Sensitive and Rapid Determination of Gliclazide in Human Plasma by UPLC-MS/MS and its Application to a Bioequivalence Study

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of gliclazide in human plasma using gliclazide-d4 as the internal standard (IS). The plasma samples were prepared by protein precipitation with acetonitrile employing 50 µL human plasma. Chromatography was performed on Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) analytical column under isocratic conditions using a mobile phase which consisted of 0.1% formic acid in water-acetonitAn ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of gliclazide in human plasma using gliclazide-d4 as the internal standard (IS). The plasma samples were prepared by protein precipitation with acetonitrile employing 50 µL human plasma. Chromatography was performed on Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) analytical column under isocratic conditions using a mobile phase which consisted of 0.1% formic acid in water-acetonitrile (10:90, v/v). The MRM transitions for gliclazide (m/z 324.2 → 127.3) and gliclazide-d4 (m/z 328.2 → 127.4) were monitored on a triple quadrupole mass spectrometer, operating in the positive ionization mode. The method was validated over a dynamic concentration range of 1.0-2000 ng/mL for gliclazide. Matrix effect was assessed by post-column analyte infusion and the mean extraction recovery was 95.7 % across six quality control levels. Stability of gliclazide in plasma was evaluated under different conditions like bench top, auto sampler, dry and wet extract, freeze-thaw and long term stability. The method was applied to a bioequivalence study with 30 mg gliclazide tablet formulation in 28 healthy subjects under fasting. Further, the assay reproducibility was confirmed by reanalysis of 129 incurred samples.  rile (10:90, v/v). The MRM transitions for gliclazide (m/z 324.2 → 127.3) and gliclazide-d4 (m/z 328.2 → 127.4) were monitored on a triple quadrupole mass spectrometer, operating in the positive ionization mode. The method was validated over a dynamic concentration range of 1.0-2000 ng/mL for gliclazide. Matrix effect was assessed by post-column analyte infusion and the mean extraction recovery was 95.7 % across six quality control levels. Stability of gliclazide in plasma was evaluated under different conditions like bench top, auto sampler, dry and wet extract, freeze-thaw and long term stability. The method was applied to a bioequivalence study with 30 mg gliclazide tablet formulation in 28 healthy subjects under fasting. Further, the assay reproducibility was confirmed by reanalysis of 129 incurred samples.