A Novel LC-ESI-MS/MS Based Bioanalytical Method for Quantification of Empagliflozin in Human Plasma: Development and Validation

A rapid, sensitive, and reproducible liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the quantitative determination of empagliflozin in human plasma. Tolbutamide was used as the internal standard (IS). Plasma proteins were precipitated using acetonitrile, followed by direct analysis of the supernatant. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (50 x 3 mm, 5 ^m) with a gradient elution of 0.2% formic acid and 10 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid in acetonitrile (mobile phase B) at a flow rate of 0.5 mL/min. Detection was performed in negative ion mode using multiple reaction monitoring (MRM) transitions of m/z 449.3 ^ 371.1 for empagliflozin and m/z 269.0 ^ 170.0 for tolbutamide. The calibration curve was linear over the concentration range of 7.81-500 ng/mL (r2 > 0.996), with a lower limit of quantification (LLOQ) of 7.81 ng/mL. Mean extraction recovery ranged from 94.6 % to 96.5 %, and intra- and inter-day precision (%CV) were within 6 %. Stability studies under various conditions confirmed analyte stability for at least 50 days at -20 °C. The method demonstrated negligible matrix effects (<5 %) and fulfilled all USFDA and ICH M10 acceptance criteria. Owing to its short analysis time (7 min), high sensitivity, and minimal sample preparation, this validated method is suitable for pharmacokinetic, bioavailability, and bioequivalence studies of empagliflozin in human plasma.