RP-HPLC Method Development and Validation for Simultaneous Estimation of Thymol, Eugenol and Alliin in Bulk and Novel Nanoformulation

Abstract

A rapid and simple HPLC method was developed to detect and quantify Thymol, Eugenol and Alliin being the potential components of Ajwain oil, Clove oil and Garlic oil respectively in herbal formulation. Liquid chromatography with a UV detector at a wavelength of 278nm, using a C18 column, was employed in this study. Isocratic elution was employed using a mixture of acetonitrile: methanol:10mM Phosphoric acid buffer (50:25:25, v/v, pH 3.0). The analytical method validation was performed in accordance with ICH guidelines, which included selectivity, range, linearity, accuracy, detection limit, quantitation limit, precision, robustness, and stability of solutions. Acceptable assay precision and accuracy (100±2.0%) were obtained at 50– 150% of the analytical concentration of Thymol, Eugenol and Alliin at the target concentration, and good linearity (0.999, 0.998 and 0.998 respectively) were achieved over a range of 10µg/mL -60µg/mL for Thymol and Eugenol and 0.25µg/mL –1.50µg/mL for Alliin. The proposed HPLC method proved to be reliable. The validation and application of this method can be adopted and used in the pharmaceutical industry for routine analysis.