A Greenness Evaluated Bioanalytical Method for Quantification of Sparsentan in Rat Plasma and Its Application to Pharmacokinetic Studies by Lc-MS

Objective: A simple, accurate, reproducible, greenness-evaluated method for bioanalytical estimation of sparsentan in rat plasma and its application to pharmacokinetic studies by liquid chromatography mass spectrometry (LC-MS) using macitentan as internal standard. Methods: The current work sought to develop and estimate sparsentan by Chromatograph, Waters 2695 high performance liquid chromatography (HPLC) with auto sampler, column oven, and degasser, and SCIEX QTRAP 5500 mass spectrometer, and with class Empower-2 software. The Mobile Phase used was acetonitrile with trifluoroacetic acid buffer (50:50) with a run time of 5 min and a flow rate of 1 ml/min. Results: Retention times of 1.933 for sparsentan and 3.270 for macitentan were found. This method was validated over a linear range of 6.0-240.0 ng/ml with a correlation coefficient of 0.9989. The intra-batch and inter-batch precision percentage coefficient of variation (%CV) across five levels of Quality parameters is less than 15.00. Bench top stability, short-term, and long-term stability studies were performed, and % CVs were found below 15%. Pharmacokinetic studies were performed by administering to rats and reported the maximum concentration (Cmax) and time to maximum concentration (Tmax). Green analysis was also performed. Conclusion: The optimized sample preparation and chromatographic conditions ensured high sensitivity and minimal matrix interference, allowing reliable quantification of sparsentan even at low concentrations. Future research can focus on method modifications to enable simultaneous quantification of sparsentan and its metabolites.