Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls

Metabolites of hydrocortisone (HC) and cortisone (C), namely tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-THF, allo-THE for the main metabolites and 11-hydroxyandrosterone, 11-hydoxyetiocholanolone, 11-ketoandrosterone, and 11-ketoetiocholanolone for the minor metabolites, as well as the two main metabolites of testosterone, androsterone and etiocholanolone, were separated from each other using HPLC fractionation of urine extracts. An isotopic ratio mass spectrometry (IRMS) analysis determined the absolute delta(13)C values of 5 alpha-androstanetrione (5 alpha-AT) and 5 beta-androstanetrione (5 beta-AT) as the oxidation products (ox-products) of the HC and C metabolites and as target compounds (TCs). We also performed IRMS analysis of 5 alpha-androstanedione (5 alpha-AD) and 50-androstanedione (5pAD) as the ox-products of etiocholanolone and androsterone and as endogenous reference compounds (ERCs). Urine samples came from two male volunteers treated with a single 10-mg oral dose and a single 100-mg intramuscular dose of HC hemisuccinate, a male volunteer treated with a single 25-mg oral dose of C acetate, and a control group of 30 drug-free athletes. The mean -3SD of delta(13)C depletion values from the controls were -1.46, -1.98, -1.78 and -2.42 for 5 beta-AT-5 beta-AD, 5 alpha-AT-5 beta-AD, 5 beta-AT-5 alpha-AD and 5 alpha-AT-5 alpha-AD, respectively, indicating -3 parts per thousand as a safe cut-off value for differentiating the pharmaceutical from the natural form. In the main metabolite fraction, delta(13)C depletion values peaked around -5 parts per thousand. and -9 parts per thousand. after oral and intramuscular administration of HC, respectively, and around -6 parts per thousand. after oral administration of C. In comparison, less impressive results were obtained when IRMS analysis focused on the ox-products of the minor metabolites. (C) 2008 Elsevier Inc. All rights reserved.