Abstract
LC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS QUANTIFICATION OF ELBASVIR AND GRAZOPREVIR IN HUMAN PLASMA

Dr. T. Shyam, K.N.Rajinikanth, Dr. V. Kiran Kumar, Dr. VVSS. Appala Raju, Dr. Macharla Venkata Ramana and Dr. N. Appala Raju*

ABSTRACT

A simple, sensitive and fast throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous estimation of Elbasvir and Grazoprevir, in human plasma, using respective deuteriated drug as internal standards. The method involved Solid Phase Extraction (SPE) of the analytes and internal standards from human plasma. The chromatographic separation was achieved on a Gemini, C18, (50×4.6mm and 5μm particle size) analytical column using isocratic mobile phase, consisting of 10mM Ammonium Acetate, Acetonitrile and Methanol (30:56:14 v/v), at a flow-rate of 1.0 mL/min with 75% flow splitting. The parent product ion transitions m/z 882.51 → 656.42 and m/z 888.49 → 662.43 for Elbasvir and Elbasvir-D6 respectively and the grazoprevir was detected at m/z 767.3/553.2 and Grazoprevir-D6 at m/z 773.3/559.2. The analytes and internal standards were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) positive ion modes. The method was validated over the concentration range of 2.00-600 ng/mL and 0.5-150 μg/mL for Elbasvir and Grazoprevir respectively. The mean recovery values for both the drugs from spiked plasma samples were reproducible. The method was rugged and rapid with a total run time of 2.0 minutes.

Keywords: Elbasvir; Grazoprevir; Elbasvir-D6; Grazoprevir-D6; LC–MS/MS; Liquid/liquid extraction.


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