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.