| Huo-Xiang-Zheng-Qi Oral Liquid(HXZQOL)is composed of ten herbs,including Pogostemonis Herba(PH),Magnoliae Officinalis Cortex(MOC),Citri Reticulatae Pericarpium(CRP),Poria(P),Angelicae Dahuricae Radix(ADR),Pinelliae Rhizoma(PR),Perillae Folium(PF),Atractylodis Rhizoma(AR),Glycyrrhizae Radix et Rhizome(GRR),Arecae Pericarpium(AP).It is widely used for the treatment of the gastroenteric cold,acute gastroenteritis,treatment of functional dyspepsia(FD)and postinfectious irritable bowel syndrome(PI-IBS),etc.However,there are few the comprehensive analysis of material basis and quality evaluation of HXZQOL.Therefore,it is urgent to apply modern instrumental analysis techniques and multi-disciplinary research methods to analyze its medicinal components and clarify the action process in vivo.Based on UPLC,LC-MS and GC-MS technology,the study comprehensively uses fingerprint,chemical composition analysis,multi-component content determination,serum pharmacochemistry and metabolism in vivo research methods to achieve comprehensive characterization of chemical components,quality control,as well as analysis of blood components and urinary and fecal metabolic transformations.Obj ectives:The purpose of this experiment is to establish a quality control method for HXZQOL,comprehensively characterize the chemical composition,clarify its blood components and metabolic processes in vivo,summarize the metabolic transformation pathways,and which provide a scientific basis for its effective substances research.Methods:1.By optimizing the mobile phase,stationary phase,column temperature and elution gradient of liquid chromatography,the UPLC fingerprint of the preparation was established.Secondly,based on the ultra-high performance liquid chromatography tandem quadrupole time-of-flight high-resolution mass spectrometry technology(UHPLC-QTOF-MS),the chemical composition of HXZQOL was comprehensively analyzed under the positive and negative ion modes.2.By optimizing the key parameters such as extraction head,extraction time,extraction temperature and analysis time,the GC-MS fingerprint of volatile components of HXZQOL was constructed;Secondly,HS-SPME-GC-MS combined with GC-IMS technology was used to identify the volatile components of HXZQOL.3.Based on the above results for compound characterization in vitro,the components in plasma,urine and fecal samples of SD rats orally administered HXZQOL were analyzed to clarify the prototype compounds and metabolites.4.Finally,a UHPLC/PRM method was used to simultaneously quantify 9 index components to realize the quality control of multiple batches of HXZQOL.Results:1.The UPLC fingerprints of 15 batches of HXZQOL were established,and 7 components in 22 common peaks were confirmed by references,namely liquiritin,magnolol,hesperidin,glycyrrhizic acid,narirutin,honokiol and oxypeucedanin hydrate.The results of methodology verification showed that the method had certain specificity,and the RSD of precision,repeatability and stability data were all less than 2.0%.The established UPLC fingerprint could evaluate the quality of HXZQOL preparations.The non-targeted MSE approach is established based on UHPLC/IM-QTOF-MS,utilized for multicomponent comprehensive characterization of HXZQOL.Combined with in house library-driven automated peak annotation and comparison of 47 reference compounds,195 components were initially identified,mainly including flavonoids,lignans,triterpenes,coumarins,alkaloids,sesquiterpenoids and others.2.A total of 61 volatile organic compounds(VOCs)were identified by comparing the mass spectral fragments with the NIST17 database,and comparing the calculated retention indices of sample components(corrected for C8-C20 n-alkanes,RI)with the RI values of standard substances in the library.Based on this method,15 batches of volatile component fingerprints were established,and 26 common peaks were confirmed,and the similarity of the fingerprints was greater than 0.95.In addition,the small molecule volatile components of C2-C10 in HXZQOL were analyzed by GC-IMS technology.According to the built-in NIST14 gas phase retention index database and the IMS migration time database,35 signal peaks(containing dimers)were finally identified.Through the analysis of GC-MS and GC-IMS technology,the VOCs in HXZQOL mainly included terpenes,esters,aldehydes and hydrocarbons,as well as small molecular ketones and alcohols.3.The prototype compounds and metabolites in plasma,urine and feces of rats after oral administration of HXZQOL were analyzed based on UHPLC/Q-Orbitrap-MS and GC-MS.Fifty-five prototype compounds were detected from the plasma samples,of which six were volatile components,mainly flavonoids,triterpenoids,coumarins,lignans and terpenoids volatile components.The Compound discover software was used to analyze biological samples by establishing Workflows,and finally 219 metabolites were identified from blood,urine and feces,and the representative components with high content were selected to summarize the metabolic pathways.The detected prototype components and metabolites may be the medicinal components of HXZQOL,which lays the foundation for medicinal efficacy research.4.Based on the above results of plasma components analysis,the representative and high content prototype components were used as the index components for the determination of the content of HXZQOL.Using UHPLC/PRM(ESI+/ESI-switching)method,the contents of 9 components were detected,including liquiritin,magnolol,hesperidin,glycyrrhizic acid,narirutin,honokiol,nobiletin,poricoic acid A and oxypeucedanin hydrate.The method validation results showed that the 9 compounds had a good linear relationship within a certain range(R2≥0.9990),the RSD of precision,stability,and repeatability data were less than 3.0%,and the sample recovery rate was between 90%and 105%.The method was applied to the content determination of 15 batches of samples,and the results showed that the content of glycyrrhizic acid was the most abundant,while the content of poricoic acid A was the lowest.In addition,the content determination results of magnolol,honokiol and hesperidin were in line with the Pharmacopoeia.Conclusions:Based on UPLC,LC-MS and GC-MS technologies,this experiment completed the comprehensive characterization of the chemical components of HXZQOL,established the UPLC fingerprint and GC-MS volatile component fingerprint,as well as determined the content of 9 index components,which clarified the chemical composition of the formula and achieved the overall quality control of the HXZQOL preparation.In addition,the prototype compounds and metabolites in plasma,urine and fecal samples of rats after administration were characterized to obtain a comprehensive metabolic profile in vivo,and the metabolic pathways were summarized.In this study,the fingerprint,chemical composition,content determination,blood-introducing composition and metabolic process of HXZQOL were systematically studied.The pharmacodynamic material basis of HXZQOL was preliminarily described,and it also provided the basis for quality control method and clinical research. |
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