CN113899843B - Method for simultaneously and quantitatively analyzing 24 ingredients of Kunxian capsule - Google Patents
Method for simultaneously and quantitatively analyzing 24 ingredients of Kunxian capsule Download PDFInfo
- Publication number
- CN113899843B CN113899843B CN202111084461.8A CN202111084461A CN113899843B CN 113899843 B CN113899843 B CN 113899843B CN 202111084461 A CN202111084461 A CN 202111084461A CN 113899843 B CN113899843 B CN 113899843B
- Authority
- CN
- China
- Prior art keywords
- epimedin
- tripterygium wilfordii
- triptolide
- acid
- solution
- 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
- 239000002775 capsule Substances 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000009740 kunxian Substances 0.000 title claims abstract description 22
- 239000004615 ingredient Substances 0.000 title claims description 21
- DFBIRQPKNDILPW-CIVMWXNOSA-N Triptolide Chemical compound O=C1OCC([C@@H]2C3)=C1CC[C@]2(C)[C@]12O[C@H]1[C@@H]1O[C@]1(C(C)C)[C@@H](O)[C@]21[C@H]3O1 DFBIRQPKNDILPW-CIVMWXNOSA-N 0.000 claims abstract description 66
- YKUJZZHGTWVWHA-UHFFFAOYSA-N triptolide Natural products COC12CC3OC3(C(C)C)C(O)C14OC4CC5C6=C(CCC25C)C(=O)OC6 YKUJZZHGTWVWHA-UHFFFAOYSA-N 0.000 claims abstract description 66
- 241000830536 Tripterygium wilfordii Species 0.000 claims abstract description 65
- 235000015398 thunder god vine Nutrition 0.000 claims abstract description 65
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims abstract description 52
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims abstract description 49
- FVQOMEDMFUMIMO-UHFFFAOYSA-N Hyperosid Natural products OC1C(O)C(O)C(CO)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 FVQOMEDMFUMIMO-UHFFFAOYSA-N 0.000 claims abstract description 45
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 claims abstract description 44
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000000243 solution Substances 0.000 claims abstract description 30
- TZJALUIVHRYQQB-XLRXWWTNSA-N icariin Chemical compound C1=CC(OC)=CC=C1C1=C(O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)C(=O)C2=C(O)C=C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(CC=C(C)C)=C2O1 TZJALUIVHRYQQB-XLRXWWTNSA-N 0.000 claims abstract description 29
- TZJALUIVHRYQQB-UHFFFAOYSA-N icariine Natural products C1=CC(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(C)O2)O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 TZJALUIVHRYQQB-UHFFFAOYSA-N 0.000 claims abstract description 29
- NGMYNFJANBHLKA-SENBMHEBSA-N Icariside II Natural products O(C)c1ccc(C2=C(O[C@H]3[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O3)C(=O)c3c(O)cc(O)c(C/C=C(\C)/C)c3O2)cc1 NGMYNFJANBHLKA-SENBMHEBSA-N 0.000 claims abstract description 27
- OVSQVDMCBVZWGM-SJWGPRHPSA-N Hyperin Natural products O[C@H]1[C@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-SJWGPRHPSA-N 0.000 claims abstract description 26
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims abstract description 26
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000003513 alkali Substances 0.000 claims abstract description 26
- KQJSQWZMSAGSHN-JJWQIEBTSA-N celastrol Chemical compound C([C@H]1[C@]2(C)CC[C@@]34C)[C@](C)(C(O)=O)CC[C@]1(C)CC[C@]2(C)C4=CC=C1C3=CC(=O)C(O)=C1C KQJSQWZMSAGSHN-JJWQIEBTSA-N 0.000 claims abstract description 26
- 235000005875 quercetin Nutrition 0.000 claims abstract description 26
- 229960001285 quercetin Drugs 0.000 claims abstract description 26
- OVSQVDMCBVZWGM-DTGCRPNFSA-N quercetin 3-O-beta-D-galactopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-DTGCRPNFSA-N 0.000 claims abstract description 26
- BBFYUPYFXSSMNV-UHFFFAOYSA-N quercetin-7-o-galactoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 BBFYUPYFXSSMNV-UHFFFAOYSA-N 0.000 claims abstract description 26
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 claims abstract description 22
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 claims abstract description 22
- LKNITMBRWDCKMG-UHFFFAOYSA-N Epimedin B Natural products COc1ccc(cc1)C2=C(OC3OC(C)C(O)C(O)C3OC4OC(CO)C(O)C4O)C(=O)c5c(O)cc(OC6OC(CO)C(O)C(O)C6O)c(CC=C(C)C)c5O2 LKNITMBRWDCKMG-UHFFFAOYSA-N 0.000 claims abstract description 22
- OCZZCFAOOWZSRX-LRHLXKJSSA-N Epimedin B Chemical compound C1=CC(OC)=CC=C1C1=C(O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)CO2)O)C(=O)C2=C(O)C=C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(CC=C(C)C)=C2O1 OCZZCFAOOWZSRX-LRHLXKJSSA-N 0.000 claims abstract description 22
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 claims abstract description 22
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 claims abstract description 22
- 229940074393 chlorogenic acid Drugs 0.000 claims abstract description 22
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 claims abstract description 22
- 235000001368 chlorogenic acid Nutrition 0.000 claims abstract description 22
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 claims abstract description 22
- NGMYNFJANBHLKA-LVKFHIPRSA-N icariside II Chemical compound C1=CC(OC)=CC=C1C1=C(O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)C(=O)C2=C(O)C=C(O)C(CC=C(C)C)=C2O1 NGMYNFJANBHLKA-LVKFHIPRSA-N 0.000 claims abstract description 22
- 235000008777 kaempferol Nutrition 0.000 claims abstract description 22
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 claims abstract description 22
- ULZLIYVOYYQJRO-JIYCBSMMSA-N Epimedin C Chemical compound C1=CC(OC)=CC=C1C1=C(O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O[C@H]2[C@@H]([C@H](O)[C@@H](O)[C@H](C)O2)O)C(=O)C2=C(O)C=C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C(CC=C(C)C)=C2O1 ULZLIYVOYYQJRO-JIYCBSMMSA-N 0.000 claims abstract description 21
- YPFSXWUDSOVOGG-UHFFFAOYSA-N epimedin C Natural products COc1ccc(cc1)C2=C(OC3OC(C)C(O)C(OC(=O)C)C3OC4OC(CO)C(O)C(O)C4O)C(=O)c5c(O)cc(OC6OC(CO)C(O)C(O)C6O)c(CC=C(C)C)c5O2 YPFSXWUDSOVOGG-UHFFFAOYSA-N 0.000 claims abstract description 21
- MQVRGDZCYDEQML-UHFFFAOYSA-N Astragalin Natural products C1=CC(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(CO)O2)O)C(=O)C2=C(O)C=C(O)C=C2O1 MQVRGDZCYDEQML-UHFFFAOYSA-N 0.000 claims abstract description 20
- JPUKWEQWGBDDQB-QSOFNFLRSA-N kaempferol 3-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=CC(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O JPUKWEQWGBDDQB-QSOFNFLRSA-N 0.000 claims abstract description 20
- GWTUHAXUUFROTF-UHFFFAOYSA-N pseudochlorogenic acid Natural products C1C(O)C(O)C(O)CC1(C(O)=O)OC(=O)C=CC1=CC=C(O)C(O)=C1 GWTUHAXUUFROTF-UHFFFAOYSA-N 0.000 claims abstract description 20
- CWVRJTMFETXNAD-NXLLHMKUSA-N trans-5-O-caffeoyl-D-quinic acid Chemical compound O[C@H]1[C@H](O)C[C@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-NXLLHMKUSA-N 0.000 claims abstract description 20
- OVSQVDMCBVZWGM-IDRAQACASA-N Hirsutrin Natural products O([C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1)C1=C(c2cc(O)c(O)cc2)Oc2c(c(O)cc(O)c2)C1=O OVSQVDMCBVZWGM-IDRAQACASA-N 0.000 claims abstract description 19
- GXMWXESSGGEWEM-UHFFFAOYSA-N isoquercitrin Natural products OCC(O)C1OC(OC2C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)C(O)C1O GXMWXESSGGEWEM-UHFFFAOYSA-N 0.000 claims abstract description 19
- OVSQVDMCBVZWGM-QSOFNFLRSA-N quercetin 3-O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-QSOFNFLRSA-N 0.000 claims abstract description 19
- TVBJKPLTBPGHDJ-ZJTKNEERSA-N COc1ccc(cc1)-c1oc2c(CC=C(C)C)c(O)cc(O)c2c(=O)c1O[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O Chemical compound COc1ccc(cc1)-c1oc2c(CC=C(C)C)c(O)cc(O)c2c(=O)c1O[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O TVBJKPLTBPGHDJ-ZJTKNEERSA-N 0.000 claims abstract description 16
- IXUUINNBVCUODQ-UHFFFAOYSA-N hexandraside F Natural products COc1ccc(cc1)C2=C(OC3C(O)C(C)OC(O)C3OC4OC(CO)C(O)C(O)C4O)C(=O)c5c(O)cc(OC6OC(CO)C(O)C(O)C6O)c(CC=C(C)C)c5O2 IXUUINNBVCUODQ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- -1 archimeside B Chemical compound 0.000 claims abstract description 11
- 238000005173 quadrupole mass spectroscopy Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 90
- 239000013558 reference substance Substances 0.000 claims description 30
- 229930183477 epimedin Natural products 0.000 claims description 26
- 150000001875 compounds Chemical class 0.000 claims description 25
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 22
- 239000002585 base Substances 0.000 claims description 20
- GYFFKZTYYAFCTR-JUHZACGLSA-N 4-O-trans-caffeoylquinic acid Chemical compound O[C@@H]1C[C@](O)(C(O)=O)C[C@@H](O)[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-JUHZACGLSA-N 0.000 claims description 19
- GYFFKZTYYAFCTR-UHFFFAOYSA-N 5-O-(6'-O-galloyl)-beta-D-glucopyranosylgentisic acid Natural products OC1CC(O)(C(O)=O)CC(O)C1OC(=O)C=CC1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-UHFFFAOYSA-N 0.000 claims description 19
- GYFFKZTYYAFCTR-LMRQPLJMSA-N cryptochlorogenic acid Natural products O[C@H]1C[C@@](O)(C[C@H](O)[C@H]1OC(=O)C=Cc2ccc(O)c(O)c2)C(=O)O GYFFKZTYYAFCTR-LMRQPLJMSA-N 0.000 claims description 19
- TZJALUIVHRYQQB-XFDQAQKOSA-N Icariin Natural products O(C)c1ccc(C2=C(O[C@H]3[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O3)C(=O)c3c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O4)c(C/C=C(\C)/C)c3O2)cc1 TZJALUIVHRYQQB-XFDQAQKOSA-N 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 15
- HOMSOWZTBJWNHP-UHFFFAOYSA-N 5-chlorothiadiazole Chemical compound ClC1=CN=NS1 HOMSOWZTBJWNHP-UHFFFAOYSA-N 0.000 claims description 14
- VDYCLYGKCGVBHN-UHFFFAOYSA-N pachymaic acid Natural products CC12CCC(OC(C)=O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC(=C)C(C)C)C(O)=O)C(O)CC21C VDYCLYGKCGVBHN-UHFFFAOYSA-N 0.000 claims description 14
- SRDNLMOBFKJOSD-UHFFFAOYSA-N pachymic acid Natural products CC12CCC(OC(C)=O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C(O)=O)C(O)CC21C SRDNLMOBFKJOSD-UHFFFAOYSA-N 0.000 claims description 14
- 238000001514 detection method Methods 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 11
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 11
- 239000005695 Ammonium acetate Substances 0.000 claims description 11
- 235000019257 ammonium acetate Nutrition 0.000 claims description 11
- 229940043376 ammonium acetate Drugs 0.000 claims description 11
- 235000019253 formic acid Nutrition 0.000 claims description 11
- 241000893536 Epimedium Species 0.000 claims description 10
- 235000018905 epimedium Nutrition 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- 241000218993 Begonia Species 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 229930185123 Baohuoside Natural products 0.000 claims description 5
- 240000002624 Mespilus germanica Species 0.000 claims description 5
- 235000017784 Mespilus germanica Nutrition 0.000 claims description 5
- 235000000560 Mimusops elengi Nutrition 0.000 claims description 5
- 235000007837 Vangueria infausta Nutrition 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 229930182470 glycoside Natural products 0.000 claims description 5
- 150000002338 glycosides Chemical class 0.000 claims description 5
- 239000012088 reference solution Substances 0.000 claims description 5
- 210000000582 semen Anatomy 0.000 claims description 5
- 239000012086 standard solution Substances 0.000 claims description 5
- 239000011550 stock solution Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 239000010282 Emodin Substances 0.000 claims description 4
- 241001514666 Kunzea Species 0.000 claims description 4
- RHMXXJGYXNZAPX-UHFFFAOYSA-N emodin Chemical compound C1=C(O)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O RHMXXJGYXNZAPX-UHFFFAOYSA-N 0.000 claims description 4
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 4
- YSCJAYPKBYRXEZ-HZPINHDXSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4ar,6ar,6bs,8as,12as,14ar,14br)-4,4,6a,6b,11,11,14b-heptamethyl-8a-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxycarbonyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3-hydroxy-4-[(2s,3r,4s, Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C(O)=O)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YSCJAYPKBYRXEZ-HZPINHDXSA-N 0.000 claims description 3
- YDZWHGJRWMQCDP-NKILCQAGSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4ar,6ar,6bs,8as,12as,14ar,14br)-8a-carboxy-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3-hydroxy-4-[(2s,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-5-[(2s,3r,4 Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(O)=O)C(O)=O)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YDZWHGJRWMQCDP-NKILCQAGSA-N 0.000 claims description 3
- KPXIBWGPZSPABK-FXAWDEMLSA-N (3bR,9bS)-6-hydroxy-9b-methyl-7-propan-2-yl-3,3b,4,5,10,11-hexahydronaphtho[2,1-e]isobenzofuran-1-one Chemical compound C1C[C@]2(C)C3=CC=C(C(C)C)C(O)=C3CC[C@H]2C2=C1C(=O)OC2 KPXIBWGPZSPABK-FXAWDEMLSA-N 0.000 claims description 3
- SMDOOINVMJSDPS-UHFFFAOYSA-N Astragaloside Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)=C1 SMDOOINVMJSDPS-UHFFFAOYSA-N 0.000 claims description 3
- VIYFRTDWJXBEDM-UHFFFAOYSA-N Triptophenolide Natural products CC(C)c1ccc2c(CCC3C4=COC(=O)C4=CCC23C)c1O VIYFRTDWJXBEDM-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- QMNWISYXSJWHRY-XWJCTJPOSA-N astragaloside Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)C4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)CC3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-XWJCTJPOSA-N 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 238000004445 quantitative analysis Methods 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000012982 microporous membrane Substances 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000012085 test solution Substances 0.000 abstract description 3
- 150000002596 lactones Chemical class 0.000 abstract 1
- 238000001819 mass spectrum Methods 0.000 description 23
- 239000003814 drug Substances 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 238000004949 mass spectrometry Methods 0.000 description 10
- 238000000605 extraction Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 229930003935 flavonoid Natural products 0.000 description 8
- 150000002215 flavonoids Chemical class 0.000 description 8
- 235000017173 flavonoids Nutrition 0.000 description 8
- IYCPMVXIUPYNHI-UHFFFAOYSA-N Icariside I Natural products C1=CC(OC)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 IYCPMVXIUPYNHI-UHFFFAOYSA-N 0.000 description 7
- 241000545405 Tripterygium Species 0.000 description 7
- 229930013930 alkaloid Natural products 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 150000003505 terpenes Chemical class 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 229930182721 icariside Natural products 0.000 description 5
- 238000011835 investigation Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000004885 tandem mass spectrometry Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- SVXJDTNFJXKATR-KQSLYFRASA-N Epimedin A Natural products O([C@@H]1[C@@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(c2ccc(OC)cc2)Oc2c(C/C=C(\C)/C)c(O[C@H]3[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O3)cc(O)c2C1=O)[C@H]1[C@@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 SVXJDTNFJXKATR-KQSLYFRASA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 150000003797 alkaloid derivatives Chemical class 0.000 description 4
- SVXJDTNFJXKATR-UHFFFAOYSA-N hexandraside A Natural products C1=CC(OC)=CC=C1C1=C(OC2C(C(O)C(O)C(C)O2)OC2C(C(O)C(O)C(CO)O2)O)C(=O)C2=C(O)C=C(OC3C(C(O)C(O)C(CO)O3)O)C(CC=C(C)C)=C2O1 SVXJDTNFJXKATR-UHFFFAOYSA-N 0.000 description 4
- NQYPTLKGQJDGTI-FCVRJVSHSA-N hyperoside Natural products OC[C@H]1O[C@@H](OC2=C(Oc3cc(O)cc(O)c3[C@H]2O)c4ccc(O)c(O)c4)[C@H](O)[C@@H](O)[C@H]1O NQYPTLKGQJDGTI-FCVRJVSHSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000002552 multiple reaction monitoring Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 235000007586 terpenes Nutrition 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 108010007005 Estrogen Receptor alpha Proteins 0.000 description 2
- 102100038595 Estrogen receptor Human genes 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940126678 chinese medicines Drugs 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical group O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- QZMAEZWZCGBZFK-AOJWCAIYSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4ar,6ar,6bs,8as,12as,14ar,14br)-4,4,6a,6b,11,11,14b-heptamethyl-8a-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxycarbonyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3,5-dihydroxy-4-[(2s,3r Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C(O)=O)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O QZMAEZWZCGBZFK-AOJWCAIYSA-N 0.000 description 1
- QZMAEZWZCGBZFK-UHFFFAOYSA-N 28-(beta-D-Glucopyranosyloxy)-28-oxoolean-12-en-3beta-yl 3-O-(beta-D-glucopyranosyl)-beta-D-glucopyranosiduronic acid Natural products C12CC(C)(C)CCC2(C(=O)OC2C(C(O)C(O)C(CO)O2)O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC(C1O)OC(C(O)=O)C(O)C1OC1OC(CO)C(O)C(O)C1O QZMAEZWZCGBZFK-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 101100136092 Drosophila melanogaster peng gene Proteins 0.000 description 1
- 206010018367 Glomerulonephritis chronic Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- OBIOZWXPDBWYHB-UHFFFAOYSA-N Nobiletin Natural products C1=CC(OC)=CC=C1C1=C(OC)C(=O)C2=C(OC)C(OC)=C(OC)C(OC)=C2O1 OBIOZWXPDBWYHB-UHFFFAOYSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 1
- 229960003805 amantadine Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000000947 anti-immunosuppressive effect Effects 0.000 description 1
- 238000004630 atomic force microscopy Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- KPJWZUAARPJYSB-UHFFFAOYSA-N glycoside C Natural products CC1(C)OC(=O)C23CCC1C2(O)CCC(C1(CCC24)C)(C)C3CCC1C2(C)CCCC4(C)COC(C(C(O)C1O)OC2C(C(O)C(CO)O2)O)OC1COC1OC(CO)C(O)C(O)C1O KPJWZUAARPJYSB-UHFFFAOYSA-N 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- LRDGATPGVJTWLJ-UHFFFAOYSA-N luteolin Natural products OC1=CC(O)=CC(C=2OC3=CC(O)=CC(O)=C3C(=O)C=2)=C1 LRDGATPGVJTWLJ-UHFFFAOYSA-N 0.000 description 1
- IQPNAANSBPBGFQ-UHFFFAOYSA-N luteolin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C(O)=C1 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 description 1
- 235000009498 luteolin Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- MRIAQLRQZPPODS-UHFFFAOYSA-N nobiletin Chemical compound C1=C(OC)C(OC)=CC=C1C1=CC(=O)C2=C(OC)C(OC)=C(OC)C(OC)=C2O1 MRIAQLRQZPPODS-UHFFFAOYSA-N 0.000 description 1
- 230000001582 osteoblastic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 210000002437 synoviocyte Anatomy 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 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
-
- 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
-
- 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
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a method for simultaneously and quantitatively analyzing 24 components of Kunxian capsules, which comprises the following steps: preparation of test solution: preparation of control solution and mixed control solution: detecting the components in the test solution, the control solution and the mixed control solution by adopting an ultra-high performance liquid chromatography-electrospray tandem triple quadrupole mass spectrometry, and simultaneously determining 24 components in the Kunzing capsule, wherein the 24 components are triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii jin alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii neone, icariine, archimeside B, baohuoside I, hyperin, tripterine, astragalin, triptolide, arrow lactone A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, chlorogenic acid, quercetin and kaempferol. The method has the characteristics of strong specificity, high sensitivity and short analysis time.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for simultaneously and quantitatively analyzing 24 components of Kunxian capsules.
Background
The Kunxian Capsule (KC) is a technological project achievement of the key Chinese medicine in the nine five countries, is a pure Chinese medicine compound preparation composed of four Chinese medicines of Kunming begonia, epimedium herb, medlar and semen cuscutae, has the effects of tonifying kidney, dredging collaterals, dispelling wind and removing dampness, and is used for treating immune diseases such as rheumatoid arthritis, systemic lupus erythematosus, chronic nephritis and the like (Zhang Ning, easy and undoubted clinical application progress [ J ]. Clinical application of Kunxian capsule, clinical research of Chinese medicine, 2014,6 (07): 147-148). Pharmacological studies also show that KC has anti-inflammatory, anti-tumor and immunosuppressive effects, and may be related to its various effective components including alkaloids, terpenes, flavonoids, glycosides, etc. For example, terpenoid triptolide, flavonoid epimedin B, icariin and the like have obvious anti-inflammatory and bone tissue injury protection effects (SuZ, sun H, ao M, zhao C.Atomic Force Microscopy Study oftheAnti-inflammatory Effects ofTriptolide on RheumatoidArthritis Fibroblast-like Synoviocytes. Microsc micro [ J ] 2017,23 (5): 1002-1012;Xiao HH,Fung CY,Mok SK,et al.Flavonoids from Herba epimedii selectively activate estrogen receptor alpha (ERalpha) and sticmulate ER-dependent osteoblastic functions in UMR-106cells [ J ] J Steroid Biochem Mol biol.2014,143: 141-51.). However, alkaloids and terpenoids in Kunming begonia show certain toxic side effects (Tang Y, zhang Y, li L, et al Kunxian Capsule for Rheumatoid Arthritis: inhibition of Inflammatory Network and Reducing Adverse Reactions Through Drug Matching [ J ]. Front Pharmacol.2020, 11:485.). Therefore, the content monitoring of the effective and toxic components in the prescription is the key for ensuring the safety and effectiveness of the medication of patients.
At present, the quality control of KC mainly uses the national drug standard (YBZ 07522006-2009Z) issued by the national food and drug administration to produce guidelines, and only monitors the content of triptolide and icariin in KC under the content measurement item. Although the characteristic spectrum of KC and the quantitative analysis method of various flavonoid components are established by adopting an HPLC method in the early stage, the chemical components such as tripterine, triptolide and the like in the Kunming begonia have weak ultraviolet absorption, and part of the chemical components are trace toxic and effective components in the formula, so that the components are difficult to detect simultaneously by adopting a common HPLC technology. In recent years, the emerging ultra-high performance liquid chromatography-electrospray tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) has been widely used in analysis of traditional Chinese medicines and compounds, especially in research of trace components, due to the characteristics of high separation speed, high sensitivity and high measurement accuracy (Pichini S, mannocchi G, gottardi M, et al Talanta.2020,209: 120537.).
The prior art is to measure the components of the Kunzian capsule, which takes hyperin as an internal reference, and adopts a multi-evaluation method to simultaneously detect flavonoid components such as the epimedium glycoside A, the epimedium glycoside C, the epimedium glycoside B, the epimedium glycoside, the luteolin, the quercetin, the nobiletin, the kaempferol, the baohuoside I and the like at most, and the detection time is 67min (Zhang Xue, peng Fuquan, what wind mine is the multi-evaluation method to measure 10 flavonoid components [ J ]. Chinese herbal medicines, 2018, 49 (24): 5823-5829). No method for measuring 24 components at one time in Kunzian capsules has been reported.
Disclosure of Invention
The invention aims to provide an analysis method for simultaneously quantifying 24 components of Kunzian capsules.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides an analysis method for simultaneously quantifying 24 ingredients of Kunxian capsules, which comprises the following steps:
first, preparing a sample solution:
adding the content of Kunxian capsule into 70% methanol, performing ultrasonic treatment for 1-30 min, performing centrifugal separation, collecting supernatant and internal standard solution, fixing volume with 70% methanol, filtering with microporous membrane, collecting filtrate to obtain sample solution;
second, preparing a reference substance solution and a mixed reference substance solution:
precisely weighing triptolide, tripterygium wilfordii secondary alkali, triptodynine, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii neone, icariine, archery side B, baohuoside I, hyperin, tripterine, astragalin, triptolide, archery side A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin, kaempferol reference substance and pachymic acid (internal standard), dissolving with methanol, and diluting to scale to obtain reference substance solutions of the respective compounds;
mixing the reference substance solution: diluting each reference substance solution prepared by the method with 70% methanol respectively to prepare mixed reference substance solutions with series of gradient concentrations;
detecting components in the sample solution, the reference solution and the mixed reference solution by adopting ultra-high performance liquid chromatography-electrospray tandem triple quadrupole mass spectrometry (UHPLC-MS/MS), and simultaneously determining 24 components in the Kunzhong capsule, wherein the 24 components are triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii advanced alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii novel alkali, icariine, arrowhead B, baohuoside I, hyperin, tripterine, astragalin, triptolide, arrowhead A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin and kaempferol;
conditions of the ultra-high performance liquid chromatography: chromatographic column Agilent ZORBAXSB C, mobile phase A is methanol, and mobile phase B is 10mmol/L ammonium acetate aqueous solution containing 0.2% formic acid; the gradient elution program is 0 to 1.0min,10 to 15 percent of A;1.0 to 2.0min,15 to 65 percent of A;2.0 to 8.5min,65 to 95 percent of A; 8.5-9.0 min,95% A, flow rate 0.4mL/min, column temperature 35 ℃ and sample injection amount 5 mu L;
conditions for electrospray tandem triple quadrupole mass spectrometry: the ion source is ESI; detection mode: positive and negative ion modes; scanning mode: multi-reaction monitoring MRM; spray voltage: 4000V; ion source temperature: 350.0 ℃.
The preparation of the sample solution in the first step is specifically as follows:
taking 0.15g of Kunxian capsule, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of 70% methanol, performing ultrasonic treatment for 20min, centrifuging at 14500rpm for 20min, taking 1mL of each of the supernatant and the internal standard solution, transferring into a 10mL volumetric flask, fixing the volume with 70% methanol, passing through a microporous filter membrane with 0.22 μm, and taking the subsequent filtrate to obtain a sample solution.
The preparation of the second step reference substance and the mixed reference substance solution is specifically as follows:
precisely weighing about 2.00mg of triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii advanced alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii novel alkali, icariine, archin B, baohuoside I, hyperin, tripterine, astragalin, triptolide A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin, kaempferol reference substances and pachymic acid (internal standard), dissolving with methanol and diluting to 2mL respectively to obtain reference substance stock solutions of the respective compounds;
diluting each reference substance solution prepared by the method with 70% methanol respectively to prepare mixed reference substance solutions with the concentration range of 75.00-3000.00 ng/mL of triptolide; 12.50-500.00 ng/mL of tripterygium wilfordii secondary alkali; jin Jian 51.50-2060.00 ng/mL of tripterygium wilfordii; epimedin A123.125-4925.00 ng/mL; epimedin A1125.00-5000.00 ng/mL; epimedin C1243.75-49750.00 ng/mL; epimedin B258.75-10350.00 ng/mL; 138.125-5525.00 ng/mL of triptolide; icariin 502.50-20100.00 ng/mL; 125.00-5000.00 ng/mL of the archery glycoside B; baohuoside I122.50-4900.00 ng/mL; hyperin 73.125-2925.00 ng/mL; 1.25-50.00 ng/mL of tripterine; astragaloside 50.75-2030.00 ng/mL; 2.575-103.00 ng/mL of triptolide; 2.3625 to 94.50ng/mL of the triptophenolide; the concentration of the archery glycoside A is 125.00-5000.00 ng/mL; 250.00-10000.00 ng/mL of 2' -O-rhamnosyl icariside II; isoquercitrin 123.125-4925.00 ng/mL; chlorogenic acid 12.4375-497.50 ng/mL; 12.50 to 500.00ng/mL of cryptochlorogenic acid; 75.00-3000.00 ng/mL of neochlorogenic acid; 90.375-3615.00 ng/mL of quercetin; 45.00-1800.00 ng/mL of kaempferol; the content of pachymic acid (internal standard) is 5000.00ng/mL.
The content of 24 ingredients in the Kunzian capsule is as follows: 2' -O-rhamnosyl icariside II 6.141-11.389 mg/g, baohuoside I3.804-8.916 mg/g, epimedin A3.566-5.164 mg/g, epimedin A13.393-4.966 mg/g, epimedin B1.775-3.018 mg/g, epimedin C40.062-55.450 mg/g, quercetin 1.828-4.172 mg, arrow side A2.837-4.311 mg/g, arrow side B1.717-5.476 mg/g, hyperin 1.028-2.619 mg/g, triptolide 0.0122-0.0195 mg/g, tripterygium wilfordii sub-base 0.138-0.441 mg/g, tripterygium wilfordii base 0.593-673 mg/g, tripterygium wilfordii red element 0.00126-0.00413 mg/g, tripterygium wilfordii Jin Jian.521-1.195 mg/g, quercetin 0.828-4.172 mg, arrow acid 0.6-6 mg, new tripterygium wilfordii glycoside 1.028-2.619 mg/g, tripterygium wilfordii side 0.0120-0.385 mg, tripterygium wilfordii base 0.385-35 mg, and new-207 mg.
The Kunxian capsule is prepared from Kunming begonia, epimedium, medlar and semen cuscutae according to the mass ratio of 2:2:1:1.
Optimally, the conditions of the ultra-high performance liquid chromatography: gradient elution procedure is 0-1.0 min,10% A+90% B;1.0 to 2.0min,15 percent of A+85 percent of B;2.0 to 8.5min,65 percent of A+35 percent of B;8.5 to 9.0min,95 percent of A+5 percent of B.
By adopting the technical scheme, the invention has the following advantages and beneficial effects:
the invention establishes an analysis method for 24 components of triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii advanced alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii neone, icariine, archimeside B, baohuoside I, hyperin, tripterine, astragalin, triptolide, arrow-A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin and kaempferol in the Kunzhongxian capsule based on UHPLC-MS/MS technology. The method can be used for rapidly and directly targeting and quantitatively detecting 24 compounds in Kunxian capsules, and has strong specificity and high sensitivity. The pretreatment process only needs simple ultrasonic and centrifugal, the chromatographic separation and mass spectrum detection process can be completed within 9min, and all the medicinal flavors in the prescription are basically covered, so that the content measurement of all the medicinal flavor and multiple components of the Kunxian capsule can be realized, and experimental reference is provided for improving the integral quality control of KC and guaranteeing the medication safety of patients.
The method can detect 24 components at one time, is rich in various active components such as alkaloid, terpenes and flavonoid, and has the detection time of 9 minutes, thereby realizing the content measurement of the multiple components of the whole medicine. The method has the main advantages of more and more comprehensive components than the components detected by the prior art and short detection time.
Drawings
FIG. 1 is a schematic diagram of the chromatogram of 24 ingredients in a mixed control solution A and a Kunzian capsule sample B.
Fig. 2 is a mass spectrum analysis chart of 24 ingredients in the Kunzhong capsule under the condition of most preferred mass spectrum parameters, wherein 1 is triptolide, 2 is triptolide, 3 is tripterygium Jin Jian, 4 is epimedin A, and 5 is epimedin A1.
FIG. 3 is a mass spectrum analysis of 24 ingredients in Kunzhong capsules under the most preferred mass spectrum parameters, 6 is epimedin C,7 is epimedin B,8 is triptolide, 9 is icariin, and 10 is icariin B.
FIG. 4 is a mass spectrum analysis of 24 ingredients in Kunzhong capsules under the most preferred mass spectrum parameters, 11 is baohuoside I,12 is hyperoside, 13 is tripterine, 14 is astragalin, and 15 is triptolide.
FIG. 5 is a mass spectrum analysis of 24 ingredients in Kunzian capsules under the most preferred mass spectrum parameters, 16 being a triptolide, 17 being a archimeside A,18 being 2 "-O-rhamnosyl icariside II, 19 being pachymic acid (internal standard), 20 being isoquercitrin.
FIG. 6 is a mass spectrum analysis of 24 ingredients in Kunzhong capsules under the most preferred mass spectrum parameters, 21 being neochlorogenic acid, 22 being cryptochlorogenic acid, 23 being chlorogenic acid, 24 being quercetin, 25 being kaempferol.
FIG. 7 is a schematic representation of the results of an optimization of the chromatograms of the different columns, A being Agilent Poroshell SB C18 (2.1X100 mm,2.7 μm).
FIG. 8 is an optimized chromatogram of different columns, B being WatersT3Column (2.1X100 mm,3 μm) examined the results.
FIG. 9 is a graph showing the results of an optimization of the chromatograms of different columns, C Agilent Poroshell ECC18 (3.0X105 mm,2.7 μm).
FIG. 10 is a schematic representation of the results of an optimization of the chromatograms of the different columns, D Agilent Eclipse XDB C (2.1X100 mm,3.5 μm).
FIG. 11 is a schematic representation of the results of an optimization of the chromatograms of the different columns, E Agilent ZORBAXSB C (2.1X100 mm,3.5 μm).
In fig. 7 to 11, 1 is kaempferol, 2 is quercetin, 3 is triptolide, 4 is triptolide, 5 is astragalin, 6 is tripterine, 7 is hyperin, 8 is baohuoside I,9 is archuloside B,10 is 2 "-O-rhamnosyl icariside ii, 11 is icariin, 12 is triptolide, 13 is epimedin B,14 is epimedin C,15 is epimedin a,16 is tripterygium Jin Jian, 17 is tripterygium wilfordii secondary base, and 18 is tripterygium wilfordii secondary base.
FIG. 12 is an optimized chromatogram of a combination of different mobile phases, A being an acetonitrile-water chromatogram.
Fig. 13 is an optimized chromatogram of different mobile phase combinations, and B is a methanol-water chromatogram.
FIG. 14 is an optimized chromatogram of a combination of different mobile phases, C being methanol-0.2% formic acid chromatogram.
FIG. 15 is an optimized chromatogram of a combination of different mobile phases, D being methanol-10 mmol/L ammonium acetate.
FIG. 16 is an optimized chromatogram of a combination of different mobile phases, E being methanol-0.2% formic acid+10 mmol/L ammonium acetate.
In fig. 12 to 16, 1 is kaempferol, 2 is quercetin, 3 is triptolide, 4 is chlorogenic acid, 5 is cryptochlorogenic acid, 6 is neochlorogenic acid, 7 is triptolide, 8 is astragalin, 9 is tripterine, 10 is isoquercitrin, 11 is hyperoside, 12 is baohuoside I,13 is pachymic acid (internal standard), 14 is archuloside B,15 is 2 "-O-rhamnosyl icariside ii, 16 is archuloside a,17 is icariine, 18 is tripterygium wilfordii new base, 19 is epimedin B,20 is epimedin C,21 is epimedin A1, 22 is tripterygium wilfordii advanced base, 23 is tripterygium wilfordii sub base, 24 is tripterygium wilfordii sub base, 25 is tripterygium wilfordii base.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Example 1
1 instrument and reagent
UHPLC-MS/MS triple quaternary lever (Agilent 1290 high performance liquid tandem mass spectrum 6460), ultrasonic instrument (SK 7200H, shanghai department ultrasonic instrument Co., ltd.), pipette gun, ten-thousandth electronic analytical balance (Sartorius Co., germany), centrifuge; the control triptolide (lot number: 111567-201404), icariin (lot number: 110737-201516), baohuoside I (lot number: 111852-201603), epimedin C (lot number: 111780-201905), hyperin (lot number: 111521-201609) are purchased from Chinese food and drug assay institute; epimedin A (lot number: MB 6562), epimedin B (lot number: MB 6564), triptolide (lot number: MB 5758), kaempferol (lot number: MB 6888), tripterine (lot number: MB 6739), quercetin (lot number: MB 2127), isoquercitrin (lot number: MB 7071), triptolide (lot number: MB 6738), neochlorogenic acid (lot number: MB 2067), cryptochlorogenic acid (lot number: MB 7041), astragalin (lot number: MB 7087) were purchased from Dalian Mei Biotechnology Co., ltd; the archery in A (lot number: DST 190415-162), archery in B (lot number: DST 190411-163), epimedin A1 (lot number: DST 190801-064), 2 "-O-rhamnosyl icariside II (lot number: DST 191010-090), triptodine (lot number: DST 190923-212), tripterygium Jin Jian (lot number: DST 190810-080), tripterygium neone (lot number: DST 191008-213) are purchased from Desmot biological limited company; chlorogenic acid (lot number: 6361) was purchased from Shanghai Shiadad Standard technical service Co., ltd; pachymic acid (lot number: J02GB 150406) is purchased from Shanghai Yuan leaf Biotechnology Co., ltd. 16 Kunxian capsule samples were supplied by the pharmaceutical Co., ltd. Of Baiyunshan Chen Liji, guangzhou (lot number: K31006, K31009-K31014, L31001-L31009); methanol is chromatographic purity, water is drohenate water, and other reagents are analytically pure.
2 methods and results
2.1 chromatography and Mass Spectrometry conditions
Chromatographic conditions: chromatographic column Agilent ZORBAXSB C (2.1X100 mm,3.5 μm), mobile phase A was methanol and B was 10mmol/L aqueous ammonium acetate with 0.2% formic acid; the gradient elution program is 0 to 1.0min,10 to 15 percent of A;
1.0 to 2.0min,15 to 65 percent of A;2.0 to 8.5min,65 to 95 percent of A; 8.5-9.0 min,95% A, flow rate 0.4mL/min, column temperature 35 ℃ and sample injection amount 5 mu L.
Mass spectrometry conditions: the ion source is ESI; detection mode: positive and negative ion modes; scanning mode: multiple Reaction Monitoring (MRM); spray voltage: 4000V; ion source temperature: 350.0 ℃. The most preferred mass spectrum parameters of the 24 compounds are shown in table 1 and fig. 2-6, fig. 2 is a mass spectrum analysis chart of 24 components in the Kunzian capsule under the condition of the most preferred mass spectrum parameters, 1 is triptolide, 2 is triptolide, 3 is tripterygium Jin Jian, 4 is epimedin A, and 5 is epimedin A1. FIG. 3 is a mass spectrum analysis of 24 ingredients in Kunzhong capsules under the most preferred mass spectrum parameters, 6 is epimedin C,7 is epimedin B,8 is triptolide, 9 is icariin, and 10 is icariin B. FIG. 4 is a mass spectrometry analysis of 24 components in Kunzea capsules under most preferred mass spectrometry parameters. 11 is baohuoside I,12 is hyperin, 13 is tripterine, 14 is astragalin, and 15 is triptolide. FIG. 5 is a mass spectrometry analysis of 24 components in Kunzea capsules under most preferred mass spectrometry parameters. 16 is a triptolide, 17 is a stigmaoside A,18 is a 2' -O-rhamnosyl icariside II, 19 is a pachymic acid (internal standard), and 20 is an isoquercitrin. FIG. 6 is a mass spectrometry analysis of 24 components in Kunzea capsules under most preferred mass spectrometry parameters. 21 is neochlorogenic acid, 22 is cryptochlorogenic acid, 23 is chlorogenic acid, 24 is quercetin, 25 is kaempferol. Under these conditions, the response of each compound was optimal, and the chromatograms of the Kunxian capsule samples and the mixed control are shown in FIG. 1. Fig. 1 is a schematic diagram of the chromatogram of 24 ingredients in a mixed control solution a and a kunzhen capsule sample B, wherein 1 is triptolide, 2 is tripterygium wilfordii secondary alkaloid, 3 is tripterygium Jin Jian, 4 is epimedin a,5 is epimedin A1,6 is epimedin C,7 is epimedin B,8 is tripterygium wilfordii new alkaloid, 9 is icariine, 10 is archimeside B,11 is baohuoside I,12 is hyperin, 13 is tripterine, 14 is astragalin, 15 is triptolide, 16 is triptolide, 17 is amantadine a,18 is 2 "-O-rhamnosyl icariside ii, 19 is pachymic acid (internal standard), 20 is isoquercitrin, 21 is neochlorogenic acid, 22 is cryptochlorogenic acid, 23 is chlorogenic acid, 24 is quercetin, 25 is kaempferol.
Table 1 mass spectral parameters
2.2 preparation of control solution and Mixed control solution:
precisely weighing Tripterine, triptolide, tripterygium wilfordii sub-alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii sub-alkali, icariine, archin B, baohuoside I, hyperin, tripterine, astragalin, triptolide, archin A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin, kaempferol reference substance and pachymic acid internal standard, respectively placing in 2mL volumetric flasks, dissolving with methanol, diluting to scale, and respectively obtaining reference substance stock solutions of the compounds.
Mixing the reference substance solution: the prepared reference substance stock solutions are respectively diluted by 70% methanol to prepare mixed reference substance solutions with serial gradient concentrations, and the specific dilution concentration values of the compounds are as follows: triptolide 75, 150, 300, 600, 1200, 2400, 3000ng/mL; 12.5, 25, 50, 100, 200, 400, 500ng/mL of tripterygium wilfordii secondary alkali; tripterygium wilfordii Jin Jian 51.5.5, 103, 206, 412, 824, 1648, 2060ng/mL; epimedin A123.125, 246.25, 492.5, 985, 1970, 3940, 4925ng/mL; epimedin a1125, 250, 500, 1000, 2000, 4000, 5000ng/mL; epimedin C1243.75, 2487.5, 4975, 9950, 19900, 39800, 49750ng/mL; epimedin B258.75, 517.5, 1035, 2070, 4140, 8280, 10350ng/mL; triptolide 138.125, 276.25, 552.5, 1105, 2210, 4420, 5525ng/mL; icariin 502.5, 1005, 2010, 4020, 8040, 16080, 20100ng/mL; about 125, 250, 500, 1000, 2000, 4000, 5000ng/mL of about; baohuoside I122.5, 245, 490, 980, 1960, 3920, 4900ng/mL; hyperin 73.125, 146.25, 292.5, 585, 1170, 2340, 2925ng/mL; 1.25, 2.5, 5, 10, 20, 40, 50ng/mL of tripterine; astragaloside 50.75, 101.5, 203, 406, 812, 1624, 2030ng/mL; triptolide 2.575, 5.15, 10.3, 20.6, 41.2, 82.4, 103ng/mL; the concentrations of the triptophenolide 2.3625, 4.725, 9.45, 18.9, 37.8, 75.6 and 94.5ng/mL; about 125, 250, 500, 1000, 2000, 4000, 5000ng/mL of archery glycoside; 2' -O-rhamnosyl icariside II 250, 500, 1000, 2000, 4000, 8000, 10000ng/mL; isoquercitrin 123.125, 246.25, 492.5, 985, 1970, 3940, 4925ng/mL; chlorogenic acid 12.4375, 24.875, 49.75, 99.5, 199, 398, 497.5ng/mL; 12.5, 25, 50, 100, 200, 400, 500ng/mL of cryptochlorogenic acid; chlorogenic acid 75, 150, 300, 600, 1200, 2400, 3000ng/mL; quercetin 90.375, 180.75, 361.5, 723, 1446, 2892, 3615ng/mL; kaempferol 45, 90, 180, 360, 720, 1440, 1800ng/mL; the pachymic acid (internal standard) is 5000ng/mL.
2.3 preparation of sample solutions
The Kunming capsule is a pure Chinese medicine compound preparation composed of four Chinese medicines of Kunming begonia, epimedium, medlar and semen cuscutae, and the prescription is prepared from the Kunming begonia, epimedium, medlar and semen cuscutae according to the mass ratio of 2:2:1:1.
Taking 0.15g of Kunxian capsule, precisely weighing, placing in a 50mL conical flask with a plug, precisely adding 70% methanol 20mL, performing ultrasonic treatment for 20min, centrifuging at 14500rpm for 20min, taking 1mL of each of the supernatant and the internal standard solution, transferring into a 10mL volumetric flask, fixing the volume with 70% methanol, passing through a microporous filter membrane with 0.22 μm, and taking the subsequent filtrate.
2.4 methodology investigation
2.4.1 linear relationship investigation
The control stock solutions prepared according to the method under item 2.2 are respectively diluted with 70% methanol to prepare control mixed solutions with serial gradient concentrations, and sample injection is carried out according to the chromatographic and mass spectrum conditions under item 2.1, so as to determine the peak area. And drawing a standard curve by taking the peak area of the reference substance as an ordinate (Y) and the concentration of the analyte as an abscissa (X), and obtaining the retention time, the linear regression equation and the linear range of each component, wherein the result shows that the r values of 24 components are between 0.9878 and 0.9974, the linear relation is good, and the result is shown in Table 2.
Table 2 Linear regression equation for each component
2.4.2 repeatability test
Taking the same batch of samples, preparing 6 parts of sample solution in parallel according to the method under the item "2.3", sampling according to the chromatographic and mass spectrum conditions under the item "2.1", recording the peak area of the component to be detected, obtaining the concentration, and calculating the RSD value. The results showed that 24 components RSD were between 1.1% and 13.4%, indicating good reproducibility of the method, as shown in table 3.
2.4.3 precision test
Precisely sucking the same mixed reference substance solution, continuously sampling for 6 times according to the chromatographic and mass spectrum conditions under the item of 2.1, recording the peak area of the component to be detected, calculating the RSD value, and examining the daily precision; the analysis was continued for 3 days, and RSD values of peak areas of 24 components were calculated to examine the daytime precision. The daily precision RSD of the instrument is 1.3-7.4%, the daily precision RSD of the instrument is 1.1-14.9%, and the daily precision RSD of the instrument is less than 15%, which indicates that the instrument precision is good, as shown in Table 3.
2.4.4 stability test
Taking the same sample solution, respectively carrying out sample injection according to chromatographic and mass spectrum conditions under the item of 2.1 for 0,2, 6, 12 and 24 hours, recording peak areas of components to be detected, obtaining concentration, calculating RSD values, and displaying that the RSD of 24 components is less than 15%, wherein the sample solution is stable within 24 hours, as shown in table 3.
TABLE 3 repeatability, precision, stability test results
2.4.5 sample recovery test
Weighing 6 parts of the determined Kunxian capsule sample, precisely adding the mixed reference substance solution with the content equivalent to each component in the sample into a 50mL volumetric flask, preparing the sample solution in parallel according to the method under the item "2.3", analyzing according to the chromatographic and mass spectrum conditions under the item "2.1", recording the peak area of the component to be detected, and calculating the recovery rate and the RSD value. The average sample recovery rate is 91.34% -105.48%, and the RSD is 1.0% -5.2%, which shows that the accuracy of the extraction method is good, as shown in Table 4.
TABLE 4 sample recovery test results
/>
/>
/>
/>
2.4.6 sample determination
16 batches of Kunxian capsules were taken, a test solution was prepared according to the method under item "2.3", the contents of 24 ingredients were calculated by sample injection measurement according to the chromatographic and mass spectrometry conditions under item "2.1", and the results are shown in tables 5 and 6.
Table 516 content of 24 Compounds in Kunzian Capsule samples
/>
Table 616 content of 24 Compounds in Kunzian Capsule samples
/>
Solvent investigation: the extraction effects of water, ethanol and methanol with different concentrations are respectively examined, and the results show that when water is used as a solvent, the extraction efficiency of most of active ingredients is lower; when ethanol and methanol are used as the extraction solvents, most of the chemical components have higher extraction efficiency, and particularly, 70% methanol has the best extraction effect, so the invention finally selects 70% methanol as the extraction solvent, and the detail is shown in Table 7.
TABLE 7 comparison of extraction Effect of different extraction solvents
/>
And (3) researching a chromatographic column: the invention respectively examines 5 chromatographic columns, firstly 3 chromatographic columns Agilent Poroshell SB C18 (2.1X100 mm,2.7 μm) and Waters with different lengths or brands are examinedT3Column (2.1X100 mm,3 μm) and Agilent Poroshell ECC18 (3.0X105 mm,2.7 μm), and as a result, it was found that the tailing of triptolide and tripterine under these 3 columns was severe, and peak-shapedAlso worse, and the peak time of some compounds (astragalin, hyperin, icariin, epimedin a, epimedin B, epimedin C) was too early, see fig. 7-9; FIG. 7 is a schematic representation of the results of an optimization of the chromatograms of the different columns, A being Agilent Poroshell SB C18 (2.1X100 mm,2.7 μm). FIG. 8 is an optimized chromatogram of different columns, B being Waters +.>T3Column (2.1X100 mm,3 μm) examined the results. FIG. 9 is a graph showing the results of an optimization of the chromatograms of different columns, C Agilent Poroshell ECC18 (3.0X105 mm,2.7 μm). Next, the present invention also examined columns of Agilent Eclipse XDB C (2.1x100 mm,3.5 μm) and Agilent ZORBAXSB C (2.1x100 mm,3.5 μm) 2 types, and the results showed that the peak shape and the degree of separation of each compound were good by using these 2 columns, but the peak time of astragalin, hyperoside, icariin, epimedin a, epimedin B, epimedin C was still earlier when using Agilent Eclipse XDB C (2.1x100 mm,3.5 μm) columns, see fig. 10 to 11, and fig. 10 is a diagram showing the results of examination of the optimum chromatograms of the different columns, D being Agilent Eclipse XDB C18 (2.1x100 mm,3.5 μm). FIG. 11 is a schematic representation of the results of an optimization of the chromatograms of the different columns, E Agilent ZORBAXSB C (2.1X100 mm,3.5 μm). In fig. 7 to 11, 1 is kaempferol, 2 is quercetin, 3 is triptolide, 4 is triptolide, 5 is astragalin, 6 is tripterine, 7 is hyperin, 8 is baohuoside I,9 is archuloside B,10 is 2 "-O-rhamnosyl icariside ii, 11 is icariin, 12 is triptolide, 13 is epimedin B,14 is epimedin C,15 is epimedin a,16 is tripterygium Jin Jian, 17 is tripterygium wilfordii secondary base, and 18 is tripterygium wilfordii secondary base. Comprehensive analysis, agilent ZORBAXSB C18 (2.1X100 mm,3.5 μm) was finally selected as the optimal chromatographic column, and the peak time and the separation degree of each compound under the chromatographic column conditions were good, and the compound response was high.
Investigation of mobile phase: the 5 different mobile phase systems were tested in sequence: acetonitrile-water, methanol-0.2% formic acid, methanol-10 mmol/L ammonium acetate and methanol-0.2% formic acid+10 mmol/L ammonium acetate, and the results show that the methanol system is superior to the acetonitrile system, and the peak shape of each component peak can be further improved when 0.2% formic acid+10 mmol/L ammonium acetate is taken as a water phase, and the effect is optimal, and the detailed figures 12-16 are shown. FIG. 12 is an optimized chromatogram of a combination of different mobile phases, A being an acetonitrile-water chromatogram.
Fig. 13 is an optimized chromatogram of different mobile phase combinations, and B is a methanol-water chromatogram. FIG. 14 is an optimized chromatogram of a combination of different mobile phases, C being methanol-0.2% formic acid chromatogram. FIG. 15 is an optimized chromatogram of a combination of different mobile phases, D being methanol-10 mmol/L ammonium acetate. FIG. 16 is an optimized chromatogram of a combination of different mobile phases, E being methanol-0.2% formic acid+10 mmol/L ammonium acetate. In fig. 12 to 16, 1 is kaempferol, 2 is quercetin, 3 is triptolide, 4 is chlorogenic acid, 5 is cryptochlorogenic acid, 6 is neochlorogenic acid, 7 is triptolide, 8 is astragalin, 9 is tripterine, 10 is isoquercitrin, 11 is hyperoside, 12 is baohuoside I,13 is pachymic acid (internal standard), 14 is archuloside B,15 is 2 "-O-rhamnosyl icariside ii, 16 is archuloside a,17 is icariine, 18 is tripterygium wilfordii new base, 19 is epimedin B,20 is epimedin C,21 is epimedin A1, 22 is tripterygium wilfordii advanced base, 23 is tripterygium wilfordii sub base, 24 is tripterygium wilfordii sub base, 25 is tripterygium wilfordii base.
Mobile phase gradient data:
mobile phase investigation is shown in table 8-1:
TABLE 8-1 gradient change 1
The obtained 24 compounds were inferior in separation degree, 5 compounds such as chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and the like were early in peak appearance time, and were inferior in peak shape due to the fact that the gradient change 1 in Table 8-1 was used for detection.
TABLE 8-2 gradient change 2
The 24 compounds obtained by the gradient change 2 detection in Table 8-2 were generally inferior in the degree of separation, and the peak shape of 3 compounds such as icariin was poor.
TABLE 8-3 gradient change 3
The obtained 24 compounds have better separation degree, better peak-out time, higher peak area and better peak shape by using the gradient change 3 detection in the table 8-3. Therefore, the gradient change 3 is selected as the final detection gradient.
Discussion 3
Because the chemical components in the formula are complex in composition, the content of each component is extremely uneven, and the isomers (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, epimedin A1 and the like) are difficult to separate, the method is characterized in that in order to realize the complete separation and rapid detection of 24 components, chromatographic columns, mobile phase combinations and mobile phase gradients are respectively optimized, agilent ZORBAXSB C (2.1X100 mm and 3.5 mu m) are finally taken as chromatographic columns, 10mmol/L ammonium acetate aqueous solution of methanol-0.2% formic acid is taken as a mobile phase, and the gradient elution program is used for 9min for analysis. Under the condition, the chromatographic separation effect of 24 components is good, the specificity is strong, and the analysis time is short. In addition, the optimal Collision Energy (CE) of the mass spectrum is optimized to obtain a chromatographic peak with higher response intensity. Under the conditions of chromatography and mass spectrometry, the linear relation of 24 components is good, and the detection effect of the components in the sample is good.
In the index component selection of content measurement, 7 terpenes and alkaloids components from Tripterygium wilfordii, tripterine, triptolide and triptolide are selected as toxic and effective components in the prescription; according to the content measurement results, the content of alkaloid components in the preparation is higher, the content of terpenoid components such as triptolide, tripterine and the like is relatively lower, and researches prove that the components have certain pharmacological effects such as anti-inflammatory and anti-tumor effects; the flavonoids components icariin, baohuoside I, epimedin A, epimedin B, epimedin C and other compounds derived from the epimedium herb have the highest content in the prescription, and have better effects of resisting inflammation, oxidation, osteoporosis and the like, so the 24 components are used as final content measurement indexes in the research.
The content measurement results of the Kunxian capsule samples of 16 batches show that the content difference of the 24 chemical components in the samples of different batches is larger, for example, the highest content of triptolide is 1.838mg/g, the lowest content of triptolide is 0.593mg/g, the difference is more than 3 times, the difference is possibly related to the quality difference and the feeding authenticity difference of the raw medicinal materials, a manufacturer is prompted to ensure the standardization of the sources of the raw medicinal materials in the process of producing the compound preparation, and each process parameter in the preparation process is refined, so that the aim of quantifying product indexes is fulfilled, and the safety and the effectiveness of the medicine application of patients are ensured.
In conclusion, the UHPLC-MS/MS method established by the invention can be used for simultaneously measuring 24 active ingredients of triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii advanced alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii novel alkali, icariine, archimeside B, baohuoside I, hyperin, tripterine, astragalin, triptolide, archimeside A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, chlorogenic acid, quercetin and kaempferol in the Kunzian capsule, and the method is simple, quick and accurate, and provides a reference for comprehensively evaluating the quality of the Kunzian capsule and ensuring the safety of the medicine.
The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.
Claims (7)
1. A method for simultaneously quantifying 24 ingredients of a Kunzian capsule, comprising the steps of:
first, preparing a sample solution:
adding the content of Kunxian capsule into 70% methanol, performing ultrasonic treatment for 1-30 min, performing centrifugal separation, collecting supernatant and internal standard solution, fixing volume with 70% methanol, filtering with microporous membrane, collecting filtrate to obtain sample solution;
second, preparing a reference substance solution and a mixed reference substance solution:
precisely weighing triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii advanced alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii neoalkali, icariine, archery side B, baohuoside I, hyperin, tripterine, astragalin, triptolide, archery side A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin, kaempferol reference substance and pachymic acid, dissolving with methanol and diluting to scale to obtain reference substance solutions of the respective compounds;
mixing the reference substance solution: diluting each reference substance solution prepared by the method with 70% methanol respectively to prepare mixed reference substance solutions with series of gradient concentrations;
detecting components in a sample solution, a reference solution and a mixed reference solution by adopting an ultra-high performance liquid chromatography-electrospray tandem triple quadrupole mass spectrometry, and simultaneously determining 24 components in the Kunzan capsule, wherein the 24 components are triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii jin alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii neone, icariine, archimeside B, baohuoside I, hyperin, tripterine, astragalin, triptolide, arrow-A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin and kaempferol;
conditions of the ultra-high performance liquid chromatography: chromatographic column Agilent ZORBAXSB C, mobile phase A is methanol, and mobile phase B is 10mmol/L ammonium acetate aqueous solution containing 0.2% formic acid; the gradient elution program is 0 to 1.0min,10 to 15 percent of A;1.0 to 2.0min,15 to 65 percent of A;2.0 to 8.5min,65 to 95 percent of A; 8.5-9.0 min,95% A, flow rate 0.4mL/min, column temperature 35 ℃ and sample injection amount 5 mu L;
conditions for electrospray tandem triple quadrupole mass spectrometry: the ion source is ESI; detection mode: positive and negative ion modes; scanning mode: multi-reaction monitoring MRM; spray voltage: 4000V; ion source temperature: 350.0 ℃.
2. The method for simultaneous quantitative analysis of 24 ingredients in a Kunzian capsule according to claim 1, wherein the first step of the preparation of the sample solution is as follows:
taking 0.15g of Kunxian capsule, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of 70% methanol, performing ultrasonic treatment for 20min, centrifuging at 14500rpm for 20min, taking 1mL of each of the supernatant and the internal standard solution, transferring into a 10mL volumetric flask, fixing the volume with 70% methanol, passing through a microporous filter membrane with 0.22 μm, and taking the subsequent filtrate to obtain a sample solution.
3. The method for simultaneously and quantitatively analyzing 24 ingredients of Kunxian capsule according to claim 1, wherein the second step of preparing the control and the mixed control solution comprises the following steps:
precisely weighing about 2.00mg of triptolide, tripterygium wilfordii secondary alkali, tripterygium wilfordii advanced alkali, epimedin A1, epimedin C, epimedin B, tripterygium wilfordii novel alkali, icariine, archin B, baohuoside I, hyperin, tripterine, astragalin, triptolide A, 2' -O-rhamnosyl icariside II, isoquercitrin, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, quercetin, kaempferol reference substance and pachymic acid, dissolving with methanol and diluting to 2mL respectively to obtain reference substance stock solutions of the respective compounds;
each control solution prepared above was diluted with 70% methanol, respectively, to prepare a mixed control solution.
4. The method for simultaneously and quantitatively analyzing 24 ingredients of Kunzhong capsules according to claim 3, wherein the triptolide is 75.00-3000.00 ng/mL in the mixed reference solution; 12.50-500.00 ng/mL of tripterygium wilfordii secondary alkali; jin Jian 51.50-2060.00 ng/mL of tripterygium wilfordii; epimedin A123.125-4925.00 ng/mL; epimedin A1125.00-5000.00 ng/mL; epimedin C1243.75-49750.00 ng/mL; epimedin B258.75-10350.00 ng/mL; 138.125-5525.00 ng/mL of triptolide; icariin 502.50-20100.00 ng/mL; 125.00-5000.00 ng/mL of the archery glycoside B; baohuoside I122.50-4900.00 ng/mL; hyperin 73.125-2925.00 ng/mL; 1.25-50.00 ng/mL of tripterine; astragaloside 50.75-2030.00 ng/mL; 2.575-103.00 ng/mL of triptolide; 2.3625 to 94.50ng/mL of the triptophenolide; the concentration of the archery glycoside A is 125.00-5000.00 ng/mL; 250.00-10000.00 ng/mL of 2' -O-rhamnosyl icariside II; isoquercitrin 123.125-4925.00 ng/mL; chlorogenic acid 12.4375-497.50 ng/mL; 12.50 to 500.00ng/mL of cryptochlorogenic acid; 75.00-3000.00 ng/mL of neochlorogenic acid; 90.375-3615.00 ng/mL of quercetin; 45.00-1800.00 ng/mL of kaempferol; the content of the pachymic acid is 5000.00ng/mL.
5. The method for simultaneously quantifying 24 ingredients of the Kunzian capsule according to claim 1, wherein the content of 24 ingredients in the Kunzian capsule is: 2' -O-rhamnosyl icariside II 6.141-11.389 mg/g, baohuoside I3.804-8.916 mg/g, epimedin A3.566-5.164 mg/g, epimedin A13.393-4.966 mg/g, epimedin B1.775-3.018 mg/g, epimedin C40.062-55.450 mg/g, quercetin 1.828-4.172 mg, arrow side A2.837-4.311 mg/g, arrow side B1.717-5.476 mg/g, hyperin 1.028-2.619 mg/g, triptolide 0.0122-0.0195 mg/g, tripterygium wilfordii sub-base 0.138-0.441 mg/g, tripterygium wilfordii base 0.593-673 mg/g, tripterygium wilfordii red element 0.00126-0.00413 mg/g, tripterygium wilfordii Jin Jian.521-1.195 mg/g, quercetin 0.828-4.172 mg, arrow acid 0.6-6 mg, new tripterygium wilfordii glycoside 1.028-2.619 mg/g, tripterygium wilfordii side 0.0120-0.385 mg, tripterygium wilfordii base 0.385-35 mg, and new-207 mg.
6. The method for simultaneously and quantitatively analyzing 24 ingredients of Kunzian capsules according to claim 1, wherein the Kunzian capsules are prepared from Kunming begonia, epimedium, medlar and semen cuscutae according to a mass ratio of 2:2:1:1.
7. The method for simultaneous quantitative analysis of 24 ingredients of a Kunzea capsule according to claim 1, wherein the conditions of the ultra-high performance liquid chromatography: gradient elution procedure is 0-1.0 min,10% A+90% B;1.0 to 2.0min,15 percent of A+85 percent of B;2.0 to 8.5min,65 percent of A+35 percent of B;8.5 to 9.0min,95 percent of A+5 percent of B.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111084461.8A CN113899843B (en) | 2021-09-16 | 2021-09-16 | Method for simultaneously and quantitatively analyzing 24 ingredients of Kunxian capsule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111084461.8A CN113899843B (en) | 2021-09-16 | 2021-09-16 | Method for simultaneously and quantitatively analyzing 24 ingredients of Kunxian capsule |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113899843A CN113899843A (en) | 2022-01-07 |
| CN113899843B true CN113899843B (en) | 2024-04-09 |
Family
ID=79028741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111084461.8A Active CN113899843B (en) | 2021-09-16 | 2021-09-16 | Method for simultaneously and quantitatively analyzing 24 ingredients of Kunxian capsule |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113899843B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187810A (en) * | 2018-10-29 | 2019-01-11 | 广州白云山陈李济药厂有限公司 | A kind of detection method for treating rheumatoid arthritis Chinese medicine composition |
| CN109528725A (en) * | 2019-01-15 | 2019-03-29 | 华中科技大学 | A kind of oxide containing di-amino-pyrimidine and the nano-composition of pyrrole alkyl amino pyrimidine oxide and the preparation method and application thereof |
| CN112730647A (en) * | 2020-12-10 | 2021-04-30 | 上海长征医院 | Identification and content determination method of triptolide in Kun Xian capsule |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2974292C (en) * | 2015-01-31 | 2024-04-16 | Constance Therapeutics, Inc. | Methods for preparation of cannabis oil extracts and compositions |
-
2021
- 2021-09-16 CN CN202111084461.8A patent/CN113899843B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187810A (en) * | 2018-10-29 | 2019-01-11 | 广州白云山陈李济药厂有限公司 | A kind of detection method for treating rheumatoid arthritis Chinese medicine composition |
| CN109528725A (en) * | 2019-01-15 | 2019-03-29 | 华中科技大学 | A kind of oxide containing di-amino-pyrimidine and the nano-composition of pyrrole alkyl amino pyrimidine oxide and the preparation method and application thereof |
| CN112730647A (en) * | 2020-12-10 | 2021-04-30 | 上海长征医院 | Identification and content determination method of triptolide in Kun Xian capsule |
Non-Patent Citations (3)
| Title |
|---|
| Efficacy and safety of Kunxian capsule in treating patients with lupus nephritis.;Li Hui等;《Chinese Journal of Integrated Traditional and Western Medicine》;20191231;全文 * |
| 不同雷公藤制剂中6个成分的含量研究;祁爱蓉;傅博;刘小燕;李顺民;文善适;郑平;陈剑平;;医学信息;20180301(第05期);全文 * |
| 昆仙胶囊HPLC特征指纹图谱和多成分定量测定研究;许春芳等;《中草药 》;20201211;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113899843A (en) | 2022-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105241980B (en) | Rapid separation liquid chromatography detection method for naoxintong capsules | |
| CN109324126B (en) | Method for simultaneously determining 9 chemical components in spina date seeds by using UPLC-MS/MS | |
| CN110907580B (en) | Detection method of HPLC (high performance liquid chromatography) characteristic spectrum of Baoyuan decoction | |
| CN109239224B (en) | Method for simultaneously and quantitatively measuring 9 blood-entering components in spina date seed water extract | |
| CN105259268A (en) | Detection method for fingerprint chromatogram of flavonoid and organic acid components in ginkgo biloba extract and application of detection method | |
| CN104614456A (en) | Method for simultaneously detecting main components of Naoxintong capsule in plasma | |
| CN104914199A (en) | Determining method for contents of twelve components in traditional Chinese medicine composition preparation | |
| Xiao et al. | Rapid and reliable method for analysis of raw and honey-processed astragalus by UPLC/ESI-Q-TOF-MS using HSS T3 columns | |
| CN103235050A (en) | Quality control method of panax notoginseng saponins injection | |
| CN113252821A (en) | Method for establishing fingerprint of flavonoid component in ginkgo leaf extraction intermediate or preparation thereof and established fingerprint | |
| CN112526047A (en) | Method for quantitatively detecting flavonoid compounds in sea buckthorn based on ultra-high performance liquid chromatography-high resolution mass spectrometry technology | |
| CN114200038A (en) | Method for detecting compound content in wrinkled gianthyssop herb zhengqi oral liquid by liquid chromatography-mass spectrometry | |
| CN107764908B (en) | Method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract | |
| CN113433232B (en) | Method for measuring ginsenoside content in ginseng traditional Chinese medicine | |
| CN112730674B (en) | Quality detection method of momordica grosvenori tea | |
| CN102879516A (en) | Method for identifying Buyang Huanwu soup and measuring content of Buyang Huanwu soup | |
| CN113899843B (en) | Method for simultaneously and quantitatively analyzing 24 ingredients of Kunxian capsule | |
| CN112684036A (en) | Fingerprint spectrum determination method of kidney-tonifying capsules containing leeches and application of fingerprint spectrum determination method | |
| CN110988198A (en) | Content determination method of bi-tong ning capsules | |
| CN109828040B (en) | Construction method and detection method of UPLC (ultra Performance liquid chromatography) characteristic spectrum of eclipta medicinal material | |
| CN110412154B (en) | Quantitative determination method for multi-component liquid quality of Qilong capsule and application thereof | |
| CN113588855A (en) | Quality detection method of lindley eupatorium herb | |
| CN103575820A (en) | Analysis method for five flavonoid glycosides in blood plasma and application of five flavonoid glycosides in pharmacokinetics | |
| CN111103365A (en) | Method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of wild jujube leaves based on MRM-IDA-EPI mode | |
| Zhao et al. | Quantitative 1H NMR for the Direct Quantification of Saikosaponins in Bupleurum chinense DC. |
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 |