Overcoming the challenge of confident identification among two related groups of 17-methyl steroids by GC-MS

Tetrahydromethyltestosterone (THMT) and 20 hydroxymethyl 18-nortetrahydromethyltestosterone (20OHnorTHMT) are metabolites of the anabolic androgenic steroids methyltestosterone and metandienone. Both molecule structures are used as markers in anti doping analysis. There are eight reasonable diastereomeric structures of each group relevant for metabolic purposes. Despite a different chemical composition, mass spectrometry reaches its limitation in the discrimination of these two groups in its entirety. Due to the low abundance of the molecular ion, high resolution mass spectrometry provides shared fragment ions challenging identification by extracted ion chromatograms out of full scan mode acquisitions. Further on, tandem mass spectrometry uses partly the same ion transitions for both groups of targeted analytes. Thus, confident identification of signals needs chromatography as an additional dimension. Therefore, a gas chromatographic method using a DB-5ms capillary column (30 m, 0.25 mm, 0.25 µm) was developed. Hence, discrimination between the two groups was enabled and a confident structural assignment between the eight diastereomers was achieved. This case study contributes to a higher quality of anti doping analysis, but even further raises awareness of the importance of chromatographic separation in cases of insufficient mass spectrometric discrimination.