1. Graduate School of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, Pittsburgh, PA, USA.
Background: Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) are the common antiretrovirals prescribed in resource limited countries due to its cost effectiveness compared to other newer antiretrovirals. Monitoring quality of such generic versions of the dosage forms is a crucial aspect. The main aim of the study is to develop a simple reversed phase (RP) HPLC-UV method to determine 3TC, AZT, NVP with some of its degradation products cytosine and thymine that can be applied in monitoring the quality of such products.
Methods: A RP gradient HPLC method was developed using following chromatographic conditions: LunaC18 150 x 4.6mm column; 50mM ammonium acetate buffer (pH = 6.8) and methanol as mobile phase; Gradient mode of elution with 0- 35%v/v methanol for first 10 minutes increased to 50%v/v till 12 minutes and isocratic to 18minutes, return to initial at 20min; sample injection volume 50μl; detection wavelength 265nm. Calibration ((n= 6) and validation samples (n=3) were prepared in triplicate for analysis and validated for specificity, linearity, range, accuracy and precision. The method was used in dissolution studies of the fixed dose combination tablets of AZT/3TC/NVP using USP Type I method.
Results: No interference between the analytes was observed. The method was found to be linear over the following concentration range for the five analytes: Cytosine and thymine (0.5-8μg/ml), 3TC (1- 50μg/ml), AZT and NVP (1-80μg/ml) with coefficient of determination (R2) value > 0•9997. Accuracy in terms of mean percent recovery for the five analytes was found to in the range of (Mean Recovery ± S.D) 101.26± 1.27%. Intra-day and Inter-day precision (%RSD) was found to be 0.49± 0.28% and 2.24 ± 0.26% respectively. There were no significant differences in the mean recovery of the APIs between the days as analyzed by two-way ANOVA at p >0•05. No matrix effect due to the presence of other excipients in the formulation was observed in the chromatograms of the dissolution samples. Conclusion: The method developed was specific, accurate, reliable and can be applied during routine pharmaceutical analyses such as drug content analysis, dissolution studies and stability of the fixed dose combination product of AZT/3TC/NVP.