Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma

•Simultaneous quantitation of three novel HCV drugs in human plasma by LC-MS/MS.•The validation results demonstrate the method is accurate, precise, and reproducible.•Cross-validation results demonstrated method equivalency with those for single drug.•The plasma method was used for pharmacokinetic assessment in many clinical studies.•Multiplexed LC-MS/MS method is time- and cost-effective, and maintains high performance in sample analysis. Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.