Important considerations for the utilisation of methanolysis in steroid analysis

The effective analysis of anabolic-androgenic steroids in urine usually requires a suitable deconjugation method for the analysis of phase II metabolites such as sulphates and glucuronides. Acid hydrolysis using methanolysis is one adopted method of deconjugation that efficiently and rapidly cleaves both sulphates and glucuronides contemporaneously. The formation of artefactual by-products is a known disadvantage of this harsh method. However, the possible promotion of deuterium-hydrogen exchange of isotopically labelled internal standards has received little attention in the literature. This report demonstrates a complete deuterium-hydrogen exchange from deuterium labelled D₉-progesterone to progesterone driven by the acidic conditions of the methanolysis. The likely mechanisms of this exchange reaction are postulated, and the results compared to other deuterated steroids. This finding highlights the importance for careful consideration when selecting labelled internal standards in a conjunction with methanolysis.