Development and validation of a method for densitometric analysis of umbelliferone, β-sitosterol and methoxsalen in polyherbal formulation

Herbal medicines are generally available as a mixture of more than one plant constituent. It is important to quantify the maximum possible number of markers in such herbal formulations by which the quality of the formulations may be assessed. TLC densitometric methods were developed using High performance thin layer chromatography for the quantification of the three marker compounds, umbelliferone, β-sitosterol and methoxsalen, from the polyherbal tablet formulation. Solvent systems were optimized to achieve best resolution of the marker compounds from the other components of the sample.  From the various solvent systems tried, the one containing toluene: ethyl acetate: methanol: formic acid (3: 3: 0.2: 0.8) gave best resolution of umbelliferone (Rf=0.35), β-sitosterol (Rf=0.47) and methoxsalen (Rf=0.6) in the presence of the other compounds in the polyherbal tablet sample and enabled the quantification of the marker compounds. The methods were validated in terms of precision, repeatability and accuracy. The method was found to be suitable for the quantification of these marker compounds in polyherbal formulation.