ABOUT AUTHORS:
Chiluba Mwila1*, Sandile S. M. M. Khamanga2, Roderick B. Walker2
1University of Zambia, School of Medicine, Department of Pharmacy, Lusaka, Zambia
2Division of Pharmaceutics, Faculty of Pharmacy, Rhodes University, Grahamstown, 6140, South Africa
*mwilachiluba@yahoo.co.uk
ABSTRACT
A simple, accurate and specific reversed phase high performance liquid chromatography (RP-HPLC) method that was developed and validated for the analysis of NVP is presented. The stationary phase used was Phenomenex® Luna C18 (2), 5 µm, 250 x 4.6 mm I.D. The mobile phase comprised of acetonitrile and water in the ratio 44: 56 (v/v) and detection was accomplished using UV detector set at 284 nm. RSM using CCD was used to facilitate method optimization. Organic phase composition, flow rate and column temperature were identified as critical factors to be studied to establish the retention times of NVP and CBZ (internal standard) and the resolution factor between the two compounds. Twenty experiments including center points were performed and quadratic models were derived for the retention times of the two compounds and the resolution factor using the experimental data. The method was validated for linearity, precision, limits of quantitation and detection, and stability indicating as per ICH guidelines. The method was found to produce sharp and well resolved peaks for NVP and CBZ with retention times of 4.30 and 7.60 minutes respectively. The method was linear in the concentration range 1 – 240 µg/ml, with the calibration curve showing a good linear relationship with a regression correlation coefficient of 0.9996. The linear regression equation was y = 0.0064x – 0.0016. The limit of quantitation (LOQ) and detection (LOD) were found to be 1.0 and 0.3 µg/ml, respectively.
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