Development and Validation of a Method for Simultaneous Estimation of Sitagliptin and Ertugliflozin in Rat Plasma by LC-MS method

Abstract

Background: The development of sound bioanalytical LC-MS (liquid chromatography-mass spectroscopy) method(s) is of paramount importance during the process of drug discovery, development and culminating in a marketing approval. The use of oral antidiabetic agents has been increased significantly from the last decades and till now no bioanalytical method is available for quantitation of sitagliptin (SG) and ertugliflozin (EG) in biological matrix which can be applied to pharmacokinetic studies using LC-MS/MS. Objective:To develop a new, rapid and sensitive LC–MS/MS method for the simultaneous estimation of sitagliptin (SG) and ertugliflozin (EG) in rat plasma by liquid–liquid extraction method (LLE) using deutereated sitagliptin (SGd6) and ertugliflozin (EGd6). Methods: Chromatographic separation was carried out on a reverse phase Waters, Xetrra C18 (150mm x 4.6mm, 2μm) column using a mixture of acetonitrile and OPA buffer (50:50v/v) at a flow rate of 1ml/min in isocratic mode. Quantification was achieved using an electrospray ion interface operating in positive mode, under Multiple Reaction Monitoring (MRM) conditions. Results:The method showed excellent linearity over the concentration range of 5.00- 75.00pg/mL for sitagliptin and 0.75- 11.35pg/mL ertugliflozin. The intra-batch and inter batch precision (%CV) was ≤ 4.3% and matrix effect (%CV) was 0.02% and 0.12% for sitagliptin at HQC and LQC, respectively. Matrix effect (%CV) was 0.08% and 0.33% for ertugliflozin at HQC and LQC, respectively. Conclusion: The simplicity of the method allows for application in laboratories, presents a valuable tool for pharmacokinetic studies. The particular assay has been proficiently put on pharmacokinetic study in rats subjects.