A simple liquid chromtographic method for the simultaneous estimation of antidiabetic drugs in spiked human plasma: Heteroscedasticity study

A precise and accurate liquid chromatography method was developed to simultaneously determine linagliptin and empagliflozin in spiked human plasma. The method utilized a C8 Eclipse Plus column (25cm X 5mm and 4.6μm) packed with L1 material, with a flow rate of 1mL/min. The mobile phase consisted of a mixture of acetonitrile, methanol, and 20mM potassium dihydrogen orthophosphate (pH 3.5) in a ratio of 26:19:55% (v/v). Detection was performed at 230nm, and the total run time was 15minutes. The retention time for linagliptin was 4.30 minutes, while for empagliflozin it was 10.35 minutes. The linear range for quantification was found to be 50-750 ng/mL for linagliptin and 30-960ng/mL for empagliflozin. The regression equations for linagliptin and empagliflozin were y = 181.24x+11241 and y = 393.64x+19552, respectively, with high regression coefficients (R²) of 0.9997 and 0.9995. Protein precipitation using a mixture of acetonitrile and methanol (70:30) was employed for extraction. The method demonstrated good recovery percentages ranging from 89.728±5.010 to 95.806±2.828 for linagliptin and 85.593±5.661 to 95.150±1.593 for empagliflozin. Extensive validation was conducted to assess linearity, accuracy, precision, recovery, and stability of the method.