Simple and rapid LC–MS/MS method for simultaneous determination of flavoxate and 3-methyl-flavone-8-carboxylic acid in human plasma: Application to a bioequivalence study

Abstract

A simple, rapid, sensitive, and reproducible LC–MS/MS method was developed for simultaneous quantification of flavoxate and 3-methyl-flavone-8-carboxylic (MFCA) in human plasma, using diphenhydramine HCl as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 – Fast LC column (100 × 2.1mmID, 2.7 μm) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1 mmID, 2.7 μm). The mobile phase consisted of 0.1 % v/v formic acid and acetonitrile (30:70, v/v) run at a flow rate of 0.40 mL/min. The standard calibration curve was linear over the concentration range of 2.00 – 2,000.31 ng/mL and 240.00 – 24,000.04 ng/mL for flavoxate and MFCA. For flavoxate and MFCA, the within-run precision was 0.81–6.67 % and 1.68–4.37 %, while accuracy was 100.21–108.25 % and 103.99–110.28 %. The between-run precision was 2.01–9.14 % and 2.31–11.11 %, and accuracy was 96.09–103.33 % and 102.37–109.52 %. The extended run precision was 7.78–11.04 % and 2.22–3.33 %, while accuracy was 100.72–101.88 % and 102.34–105.60 %. Flavoxate and MFCA in plasma were stable 4 h at bench top (short term), 24 h in autosampler and instrumentation room (post-preparative), after 7 freeze-thaw cycles, and 89 days in the freezer. Both analytes and IS stock solutions were stable for 31 days when kept at room temperature (25 ± 4 °C) and refrigerated (2−8 °C). The validated method was successfully applied to a bioequivalence study of two flavoxate formulations involving 24 healthy volunteers.