Wilhelm Schänzer

Factors influencing the steroid profile in doping control analysis.

Steroid profiling is one of the most versatile and informative screening tools for the detection of steroid abuse in sports drug testing. Concentrations and ratios of various endogenously produced steroidal hormones, their precursors and metabolites including testosterone (T), epitestosterone (E), dihydrotestosterone (DHT), androsterone (And), etiocholanolone (Etio), dehydroepiandrosterone (DHEA), 5alpha-androstane-3alpha,17beta-diol (Adiol), and 5beta-androstane-3alpha,17beta-diol (Bdiol) as well as androstenedione, 6alpha-OH-androstenedione, 5beta-androstane-3alpha,17alpha-diol (17-epi-Bdiol), 5alpha-androstane-3alpha,17alpha-diol (17-epi-Adiol), 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo), 5alpha-androstanedione (Adion), and 5beta-androstanedione (Bdion) add up to a steroid profile that is highly sensitive to applications of endogenous as well as synthetic anabolic steroids, masking agents, and bacterial activity. Hence, the knowledge of factors that do influence the steroid profile pattern is a central aspect, and pharmaceutical (application of endogenous steroids and various pharmaceutical preparations), technical (hydrolysis, derivatization, matrix), and biological (bacterial activities, enzyme side activities) issues are reviewed.

Current role of LC–MS(/MS) in doping control

AbstractLiquid chromatography–(tandem) mass spectrometry [(LC-MS(/MS)] has become an integral part of modern sports drug testing as it offers unique capabilities complementing immunological and gas chromatography–(tandem) mass spectrometry [(GC-MS(/MS)]-based detection methods for prohibited compounds. The improved options of fast and sensitive targeted analysis as well as untargeted screening procedures utilizing high resolution/high accuracy mass spectrometry have considerably expanded the tools available to anti-doping laboratories for initial testing and confirmation methods. One approach is to focus on pre-selected target analytes that are measured with utmost specificity and sensitivity using diagnostic precursor–product ion pairs in low resolution tandem mass spectrometers. The other scenario is to measure and plot extracted ion chromatograms of protonated or deprotonated molecules as well as product ions as recorded in the full scan mode with high resolution/high accuracy mass spectrometry. Examples of recent applications of sports drug testing procedures published between 2007 and 2010 are presented and discussed, outlining the particular advantages of the selected approaches as well as their limitations in a short- and long-term perspective. FigureThe utility of LC-MS(/MS) in current sports drug testing programs is discussed and trends in doping control methods as well as data mining are presented

Determination of 13C/12C ratios of endogenous urinary steroids : method validation, reference population and application to doping control purposes

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values.

Quantification of human insulin-like growth factor-1 and qualitative detection of its analogues in plasma using liquid chromatography/electrospray ionisation tandem mass spectrometry

Human insulin-like growth factor-1 (IGF-1) is a peptide hormone that acts as a mediator of most of the somatotropic effects of growth hormone (GH). Therefore, it is supposed to be a biomarker indicating GH abuse in sports as well as diseases associated with a change in IGF-1 plasma concentration. It can be applied locally by injection to increase total protein and DNA content in tissues such as skeletal muscle--a highly desirable effect in various sports disciplines. In order to improve its growth-promoting properties, the primary structure of IGF-1 has been modified, yielding analogues such as des(1-3)IGF-1 and LONGR3IGF-1, which show a considerably reduced affinity to the respective binding proteins in plasma and, thus, an increased bioavailability at target tissues. Due to their capability to enhance performance, IGF-1 and its analogues belong to the prohibited list of the World Anti-Doping Agency. Hence, it was necessary to develop a reliable assay for the quantification of human IGF-1 as well as the detection of its derivatives. Immunoaffinity isolation and purification from 60 microL of plasma followed by liquid chromatography/electrospray ionisation tandem mass spectrometry enabled the unequivocal determination of all target analytes. Diagnostic product ions were characterised utilising an Orbitrap mass spectrometer with high resolution/high accuracy properties and employed for triple quadrupole MS/MS analysis. The described assay provided lower limits of detection (LLODs) between 20 and 50 ng/mL, recovery rates between 34-43% and a precision <15% at the LLOD as well as higher concentration levels. In order to prove the applicability of the developed assay, human plasma samples were analysed and the results were compared with the values obtained from a commercially available immunoradiometric assay (IRMA). Four of six samples resulted in concentration ratios with good correlation between both assays, whereas the absolute concentrations were lower for the presented procedure.

Annual banned-substance review: analytical approaches in human sports drug testing

The timely update of the list of prohibited substances and methods of doping (as issued by the World Anti-Doping Agency) is an essential aspect of international anti-doping efforts and represents consensual agreement by expert panels regarding substances and the methods of performance manipulation in sports. The annual banned-substance review for human doping controls critically summarizes recent innovations in analytical approaches; its purpose is to improve the quality of doping controls by reporting emerging and advancing methods that focus on detecting known and recently outlawed substances. This review surveys new and/or enhanced procedures and techniques of doping analysis together with information relevant to doping control that has been published in the literature between October 2009 and September 2010.

ESC study group of sports cardiology position paper on adverse cardiovascular effects of doping in athletes.

The use of doping substances and methods is extensive not only among elite athletes, but also among amateur and recreational athletes. Many types of drugs are used by athletes to enhance performance, to reduce anxiety, to increase muscle mass, to reduce weight or to mask the use of other drugs during testing. However, the abuse of doping substances and methods has been associated with the occurrence of numerous health side-effects. The adverse effects depend on the type of the consumed drug, as well as the amount and duration of intake and the sensitivity of the body, since there is a large inter-individual variability in responses to a drug. Usually the doses used in sports are much higher than those used for therapeutic purposes and the use of several drugs in combination is frequent, leading to higher risk of side-effects. Among biomedical side-effects of doping, the cardiovascular ones are the most deleterious. Myocardial infarction, hyperlipidemia, hypertension, thrombosis, arrythmogenesis, heart failure and sudden cardiac death have been noted following drug abuse. This paper reviews the literature on the adverse cardiovascular effects after abuse of prohibited substances and methods in athletes, aiming to inform physicians, trainers and athletes and to discourage individuals from using drugs during sports.

Reference ranges for urinary concentrations and ratios of endogenous steroids, which can be used as markers for steroid misuse, in a Caucasian population of athletes.

The detection of misuse with naturally occurring steroids is a great challenge for doping control laboratories. Intake of natural anabolic steroids alters the steroid profile. Thus, screening for exogenous use of these steroids can be established by monitoring a range of endogenous steroids, which constitute the steroid profile, and evaluate their concentrations and ratios against reference ranges. Elevated values of the steroid profile constitute an atypical finding after which a confirmatory IRMS procedure is needed to unequivocally establish the exogenous origin of a natural steroid. However, the large inter-individual differences in urinary steroid concentrations and the recent availability of a whole range of natural steroids (e.g. dehydroepiandrosterone and androstenedione) which each exert a different effect on the monitored parameters in doping control complicate the interpretation of the current steroid profile. The screening of an extended steroid profile can provide additional parameters to support the atypical findings and can give specific information upon the steroids which have been administered. The natural concentrations of 29 endogenous steroids and 11 ratios in a predominantly Caucasian population of athletes were determined. The upper reference values at 97.5%, 99% and 99.9% levels were assessed for male (n=2027) and female (n=1004) populations. Monitoring minor metabolites and evaluation of concentration ratios with respect to their natural abundances could improve the interpretation of the steroid profile in doping analysis.

δC13-Values of endogenous urinary steroids

By means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) urinary steroids obtained from a reference population of 56 subjects were analyzed for their (13)C/(12)C-ratios. The analytes encompassed androsterone (A), etiocholanolone (E), 11beta-hydroxyetiocholanolone (OHE), 11beta-hydroxyandrosterone (OHA), and 5beta-pregnane- 3alpha,20alpha-diol (PD). A and E represent androgen metabolites (AM). PD, OHE, and OHA have sources independent from androgen metabolism. The delta(13)C-values of the latter compounds may be compared to those of AM in order to detect doping with synthetic androgens and thus may serve as endogenous reference compounds (ERC). In order to allow for classification of conspicuous samples, reference ranges and limits were calculated for delta(13)C-values of selected steroids and differences hereof (Delta(13)C-values). When A is compared to ERCs, Delta(13)C-values larger than 3 per thousand are very unlikely. A set of additional parameters was surveyed by a questionnaire. Several factors turned out to exert significant influence on the delta(13)C-values of urinary steroids. These encompass the identity of the steroid itself, sex, oral contraception, travels, and physical activity.

Aryl-Propionamide-Derived Selective Androgen Receptor Modulators: Liquid Chromatography-Tandem Mass Spectrometry Characterization of the in Vitro Synthesized Metabolites for Doping Control Purposes

Selective androgen receptor modulators (SARM) are a prominent group of compounds for being misused in sports owing to their advantageous anabolic properties and reduced side effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM, an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and the structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, monohydroxylation, and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization and detected as deprotonated species [M-H]-. The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control.

Immunoaffinity purification of peptide hormones prior to liquid chromatography-mass spectrometry in doping controls.

For most peptide hormones prohibited in elite sports the concentrations in plasma or urine are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to mass spectrometric detection are required to obtain sufficient doping control assays. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra, Lantus DesB30-32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1-29)) and CJC-1295 (GH-RH analogue), LongR(3)-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. Due to the fast and simple sample preparation procedure (∼25 samples per day) and the fact that all materials are commercial available, the implementation of the methodology to laboratories from other analytical fields (forensics, pharmacokinetic sciences, etc.) is enabled.