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).