Abstract
A simple, precise and accurate high performance thin layer chromatographic (HPTLC) method was developed and validated for the simultaneous determination of Capsaicin, Gamma Linolenic Acid (GLA) and Ferulic acid in the developed herbal formulation. The separation was carried out on Merck TLC aluminum sheets of silica gel G60 F254 of 200 µm thickness using Toluene: Ethyl acetate: Diethyl ether (7:3:1 v/v/v) as mobile phase and densitometric analysis of compound was carried out in absorbance mode at254 nm. The aforesaid mobile phase gave well defined peaks at Rfvalue of 0.31 ± 0.03 for Capsaicin, 0.81 ± 0.03 for Gamma Linolenic acid and 0.54 ± 0.03 for Ferulic acid. The linear regression analysis data for the calibration plots for Capsaicin, GLA and Ferulic acid showed good linear relationship with regression coefficient (r2) of 0.999,0.999 and 0.995 respectively; in the concentration range of 100-500 ng/spot,200-5000 ng/spot and 200-1000 ng/spot respectively. The limit of detection and quantitation were 116.7 and 353.7 ng/spot, respectively for Capsaicin and 40.5 and 126.4 ng/spot, respectively for GLA and 45.8 and 38.27 ng/spot, respectively for Ferulic acid. The method was validated for linearity, accuracy, precision, specificity, robustness, limit of detection (LOD)and limit of quantification (LOQ) as per ICH guidelines. In conclusion, the statistical analysis of the data showed that the method is reproducible and selective for estimation of the three compounds. The proposed developed HPTLC method can be applied for identification and quantitative determination of Capsaicin, GLA and Ferulic acid in their extracts and herbal formulation.
