Validation of an HPLC method for the simultaneous determination of DZ5 and its active metabolite in dog plasma and its pharmacokinetics

A validated HPLC method is described for the simultaneous determination of daidzein 7,4′-di-succinic acid mon-ester-O-ethoxy (DZ5) and its active metabolite daidzein 7,4′-dioxy-ethoxy (DZ4) in dog plasma. DZ5 and DZ4 were determined by reversed-phase HPLC (column: Hypersil C18 5 μm silica, 200 mm × 4.6 mm i.d.; eluent: 400 ml water, 500 μl 85% phosphoric acid, 600 ml methanol) and photometric detection (250 nm), with Kaempferol as the internal standard. The calibration curve was linear over the range 0.1-50.0 μg/ml in dog plasma. The average extraction recoveries were 84.6% (DZ5), 82.7% (DZ4) and the within-day and between-day precisions were less than 10.93%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration and intravenous administration of DZ5, DZ5 was eliminated rapidly from the plasma and DZ4 was found in plasma. The absolute bioavailability of total DZ5 and DZ4 was 41.5%. The method was demonstrated to be feasible for pharmacokinetic studies of DZ5 in dogs.