Simultaneous Determination Of Isoflavone In Rat Plasma After Oral Administration Of Semen Sojae Praeparatum Extract For Pharmacokinetic Study

Soy isoflavones,one kind of naturally occurring phenolic compound,are also present in Traditional Chinese Medicine Semen Sojae Praeparatum,which are believed to have beneficial effects in preventive action against most chronic diseases.However,The isoflavones were be found in the common rat food.Consequently,the substances in animal food might have interference with the isoflavones when the animal was administrated with them in the pharmacokinetic study.Therefore,For eliminating the influence by the commercial food pellets,an LC-MS/MS method for simultaneous determination of daidzin,daidzein,glycitin,glycitein,genistin and genistein in rat plasma was develop in this paper.The baseline levels of six isoflavones in blank plasma obtained from rats consuming commercial food containing some isoflavones were measured for the correction of the plasma concentrations.And the principle pharmacokinetic parameters were calculated from rats with or without regular commercial food,and the effects of different diets on the determination of isoflavones in plasma of rats were compared.1.The development and validation of analytical method for six isoflavones in rats plasma.Rapid,sensitive and selective LC–MS/MS method has been developed and validated for the simultaneous determination of the six isoflavones of SSP in rat plasma.It was successfully applied to the pharmacokinetic study of these constituents after oral administration of SSP extract.The excellent selectivity,sensitivity,precision,accuracy and linear range proved that the method is suitable for pharmacokinetic study.Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate with quercetin as the internal standard(IS).LC separation was achieved on a Symmetry C18 column(100 mm × 3.0 mm)using a gradient mobile phase consisting of acetonitril and water(containing 0.1% formic acid)with a run time of 13.0 min at a flow rate of 0.4m L/min.The detection was carried out by a triple–quadrupole tandemmass spectrometer in multiple reaction monitoring(MRM)mode via polarity switching between negative(for daidzein,glycitein,genistein and glycitin)and positive(for daidzin,genistin and IS)ionization mode.All calibration curves exhibited good linearity(r> 0.99)over a wide concentration range for all components.The intra-day and inter-day precisions(RSD)at three different levels were both less than 14.9 % and the accuracies(RE)ranged from-9.3 % to 14.5 %.The extraction recoveries of the analytes and the IS ranged from 85.7 % to 117 %.The excellent selectivity,sensitivity,precision,accuracy and linear range proved that the method is suitable for pharmacokinetic study.2.The pharmacokinetics study of isoflavone in SD rats with isoflavones-free feed.The method of correcting the dynamic baseline concentration was put forward in this paper.The validated LC-MS/MS method was used to investigate the quantification of daidzin,daidzein,glycitin,glycitein,genistin and genistein after oral administration of Semen Sojae Praeparatum(SSP)extract in SD rats with isoflavones-free feed.Six kinds of soybean isoflavone drug concentration time curve were plotted.And the principle pharmacokinetic parameters were calculated.The pharmacokinetic parameters,based on the measured density data,were calculated with the non-compartmental model.It was successfully applied to the pharmacokinetic study of these constituents after oral administration of SSP extract.3.The pharmacokinetics study of isoflavone in SD rats with isoflavones-free feed.we investigated the dynamic baseline plasma concentrations of isoflavones in blank rats fed with the commercial food pellets merely.And then the corrected plasma concentrations should be obtained by subtracting the baseline concentrations from the plasma concentrations of the analyte at each corresponding time point.In the following,the corrected plasma concentrations were used to determine the principle parameters in the pharmacokinetic study in rats after oral administration of SSP,evaluating the influence of the dietary isoflavones on them.This approach has been proven an alternative and effective strategy in the pharmacokinetic study of isoflavones in ratswithout changing the dietary composition and could be applied to assess the pharmacokinetic profiles of other compounds from food.4.Comparative pharmacokinetics of rats with or without regular commercial feed were given SSP extract.Compared with rats fed with isoflavones-free diet,rats consuming commercial food exhibited the different pharmacokinetic parameters,demonstrating the dietary intake of commercial food containing some isoflavones might have an impact on the pharmacokinetic behavior of the six isoflavones in the rats after oral administration of SSP extract.It also suggests that the effects of different diets on the pharmacokinetics of the SSP in rats can not be ignored.