Simple and rapid LC-MS/MS method for determination of sitagliptin in human plasma and application to bioequivalence study

Abstract A simple, rapid, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine sitagliptin in human plasma. Diphenhydramine HCl was used as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 – Fast LC column (100 × 2.1mmID, 2.7) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1mmID, 2.7 µm). The mobile phase consisted of 0.1% v/v formic acid and methanol (45:55, v/v) run at a flow rate of 0.45 mL/min at 30 °C. Methanol produced relatively cleaner plasma sample as deproteinization agent. Polytetrafluoroethylene membrane was preferred over nylon membrane as the former produced clear plasma samples. The standard calibration curve was linear over the concentration range of 5–500.03 ng/mL. The within-run precision was 0.53–7.12% and accuracy 87.09–105.05%. The between-run precision was 4.74–11.68% and accuracy 95.02–97.36%. The extended run precision was 3.60–6.88% and accuracy 93.18–95.82%. The recovery of analyte and IS was consistent. Sitagliptin in plasma was stable at benchtop (short term) for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h (post-preparative), after 7 freeze-thaw cycles (−20 ± 10 °C), and 62 days in the freezer (−20 ± 10 °C). Both sitagliptin (analyte) and IS stock solutions were stable for 62 days when kept at room temperature (25 ± 4 °C) and in chiller (2–8 °C). The validated method was successfully applied to a bioequivalence study of two sitagliptin formulations involving 26 healthy Malaysian volunteers.