Simple Reversed-Phase High Performance Liquid Chromatographic Estimation of the Antiretroviral Agent Efavirenz from Human Plasma
Benjamin U. Ebeshi *
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Nigeria.
Oluseye O. Bolaji
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
Margaret Oluka
Department of Pharmacology and Pharmacognosy, School of Pharmacy, University of Nairobi, Kenya.
Vaikosen N. Edebi
Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Nigeria.
Julius O. Soyinka
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
Anastasia Guantai
Department of Pharmacology and Pharmacognosy, School of Pharmacy, University of Nairobi, Kenya.
*Author to whom correspondence should be addressed.
Abstract
Aims: Sequel to the resurgence of TB co-infection in HIV/AIDS patients in sub-Saharan Africa, efavirenz has become an important component of the highly active antiretroviral treatment (HAART). The objective of this study therefore is to provide a simple reversed-phase high performance liquid chromatographic (HPLC) method for the determination of efavirenz in human plasma.
Study Design: Method development and experimental study.
Place and Duration of Study: School of Pharmacy, University of Nairobi, Nairobi, Kenya, between October 2009 and September 2010.
Methodology: A 500µl drug-free plasma sample was each placed in six different centrifuge tubes (2ml) and varying aliquots of the stock solution (100μg/ml) of efavirenz were spiked and vortexed for 60sec to give concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16µg/ml for calibration standards and 2.0, 4.0, 8.0 and 16.0µg/ml for quality control samples. The off-column sample pretreatment was carried out by protein precipitation using ice-cold acetonitrile. The samples were chromatographed in a phenomenex (C18) 5µm particle size column with 250x4.6mm I.D and UV detection at 254nm using a mobile phase, which was made up of a mixture of solutions A and B. Both consisted of acetonitrile, 25mM ammonium acetate buffer and glacial acetic acid in proportions of 90:10:0.1 and 10:90:0.1(v/v), respectively. The analytical technique was validated for precision, accuracy and analyte recovery.
Results: The calibration plot for efavirenz was found to be linear over the concentration range of 0.5 to 16.0µg/ml with the regression line equation obtained as y=26842x–409.4 and the regression coefficient (R2=0.999), which allows for accurate reading of the concentrations of the test samples. The RSD (%) in intraday and interday assays ranged from 0.44 to 0.78%. Accuracy ranged from 92 to 110% and the recovery was >97%.
Conclusion: This new HPLC method is simple, reproducible and cost-effective and can be used for therapeutic drug monitoring of efavirenz in HIV/AIDS patients on HAART as demonstrated in this study.
Keywords: Efavirenz, HPLC, pharmacokinetics, plasma, HIV/AIDS.