Simultaneous Determination Of Flavonol Glycoside And Its Major Metabolites In Rat Plasma By Liquid Chromatography-tandem Mass Spectrometry

Background Flavonol glycoside?GDH?is a synthetic flavonol glycoside obtained by structural modification on the basis of 3,3',4'-trihydroxy flavone.It has good activity in reducing blood lipid,can improve blood circulation and reduce cholesterol.Flavonol glycoside is currently under the clinical development.Objective To develop and validate a sensitive and accurate liquid chromatography-tandem mass spectrometry?LC-MS/MS?with a short time-consuming for the simultaneous determination of flavonol glycoside?M0?,aglycone?M1?and glucuronide conjugate?M2?.The method was to be validated and applied to the pre-clinical pharmacokinetic study of flavonol glycoside,so that can provide a better basis for clinical pharmacokinetic study.Method After a protein precipitation using methanol?0.2%formic acid?as the precipitation solvent,analytes and IS were separated on a XDB C₁₈?50×4.6 mm,1.8?m?column using a gradient elution with mobile phase consisting of water-0.2%formic acid in methanol at a flow rate of 0.6 mL·min^-1.Positive electrospray ionization was performed using multiple reaction monitoring?MRM?with transitions of m/z 461.3?m/z 299.1?flavonol glycoside,M0?,m/z 299.1?m/z 283.1?aglycone,M1?and m/z 475.0?m/z 299.1?glucuronide conjugate,M2?,and transitions of m/z 467.3?m/z 305.1 for the internal standard d₆-flavonol glycoside.Results The linear calibration for flavonol glycoside,aglycone and glucuronide conjugate were obtained in the concertration range of2.00-400 ng·mL^-1,2.00 ng.mL^-1-400 ng·mL^-11 and 4.00-1000 ng·mL^-1,respectively.The precision and accuracy values of the method were within the acceptable range of guidances for bioanalysis.Stabilities of flavonol glycoside,aglycone and glucuronide conjugate in blood samples were evaluated after storage at room temperature for 2 h,in rat plasma were evaluated after storage at room temperature for 6 h,after storage-70°C for 50 days,and after storage-20°C for 50 days.The post-preparative stability was measured after exposure of the processed samples at room temperature for 6 h.Stability of plasma samples were evaluated after three freeze-thaw cycles from-70°C to room temperature and from-20°C to room temperature.The results showed that the blood and plasma samples were stable under the above storage conditions.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_maxax of M0 was?341±106?ng·mL^-1 and AUC_0-t-t was?1960±725?h·ng·mL^-1,the plasma concentration of M1 was lower than LLOQ so that C_max and AUC_0-t could not be calculated,and the C_maxax of M2 was?1720±843?ng·mL^-1and AUC_0-t-t was?8510±2920?h·ng·mL^-1.Conclusion An accurate liquid chromatography-tandem mass spectrometry?LC-MS/MS?method was developed and validated for the simultaneous determination of flavonol glycoside,aglycone and glucuronide conjugate in rat plasma.The method was then successfully applied to the non-clinical pharmacokinetics study of flavonol glycoside in SD rats.The results suggested that flavonol glycoside presented mainly in the form of M0 and M2 in plasma.After flavonol glycoside being hydrolyzed by the intestinal flora,it was absorbed in the form of aglycone and further metabolized to glucuronide conjugate after a first-pass effect.In this paper,the main metabolites of flavonol glycoside in rat plasma were determined for the first time,which provided the basis for the design of clinical pharmacokinetic studies.The pharmacokinetic study of flavonoids should not only focus on the original drug,but also pay attention to its metabolites,which usually have much higher plasma exposures than the former.