A validated RP-HPLC-UV/DAD method for simultaneous quantitative determination of rosmarinic acid and eugenol in Ocimum sanctum L.

Background: Ocimum sanctum L. is present as an ingredient in many herbal formulations and hence standardization of its raw materials and extracts is significant. The study was aimed at developing and validating a RP-HPLC method for the standardization of O. sanctum L. raw material and extracts by selecting rosmarinic acid and eugenol as markers. Methods: The developed method used a Phenomenex Luna C 18 column (250
× 4.6 mm; 5 µm) using a gradient elution of 1 mM ammonium acetate buffer and acetonitrile as the mobile phase with a flow rate of 1.5 mL/min. The analytes were monitored at 278 nm. The method was validated according to the ICH guidelines. Results: The calibration curves showed good linear regression (r 2 > 0.999) within test ranges. Specificity of the method was assessed using PDA profile and the peak purity of all the analytes was greater than 99%. This method showed good reproducibility for the quantification of both the compounds in samples with an RSD of less than 2.5% respectively. Percentage recovery exceeded 90% for all analytes. Conclusion: The validated method was successfully applied to quantify rosmarinic acid and eugenol in O. sanctum raw material and extracts which provides a new basis for overall assessment on quality. Key words: Eugenol, Ocimum sanctum ,RP-HPLC,Rosmarinic acid,Validation.