A Rabit Method for Quantification of Selexipag in Human Plasma Using High Performance Liquid Chromatography with Electron Spray Ionzationtendem Mass Spectrometry

Abstract

A simple, accurate and rabit method was developed using high performance liquid chromatography with electron spray ionzationtendem mass spectrometry (HPLC-ESI-MS) to quantify the concentration of selexipag in human plasma with K₂EDTA anticoagulant was developed and fully validated. Stable isotobically labelled compound selexipag D7 was used as an internal standard (ISTD). The sample extraction procedure utilized protein precipitation method. The chromatographic analysis was conducted on a Zorbax C18 XDB column (100x 4.6mm,i.d 3.5µm) within 5 min, using methanol with 5mM ammonium acetate (75:25%, v/v) was used as mobile phase at the flow rate of 0.7mL/min under an isocratic condition. The ionization was performed on electron spray ionization interference with positive mode by multiple reaction monitoring(MRM). The mass transions were 497.100→455.200 m/z for selexipag and 504.300→456.200 m/z for ISTD. Method validated as per USFDA guidelines and calibration curve was found to be linear in the range of 0.100-50.869 ng/mL.The results were within the acceptance limits. The extraction efficiency was 93.45% at the three quality control levels. The lower limit of detection (LLOQ) was found to be 0.104ng/mL.Stability studies demonstrated that selexipag was stable in plasma during Bench-Top (7hr at room temperatore), Auto-sampler (26hr 30 min at 4^oC), Freeze-Thaw (5cycles ) and Long term analyte stability in plasma (41days at -20^oC).