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

• Published in: Drug testing and analysis Vol. 14; no. 11-12; pp. 1864 - 1870
• Main Authors: Thieme, Detlef, Anielski, Patricia, Rzeppa, Sebastian, Wolf, Clemens A., Wolber, Gerhard, Keiler, Annekathrin M.
• Format: Journal Article
• Language: English
• Published: England 01.11.2022
• Subjects: 18‐methyl steroids; Androgens; Dietary Supplements; doping analysis; Doping in Sports; Humans; methoxydienone; molecular modelling; Nandrolone - analysis; nutritional supplements; Receptors, Androgen; Steroids - analysis; doping analysis; nutritional supplements; methoxydienone; 18-methyl steroids; molecular modelling
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.3389

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. The detection of putative 18‐methyl‐19‐norandrosterone in a forensic urine sample has triggered a follow‐up investigation. Doping substances in the possession of the suspect were examined with regard to possible precursor steroids. 18‐Methyl‐19‐nortestosterone proved to be a suitable marker for reliable detection in doping analysis.