Development and validation of a normal-phase HPTLC method for the simultaneous analysis of lamivudine, stavudine and nevirapine in fixed-dose combination tablets

This paper presents the development and validation of an improved method for the simultaneous analysis of lamivudine (LVD), stavudine (STV) and nevirapine (NVP) using high-performance thin-layer chromatography (HPTLC) with densitometric detection. Separation was performed on silica gel 60F254 plates. The mobile phase is comprised of ethylacetate, methanol, toluene and concentrated ammonia (38.7:19.4:38.7:3.2, v:v:v:v). Detection wavelength was 254 nm. The Rf values were 0.24 ± 0.03, 0.38 ± 0.04 and 0.69 ± 0.04 (n = 8) for LVD, STV and NVP, respectively. An F-test indicated that calibration graphs were adequately linear at the evaluated concentration ranges. The pooled %RSD for repeatability of the percentage amount recovered for LVD, STV and NVP were found to be 0.62, 0.54, and 0.79, and the pooled %RSD for time-different intermediate precision were 1.66, 1.27 and 1.21. The percentage recoveries for the trueness were 99.2% ± 1.5 for LVD, 98.6% ± 1.5 for STV and 99.3% ± 1.7 for NVP (n = 3). Most factors evaluated in the robustness test were found to have an insignificant effect on the selected responses at 95% confidence level. This method was successfully used to analyze fixed-dose tablets samples of LVD, STV and NVP.