Validated High-Performance Thin-Layer Chromatographic Methods for the Quantitative Estimation of Bioactive Constituents in Methiorep Premix, a Feed Additive Polyherbal Formulation

Herbal formulations have reached extensive acceptability as therapeutic agents for several diseases. The development of an authentic analytical method which can reliably profile the phytochemical composition, including quantitative analysis of marker/bioactive compounds and other major constituents, is a major challenge to scientists. With the realization of the growing popularity and demand for phytopharmaceuticals, standardization is becoming a mandatory part for the regulation of herbal drug industry. Simple, precise, and rapid high-performance thin-layer chromatographic methods were developed for the estimation of l-3,4-dihydroxyphenylalanine (l-DOPA) (I) and rutin (II), the two bioactive phytoconstituents in Methiorep premix, a phytoadditive formulation containing herbal ingredients that mimics methionine-like activity. Separation of I and II was carried out on silica gel F254 thin-layer chromatography (TLC) with butan-1-ol-acetic acid-water (8:2:2, v/v) and ethyl acetate-butan-1-ol-formic acid-water (5:3:1:1, v/v), respectively. Visualization, scanning, and quantification of analytes were performed at 288 nm and 254 nm, respectively. Linear regression analysis was used to calculate the slope, intercept, and coefficient of determination/regression coefficient (r2) for calibration plot. The response was linear in the ranges investigated. Evaluation was on the basis of peak area. The average content of these markers in different batches of the formulation was 0.28% and 0.02% (w/w), respectively. The methods were validated for linearity, accuracy, and precision in accordance with the statistical method of validation and are useful for establishing the batch-to-batch consistency.