Simultaneous determination of flavonol glycoside and its major metabolites by LC-MS/MS in rat plasma

Flavonol glycoside is in clinical trials for treatment of hyperlipidemia. An accurate and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of flavonol glycoside (M0), aglycone (M1) and glucuronide conjugate (M2) in rat plasma. d₆-Flavonol glycoside was used as internal standard (IS). After extraction from the plasma by protein precipitation, the analytes and internal standard were separated on a XDB C₁₈ column (50 mm×4.6 mm, 1.8 μm) using a gradient elution procedure. The mobile phase consisted of methanol and water (0.2% formic acid) at a flow rate of 0.6 mL·min⁻¹. The total run time was 4.5 min. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 461.3 → m/z 299.1 for M0, m/z 299.1 → m/z 283.1 for M1, m/z 475.0 → m/z 299.1 for M2, and m/z 467.3 → m/z 305.1 for d₆-flavonol glycoside. The method was validated and successfully applied to the pharmacokinetics study of flavonol glycoside in SD rats which were given flavonol glycoside (30 mg·kg⁻¹) by gavage. The C_maxof M0 is (341 ±106) ng·mL⁻¹ and AUC_0−t is (1 960 ±725) h·ng·mL⁻¹, while the C_maxof M2 is (1 720 ±843) ng·mL⁻¹and AUC_0−t is (8 510 ±2 920) h·ng·mL⁻¹. The results suggest that flavonol glycoside existed mainly in the form of M0 and M2 in rats. After flavonol glycoside being hydrolyzed by the intestinal flora, it was absorbed in the form of aglycone and further metabo­lized to M2 after the first-pass effect. In this paper, the main metabolites of flavonol glycoside in rat plasma were determined for the first time, which provided a basis for the design of clinical pharmacokinetic experiment.