Case Study: Atypical δ13 C values of urinary norandrosterone.

Isotope ratio mass spectrometry (IRMS) has been established in doping control analysis to identify the endogenous or exogenous origin of a variety of steroidal analytes including the 19-norsteroid metabolite norandrosterone (NorA). NorA can be found naturally in human urine in trace amounts due to endogenous demethylation or in situ microbial degradation. The administration of nortestosterone (nandrolone) or different prohormones results in the excretion of urinary NorA. Usually, this can be detected by IRMS due to differing δ13 C values of synthetic 19-norsteroids compared to endogenous reference compounds. The consumption of uncastrated pig edible parts like offal or even meat may also lead to a urinary excretion of NorA. In order to determine the δ13 C values of such a scenario, urine samples collected after consumption of a wild-boar-testicle meal were analyzed. IRMS revealed highly enriched δ13 C values for urinary NorA, which could be related to the completely corn-based nutrition of the animal. Isotopic analysis of the boar's bristles demonstrated a dietary change from C3 -based forage, probably in winter and spring, to a C4 -based diet in the last weeks to months prior to death. These results supported the interpretation of an atypical test result of a Central European athlete's doping control sample with δ13 C values for NorA of -18 ‰, most probably caused by the consumption of a wild boar ragout. As stated before, athletes should be fully aware of the risk that consumption of wild boar may result in atypical or even adverse analytical findings in sports drug testing.

Screening for benfluorex and its major urinary metabolites in routine doping controls.

Benfluorex [1-(m-trifluoromethylphenyl)-2-(ß-benzoyloxyethyl)aminopropane] has been widely used for the treatment of atherogenic metabolic disorders and impaired carbohydrate metabolism (particularly in obese type-II diabetic patients) as well as an anorectic drug. Due to its potentially performance-enhancing properties, benfluorex has been added to the list of prohibited compounds and methods of doping by the World Anti-Doping Agency (WADA) in 2010, necessitating the implementation of the drug as well as its major metabolites into routine doping control procedures. In the present study, human urinary metabolites of benfluorex were characterized by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) as well as liquid chromatography-electrospray ionization-high resolution/high accuracy tandem mass spectrometry (LC-ESI-MS/MS). Commonly employed sports drug testing approaches consisting of liquid-liquid extraction followed by GC-MS or urine dilution and immediate LC-MS/MS analysis were expanded and validated with regard to specificity, recovery (48-54%, GC-MS only), intra- and interday precision (<25%), limits of detection (5-8 ng/mL for LC-MS/MS and 80 ng/mL for GC-MS), and ion suppression (for LC-ESI-MS/MS only) to allow the detection of benfluorex metabolites 1-(m-trifluoromethylphenyl)-2-(2-hydroxyethyl)aminopropane (M1), 1-(m-trifluoromethylphenyl)-2-(2-carboxymethyl)aminopropane (M2), and 1-(m-trifluoromethylphenyl)-2-aminopropane (M3) as well as the glucuronic acid conjugate of M1.

Determination of (13)C/(12)C ratios of urinary excreted boldenone and its main metabolite 5beta-androst-1-en-17beta-ol-3-one.

Boldenone (androsta-1,4-dien-17beta-ol-3-one, Bo) is an anabolic steroid known to have been used in cattle breeding or equine sport as a doping agent for many years. Although not clinically approved for human application, Bo or its main metabolite 5beta-androst-1-en-17beta-ol-3-one (BM1) were detected in several doping control samples. For more than 15 years the possibility of endogenous Bo production in human beings has been discussed. This is a challenging issue for doping control laboratories as Bo belongs to the list of prohibited substances of the World Anti-Doping Agency and therefore the chance for false positive testing is significant. By GC/C/IRMS (gas chromatography/combustion/isotope ratio mass spectrometry) it should be possible to analyze the (13)C/(12)C ratio of either Bo or BM1 and to distinguish whether their source is endogenous or exogenous. Therefore a method was developed to determine the (13)C/(12)C ratios of Bo, BM1, pregnanediol, androsterone, etiocholanolone, and testosterone from a single urine specimen. The validity of the method was ensured by repeated processing of urine fortified with 2-50 ng/mL Bo and BM1. The specificity of the method was ensured by gas chromatography/mass spectrometry determinations. Out of 23 samples investigated throughout the last four years, 11 showed (13)C/(12)C ratios of Bo or BM1 inconsistent with an exogenous origin. Two of these samples were collected from the same athlete within a one-month interval, strongly indicating the chance of endogenous Bo production by this athlete.

Doping control analysis of metamfepramone and two major metabolites using liquid chromatography-tandem mass spectrometry.

The sympathomimetic agent metamfepramone (2-dimethylamino-1-phenylpropan-1-one, dimethylpropion) is widely used for the treatment of the common cold or hypotonic conditions. Due to its stimulating properties and its rapid metabolism resulting in major degradation products such as methylpseudoephedrine and methcathinone, it has been considered relevant for doping controls by the World Anti-Doping Agency (WADA). The rapid degradation of the active drug complicates the detection of metamfepramone itself but the metabolites methylpseudoephedrine and methcathinone can be monitored, and the finding of the latter in particular allows the inference of a metamfepramone administration. In order to improve sports drug testing procedures, metamfepramone, methylpseudoephedrine and methcathinone were characterized using electrospray ionization-high resolution/high accuracy mass spectrometry, and a method employing liquid chromatography/tandem mass spectrometry was established that allowed the analysis of these three analytes by direct injection of 2 microL of urine specimens. The assay was validated with regard to specificity, lower limits of detection (2-10 ng mL(-1)), intraday and interday precision (3-17%) and ion suppression/enhancement effects. The developed procedure has been used to verify or falsify suspicious signals observed in routine screening procedures based on gas chromatography/mass spectrometry and yielded an adverse analytical finding concerning a metamfepramone administration in an authentic doping control sample. Although the active drug was not detected, the indicative metabolites methylpseudoephedrine and methcathinone were considered sufficient to infer the application of the prohibited drug.