A Simple, Sensitive, and Stable LC–MS/MS Method for the Simultaneous Determination and Pharmacokinetic Study of Dapagliflozin and Its Metabolite D3OG in Human Plasma

Abstract

Dapagliflozin is a widely used sodium-glucose cotransporter 2 inhibitor that has been approved for the treatment of Type 2 diabetes. In this study, we present a simple and sensitive high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method for the simultaneous quantification of dapagliflozin and its metabolite, dapagliflozin 3-O-glucuronide (D3OG). The experiments were conducted using an Agilent G6495B triple quadrupole mass spectrometer coupled with an Agilent 1290 Infinity II HPLC system, featuring a Poroshell 120 EC-C18 column. Gradient elution was performed with ammonium formate (10 mM) and methanol as the mobile phase. The G6495B was operated in negative ion mode with electrospray ionization and multiple reaction monitoring. The quantitative method was validated according to FDA and EMA guidelines, assessing parameters such as selectivity, linearity, accuracy, precision, dilution integrity, stability, and recovery. Methanol was used as a protein precipitant during sample preparation, resulting in consistent extraction recoveries ranging from 91% to 96% for all analytes. The linear range for the analytes was established at 2–800 μg/L with a sample volume of 100 μL. This validated method is sufficient for the simultaneous quantification of dapagliflozin and D3OG in plasma and has been successfully applied in pharmacokinetic studies, bioequivalence assessments, and clinical therapeutic monitoring.

Trial Registration: Chinese Clinical Trial identifier: ChiCTR2100044600