High‐volume steroid isotopic standards developed as working standards for gas chromatography–combustion–isotope ratio mass spectrometry

High‐precision carbon isotope ratio analysis of urinary steroids by gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) is the official test to detect illicit doping of synthetic versions of endogenous steroids, such as testosterone. Our group created the first steroid isotopic standards (SIS) specifically for World Anti‐Doping Agency (WADA) accredited laboratories. The standards contain mixtures of steroids as acetates or free steroids at ~400 μg each per ampoule and have been widely distributed to anti‐doping laboratories to facilitate comparability of inter‐laboratory results. Here we report on the creation and characterization of 3 new high‐volume single component SIS suitable for use as working standards. They contain ~50 times more steroid mass per ampoule than previous SIS. The new SIS, coded CU/PCC 40‐1, CU/PCC 41‐1, & CU/PCC 42‐1, contain ~20 mg of androsterone, androsterone‐AC, and 5α‐cholestane, with determined isotopic values of ‐27.09 ± 0.07 mUr, ‐32.82 ± 0.01 mUr, ‐25.03 ± 0.01 mUr, respectively. We used our previously developed protocol to calibrate the isotopically uniform steroids against the isotopic standard gases methane and ethane in NIST RM 8559 that are traceable to the international standard Vienna PeeDee Belemnite (VPDB). Two sets of data, acquired 7 months apart, of absolute δ¹³C_VPDB and ?Δδ¹³C_VPDB values from 8 randomly selected ampoules of all 3 SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ¹³C) < 0.2 mUr Our results show that protocols for SIS extend to creation of high volume working standards that can also be used as internal standards under appropriate GC conditions.