Steroid isotopic standards for gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS)

Carbon isotope ratio (CIR) analysis of urinary steroids using gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit doping with synthetic testosterone. There are currently no universally used steroid isotopic standards (SIS). We adapted a protocol to prepare isotopically uniform steroids for use as a calibrant in GCC-IRMS that can be analyzed under the same conditions as used for steroids extracted from urine. Two separate SIS containing a mixture of steroids were created and coded CU/USADA 33-1 and CU/USADA 34-1, containing acetates and native steroids, respectively. CU/USADA 33-1 contains 5 alpha-androstan-3 beta-ol acetate (5 alpha-A-AC), 5 alpha-androstan-3 alpha-ol-17-one acetate (androsterone acetate, A-AC), 5 beta-androstan-3 alpha-ol-11, 17-dione acetate (11-ketoetiocholanolone acetate, 11k-AC) and 5 alpha-cholestane (Cne). CU/USADA 34-1 contains 5 beta-androstan-3 alpha-ol-17-one (etiocholanolone, E), 5 alpha-androstan-3 alpha-ol-17-one (androsterone, A), and 5 beta-pregnane-3 alpha, 20 alpha-diol (5 beta P). Each mixture was prepared and dispensed into a set of about 100 ampoules using a protocol carefully designed to minimize isotopic fractionation and contamination. A natural gas reference material, NIST RM 8559, traceable to the international standard Vienna PeeDee Belemnite (VPDB) was used to calibrate the SIS. Absolute delta(13) C-VPDB and Delta delta C-13(VPDB) values from randomly selected ampoules from both SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(delta C-13) < 0.2%. This procedure for creation of isotopic steroid mixtures results in consistent standards with isotope ratios traceable to the relevant international reference material. (C) 2008 Elsevier Inc. All rights reserved.