Picogram Level Quantification of Grazoprevir and Elbasvir with Deuterated Internal Standards in Human Plasma Samples by LC–ESI-MS/MS

  A fixed oral dose combination of grazoprevir and elbasvir is used for the treatment of patients with genotypes 1&4 HCV infections. In this manuscript, authors developed a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method used for quantification of grazoprevir and elbasvir in human plasma. Agilent TC-C18, 4.6 x 75 mm, 3.5 μm, 80 Å column, and 5 mM ammonium acetate: acetonitrile (20:80 v/v) mobilephase was used for Chromatographic separation. MRM positive mode was used to detect the grazoprevir and elbasvir at m/z 767.3/553.2, 883.4/656.3 respectively. Liquid-liquid extraction was employed in the extraction of analytes and internal standard from human plasma. This method is validated over a linear concentration range of 50.0 – 10000.0 pg/ml for grazoprevir and elbasvir. Calibration functions, lower limit of quantitation (LLOQ), stability, intra and inter-day reproducibility, accuracy, and recovery are estimated. Both drugs were stable in plasma samples. This method was free from matrix effects and any abnormal ionization.