Simultaneous determination of acetylpuerarin and puerarin in rat plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study following intravenous and oral administration

• The study supported the metabolic profile of acetylpuerarin in vivo.
• Acetylpuerarin and the metabolite puerarin were simultaneously determined by LC–MS/MS.
• The method was fully validated for both acetylpuerarin and puerarin.
• The method was successfully applied to a pharmacokinetic study of acetylpuerarin and its major metabolite puerarin.

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the simultaneous determination of acetylpuerarin (AP) and its major metabolite puerarin (PUE) in rat plasma using genistein as the internal standard (IS). Plasma samples were pretreated by protein precipitation with a mixture of methanol and acetonitrile. Chromatographic separation was performed on a CAPCELL PAK C18 MGШ column with a mixture of 0.1% formic acid in water and methanol (35:65, v/v) as the mobile phase. The analytes were detected using a tandem mass spectrometer in the positive ionization and multiple-reaction monitoring mode. The ion transition of m/z 669.4 → 627.3, 417.5 → 297.6 and 271.3 → 153.0 was utilized to quantify AP, PUE and the IS, respectively. The calibration curves showed good linearity over the plasma concentration range of 1–2000 ng/mL for AP and 2.5–5000 ng/mL for PUE. The intra- and inter-day precisions (RSD %) for each analyte were less than 6.91%, and the accuracies ranged from −2.17% to 2.93%. The validated LC–MS/MS method was further successfully applied to a pharmacokinetic study of AP and PUE in rats following intravenous and oral administration.