Determination of pioglitazone, its metabolite and alogliptin in human plasma by a novel LC-MS/MS method; application to a pharmacokinetic study.

A novel, high throughput and sensitive LC-MS/MS assay method was developed and fully validated for quantitative determination of pioglitazone, its hydroxyl metabolite and alogliptin in human plasma. A simple and rapid sample preparation procedure based on protein precipitation technique with acetonitrile was utilized. Chromatographic separation was achieved on C (50 × 4.6 mm, 5 μm) Kinetex® analytical column using methanol and 0.1% formic acid in a gradient elution mode at a flow rate of 0.7 mL/min with injection volume of 8 μL. Detection was performed on a triple quadrupole mass spectrometer accompanied with electrospray ionization (ESI) technique in positive mode, operating in multiple reaction monitoring, with the transitions of 357.2 → 119.1, 373.1 → 150.1, 340.3 → 116.1, 361.1 → 138.1 and 343.2 → 116.1 m/z for pioglitazone, its hydroxyl metabolite, alogliptin, pioglitazone-d4 (IS-1) and alogliptin-d3 (IS-2), in order. Analysis was achieved within 4 min over a linear concentration range of 10-3000 ng/mL, 5-2000 ng/mL and 3-300 ng/mL, for pioglitazone, hydroxyl pioglitazone and alogliptin, in order. The method was fully validated according to FDA guidelines. The developed method was used for estimation of the three studied analytes in human plasma and pharmacokinetic parameters were demonstrated after oral dose administration of Oseni® tablets to Egyptian healthy volunteers.