Development and Validation of a Novel RP-HPLC Analytical Method for Sitagliptin Determination in Human Plasma

Abstract

Background: Different bio-analytic methods have been developed for determining drug concentration in plasma, but methods for sitagliptin determination are still very rare. In this study, RP-HPLC based method has been developed for assessing sitagliptin concentration in plasma. Aim: To develop and validate RP-HPLC based analytical method for estimating sitagliptin in human plasma for pharmacokinetic applications. Methods: In the present study, the mobile phase composed of acetonitrile: 0.5% triethanolamine (20:80) with pH 6.5 has been utilized. Samples of plasma containing sitagliptin and internal standard (IS)-rosiglitazone were extracted with dichloromethane:diethyl ether (4:6; v/v) at pH 7.4. The rate of flow was 1 ml/min. The retention time was about 5,232 and 6,903 minutes respectively for sitagliptin and rosiglitazone. Results: At concentrations of 100-3200 ng/ml in plasma, calibration curves of sitagliptin were linear. The inter- and intra-day precision and accuracy ranged in between 93.56-98.56% and 1.09-4.55% respectively. For specificity, the study findings showed no co-eluting peaks occurring with IS drug (rosiglitazone) and confirmed that no percentage of interferences at analyte (sitagliptin) retention in presence of rosiglitazone. The sitagliptin recovery was 96.442%. Chromatographic separations were performed on HI Qsil C-18 HS column (250mm x 4.6mm x 5μm). The stability of stock solutions of sitagliptin and IS at room temperature was 98.06% and 100.79% respectively while under refrigerated conditions stability was 98.19% and 96.59% respectively. Freeze-thaw stability for sitagliptin was performed with low & high QC and shown as 98.26% for and 97.45% respectively. The limits of detection and quantification were 8.592 ng/ml and 28.641 ng/ml respectively. Conclusion: A simple, sensitive and accurate method was developed for bio analytical estimation of sitagliptin in human plasma using liquid-liquid extraction technique. The validation results of linearity, accuracy, precision, stability, selectivity, ruggedness, LOD and LOQ were good under acceptable limit and can be applied for pharmacokinetic studies.