Development and Validation of an HPLC-UV-MS-MS Method for Identification and Quantification of Polyphenols in Artemisia pallens L.

Artemisia pallens L. (Compositae) is used in Indian traditional medicine to treat diabetes mellitus, jaundice, hysteria, body pain, and bacterial and fungal infections. A major cause of a variety of diseases is oxidative stress which is reduced by antioxidants such as polyphenols. These secondary metabolites are generally ubiquitous in plants and extensively used in the pharmaceutical, cosmetic, and food industries. In this study a simple and sensitive HPLC-UV-MS-MS-based method was developed for separation, identification, and quantification of polyphenols, for example gallic, protocatechuic, chlorogenic, caffeic, and ferulic acids, rutin, quercetin, and kaempferol. Amounts of polyphenols detected in 50% methanol-water extracts of the plant varied from 0.005% (kaempferol) to 0.24% (protocatechuic acid). Separation of the polyphenols was achieved on a reversed-phase C-18 with a mobile phase prepared from 1% aqueous with acetic acid and acetonitrile at a flow rate of 0.6 mL min(-1). The phenolic compounds were detected by UV absorption at 254 nm. The method was validated for linearity, accuracy, precision, LOD, LOQ, specificity, selectivity, and compound stability. Results from intra and inter-day validation (n = 6) showed the method was efficient and rapid. The optimized method was applied to extracts of A. pallens for identification and quantification of the polyphenols. The reference standards and their presence in A. pallens were confirmed by mass spectrometry.