Detection of 18‐methyl steroids: Case report on a forensic urine sample and corresponding dietary supplements

Abstract

The detection of a putative 18‐methyl‐19‐nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow‐up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC–MSMS and LC‐HRMSMS. As neither 18‐methyl‐testosterone, nor 18‐methyl‐19‐nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18‐methyl‐19‐nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18‐methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18‐methyl‐19‐nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound‐specific intermediates were identified that allowed differentiation from other 18‐methyl steroids, the 18‐methyl‐19‐nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

Annual banned‐substance review 16th edition—Analytical approaches in human sports drug testing 2022/2023

“…The fact that, eventually, confirmation of in vitro metabolism study data with authentic elimination study specimens is vital for routine doping controls was demonstrated in a case report by Thieme et al 31 In the context of a forensic investigation, the biotransformation of the AAS methoxydienone (Figure 1, 2 ) was studied employing the aforementioned HepG2‐based in vitro metabolism approach as well as urine samples collected after oral administration to a human volunteer, and the urinary metabolite observed by means of routine doping control analytical procedures, presumably 18‐methylnorandrosterone, was not generated in vitro but abundantly observed in post‐administration urine specimens.…”

The 40th Manfred Donike workshop on doping analysis

“…Improvements regarding test methods and enhanced knowledge concerning metabolic and degradation processes concerning the class of anabolic‐androgenic steroids (AAS) were presented by various examples, for example, by the first report on findings attributed to “methoxydienone” (13β‐ethyl‐3‐methoxygona‐2,5(10)‐dien‐17‐one) and its biotransformation product in human urine 6 . The fact that, besides relevant human metabolic reactions, also microbial degradation of urinary steroids necessitates monitoring and consideration was addressed by the suggestion of employing a degradation marker compatible with routine AAS screening protocols.…”