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Simultaneous HPLC Determination of Efavirenz, 8-hydroxy Efavirenz, Neostigmine and Comparison of their Separation Using a C18 and Biphenyl Column through Pharmacological Evaluation

Author(s): S Kumar*, P. J. Bouic and B. Rosenkranz
Department of Medicine, Division of Clinical Pharmacology, University of Stellenbosch, Cape Town, 7505, South Africa, Synexa Life Sciences, Montague Gardens, Cape Town, 7441, South Africa, Department of Medicine, Division of Medical Microbiology, University of Stellenbosch, Cape Town, 7505, South Africa

Correspondence Address:
Department of Medicine, Division of Clinical Pharmacology, University of Stellenbosch, Cape Town, 7505, South Africa E-mail:

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 (150ï?´4.6 mm, 2.6 μ), by gradient elution using a mobile phase consisting of water:acetonitrile at 0.6 ml min-1 flow 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 (150ï?´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 coefficient=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 quantification at 32 μM and 96.97 μM for efavirenz and 29.49 μM and 89.38 μM for 8-hydroxy efavirenz, respectively. Pharmacological drug metabolism evaluation was done on the method specificity using in vitro incubations using human liver microsomes and linearity was established for the 30, 45 and 60 min incubations (correlation coefficient=0.97). The method provides a necessary tool to investigate herb and drug-drug interactions and can be applied for quantifying the drugs in in vitro drug metabolism assays.

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