Synthesis of 17ß-hydroxymethyl-17a-methyl-18-nor-2-oxa-5a-androsta-13-en-3-one, a long-term marker for oxandrolone abuse

Code: R11A01MT

The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one to routine doping controls resulted in hundreds of adverse analytical findings for metandienone worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methylated steroids, analogues to this metabolite have been described, but comprehensive characterization and pharmacokinetic data were still missing and dersirable. In this project, the synthesis of the two epimeric oxandrolone metabolites 17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α–androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α–androsta-13-en-3-one using a fungus (Cunninghamella elegans) based protocol was conducted followed by full characterization of the obtained reference material by means of liquid chromatography-nuclear magnetic resonance spectroscopy and -high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete’s urine samples, different analytical approaches were followed, such as LC-MS/MS (QqQ and Q-Orbitrap) and GC-MS/MS to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R2 > 0.99) was tested and precise results were obtained for the detection of the analytes (CV < 20%). Limits of detection (S/N) for confirmatory and screening analysis were estimated at one and two nanogram per milliliter of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

Main findings

According to the WADA statistics, anabolic androgenic steroids are still the most frequently detected substances in sports drug testing. Nevertheless, for some anabolic steroids as oxandrolone the number of positive findings is relativly low despite of very potent anabolic activity and only weak side effects making the steroid very attractive for cheating athletes. To improve sports drug testing and to develop comprehensive analytical strategies for an effective detection of an oxandrolone abuse, two isomeric oxandrolone metabolites Ox M1 and its epimer Ox M2 were successfully synthesized and fully characterized. Within an oxandrolone post administration study, this reference material was applied to obtain data on approximate detection windows. The first time, it has been demonstrated that Ox M1 and M2 provided significantly prolonged detection periods compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone. Based on these results, a very comprehensive, sensitive and reliable methodology based on LC-(ESI)-MS/MS and GC-MS/MS for screening and confirmatory analysis of the new long term metabolites Ox M1 and M2 was developed presenting a powerful tool in the fight against doping.