Determination of D/H ratios of endogenous urinary steroids : method validation and first results

The development and application of a combined gas chromatography/thermal combustion/isotope ratio mass spectrometry (GC/TC/IRMS) method for D/H ratio determination of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive clean up with high performance liquid chromatography (HPLC) of foremost free and then acetylated steroids. So it is possible to achieve sufficient clean up of all analytes and to determine their D/H ratio. Ten steroids (11ß-hydroxy-androsterone OHA, 5α-androst-l6-en-3α-ol 16EN, pregnanediol PD, androsterone A, etiocholanolone E, leslosterone T, epitestosterone EpiT, 5α-androstan-3α, 17ß-diol 5a, 5ß-androstan-3α, 17ß-diol 5b and dehydroepiandrosterone DHEA) are measured from a single urine specimen. Depending on the biological background the determination limit for all steroids ranges from 10 to 15 ng/mL for a 20 mL specimen. The method is validated by application of linear mixing models on each steroid which covers repeatability and reproducibility. The specificity for each steroid was ensured by gas chromatography/mass spectrometry (GC/MS) determinations. Within sample preparation no isotopic fractionation was observed and no amount-depending shift for the D/H ratios during measurement was noticed. Possible memory effects occurring during IRMS measurements were corrected for by applying a simple rule of proportion. In order to determine the naturally occurring D/H ratios of all the implemented steroids a reference population of n = 18 subjects was established. By means of this it is possible to calculate preliminary reference limits for relevant ∆ values among the steroids and to test the method for its possible application for doping control purposes. Verf.-Referat