Development and validation of a high-performance thin-layer chromatography method for the simultaneous quantification of rosmarinic acid, quercetin, glycyrrhizin and betulinic acid in polyherbal immunostimulant formulation

Quantification of bioactive markers through modern analytical methods is very essential for establishing the authenticity and creditability of prescription and usage of herbal drugs formulations. In the present study, simultaneous quantification of rosmarinic acid (RA), quercetin (Q), glycyrrhizin (G) and betulinic acid (BA), in polyherbal formulation by high-performance thin-layer chromatography (HPTLC) method was developed. Mobile phase of ethyl acetate-toluene-methanol-formic acid (7:1:0.5:0.5, V/V) was used on pre-coated plate of silica gel 60F(254) and quantified by densitometric method. Validation of the method was done to demonstrate its selectivity, linearity, precision and accuracy as per the International Council for Harmonization (ICH) guidelines. Intra-day assay and inter-day assay precision of the analytes were less than 2%, and the average recovery rates obtained were in the range of 98.11-101.14% for all with percent relative standard deviation (%RSD) below 2%. Correlation coefficient shows that the developed method was accurate and precise. This method can be used in the routine analysis of polyherbal formulations.