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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
Author(s) -
Angelis Yiannis S.,
Kioussi Maroula K.,
Kiousi Polyxeni,
Brenna J. Thomas,
Georgakopoulos Costas G.
Publication year - 2012
Publication title -
drug testing and analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.065
H-Index - 54
eISSN - 1942-7611
pISSN - 1942-7603
DOI - 10.1002/dta.408
Subject(s) - chemistry , acetic anhydride , derivatization , reagent , chromatography , kinetic isotope effect , isotope ratio mass spectrometry , mass spectrometry , analytical chemistry (journal) , organic chemistry , deuterium , physics , quantum mechanics , catalysis
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. Copyright © 2012 John Wiley & Sons, Ltd.