Development and Validation of LC-MS/MS Method for Simultaneous Estimation of Emtricitabine, Tenofovir Alafenamide and Dolutegravir in Human Plasma

Abstract

A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of Emtricitabine (EMT), Tenofovir alafenamide (TAF) and Dolutegravir (DTG) in human plasma. The analytes were extracted with 0.1% v/v formic acid in water using solid phase extraction technique. The chromatographic separation was developed using Kinetex biphenyl 100 A column C18 (100 mm × 4.6 mm, 5μm) with mobile phase of 0.1% v/v formic acid in water and 0.1% v/v formic acid in acetonitrile in gradient elution. The chromatographic run time for EMT, TAF and DTG was obtained at 0.991, 1.287, 1.854 min, respectively. The mass transitions have been monitored in electron spray ionization (Positive mode and triple quadrupole as m/z 248.05→130.15 for EMT, m/z 477.10→270.10 for TAF, m/z 420.15→276.95 for DTG. The proposed method has been validated with a linear range of 20 to 5000 ng/ml for EMT, 2 to 500 ng/ml for TAF and 30 to7500 ng/ml for DTG. The intrarun and interrun precision are 3.64% for EMT, 4.36% for TAF, 4.5% for DTG at their respective LOQ levels. The method was validated for linearity, accuracy, precision, specificity and stability. The developed LC-MS/MS method has been successfully applied to simultaneous quantification of Emtricitabine, Tenofovir alafenamide and Dolutegravir in human plasma without any interference.