LC-MS/ MS analysis of metformin, saxagliptin and 5-hydroxy saxagliptin in human plasma and its pharmacokinetic study with a fixed- dose formulation in healthy Indian subjects

A specific and rapid liquid chromatography-tandem mass spectrometry method is proposed for the simultaneous determination of metformin (MET), saxagliptin (SAXA) and its active metabolite, 5-hydroxy saxagliptin (5-OH SAXA) in human plasma. Sample preparation was accomplished from 50 mu L plasma sample by solid-phase extraction using sodium dodecyl sulfate as an ion-pair reagent. Reversed-phase chromatographic resolution of analytes was possible within 3.5 min on ACE 5CN (150 x 4.6 mm, 5 mu m) column using acetonitrile and10.0mM ammonium formate buffer, pH 5.0 (80: 20, v/ v) as the mobile phase. Triple quadrupole mass spectrometric detection was performed using electrospray ionization in the positive ionization mode. The calibration curves showed good linearity (r(2) >= 0.9992) over the established concentration range with limit of quantification of 1.50, 0.10 and 0.20 ng/ mL for MET, SAXA and 5-OH SAXA respectively. The extraction recoveries obtained from spiked plasma samples were highly consistent for MET (75.1277.84%), SAXA (85.90-87.84%) and 5-OH SAXA (80.32-82.69%) across quality controls. The validated method was successfully applied to a bioequivalence study with a fixed-dose formulation consisting of 5mg SAXA and 500 mg MET in 18 healthy subjects. The reproducibility of the assay was demonstrated by reanalysis of 87 incurred samples.