Study On The Analysis Method Of Alkaloid By Comprehensive 2D-LC With PH Modulation

Alkaloids are an important class of natural compounds and are one of the active ingredients of the medicinal plants.Most alkaloids have remarkable pharmacological and physiological activity,such as anti-cancer、anti-inflammatory、anti-hypotension、anti-virus,and so on.In recent years,hundreds of alkaloid can be used as clinical drugs.Due to the alkaloids has its unique effect,so the study and development has aroused people,s widespread concern for alkaloids.The alkaloids on separation and analysis methods of the literature has lots of reports,such as thin-layer chromatography,gas chromatography,liquid chromatography,high performance liquid chromatography.Alkaloids are usually existed in the complex system of plants and are low in content.In the face of such a complicated system,limited peak capacity,resolution and selectivity of one-dimensional chromatography is difficult to achieve efficient separation and analysis of alkaloids.In contrast,two-dimensional or ever multidimensional chromatography is one of powerful tools to solve the complex material system of traditional Chinese medicine.In this paper,the total alkaloids in the Chinese traditional medicine materials Corydalis yanhusuo W.T.Wang,Corydalis decumbens(Thunb.)Pers.and Aconitum carmichaeli Debx.were taken as the research object,the method that the analysis of alkaloids of based on p H-modulated two-dimensional liquid chromatography(2D-LC)was successfully established and the ingredients was efficient separation.The target peaks in the three herbs were identified by liquid chromatography-mass spectrometry(LC-MS),according to the chromatographic and mass spectrometric information of the reference standard and the data reported in the literature,the aim was to find and discover new compounds to provide a new approach.Main content and the results of this study are as follows:1.Study on analysis method of alkaloids in Corydalis yanhusuo W.T.Wang by two-dimensional liquid chromatography.A new two-dimensional(2D)reversed-phase chromatography(RP x RP)method based on p H modulation was used to analyze the alkaloids components in Corydalis yanhusuo.The first dimension chromatogram was separated byacetonitrile and 0.6% ammonia buffer solution as mobile phase and reversed phase(RP)column,the peak capacity was 94;The second dimension chromatographic was carried out using acetonitrile and 0.5% formic acid buffer as the mobile phase and strong cation exchange column to obtain a peak capacity of 205;The total capacity in Corydalis yanhusuo was 9090,and 16 compounds in the extract was identified by UV and MS,the compounds were Tetrahydroprotopapaverine(1),13-methyl-dehydrocorydalmine(2),Dehydrocorydaline(3),Demethylcorydalmine(4),Coptisine(5),Columbamine(6),Tetrahydrocolumbamine(7),13-methypalmatrubine(8),Protopine(9),D-glaucine(10),Dehydrocorybulbine(11),Dl-tetrahydropalmatine(12),Palmatine(13),Canadine(14),Dl-tetrahydrocoptisine(15),and Fumaricine(16).2.Study on analysis method of alkaloids in Corydalis decumbens(Thunb.)Pers.by two-dimensional liquid chromatography.The same method was used to analyze the alkaloid composition in Corydalis decumbens 0.6% ammonia buffer solution and acetonitrile as the mobile phase and reversed-phase(RP)columns for the first dimensional chromatogram separation,the peak capacity was 236;Acetonitrile and 0.5% formic acid buffer as the mobile phase and strong cation-exchange column was used to separate for the second dimension,the peak capacity was 88;The total capacity in Corydalis decumbens was 6925,and 24 compounds in the extract was identified by UV and MS,the compounds were Tetrahydroberberine(1),Bicuculline(2),Berberine(3),Corydaline(4),Coptisine(5),Columbamine(6),Tetrahydrocolumbamine(7),Scoulerine(8),Protopine(9),D-glaucine(10),Dehydrocorybulbine(11),Dl-tetrahydropalmatine(12),Palmatine(13),Cryptopine(14),Papaverine(15),and Fumaricine(16),Bulbocapnine(17),Allocryptopine(18),Kikemanine(19),Muramine(20),Egenine(21),13-methyl-dehydrocorydalmine(22),Tetrahydrocoptisine(23),and 13-methypalmatrubine(24).3.Study on analysis method of alkaloids in Aconitum carmichaeli Debx.by two-dimensional liquid chromatography.The same method was used to analyze the alkaloid composition in Aconitum carmichaeli.The first dimension chromatogram was separated by methanol and 0.5%ammonia buffer solution as mobile phase and reversed phase(RP)column,the peak capacity was141;The second dimension chromatographic was carried out using methanol and 0.1% formic acid buffer as the mobile phase and strong cation exchange column to obtain a peak capacity of131;The total capacity in Aconitum carmichaeli was 5700,and 10 compounds in the extract was identified by UV and MS,the compounds were senbusine A(1),9-hydroxysenbusine(2),14-dehydro-19-oxodelcosin(3),8-OEt-14-benzoylmesac Onitine(4),Mesaconitine(5),Hypaconine(6),Neoline(7),Delsoline(8),N-deethyl-14-benzoylaconine(9),14-acetylkarakoline(10).