Studies On Quality Control,Pharmacokinetics And Metabolomics Of Shuang-Huang-Lian Injection Based On Multidimensional Fusion Technology

Shuang-Huang-Lian Injection（lyophilized powder）（SHLI）is the earliest developed pure traditional Chinese medicine compound preparation for injection in China.It is composed of honeysuckle,forsythia and scutellaria baicalensis.Its main components are baicalin and chlorogenic acid.It has both antiviral and antibacterial effects.Its antibacterial effect is different from other commonly used anti-infective drugs.SHLI mainly including flavonoids,organic acids,iridoids,phenylethanol and lignin.Modern pharmacology shows that SHL has the effect of resisting influenza A,B and parainfluenza viruses（typesⅰ～ⅳ）,and has a positive regulating effect on human immune system.However,at present,the overall quality control,multi-component pharmacokinetics and metabolomics of SHLI are not perfect.Therefore,it is urgent to establish accurate,efficientive and sensitive modern instruments and methods to realize the overall quality control,multi-component pharmacokinetics and metabolomics of SHLI.Objective:this subject takes SHLI as the research object.Firstly,a method based on UPLC/Vion IMS-QTOF-MS was constructed to fully characterize and identify the chemical components of SHLI.Secondly,10 quality markers（chlorogenic acid,neochlorogenic acid,isochlorogenic acid B,isochlorogenic acid C,baicalin,baicalein,forsythin,forsythin A,luteolin and swertia glycoside）for quality control in SHLI were selected.Next,according to the chemical composition of SHLI and the main pharmacodynamic components specified in the Chinese Pharmacopoeia,the pharmacokinetics of 6 representative compounds in the five categories were studied;Finally,based on the idea of component traditional Chinese medicine,using the research strategy of"representative compound"-"component traditional Chinese medicine"-"compound preparation".Methods:Firstly,the liquid chromatography system was constructed by optimizing the stationary phase,mobile phase and mobile phase elution gradient.Then,the mass spectrum parameters are optimized,including capillary voltage,conical hole voltage and normalized collision energy.Based on UPLC/Vion IMS-QTOF-MS,it was further analyzed in negative ion mode.Honeysuckle,forsythia suspensa and scutellaria baicalensis,a"self built database of chemical components of SHL"was established,and a total of 326 known structural information of the relevant chemical components of SHL were recorded.The database is imported into UNIFI^TM software,and an efficient identification process based on UNIFI^TM automatic annotation of MSE data（including analysis method setting,data acquisition,data correction,data processing and identification,and reconfirmation of identification results）is established,combined with self built database,standard reference material information,reported literature and online database retrieval.The chemical constituents of SHLI were characterized.Secondly,the content of several quality markers in SHLI was determined simultaneously by using parallel reaction detection mode（PRM）in UPLC/Q-Orbitrap-MS.The quality control of multiple batches of SHLI samples was realized.In addition,21 sample pretreatment methods were used to optimize the best sample pretreatment method for the simultaneous determination of several effective components（chlorogenic acid,swertia glycoside,forsythin A,luteolin,baicalin and forsythin）with good pharmacokinetic behavior in rat plasma.Finally,the established UPLC/Vion IMS-Qtof-MS combined with unifitm metabolic platform method was used to determine the blood components and metabolites of SHLI.Conclusion:in order to realize the overall quality control of TCMs,taking SHLI as the research object,a variety of fast,effective and accurate methods have been established for comprehensive research.Firstly,UPLC/Vion IMS-Qtof-MS was used to quickly and comprehensively characterize the chemical components in SHLI.As a result,90 chemical components were identified or preliminarily identified;Secondly,based on the established PRM mode quantitative method in UPLC/Q-Orbitrap-MS,10 quality markers in SHLI were analyzed simultaneously;Next,a method was established for the simultaneous determination of several effective components（chlorogenic acid,swertia glycoside,forsythin a,luteolin,baicalin and forsythin）with good pharmacokinetic behavior in rat plasma;Finally,after the tail vein injection of SHL in rats,the blood components were preliminarily identified,including 62prototype compounds and 96 related metabolites.The identification and structure elucidation of these compounds provided necessary data for the study of pharmacology and pharmacokinetics of SHLI.To sum up,this study expounds the material basis of SHLI.It provides a method for its quality control.The above methods are helpful for the development of clinical dosage forms.