Degradation of urine samples and its influence on the 13C/12C ratios of excreted steroids

The degradation processes in deficiently stored urine samples are well investigated regarding steroid concentrations and diagnostic ratios, such as the quotient of testosterone divided by epitestosterone. In contrast, nothing is known about the influence on carbon isotope ratios (CIR) by inappropriate storage conditions. In general, it is assumed that degradation, i.e. deconjugation or dehydrogenation, does not change CIR and thus CIR can be used in cases where the steroid profile turns out to be invalid. Therefore, the CIR of urinary steroids was investigated in different urine samples during the course of degradation over a time period of six months. Several steroids excreted as glucuronides (androsterone (A), etiocholanolone (E), testosterone, pregnanediol (PD) and 5 alpha- and 5 beta-androstane-3 alpha,17 beta-diol) or sulfo-conjugated (A, E and androst-5-ene-3 beta,17 beta-diol (5EN17b)) were investigated together with their unconjugated correspondents (A, E, PD and 5EN17b) and the main dehydrogenation products (5a- and 5 beta-androstane-3,17-diol and androst-4-ene-3,17-diol). For this purpose, the exiting methods for CIR determination were extended and validated. In addition, the urinary concentrations of all investigated steroids were monitored. Particular attention was paid to dehydroepiandrosterone conjugated and unconjugated together with its degradation product 3 alpha,5-cyclo-5 alpha-androstan-6 beta-ol-17-one as here the strongest influence on CIR was expected. Copyright (C) 2010 John Wiley & Sons, Ltd.