External calibration in Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry measurements of endogenous androgenic anabolic steroids in sports doping control

An alternative calibration procedure for the Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry (GC–C–IRMS) measurements of the World Antidoping Agency (WADA) Accredited Laboratories is presented. To alleviate the need for externally calibrated CO2 gas for GC–C–IRMS analysis of urinary steroid metabolites, calibration using an external standard mixture solution of steroids with certified isotopic composition was investigated. The reference steroids of the calibration mixture and routine samples underwent identical instrumental processes. The calibration standards bracketed the entire range of the relevant δ13C values for the endogenous and exogenous steroids as well as their chromatographic retention times. The certified δ13C values of the reference calibrators were plotted in relation to measured m/z13CO2/12CO2 (i.e. R(45/44)) mass spectrometric signals of each calibrator. δ13C values of the sample steroids were calculated from the least squares fit through the calibration curve. The effect of the external calibration on δ13C values, using the same calibration standards and set of urine samples but different brands of GC–C–IRMS instruments, was assessed by an interlaboratory study in the WADA Accredited Laboratories of Sydney, Australia and Athens, Greece. Relative correspondence between the laboratories for determination of androsterone, etiocholanolone, 5β-androstane-3α,17β-diacetate, and pregnanediacetate means were SD(δ13C) = 0.12‰, 0.58‰, −0.34‰, and −0.40‰, respectively. These data demonstrate that accurate intralaboratory external calibration with certified steroids provided by United States Antidoping Agency (USADA) and without external CO2 calibration is feasible and directly applicable to the WADA Accredited Laboratories for the harmonization of the GC–C–IRMS measurements.