Rapid and sensitive method for determination of withaferin-A in human plasma by HPLC

Abstract

Background: To develop and validate a rapid and sensitive high-performance liquid chromatographic method for determination of withaferin-A in human plasma. Withaferin-A, the active molecule of a traditional Indian herb, has demonstrated several biological activities in preclinical models. A validated bioassay is not available for its pharmacokinetic evaluation. Results: The chromatographic system used a reverse-phase C18 column with UV-visible detection at 225 nm. The mobile phase consisted of water and acetonitrile applied in a gradient flow. Withaferin-A was extracted by simple protein-precipitation technique. The calibration curve was linear in the concentration range of 0.05–1.6 µg/ml. The method has the desired sensitivity to detect the plasma concentration range of withaferin-A that is likely to show biological activity based on in vitro data. Conclusion: This is the first HPLC method ever described for the estimation of withaferin-A in human plasma which could be applied for pharmacokinetic studies.