Development of a rapid resolution HPLC method for the quantitative determination of sitagliptin in Human plasma.

A new high performance liquid chromatography (HPLC) method for the quantitative determination of sitagliptin in human plasma was developed and validated for pharmacokinetics study. The plasma was spiked with the internal standard (Salbutamol, IS), extracted with trichloro acetic acid. The extracted analyte was injected into a Symmetry® ODS C18 column (250mm×4.5mm, 5m) and the flourometric detector was operated at 267nm for excitation and 575nm for emission. The mobile phase consisting of Potassium dihydrogen phosphate buffer pH (4.9)-Acetonitrile-Methanol (30:50:20 v/v) at flow rate of 1.0mL/min. The method showed high specificity. Calibration curves of the peak area ratio of each analyte/IS versus sitagliptin concentration were linear in the range of 0.122-31.25μg/mL (r>0.989) for plasma and 0.012-25ug/ml for QC solution(r>0.995). The lower limit of quantification (LLOQ) was 0.122μg/mL in plasma and 0.012 in QC solution. The intraday and interday coefficient of variation was lower than 10%. The accuracy (relative recovery) at three levels was 100.95%, 101.03% and 97.79% respectively. The extraction recovery was 97.6%, 92.2% and 91.96% at the concentrations of 6.25, 25 and 100μg/mL, respectively. Short term and long term, freeze thaw stability of standard solutions and plasma samples were satisfactory. The optimized HPLC method was validated and proved to be specific, robust and accurate for determination of Sitagliptin in human plasma.