Quantification of levornidazole and its metabolites in human plasma and urine by ultra-performance liquid chromatography–mass spectrometry

•We developed an UPLC–MS/MS method for simultaneous determination of levornidazole and its three metabolites M1, M2 and M4 in human plasma and urine.•The method is fully validated in terms of linearity, specificity, precision and accuracy, recovery and stability under various conditions.•The method is successfully applied in a clinical pharmacokinetic study.

We developed and validated an ultra-performance liquid chromatographic (UPLC) method coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry for simultaneous determination of levornidazole and its first-pass metabolites, l-chloro-3-(2-hydroxymethyl-5-nitro-l-imidazolyl)-2-propanol (Ml), 2-methyl-5-nitroimidazole (M2) and 3-(2-methyl-5-nitro-1-imidazolyl)-1,2-propanediol (M4), in human plasma and urine. The biological samples were pretreated by protein precipitation and liquid–liquid extraction and analyzed using an ACQUITY UPLC CSH C18 column (2.1 × 50 mm, 1.7 μm) and a QTRAP mass spectrometer in multiple reaction monitoring mode via APCI. Acetonitrile and 0.1% formic acid in water was used as the mobile phase in gradient elution at a flow rate of 0.6 mL/min. The lower limit of quantification of this method was 0.0100, 0.00500, 0.0200 and 0.00250 μg/mL for levornidazole, M1, M2 and M4, respectively. The linear calibration curves were obtained for levornidazole, M1, M2, and M4 over the range of 0.0100–5.00, 0.00500–2.50, 0.0200–10.0 and 0.00250–1.25 μg/mL, respectively. The intra- and inter-batch precision was less than 12.2% in plasma and less than 10.8% in urine. The intra- and inter-batch accuracy was 87.8–105.7% in plasma and 92.8–109.2% in urine. The mean recovery of levornidazole, M1, M2 and M4 was 91.1–105.1%, 95.8–103.8%, 87.8–96.8%, 96.8–100.6% from plasma and 96.0–100.9%, 96.9–107.9%, 95.1–102.7%, 103.7–105.9% from urine respectively. This method was validated under various conditions, including room temperature, freeze–thaw cycles, long-term storage at −40 ± 5 °C, after pretreatment in the autosampler (at 10 °C), and 10- and 100-fold dilution. This newly established analytical method was successfully applied in a pharmacokinetic study following single intravenous infusion of levornidazole in 24 healthy Chinese subjects.