Enantioselective determination of ornidazole in human plasma by liquid chromatography–tandem mass spectrometry on a Chiral-AGP column

Abstract

A rapid, sensitive, and enantioselective method was developed and validated for determination of ornidazole enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. Ornidazole enantiomers were extracted from 100μl of plasma using ethyl acetate. Baseline chiral separation (Rs=2.0) was obtained within 7.5min on a Chiral-AGP column (150mm×4.0mm, 5μm) using an isocratic mobile phase of 10mM ammonium acetate/acetic acid (100/0.01, v/v). Stable isotopically labeled R-(+)-d5-ornidazole and S-(−)-d5-ornidazole were synthesized as internal standards. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode via positive electrospray ionization, using the transitions of m/z 220→128 for ornidazole enantiomers, and m/z 225→128 for d5-ornidazole enantiomers. The method was linear in the concentration range of 0.030–10.0μg/ml for each enantiomer. The lower limit of quantification for each enantiomer was 0.030μg/ml. The relative standard deviation values of intra- and inter-day precision were 1.8–6.2% and 1.5–10.2% for R-(+)-ornidazole and S-(−)-ornidazole, respectively. The relative error values of accuracy ranged from −4.5% to 1.2% for R-(+)-ornidazole and from −5.4% to −0.8% for S-(−)-ornidazole. The validated method was successfully applied to a stereoselective pharmacokinetic study of ornidazole after oral administration of 1000mg racemic ornidazole.