Determination of 13C/12C ratios of urinary excreted boldenone and its main metabolite 5β‐androst‐1‐en‐17β‐ol‐3‐one

Boldenone (androsta–1,4–dien–17β–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 5β‐androst‐1‐en‐17β‐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 ¹³C/¹²C ratio of either Bo or BM1 and to distinguish whether their source is endogenous or exogenous. Therefore a method was developed to determine the ¹³C/¹²C 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 ¹³C/¹²C 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. Copyright © 2010 John Wiley & Sons, Ltd.