Validation of a novel UPLC-MS/MS method for estimation of metformin and empagliflozin simultaneously in human plasma using freezing lipid precipitation approach and its application to pharmacokinetic study
Abstract
A fast, sensitive one step UPLC ESI-MS/MS method was successfully applied for the simultaneous estimation of two concurrently administrated antidiabetic drugs, Metformin (MET) and Empagliflozin (EMPA) in human plasma. Metformin-d6 (MET-d6) and Empagliflozin-d4 (EMPA-d4) were utilized as internal standards. Extraction of the analytes from the human plasma was performed through acetonitrile precipitation technique followed by freezing the precipitated plasma proteins and lipids to minimize the matrix effect. Chromatographic analysis was developed on Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm) using isocratic elution mode. A mobile phase of formic acid (0.01 %): acetonitrile (70:30 v/v) with a flow rate of 0.3 mL/min achieved optimum separation. Multiple reaction monitoring (MRM) in positive ion mode, with transitions at (m/z) 130.14 →71.08 for (MET), 451.72 →71.29 for (EMPA), 136.03 →77.02 for (MET-d6), and 455.43 → 75.05 for (EMPA-d4) was used for quantification. The obtained linearity covered the concentration ranges of 10-1500 ng/mL and 2.0-250.0 ng/mL for MET and EMPA, respectively. The run time of the proposed Method didn't exceed 3.0 min allowing faster analysis and determination of larger number of samples per day without affecting accuracy and sensitivity. The presented chromatographic method could be successfully applied in pharmacokinetics studies and therapeutic monitoring of MET and EMPA in patients' plasma administrating fixed dose combination of both drug…
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