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

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.