May-June 2017 Indian Journal of Pharmaceutical Sciences 353 Research Paper Efavirenz (EFV, C 14 H 9 ClF 3 NO 2 ) is an antiretroviral (ARV) agent employed in the treatment of human immunodefciency virus (HIV) type 1 in combination with other drugs. It is an analogue of non-nucleoside ARV agent and a non-competitive reverse transcriptase inhibitor and is directly connected to the enzyme and blocks RNA and DNA-dependent, DNA-polymerase activities, causing destruction of the enzyme catalytic site. Chemically it is (S)-6-chloro-(cyclopropylethynyl)- 1,4-dihydro-4-(trifuoromethyl)-2H-3,1-benzoxazin- 2-one and is optically active with a molecular weight of 315.68 g/mol and melting point in the range 139- 141°. This crystalline powder has a white or slightly yellowish appearance. The substance has a melting point ranging from 136-141°, is nearly water insoluble but soluble in methanol and dichloromethane [1] . EFV is predominantly cleared by hepatic metabolism [2] . The metabolites identifed in human plasma and urine (almost exclusively as glucuronide or sulphate conjugates) were 7- and 8-hydroxy efavirenz (primary metabolites) and 8,14-dihydroxy efavirenz (secondary metabolite). 8-hydroxy efavirenz (E8H, C1 4 H 9 ClF 3 NO 3 ) is the major metabolite from EFV primarily formed via CYP2B6 of the cytochrome P 450 system [3] . It has the IUPAC name (4S)-6-chloro-4-(2- Simultaneous HPLC Determination of Efavirenz, 8-Hydroxy Efavirenz, Neostigmine and Comparison of Separation Using a C18 and Biphenyl Column through Pharmacological Evaluation S. KUMAR*, P. J. BOUIC 1,2 AND B. ROSENKRANZ Division of Clinical Pharmacology, Department of Medicine, University of Stellenbosch, Cape Town, 7505, 1 Synexa Life Sciences, Montague Gardens, Cape Town, 7441, 2 Division of Medical Microbiology, Department of Medicine, University of Stellenbosch, Cape Town, 7505, South Africa Kumar, et al.: HPLC Determination of Efavirenz, 8-Hydroxy Efavirenz and Neostigmine A simple, rapid and stable high performance liquid chromatography method for a combination of efavirenz, its major metabolite 8-hydroxy efavirenz and neostigmine was developed and validated. The drugs individually and in combination, were analysed using an Agilent 1260 high performance liquid chromatography coupled with variable wavelength detector. Successful separation of combined drugs were achieved by gradient elution on a reverse-phase C18 Phenomenex Evo 100A column (150×4.6 mm, 2.6 μ), by gradient elution using a mobile phase consisting of water:acetonitrile at 0.6 ml×min -1 fow rate, detection wavelength 245 nm, column oven temperature 27° and injection volume 15 μl. The same method was also deployed on a Restek Ultra biphenyl column (150×4.6 mm, 5 μ) to compare the levels of separation of the drugs and the metabolite. The chromatographic retention times were consistent at 8.75, 9.33 and 10.20 min for efavirenz, 8-hydroxy efavirenz and neostigmine, respectively. Polynomial regression data for the calibration plots exhibited linear relationship (correlation coeffcient=0.999) in the range of 2.5-150 μM for both efavirenz and 8-hydroxy efavirenz, and the lower limit of detection and lower limit of quantifcation at 32 and 96.97 μM for efavirenz and 29.49 and 89.38 μM for 8-hydroxy efavirenz, respectively. Evaluation of drug metabolism using human liver microsomes could be achieved with this method and linearity was established for the 30, 45 and 60 min incubations (correlation coeffcient=0.97). The method could be a potential tool to investigate herb and drug- drug interactions as well as for quantifying drugs in in vitro drug metabolism assays. Key words: HPLC, C18, efavirenz, 8-hydroxy efavirenz, neostigmine, biphenyl column, human liver microsomes *Address for correspondence E-mail: saneesh.7.kumar@gmail.com This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms Accepted 06 April 2017 Revised 31 December 2016 Received 13 September 2016 Indian J Pharm Sci 2017;79(3):353-360