scan REC: 82–98% [22]
Theophylline, Develosil ODS-HG- acetic acid–glycerol– LOD: 2–
theobromine, caffeine 15/30 (3530.3 mm water (0.1:0.5:99.4); 10 ng/ml
I.D., 15–30 mm) 17 M acetic acid– LIN: 10–
Analytical column: glycerol–water 1000 ng/ml
Develosil ODS-HG-5 (0.5:0.5:99–99:89–0)
(15030.3 mm I.D., 5 mm)
Miscellaneous drugs:
Bromisovalum P, B SPE Hypersil ODS MeOH-water (50:50) PB SIM REC:? [20]
(10032.1 mm I.D., LOD: 100 ng/ml
5 mm), 408C LIN: 500–
5000 ng/ml)
Colchicine B, P, U LLE Alltech C 18 (25031 mm 2 mM ammonium ES SIM REC: 86–91% [42]
I.D., 5 mm) with a acetate (pH 3.0)– LOD: 0.6 ng/ml
guard column (MGU- ACN (25:75) LIN: 5–
80 C 18 (130.8 mm 200 ng/ml
I.D., 5 mm))
Cyclobenzaprine P, U EHY (U), Keystone BDS C 18 10 mM ammonium APCI MS-MS, SRM REC: 92–104% (P, U) [63]
LLE (5034.6 mm I.D., acetate/0.1 formic LIN: 0.1–
5 mm) with a guard acid–ACN (10:90) 50 ng/ml (P);
column (BDS C 18 10–1000 ng/ml (U)
(2034.6 mm I.D.))
Glibenclamide S LLE Nucleosil C 18 (length 0.05% aqueous acetic APCI SIM REC: 98–100% [4]
and I.D. not reported, acid–MeOH (20:80) LOD: 5 ng/ml
7 mm) LIN: 50–
1500 ng/ml
Granisetron1M (hydroxy-) P (dog) LC-LC (on-line Inertsil C 8 (5034.6 mm 50 mM ammonium APCI MS-MS, SRM REC: 93–105% [64]
sample enrichment) I.D., 5 mm) acetate (pH 5)–ACN LIN: 0.05–
with two guard (73:27); initial loading 50 ng/ml (from
columns: Regis solvent: water–ACN 80 ml P!)
Chemicals ISRP GFF (95:5)
II (1033 mm I.D.)
Hyoscyamine ( L-atropine) P LLE Keystone BDS C 18 10 mM ammonium ES MS-MS, SRM REC: 110% [45]
(5033 mm I.D., 3 mm) acetate–MeOH–ACN LIN: 0.02–
with a guard (13:32.7:54.3) 0.5 ng/ml
column (BDS C 18 LOQ: 0.05 ng/ml
(1032 mm I.D.,
5 mm))

(Cont.)
14 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

Table 2. Continued
Compound Sample Work-up Stationary phase Mobile phase Interface Detection mode Validation data Ref.

Ifosfamide1M S LLE Spherisorb C 18 For FAB: FAB, ES scan, SIM REC: 93–99% [5]
(2031.6 mm I.D.) MeOH–water– LOD scan :
glycerol (50:40:10), 35 ng/ml
12 ml/min; LOD SIM : 1.5 ng/ml
For ES: LIN: 50–
ACN–water (90:10) 120.000 ng/ml
Iloperidone1M (dihydro-) P SPE Hypersil C 18 2.5 mM ammonium ES SIM for QU; REC: 82–101%, [46]
(10034.6 mm I.D., formate (pH 3.5)– MS-MS, DIS 73–97 (M)
5 mm) ACN (30:70) for ID LIN: 0.25–
20 ng/ml
Indinavir P LLE Hypersil BDS C 8 7 mM ammonium APCI MS-MS, SRM REC: 94% [6]
(5032 mm I.D., acetate (pH 4.9)– LIN: 1–
3 mm) ACN (60:40) 200 ng/ml
Mesocarb1M U (rat) LLE Capcell PAK C 18 Gradient: 150 mM TS ID: scan REC: ? [7]
(6034.6 mm I.D., ammonium acetate– QU: SIM LOD: ?
3 mm) MeOH (80:20–40:60)
Oxybutynin1M (desethyl-) P LLE Chiralpak AD CSP n-Hexane–2-propanol- ES MS-MS, SRM The authors [43]
(10032.1 mm I.D., -diethylamine state that
10 mm), 208C; chiral (90:10:0.1), addition validation is
separation of 800 ml/min of in progress
25 mM ammonium
acetate–2-propanol
(25:75) post-column to
improve the ionization
and to reduce the risk
of explosion
Oxymetazoline B (rat) LLE Zorbax C 18 (15032.1 5 mM ammonium ES SIM REC: 68–89% [48]
mm I.D., 5 mm) acetate (pH 6.5)– LIN: 0.67–
ACN (40:60) 167 ng/g
LOQ: 1ng/g
Pirenzepine P LLE BDS-Hypersil C 18 100 mM ammonium ES MS-MS, SRM REC: 78% [49]
(5034.6 mm I.D., acetate–ACN LIN: 1–
5 mm) (60:40) 100 ng/ml
With a guard column
(BDS-Hypersil C 18
(2034.6 mm I.D.,
5 mm))
Pramipexole P LLE Zorbax SB-CN Water–0.1 mM APCI MS-MS, SRM REC: 82% [65]
(15034.6 mm I.D., ammonium acetate– LIN: 0.05–
5 mm) MeOH (15:5:80) 5 ng/ml
Sumatriptan P SPE Beckman CN 0.1% aqueous APCI MS-MS, SRM REC: 87–113% [66]
(25034.6 mm I.D., trifluoroacetic acid– LIN: 0.5–
5 mm) MeOH–ACN 50 ng/ml
(58:6:36)
Terfenadine P LLE TSK gel ODS-80TS 10 mM ammonium ES MS-MS, SRM REC: 125% [51]
(15032 mm I.D., acetate (pH 4)– LIN: 0.2–
5 mm) formic acid (1%)– 50 ng/ml
ACN (2:13:85)

variable pH and organic modifiers such as methanol added [27,43]. Volatile acids, such as formic
(MeOH) or acetonitrile (ACN) used for isocratic or [8,27,38,51], acetic [4,9,21,61,67] or trifluoroacetic
gradient elution. Since basic drugs often give poor acid [66], were used to improve the ionization. In our
peak shapes on reversed-phase columns, minor experience, there is no general rule for the choice of
amounts of an amine such as triethylamine were the acid. It should be tested for every analyte and
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 15

Table 3
LC–MS procedures for the identification and / or quantification of poisons and / or their metabolites in biosamples
Compound Sample Work-up Stationary phase Mobile phase Interface Detection mode Validation data Ref.

Aconitum alkaloids: B, U SPE Inertsil ODS-2 (15034.6 mm Tetrahydrofuran–0.3% FAB SIM REC: 94–100% [12]
Aconitine, mesaconitine, I.D.), 408C trifluoroacetic acid– LOD: 50 ng/ml (UV)
hypaconitine, jesaconitine glycerol (19:81:0.3) LIN: 100–10 000 ng/ml (UV)
Aflatoxins: U (dust) LLE Supelco C 18 (25034.6 mm ACN–MeOH–water (20:20:60) ES MS-MS, REC: ? [13]
B 1 , B 2 , G 1 and G 2 I.D., 5 mm) SRM LOD: 0.05 ng/ml
LIN: 0.05–0.2 ng/ml
Amanita toxins: U SPE Kromasil C 18 (12532 mm I.D., 20 mM ammonium acetate ES SIM REC: 63–65%; [14]
a-, b-Amanitin 5 mm) with a guard column (pH 5)-MeOH (78:22) LOD: 10 ng/ml
(Kromasil C 18 (1032 mm I.D.,
5 mm))
Nicotiana alkaloids: P LLE BDS-Hypersil C 18 (10033 mm 10 mM ammonium acetate– APCI MS-MS, SRM For nicotine: [68]
Nicotine1M (cotinine) I.D., 3 mm) with a guard MeOH–ACN (15:32:53) REC: 106%
column (BDS-Hypersil C 18 LIN: 1–50 ng/ml
(1032 mm I.D.)) For cotinine:
REC: 86%
LIN: 10–500 ng/ml
Pesticides: U LLE (ID), alkaline Whatman ODS-3 (25034.6 mm 0.1% aqueous acetic acid– APCI MS-MS, REC: 75% [67]
Alachlor1M (mercapturate) HY to form diethyl I.D.) MeOH (30:70) DIS (ID), LOD: ?
aniline SPE (QU) SRM (QU)
Pesticides: S, U LLE Nova-Pak C 18 (15033.9 mm Gradient: 100 mM ammonium APCI scan, SIM REC: ? [69]
Propoxur, and other I.D., 4 mm) acetate–MeOH (35:65–55:45) LOD: ?
organophosphorus or
carbamate pesticides

chromatographic system. In some papers, post-col- reported. The interface type and the mass spectral
umn addition of ammonium acetate solutions was detection mode are listed. Since in most papers both
recommended to improve the ionization in the MS mass spectra and chromatograms / retention times are
without influencing the chromatographic separation given, these data were not specified in the tables.
[25,29,43]. Validation data, such as recovery (REC), limit of
detection (LOD) or linearity (LIN), are summarized
2.3. LC–MS procedures for easy evaluation, whether or not a procedure can
be useful to solve an actual toxicological case.
The drugs and poisons are listed in the tables However, only data recorded in biosamples are
according to drug classes and to the international given, since data recorded in methanolic solutions
non-proprietary names (INN) or the common names. are of minor value for toxicological analysis. The
If only metabolites were determined ‘2M’ is added limit of quantification (LOQ) is given only if not
to the name. If metabolites were determined addi- identical to the lowest linearity value. Since preci-
tionally, ‘1M’ is given in the Compound column. sion of all the reviewed procedures was better than
The information concerning quantification, if avail- 20% as recommended for analysis in biosamples,
able, can be found in the Validation column. The these data were omitted in order to save space.
type of biosample used is given in the Sample
column (B, blood; P, plasma; S, serum; U, urine, 2.3.1. Illicit drugs of abuse
etc.). If samples from animals were studied, the
species is given in brackets. The sample preparation 2.3.1.1. Amphetamines. Amphetamine derivatives
is concisely summarized in the Work-up column. The are often misused as central stimulants. Immuno-
principal information on the stationary and mobile assays are available for amphetamine, metham-
phases is given. The flow-rate data were omitted, phetamine and some ring or side chain-modified
since in many cases the eluent was split before derivatives, such as the methylenedioxy designer
entering the interface, but the split ratio was not drugs. For confirmation of the immunoassay result or
16 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

for differentiation, series of GC–MS procedures volume [106]. However, the detection of LSD is
were published [101], since amphetamine derivatives rather complicated because the concentrations are
are volatile in GC. Nevertheless, three LC–MS very low and the molecule is not very volatile.
procedures for serum or urine analysis have been Therefore, since 1996 five papers appeared describ-
recently published [23,24,53]. Bogusz et al. [53] ing ES-LC–MS(–MS) detection in urine [32–36].
described the APCI-LC–MS identification and After solid-phase extraction, the detection limits
quantification of amphetamines, including designer ranged between 0.5 and 0.025 ng / ml, which is
drugs, in serum and urine after derivatization. The sufficient to confirm positive immunoassay results.
mass spectra of the 12 drugs tested are quite different However, after immunoaffinity extraction (IAE) and
allowing their differentiation even if the chromato- LC–MS–MS detection the LOD was 10 times lower
graphic peaks are not completely separated. Due to (0.0025 ng / ml) [35]. As discussed above, IAE and /
the use of deuterated internal standards, the valida- or MS–MS detection are powerful procedures for the
tion data promise reliable quantification in the SIM detection of very low concentrations of analytes in
mode. However, Bogusz himself has warned, that in biomatrices.
APCI-LC–MS large amounts of analyte may con-
siderably influence the peak areas of their coinjected 2.3.1.4. Opiates. Heroin is widely abused by drug
deuterated analogues used as internal standard [105]. addicts for euphoriant and anxiolytic effects, while
Katagi et al. [24] have described enantioselective morphine is therapeutically used as a potent analge-
TS-LC–MS detection and LC–UV quantification of sic especially in the final stage of cancer diseases. If
amphetamines in urine for differentiation between heroin is not available, addicts often take morphine
ingestion of illicit and therapeutic drugs. Again, such or other opioid medicaments. For legal reasons, the
differentiation can also be performed by GC–MS application of heroin must be differentiated ana-
[101]. lytically from an intake of other opioids. Therefore,
Tatsuno et al. [23] have developed a reliable 6-monoacetylmorphine (MAM), the only heroin-spe-
TS-LC–MS procedure for simultaneous determina- cific metabolite, must be detected in body samples.
tion of different illicit drugs, such as amphetamines, The TS-LC–MS procedure of Tatsuno et al. [23], as
cocaine, morphine and their metabolites, in urine. well as the recent APCI-LC–MS procedure of
The mass spectra of the 10 drugs tested are quite Bogusz et al. [55], allow the detection of MAM as
different allowing their differentiation with the ex- well as morphine and its glucuronides. Determi-
ception of that of morphine-3- and -6-glucuronide. nation of morphine and its two glucuronides in
Both glucuronides can only be differentiated by their serum was further described using ES-LC–MS
retention time. [37,38]. Although only the 6-glucuronide is pharma-
cologically active and therefore toxicologically rel-
2.3.1.2. Cocaine. As just discussed, cocaine and its evant, Bogusz et al. [55] have discussed the possible
main metabolite benzoylecgonine (BZE) can be relevance in correlating the blood levels of morphine
determined in urine by TS-LC–MS [23]. Sosnoff et and its two glucuronides, e.g. in fatal cases for the
al. [54] have described APCI-LC–MS–MS detection estimation of the survival time after drug intake.
of traces of BZE eluted from blood spots of new-
borns collected on filter paper for epidemiological 2.3.2. Therapeutic drugs relevant to forensic or
studies of the prevalence of cocaine abuse in late clinical toxicology
pregnancy. The LOD was about 2 ng / ml based on a
12-ml sample volume. 2.3.2.1. Anabolic steroids. Anabolic steroids are
misused in sports and in cattle breeding. Since GC–
2.3.1.3. Lysergide (LSD). LSD is less often abused MS procedures are time-consuming, Barron et al.
than other illicit drugs, but with increasing fre- [19] have developed a fully automated LC–MS
quency, at least in Europe. Immunoassays are avail- procedure for anabolics in urine. Twenty-eight ster-
able for screening (cut-off value, 0.5 ng / ml) and oids and metabolites could be detected after en-
some GC–MS procedures are described for con- zymatic hydrolysis, on-line SPE, reversed-phase
firmation as reviewed by Kuffer et al. in this Special chromatography and PB interfacing by electron
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 17

impact mass spectrometry. The relatively low sen- [56]. Senda et al. [57] have published a very
sitivity of PB was sufficient to detect therapeutic sensitive APCI-LC–MS–MS procedure for the low-
concentrations of the tested anabolics even if only dosed triazolam with a LOD of 0.02 ng / ml. Verweij
small amounts of urine were available, because the et al. [25] described TS-LS–MS–MS quantification
whole sample could be injected onto the on-line SPE of benzodiazepines, neuroleptics and opioids in
column. Bowers and Sanaullah [9] described the whole blood. Unfortunately, this paper is of less
direct detection of several steroid glucuronide and value for toxicological analysis since no validation
sulfate conjugates in urine by ES-LC–MS and ES- data recorded in biosamples were reported. The
LC–MS–MS to avoid the enzymatic hydrolysis of conversion of on-column LODs of methanolic solu-
the conjugates, which is less reproducible and may tions to LODs in blood is very daring. Every
lead to artifacts. Considering the measured on-col- bioanalyst knows that this is unrealistic.
umn LODs of the reference solutions the authors
conclude that they were readily compatible with 2.3.2.4. b2 -Agonists. b 2 -Adrenoceptor agonists (b 2 -
testing in urine. In the MS–MS mode spectral agonists) are widely used as bronchodilators or
differences appeared to be sufficient for differentia- tocolytics. Because of their acclaimed anabolic effect
tion. at higher doses, they are misused in sports or in
livestock production. Doerge et al. [59] have de-
2.3.2.2. Antihypertensive drugs. Antihypertensive scribed APCI-LC–MS and LC–MS–MS procedures
drugs, such as a 1 -blockers, b-blockers or calcium for sensitive and specific determination of five b 2 -
channel blockers, may lead to cardiovascular dis- agonists in human plasma. Salbutamol could not be
orders when they are incorrectly taken. For moni- determined by LC–MS–SIM because of matrix
toring such patients, procedures published for phar- interferences. Therefore, MS–MS was tested for
macokinetic studies can be used. The calcium chan- determination of clenbuterol, fenoterol and sal-
nel blocker, amlodipine, can very sensitively be butamol. The authors stated that lower LODs could
quantified in serum by APCI-LC–MS–MS [58], so be reached by LC–MS–MS, but no detailed data
that only small amounts of blood are needed. Chiral were given.
determination in plasma by normal-phase ES-LC– Determination of b 2 -agonists in bovine urine was
MS–MS was described for the a 1 -blocker, dox- described using APCI-LC–MS–MS [60,61] or TS-
azosin [43], the b-blocker, sotalol, and the calcium LC–MS–MS [27] with LODs of 0.05 ng / ml. Hagen-
channel blockers, verapamil and nimodipine [47]. doorn et al. [27] and Cai and Henion [61] have
Problems arising from normal-phase LC were al- developed on-line extraction as discussed in detail in
ready discussed in Section 2.2. Reserpine used in Section 2.1. Further methods for b 2 -agonists were
humans as antihypertensive drug is also used as a recently reviewed by Polettini [108].
tranquillizer in horses. To monitor plasma levels for
both indications, the ES-LC–MS–MS procedure 2.3.2.5. Cardiac glycosides. Cardiac glycosides are
validated for equine plasma can be applied [50]. used in the treatment of congestive heart failure.
Because of their narrow margin of therapeutic safety
2.3.2.3. Benzodiazepines. Benzodiazepines are wide- they are routinely monitored in plasma by immuno-
ly used and they may lead to addiction or severe assay. In some circumstances (e.g. forensic cases),
intoxication, especially in combination with alcohol. the immunoassay results must be confirmed and / or
Therefore, screening for benzodiazepines is neces- the glycoside that was actually applied must be
sary in clinical and forensic toxicology. While benzo- identified. Tracqui et al. [2] developed an ES-LC–
diazepines can easily be screened in urine, e.g. after MS procedure for the detection of the most important
acid hydrolysis by GC–MS [1,107], GC–MS quanti- cardiac glycosides in plasma. The precision of the
fication in blood suffers, e.g. from thermal instability method is acceptable. Although the LC–MS LODs
and low volatility of some of the parent compounds. were higher than the immunoassay LODs, they
LC–MS provides good precision, specificity and should be sufficient for overdose cases. As shown in
sensitivity. The LOQs ranged between 1 and 2 ng / Fig. 2, the authors were able to identify oleandrin in
ml using ES-LC–MS–MS [41] or APCI-LC–MS a plasma extract of a patient who ingested a tea made
18 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

Fig. 2. Total ion chromatogram from a plasma extract of a patient suffering from an oleandrin intoxication (upper part). Reconstructed mass
chromatogram of ion m /z 594 (ammonium adduct ion) indicating the presence of oleandrin (lower part). The inset shows the peak
underlying ES mass spectrum (taken from Ref. [2]).

from parts of the toxic plant Nerium oleander. As the analyzed fatal forensic cases due to shock caused by
total ion chromatogram indicates, this case could not intake of such antibiotics. A lot of mass spectral and
have been solved without mass spectral information. retention data are published, but no validation data
Using reconstructed mass chromatography with the recorded in biosamples were given. Only ceftriaxone
ammonium adduct ion [M1NH 4 ] 1 (C 32 H 48 O 9 5 and cefazolin have already been successfully de-
5761185594), the presence of oleandrin was indi- termined in serum of patients treated intravenously
cated. The peak underlying ES mass spectrum (inset with the corresponding antibiotic.
in Fig. 2) was compared for confirmation with the
reference spectrum shown in the original paper [2]. 2.3.2.7. Corticosteroids. Synthetic corticosteroids
are illegally used as growth promotors in livestock
2.3.2.6. Cephem antibiotics. Kobayashi et al. [3] breeding or as anti-inflammatory drugs for unfit race
developed a FAB-LC–MS procedure for the de- horses. An APCI-LC–MS–MS procedure was de-
tection of cephem antibiotics. For example, they veloped to control corticosteroid misuse in bovine
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 19

urine [62] and an ES-LC–MS–MS procedure to analyzed in body samples. Most of the opioids can
control triamcinolone doping in equine urine or be analyzed by GC–MS [1]. Low-dosed buprenor-
serum [52]. phine and its nor metabolite can sensitively be
detected in urine by GC–MS [109], but for de-
2.3.2.8. Immunosuppressants. For detailed therapeu- termination in plasma, hair [40] or whole blood [39],
tic drug monitoring of immunosupressant drugs and ES-LC–MS has been proved to be the method of
their metabolites in blood, PB-LC–MS [10] or ES- choice.
LC–MS–MS [11] assays were described for tac- Verweij et al. [26] described a TS-LC–MS–MS
rolimus and an ES-LC–MS [8] assay for sirolimus. assay for some opioids (and neuroleptics), but they
Surprisingly, the linearity range of the PB-LC–MS have validated the assay in blood only for dex-
assay was the same as that of the ES assay combined tromoramide. The second publication of these au-
with MS–MS detection. thors with quite similar contents was critically
discussed in Section 2.3.2.3.
2.3.2.9. Neuroleptics (butyrophenones and phenothi-
azines). Neuroleptics may lead to severe intoxica- 2.3.2.12. Quaternary ammonium drugs. For determi-
tions, so that fast diagnosis is required. Immuno- nation of quaternary ammonium drugs in plasma a
assays are not available. Neuroleptics can easily be TS-LC–MS procedure using a resin-based stationary
screened by GC–MS [1]. For quantification in blood, phase was reported [28]. Using such a column,
LC–MS procedures were described. Hoja et al. [44] ion-pairing reagents often incompatible with the LC–
described a precise ES assay for haloperidol and its MS ionization are not necessary. The method covers
metabolite. Verweij et al. [26] described a TS-LC– eight drugs, but it was only validated for antrenyl.
MS–MS assay for butyrophenone and bis-
fluorophenyl neuroleptics (and opioids), but they 2.3.2.13. Xanthins. The xanthin derivatives, theo-
have validated the assay in blood only for halo- phylline, theobromine and caffeine, are ingredients
peridol. The second publication of these authors with of foods and beverages. Theophylline and caffeine
quite similar contents was critically discussed in are also used as therapeutic or doping agents and
Section 2.3.2.3. suicides occur. The xanthins can easily be detected
in GC–MS drug screenings. A FAB-LC–MS assay
2.3.2.10. Non-steroidal anti-inflammatory drugs was described for precise quantification in plasma
(NSAIDs). Non-steroidal anti-inflammatory drugs [28]. This procedure using double-focussing MS may
have been misused in horse doping. For doping be a nice scientific application, but I cannot imagine
control, PB- and ES-LC–MS procedures were de- that anyone would use it routinely.
scribed for 40 NSAIDs, but validated only for
flunixin and its metabolites [21]. The authors stated 2.3.2.14. Miscellaneous drugs. A number of LC–
that ES was more sensitive than PB. Since NSAIDs MS papers appeared dealing with the determination
form anions or cations, the ES apparatus must of of single therapeutic drugs. As mentioned above they
course be switched for the screening into the positive are not discussed in detail. However, procedures
or negative mode. concerning single therapeutic drugs relevant to foren-
sic or clinical toxicology are also summarized in
2.3.2.11. Opioids. Opioids, often named narcotics, Table 2, while those concerning future drugs will
are potent analgesics especially used in a postopera- only be mentioned in Section 2.3.4.
tive state or in the final stage of cancer diseases.
Furthermore, they are abused (typically by medical 2.3.3. Poisons
staff) for their euphoriant and anxiolytic effects.
Heroin addicts also take opioids, if heroin is not 2.3.3.1. Aconitum alkaloids. Aconitum sp. alkaloids
available. Finally, some opioids, such as methadone are toxic and may lead to severe intoxications. For
or buprenorphine, are used for substitution therapy of diagnosis, a LC–UV procedure was recently pub-
heroin addicts. For all these reasons, opioids must be lished [12]. The authors stated that using FAB-LC–
20 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

MS the results could be confirmed and that they phalloides) the amatoxins, a- and b-amanitin, may
could detect ‘therapeutic’ levels of the alkaloids in cause severe gastrointestinal disorders and fatal liver
blood and urine. Unfortunately, ‘therapeutic’ levels damage. Since diagnosis of an intoxication entails a
were not defined and authentic cases were not large scale of invasive and expansive therapy, a
reported. highly specific detection of amanitins in body fluids
is necessary. Determination of amanitins in urine by
2.3.3.2. Aflatoxins. Aflatoxins are carcinogenic radioimmunoassay has several disadvantages, while
mycotoxins. They can be ingested via contaminated ES-LC–MS was suitable for sensitive and specific
food or inhaled via dust generated by mould-infected detection of amatoxins in urine, as described by
products. Kussak at al. [13] developed an ES-LC– Maurer et al. [14]. In the meantime, this procedure
MS–MS procedure for the determination of afla- has been improved [103,104,110]. In Fig. 3, a cutout
toxins in urine of feed factory workers. Unfortuna- of the ES spectra of the amanitins, the structures, the
tely, no data are given on tolerable urine levels and empirical formulae and the molecular masses are
so it cannot be concluded whether this procedure shown. As shown in Fig. 4, the analysis time could
actually can be used for biomonitoring. be shortened using gradient instead of isocratic
elution, and the specificity could be improved using
2.3.3.3. Amanita toxins. After ingestion of the toxic six instead of two selected ions. We have been
mushrooms of the Amanita species (e.g. Amanita developing an immunoaffinity extraction procedure

Fig. 3. Cutout of the ES spectra, structures, empirical formulae and molecular masses of a- and b-amanitin.
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 21

fortunately, no validation data recorded in biosam-

ples were reported. On-column LODs of methanolic
solutions are of minor value for toxicological analy-
sis.

2.3.4. Applications in studies on pharmacokinetics

or metabolism of future drugs
During the development of new drugs, studies on
pharmacokinetics, including metabolism, must be
performed. HPLC is a versatile method for such
studies and is still commonly used. Classical detec-
tors, such as UV, diode array (DAD) or fluorescence
detectors, are selective and sensitive enough for
many pharmacokinetic applications. However, for
determination of low-dosed drugs (especially when
they have low UV absorbance or no natural fluores-
Fig. 4. Selected-ion chromatograms indicating a- and b-amanitin cence), mass spectral detection is needed, especially
in urine (conc. 100 ng / ml). in the MS–MS mode. Today, many pharmaceutical
companies are using LC–MS and LC–MS–MS
techniques for series of routine quantifications during
to decrease the LOD and to make the procedure also pharmacokinetic studies [111]. Using LC–MS–MS,
suitable for other biosamples [103,104]. the sample preparation can be simple and fast, the
(MS) separation very fast and the detection very
2.3.3.4. Nicotiana alkaloids. Ingestion of cigarettes specific and sensitive, so that this technique will
may lead to life-threatening situations especially in become the golden standard in pharmacokinetics
children. Nicotine and its metabolites can easily be [111]. For the identification of new metabolites,
screened by GC–MS. In smoking-cessation therapies especially of conjugates, LC–MS [93,96,112] or
under nicotine substitution (e.g. via patches), nicotine LC–MS–MS [29–31,113–115] are widely used
plasma levels should be monitored to counteract the today.
craving for cigarettes. Xu et al. [68] have described a For reasons of space, papers concerning the
sensitive, precise and fast APCI-LC–MS–MS pro- quantification of future therapeutic drugs as part of
cedure for quantification of nicotine and its metabo- pharmacokinetic or metabolic studies are not re-
lite cotinine in plasma, that was suitable for this viewed in detail, but they are mentioned here. They
purpose and that would also be suitable for clinical were not omitted for two reasons: firstly such
or forensic problems. therapeutic drugs may also occur in future tox-
icological cases and secondly they may give hints for
2.3.3.5. Pesticides. Pesticides must be analyzed in developing new procedures for similar compounds of
body fluids for diagnosis of an intoxication or for toxicological interest. Only ES or APCI procedures
biological monitoring of occupationally exposed are mentioned, since these techniques have become
persons. Driskell et al. [67] applied APCI-LC–MS– standard. ES-LC–MS quantification was described
MS for identification of metabolites of the herbicide for the HIV protease inhibitor BMS-186318 [88] and
alachlor in urine for biomonitoring purposes. The the platelet inhibitor Ro 44-3888 [89]. APCI-LC–
urine samples used for this study were collected from MS quantification was described for the antianginal
alachlor-exposed workers, but no data were given on drug ranolazine [90], the antibiotic azithromycin and
tolerable urine levels. the epimers of the glucocorticoid budesonide [91].
Itoh et al. [69] described an APCI-LC–MS meth- ES-LC–MS–MS quantification was described for the
od for the detection of 30 pesticides, but only antiarrhythmic MK-0499 [92], the antimigraine
propoxur was studied in biosamples (serum). Un- drugs rizatriptan [75] and GR-151004 [76], a col-
22 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25

lagenase-inhibiting antirheumatic [78], the endo- unknown drugs or poisons, because neither its sepa-
thelin receptor antagonist bosetan [77], the HIV ration power nor the mass spectral information are
protease inhibitor saquinavir [79], mevinolinic acid, comparable to that of capillary GC and electron
a metabolite of the antihypercholesterolemic lovas- impact full-scan MS. However, LC–MS will ‘play
tatin [80], and the muscarinic agent LY-297802 [81]. the first violin’ in analyzing peptide or nucleotide
APCI-LC–MS–MS quantification was described for drugs, interesting drugs of the future.
the the antihypercholesterolemic 447C88 [82], the
antifungal fenticonazole [83,84], the M 3 blocker
darifenacin [85], and the muscarinic agent 4. List of abbreviations
xanomeline [86].
In drug metabolism, LC–MS or LC–MS–MS ACN acetonitrile
provide new aspects in the identification and quanti- AM amphetamine
fication of phase II metabolites, such as glucuronide APCI atmospheric-pressure chemical ionization
[9,23,29–31,37,38,55,72,93–98], glycine [73] or es- API atmospheric-pressure ionization
pecially sulfate conjugates [9,30,93]. B blood
CID collision-induced dissociation
CNLS constant neutral-loss scanning
3. Conclusions and perspectives DAD diode array detection
DIS daughter-ion scanning
In the last 2 years an increasing number of papers EHY enzymatic hydrolysis for cleavage of
appeared concerning LC–MS identification and / or conjugates
quantification of drugs, poisons and / or their metabo- ES (atmospheric-pressure) electrospray ioni-
lites in biosamples relevant to forensic and clinical zation
toxicology. Sample preparation was performed either FAB fast atom bombardment ionization
by classical off-line liquid–liquid or solid-phase GC gas chromatography
extraction, or by modern on-line extraction on re- GC–MS gas chromatography–mass spectrometry
versed-phase or immunoaffinity columns. The chro- IA(E) immunoaffinity (extraction)
matographic systems used were rather similar and ID identification
classical: isocratic or gradient elution with C 18 INN international non-proprietary name
columns and mixtures of volatile buffers, such as (WHO)
ammonium acetate, with variable pH and organic LC liquid chromatography
modifiers, such as methanol or acetonitrile. Pre- or LC–MS liquid chromatography–mass spec-
post-column addition of volatile acids, such as trometry
formic, acetic or trifluoroacetic acid, improved the LIN linearity
ionization of the analyte. Different types of LC–MS LLE liquid–liquid extraction
interfaces, mass analyzers and detection modes were LOD limit of detection
used. However, the more recent papers on analytical LOQ limit of quantification
toxicology focus on ES and APCI in combination LSD lysergic acid diethylamide (INN, lyser-
with MS or MS–MS detection. It can be concluded, gide)
that the two relatively robust atmospheric-pressure M mol / l
ionization techniques ES and APCI will become the M metabolite
golden standard in LC–MS. This review documents MA methamphetamine
that LC–MS has become a powerful tool in ana- MAM 6-monoacetylmorphine
lytical toxicological science and practice. However, MDA methylenedioxyamphetamine
at least at present, LC–MS is a complementary but MDE methylenedioxyethylamphetamine
not an alternative technique to GC–MS, still the MDMA methylenedioxymethamphetamine
golden standard in analytical toxicology. For exam- MeOH methanol
ple, LC–MS is not suitable for a broad screening for M3G morphine-3-glucuronide
 H.H. Maurer / J. Chromatogr. B 713 (1998) 3 – 25 23

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Weber, J. Chromatogr. B 689 (1997) 81–89.
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