Synthesis of stable isotope labelled steroid bis(sulfate) conjugates and their behaviour in collision induced dissociation experiments

Steroid bis(sulfate) metabolites derived from the two-fold sulfation of unconjugated precursors represent an important yet understudied portion of the steroid profile. The investigation of these compounds in fields such as medicine or anti-doping science relies on mass spectrometry (MS) as the principal tool to identify and quantify biomarkers of interest and depends in turn on access to steroid reference materials and their stable isotope labelled (SIL) derivatives. A new [18O] stable isotope label for sulfate metabolites is reported, which allows for the selective, late-stage and ‘one-pot’ synthesis of a variety of SIL–steroid conjugates suitable as MS probes and internal standards. The method is applied to more comprehensively study the MS behaviour of steroid bis(sulfate) compounds through collision-induced dissociation (CID) experiments.