Monitoring Anabolic Steroids in Meat-Producing Animals. Review of
Current Hyphenated Mass Spectrometric Techniques |
| B. Le Bizec, P.
Marchand, D. Maume, F. Monteau, F. André |
S-3 |
| Monitoring
anabolic steroids in meat-producing animals is a challenging task. It implies very
specific and sensitive analytical methods able to detect and identify sub-µg kg–1
residue levels in complex biological matrices such as meat, urine, or hair. Gas and liquid
chromatography coupled to mass spectrometry are the most efficient means of achieving
these objectives. In this paper we review how developments in mass spectrometry have been
rapidly applied to this problem, how efficient analysis of anabolic steroids in urine,
edible tissue, and hair has been achieved, and, later, how measurement of conjugate
steroids and determination of the origin of natural steroid hormones has been achieved.
The performance characteristics of different mass spectrometers (quadrupole, ion-trap,
electromagnetic, isotope-ratio, tandem, and hybrid instruments), the efficiency of
different acquisition techniques (LR-SIM, HR-SIM, MRM), and, finally, sample introduction
(gas chromatography and liquid chromatography, with discussion of alternative interfaces)
are discussed, with numerous applications. |
|
Application of Hyphenated Mass Spectrometric Techniques to the
Determination of Corticosteroid Residues in Biological Matrices |
| J.-P. Antignac,
F. Monteau, J. Négriolli, F. André , B. Le Bizec |
S-13 |
| Fifty
years after the discovery of natural corticosteroid hormones and their anti-inflammatory
properties many synthetic derivatives of these molecules are now available. Most are
widely used in human and veterinary medicine, legally but under regulated conditions.
These compounds can also be used as growth promoters in animal breeding, although such use
is illegal in Europe. Consequently, analytical methods have been developed to monitor use
of corticosteroids in cattle. This paper, based on the authors experience and the main
relevant literature, describes the different mass spectrometric approaches used for
measurement of corticosteroid residues (parent drug, metabolites, and esters) in
biological matrices (urine, meat, hair), including gas chromatography– mass
spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry
(LC–MS2). The respective advantages of liquid chromatography and gas
chromatography, in conjunction with different derivatisation reactions, are discussed. The
behavior of corticosteroids with different ionization techniques is also discussed.
Application to monitoring corticosteroid misuse and to investigation of pharmacokinetics
and metabolism in bovine species is described and new data are presented relating to
elimination and hair fixation kinetics for free and ester forms and the nature and
proportions of corticosteroid phase I and phase II metabolites. Finally, this work reviews
ten years experience of the use of a variety of mass spectrometric techniques for analysis
of corticosteroids in animals produced as food. |
|
Receptor-Based Screening Assays: New Perspectives in Anti-Doping
Control |
| M.-L. Scippo, P.
Willemsen, S. Danyi, V. Helbo, M. Muller, J. Martial, G. Maghuin-Rogister |
S-23 |
| The
so-called ‘‘growth promoters’’, steroid hormones and b-agonists, are
currently controlled by using hyphenated analytical methods (chromatography coupled to
mass spectrometry) or, sometimes for screening purposes, on immunoassays. These methods
are often too specific to allow an effective multianalyte control. To develop more
efficient assays, the use of hormonal receptors as detection tools (receptor-based binding
assays and cell-based assays) is proposed. Receptor-based assays represent useful tools in
screening of hormonal residues in food, but they could also be applied in doping control
(to detect ‘‘new’’ hormonal substances). Furthermore, these assays
could be used to monitor the human exposure to endocrine disruptors. |
|
Detection of Anti-Ulcer Drugs and Their Metabolites in Horse Urine
by Liquid Chromatography – Mass Spectrometry |
| E. W. Chung, E.
N. M. Ho, D. K. K. Leung, F. P. W. Tang, K. C. H. Yiu, T. S. M. Wan |
S-29 |
| This
paper describes a convenient method for the extraction and detection of eight anti-ulcer
drugs simultaneously in horse urine, a relatively complex and viscous matrix, using a
single-step liquid-liquid extraction followed by high-performance liquid chromatography
– mass spectrometry (LC-MS). Anti-ulcer drugs were isolated from horse urine by
salting out and liquidliquid extraction. Detection of these drugs at concentrations below
1 ng mL–1 could be achieved using LC-MS-MS in the positive atmospheric
pressure chemical ionisation (APCI) mode. The above analysis was also extended to
elimination studies of horses administered with ranitidine and omeprazole. The urinary
elimination data of these two drugs suggest that ranitidine was eliminated from horse
urine at a much slower rate than omeprazole. In addition, three ranitidine metabolites and
six omeprazole metabolites were identified for the first time in equine urine samples. |
|
Criteria in Chromatography and Mass Spectrometry – a Comparison
Between Regulations in the Field of Residue and Doping Analysis |
| P. Van Eenoo, F.
T. Delbeke |
S-39 |
| Residue
analysis and doping control aim at the detection of prohibited substances (often
registered drugs) in biological matrices and are hence related fields of analysis. In both
fields, the detection of such a prohibited substance has legal and economical implications
for the persons involved (farmer, horse-owner, trainer or athlete). Hence, the necessary
precautions need to be taken to ensure that the unequivocal presence of a substance has
indeed been established. In each of these fields of analysis, chromatography and mass
spectrometry are the primary techniques used in the identification process and, not
surprisingly, the criteria to which these techniques must comply depend on the regulatory
authority. It seems illogical that different sets of criteria exist for fields of analysis
that are so closely related. However, because of the complexity encountered in these
analyses, the creation of an ‘‘ideal’’ set of criteria encompassing
and foreseeing all possible diffi- culties met by the analyst during his work seems
impossible. This paper tries to give an overview of the similarities and differences in
each set of regulations, while critically pointing out and illustrating pitfalls and
positive aspects of each set of regulations in an attempt to aid the analyst in the
decision process when regulations leave room for interpretation. The need for the analyst
to critically evaluate the regulations is illustrated in two examples. |
|
Detection of Quaternary Ammonium Drugs in Equine Urine by Liquid
Chromatography-Mass Spectrometry |
| K. C. H. Yiu, E.
N. M. Ho, T. S. M. Wan |
S-45 |
| Quaternary
ammonium drugs are anticholinergic agents and some of which have been known to be abused
in equine sports. A general screening method for this class of drugs in equine urine by
liquid chromatography-mass spectrometry (LC-MS) has not been reported. The paper describes
an efficient LC-MS-MS method for the simultaneous detection and confirmation of twenty
quaternary ammonium drugs at low ng mL–1 in equine urine after solid-phase
extraction. Quaternary ammonium drugs were extracted from equine urine by solid-phase
extraction using ISOLUTE® CBA SPE Columns and analysed by LC-MS-MS in the
positive electrospray ionisation mode. Separation of twenty quaternary ammonium drugs (the
quaternary ammonium ions of edrophonium chloride, pyridostigmine bromide, neostigmine
bromide, bretylium tosylate, ipratropium bromide, tubocurarine chloride, N-butylscopolammonium
bromide, mepenzolate bromide, rocuronium bromide, clidinium bromide, pipenzolate bromide,
isopropamide iodide, glycopyrronium bromide, alcuronium chloride, oxyphenonium bromide,
propantheline bromide, tridihexethyl chloride, vecuronium bromide, mivacurium chloride and
pancuronium bromide) was achieved in a reversed phase column with a mixture of aqueous
ammonium formate (pH 3.0, 10 mM) and acetonitrile as the mobile phase. Detection and
confirmation of the twenty quaternary ammonium drugs at about 5 ng mL–1 in
equine urine could be achieved within 22 min using product-ion scan MS-MS. The target
analytes were detected by examination of extracted-ion chromatograms of their product
ions. Drugs spiked in different equine urine (n = 15) were consistently detected.
Negative samples (n = 30) of normal post-race equine urine have also been
analysed, no matrix interference at the targeted masses and retention times was observed.
The method was successfully applied to the analyses of drugadministration samples. Other
method validation data including reproducibility and recovery will also be presented. An
LC-MS-MS method for the simultaneous detection and confirmation of twenty quaternary
ammonium drugs in equine urine was developed. The methodology should be applicable to
other biological matrices such as human urine. |
|
Approaches to the Solid-Phase Extraction of Equine Urine |
P. M.
Wynne, D. C. Batty, J. H. Vine, N. J. K. Simpson |
S-51 |
| Three
approaches to the solid-phase extraction of horse urine are reviewed. Simple reversedphase
SPE may be coupled with a chromatographic technique that provides a second stage of
separation in the presence of a high matrix background. Pre-extraction of the urine or
hydrolysed urine allows the elimination of salts and other matrix related interferences
prior to the primary extraction. Serial processing of urine, hydrolysed urine or
pre-extracted samples through specialty sorbents allow speciation of a single sample into
many different classes suitable for Systematic Toxicological Analysis analysis by GC-MS or
LC-MS. The multi-eluate approach allows significant reduction of the matrix background
when compared with parallel SPE of a sample. |
|
Quantitative Determination of Carisoprodol and its Metabolites in
Equine Urine and Serum by Liquid Chromatography-Tandem Mass Spectrometry |
| W. Skinner, D.
McKemie, S. StanleyP. Wójcik / K. Pyrzynska / M. Biesaga |
S-61 |
| A liquid
chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for the
quantitation of carisoprodol and its metabolites, meprobamate and hydroxycarisoprodol, in
equine urine and serum. The method consists of solid phase extraction followed by LC-MS-MS
using electrospray ionization (ESI) and multiple reaction monitoring. Validation of the
method showed excellent sensitivity, selectivity, accuracy, precision and ruggedness. The
urinary excretion and serum profiles of these analytes were determined after oral
administration of carisoprodol and meprobamate. The determination of hydroxycarisoprodol
in urine and/or intact carisprodol in serum allows one to effectively distinguish between
specimens arising from administration of carisoprodol (a class 4 drug violation) and
meprobamate (a class 2 violation). |
|
Retrospective Detection and Deposition Profiles of Potentiated
Sulphonamides in Equine Hair by Liquid Chromatography |
| M. Dunnett, P.
Lees |
S-69 |
| Despite
potential benefits of hair analysis to the equine industry, deposition of drugs in equine
hair has been little studied. Trimethoprim/sulphonamides were selected as experimental
‘target’ drugs to investigate factors affecting drug uptake and distribution in
equine hair. Hair samples were analysed by HPLC after extraction (0.2 M ammonium
hydroxide, 60 °C, 24 h) and SPE (mixed-mode sorbent, C18/SCX). Analytes were resolved on
a C18 column (150 x 2.1 mm, 4 µm) by gradient elution (25 mM triethylammonium phosphate,
pH 3.0)/ acetonitrile), detected at 240/270 nm, and identified by absorption spectra
(225–360 nm). Calibration was linear from 0.125–25.0 ng.mg–1 .
Recoveries were 71–93%. Sulphadimidine, sulphadiazine and trimethoprim were detected
in hair from 12 horses. Hair colour and dose size affected the magnitude of drug uptake.
Longitudinal distribution profiles of these drugs in tail hair were determined. |
|
Determination of Salmeterol in Equine Urine and Serum |
| A. F. Lehner, C.
G. Hughes, W. Karpiesiuk, F. C. Camargo, D. J. Harkins, W. E. Woods, J. Bosken, J. Boyles,
A. Troppmann, T. Tobin |
S-79 |
| Salmeterol
is a b2-adrenergic agonist and an Association of Racing Commissioners
International (ARCI) class 3 drug. Trade names of its xinafoate salt are Arial (Dompé),
Salmetedur (Menarini), and Serevent (Glaxo). Salmeterol is routinely used to increase ease
of breathing in race horses during their training. Due to its bronchodilating and central
nervous system stimulant properties, its administration to a horse just prior to race time
has the potential to affect the horse’s performance, therefore a reliable method of
analysis for this compound is necessary. This paper describes a method for the
identification and quantitation of salmeterol in equine urine using liquid-liquid
extraction followed by liquid chromatography and tandem mass spectrometry (LC-MS-MS).
Urine salmeterol concentrations peaked at about 2 h post-dose following administration of
500 ug both intravenously and intratracheally at concentrations of 14 ng mL–1
and 4 ng mL–1, respectively. Serum concentrations at 30 min were below the
minimum level of quantitation. |
|
Norchlorotestosterone Acetate: An Alternative Metabolism Study and
GC–MS2 Analysis in Kidney Fat, Urine, and Faeces |
| N. Van Hoof, K.
De Wasch, S. Poelmans, D. Bruneel, S. Spruyt, H. Noppe, C. Janssen, D. Courtheyn, H. De
Brabander |
S-85 |
| Norchlorotestosterone
acetate (NClTA) is an anabolic steroid which resembles chlorotestosterone acetate. It
cannot yet be detected by routine methods used for anabolic steroids, because there is no
knowledge of its metabolic pathway. The invertebrate Neomysis integer has been
used as an alternative model to study the metabolism of NClTA. The experimental results
indicated the presence of 4-norchloroandrost-4-ene-17-ol-3-one (NClT) and
4-norchloroandrost- 4-ene-3,17-dione (NorClAD) as possible metabolites of NClTA.
Subsequently NClTA and the synthesised metabolites NClT and NorClAD were incorporated into
the routine multi-residue method for detection of anabolic steroids in kidney fat, urine,
and faeces. |
|
Detection and Identification of Dizocilpine and its Major Urinary
Metabolites in the Horse: A Preliminary Report |
| A. F. Lehner, C.
G. Hughes, J. D. Harkins, W. Karpiesiuk, F. Camargo, J. Boyles, W. E. Woods, T. Tobin |
S-95 |
Dizocilpine
([+]-10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine), is a potent and
selective NMDA (N-methyl-D-aspartate) receptor antagonist, which acts by blocking receptor
ion channels. Dizocilpine is pharmacologically related to ketamine and phencyclidine; as
such, it has the potential to affect behavior and performance in horses, with particular
efficacy at lower concentrations.
We now report development of a sensitive method for the detection of dizocilpine and
preliminary characterization of its urinary metabolites in the horse. Dizocilpine (MW 221)
readily produces a protonated species [M+H]+ in formic acid, and yields a m/z
205 product ion in Multiple Reaction Monitoring (MRM), allowing highly sensitive detection
of parent drug. The 17 AMU loss most likely represents an unusual loss of CH5
from the exocyclic methyl group. No unchanged dizocilpine was identified in unhydrolysed
urine, and the presence of hydroxymethyl and carboxydizocilpine glucuronide metabolites
were supported by observation of m/z 414®238
and 428®235
transitions.
Urine samples from horses dosed with dizocilpine (0.0132 and 0.0656 mg kg–1,
iv) were hydrolysed with glucuronidase and were found to contain dizocilpine and
OH-dizocilpine. Tentatively identified phase I post-hydrolysis compounds include
dizocilpine itself, an hydroxymethyl metabolite, two ring-hydroxylated metabolites, a
di-hydroxy metabolite, and a carboxy-dizocilpine metabolite. Corresponding Phase II
glucuronidated metabolites were also identified as well as a number of combination
metabolites and a posssible n-glucuronide metabolite for a total of at least six
identifiable urinary glucuronide metabolites. Among the phase I metabolites, the
hydroxymethyl metabolite apparently predominated, especially at the 0.0132 mg kg–1
dose.
The goal of this research was to identify a target analyte for dizocilpine in
post-administration equine urine, so that work may begin on development of a forensically
validated qualitative method for this target analyte. Given the likelihood that the doses
of dizocilpine used in attempts to influence the behavior or performance of horses, either
alone or in combination with other agents, are expected to be in the order of 0.02 mg kg–1
or less, these results suggest selection of the phase I hydroxymethyl metabolite of
dizocilpine as the optimal target analyte for regulatory control of dizocilpine in
performance horses. |
|
Gas Chromatographic – Mass Spectrometric Cardiotonic Glycosides
Detection in Equine Urine Doping Analysis |
| P. Kiousi, Y. S.
Angelis, M. Koupparis, D. Kouretas, N. Diakakis, A. Desiris, C. G. Georgakopoulos |
S-105 |
| A
screening method for the detection of cardiotonic glycosides in horse’s urine is
presented in this paper. This method is based on the detection by gas chromatography-mass
spectrometry (GC-MS) and gas chromatography-high resolution mass spectrometry (GC-HRMS) of
the trimethylsilyl (TMS) derivatives of the aglycon moieties of these compounds after
solid phase extraction and methanolysis, according to the standard procedure currently
used for the screening of anabolic steroids in horses, in the Athens Doping Control
Laboratory. This validated method was also applied for the excretion study of digoxin in
two mare horses after a single administration of a 75 lg Kg–1 oral dose,
by GC-MS and GC-HRMS. |
|
Development and Validation of a Quantitative Gas Chromatography
– Mass Spectrometry Confirmatory Method for Phenylbutazone in Equine Plasma |
| S. Hines, C.
Pearce, J. Bright, P. Teale |
S-109 |
| The
non-steroidal anti-inflammatory drug phenylbutazone is known to have a number of adverse
side effects, however, its use in the equine remains widespread. To protect the human
consumer, an action level of 50 ng mL–1 in plasma has been agreed by the
European Union and regulatory compliance requires a methodology validated to the EU
guidelines SANCO/ 1805/200 revision 6. Accordingly, a GC-MS method has been developed and
validated to serve this purpose. The method counters the adverse effects of strong protein
binding and the instability of phenylbutazone at low pH. Selected ion monitoring (SIM)
allows the acquisition of three diagnostic ions and the use of a deuterated internal
standard secures good quantitative performance. |
|
Detection of Eltenac in the Horse: Identification of Phase I
Metabolites in Urine by Capillary Gas Chromatography-Mass Spectrometry and the
Determination of Excretion Profile by Liquid Chromatography-Mass Spectrometry |
| M. C. Dumasia, I.
Morelli, P. Teale |
S-115 |
| Telzenac®
(Eltenac; 0.5 mg kg–1) was administered intravenously to two
thoroughbred horses. After initial alkaline saponification followed by enzymolysis of the
urinary phase II conjugates, the combined unconjugated compounds and aglycones were
isolated by mixed mode solid phase extraction (SPE). The acidic isolate was either
methylated or silylated (trimethylsilyl ether, TMS) and analysed by positive ion electron
ionisation gas chromatography-mass spectrometry (GC-EI+-MS). Eltenac and two isobaric
metabolites, hydroxyeltenac (aromatic oxidation) and eltenac sulfoxide were tentatively
identified. Base peaks in the EI+ spectra of underivatised, methylated and TMS derivatised
eltenac are formed by an initial loss of H2O, CH3OH or (CH3)3-Si-OH
respectively, followed by successive losses of a chlorine atom and a carbonyl group.
Similar fragmentation patterns were observed for the methyl and TMS derivatives of the two
metabolites. Triamcinolone acetonide was used as the internal marker for the
semiquantification of eltenac. Selected samples were base-hydrolysed and extracted on-line
on a C2 SPE column using a Prospekt sample handler. The retained analytes were eluted
directly on to an analytical LC column and analysed by high performance liquid
chromatography positive ion atmospheric pressure chemical ionisation MS in the selective
ion recording mode. Most of the drug was excreted in less than 24 h. However it could
still be detected in urine by full-scan GCEI+-MS for over 96 h |
|
Application of Oestrogen Receptor Ligand Binding Domain to the
Generic Isolation of Oestrogens by Receptor Affinity Chromatography |
| M. F. Byford, M.
J. Sauer |
S-123 |
|
Human
oestrogen receptor (alpha) ligand binding domain (hER-LBD) was expressed in E. coli
and isolated using a novel approach. The solubilised recombinant receptor had the expected
biological activity in terms of ligand binding affinity and selectivity, indicating the
potential for use in the proposed receptor affinity chromatography (RAC) application.
Subsequent covalent binding of hER-LBD to agarose support provided an affinity matrix
capable of selective binding of oestrogenic ligands, with a capacity for 17b-oestradiol of ~6 ng/mL
wet gel. In initial studies, a yield of ~75% of bound ligand from the affinity
matrix was obtained by elution with aqueous ethanol. Immobilised hER-LBD eluted with
ethanol retained the majority of its capability to bind 17b-oestradiol (E2), indicating the
possibility of reuse of the receptor matrix. In ligandreceptor displacement studies, using
[3H]E2-saturated immobilised hER-LBD, direct extraction of the xenoestrogen
2',3',4',5'-tetrachloro-4-biphenylol (TeCBol) from a model food (aqueous gelatin solution)
was inhibited at the highest concentration of gelatin tested (1%), however, prior
precipitation and extraction with ethanol enabled dose dependent binding of TeCBol.
The present studies thus provide preliminary proof of principle for the application of
hERLBD for the purpose of RAC and for the generic extraction of oestrogens and
xenoestrogens from biological matrices. |
|
Case Study: Doping Substances in Equestrian Food Supplements |
| M. Machnik, M.
Düe, M. Parr, C. von Kuk, W. Schänzer |
S-131 |
| In the
course of investigations on equestrian supplemental products for the presence of doping
substances, two products were found to contain forbidden substances. As reported earlier a
plant extract (Mexican cactus extract) named ‘‘Energy 5’’ contained
the anabolic androgenic steroids (AAS) stanozolol, 17b-hydroxy-17a-methyl-5a-androstane-3b-ol (3b,5a-THMT) as well as mestanolone not
declared on the label. In the present study, a product called ‘‘Super Kalm
Paste’’ was tested. Analysis by gas chromatography - mass spectrometry (GC-MS)
revealed that the preparation contained the class I anti-arrhythmics quinine (trade names
KinidinTM,
Durules) and cinchonine. The samples were prepared according to a sample preparation
procedure established for anabolic steroids in nutritional supplements for humans. The
sample treatment comprised the extraction and purification of the analytes as well as the
chemical conversion with N-methyl-N-trimethylsilyl-trifluoracetamide
(MSTFA) to yield the trimethylsilyl (TMS)-derivatives. To verify whether the
administration of such products could lead to positive doping tests, a pilot excretion
study on ‘‘Energy 5’’ was conducted with two geldings, and urine
samples were collected. Gas chromatography - high resolution mass spectrometry (GC-HRMS)
after solid phase extraction and mixed derivatisation has demonstrated the presence of the
stanozolol metabolite 16b-hydroxy-stanozolol in urine samples after ‘‘Energy
5’’ application. |
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