Abstract
Direct-acting antivirals (DAA) have markedly improved the treatment of hepatitis C, with a series of DAA combinations available for treatment. A sensitive method by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous quantification of seven commonly used DAAs, daclatasvir (DAC), elbasvir (ELB), grazoprevir (GZR), ledipasvir (LDV), simeprevir (SIM), sofosbuvir (SOF), velpatasvir (VEL) and the primary analyte of interest of SOF (GS-331007) in EDTA plasma. Adequate chromatographic separation with well-defined peaks for all eight analytes was achieved with an Acquity UPLC BEH C18 column (1.7 μm 2.1 × 50 mm). Runtime for the final assay was 12 min. Protein precipitation in the sample preparation and stable isotope- labelled internal standards resulted in a robust sensitive and rapid method. Method validation was performed in accordance with the "guideline on bioanalytical method validation" of the European Medicines Agency (EMA). No interferences from endogenous substances were observed and carry-over in the blank sample was <20% of the LLOQ of each analyte and <5% of the IS after injection of the HLOQ sample. Validation was established over the range of 10-5000 μg/L for DAC and SIM, 3.0-1500 μg/L for ELB and GZR, 7.5-1500 μg/L for LDV and VEL, 5.0-2500 μg/L for SOF and 25-5000 μg/L for GS-331007. Within-day accuracy values for all analytes, ranged from 94% to 109%, and precision expressed as residual standard deviation % (RSD) was <9.3%. Between-day accuracy ranged from 99 to 107% with a RSD of <5.3%. For the lower limit of quantification, the percent deviation from the nominal concentration and the relative standard deviation was <20%. The method has been applied successfully in clinical evaluation of patients treated with DAA and to describe the pharmacokinetics of DAAs in clinical studies.