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

• The first chiral LC–MS/MS method for ornidazole quantification in human plasma.
• Separation was performed on a Chiral-AGP column operated in reverse-phase mode.
• Baseline resolution within 7.5 min leads to a huge reduction of total run time.
• Deuterium labeled ISs were used to compensate for matrix effects.
• Sensitivity was highly improved with smaller amount of plasma.

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 100 μl of plasma using ethyl acetate. Baseline chiral separation (Rs = 2.0) was obtained within 7.5 min on a Chiral-AGP column (150 mm × 4.0 mm, 5 μm) using an isocratic mobile phase of 10 mM 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 0.030–10.0 μg/ml for each enantiomer. The lower limit of quantification for each enantiomer was 0.030 μg/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 1000 mg racemic ornidazole.

Representative enantioselective MRM chromatograms for R-(+)-ornidazole (I), S-(−)-ornidazole (II), R-(+)-d5-ornidazole (IS; III), and S-(−)-d5-ornidazole (IS; IV) in human plasma. (A) Blank plasma sample. (B) Blank plasma sample spiked with R-(+)-ornidazole (0.030 μg/ml), S-(−)-ornidazole (0.030 μg/ml), R-(+)-d5-ornidazole (1.00 μg/ml), and S-(−)-d5-ornidazole (1.00 μg/ml). (C) Plasma sample at 1.5 h after oral administration of 1000 mg racemic ornidazole to a volunteer.Figure optionsDownload as PowerPoint slide