Development and validation of an ultrasensitive LC–MS/MS method for the quantification of cetagliptin in human plasma and its application in a microdose clinical trial

This study established and validated an LC-MS/MS method for the ultrasensitive determination of cetagliptin in human plasma. Sample pretreatment was achieved by liquid-liquid extraction with ethyl acetate, and chromatographic separation was performed on an XB-C18 analytical column (50 × 2.1 mm, 5 μm) with gradient elution (0.1% formic acid in acetonitrile and 0.1% formic acid) at a flow rate of 1.0 mL/min. For mass spectrometric detection, multiple reaction monitoring was used, and the ion transitions monitored were m/z 421.2-86.0 for cetagliptin and m/z 424.2-88.0 for cetagliptin-d3. Method validation was performed according to the U.S. Food and Drug Administration Bioanalytical Method Validation Guidance, for which the calibration curve was linear in the range of 50.0-2000 pg/mL. All of the other results, such as selectivity, lower limit of quantitation, precision, accuracy, matrix effect, recovery, and stability, met the acceptance criteria. The validated method was successfully applied in a microdose clinical trial to systematically investigate the pharmacokinetic profile of cetagliptin in healthy subjects. Both rapid absorption and prolonged duration demonstrate the potential value of cetagliptin for diabetes treatment.