title: External calibration in gas chromatography-combustion-isotope ratio mass spectrometry measurements of endogenous androgenic anabolic steroids in sports doping control.
abstract: 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/z ¹³CO₂/¹²CO₂ (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.
authors.affiliation: Doping Control Laboratory of Athens, Olympic Athletic Centre of Athens (OAKA), Kifissias 37, 15123 Maroussi, Greece.
authors.forename: Maroula K
authors.initials: MK
authors.lastname: Kioussi
authors.forename: Yiannis S
authors.initials: YS
authors.lastname: Angelis
authors.forename: Adam T
authors.initials: AT
authors.lastname: Cawley
authors.forename: Michalis
authors.initials: M
authors.lastname: Koupparis
authors.forename: Rymantas
authors.initials: R
authors.lastname: Kazlauskas
authors.forename: J Thomas
authors.initials: JT
authors.lastname: Brenna
authors.forename: Costas G
authors.initials: CG
authors.lastname: Georgakopoulos
mesh_terms: D045930:Anabolic Agents / Q000652:urine*; D002138:Calibration; D002247:Carbon Isotopes / Q000032:analysis; D004300:Doping in Sports; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods* / Q000592:standards; D006801:Humans; D008297:Male; D013256:Steroids / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods* / Q000592:standards
chemical_list: D045930:Anabolic Agents; D002247:Carbon Isotopes; D013256:Steroids