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Robert A. A. Maes - One of the best experts on this subject based on the ideXlab platform.

  • Artifact formation due to ethyl thio-incorporation into silylated steroid structures as determined in Doping Analysis.
    Journal of Chromatography A, 2002
    Co-Authors: D.h. Van De Kerkhof, Jos H.h. Thijssen, D. De Boer, R.d Van Ooijen, Roel H. Fokkens, Nico M. M. Nibbering, Jan W. Zwikker, Robert A. A. Maes
    Abstract:

    Trimethylsilylation of target substances in a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and ethanethiol is frequently applied for the application of gas chromatography–mass spectrometry (GC–MS) in steroid Analysis. However, artifacts were formed when using this mixture to silylate the steroids androsterone and etiocholanolone obtained from a urine matrix. The artifacts were identified as ethyl thio-containing products of the respective trimethylsilyl derivatives. The conversion of the studied products increased slowly as a function of time, was dependent on the presence of the urine matrix and was significantly accelerated by adding diethyl disulfide to the reagent before incubation. Also ethyl thio-incorporation into testosterone and epitestosterone was established. A mechanism for ethyl thio-incorporation is proposed. The conversion achieved after 120-h sample storage at room temperature was insufficient to significantly influence the Analysis of androsterone and etiocholanolone under the studied conditions. However, the results provide fundamental insight into the mechanism of silylation and the occurring side-reactions. Moreover, when investigating the formation of new metabolites, the ethyl thio-incorporation can lead to misinterpretation.

  • Evaluation of Testosterone/Epitestosterone Ratio Influential Factors as Determined in Doping Analysis
    Journal of Analytical Toxicology, 2000
    Co-Authors: D.h. Van De Kerkhof, Jos H.h. Thijssen, D. De Boer, Robert A. A. Maes
    Abstract:

    The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the

  • evaluation of testosterone epitestosterone ratio influential factors as determined in Doping Analysis
    Journal of Analytical Toxicology, 2000
    Co-Authors: D.h. Van De Kerkhof, Jos H.h. Thijssen, D. De Boer, Robert A. A. Maes
    Abstract:

    The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in Doping Analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair Analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.

  • Evaluation of Testosterone/Epitestosterone Ratio Influential Factors as Determined in Doping Analysis
    Journal of Analytical Toxicology, 2000
    Co-Authors: D.h. Van De Kerkhof, Jos H.h. Thijssen, D. De Boer, Robert A. A. Maes
    Abstract:

    The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in Doping Analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair Analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.

  • The detection of danazol and its significance in Doping Analysis.
    Journal of Analytical Toxicology, 1992
    Co-Authors: D. De Boer, E. G. De Jong, Robert A. A. Maes
    Abstract:

    The use of anabolic steroids and related compounds in sport is forbidden by the International Olympic Committee (IOC). Because danazol (17 alpha-pregna-2,4-dien-20-yno[2,3-D] isoxazol-17 beta-ol) is structurally related to the anabolic steroid stanozolol, its use should be questioned. Therefore, the detection and the significance of danazol in Doping Analysis are discussed. A urine specimen suspected of containing danazol metabolites was analyzed in order to characterize the metabolites. After isolation and conversion into three different derivatives, the metabolites were subjected to gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. The structure assignment was based on the molecular ions, fragmentation patterns observed for the three different derivatives, and the possible metabolite structures given in the literature. Ethisterone was identified as a nonconjugated metabolite. 2-Hydroxymethylethisterone was observed in two stereoisomeric forms. One stereoisomer was found mainly in the nonconjugated steroid fraction and the other in the conjugated fraction. The results were confirmed by analyzing urine specimens of a volunteer who was known to have taken danazol. Derivatization methods and GC/MS data are given to implement danazol detection in routine screening and confirmation procedures.

Koen Deventer - One of the best experts on this subject based on the ideXlab platform.

  • Validation of an ultra-sensitive detection method for steroid esters in plasma for Doping Analysis using positive chemical ionization GC-MS/MS.
    Journal of Chromatography B, 2020
    Co-Authors: Pieter Van Renterghem, Koen Deventer, Wouter Viaene, Wim Van Gansbeke, Juliana Barrabin, Michele Iannone, Michaël Polet, Guy T'sjoen, Peter Van Eenoo
    Abstract:

    Abstract The standard approach to detect misuse with testosterone in sport is based on the determination and evaluation of the urinary steroid profile followed by the confirmation of atypical profiles using isotope ratio mass spectrometry. The detection capacity of these methods can be attenuated by confounding factors or testosterone preparations with endogenous isotopic fingerprints. An alternative detection method for misuse of an endogenous steroid in sports is the direct detection of the administered steroid ester present in most preparations. Thus unambiguous proof for Doping misuse can be delivered. In this work, the sensitivity of gas chromatography coupled to a triple quadrupole with chemical ionization (GC-CI-MS/MS) is applied to detect trace levels of 10 testosterone and 2 nandrolone esters in plasma for in human Doping Analysis. The detection method was developed employing a liquid-liquid extraction and HPLC cleanup step before Analysis on the GC-CI-MS/MS. The quantitative method was validated in a linear range of 100–2000 pg/ml and proved to be selective, reproducible and very sensitive with limits of detection as low as to 10 pg/ml. A clinical study with the administration of testosterone undecanoate in 3 volunteers was carried out and the compound was detectable up to 86 days after administration.

  • Detection and characterization of anabolic steroids in Doping Analysis by LC-MS
    TrAC Trends in Analytical Chemistry, 2008
    Co-Authors: Oscar J. Pozo, Koen Deventer, Peter Van Eenoo, Frans Delbeke
    Abstract:

    Abstract The detection of target anabolic steroids in Doping Analysis has generally been performed by gas chromatography combined with mass spectrometry (GC-MS). However, liquid chromatography combined with tandem mass spectrometry (LC-MS2) is gradually becoming more important for this purpose. Also, since non-commercially available anabolic steroids have been found in some Doping-control samples, detection and structural determination of unknown steroids has become a challenge for Doping-control laboratories. We discuss the potential of different LC-MS and LC-MS2 scan modes for detection of both target and unknown anabolic steroids. Several modes (e.g., selected reaction monitoring, full scan and product-ion scan) can be successfully used for the detection of target analytes. In order to detect and to characterize unknown steroids and metabolites, the most powerful approach seems to be to combine several scan modes. We use a practical case to illustrate the potential of LC-MS and LC-MS2 for this purpose.

  • screening for anabolic steroids in Doping Analysis by liquid chromatography electrospray ion trap mass spectrometry
    Biomedical Chromatography, 2006
    Co-Authors: Koen Deventer, P. Van Eenoo, F T Delbeke
    Abstract:

    A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human urine has been developed and validated. Liquid-liquid extraction with diethyl ether was applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds. The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10 ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-hydroxystanozolol, were assessed using the described method.

  • screening for amphetamine and amphetamine type drugs in Doping Analysis by liquid chromatography mass spectrometry
    Rapid Communications in Mass Spectrometry, 2006
    Co-Authors: Koen Deventer, P. Van Eenoo, F T Delbeke
    Abstract:

    A selective and sensitive method for the qualitative screening of urine samples for 27 amphetamine and amphetamine-type drugs in the field of Doping Analysis is described. The method consists of a liquid-liquid extraction with diethyl ether at pH 14 and Analysis of the extracts with a LCQ-Deca® mass spectrometer equipped with an atmospheric pressure chemical ionisation interface, operated in positive ionisation mode. The total run time was 15 min. All compounds were analysed in MS2 or MS3. The detection limit for all compounds was lower than 25 ng/mL except for chlorphentermine (detection limit: 250 ng/mL). Copyright © 2006 John Wiley & Sons, Ltd.

  • Screening for amphetamine and amphetamine-type drugs in Doping Analysis by liquid chromatography/mass spectrometry.
    Rapid Communications in Mass Spectrometry, 2006
    Co-Authors: Koen Deventer, P. Van Eenoo, Frans Delbeke
    Abstract:

    A selective and sensitive method for the qualitative screening of urine samples for 27 amphetamine and amphetamine-type drugs in the field of Doping Analysis is described. The method consists of a liquid-liquid extraction with diethyl ether at pH 14 and Analysis of the extracts with a LCQ-Deca® mass spectrometer equipped with an atmospheric pressure chemical ionisation interface, operated in positive ionisation mode. The total run time was 15 min. All compounds were analysed in MS2 or MS3. The detection limit for all compounds was lower than 25 ng/mL except for chlorphentermine (detection limit: 250 ng/mL). Copyright © 2006 John Wiley & Sons, Ltd.

Frans Delbeke - One of the best experts on this subject based on the ideXlab platform.

  • Detection and characterization of anabolic steroids in Doping Analysis by LC-MS
    TrAC Trends in Analytical Chemistry, 2008
    Co-Authors: Oscar J. Pozo, Koen Deventer, Peter Van Eenoo, Frans Delbeke
    Abstract:

    Abstract The detection of target anabolic steroids in Doping Analysis has generally been performed by gas chromatography combined with mass spectrometry (GC-MS). However, liquid chromatography combined with tandem mass spectrometry (LC-MS2) is gradually becoming more important for this purpose. Also, since non-commercially available anabolic steroids have been found in some Doping-control samples, detection and structural determination of unknown steroids has become a challenge for Doping-control laboratories. We discuss the potential of different LC-MS and LC-MS2 scan modes for detection of both target and unknown anabolic steroids. Several modes (e.g., selected reaction monitoring, full scan and product-ion scan) can be successfully used for the detection of target analytes. In order to detect and to characterize unknown steroids and metabolites, the most powerful approach seems to be to combine several scan modes. We use a practical case to illustrate the potential of LC-MS and LC-MS2 for this purpose.

  • Implementation of gas chromatography combined with simultaneously selected ion monitoring and full scan mass spectrometry in Doping Analysis
    Journal of Chromatography A, 2008
    Co-Authors: W. Van Thuyne, P. Van Eenoo, Frans Delbeke
    Abstract:

    Abstract A comprehensive screening method for the detection of prohibited substances in Doping control is described and validated. This method is capable of detecting over 150 components mentioned on the list of the World Anti-Doping Agency including anabolic androgenic steroids, stimulants and all narcotic agents that are currently analysed using different analytical methods. The analytes are extracted from urine by a combined extraction procedure using freshly distilled diethyl ether and tert -butyl methyl ether as extraction solvents at pH 9.5 and 14 respectively. Prior to GC–MS Analysis the residues are combined and derivatised using a mixture of N -methyl- N -trimethylsilyltrifluoroacetamide, NH 4 I and ethanethiol. The mass spectrometer is simultaneously operated in the full scan mode (mass range varies along with GC-oven temperature program) and in the selected ion monitoring mode. The obtained limits of detection are in compliance with the requirements set by the World Anti-Doping Agency. Besides narcotics, stimulants and anabolic androgenic agents, this method is also capable of detecting several agents with anti-estrogenic activity and some beta-agonists. This comprehensive screening method reduces the amount of urine needed and increases the sample throughput without a loss in sensitivity and selectivity.

  • Screening for amphetamine and amphetamine-type drugs in Doping Analysis by liquid chromatography/mass spectrometry.
    Rapid Communications in Mass Spectrometry, 2006
    Co-Authors: Koen Deventer, P. Van Eenoo, Frans Delbeke
    Abstract:

    A selective and sensitive method for the qualitative screening of urine samples for 27 amphetamine and amphetamine-type drugs in the field of Doping Analysis is described. The method consists of a liquid-liquid extraction with diethyl ether at pH 14 and Analysis of the extracts with a LCQ-Deca® mass spectrometer equipped with an atmospheric pressure chemical ionisation interface, operated in positive ionisation mode. The total run time was 15 min. All compounds were analysed in MS2 or MS3. The detection limit for all compounds was lower than 25 ng/mL except for chlorphentermine (detection limit: 250 ng/mL). Copyright © 2006 John Wiley & Sons, Ltd.

  • Screening for anabolic steroids in Doping Analysis by liquid chromatography/electrospray ion trap mass spectrometry.
    Biomedical Chromatography, 2006
    Co-Authors: Koen Deventer, P. Van Eenoo, Frans Delbeke
    Abstract:

    A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human urine has been developed and validated. Liquid-liquid extraction with diethyl ether was applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds. The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10 ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-hydroxystanozolol, were assessed using the described method.

  • Validation of a screening method for corticosteroids in Doping Analysis by liquid chromatography/tandem mass spectrometry.
    Rapid Communications in Mass Spectrometry, 2003
    Co-Authors: Koen Deventer, Frans Delbeke
    Abstract:

    A selective and sensitive method for the screening of nine corticosteroids in human urine has been validated. Analyses were performed using an ion trap instrument equipped with an electrospray ionisation (ESI) interface. All corticosteroids were separated in less than 20 min after liquid/liquid extraction with diethyl ether. The limit of detection for all substances was 4 ng/mL or lower. The method was applied to detect betamethasone after the intramuscular injection of Diprophos®. Betamethasone could be detected for up to 12 days after administration. Validation of the chromatographic separation and mass spectrometric identification of mixtures of betamethasone and dexamethasone are also presented. Copyright © 2003 John Wiley & Sons, Ltd.

F T Delbeke - One of the best experts on this subject based on the ideXlab platform.

  • screening for anabolic steroids in Doping Analysis by liquid chromatography electrospray ion trap mass spectrometry
    Biomedical Chromatography, 2006
    Co-Authors: Koen Deventer, P. Van Eenoo, F T Delbeke
    Abstract:

    A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human urine has been developed and validated. Liquid-liquid extraction with diethyl ether was applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds. The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10 ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-hydroxystanozolol, were assessed using the described method.

  • screening for amphetamine and amphetamine type drugs in Doping Analysis by liquid chromatography mass spectrometry
    Rapid Communications in Mass Spectrometry, 2006
    Co-Authors: Koen Deventer, P. Van Eenoo, F T Delbeke
    Abstract:

    A selective and sensitive method for the qualitative screening of urine samples for 27 amphetamine and amphetamine-type drugs in the field of Doping Analysis is described. The method consists of a liquid-liquid extraction with diethyl ether at pH 14 and Analysis of the extracts with a LCQ-Deca® mass spectrometer equipped with an atmospheric pressure chemical ionisation interface, operated in positive ionisation mode. The total run time was 15 min. All compounds were analysed in MS2 or MS3. The detection limit for all compounds was lower than 25 ng/mL except for chlorphentermine (detection limit: 250 ng/mL). Copyright © 2006 John Wiley & Sons, Ltd.

  • Criteria in Chromatography and Mass Spectrometry – a Comparison Between Regulations in the Field of Residue and Doping Analysis
    Chromatographia, 2004
    Co-Authors: P. Van Eenoo, F T Delbeke
    Abstract:

    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 difficulties 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.

  • criteria in chromatography and mass spectrometry a comparison between regulations in the field of residue and Doping Analysis
    Chromatographia, 2004
    Co-Authors: P. Van Eenoo, F T Delbeke
    Abstract:

    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 difficulties 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.

  • validation of a screening method for corticosteroids in Doping Analysis by liquid chromatography tandem mass spectrometry
    Rapid Communications in Mass Spectrometry, 2003
    Co-Authors: Koen Deventer, F T Delbeke
    Abstract:

    A selective and sensitive method for the screening of nine corticosteroids in human urine has been validated. Analyses were performed using an ion trap instrument equipped with an electrospray ionisation (ESI) interface. All corticosteroids were separated in less than 20 min after liquid/liquid extraction with diethyl ether. The limit of detection for all substances was 4 ng/mL or lower. The method was applied to detect betamethasone after the intramuscular injection of Diprophos®. Betamethasone could be detected for up to 12 days after administration. Validation of the chromatographic separation and mass spectrometric identification of mixtures of betamethasone and dexamethasone are also presented. Copyright © 2003 John Wiley & Sons, Ltd.

Peter Hemmersbach - One of the best experts on this subject based on the ideXlab platform.

  • Pharmacokinetics of nebulized and oral procaterol in asthmatic and non‐asthmatic subjects in relation to Doping Analysis
    Drug Testing and Analysis, 2016
    Co-Authors: Nanna Krogh, Peter Hemmersbach, Vibeke Backer, Sebastian Rzeppa, Morten Hostrup
    Abstract:

    The purpose of the present study was to investigate pharmacokinetics of procaterol in asthmatics and non-asthmatics after nebulized and oral administration in relation to Doping. Ten asthmatic and ten non-asthmatic subjects underwent two pharmacokinetic trials. At first trial, 4 µg procaterol was administered as nebulization. At second trial, 100 µg procaterol was administered orally. Serum and urine samples were collected before and after administration of procaterol. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum and urine concentrations of procaterol were markedly higher after oral administration compared to nebulized administration. After oral administration, serum procaterol concentration-time area under the curve (AUC) was higher (P ≤ 0.05) for asthmatics than non-asthmatics. Likewise, urine concentrations were higher (P ≤ 0.01) for asthmatics than non-asthmatics 4 (47 ± 12 vs. 28 ± 9 ng/mL) and 8 h (39 ± 9 vs. 15 ± 5 ng/mL) after oral administration. Detection of serum procaterol was difficult after nebulized administration with 38 samples (27%) below limit of quantification (LOQ) and only trends were observed. No differences were observed between asthmatics and non-asthmatics in the urine concentrations of procaterol after nebulized administration. In summary, our data showed that asthmatics had higher urine concentrations of procaterol than non-asthmatics after oral administration of 100 µg, whereas no difference was observed between the groups after nebulized administration. For Doping control purposes, our observations indicate that it is possible to differentiate therapeutic nebulized administration of procaterol from prohibited use of oral procaterol. Copyright © 2016 John Wiley & Sons, Ltd.

  • Pharmacokinetics of inhaled terbutaline in relation to Doping Analysis: Impact of exercise and heat
    4.1 Clinical respiratory physiology exercise and functional imaging, 2015
    Co-Authors: Michael Kreiberg, Peter Hemmersbach, Vibeke Backer, Victoria Becker, Sebastian Rzeppa, Morten Hostrup
    Abstract:

    The World Anti-Doping Agency (WADA) has loosened their restrictions towards inhaled β2-agonists. Terbutaline (TER) still requires athletes to provide evidence of asthma to get a dispensation (i.e. TUE) for inhaled use. Limited data exist on the pharmacokinetics of inhaled TER during exercise. The purpose of this study was thus to investigate the influence of exercise in normal and in hot ambient conditions on the pharmacokinetics of 8x500 µg inhaled TER in relation to Doping Analysis. Methods: Thirteen trained endurance athletes, all males and non-asthmatics, aged 23.5±4.5 yrs (Mean±SD), VO2max 55.3±7 ml/min/kg, participated in the study. Urine and serum concentrations of TER were determined during three conditions: Exercise (EX), exercise in hot ambient conditions (30-35°C) (EXH), and rest (R). Exercise consisted of submaximal work at 60% of subjects VO2max on a bike ergometer for 124 min, and then a VO2max test. Urine and blood samples were were analyzed for TER. Results: A significant(P Conclusion: Exercise in hot ambient conditions results in higher urine concentrations of TER in trained men. This should be taken into account when evaluating Doping cases of TER.

  • Comparison of newly developed immuno-MS method with existing DELFIA® immunoassay for human chorionic gonadotropin determination in Doping Analysis
    Bioanalysis, 2013
    Co-Authors: Hanne Lund, Peter Hemmersbach, Trine Grønhaug Halvorsen, Ann Helene Snilsberg, Léon Reubsaet
    Abstract:

    Background: The performance of a method for MS determination of human chorionic gonadotropin (hCG) was compared with a reference method currently used in World Anti-Doping Agency accredited Doping laboratories – the DELFIA® immunoassay. Results: A strong correlation was demonstrated for the serum samples. However, for the urine samples, DELFIA reported significantly lower quantitative hCG measurements than the MS method. This was explained by the relatively unstable content of intact hCG-heterodimer in urine during storage compared with in serum. Discrepancies observed for the urine analyses might be related to the molecular dissociation of intact hCG-heterodimer into free subunits during storage, and the direct effect this has on the intact hCG measurements provided by DELFIA. The MS method quantified both intact hCG and free hCG β-subunit simultaneously, and was thus less susceptible to this problem. However, both methods detected illicit levels of serum hCG an equally long time after administration. Co...

  • Sports drug testing using immuno-MS: clinical study comprising administration of human chorionic gonadotropin to males
    Analytical and Bioanalytical Chemistry, 2012
    Co-Authors: Hanne Lund, Peter Hemmersbach, Elisabeth Paus, Trine Grønhaug Halvorsen, Ann Helene Snilsberg, Léon Reubsaet
    Abstract:

    The applicability of a mass spectrometry (MS)-based method for determination of various forms of human chorionic gonadotropin (hCG) in Doping Analysis was demonstrated. A clinical study involving the hCG-containing pharmaceuticals Pregnyl and Ovitrelle was carried out, comprising a single injection of one pharmaceutical per participant to a total of 24 healthy male voluntaries. Hereafter, serum and urine samples were collected over a period of 14 days. The Analysis of the samples using immuno-MS demonstrated elimination profiles of intact hCG for both pharmaceuticals, with last day of detection following administration at day 7 in serum, and at day 10 in urine, at limit of detections as defined by the World Anti-Doping Agency. Furthermore, the method allowed detection and differentiation of the various forms of hCG known to be present in serum and urine as a function of metabolism. For both pharmaceuticals, only the intact hCG was detected in serum, whereas in urine the injection of Pregnyl as hCG source (containing urinary hCG, i.e., most hCG variants) was shown to generate a more complex hCG variant pattern compared to Ovitrelle (contains only intact hCG). By detecting hCG using this MS-based approach in Doping Analysis, strong analytical evidence is provided minimizing the risk of false-positive and false-negative results.

  • Intra-Individual Variability in the Urine Concentrations of Inhaled Salmeterol in Male Subjects with Reference to Doping Analysis?Impact of Urine Specific Gravity Correction
    Journal of Sports Medicine & Doping Studies, 2012
    Co-Authors: Morten Hostrup, Anders Kalsen, Peter Hemmersbach, Vibeke Backer
    Abstract:

    Since 2010, the World Anti-Doping Agency (WADA) has introduced urinary thresholds for some beta2-agonists. In Doping Analysis urine samples of beta2-agonists are not corrected for the Urine Specific Gravity (USG) by the WADA laboratories. Several studies have observed high differences in the urine concentrations of beta2-agonists when correction for USG is compared with no correction, as well as high inter-individual variability between subjects. However, no studies have measured the intra-individual variability after inhalation of the long-acting beta2-agonist salmeterol. As such, the purpose of this study was to measure the intra-individual variability in the urine concentrations of salmeterol and its metabolite α-hydroxysalmeterol. Furthermore, to highlight the variability between corrected and uncorrected urine samples for USG. Urine samples from 20 subjects were analyzed for USG, urine excretion and urine concentrations of salmeterol and α-hydroxysalmeterol. Seven of the subjects underwent a second visit with the same procedures. At each visit 100 μg salmeterol was administered by inhalation. Urine samples were collected before administration of the drug (T0) and 4 (T4), 8 (T8) and 12 (T12) hours after administration. The mean relative differences in the urine concentrations of salmeterol and α-hydroxysalmeterol between USG corrected and uncorrected samples were 43 ± 44, 27 ± 42 and 56 ± 87% at T4, T8 and T12, respectively. The intra-individual variability in the urine excretion of salmeterol and α-hydroxysalmeterol during visits one and two were 12.6 and 21.8%, respectively. The intra-individual variability of salmeterol and α-hydroxysalmeterol in the urine concentrations were significantly higher when uncorrected for USG with 43.0 and 43.7% versus 20.4% (p