The present application wish to seem at the event of validation of bio analytical method and pharmacokinetic study of selexipag and its related impurities in rat plasma using LC–MS/MS. The optimized method contains gradient elution of selexipag with a flow rate of 1 ml/min and X-Bridge phenyl column (150 × 4.6 mm, 3.5 µ). A buffer of 1 mL formic acid in l liter water and acetonitrile mixture is used as mobile phase. 30 min run time was used for separation of selexipag and its related impurities with Ambrisentan as internal standard and impurity-D as active metabolite. The linearity curves are linear in between the percentages of 10 to 200% of rat plasma and R2 value of each analyte was observed as 0.999. This application denotes all the parameters like precision, accuracy, recovery and stability were got the results within the limit of USFDA guidelines. This method applies effectively for the investigation of pharmacokinetic studies using rat plasma.

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A new RP-HPLC method with a quick, sensitive and stable indication for the quantitative measurement of selexipag and its associated substances was developed and validated in the present study. In this new method, using the impurity-spiked solution, the chromatographic approach was optimized. Similarly, using the X-bridge phenyl column with isocratic elution of mobile phase containing acetonitrile and formic acid, selexipag and its impurities were separated. Recovery experiments obtained were satisfactory, and also the calibration graphs plotted for selexipag and its five impurities were found to be linear. The system validation parameters such as specificity, linearity, precision, accuracy and robustness were determined successfully. The obtained results indicated that the developed method was found to be useful for analyzing selexipag from its impurities. Further, using stress tests against acid, alkali, peroxide, reduction, thermal, hydrolysis and UV conditions, the present established method of HPLC was assessed and validated as per ICH Q2(R1) guidelines.

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