CN117330692B - 基于uplc-q-tof-ms/ms技术分析甘草多糖的方法 - Google Patents
基于uplc-q-tof-ms/ms技术分析甘草多糖的方法 Download PDFInfo
- Publication number
- CN117330692B CN117330692B CN202311628162.5A CN202311628162A CN117330692B CN 117330692 B CN117330692 B CN 117330692B CN 202311628162 A CN202311628162 A CN 202311628162A CN 117330692 B CN117330692 B CN 117330692B
- Authority
- CN
- China
- Prior art keywords
- uplc
- tof
- analyzing
- mass spectrum
- polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 72
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 72
- -1 liquorice polysaccharide Chemical class 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000005516 engineering process Methods 0.000 title claims abstract description 28
- 238000004885 tandem mass spectrometry Methods 0.000 title claims abstract description 27
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 title claims abstract description 12
- 244000303040 Glycyrrhiza glabra Species 0.000 title claims abstract description 8
- 235000011477 liquorice Nutrition 0.000 title claims abstract description 8
- 150000002500 ions Chemical class 0.000 claims abstract description 118
- 238000001819 mass spectrum Methods 0.000 claims abstract description 59
- 239000000126 substance Substances 0.000 claims abstract description 47
- 241000202807 Glycyrrhiza Species 0.000 claims abstract description 46
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 238000010828 elution Methods 0.000 claims abstract description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 57
- 238000003756 stirring Methods 0.000 claims description 26
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 25
- 150000004676 glycans Chemical class 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 19
- 238000004458 analytical method Methods 0.000 claims description 17
- 238000009210 therapy by ultrasound Methods 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 14
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 14
- 238000011049 filling Methods 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 14
- 238000005336 cracking Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 12
- 235000019253 formic acid Nutrition 0.000 claims description 12
- 239000003921 oil Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 9
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 7
- 230000005587 bubbling Effects 0.000 claims description 7
- 229950005499 carbon tetrachloride Drugs 0.000 claims description 7
- 230000014759 maintenance of location Effects 0.000 claims description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- 230000000087 stabilizing effect Effects 0.000 claims description 7
- 229910001220 stainless steel Inorganic materials 0.000 claims description 7
- 239000010935 stainless steel Substances 0.000 claims description 7
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 7
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 7
- 239000012498 ultrapure water Substances 0.000 claims description 7
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 6
- 238000001704 evaporation Methods 0.000 claims description 6
- 239000012621 metal-organic framework Substances 0.000 claims description 6
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 235000010413 sodium alginate Nutrition 0.000 claims description 6
- 229940005550 sodium alginate Drugs 0.000 claims description 6
- 239000000661 sodium alginate Substances 0.000 claims description 6
- XDLMVUHYZWKMMD-UHFFFAOYSA-N 3-trimethoxysilylpropyl 2-methylprop-2-enoate Chemical compound CO[Si](OC)(OC)CCCOC(=O)C(C)=C XDLMVUHYZWKMMD-UHFFFAOYSA-N 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 claims description 5
- 238000004132 cross linking Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000006116 polymerization reaction Methods 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 4
- 238000000889 atomisation Methods 0.000 claims description 3
- 238000007405 data analysis Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 2
- 239000012982 microporous membrane Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 21
- 238000004704 ultra performance liquid chromatography Methods 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 5
- 238000012545 processing Methods 0.000 abstract description 4
- 239000003643 water by type Substances 0.000 abstract description 4
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 description 50
- 238000012360 testing method Methods 0.000 description 37
- 238000003776 cleavage reaction Methods 0.000 description 32
- 230000007017 scission Effects 0.000 description 32
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 28
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 description 27
- 229960004949 glycyrrhizic acid Drugs 0.000 description 27
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 description 27
- 235000019410 glycyrrhizin Nutrition 0.000 description 27
- 239000004378 Glycyrrhizin Substances 0.000 description 18
- 230000037361 pathway Effects 0.000 description 16
- 238000000926 separation method Methods 0.000 description 14
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 description 12
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 12
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 description 9
- 239000001685 glycyrrhizic acid Substances 0.000 description 9
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 8
- JBQATDIMBVLPRB-UHFFFAOYSA-N isoliquiritigenin Natural products OC1=CC(O)=CC=C1C1OC2=CC(O)=CC=C2C(=O)C1 JBQATDIMBVLPRB-UHFFFAOYSA-N 0.000 description 8
- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 description 7
- 229940117954 naringenin Drugs 0.000 description 7
- 235000007625 naringenin Nutrition 0.000 description 7
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 6
- 239000001606 7-[(2S,3R,4S,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)chroman-4-one Substances 0.000 description 6
- 239000004475 Arginine Substances 0.000 description 6
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 6
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 6
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 description 6
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 description 6
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 6
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 6
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 229960003720 enoxolone Drugs 0.000 description 6
- 229930003935 flavonoid Natural products 0.000 description 6
- 235000017173 flavonoids Nutrition 0.000 description 6
- 229940029575 guanosine Drugs 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- DXDRHHKMWQZJHT-FPYGCLRLSA-N isoliquiritigenin Chemical compound C1=CC(O)=CC=C1\C=C\C(=O)C1=CC=C(O)C=C1O DXDRHHKMWQZJHT-FPYGCLRLSA-N 0.000 description 6
- 235000008718 isoliquiritigenin Nutrition 0.000 description 6
- 229960004488 linolenic acid Drugs 0.000 description 6
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 6
- 238000004949 mass spectrometry Methods 0.000 description 6
- DFPMSGMNTNDNHN-ZPHOTFPESA-N naringin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC=2C=C3O[C@@H](CC(=O)C3=C(O)C=2)C=2C=CC(O)=CC=2)O[C@H](CO)[C@@H](O)[C@@H]1O DFPMSGMNTNDNHN-ZPHOTFPESA-N 0.000 description 6
- 229930019673 naringin Natural products 0.000 description 6
- 229940052490 naringin Drugs 0.000 description 6
- 229940100243 oleanolic acid Drugs 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 6
- 238000002791 soaking Methods 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 238000000921 elemental analysis Methods 0.000 description 5
- 150000002215 flavonoids Chemical class 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 4
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 description 4
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 229940097043 glucuronic acid Drugs 0.000 description 4
- 229940010454 licorice Drugs 0.000 description 4
- 239000002777 nucleoside Substances 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical group C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 229930194248 Licoflavone Natural products 0.000 description 3
- MEHHCBRCXIDGKZ-UHFFFAOYSA-N Licoflavone C Natural products CC(C)=CCC1=C(O)C=C(O)C(C(C=2)=O)=C1OC=2C1=CC=C(O)C=C1 MEHHCBRCXIDGKZ-UHFFFAOYSA-N 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 229960000956 coumarin Drugs 0.000 description 3
- 235000001671 coumarin Nutrition 0.000 description 3
- 150000004775 coumarins Chemical class 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000000017 hydrogel Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 229930182493 triterpene saponin Natural products 0.000 description 3
- 150000003648 triterpenes Chemical class 0.000 description 3
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 2
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 2
- FURUXTVZLHCCNA-UHFFFAOYSA-N Liquiritigenin Natural products C1=CC(O)=CC=C1C1OC2=CC(O)=CC=C2C(=O)C1 FURUXTVZLHCCNA-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004896 high resolution mass spectrometry Methods 0.000 description 2
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 2
- FURUXTVZLHCCNA-AWEZNQCLSA-N liquiritigenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC=C2C(=O)C1 FURUXTVZLHCCNA-AWEZNQCLSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 2
- 238000007142 ring opening reaction Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- GSZUGBAEBARHAW-UHFFFAOYSA-N sophoraflavone B Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C=2OC3=CC(O)=CC=C3C(=O)C=2)C=C1 GSZUGBAEBARHAW-UHFFFAOYSA-N 0.000 description 2
- 150000008130 triterpenoid saponins Chemical class 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- IGJIPNOEJDYNRR-UHFFFAOYSA-N 9-(1,2-dihydroxyethyl)-6,9,11-trihydroxy-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione Chemical group C1C(O)(C(O)CO)CCC2=C1C(O)=C1C(=O)C(C=CC=C3OC)=C3C(=O)C1=C2O IGJIPNOEJDYNRR-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N flavone Chemical class O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182480 glucuronide Natural products 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000004190 ion pair chromatography Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- NWMIYTWHUDFRPL-UHFFFAOYSA-N sapogenin Natural products COC(=O)C1(CO)C(O)CCC2(C)C1CCC3(C)C2CC=C4C5C(C)(O)C(C)CCC5(CCC34C)C(=O)O NWMIYTWHUDFRPL-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
- B01J20/045—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium containing sulfur, e.g. sulfates, thiosulfates, gypsum
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/223—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material containing metals, e.g. organo-metallic compounds, coordination complexes
- B01J20/226—Coordination polymers, e.g. metal-organic frameworks [MOF], zeolitic imidazolate frameworks [ZIF]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
- B01J20/267—Cross-linked polymers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
本发明公开了一种基于UPLC‑Q‑TOF‑MS/MS技术分析甘草多糖的方法,采用Waters Acquity UPLC BEH‑C18色谱柱;乙腈‑甲酸水溶液为流动相进行梯度洗脱,固定相为改性固定相;采用电喷雾离子源,在正、负离子模式下对甘草多糖进行质谱扫描;通过软件进行数据采集和数据处理;根据高分辨质谱提供的化合物相对分子质量、离子碎片信息,对甘草多糖中化学成分进行分析。与现有技术相比,本发明采用的UPLC‑Q‑TOF‑MS/MS技术能有效分析出甘草多糖中化学成分,可为甘草多糖后续实验提供支撑。
Description
技术领域
本发明涉及检测技术领域,尤其涉及一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法。
背景技术
基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,目前已经有一些现有的技术和方法。以下是其中几种常用的方法:离子对色谱法,该方法通过添加离子对试剂,如三氟乙酸、甲酸等,以增强多糖化合物在UPLC柱上的保留和分离效果。离子对试剂与多糖之间形成离子对复合物,提高了多糖的检测灵敏度和分离度。聚合物逆向相色谱法,该方法使用聚合物修饰的反相色谱柱,如聚合物甲基丙烯酸酯柱、聚合物乙烯基苯柱等,增强了多糖的保留和分离效果,并提高了质谱的信号强度。阴离子交换色谱法,该方法使用具有阴离子交换功能的色谱柱,如强阴离子交换柱,通过控制流动相中的pH值和离子强度,实现多糖的分离和定量分析。聚合物凝胶色谱法,该方法使用聚合物凝胶柱,如聚丙烯酰胺凝胶,通过多糖在凝胶柱中的分子大小和形状的差异,实现多糖的分离和纯化。质谱联用技术,将UPLC与高分辨质谱仪联用,实现多糖的分子式、分子量和结构鉴定。通过质谱的高灵敏度和高分辨率,可以准确分析复杂的多糖混合物。
这些方法需要根据实际需要进行选择和优化,以获得高效、灵敏和准确的甘草多糖分析结果。
中国发明授权专利CN109553654B公开了一种从甘草中提取甘草甜素、甘草黄酮及甘草多糖的方法,所述从甘草中提取甘草甜素的方法,包括以下步骤:(1)将干燥的甘草根或根茎粉碎,投入渗漉器中,压紧,加水,在室温下,渗漉提取,得渗漉液;(2)超滤,收集超滤膜透过液;(3)上阴离子交换树脂柱,用洗脱剂洗脱,洗脱液减压浓缩,调节pH值至酸性,在室温下,搅拌析晶,离心过滤,冰水洗晶,干燥,得甘草甜素。该发明方法还公开了提取甘草黄酮及甘草多糖的方法。该发明方法所得甘草甜素、甘草黄酮和甘草多糖的纯度和收率高;该发明方法可提取多种活性成分,工艺过程可操作性强,成本低,不使用有毒有害化工溶剂、绿色环保,甘草资源高效综合利用,适宜于工业化生产。但是该发明提取方法分离性能差,不能完全提取甘草中的各种成分。
发明内容
有鉴于现有技术中甘草多糖的提取方法分离性能差的缺点,本发明所要解决的技术问题是提供一种分离性能好的基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法。
为了实现上述发明目的,本发明采用了如下的技术方案:
一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法:
步骤1、供试品溶液配制:称取甘草多糖,加入超纯水,超声处理溶解后,摇匀,制得甘草多糖溶液,经微孔滤膜过滤,得到供试品溶液;
步骤2、色谱条件:采用Waters ACQUITY UPLC BEH C18色谱柱(2.1mm×100 mm,1.7μm);采用流动相梯度洗脱,固定相为改性固定相;
步骤3、质谱条件:采用电喷雾离子源(ESI),正、负离子2种模式扫描,质谱扫描;正离子模式下,负离子模式下,毛细管电压-4000~-5000kV,裂解电压-70~-90V,碰撞能量30~40eV;
步骤4、数据分析、采用Analyst TF 1.8.1数据采集软件(美国 AB SCIEXI 公司),采集时间20~40min;采用SCIEX OS 2.0.0软件进行数据处理(美国AB SCIEXI 公司),化学成分依据相对分子质量、离子碎片信息、保留时间,结合数据库及文献报道数据结果进行分析。
优选的,所述步骤1中为称取0.5~2重量份甘草多糖,加入超纯水,超声溶解后,摇匀,制得浓度为4~6mg/mL的甘草多糖溶液。
优选的,所述步骤1中微孔滤膜的孔径为0.2~0.25μm。
优选的,所述步骤2中流动相为乙腈和0.05~0.2wt%甲酸水溶液配置而成。
优选的,所述步骤2中流动相梯度洗脱参数为:0~2min,流动相由5%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;2~42min,流动相由5%~95%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;42~47min,流动相由95%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;47~47.1min,流动相由5%~95%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;47.1~50min,流动相由5%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;流速0.2~0.4mL/min;柱温:35~45℃;进样量4~6μL。
优选的,所述步骤3中质谱扫描范围m/z 100~1500Da,雾化气压50~60PSI,辅助气压50~60PSI,气帘气压30~40PSI。
优选的,所述步骤3中正离子模式下参数为:毛细管电压5000~6000V,裂解电压80~120V,碰撞能量30~40eV。
优选的,所述步骤3中负离子模式下参数为:毛细管电压-4000~-5000kV,裂解电压-70~-90V,碰撞能量30~40eV。
所述改性固定相的制备方法如下,以重量份计:
S1、将4~6份二氧化硅粉末、8~12份3-(异丁烯酰氧)丙基三甲氧基硅烷、8~12份水和0.5~2份乙酸加入到18~22份无水乙醇中,在50~70℃油浴中搅拌20~40min,静置3~5h,得到预处理物;
S2、将0.1~0.3份甲基丙烯酰胺、0.04~0.06份海藻酸钠和0.04~0.06份N,N'-亚甲基-双丙烯酰胺加入到30~50份水中,进行超声处理,然后加入2~4份步骤S1制备的预处理物,100~500rpm磁力搅拌0.5~2h,静置8~15h,再加入0.04~0.06份过硫酸铵、0.004~0.006份硫酸钙和0.1~0.2份金属有机骨架材料,在50~70℃油浴中进行交联聚合1~3h,用氮气鼓泡除去混合物中溶解的空气,然后加热至50~70℃,100~300rpm搅拌回流冷凝4~6h,随后,温度加热到70~90℃,在100~200rpm搅拌下完全蒸发溶剂,然后,加入40~60份水浸泡一夜,最后用水洗涤1~3次,在70~90℃真空烘箱中干燥5~15h,得到后处理物;
S3、将2~3份后处理物在20~30份四氯甲烷中超声处理,以正己烷为推进液,在30~50Mpa的压力下将混合物填充到140~160×4~5mm不锈钢管中,当流出的液体体积超过100mL时,床层稳定,充填完成,得到改性固定相。
优选的,所述超声处理时间各自独立地为10~30min、超声功率各自独立地为200~500W、超声频率各自独立地为20~60kHz。
本发明采用UPLC-Q-TOF-MS/MS技术对甘草多糖化学成分进行定性分析,以明确甘草多糖中化学成分。
采用Waters Acquity UPLC BEH-C18色谱柱(2.1 mm×100 mm,1.7 μm);乙腈-甲酸水溶液为流动相进行梯度洗脱,流速0.3 mL/min,柱温40℃,进样量5μL,固定相为改性固定相。采用电喷雾离子源,在正、负离子模式下对甘草多糖进行质谱扫描。通过Analyst TF1.8.1软件和SCIEX OS 2.0.0软件进行数据采集和数据处理。根据高分辨质谱提供的化合物相对分子质量、离子碎片信息,参考文献报道的质谱数据并对比数据库分析结果,对甘草多糖中化学成分进行分析。
在正、负离子模式下共分析出95种化学成分,其中包括57种黄酮类成分,18种三萜类成分,4种香豆素类成分,3种有机酸类成分,其他类包括7种氨基酸类成分、3种糖类成分、3种核苷类成分,共13种。UPLC-Q-TOF-MS/MS技术能有效分析出甘草多糖中化学成分,可为甘草多糖后续实验提供支撑。
与现有技术相比,本发明的有益效果:
1)本发明通过使用UPLC-Q-TOF-MS/MS技术能有效分析出甘草多糖中化学成分,可为甘草多糖后续实验提供支撑。
2)本发明采用的改性固定相中存在多模式相互作用,具有更好的分离性能和良好的稳定重复性,在实际应用中具有重要作用,拓展了在色谱分离领域的潜在应用。
附图说明
图1为测试例1中正离子模式甘草多糖总离子流图。
图2为测试例1中负离子模式甘草多糖总离子流图。
图3为测试例2中柚皮苷二级质谱图。
图4为测试例2中柚皮素二级质谱图。
图5为测试例2中柚皮苷、柚皮素的裂解途径。
图6为测试例2中甘草素二级质谱图。
图7为测试例2中甘草素的裂解途径。
图8为测试例2中异甘草素二级质谱图。
图9为测试例2中异甘草素的裂解途径。
图10为测试例3中甘草酸二级质谱图。
图11为测试例3中甘草酸的裂解途径。
图12为测试例3中齐墩果酸二级质谱图。
图13为测试例3中齐墩果酸的裂解途径。
图14为测试例3中甘草皂苷H2二级质谱图。
图15为测试例3中甘草皂苷H2的裂解途径。
图16为测试例3中甘草次酸二级质谱图。
图17为测试例3中甘草次酸的裂解途径。
图18为测试例4中甘草酚二级质谱图。
图19为测试例4中甘草酚的裂解途径。
图20为测试例5中亚麻酸二级质谱图。
图21为测试例5中亚麻酸的裂解途径。
图22为测试例6中精氨酸二级质谱图。
图23为测试例6中精氨酸的裂解途径。
图24为测试例6中蔗糖二级质谱图。
图25为测试例6中蔗糖的裂解途径。
图26为测试例6中鸟苷二级质谱图。
图27为测试例6中鸟苷的裂解途径。
具体实施方式
主要物质来源:
甘草多糖(批号190201)由康奇生物技术股份有限公司提供;
甲酸(色谱纯,批号158673)、乙腈(色谱纯,批号215625)购自美国FisherChemical 公司;
超纯水(Milli-Q超纯水仪制备,德国Merck Milipore公司);
Triple TOF 5600高分辨质谱仪(美国AB SCIEXI 公司);
ExionLC AD型液相色谱仪(美国AB SCIEXI 公司);
FA2004B电子天平(上海天美天平仪器有限公司);
JP-060S 结盟牌超声波清洗机(深圳市结盟清洗设备有限公司);
ST16R型高速低温离心机(美国Thermofisher Scientific公司);
二氧化硅粉末:济南卡松化工有限公司,型号:A300,目数:100-3000目;
金属有机骨架材料:西安瑞禧生物科技有限公司,货号:1237123。
实施例1
一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法:
步骤1、供试品溶液配制:称取1g甘草多糖,加入超纯水,超声溶解后,超声功率为300W、超声频率为40kHz,摇匀,制得浓度为5mg/mL的甘草多糖溶液,经0.22μm微孔滤膜过滤,得到供试品溶液;
步骤2、色谱条件:采用Waters ACQUITY UPLC BEH C18色谱柱(2.1mm×100 mm,1.7μm);乙腈和0.1wt%甲酸水溶液配置为流动相,梯度洗脱(1min,流动相由5%乙腈、0.1wt%甲酸水溶液补足100%组成;20min,流动相由55%乙腈、0.1wt%甲酸水溶液补足100%组成;45min,流动相由95%乙腈、0.1wt%甲酸水溶液补足100%组成;47min,流动相由55%乙腈、0.1wt%甲酸水溶液补足100%组成;48min,流动相由5%乙腈、0.1wt%甲酸水溶液补足100%组成);流速0.3mL/min;柱温:40℃;进样量5μL,固定相为改性固定相;
步骤3、质谱条件:采用电喷雾离子源(ESI),正、负离子2种模式扫描,质谱扫描范围m/z 100~1500Da,雾化气压55PSI,辅助气压55PSI,气帘气压35PSI;正离子模式下,毛细管电压5500V,裂解电压100V,碰撞能量35eV;负离子模式下,毛细管电压-4500kV,裂解电压-80V,碰撞能量35eV;
步骤4、数据分析、采用Analyst TF 1.8.1数据采集软件(美国 AB SCIEXI 公司),采集时间30 min;采用SCIEX OS 2.0.0软件进行数据处理(美国AB SCIEXI 公司),化学成分依据相对分子质量、离子碎片信息、保留时间,结合数据库及文献报道数据结果进行分析。
所述改性固定相的制备方法如下:
S1、将5g二氧化硅粉末、10g 3-(异丁烯酰氧)丙基三甲氧基硅烷、10g水和1g乙酸加入到20g无水乙醇中,在60℃油浴中搅拌30min,静置4h,得到预处理物;
S2、将0.2g甲基丙烯酰胺、0.05g海藻酸钠和0.05g N,N'-亚甲基双丙烯酰胺加入到40g水中,进行超声处理20min、超声功率为300W、超声频率为40kHz,然后加入3g步骤S1制备的预处理物,300rpm磁力搅拌1h,静置12h,再加入0.05g过硫酸铵、0.005g硫酸钙和0.15g金属有机骨架材料,在60℃油浴中进行交联聚合2h,用氮气鼓泡除去混合物中溶解的空气,然后加热至60℃,150rpm搅拌回流冷凝5h,随后,温度加热到80℃,在150rpm搅拌下完全蒸发溶剂,然后,加入50g水浸泡一夜,最后用水洗涤3次,在80℃真空烘箱中干燥12h,得到后处理物;
S3、将2.2g后处理物在22g四氯甲烷中超声处理10min、超声功率为400W、超声频率为40kHz,以正己烷为推进液,在40Mpa的压力下将混合物填充到150×4.6mm不锈钢管中,当流出的液体体积超过100mL时,床层稳定,充填完成,得到改性固定相。
对比例1
一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法与实施例1基本相同,唯一区别仅仅在于:所述改性固定相的制备方法不同。
所述改性固定相的制备方法如下:
S1、将0.2g甲基丙烯酰胺、0.05g海藻酸钠和0.05g N,N'-亚甲基双丙烯酰胺加入到40g水中,进行超声处理20min、超声功率为300W、超声频率为40kHz,然后加入3g二氧化硅粉末,300rpm磁力搅拌1h,静置12h,再加入0.05g过硫酸铵、0.005g硫酸钙和0.15g金属有机骨架材料,在60℃油浴中进行交联聚合2h,用氮气鼓泡除去混合物中溶解的空气,然后加热至60℃,150rpm搅拌回流冷凝5h,随后,温度加热到80℃,在150rpm搅拌下完全蒸发溶剂,然后,加入50g水浸泡一夜,最后用水洗涤3次,在80℃真空烘箱中干燥12h,得到后处理物;
S2、将2.2g后处理物在22g四氯甲烷中超声处理10min、超声功率为400W、超声频率为40kHz,以正己烷为推进液,在40Mpa的压力下将混合物填充到150×4.6mm不锈钢管中,当流出的液体体积超过100mL时,床层稳定,充填完成,得到改性固定相。
对比例2
一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法与实施例1基本相同,唯一区别仅仅在于:所述改性固定相的制备方法不同。
所述改性固定相的制备方法如下:
S1、将5g二氧化硅粉末、10g 3-(异丁烯酰氧)丙基三甲氧基硅烷、10g水和1g乙酸加入到20g无水乙醇中,在60℃油浴中搅拌30min,静置4h,得到预处理物;
S2、在40g水中加入3g步骤S1制备的预处理物,300rpm磁力搅拌1h,静置12h,再加入0.05g过硫酸铵、0.005g硫酸钙和0.15g金属有机骨架材料,在60℃油浴中保存2h,用氮气鼓泡除去混合物中溶解的空气,然后加热至60℃,150rpm搅拌回流冷凝5h,随后,温度加热到80℃,在150rpm搅拌下完全蒸发溶剂,然后,加入50g水浸泡一夜,最后用水洗涤3次,在80℃真空烘箱中干燥12h,得到后处理物;
S3、将2.2g后处理物在22g四氯甲烷中超声处理10min、超声功率为400W、超声频率为40kHz,以正己烷为推进液,在40Mpa的压力下将混合物填充到150×4.6mm不锈钢管中,当流出的液体体积超过100mL时,床层稳定,充填完成,得到改性固定相。
对比例3
一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法与实施例1基本相同,唯一区别仅仅在于:所述改性固定相的制备方法不同。
所述改性固定相的制备方法如下:
S1、将5g二氧化硅粉末、10g 3-(异丁烯酰氧)丙基三甲氧基硅烷、10g水和1g乙酸加入到20g无水乙醇中,在60℃油浴中搅拌30min,静置4h,得到预处理物;
S2、将0.2g甲基丙烯酰胺、0.05g海藻酸钠和0.05g N,N'-亚甲基双丙烯酰胺加入到40g水中,进行超声处理20min、超声功率为300W、超声频率为40kHz,然后加入3g步骤S1制备的预处理物,300rpm磁力搅拌1h,静置12h,再加入0.05g过硫酸铵、0.005g硫酸钙,在60℃油浴中保存2h,用氮气鼓泡除去混合物中溶解的空气,然后加热至60℃,150rpm搅拌回流冷凝5h,随后,温度加热到80℃,在150rpm搅拌下完全蒸发溶剂,然后,加入50g水浸泡一夜,最后用水洗涤3次,在80℃真空烘箱中干燥12h,得到后处理物;
S3、将2.2g后处理物在22g四氯甲烷中超声处理10min、超声功率为400W、超声频率为40kHz,以正己烷为推进液,在40Mpa的压力下将混合物填充到150×4.6mm不锈钢管中,当流出的液体体积超过100mL时,床层稳定,充填完成,得到改性固定相。
对比例4
一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法与实施例1基本相同,唯一区别仅仅在于:所述改性固定相的制备方法不同。
所述改性固定相的制备方法如下:
S1、在40g水中加入3g二氧化硅粉末,300rpm磁力搅拌1h,静置12h,再加入0.05g过硫酸铵、0.005g硫酸钙,在60℃油浴中保存2h,用氮气鼓泡除去混合物中溶解的空气,然后加热至60℃,150rpm搅拌回流冷凝5h,随后,温度加热到80℃,在150rpm搅拌下完全蒸发溶剂,然后,加入50g水浸泡一夜,最后用水洗涤3次,在80℃真空烘箱中干燥12h,得到后处理物;
S2、将2.2g后处理物在22g四氯甲烷中超声处理10min、超声功率为400W、超声频率为40kHz,以正己烷为推进液,在40Mpa的压力下将混合物填充到150×4.6mm不锈钢管中,当流出的液体体积超过100mL时,床层稳定,充填完成,得到改性固定相。
测试例1
甘草多糖化学成分分析
将实施例1和对比例1~4采用UPLC-Q-TOF-MS/MS技术对甘草多糖进行质谱检测,按照色谱、质谱条件进行分析,得到正、负离子模式下甘草多糖总离子流图见图1和图2,根据所分析化学成分相对分子质量、二级质谱裂解碎片信息,甘草多糖中所分析化学成分的分子式、相对分子质量、二级质谱碎片离子信息和成分类别。其中实施例1和对比例1~4分析出的化学成分种类统计如表1。
表1 甘草多糖化学成分测试结果
从实施例1的测试数据可以看出,本发明实施例1的方法测出的种类成分最多,实施例1与对比例1相比可能原因在于本发明得到的预处理物为八面体结构,尺寸均匀,由于预处理物的掺入,得到的改性固定相呈现出粗糙的表面,具有核壳结构,改性固定相中存在多模式相互作用。比二氧化硅具有更好的分离性能。
实施例1与对比例2-3相比,可能原因在于丙烯酰胺、海藻酸钠和N,N'-亚甲基-双丙烯酰胺可以形成复合水凝胶结构,复合水凝胶的修饰改善了改性固定相上的色谱峰形,具有很好的选择性。此外,保留时间随苯单元数的增加而增加,这表明相互作用可能在多环芳烃的分离中起关键作用。改性固定相由于加入了含有丰富芳环并能提供相互作用的预处理物,具有更好的峰形和选择性。将预处理物加入到水凝胶聚合物网络中,不仅可以解决柱压不稳定的缺点,还可以通过提供额外的相互作用位点(如金属位点和苯单元)提高分离选择性,改性固定相在分离样品过程中具有亲水性、疏水性、π-π等多重相互作用。此外,改性固定相具有良好的稳定性和重现性,在实际应用中具有重要作用,拓展了在色谱分离领域的潜在应用。
测试例2
黄酮类成分分析
黄酮类化合物是指母核为2-苯基色原酮的一类化合物,在质谱裂解中常易发生糖苷键断裂或C环的RDA裂解以及失去-CH3、CO、CO2、H2O等碎片分子。通过与数据库和文献报道化合物比对,共分析出57种黄酮类化合物。本发明以柚皮苷和柚皮素、甘草素和异甘草素为例,分析黄酮类化合物裂解规律。
柚皮苷,负离子模式下,一级质谱图显示其准分子离子峰为m/z 579.1753 [M-H]-,tR=7.19 min,分子式为C27H32O14,观察其二级高分辨质谱,主要碎片离子包括m/z255.0681 [M-H-C6H10O4-C6H10O5-H2O]-、m/z 135.0095 [M-H-C8H8O2]-、m/z 119.0504 [M-H-C7H4O4]-。m/z 255.0681为准分子离子峰脱去一分子鼠李糖和葡萄糖后所得苷元进一步脱去1分子H2O产生的碎片离子,m/z 135.0095、m/z 119.0504碎片离子均为苷元C环发生RDA裂解产生,分析该化合物为柚皮苷。
柚皮素,一级质谱准分子离子峰为m/z 271.0632 [M-H]-,tR=11.46 min,推测其分子式为C15H12O5,二级高分辨质谱显示主要碎片离子包括m/z 177.0203 [M-H-C6H6O]-、m/z 151.0051 [M-H-C8H8O]-、m/z 135.0465 [M-H-C8H8O2]-、m/z 119.0515 [M-H-C7H4O4]-、m/z 107.0147 [M-H-C8H8O2-CO]-。准分子离子失去1分子C6H6O得到m/z177.0203碎片离子,失去1分子C8H8O得到m/z 151.0051碎片离子,m/z 135.0465 与m/z119.0515碎片离子为化合物C环发生RDA裂解产生,m/z 107.0147碎片离子为m/z 135.0465碎片进一步失去1分子CO产生,经与文献中柚皮素特征峰比对,分析该化合物为柚皮素。柚皮苷、柚皮素二级质谱图及裂解过程见图3-5。
甘草素,正离子模式下,一级质谱显示其准分子离子峰为m/z 257.0813 [M+H]+ ,tR=12.74 min,依据元素分析结果推测其分子式为C15H12O4。二级质谱图中主要碎片离子有m/z 137.0227 [M+H-C7H4O2]+、m/z 119.0493 [M+H-C7H4O3]+,以上均为化合物C环发生RDA裂解产生的特征碎片离子,故推断该化合物为甘草素。异甘草素,准分子离子峰为m/z257.0809 [M+H]+,tR=12.75 min,分子式C15H12O4,出峰略晚于甘草素,为甘草素C环开环后产物,与甘草素互为同分异构体,二级质谱显示其与甘草素具有相同裂解途径,分析该化合物为为异甘草素。甘草素和异甘草素二级质谱图及裂解途径见图6-9。
测试例3
三萜皂苷类成分分析
三萜皂苷类化合物由三萜皂苷元和糖类部分组成,在质谱裂解中三萜皂苷类化合物受离子源轰击后易失去中性葡萄糖醛酸分子,其余糖苷配基上易失去CH3、H2O、CO等中性小分子。通过与数据库和文献报道化合物比对,共分析出18种三萜皂苷类成分。本发明中以甘草酸、齐墩果酸、甘草皂苷H2和甘草次酸为例,分析三萜皂苷类化合物裂解规律。
甘草酸,正离子模式下其准分子离子峰为m/z 823.4103[M+H]+,tR=15.63 min,分析其元素组成为C42H62O16,二级质谱图显示其碎片离子有m/z 647.3794 [M+H-C6H9O6]+、m/z471.3479 [M+H-C6H9O6-C6H8O6]+、m/z 453.3357 [M+H-C6H9O6-C6H8O6-H2O]+。准分子离子失去1分子葡萄糖醛酸产生m/z 647.3794碎片离子,失去2分子葡萄糖醛酸产生m/z 471.3479碎片离子 ,随后再失去1分子水产生m/z 453.3357碎片离子,分析该化合物为甘草酸。甘草酸二级质谱图及裂解途径见图10-11。
齐墩果酸,正离子模式下显示其准分子离子峰为m/z 457.3703 [M+H]+,tR=16.27min,分子式为C30H48O3,二级质谱图中主要碎片离子有m/z 439.3470 [M+H-H2O]+、m/z191.1721 [M+H-H2O-C15H21-COOH]+。准分子离子峰失去1分子H2O得到m/z 439.3470碎片离子,随后,化合物C环发生RDA裂解并进一步失去1分子COOH产生的m/z 191.1721碎片离子,分析该化合物为齐墩果酸。齐墩果酸二级质谱图及裂解途径见图12-13。
甘草皂苷H2,负离子模式下检测其准分子离子峰分别为m/z 823.4139[M-H]-,tR=17.11 min,二级质谱图中显示其碎片离子m/z 647.3826 [M-H-C6H8O6]-、m/z 351.0594[M-H-C30H46O4]-、m/z 193.0378 [M-H-C30H46O4-C6H6O5]-。m/z 647.3826为准分子离子峰失去1分子葡萄糖醛酸所产生碎片离子,化合物结构中两分子葡萄糖醛酸苷键断裂后再失去1分子H2O产生m/z 351.0594碎片离子 ,m/z 351.0594碎片离子脱去1分子C6H6O5得到m/z193.0378碎片离子,分析该化合物为甘草皂苷H2。甘草皂苷H2二级质谱图及裂解途径见图14-15。
甘草次酸,正离子模式下其准分子离子峰为m/z 471.3476 [M+H]+,tR=20.56min,分子式为C30H46O4,二级质谱图显示其主要碎片离子m/z 453.3312 [M+H-H2O]+、m/z407.3311 [M+H-H2O-COOH]+ 、m/z 235.1680 [M+H-H2O-COOH-C13H17]+、m/z 217.1614 [M+H-H2O-COOH-C13H17-H2O]+。m/z 453.3312为准分子离子失去1分子H2O产生的碎片离子,随后再失去1分子COOH得到m/z 407.3311碎片离子 ,进而失去1分子C13H17得到m/z 235.1680碎片离子,随后失去1分子H2O得到m/z 217.1614碎片离子,分析该化合物为甘草次酸。甘草次酸二级质谱图及裂解途径见图16-17。
测试例4
香豆素类成分分析
香豆素类化合物是指母核为苯骈α-吡喃酮的一类化合物,在质谱裂解中易失去-OCH3、-CH3、CO、CO2等碎片分子,通过与数据库和文献报道化合物比对,分析出4种香豆素类化合物。本文以甘草酚为例,分析香豆素类化合物裂解规律。
甘草酚,正离子模式下观察其一级高分辨质谱,准分子离子峰为m/z 367.1254,tR=16.78 min,推测其分子式为C21H18O6。观察其二级高分辨质谱,确定其主要碎片离子为m/z352.0820 [M+H-CH3]+、m/z 296.0254 [M+H-CH3-C4H8]+。m/z 352.0820 为准分子离子失去1分子CH3得到的碎片离子,进一步脱去1分子C4H8得到m/z 296.0254碎片离子,分析该化合物为甘草酚。甘草酚二级质谱图及裂解途径见图18-19。
测试例5
有机酸类成分分析
有机酸类化合物是一种含有羧基的酸性化合物,在质谱裂解中常脱落H2O、CO2中性小分子和-CH3、-CH2、-COOH等碎片分子。通过与数据库和文献报道化合物比对,分析出3种有机酸类化合物。本发明以亚麻酸为例,分析有机酸类成分裂解规律。
亚麻酸,正离子模式下,检测其准分子离子峰m/z 279.2342 [M+H]+,tR=19.93min,元素分析其化学式为C18H30O2。二级质谱图中其主要碎片离子有m/z 149.0229 [M+H-C7H14O2]+、m/z 121.0281 [M+H-C7H14O2-C2H2]+、m/z 65.0383 [M+H-C7H14O2-C2H2-C4H8]+。准分子离子峰失去1分子C7H14O2得到m/z 149.0229碎片离子,随后失去1分子C2H2得到m/z121.0281碎片离子,失去1分子C4H8得到m/z 65.0383碎片离子,分析该化合物为亚麻酸。亚麻酸二级质谱图及裂解途径见图20-21。
测试例6
其他类成分分析
本研究还在甘草多糖中分析出13种其它类化合物,主要包括7种氨基酸类化合物,3种糖类化合物和3种核苷类化合物。本文以化合物精氨酸、蔗糖、鸟苷为例,分析氨基酸类、糖类、核苷类化合物裂解规律。
精氨酸,正离子模式下,检测其准分子离子峰为m/z 175.1189 [M+H]+,tR=0.78min,元素分析其化学式为C6H14N4O2。二级质谱图中其主要碎片离子有m/z 130.0967 [M+H-COOH]+、m/z 116.0710 [M+H-NH]+、m/z 70.0644 [M+H-CH6N2]+。准分子离子峰失去1分子COOH得到m/z 130.0967碎片离子,随后脱去1分子NH得到m/z 116.0710碎片离子,进一步丢失1分子CH6N2得到m/z 70.0644碎片离子,分析该化合物为精氨酸。精氨酸二级质谱图及裂解途径见图22-23。
蔗糖,负离子模式下检测其准分子离子峰m/z 341.0146 [M-H]-,tR=0.93 min,元素分析其化学式为C12H22O11。二级质谱图中其主要碎片离子有m/z 179.0571 [M-H-C5H8O6]-、m/z 161.0447 [M-H-H2O]-。准分子离子峰失去1分子果糖得到m/z 179.0571碎片离子,随后丢失1分子H2O得到m/z 161.0477碎片离子,分析该化合物为蔗糖。蔗糖二级质谱图及裂解途径见图24-25。
鸟苷,正离子模式下检测其准分子离子峰为m/z 284.0996 [M+H]+,tR=1.54min,元素分析其化学式为C10H13N5O5。二级质谱图中其主要碎片离子有m/z 152.0561[M+H-C10H8O4]+、135.0302 [M+H-O]+、110.0358 [M+H-CHN]+。准分子离子峰失去1分子核糖得到m/z 152.0561碎片离子,随后酰胺键水解得到m/z 135.0302碎片离子,进一步开环脱去CHN得到m/z 110.0358碎片离子,分析该化合物为鸟苷。鸟苷二级质谱图及裂解途径见图26-27。
通过测试例1~6的测试可以看出本实验通过建立UPLC-Q-TOF-MS/MS技术对甘草多糖化学成分进行分析,依据高分辨质谱提供的一级质谱图确认其准分子离子峰和保留时间,借助二级质谱图结合文献报道数据指认出主要碎片离子,最终共确定出95种化学成分。将95种成分归属后显示,甘草多糖化学成分包括57种黄酮类成分,18种三萜类成分,4种香豆素类成分,3种有机酸类成分,其他类包括7种氨基酸类成分、3种糖类成分、3种核苷类成分,共13种。因后续将进行甘草多糖对癌因性疲乏的药效学研究,而多糖的纯度又是影响其药效的因素之一,所以对甘草多糖进行分离纯化从而得到高纯度多糖是下一步研究关键。综上,本实验分析分析出甘草多糖中包括多糖在内的共7类成分,以期为甘草多糖后续分离纯化研究提供参考。
Claims (9)
1.一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,步骤如下:
步骤1、供试品溶液配制:称取甘草多糖,加入超纯水,超声处理溶解后,摇匀,制得甘草多糖溶液,经微孔滤膜过滤,得到供试品溶液;
步骤2、色谱条件:采用WatersACQUITYUPLC BEH C18色谱柱;采用流动相梯度洗脱,固定相为改性固定相;
步骤3、质谱条件:采用电喷雾离子源(ESI),正、负离子2种模式扫描,质谱扫描;正离子模式下,负离子模式下,毛细管电压-4000~-5000kV,裂解电压-70~-90V,碰撞能量30~40eV;
步骤4、数据分析、采用Analyst TF 1.8.1数据采集软件,采集时间20~40min;采用SCIEX OS2.0.0软件进行数据处理,化学成分依据相对分子质量、离子碎片信息、保留时间,结合数据库及文献报道数据结果进行分析;
所述改性固定相的制备方法如下,以重量份计:
S1、将4~6份二氧化硅粉末、8~12份3-(异丁烯酰氧)丙基三甲氧基硅烷、8~12份水和0.5~2份乙酸加入到18~22份无水乙醇中,在50~70℃油浴中搅拌20~40min,静置3~5h,得到预处理物;
S2、将0.1~0.3份甲基丙烯酰胺、0.04~0.06份海藻酸钠和0.04~0.06份N,N'-亚甲基双丙烯酰胺加入到30~50份水中,进行超声处理,然后加入2~4份步骤S1制备的预处理物,100~500rpm磁力搅拌0.5~2h,静置8~15h,再加入0.04~0.06份过硫酸铵、0.004~0.006份硫酸钙和0.1~0.2份金属有机骨架材料,在50~70℃油浴中进行交联聚合1~3h,用氮气鼓泡除去混合物中溶解的空气,然后加热至50~70℃,100~300rpm搅拌回流冷凝4~6h,随后,温度加热到70~90℃,在100~200rpm搅拌下完全蒸发溶剂,然后,加入40~60份水浸泡一夜,最后用水洗涤1~3次,在70~90℃真空烘箱中干燥5~15h,得到后处理物;
S3、将2~3份后处理物在20~30份四氯甲烷中超声处理,以正己烷为推进液,在30~50Mpa的压力下将混合物填充到140~160×4~5mm不锈钢管中,当流出的液体体积超过100mL时,床层稳定,充填完成,得到改性固定相。
2.如权利要求1所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述步骤1中为称取0.5~2重量份甘草多糖,加入超纯水,超声溶解后,摇匀,制得浓度为4~6mg/mL的甘草多糖溶液。
3.如权利要求1所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述步骤1中微孔滤膜的孔径为0.2~0.25μm。
4.如权利要求1所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述步骤2中流动相为乙腈和0.05~0.2wt%甲酸水溶液配置而成。
5.如权利要求1所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述步骤2中流动相梯度洗脱参数为:0~2min,流动相由5%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;2~42min,流动相由5%~95%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;42~47min,流动相由95%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;47~47.1min,流动相由5%~95%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;47.1~50min,流动相由5%乙腈、0.05~0.2wt%甲酸水溶液补足100%组成;流速0.2~0.4mL/min;柱温:35~45℃;进样量4~6μL。
6.如权利要求1所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述步骤3中质谱扫描范围m/z100~1500Da,雾化气压50~60PSI,辅助气压50~60PSI,气帘气压30~40PSI。
7.如权利要求1所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述步骤3中正离子模式下参数为:毛细管电压5000~6000V,裂解电压80~120V,碰撞能量30~40eV。
8.如权利要求1所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述步骤3中负离子模式下参数为:毛细管电压-4000~-5000kV,裂解电压-70~-90V,碰撞能量30~40eV。
9.如权利要求1-8任一项所述的一种基于UPLC-Q-TOF-MS/MS技术分析甘草多糖的方法,其特征在于,所述超声处理时间各自独立地为10~30min、超声功率各自独立地为200~500W、超声频率各自独立地为20~60kHz。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311628162.5A CN117330692B (zh) | 2023-12-01 | 2023-12-01 | 基于uplc-q-tof-ms/ms技术分析甘草多糖的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311628162.5A CN117330692B (zh) | 2023-12-01 | 2023-12-01 | 基于uplc-q-tof-ms/ms技术分析甘草多糖的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117330692A CN117330692A (zh) | 2024-01-02 |
CN117330692B true CN117330692B (zh) | 2024-02-20 |
Family
ID=89293802
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311628162.5A Active CN117330692B (zh) | 2023-12-01 | 2023-12-01 | 基于uplc-q-tof-ms/ms技术分析甘草多糖的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117330692B (zh) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109030663A (zh) * | 2018-09-25 | 2018-12-18 | 昆药集团股份有限公司 | 一种uhplc法检测复方甘草片中化学成分的方法 |
CN112444592A (zh) * | 2020-11-11 | 2021-03-05 | 谱尼测试集团北京检验认证科学研究院有限公司 | 一种uplc-q-tof-ms快速筛查甘草中活性成分的方法 |
WO2021073175A1 (zh) * | 2019-10-16 | 2021-04-22 | 石家庄以岭药业股份有限公司 | 一种中药组合物中多种成分鉴别及含量的测定方法 |
CN113267578A (zh) * | 2021-05-17 | 2021-08-17 | 中国中医科学院中药研究所 | 芍药甘草汤的质量控制方法 |
CN113866293A (zh) * | 2021-09-17 | 2021-12-31 | 广东省第二中医院(广东省中医药工程技术研究院) | 一种快速识别分析洋甘菊中化学成分的方法 |
CN115980221A (zh) * | 2022-12-29 | 2023-04-18 | 浙江工业大学 | 一种基于分子印迹-hplc联用检测生物样品中痕量药物浓度的方法 |
-
2023
- 2023-12-01 CN CN202311628162.5A patent/CN117330692B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109030663A (zh) * | 2018-09-25 | 2018-12-18 | 昆药集团股份有限公司 | 一种uhplc法检测复方甘草片中化学成分的方法 |
WO2021073175A1 (zh) * | 2019-10-16 | 2021-04-22 | 石家庄以岭药业股份有限公司 | 一种中药组合物中多种成分鉴别及含量的测定方法 |
CN112444592A (zh) * | 2020-11-11 | 2021-03-05 | 谱尼测试集团北京检验认证科学研究院有限公司 | 一种uplc-q-tof-ms快速筛查甘草中活性成分的方法 |
CN113267578A (zh) * | 2021-05-17 | 2021-08-17 | 中国中医科学院中药研究所 | 芍药甘草汤的质量控制方法 |
CN113866293A (zh) * | 2021-09-17 | 2021-12-31 | 广东省第二中医院(广东省中医药工程技术研究院) | 一种快速识别分析洋甘菊中化学成分的方法 |
CN115980221A (zh) * | 2022-12-29 | 2023-04-18 | 浙江工业大学 | 一种基于分子印迹-hplc联用检测生物样品中痕量药物浓度的方法 |
Non-Patent Citations (1)
Title |
---|
柱前衍生化高效液相色谱法分析9 种多糖中的单糖组成;王媛媛;《济宁医学院学报》;第39卷(第4期);第241-244页 * |
Also Published As
Publication number | Publication date |
---|---|
CN117330692A (zh) | 2024-01-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Xie et al. | Rapid identification of ophiopogonins and ophiopogonones in Ophiopogon japonicus extract with a practical technique of mass defect filtering based on high resolution mass spectrometry | |
Wang et al. | A strategy for identification and structural characterization of compounds from Gardenia jasminoides by integrating macroporous resin column chromatography and liquid chromatography-tandem mass spectrometry combined with ion-mobility spectrometry | |
Liu et al. | Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry | |
CN102030795B (zh) | 葫芦烷型苦瓜皂苷的制备方法 | |
Zhang et al. | Simultaneous characterization of isoflavonoids and astragalosides in two Astragalus species by high‐performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry | |
Van Le et al. | Ginseng saponins in different parts of Panax vietnamensis | |
Fang et al. | Rapid analysis of steroidal saponin mixture using electrospray ionization mass spectrometry combined with sequential tandem mass spectrometry | |
CN105203654A (zh) | 一种用于测定中兽药散剂中11种非法添加药物含量的方法 | |
Li et al. | Fast screening of flavonoids from switchgrass and Mikania micrantha by liquid chromatography hybrid-ion trap time-of-flight mass spectrometry | |
Jia et al. | Identification of glycoside compounds from tobacco by high performance liquid chromatography/electrospray ionization linear ion-trap tandem mass spectrometry coupled with electrospray ionization orbitrap mass spectrometry | |
CN105572284A (zh) | 一种快速检测与鉴定钩吻中化学成分的分析方法 | |
Li et al. | Analysis of iridoid glucosides in Hedyotis diffusa by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry | |
CN117330692B (zh) | 基于uplc-q-tof-ms/ms技术分析甘草多糖的方法 | |
Lee et al. | Isolation and tandem mass fragmentations of an anti-inflammatory compound from Aralia elata | |
CN108037197B (zh) | 一种鱼腥草破壁饮片非挥发类次生代谢产物化学成分定性及定量分析的方法 | |
Jin et al. | Novel analysis procedure for red ginseng polysaccharides by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry | |
CN110095524B (zh) | 一种三萜皂苷质谱结构解析方法 | |
Li et al. | Characterization and identification of chemical components in Neopicrorhiza scrphulariiflora roots by liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry | |
CN108982703B (zh) | 一种多酚类物质的液质联用检测方法 | |
Sui et al. | Removal and recovery of deep eutectic solvent with membrane-based methodology: A promising strategy to enhance extraction and purification of Dendrobium officinale flavonoids | |
Hu et al. | Application of high-speed counter-current chromatography for the isolation of 5 alkaloids from lotus (Nelumbo nucifera Gaertn.) leaves | |
CN111337610A (zh) | 一种复杂环境基质中痕量雌激素、壬基酚及双酚a的检测方法 | |
Guo et al. | Analysis of saponins from leaves of Aralia elata by liquid chromatography and multi-stage tandem mass spectrometry | |
CN109142571B (zh) | 一种测定蒽醌类成分含量的方法 | |
Zhong et al. | Rapid analysis and identification of the main constituents in Patrinia scabiosaefolia Fisch. by UPLC/Q-TOF-MS/MS |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |