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 molecular structures are used as markers in anti-doping analysis. There are eight reasonable diastereomeric structures of each group relevant for metabolic purposes. Highly sophisticated mass spectrometers fail to confidently differentiate these closely related, yet non-isomeric and non-isobaric groups of molecules. Due to the low abundance of the molecular ion, high-resolution mass spectrometry provides shared fragment ions that challenge 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, a reliable chromatographic separation is absolutely necessary. Therefore, a gas chromatographic method using a DB-5 ms capillary column (30 m, 0.25 mm, and 0.25 µm) was developed. Hence, discrimination between the two groups was enabled, and a confident structural assignment among 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.