SFC‐MS/MS for orthogonal separation of hydroxylated 17α‐methyltestosterone isomers

Abstract

AbstractBecause of their performance‐enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase‐I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC‐MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC‐MS/MS) methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC‐MS[/MS]) methods have a lower separation efficiency than GC‐MS methods. Both techniques lack selectivity for hydroxylated 17α‐methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometry method was developed to separate four hydroxy metabolites of 17α‐methyltestosterone (2α‐/2β‐/4‐/6β‐hydroxy‐17α‐methyltestosterone). This project aimed to get a more in‐depth look at the metabolization and analysis of 17α‐methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6 ng/ml at an accuracy of 85–115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β‐hydroxy‐17α‐methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β‐hydroxy‐17α‐methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.