Estimation and quantitation of β-asarone from Acorus calamus rhizome and its formulations using validated RP-HPLC method

Introduction: Acorus calamus Linn. ( A. calamus ) has been found use in medicines to cure fevers, for asthma, bronchitis and as an all-round sedative. β-asarone is an important phytochemical compound present richly in the rhizomes of Acorus calamus that imparts several therapeutic properties to the plant by the virtue of which the plant has occupied a significant therapeutic acclaim in ancient Ayurvedic text and is employed as one of the key ingredients in several traditional and herbal formulations. Thus, the study aims to develop and validate an efficient Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for quantification of β-asarone from rhizomes of A. calamus of the wild and marketed variety and also intends to apply the validated methods for the estimation of the biomarker from different formulations containing the rhizome as one of its ingredients. Methods: Separation was carried out on Cosmosil C 18 column eluted with mobile phase of methanol: distilled water (50:50, v/v) at flow rate of 1 mL/min. Detection was carried out at 304 nm using a photodiode array detector (PDA) and the method was validated as per International Conference on Harmonization (ICH) guidelines. Rhizome was collected from Kerala and also procured from the market.  Commercial traditional and herbal formulations like Sarasvata Curna, Maanasmitra Vatakam, Khadiradi Vati, Chandra Prabha Vati, Sanjeevani Vati, Mahashankha Vati, Smritisagara Rasa, Abana, Vacadi Taila, and Ashwagandharishtha were further subjected to RP-HPLC for separation and estimation of β-asarone. Results: The limit of detection (LOD) and limit of quantitation (LOQ) levels were found to be 0.025 µg/mL and 0.1 µg/mL, respectively. The content of β-asarone was found to be maximum in the sample collected from Kerala which was 0.2946±0.0152 mg/g. Conclusion: The developed method can be recommended for marker-based standardization and quality assurance of A. calamus and its formulations. Key words: Acorus calamus , Formulations, Rhizome, RP-HPLC, Validation.