title: Examination of the kinetic isotopic effect to the acetylation derivatization for the gas chromatographic-combustion-isotope ratio mass spectrometric doping control analysis of endogenous steroids.
abstract: In gas chromatographic-combustion-isotope ratio mass spectrometry (GC-C-IRMS) doping control analysis, endogenous androgenic anabolic steroids and their metabolites are commonly acetylated using acetic anhydride reagent, thus incorporating exogenous carbon that contributes to the measured isotope ratio. Comparison of the endogenous δ(13)C of free, mono-, and di-acetylated steroids requires application of corrections, typically through straightforward use of the mass balance equation. Variability in kinetic isotope effects (KIE) due to steroid structures could cause fractionation of endogenous steroid carbon, resulting in inaccurate results. To test for possible KIE influence on δ(13)C, acetic anhydride of graded isotope ratio within the natural abundance range was used under normal derivatization conditions to test for linearity. In all cases, plots of measured steroid acetate δ(13)C versus acetic anhydride δ(13)C were linear and slopes were not significantly different. Regression analysis of the Δδ(13)C of enriched acetic anhydrides versus Δδ(13)C of derivatized steroids shows that KIE are similar in all cases. We conclude that δ(13)C calculated from the mass balance equation is independent of the δ(13)C of the acetic anhydride reagent, and that net KIE under normal derivatization conditions do not bias the final reported steroid δ(13)C.
authors.affiliation: Doping Control Laboratory of Athens, Olympic Athletic Center of Athens, Maroussi, Greece. y_angelis@yahoo.gr
authors.forename: Yiannis S
authors.initials: YS
authors.lastname: Angelis
authors.forename: Maroula K
authors.initials: MK
authors.lastname: Kioussi
authors.forename: Polyxeni
authors.initials: P
authors.lastname: Kiousi
authors.forename: J Thomas
authors.initials: JT
authors.lastname: Brenna
authors.forename: Costas G
authors.initials: CG
authors.lastname: Georgakopoulos
mesh_terms: D000089:Acetic Anhydrides / Q000737:chemistry; D000107:Acetylation; D045930:Anabolic Agents / Q000652:urine*; D015415:Biomarkers / Q000652:urine; D002138:Calibration; D002247:Carbon Isotopes / Q000652:urine*; D004300:Doping in Sports; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D058573:Performance-Enhancing Substances / Q000652:urine*; D011237:Predictive Value of Tests; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D013256:Steroids / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods*
chemical_list: D000089:Acetic Anhydrides; D045930:Anabolic Agents; D015415:Biomarkers; D002247:Carbon Isotopes; D058573:Performance-Enhancing Substances; D013256:Steroids; C031800:acetic anhydride