Simultaneous Separation and Determination of Lamivudine and Zidovudine in Pharmaceutical Formulations Using the HPTLC Method

Abstract

A high-performance thin-layer chromatographic method (HPTLC) for the simultaneous determination of lamivudine and zidovudine in a binary mixture has been developed. The method developed was based on HPTLC separation of the two drugs followed by densitometric measurements of spots at 276 and 271 nm for lamivudine and zidovudine, respectively. Separation was carried out on Merck HPTLC silica-gel 60 F₂₅₄ plates, using toluene/chloroform/methanol (1:6:3 v:v) as the mobile phase. Validation of the method was performed based on The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Second-order polynomial equations were obtained for the regression line in the ranges of 250–1400 and 250–1700 ng/spot for lamivudine and zidovudine respectively. Correlation coefficient (r) values were 0.9998 for both analytes. The method provides sufficient accuracy as indicated by recovery percentages given for lamivudine and zidovudine. For system precision study, the low coefficient of variation values (<2%) for both lamivudine and zidovudine ensured reproducible performance of the instrument. In the method precision study, coefficients of variation <2% were obtained, which showed that the proposed method provides acceptable intraday and interday variation. The detection and quantification limits and were 3.06 and 9.28 ng/spot for lamivudine and 3.34 and 10.13 ng/spot for zidovudine, respectively. Parameters such as mobile-phase composition, volume of mobile phase, time from spotting to development, and time from development to scanning were employed while testing for robustness of the method, and the standard deviation of peak areas was calculated for each parameter. The low coefficient of variation values indicated the robustness of the method. Statistical manipulation did not show any significant effect of one parameter over the others on the robustness of the method.

Keywords:

• Densitometric detection
• HPTLC
• lamivudine and zidovudine combinations
• simultaneous determination

Notes

Note. All calculations were done using a 95% confidence limit.

^a Percentage of the labelled amount.

^b Coefficient of variation.

^c Calculated t value is 2.57 (P = 0.05).

^a Lamivudine.

^b Zidovudine.

^c Standard deviation.

^d Coefficient of variation.

^a Lamivudine.

^b Zidovudine.

^c Standard deviation.

^d Coefficient of variation.

^a Standard deviation.

^b Coefficient of variation.

^a Standard deviation of an average of three concentrations (400, 500, 600 ng/spot and 800, 1000, 1200 ng/spot for lamivudine and zidovudine, respectively).

^b Coefficient of variation.

^a Standard deviation.

^b Coefficient of variation.

^a Standard deviation.

^b The theoretical t and F values are equal to 2.57 and 5.05, respectively (P = 0.05).

^a Coefficient of variation.

^b The theoretical t and F values are equal to 2.57 and 5.05, respectively (P = 0 .05).