LC-MS/MS method for quantification of avacopan in human plasma from patients treated for antineutrophil cytoplasmic antibody-associated vasculitis

Abstract

Avacopan is a new treatment for antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-AAV). To date, there has been a lack of published bioanalytical methods to assay it in plasma, resulting in sparse pharmacokinetic data in clinical settings. The objective of this study was to develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of avacopan in human plasma. [²H₄]-Avacopan was as used as internal standard (IS). Samples were prepared by protein precipitation and separated was on an Accurore® C18 column (2.1 ×50 mm, 2.6 µm), with an elution gradient at a flow rate of 0.5 mL/min. The mobile phase consisted of acetonitrile (0.1 % formic acid) and water (0.1 % formic acid). The analysis run time was 6 min. Avacopan was detected by electrospray ionization on a TSQ Quantis® triple quadrupole mass spectrometer (ThermoFisher Scientific). The linearity of method ranged from 10 to 800 ng/mL. The within-run and between-run relative standard deviations were < 10.2 %. The within-run and between-run relative errors ranged from 2.4 % to 14.4 %. The IS-normalized matrix effect ranged from 2.2 % to 5.1 %, and the IS-normalized extraction recovery ranged from 104.3 % to 109.7 %. The method was fully validated, including checks on linearity, dilution integrity and carry-over. Plasma samples from 16 patients undergoing treatment for ANCA-AAV were then successfully treated with the method. The reanalysis of the samples incurred was below 14 %. This method is suitable for plasma drug monitoring of avacopan because it is both accurate and precise, and meets all validation criteria.