Liquid chromatography-tandem mass spectrometry to assess the pharmacokinetics and tissue distribution of withaferin A in rats

Withaferin A (WA) is a bioactive ingredient in the medicinal Indian herb Withania somnifera (WS). In this study, we developed a rapid and accurate Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) method to determine the concentration of WA in rat plasma and tissue following intravenous (i.v., 4.5 mg/kg) and oral (i.g, 0.5, 1.5 and 4.5 mg/kg) administration. WA was isolated on a Hypurity C18 (50 × 4.6 mm, 5 μm) column by isocratic elution at a flow rate of 0.5 mL/min using acetonitrile and water as the mobile phase (35:65, v/v). The retention time was 4 min. Ethyl acetate containing 5% ascorbic acid was used as the extraction solvent through simple liquid-liquid extraction (LLE). Withanolide A (WLD) was used as the internal standard (IS). Quantification was performed through multiple reaction monitoring (MRM) modes of m/z 471.1 → 281 for WA and m/z 488.1 → 263 for IS in the positive-ion mode. This revealed no significant effects of the WA concentration or administration route on the T1/2. The distribution of WA in the various tissues was in the order: stomach > heart > lung > kidney > small intestinal > spleen > following i.g administration (4.5 mg/kg). These data provide valuable insight into the clinical parameters of WA.