Fluorimetric derivatization-based HPLC-FL method for the prototype pharmacokinetic analysis of selexipag in human plasma

A simple and cost-effective HPLC-FL method has been developed for measuring selexipag in human plasma, showcasing its suitability for pharmacokinetic research. Selexipag was precolumn derivatized with 7-chloro-4-nitrobenzofurazan (NBD-Cl) and the fluorescent derivative was separated on a C18 (150 mm × 4.6 mm × 2.6 μm) analytical column at 30 ºC using a mobile phase composed of acetonitrile – 0.1% o-phosphoric acid in water (70:30, v/v) by isocratic elution with flow rate of 1.0 mL min-1. The method was based on measuring the derivative using fluorescence detection (λex = 380 nm, λem = 420 nm). The retention time of selexipag is 6.40 ± 0.01 min. This currently developed method was validated according to EMA criteria by evaluating the specificity, linearity, precision, accuracy, and robustness. The method was determined to be linear in a concentration range of 0.01-20 ng mL-1 with a correlation coefficient of 0.9998. LOD and LOQ were found to be 0.003 and 0.01 ng mL-1, respectively. Intraday and interday RSD values were less than 1.75%. The plasma concentration-time profile and pharmacokinetic parameters such as AUC0–t, AUC0–∞, Cmax, tmax, t1/2, were calculated according to the assays. The presented method can be effectively used for bioequivalence and bioavailability investigations, as well as for routine analysis of the drug in plasma.