Objective: To develop and Validate a Method for the estimation of Efavirenz in Human Plasma.

Introduction: Efavirenz EFA; (4S)-6-chloro-4-(cyclopropylethynyl)-4 (trifluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one) belongs to theNon-nucleotide analogue reverse transcriptaseinhibitors (NNRTIs) class of antiretroviral drugs. Different methods have been reported in the literature for monitoring plasma levels of EFA.A. E. Mutlib et al., Identified and Characterized Efavirenz Metabolites by Liquid Chromatography / Mass Spectrometry and High field NMR: Species differences in the metabolism of Efavirenz. Mathias AA et al., 14 have done the Bioequivalence study of efavirenz / emtricitabine / tenofovir disoproxil fumarate single-tablet regimen. Hsieh Y et al., were examined the Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry. Since there is no specific method available in the literature for the SolidPhase Extractionof human plasma and estimation using LC–MS/MS as the detection system with a sufficiently short run time.

Method: A rapid and sensitive LC–MS–MS method has been developed and validated for analysis of Efavirenz(EFA) in human plasma. The analytes were extracted from human plasma by SPE. Nevirapine(NEV) is used as the internal standards for EFA. An RP18 column enabled chromatographic separation of the analytes. The method involves simple isocratic chromatography and MS detection in Negative-ionization mode. Validation of the method showed response was a linear function of concentration in the ranges 50-5000 ng mL-1.

Results: At the LOQ levels, inter-run and intra-run precision were within4.56 and 5.70 and respectively, for. The mean recovery of Efavirenz at LQC, MQC, HQC levels were 89.72 %, 91.27%, 85.49% with the R.S.D. of 7.48, 6.00, 2.45 respectively . Total elution time was 2 min only, which enabled quantification of more than 200 plasma samples per day. This validated method was used successfully for analysis of plasma samples from a bioequivalence study.