Validated, Rapid and Sensitive HPLC-MS/MS Method for Determination of 7, 4'-Dihydroxylflavone in Rat Plasma and Its Application to Preliminary Pharmacokinetics

A sensitive, specific and rapid high-performance liquid chromatography-electronic spray ionization-tandem mass spectrometric method was developed and validated for the determination of 7, 4'-dihydroxylflavone (7, 4'-DHF) in rat plasma. Genistein (internal standard, IS) was added in the collected plasma samples and subsided together by a simple one-step protein precipitation using acetonitrile-methanol (1:1, v/v). Chromatographic separation was performed on an Agilent Zorbax XDB C₁₈ chromatography column and gradient elution with the mobile phase consisting of methanol and 0.1% formic acid was used. The mass spectrometric detection was performed by negative ion electro-spray ionization in multiple selected reactions monitoring (MRM) mode, with the transitions of m/z 253.1→113.0 for 7, 4'-DHF and m/z 268.9→158.8 for IS. The calibration curve has liner relationship over the concentration range of 0.1-50 ng/mL (r=0.995 4). The intra- and inter-day precision (RSD %) was less than 10%, and the accuracy (RE%, relative error) ranged from -5.2% to 8.0%. The fully validated method was applied to the pharmacokinetics (PK) of 7, 4'-dihydroxylflavone (7, 4'-DHF) in rat plasma after oral administration (two doses: 15 and 30 mg/kg) and intravenous injection (5 mg/kg). The result showed that T_max and C_max was 1.33±0.29 h and 0.12±0.02 ng/mL (15 mg/kg), and 1.17±0.29 h and 0.17±0.04 ng/mL (30 mg/kg), respectively. The bioavailability was 0.078% (15 mg/kg) and 0.070% (30 mg/kg), respectively.