Bioanalytical Method
The levels of nemonoxacin in plasma and urine were assayed using a validated liquid chromatography-tandem mass spectrometry (ACQUITY UPLC, Waters, USA and 4000 QTRAP, AB Sciex, USA) method modified from previous report [17]. Liquid chromatographic separation was achieved on an ACE UltraCore 2.5 super C18 column (4.6 mm × 50 mm, 2.5 µm, Advanced Chromatography Technologies Ltd, UK) using the mobile phase composed of A (0.3% formic acid aqueous solution) and B (methanol) at a flow rate of 0.6 mL/min. The elution program was 60% phase A, 0–0.3 min; 60% to 30% phase A, 0.3–0.9 min; 30% phase A, 0.9–1.5 min; 30% to 60% phase A, 1.5–1.8 min; and 60% phase A, 1.8–2.0 minutes. The column temperature was 40°C. Nemonoxacin was analyzed using positive multiple reaction monitoring mode and operating in electrospray ionization (ESI) ion source. The ion pairs for quantitative analysis of nemonoxacin and its stable isotope-labeled internal standard (nemonoxacin-d3) were m/z 372.5 → 354.5 andm/z 375.5 → 357.5 at 12 eV collision energy. An aliquot of 50 μL PK sample (plasma or urine) was precipitated by 450 μL acetonitrile. After centrifugation at 3000 × g for 10 min, the supernatant was diluted with 50% methanol/0.1% formic acid aqueous solution in a ratio of 1:1 or 1:40, respectively. The intra- and inter-batch accuracy of nemonoxacin in plasma varied from 98.0% to 104.0% and 100.0% to 101.3% (coefficient of variation ≤ 8.6%). Likewise, the intra- and inter-batch accuracy of urine assay varied from 95.5% to 107.5% and 97.2% to 104.2% (coefficient of variation ≤ 6.2%). The calibration curves were linear in the range of 0.0500-20.0 µg/mL for plasma (r2 ≥ 0.995) and 2.00-1000 µg/mL for urine (r2 ≥ 0.998).