Supercritical Fluid Chromatography for Expedited Sample Clean-Up in Isotope Ratio Mass Spectrometry Including Cholesterol as New Endogenous Reference Compound.

Doping control samples showing elevated urinary concentrations and concentration ratios of testosterone or testosterone metabolites are forwarded to isotope ratio mass spectrometry-based determinations in order to differentiate between naturally elevated concentrations and doping offenses. Routine sample preparation protocols commonly encompass liquid-liquid and solid-phase extraction steps and enzymatic deconjugation of steroid glucuronides. In order to separate all steroids of interest from matrix components and to obtain sufficiently clean urinary extracts, high performance liquid chromatography (HPLC) with fraction collection is a frequently employed method of choice. This preparation step is time consuming as each HPLC run requires approx. 45 min, and the evaporation of collected fractions containing water and acetonitrile necessitates up to 60 min. Supercritical fluid chromatography employs supercritical carbon dioxide as eluent combined with other organic solvents as modifiers. The unique physiochemical properties of supercritical carbon dioxide enable the acceleration of the chromatographic separation of different compounds, and collected fractions only contain the modifier and additional methanol as make-up solvent, which allows for rapid evaporation within 10 min. Therefore, a method was developed and validated in-line with current World Anti-Doping Agency-stipulated regulations encompassing testosterone, epitestosterone (E), dehydroepiandrosterone, androsterone, etiocholanolone, 5α- and 5β-androstanediol as target analytes and cholesterol, pregnanediol, 16-androstenol, and 11-oxo-etiocholanolone as endogenous reference compounds. Reference population data encompassed 40 female and 40 male routine doping control samples. Cholesterol as a new endogenous reference compound was carefully investigated. As proof-of-concept, samples derived from a testosterone-gel administration trial were investigated, demonstrating an impact of the drug intervention on E.