Simultaneous determination of bioactive flavonoids of Hoveniae Semen in rat plasma by LC-MS/MS: Application to a comparative pharmacokinetic study.

A selective, sensitive and fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the determination of dihydromyricetin, dihydroquercetin, myricetin and quercetin in rat plasma after intragastric administration of Hoveniae Semen total flavonoids. Baicalin was selected as the internal standard. Analytes were extracted from the rat plasma by protein precipitation with acetonitrile and separated on a C18 chromatographic column (Agilent ZORBAX Eclipse Plus, 4.6 mm × 100 mm, 3.5 μm) using the mobile phase containing acetonitrile (A) and 0.1% formic acid-water (B) by gradient elution at 0.5 mL/min flow rate. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used to detect analytes. The analytes were measured by multiple reaction monitoring (MRM) in the negative ionization mode. The lower limit of quantification of dihydromyricetin, dihydroquercetin, myricetin and quercetin were 0.70, 8.16, 1.62 and 0.56 ng/mL, respectively. The accuracy, intra-day and inter-day precision and recovery were all satisfactory and the compounds were stable in rat plasma under all tested conditions. The approach was successfully applied to study pharmacokinetic characteristics of the four bioactive flavonoids in plasma after administering Hoveniae Semen total flavonoids intragastrically to rat. Further investigation was carried out to assess pharmacokinetic comparability of the four bioactive flavonoids after intragastric administration of Hoveniae Semen total flavonoids to mixture of flavonoids.