ANALYTICAL AND BIOANALYTICAL UHPLC-MS METHOD VALIDATION FOR DETERMINATION OF METFORMIN, A BIGUANIDE AND SITAGLIPTIN, A DPP-4 INHIBITOR

A very sensitive, rapid and simple Ultra High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) method has been developed and validated for the simultaneous estimation of Metformin Hydrochloride and Sitagliptin Phosphate in standard and tablet formulation. The chromatographic separation of the drugs was achieved on a Hypersil Gold C18 analytical column (150x3mm, 5µ) with an isocratic elution of methanol and water with 0.2% formic acid (48:52 v/v) at a constant flow rate of 500µL/min on a UHPLC and the retention time of Metformin Hydrochloride and Sitagliptin Phosphate were 1.40±0.01 and 2.38±0.01 min respectively. Elutes were detected online on an Orbitrap mass spectrometer in ESI (+) mode. The method was validated according to ICH guidelines. The calibration plot was linear with correlation coefficient (r²) of 0.9998 for Metformin Hydrochloride in the concentration range of 0.04-0.28ng/mL and 0.9992 for Sitagliptin Phosphate in the concentration range 0.04-0.28 ng/mL respectively. The LLOD of Metformin Hydrochloride and Sitagliptin Phosphate were 0.2 and 0.02pg/mL respectively. This method was validated with respect to linearity, accuracy, precision, specificity and robustness. The developed method was successfully applied and validated for the bioanalytical study in rat plasma according to US-FDA guidelines.