B-172 Development and Validation of an LC-MS/MS Assay for Quantification of Glecapravir/Pibrentasvir in Human Plasma

Abstract

Abstract Background The global prevalence of chronic hepatitis C virus (HCV) infection is 58 million. Untreated chronic HCV infections can lead to serious health complications, including liver cirrhosis, damage, and disease. Direct acting antiviral (DAA) therapies for HCV infections are effective in the treatment and management of chronic infections. Glecapravir (GLE) and pibrentasvir (PIB) are DAAs that may be delivered alone or as a fixed-dose oral formulation to treat adolescent and adult patients with chronic HCV infections (genotypes 1–6) with or without cirrhosis. Here, we describe a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assay for the multiplexed quantification of GLE and PIB in human plasma, with future applications in clinical trials to evaluate the pharmacokinetics of intramuscularly delivered GLE/PIB. Methods Calibrators were prepared in human K2EDTA plasma at final concentrations ranging from 0.25 to 2000 ng/mL GLE and 0.25 to 1000 ng/mL PIB. Samples at the lower limit of quantification (LLOQ), low, mid, high, and dilution quality control (QC) levels were prepared at 0.25, 0.75, 800, 1800, and 20 000 ng/mL for GLE and 0.25, 0.75, 400, 800 ng/mL, and 10 000 ng/mL for PIB, respectively.Plasma samples containing GLE and PIB were combined with isotopically labeled internal standards and subjected to protein precipitation. Samples were evaporated to dryness, reconstituted in 50:50 mobile phase A (MPA, 0.1% formic acid in water): mobile phase B (MPB, 0.1% formic acid in 80:20 acetonitrile:water) and subjected to LC-MS/MS analysis.Chromatographic separation and analyte quantification occurred on an API 6500+ (Sciex, Foster City, CA) interfaced with an LC-30 (Shimadzu, Kyoto, Japan) operated in positive ionization and selective reaction monitoring modes. Chromatographic separation occurred using a Zorbax Eclipse C18 column under a gradient elution from MPA to MPB. Transitions monitored for analytes of interest were as follows: GLE (839.4 à 684.3 m/z), PIB (557.5 à 871.4 m/z), and their internal standards (GLE -IS 843.5 à 684.3 m/z; PIB-IS 561.5 à 629.3 m/z).The LC-MS/MS assay was validated in accordance with Food and Drug Administration Bioanalytical Method Validation Guidance for Industry recommendations. Results Inter-assay precision and accuracy ranged from 4.34% to 15.6% and −4.90% to 10.6% for GLE and 3.20% to 9.80% and −7.48% to −1.46% for PIB, respectively. GLE and PIB in plasma are stable following six freeze thaw cycles at <−70 °C, with a percent difference (%DIF) ≤±10.8% for GLE and ≤±12.4 for PIB. Additionally, GLE and PIB are stable at room temperature for up to 67 h and demonstrated re-injection stability at 4 °C for up to 101 h (GLE: %DIF ≤±4.1%; PIB %DIF: ≤±7.8%). Matrix effects were assessed quantitatively; negligible percent matrix effects were observed for PIB (106%) and PIB-IS (111%) across the measuring range of the assay. Significant ion suppression was observed for GLE (59.9%) and GLE-IS (63.9%); however, relative matrix effects were <5% between drug and internal standard and deemed acceptable. Other assay validation assessments in alternative matrices were also deemed acceptable. Conclusion An LC-MS/MS method for GLE and PIB quantification in plasma has been developed and validated and may be used in downstream applications to characterize DAA pharmacology for HCV treatment.