Bioanalytical LC-MS/MS method for Determination and comparison of Selexipag Assay in various Biological materials and its Application to Pharmacokinetics Studies in Rat plasma

Abstract

A rapid, sensitive and selective bioanalytical method was developed and validated by Liquid Chromatography - Mass spectrometry (LC-MS/MS) for determination and comparison of Selexipag% assay in various biological materials. Selexipag wasextractedand compared its % assay after protein precipitation technique from various biological materials such as rat plasma, rabbit plasma, human plasma and urine. Ambrisentan was selected as internal standard. Selected analytical column Waters, X-Bridge C18 3.5µ (50 x 4.6 mm), mobile phase consists of Hexane sulfonic acid and Acetonitrile (80:20 v/v) at a flow rate of 1.0 mL /minin isocratic modeand Selexipag was determined by the +ve mode of electrospray ionization by using Mass detector. The method was developed to assess the lowerlimit of detection (LLOD)(0.5 ng/mL), lower limit of quantification(LLOQ) (5 ng/mL) concentrations and Linearity range of 1 ng/mL to 20 ng/mLconcentration with regression correlation coefficient 0.999 were observed for Selexipag in Rat plasma. The test samples at lower, medium and higher concentrationsof Selexipag shows precision (% CV was 0.8 to 1.11) and accuracy results (97.3 % to 100.6%) for inter-day and intra-day analysis at0.5, 5, 10, 15 ng/mL concentrationsof Selexipag. Appreciable recoveries for Selexipag were observed when extracted in Rat plasma compared with other biological materials. Stability of Selexipag exists in all conditions like wet extract, bench top, freeze-thaw and in instrument auto sampler as per FDA guidelines. This method indicates good results of accuracy, precision, linearity, recovery, stability and pharmacokinetic studies in rat plasma for clinical trials.