Studies On Imperatorin Metabolism In Vitro And In Vivo By HPLC-MS Technologies And The Quality Control Of Herba Scutellariae Barbatae

Imperatorin is a bioactive linear-type furanocoumarin originating frommany traditional Chinese herbal medicines such as Angelica Dahurica, RadixGlehniae and Angelica archangelica (Umbelliferae). It possesses antibacterial,antiviral and antitumor activities. Previous research showed that imperatorinwas mainly excreted in the form of metabolites, and the excretion of prototypewas very few. This was indicated that imperatorin was widelybiotransformated in vivo. In the present study, HPLC-MS methods wereperformed to investigate the metabolism of imperatorin in vitro and in vivo. AHPLC-QTrap-MS method was developed for the identification of metabolitesin rat urine, feces and bile after oral administration of imperatorin, and thetransformation products of imperatorin from the incubation of rat livermicrosome. Meanwhile, A HPLC-QTof-MS method was estabolished forfurther analysis of imperatorin metabolites in rat urine. According to theresults of the two HPLC-MS methods, mass fragmentation rules ofmetabolites were summarized, and metabolic pathways were also illustrated.Herba Scutellariae Barbatae is originated from the dried whole plant ofan herb Scutellaria barbata D.Don. It is a famous traditional Chinesemedicine called 'Ban-Zhi-Lian' and has been used for thousands of years forthe treatment of cancer, inflammation and urinary disease. Because ofdifferent cultivation areas and climatic conditions, its chemical constituentsmay vary substantially. Meanwhile, substitution use of Herba ScutellariaeBarbatae is not uncommon. It is absolutely necessary to establish an analyticalmethod to differentiate Herba Scutellariae Barbatae and its confused plants. Inthe present study, a sensitive and selective high performance liquidchromatography tandem mass spectrometric method (HPLC-MS/MS) was developed for the simultaneous analysis of11components (7flavonoids and4phenolic acids) in Herba Scutellariae Barbatae. In addition,18batches ofHerba Scutellariae Barbatae and3batches of its confused plants from differentsources were compared using the developed method. This method can beeffectively applied for the quality control and evaluation of Herba ScutellariaeBarbatae and its confused plants.Part one Studies on imperatorin and its metabolism in vitro and in vivoby HPLC-MS technologiesObjective:1. To identify the metabolites of imperatorin in rat urine,feces and bile after oral administration, as well as the transformation productsof imperatorin from the incubation of rat liver microsome with HPLC-QTrap-MS technology.2. To develop HPLC-QTof-MS method for the detection andcharacterization of metabolites in rat urine of imperatorin to illustrate itsmetabolic pathways rule.Methods:1. The combination use of HPLC-QTrap-MS scanning modeincluding multiple ion monitoring-information dependentacquisition-enhanced product ion (MRM/MIM-IDA-EPI) mode, precursorscan-enhanced resolution-information dependent acquisition-enhancedproduct ion (PREC-ER-IDA-EPI) mode and enhanced product ion (EPI) modewere performed for the identification of metabolites in rat urine, feces and bilesamples, and the incubation products of imperatorin from rat liver microsome.Based on the simultaneous appearance of [M+H]+and [M+NH+4]in thespectrum of PREC, the molecular weight could be unambiguously identified.The structures of compounds were then indentified by the fragment ionsgenerated from these three modes.2. Urine sample was analyzed byHPLC-QTof-MS in the scanning mode of TOF-MS-IDA-4MS/MS to acquireexact mass and fragment ions of the unknown metabolites. Then, thecombination use of post-acquisition data mining tools including XIC(extracted ion chromatography), MDF (mass defects), PIF (product ion filter)and NLF (neutral loss filter) were chosen to identify the metabolites ofimperatorin and deduce its pathways rule. Urine, feces and bile samples were collected after single oral administration of imperatorin (80mg/kg) to rats.Then the samples were pretreated by liquid-liquid extraction with ethyl acetate.The chromatographic separation was performed on an Agilent Zorbax EclipseXDB-C18(150mm×4.6mm,5μm) column, with1mmol/L ammoniumacetate and methanol as gradient eluents. The mobile phase flow rate was setat1.0mL/min.Results:1. With the HPLC-QTrap-MS method,32metabolites in urinesample,14metabolites in faces sample,6metabolites in bile sample and17transformation products from the rat liver microsome sample were detected.2.With the HPLC-QTof-MS method,51metabolites were identified in rat urine,including44phase I metabolites and7phase II metabolites. Meanwhile, theprobably metabolite pathway of imperatorin were illustrated.Conclusion:1. HPLC-QTrap-MS method was developed for thequalitative analysis of the metabolites of imperatorin. The unprecedentedadvantage of the combination use of the MRM/MIM-IDA-EPI,PREC-ER-IDA-EPI and EPI modes were also displayed which could easilyand accurately characterize the metabolites in complex system.2.HPLC-QTof-MS was high sensitive, accurate and selective for the drugmetabolite identification. In this study we concluded that (1) Imperatorin wastransformed extensively in vivo and in vitro.(2) Imperatorin was metabolizedmainly in liver and excreted through kidney.(3) The metabolic pathways rulesof imperatorin were illustrated.(4) The metabolic profile of imperatorin invivo and in vitro had good correlation.Part two Simultaneous quantification of flavonoids and phenolic acids inHerba Scutellariae Barbatae and its confused plants byHPLC-MS/MSObjective: To develop a HPLC-MS/MS method for the simultaneousanalysis of11components (7flavonoids and4phenolic acids) in HerbaScutellariae Barbatae and its confused plants. Hierarchical cluster analysis wasused for its species authentication.Methods: The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turboion spray interface in negative mode using multiple-reaction monitoring(MRM). The ion spray voltage was set to-4500V. The turbo spray temperaturewas maintained at650°C. The precursor-to-product ion pairs of11analyteswere153.0/108.9for protocatechuic acid,353.1/191.0for chlorogenic acid,121.0/91.9for p-hydroxybenzaldehyde,461.1/285.0for scutellarin,609.3/301.1for hesperidin,147.0/103.0for cinnamic acid,271.0/151.0fornaringenin,285.0/133.0for luteolin,269.0/117.0for apigenin,269.0/223.0forbaicalein and283.0/267.9for wogonin, respectively. The11constituents wereseparated within13.0min on an Agilent Zorbax SB-C18column (150mm×4.6mm,5μm) using a mobile phase consisted of methanol and0.1‰acetic acidin water with gradient elution at a flow rate of0.8mL/min. The columntemperature was maintained at25°C. The method was successfully applied todifferentiate18batche of Herba Scutellariae Barbatae samples and3batchesof its confused plants from different sources.Results: The average recovery was found between94.2and110.0%andthe precision in terms of RSD was in the range of1.0~4.8%. The linearrelationships, linearity range, precision, stability, LOD and LOQ of themethod were good for the11components. The results demonstrated thatdifferent localizations and harvesting time might contribute to the differencesin the level of active constituents among various source of Herba ScutellariaeBarbatae samples. Also, there is variation in composition between HerbaScutellariae Barbatae and its confused speciesConclusion: The method was sensitive and suitable for the simultaneousanalysis of11constituents in Herba Scutellariae Barbatae and its confusedplants. It provided an effective method for the quality control of HerbaScutellariae Barbatae.