Bioequivalence and Analytical Validation of Empagliflozin in Human Plasma Using LC -MS/MS

This study presents the development and validation of a highly sensitive and reliable analytical method for the quantification of empagliflozin in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Employing a Zorbax SB-C۱۸ column and an optimized mobile phase, the method was developed to achieve superior separation and quantitation, adhering to regulatory guidelines outlined by EMEA and ICH. The method incorporates an internal standard, hydrocortisone, to reduce variability, ensuring high accuracy and precision. Comprehensive validation was conducted, including assessments of specificity, carry-over, linearity, accuracy, precision, matrix effects, and analyte stability. The calibration curve exhibited excellent linearity over the range of ۵–۲۴۰۰ ppb (R² > ۰.۹۹۹). The method's limit of detection was determined to be ۱.۵ ppb, while the lower limit of quantification (LLOQ) was established at ۵ ppb, ensuring its suitability for detecting low plasma concentrations. Stability evaluations, including short-term, freeze-thaw, and long-term studies, demonstrated the analyte's robustness under various storage and handling conditions, with deviation percentages within acceptable limits. The method's application was further validated through a bioequivalence study conducted on ۲۴ healthy volunteers in a two-period, crossover design. Pharmacokinetic parameters, including maximum plasma concentration (Cmax) and time to reach Cmax (Tmax), were consistent across participants, with minimal inter- and intra-subject variability. These results highlight the method’s applicability in therapeutic drug monitoring, bioavailability studies, and regulatory compliance for generic drug development.