Determination of (13)C/(12)C ratios of endogenous urinary steroids excreted as sulpho conjugates

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry-based method for the measurement of stable carbon isotopes of endogenous urinary steroids excreted as sulphates is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography of underivatized and acetylated steroids, which allows the isolation of seven analytes (pregn-5-ene-3β,17α,20α-triol, etiocholanolone, androsterone, epiandrosterone, dehydroepiandrosterone (DHEA), androst-5-ene-3β,17β-diol and androst-5-ene-3β,17α-diol) from a single urine specimen. These steroids are of particular importance to doping controls as they should enable the sensitive and retrospective detection of DHEA abuse by athletes.Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers the items, repeatability and reproducibility. The specificity was further demonstrated by gas chromatography/mass spectrometry for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios and urinary concentrations of all implemented steroids, a reference population of n = 67 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values.The applicability of the developed method was tested by means of a DHEA excretion study. Despite the fact that orally ingested DHEA is preferentially converted into sulphated metabolites and that the renal clearance of sulphated steroids is slow, only the (13)C/(12)C ratio of EpiA demonstrated the potential to prolong the detection time for DHEA misuse.