SPE-HPLC method for the determination of four flavonols in rat plasma and urine after oral administration of Abelmoschus manihot extract

A SPE-HPLC method was developed and validated for the simultaneous determination of flavonols, isoquercitrin (1), hibifolin (2), myricetin (3), quercetin-3′-O-d-glucoside (4) and quercetin (5) in rat plasma and urine after oral administration of the total flavonoids from Abelmoschus manihot (TFA). The astragalin (6) and kaempferol (7) were used as internal standards (IS). Plasma and urine samples were pretreated by solid-phase extraction using Winchem™ C18 reversed-phase cartridges. Analysis of the plasma and urinary extract was performed on YMC-Pack ODS-A C18 and Thermo ODS-2HYEPRSIL C18 reversed-phase column, respectively and a mobile phase of acetonitrile–0.1% phosphoric acid was employed. HPLC analysis was conducted with different elution gradients. The flow rate was 1.0 mL/min and the detection wavelength was set at 370 nm. Calibration ranges in plasma for flavonols 2–5 were at 0.011–2.220, 0.014–2.856, 0.022–4.320, and 0.028–5.600 μg/mL, respectively. In urine calibration ranges for flavonols 1, 2, 4 and 5 were at 2.00–16.00, 8.56–102.72, 2.70–21.60, and 3.00–24.00 μg/mL, respectively. The RSD of intra- and inter-day was less than 5.40% and 4.89% in plasma, and less than 3.96% and 6.85% in urine for all the analyses. A preliminary experiment to investigate the plasma concentration and urinary excretion of the flavonols after oral administration of TFA to rats demonstrated that the present method was suitable for determining the flavonols in rat plasma and urine.