Abstract

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 versions of normally endogenous steroids such as testosterone. However, there are currently no universally used steroid isotopic standards (SIS). We developed a protocol to prepare isotopically uniform steroids for use as a calibrant in GCC-IRMS. These SIS can be analyzed under the same conditions as used to analyze 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. CU/USADA 33-1 contains 5α-androstan-3α-o1 acetate, 5α-androstan-3α-o1-17-one acetate, 5α-androstan-3α-o1-11, 17-dione acetate and 5α-cholestane. CU/USADA 34-1 contains 5ß-androstan-3α-o1-17-one, 5α-androstan-3α-o1-17-one, and 5ß-pregnane-3α, 20α-diol. Each mixture was prepared and dispensed into a large set of ampoules using a protocol carefully designed to minimize isotopic fractionation and contamination. The ampoules were subsequently flame sealed. A natural gas reference material, which is traceable to the international standard NIST RM 8559, was used to calibrate the SIS. Eight ampoules were randomly selected from both SIS CU/USADA 33-1 and SIS CU/USADA 34-1 batches for analysis by GCC-IRMS. The absolute δ13 CVPDB and the ∆δ13 values measured for the steroids in both CU/USADA 33-1 and CU/USADA 34-1 indicate uniformity of steroid isotopic composition within measurement reproducibility among the ampoule sets prepared. Verf.-Referat