Preet Kawal Kaur, Karan Vasisht and Maninder Karan
A simple, selective, precise, robust, rapid and reliable high-performance thin layer chromatographic method of analysis for simultaneous determination of acetylbarlerin, barlerin and shanzhiside methyl ester (the major iridoids of Barleria) was developed and validated. The three iridoid markers were chromatographed on aluminium base TLC plates precoated with silica gel 60F254 using chloroform-ethylacetate- methanol-acetic acid (3.0:3.0:3.0:1.0, v/v/v/v) as mobile phase having pH of 5.01. The compounds were quantified by quantitative analysis in absorbance mode at 233 nm. The system gave compact spots for acetylbarlerin, barlerin and shanzhiside methyl ester with optimum resolution (Rf 0.71, 0.61 and 0.50 respectively) in single development. The linear regression analysis data with 95 % confidence limits by the Software package for Statistical Analysis (SPSS software, version 16) for the calibration plots for acetylbarlerin, barlerin and shanzhiside methyl ester showed good linear relationship with r2 =0.997, 0.995 and 0.992 in the concentration range of 1.42-4.95, 0.28-1.67 and 0.60-3.60 μg/spot respectively for the three markers. The mean value of slope and intercept were 0.98 ± 0.013 and -203.14 ± 42.7 for acetylbarlerin, 2.29 ± 0.04 and 249.52 ± 40.5 for barlerin and 0.96 ± 0.021 and 53.82 ± 49.89 for shanzhiside methyl ester respectively. The method was validated for precision, recovery, repeatability and robustness as per the International Conference on Harmonisation (ICH) guidelines. The limit of detection and limit of quantification were 0.07, 0.21; 0.05, 0.15 and 0.05, 0.15 μg/spot respectively for acetylbarlerin, barlerin and shanzhiside methyl ester respectively. Statistical analysis showed the method to be repeatable and selective for the estimation of the three iridoid markers. Since the proposed mobile phase effectively resolves acetylbarlerin, barlerin and shanzhiside methyl ester, the developed method can be successfully applied for the identification and simultaneous quantification of these markers. The method will be of particular use for crude drug/herbal extracts quality testing etc in especially resource constrained countries and laboratories of Asia and Africa where this plant is known to grow in abundance.