The Study On Metabolism In Vitro And In Vivo And Pharmacokinetics Of Eupatorin And Eriocitrin Based On LC-MS/MS Technology

Objective:UHPLC-Q-TOF-MS/MS strategies were applied to identify the metabolites of eupatorin and eriocitrin in vitro and in vivo.LC-MS/MS methods were developed for the pharmacokinetic studies of eupatorin and eriocitrin.Methods:The chromatographic separation was performed on Poroshell120 EC-C₁₈ column?2.1×100 mm,2.7?m?.The e cient online data acquisition using full scan,negative ion mode monitoring combined with multi-mass defect filter?MMDF?and dynamic background subtraction?DBS?was employed to monitor all possible metabolites.In addition,multiple data processing techniques were applied for metabolites analysis and identification.Chromatographic separation was carried on a Wonda Cract ODS-2 C₁₈Column?150×4.6 mm,5?m?.The detection of analytes and internal standard was achieved in multiple reaction monitoring?MRM?mode using QTRAP-3200 system.Results:In this study,a total of 71 metabolites of eupatorin were structurally characterized:51 metabolites were identified in vivo?8metabolites in the plasma,5 metabolites in the bile,36 metabolites in the urine and 32 metabolites in the feces?,while 60 metabolites were detected in vitro?22 metabolites in the rat liver microsomes and 53 metabolites in rat intestinal flora?.Oxidation,hydrogenation,acetylation,methylation and sulfate combination were the main metabolic pathways of eupatorin.A total of 41 metabolites of eriocitrin were characterized:32 metabolites were identified in vivo?6 metabolites in the plasma,14 metabolites in the bile,19 metabolites in the urine and 13 metabolites in the feces?,while 27metabolites were detected in vitro?12 metabolites in the rat liver microsomes and 20 metabolites in rat intestinal flora?.The main metabolic pathways of eriocitrin include the loss of glucose groups,oxidation,methylation,hydrogenation,acetylation and sulfate combination reactions.The pharmacokinetic study showed that the eupatorin in rat plasma had good linear relationship and high precision and accuracy.After oral administration of eupatorin,it was absorbed rapidly in vivo.The maximum corresponding time to reach the C_max(T_max)was 0.25 h.In addition,double peaks were detected in mean plasma concentration profiles.Experimental study indicated that eriocitrin was quickly absorbed after oral administration.The maximum plasma concentration(C_max)was 299.833±16.743?g/L,and the produced a double peak phenomenon.Conclusions:In this study,UHPLC-Q-TOF-MS/MS combined with a variety of efficient data processing methods was used for the first time to systematically identify the metabolites of eupatorin and eriocitrin in vivo and in vitro,which provided the basis for the further investigation of the pharmacological mechanism and the development of new drugs,and laid a theoretical foundation for clinical testing and application.In addition,rapid and sensitive LC/MS/MS analytical methods were developed and validated for eupatorin and eriocitrin quantification in rat plasma for the first time,and the present methods were successfully applied to pharmacokinetic studies.The pharmacokinetic results showed that both eupatorin and eriocitrin reached their C_max quickly and had secondary absorption.The results laid the foundation for further pre-clinical and clinical research of eupatorin and eriocitrin.