The identification and confirmation of steroid sulfate metabolites in biological samples are essential to various fields, including anti-doping analysis and clinical sciences. Ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) is the leading method for the detection of intact steroid conjugates in biofluids, but because of the inherent complexity of biological samples and the low concentration of many targets of interest, metabolite identification based solely on mass spectrometry remains a major challenge. The confirmation of new metabolites typically depends on a comparison with synthetically derived reference materials that encompass a range of possible conjugation sites and stereochemistries. Herein, energy-resolved collision-induced dissociation (CID) is used as part of UHPLC-HRMS/MS analysis to distinguish between regio- and stereo-isomeric steroid sulfate compounds. This wholly MS-based approach was employed to guide the synthesis of reference materials to unambiguously confirm the identity of an equine steroid sulfate biomarker of testosterone propionate administration.

The concept of biomarker measurements in the form of a ratio has not been explored in detail. This is surprising considering the current and future potential for biomarkers incorporating endogenous reference compounds (ERCs) in a range of fields. A selection of these relating to clinical and forensic applications, human antidoping, equine antidoping and veterinary residues are discussed.

Equine urine analysis has evolved over time to detect thousands of urinary compounds for doping control in the horse racing industry. The longitudinal assessment of 3‐methoxytyramine to tyramine ratio (3‐MT/T) values in equine urine by GC–MS profiling was investigated to support the Racing NSW Equine Biological Passport (EBP) for detection of dopaminergic manipulation in racehorses. This involved comparison of routine urine samples to administration studies of Sinemet, a common Parkinson's disease medication containing levodopa. Using an endogenous reference compound (ERC) in a urinary ratio enabled greater confidence to provide intelligence of pharmaceutical manipulation as distinct from physiological variation. Population reference limits (PRLs) of 776 ng/ml for urinary 3‐MT and 5.3 for 3‐MT/T, together with the use of individual reference limits (IRLs), are proposed.

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