Chemical Composition Analysis And Quality Standard Study Of Qili Powder And Guixi Single Herbs

Qili San is a famous recipe commonly used in wound medicine,which is often used to treat various kinds of bruises and battering injuries and blood stasis pain.The main ingredients of Qili San are Draconis Sanguis,Olibanum,and Myrrha,which are commonly used in clinical practice.At present,there are deficiencies in the quality control means for the authenticity and safety of Qili San,and the 2020 edition of the Chinese Pharmacopoeia includes Draconis Sanguis,Olibanum,and Myrrha,but there are deficiencies in their identification and content determination,which are not conducive to their effective quality control.In this paper,based on the research idea from single drug to compound,a comprehensive qualitative analysis method of chemical composition was established based on the current pharmacopoeia quality standards,with the aim of improving the quality standards of Qili San and Draconis Sanguis,Olibanum,and Myrrha.In view of the fact that the chemical composition of Draconis Sanguis has not been fully elucidated,a comprehensive characterization of the chemical composition of Draconis Sanguis was first performed using an ultra-high performance liquid chromatography coupled with quadrupole-Orbitrap high resolution mass spectrometry(UHPLC-QE Orbitrap HRMS).A total of 58 chromatographic signals were identified,and 47 compounds were initially identified.The existing thin-layer chromatography(TLC)method was improved with the qualitative identification index components added,and high performance thin-layer chromatography(HPTLC)analysis was performed with dichloromethane-methanol-formic acid(40:1:1)as the unfolding agent.Each spot observed under 254 nm and 365 nm UV lamps,and colored by each vanillin was well separated and clearly developed.Four batches of Draconis Sanguis bought from market were identified of adulterating Resina Draconis.The characteristic spectra and multi-component content determination methods were established by UHPLC-DAD,and 23 common peaks including dracorhodin and(2S)-5-methoxy-6-methyflavanl-7-ol(MMF)were matched.The clustering analysis(CA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)were used to identify the common peaks with differences.As a result,MMF was highly correlated with the quality of Draconis Sanguis.Therefore,the content of dracorhodin and MMF was determined,and the content of dracorhodin in each batch was greater than 1%.Based on the results,it was determined that the content of MMF in Draconis Sanguis should not be less than 2%,therefore,a draft upgrading to the existing standards for Draconis Sanguis was proposed.To address the shortcomings of existing studies and quality standards of Olibanum.UHPLC-QE Orbitrap HRMS was used for the qualitative analysis of the non-volatile components,and 75 compounds were initially characterized from 95 peaks.A HPTLC method was established for the identification of Olibanum,and hexane-ethyl acetate-formic acid(4:1:0.1)was used as the unfolding agent.The separation of the spots was great under 254 nm UV lamp and after the color development of vanillin.A total of 38 compounds were identified by gas chromatography-mass spectrometry(GC-MS).Meanwhile.the GC.-MS fingerprint and content determination methods for incensole and incensole acetate of Olibanum was developed,and 19 common peaks including incensole and incensole acetate were identified from the fingerprint.The content of incensole in Olibanum should not be less than 2%and the content of incensole acetate should not be less than 1%.A draft upgrading to the existing standards for Olibanum was proposed.To address the shortcomings of the existing research and quality standards of Myrrha,the qualitative analysis of the non-volatile components of Myrrha based on UHPLC-QE Orbitrap HRMS was carried out,and 46 compounds were initially identified from 62 mass spectrometric peaks;The existing TLC identification method of Myrrha was improved by adding qualitative identification index components.Eleven batches of commercially available Myrrha were analyzed by HPTLC using hexane-ethyl acetate-formic acid(6:1:0.2)as the unfolding agent.The results showed that there were significant differences between the TLC spectra of Opoponax and Myrrha,and three batches of Opoponax and seven batches of Myrrha were identified from them,while another batch of Myrrha could not be identified for the time being because of the low content of each chemical component and inconspicuous spots.A GC-MS method was established for the qualitative analysis of chemical components,fingerprint and content determination of Myrrha,from which 52 compounds were initially identified,13 peaks were identified from Myrrha and 15 peaks from Opoponax,and rel-2R-methyl-5S-acetoxy-4Rfuranogermacr-1(10)Z-en-6-one(MAFEO)was identified from Myrrha.the content of which in natural myrrh was determined to be not less than 0.1%.On this basis,a draft upgraded of the current standard for Myrrha was proposed.To provide a basis for the comprehensive analysis of the chemical composition of Qili San,qualitative analysis of the chemical composition of the remaining single drugs of Qili San was carried out.A qualitative method of UHPLC-QE Orbitrap HRMS was established for catechu and safflower,from which 40 and 34 compounds were identified,respectively.The chemical composition of natural,musk and artificial musk were analyzed by GC-MS for comparison.The chemical composition of natural and cultured musk had the highest content of muscone,and the cultured musk also contained more steroidal components such as cholestan-3-ol,etc.The artificial musk had fewer components,mainly muscone and 5?-androstane-3,17-dione.Based on the systematic qualitative analysis of the chemical composition of each single drug of Qili san,UHPLC-QE Orbitrap HRMS was used to comprehensively characterize the chemical composition of Qili San,from which 91 compounds were initially identified.To address the shortcomings of the current TLC identification method of Qili San,a"one-plate,multi-test" HPTLC method was established to rapidly identify Draconis Sanguis,Catechu,Olibanum and Myrrha from Qili San;The characteristic spectra of Qili San were established by HPLC,from which 14 common peaks including catechin,epicatechin,dracorhodin and MMF were identified.The contents of catechin,epicatechin,dracorhodin,MMF and AKBA were determined simultaneously,and the contents of which should not be less than 3%,0.015%,0.5%,0.5% and 0.1%,respectively,in Qili San.A method for the determination of Borneol,muscone,incensole and incensole acetate in Qili San was established.In this regard,it was stipulated that Qili San shall not contain less than 0.1% of borneol,not less than 50 ppm of muscone,not less than 0.1% of incensole,and not less than 0.1% of incensole acetate;The inductively coupled plasma mass spectrometry(ICP-MS)was used for the determination of Hg,Pb,As,Cd,Cu and other heavy metals,harmful and trace elements in Qili San,among which two batches of Qili San had excessive Pb,and the content of Fe and manganese was relatively high.On the basis of the results of the above study,the draft quality standards of Qili San and the main noble herbs Draconis Sanguis,Olibanum,Myrrha were proposed to be improved.It provided a feasible analytical method for the improvement of the quality control level of the famous Chinese patent medicine Qili San and the commonly used precious fine drugs Draconis Sanguis,Olibanum,Myrrha,and provided a large amount of effective experimental data to ensure the safety and effectiveness of the clinical use.