CN107991399B - Method for simultaneously determining 31 components in compound red sage root extract or related medicinal materials - Google Patents
Method for simultaneously determining 31 components in compound red sage root extract or related medicinal materials Download PDFInfo
- Publication number
- CN107991399B CN107991399B CN201610969524.0A CN201610969524A CN107991399B CN 107991399 B CN107991399 B CN 107991399B CN 201610969524 A CN201610969524 A CN 201610969524A CN 107991399 B CN107991399 B CN 107991399B
- Authority
- CN
- China
- Prior art keywords
- solutions
- concentration
- solution
- sample
- mixed
- 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
- 150000001875 compounds Chemical class 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 57
- 239000000284 extract Substances 0.000 title claims abstract description 48
- 239000000463 material Substances 0.000 title claims abstract description 27
- 240000007164 Salvia officinalis Species 0.000 title claims description 26
- 235000005412 red sage Nutrition 0.000 title claims description 20
- 239000000243 solution Substances 0.000 claims abstract description 65
- 239000012488 sample solution Substances 0.000 claims abstract description 28
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 claims abstract description 26
- 239000012086 standard solution Substances 0.000 claims abstract description 26
- 230000003993 interaction Effects 0.000 claims abstract description 18
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 10
- 238000010586 diagram Methods 0.000 claims abstract description 9
- 239000012085 test solution Substances 0.000 claims abstract description 8
- 239000013582 standard series solution Substances 0.000 claims abstract description 5
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 78
- AIGAZQPHXLWMOJ-UHFFFAOYSA-N Tanshinone I Chemical compound C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)=CO1 AIGAZQPHXLWMOJ-UHFFFAOYSA-N 0.000 claims description 49
- 239000000126 substance Substances 0.000 claims description 44
- 150000002500 ions Chemical class 0.000 claims description 36
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 33
- 239000013558 reference substance Substances 0.000 claims description 31
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 28
- 244000132619 red sage Species 0.000 claims description 25
- 238000005303 weighing Methods 0.000 claims description 25
- 238000002360 preparation method Methods 0.000 claims description 20
- 239000011550 stock solution Substances 0.000 claims description 20
- 239000000523 sample Substances 0.000 claims description 19
- 238000004458 analytical method Methods 0.000 claims description 17
- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 claims description 16
- 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 14
- 235000019253 formic acid Nutrition 0.000 claims description 14
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 14
- 244000131316 Panax pseudoginseng Species 0.000 claims description 12
- 235000003181 Panax pseudoginseng Nutrition 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 11
- ODHCTXKNWHHXJC-GSVOUGTGSA-N Pyroglutamic acid Natural products OC(=O)[C@H]1CCC(=O)N1 ODHCTXKNWHHXJC-GSVOUGTGSA-N 0.000 claims description 11
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 claims description 11
- KDXKERNSBIXSRK-OZDFLOAESA-N (2s)-2,6-diamino-4,4,5,5-tetradeuteriohexanoic acid Chemical compound NCC([2H])([2H])C([2H])([2H])C[C@H](N)C(O)=O KDXKERNSBIXSRK-OZDFLOAESA-N 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 10
- 239000012895 dilution Substances 0.000 claims description 10
- 229930182494 ginsenoside Natural products 0.000 claims description 10
- 229940089161 ginsenoside Drugs 0.000 claims description 10
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 9
- 229960005091 chloramphenicol Drugs 0.000 claims description 9
- STCJJTBMWHMRCD-UHFFFAOYSA-N salvianolic acid B Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=O)C=Cc2cc(O)c(O)c3OC(C(C(=O)OC(Cc4ccc(O)c(O)c4)C(=O)O)c23)c5ccc(O)c(O)c5 STCJJTBMWHMRCD-UHFFFAOYSA-N 0.000 claims description 9
- 150000004043 trisaccharides Chemical class 0.000 claims description 9
- 239000003643 water by type Substances 0.000 claims description 9
- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 claims description 8
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 claims description 8
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 claims description 8
- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 claims description 7
- SNKFFCBZYFGCQN-VWUOOIFGSA-N Lithospermic acid B Natural products C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2[C@H](C(=O)O[C@H](CC=3C=C(O)C(O)=CC=3)C(O)=O)[C@H](OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-VWUOOIFGSA-N 0.000 claims description 7
- AAOVKJBEBIDNHE-UHFFFAOYSA-N diazepam Chemical compound N=1CC(=O)N(C)C2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 AAOVKJBEBIDNHE-UHFFFAOYSA-N 0.000 claims description 7
- 229960003529 diazepam Drugs 0.000 claims description 7
- 239000007789 gas Substances 0.000 claims description 7
- 150000004044 tetrasaccharides Chemical class 0.000 claims description 7
- IBGBGRVKPALMCQ-UHFFFAOYSA-N 3,4-dihydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1O IBGBGRVKPALMCQ-UHFFFAOYSA-N 0.000 claims description 6
- 229930024421 Adenine Natural products 0.000 claims description 6
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 235000017276 Salvia Nutrition 0.000 claims description 6
- YMGFTDKNIWPMGF-QHCPKHFHSA-N Salvianolic acid A Natural products OC(=O)[C@H](Cc1ccc(O)c(O)c1)OC(=O)C=Cc2ccc(O)c(O)c2C=Cc3ccc(O)c(O)c3 YMGFTDKNIWPMGF-QHCPKHFHSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims 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 claims description 6
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims description 6
- 229960000643 adenine Drugs 0.000 claims description 6
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229930189092 Notoginsenoside Natural products 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- 238000000132 electrospray ionisation Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000001819 mass spectrum Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 239000004475 Arginine Substances 0.000 claims description 4
- AGBCLJAHARWNLA-DQUQINEDSA-N Ginsenoside RG2 Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@@](C)(O)CCC=C(C)C)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O AGBCLJAHARWNLA-DQUQINEDSA-N 0.000 claims description 4
- 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 claims description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 4
- UMPZKDHDIZUVTO-UHFFFAOYSA-N Salvianolic acid D Natural products Cc1ccc(C=CC(=O)OC(Cc2ccc(O)c(O)c2)C(=O)O)c(CC(=O)O)c1O UMPZKDHDIZUVTO-UHFFFAOYSA-N 0.000 claims description 4
- KFCMFABBVSIHTB-WUTVXBCWSA-N Salvianolic acid D Chemical compound OC(=O)CC1=C(O)C(O)=CC=C1\C=C\C(=O)O[C@@H](C(O)=O)CC1=CC=C(O)C(O)=C1 KFCMFABBVSIHTB-WUTVXBCWSA-N 0.000 claims description 4
- YURJSTAIMNSZAE-UHFFFAOYSA-N UNPD89172 Natural products C1CC(C2(CC(C3C(C)(C)C(O)CCC3(C)C2CC2O)OC3C(C(O)C(O)C(CO)O3)O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O YURJSTAIMNSZAE-UHFFFAOYSA-N 0.000 claims description 4
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 4
- JURZHOVRCOWZFN-UHFFFAOYSA-N notoginsenoside R1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5C(CC34C)OC6OC(COC7OCC(O)C(O)C7O)C(O)C(O)C6O)C JURZHOVRCOWZFN-UHFFFAOYSA-N 0.000 claims description 4
- YMGFTDKNIWPMGF-AGYDPFETSA-N 3-(3,4-dihydroxyphenyl)-2-[(e)-3-[2-[(e)-2-(3,4-dihydroxyphenyl)ethenyl]-3,4-dihydroxyphenyl]prop-2-enoyl]oxypropanoic acid Chemical compound C=1C=C(O)C(O)=C(\C=C\C=2C=C(O)C(O)=CC=2)C=1/C=C/C(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 YMGFTDKNIWPMGF-AGYDPFETSA-N 0.000 claims description 3
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims description 3
- GVKKJJOMQCNPGB-UHFFFAOYSA-N Cryptotanshinone Natural products O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)CO2 GVKKJJOMQCNPGB-UHFFFAOYSA-N 0.000 claims description 3
- GVKKJJOMQCNPGB-JTQLQIEISA-N Cryptotanshinone Chemical compound O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1[C@@H](C)CO2 GVKKJJOMQCNPGB-JTQLQIEISA-N 0.000 claims description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 3
- GCJWPRRNLSHTRY-UHFFFAOYSA-N Salvianolic acid C Natural products C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)=CC=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 GCJWPRRNLSHTRY-UHFFFAOYSA-N 0.000 claims description 3
- GCJWPRRNLSHTRY-VURDRKPISA-N Salvianolic acid C Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2C=C(OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 GCJWPRRNLSHTRY-VURDRKPISA-N 0.000 claims description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 3
- HYXITZLLTYIPOF-UHFFFAOYSA-N Tanshinone II Natural products O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)=CO2 HYXITZLLTYIPOF-UHFFFAOYSA-N 0.000 claims description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 3
- 229960005305 adenosine Drugs 0.000 claims description 3
- 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 claims description 3
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims description 3
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 3
- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 claims description 3
- AOGZLQUEBLOQCI-UHFFFAOYSA-N ginsenoside-Re Natural products CC1OC(OCC2OC(OC3CC4(C)C(CC(O)C5C(CCC45C)C(C)(CCC=C(C)C)OC6OC(CO)C(O)C(O)C6O)C7(C)CCC(O)C(C)(C)C37)C(O)C(O)C2O)C(O)C(O)C1O AOGZLQUEBLOQCI-UHFFFAOYSA-N 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- LLPWNQMSUYAGQI-OOSPGMBYSA-N notoginsenoside R1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)CO2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O LLPWNQMSUYAGQI-OOSPGMBYSA-N 0.000 claims description 3
- 229960003371 protocatechualdehyde Drugs 0.000 claims description 3
- AZEZEAABTDXEHR-UHFFFAOYSA-M sodium;1,6,6-trimethyl-10,11-dioxo-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-2-sulfonate Chemical compound [Na+].C12=CC=C(C(CCC3)(C)C)C3=C2C(=O)C(=O)C2=C1OC(S([O-])(=O)=O)=C2C AZEZEAABTDXEHR-UHFFFAOYSA-M 0.000 claims description 3
- 229930189533 tanshinol Natural products 0.000 claims description 3
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims description 3
- 229940045145 uridine Drugs 0.000 claims description 3
- 239000004474 valine Substances 0.000 claims description 3
- AGBCLJAHARWNLA-UHFFFAOYSA-N (20R)-ginsenoside Rg2 Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C3C(C)(C)C(O)CCC3(C)C3C(C4(CCC(C4C(O)C3)C(C)(O)CCC=C(C)C)C)(C)C2)OC(CO)C(O)C1O AGBCLJAHARWNLA-UHFFFAOYSA-N 0.000 claims description 2
- RAQNTCRNSXYLAH-RFCGZQMISA-N (20S)-ginsenoside Rh1 Chemical compound O([C@@H]1[C@H]2C(C)(C)[C@@H](O)CC[C@]2(C)[C@H]2C[C@@H](O)[C@H]3[C@@]([C@@]2(C1)C)(C)CC[C@@H]3[C@@](C)(O)CCC=C(C)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RAQNTCRNSXYLAH-RFCGZQMISA-N 0.000 claims description 2
- FBFMBWCLBGQEBU-RXMALORBSA-N (2s,3r,4s,5s,6r)-2-[(2r,3r,4s,5s,6r)-2-[[(3s,5r,6s,8r,9r,10r,12r,13r,14r,17s)-3,12-dihydroxy-4,4,8,10,14-pentamethyl-17-[(2s)-6-methyl-2-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhept-5-en-2-yl]-2,3,5,6,7,9,11,12,13,15,16,17-dodecah Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O FBFMBWCLBGQEBU-RXMALORBSA-N 0.000 claims description 2
- FBFMBWCLBGQEBU-GYMUUCMZSA-N 20-gluco-ginsenoside-Rf Natural products O([C@](CC/C=C(\C)/C)(C)[C@@H]1[C@H]2[C@H](O)C[C@H]3[C@](C)([C@]2(C)CC1)C[C@H](O[C@@H]1[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O2)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1C(C)(C)[C@@H](O)CC[C@]31C)[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 FBFMBWCLBGQEBU-GYMUUCMZSA-N 0.000 claims description 2
- LLPWNQMSUYAGQI-QBQUQATFSA-N Ginsenoside R1 Natural products O([C@](CC/C=C(\C)/C)(C)[C@@H]1[C@H]2[C@@H](O)C[C@H]3[C@](C)([C@]2(C)CC1)C[C@H](O[C@@H]1[C@H](O[C@@H]2[C@@H](O)[C@@H](O)[C@@H](O)CO2)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1C(C)(C)[C@@H](O)CC[C@]31C)[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 LLPWNQMSUYAGQI-QBQUQATFSA-N 0.000 claims description 2
- UFNDONGOJKNAES-UHFFFAOYSA-N Ginsenoside Rb1 Natural products CC(=CCCC(C)(OC1OC(COC2OC(CO)C(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CC(O)C45C)C UFNDONGOJKNAES-UHFFFAOYSA-N 0.000 claims description 2
- HYPFYJBWSTXDAS-UHFFFAOYSA-N Ginsenoside Rd Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C4CCC5C(C)(C)C(CCC5(C)C4CC(O)C23C)OC6OC(CO)C(O)C(O)C6OC7OC(CO)C(O)C(O)C7O)C HYPFYJBWSTXDAS-UHFFFAOYSA-N 0.000 claims description 2
- RAQNTCRNSXYLAH-UHFFFAOYSA-N Ginsenoside Rh1 Natural products CC(C)=CCCC(C)(O)C1CCC(C2(C3)C)(C)C1C(O)CC2C1(C)CCC(O)C(C)(C)C1C3OC1OC(CO)C(O)C(O)C1O RAQNTCRNSXYLAH-UHFFFAOYSA-N 0.000 claims description 2
- GZYPWOGIYAIIPV-JBDTYSNRSA-N ginsenoside Rb1 Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GZYPWOGIYAIIPV-JBDTYSNRSA-N 0.000 claims description 2
- TXEWRVNOAJOINC-UHFFFAOYSA-N ginsenoside Rb2 Natural products CC(=CCCC(OC1OC(COC2OCC(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CCC45C)C TXEWRVNOAJOINC-UHFFFAOYSA-N 0.000 claims description 2
- YURJSTAIMNSZAE-HHNZYBFYSA-N ginsenoside Rg1 Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YURJSTAIMNSZAE-HHNZYBFYSA-N 0.000 claims description 2
- CBEHEBUBNAGGKC-UHFFFAOYSA-N ginsenoside Rg1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5CC(OC6OC(CO)C(O)C(O)C6O)C34C)C CBEHEBUBNAGGKC-UHFFFAOYSA-N 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- UOJAEODBOCLNBU-UHFFFAOYSA-N vinaginsenoside R4 Natural products C1CC(C2(CC(O)C3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O UOJAEODBOCLNBU-UHFFFAOYSA-N 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims 2
- 230000001681 protective effect Effects 0.000 claims 2
- 239000009724 Salvia extract Substances 0.000 abstract description 7
- 241000304195 Salvia miltiorrhiza Species 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 53
- 230000004044 response Effects 0.000 description 24
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 20
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 20
- 229930183118 Tanshinone Natural products 0.000 description 19
- 150000001413 amino acids Chemical class 0.000 description 18
- 241000180649 Panax notoginseng Species 0.000 description 17
- 235000003143 Panax notoginseng Nutrition 0.000 description 17
- 150000007965 phenolic acids Chemical class 0.000 description 16
- 229930182490 saponin Natural products 0.000 description 16
- 235000017709 saponins Nutrition 0.000 description 16
- 239000012491 analyte Substances 0.000 description 15
- 239000012074 organic phase Substances 0.000 description 15
- 150000007949 saponins Chemical class 0.000 description 15
- 229920001542 oligosaccharide Polymers 0.000 description 14
- 150000002482 oligosaccharides Chemical class 0.000 description 14
- 238000000926 separation method Methods 0.000 description 14
- 239000002777 nucleoside Substances 0.000 description 13
- 238000001514 detection method Methods 0.000 description 11
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 11
- 239000003814 drug Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 150000003833 nucleoside derivatives Chemical class 0.000 description 9
- 150000007524 organic acids Chemical class 0.000 description 9
- 150000001408 amides Chemical class 0.000 description 8
- 238000010828 elution Methods 0.000 description 8
- 230000005526 G1 to G0 transition Effects 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- -1 phenolic acid compound Chemical class 0.000 description 7
- 239000000654 additive Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 235000009048 phenolic acids Nutrition 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 4
- 238000002552 multiple reaction monitoring Methods 0.000 description 4
- 125000003835 nucleoside group Chemical group 0.000 description 4
- 239000006187 pill Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- YMGFTDKNIWPMGF-UCPJVGPRSA-N Salvianolic acid A Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C(=C(O)C(O)=CC=1)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 YMGFTDKNIWPMGF-UCPJVGPRSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 235000005985 organic acids Nutrition 0.000 description 3
- 229930010796 primary metabolite Natural products 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229930183842 salvianolic acid Natural products 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 229930000044 secondary metabolite Natural products 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- UJZQBMQZMKFSRV-RGKBJLTCSA-N (2s,3s)-4-[(e)-3-[(1r)-1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]-3-oxoprop-1-enyl]-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-3-carboxylic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2[C@H](C(O)=O)[C@H](OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 UJZQBMQZMKFSRV-RGKBJLTCSA-N 0.000 description 2
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 2
- 206010008479 Chest Pain Diseases 0.000 description 2
- UJZQBMQZMKFSRV-PHQFMFTGSA-N Lithospermic acid Natural products O([C@@H](C(=O)O)Cc1cc(O)c(O)cc1)C(=O)/C=C/c1c2[C@@H](C(=O)O)[C@H](c3cc(O)c(O)cc3)Oc2c(O)cc1 UJZQBMQZMKFSRV-PHQFMFTGSA-N 0.000 description 2
- NFOCYHUCMXEHDG-UHFFFAOYSA-N Monomethyl lithospermate Natural products COC(=O)C1C(C=2C=C(O)C(O)=CC=2)OC(C(=CC=2)O)=C1C=2C=CC(=O)OC(C(O)=O)CC1=CC=C(O)C(O)=C1 NFOCYHUCMXEHDG-UHFFFAOYSA-N 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012156 elution solvent Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- DTGKSKDOIYIVQL-WEDXCCLWSA-N (+)-borneol Chemical compound C1C[C@@]2(C)[C@@H](O)C[C@@H]1C2(C)C DTGKSKDOIYIVQL-WEDXCCLWSA-N 0.000 description 1
- PAFLSMZLRSPALU-MRVPVSSYSA-N (2R)-3-(3,4-dihydroxyphenyl)lactic acid Chemical compound OC(=O)[C@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-MRVPVSSYSA-N 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- PAFLSMZLRSPALU-QMMMGPOBSA-N Danshensu Natural products OC(=O)[C@@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-QMMMGPOBSA-N 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 239000009277 Panax notoginseng extract Substances 0.000 description 1
- PAFLSMZLRSPALU-UHFFFAOYSA-N Salvianic acid A Natural products OC(=O)C(O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-UHFFFAOYSA-N 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 150000002337 glycosamines Chemical class 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000001474 liquid chromatography-evaporative light scattering detection Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920006112 polar polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 208000023482 precordial pain Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229940112950 sage extract Drugs 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
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
-
- 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
- G01N30/89—Inverse chromatography
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)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for simultaneously measuring 31 components in a compound salvia extract or related medicinal materials, which adopts a direct tandem-reversed phase-hydrophilic interaction chromatography-mass spectrometry combined method and comprises the following steps: step 1, preparing a sample solution; step 2, preparing standard series solution; step 3, preparing an internal standard solution; step 4, preparing a test solution; and 5, injecting the solution obtained in the step 4 into a direct tandem reverse phase-hydrophilic interaction liquid chromatography-mass spectrometry combined system to obtain an extracted ion flow diagram of 31 analytes, and calculating the content of 31 components in the compound salvia miltiorrhiza extract or related medicinal materials according to the extracted ion flow diagram.
Description
The technical field is as follows:
the invention relates to a detection method of effective components of traditional Chinese medicines, in particular to a method for simultaneously determining 31 components in a compound salvia miltiorrhiza extract or related medicinal materials.
Background art:
the study of mass balance is of great significance for the elucidation of the composition of the chemical substance group of traditional Chinese medicine. The chemical composition of Chinese medicine is very complex, and contains numerous primary metabolites and secondary metabolites.
Earlier studies show that compound Saviae Miltiorrhizae radix dripping pill (prepared from Saviae Miltiorrhizae radix, Notoginseng radix, and Borneolum Syntheticum, and its preparation method is shown in "Chinese pharmacopoeia" 2015 edition) mainly contains oligosaccharide, amino acids, nucleosides, organic acids, phenolic acids, saponins and tanshinone compounds. Due to the large differences in the structure and physicochemical properties of these compounds, researchers often use a variety of analytical methods to perform quantitative analysis on different types of compounds, such as detecting amino acids using an amino acid analyzer, detecting oligosaccharides and saponins using a high performance liquid chromatography-evaporative light scattering detector, and detecting phenolic acids and tanshinone using a high performance liquid chromatography-ultraviolet detector or a liquid chromatography-mass spectrometer. The application of multiple analytical methods brings great inconvenience to the study of mass balance.
Reversed phase chromatography: the case where the mobile phase is more polar than the stationary phase is called reverse phase chromatography. The nonpolar bonding phase chromatography can be reversed phase chromatography. Most widely used in modern liquid chromatography, over 70% of modern liquid chromatography work is done on non-polar bonded stationary phases.
Hydrophilic interaction chromatography: can be regarded as a continuation of the field of normal phase chromatography towards aqueous mobile phases. The mobile phase is aqueous buffer (< 40%) and organic solvent. The stationary phase is a polar adsorbent with strong hydrophilicity, such as silica gel bonded phase, polar polymer filler or ion exchange adsorbent. A common feature of these stationary phases is that they are very reactive with water and therefore are "hydrophilic". The gradient and antiphase modes used are reversed. The initial conditions include a high proportion of an organic phase, typically a 95% strength organic phase such as acetonitrile, which is gradually reduced to an aqueous phase. Therefore, it is also called reverse phase chromatography.
Direct tandem-reversed phase-hydrophilic interaction chromatography-mass spectrometry combined system: two chromatographic columns in the separation mode are directly connected in series, so that substances from strong polarity to weak polarity can be well reserved, and the purpose of separation is achieved.
The compound Saviae Miltiorrhizae radix extract or related medicinal materials are conventional medicines, and are prepared from Notoginseng radix, Saviae Miltiorrhizae radix and Borneolum. Has the effects of promoting blood circulation, removing blood stasis, regulating qi and relieving pain. Can be used for treating thoracic obstruction due to qi stagnation and blood stasis, with symptoms of chest distress and precordial pain; coronary heart disease and angina pectoris with the above syndrome.
The compound salvia extract or related medicinal materials comprise a compound salvia preparation sold in the market, such as a compound salvia dropping pill or a compound salvia tablet, and also comprise common extracts of panax notoginseng and salvia miltiorrhiza or extracts extracted respectively and then mixed, wherein the extracts are sold in the market, such as a compound salvia extract, a salvia miltiorrhiza extract and a panax notoginseng extract, and also can be prepared by referring to a preparation method in the prior art, and also comprise panax notoginseng and salvia miltiorrhiza medicinal materials, such as salvia miltiorrhiza powder and panax notoginseng powder.
The compound salvia miltiorrhiza extract or related medicinal materials of the invention respectively comprise 31 effective components: adenine, succinic acid, pyroglutamic acid, adenosine, uridine, tanshinol, valine, lysine, proline, protocatechualdehyde, glucose, sucrose, arginine, salvianolic acid D, notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, rosmarinic acid, trisaccharide, alkannic acid, salvianolic acid B, salvianolic acid A, ginsenoside Rb1, salvianolic acid C, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rd, tetrasaccharide, cryptotanshinone, tanshinone I, tanshinone IIA.
Aiming at the defects of the existing mass balance research method, the research establishes a direct tandem-reversed phase-hydrophilic interaction chromatography-mass spectrometry combined system, and can simultaneously determine 7 major types and 31 types of components in the compound salvia miltiorrhiza extract or related medicinal materials.
The invention establishes a direct tandem-reversed phase-hydrophilic interaction chromatography-mass spectrometry combined system, measures 7 and 31 components with great difference in properties in the compound salvia extract or related medicinal materials, can be applied to quality evaluation of the compound salvia extract or related medicinal materials, and has the characteristics of comprehensiveness, accuracy and rapidness.
The invention content is as follows:
the invention provides a method for simultaneously measuring 31 components in a compound salvia miltiorrhiza extract or related medicinal materials, which adopts direct tandem-reversed phase-hydrophilic interaction chromatography-mass spectrometry combination.
The method provided by the invention comprises the following steps:
step 1 preparation of sample solution: preparing compound Saviae Miltiorrhizae radix extract or related medicinal materials into methanol solution with methanol;
step 3 preparation of internal standard solution: taking an internal standard substance L-lysine-4, 4,5,5-d4Diazepam and chloramphenicol, respectively preparing methanol solutions with methanol;
and 5, injecting the test solution obtained in the step 4 into a direct tandem reverse phase-hydrophilic interaction liquid chromatography-mass spectrometry system to obtain an extracted ion flow diagram of 31 analytes, and calculating the content of 31 components in the compound salvia miltiorrhiza extract or related medicinal materials according to the extracted ion flow diagram.
The order of steps 1-4 above is not a chronological limitation and may be interchanged with one another.
In the above method, the compound red sage root extract or related medicinal materials are selected from: compound red sage root extract, notoginseng extract, red sage root or notoginseng.
Preferably, the step 1: the sample solution was prepared as follows: respectively taking 0.10g of compound red sage root extract, pseudo-ginseng extract, red sage root powder or pseudo-ginseng powder, precisely weighing, adding 8-12mL of 60-80% methanol, carrying out ultrasonic treatment for 20-40min, centrifuging for 8-12min at 16,000rpm, filtering with a 0.22 mu m filter membrane, and storing all solutions in a refrigerator at 4 ℃ before analysis;
further preferably, step 1: the sample solution was prepared as follows: respectively taking 0.10g of compound red sage root extract, pseudo-ginseng extract, red sage root powder and pseudo-ginseng powder, precisely weighing, adding 10mL of 70% methanol, carrying out ultrasonic treatment for 30min, centrifuging for 10min at 16,000rpm, filtering with a 0.22 mu m filter membrane, and storing all solutions in a refrigerator at 4 ℃ before analysis;
preferably, step 2: the standard series of solutions was prepared as follows: weighing appropriate amount of standard substance of the object to be tested, precisely weighing, respectively adding 60-80% methanol water to prepare solution with concentration of 450-1Glucose with concentration of 6000ng/mL, trisaccharide with concentration of 4000ng/mL and ginsenoside Rb with concentration of 4000ng/mL1Ginsenoside Rd with the concentration of 4000ng/mL, pyroglutamic acid with the concentration of 2000ng/mL, rosmarinic acid with the concentration of 2000ng/mL, and notoginsenoside R with the concentration of 2000ng/mL1And mixed standard stock solution of other substances to be detected with the concentration of 1000 ng/mL; taking a proper amount of the mixed standard stock solution, preparing a series of mixed standard solutions with concentration by using 60-80% methanol according to the dilution times of 2, 5, 10, 20, 50, 100, 200, 500 and 1000, and storing the mixed standard solutions in a refrigerator at 4 ℃ for later use;
further preferably, step 2: sign boardThe preparation of the quasi-series solution was: weighing appropriate amount of standard substance to be tested, adding 70% methanol water to obtain solutions with concentration of 500 μ g/mL, precisely measuring appropriate amount of stock solutions of each to-be-tested substance, respectively, preparing tetrasaccharide with concentration of 70000ng/mL, sucrose with concentration of 50000ng/mL, salvianolic acid B with concentration of 12000ng/mL, and ginsenoside Rg with concentration of 8000ng/mL with 70% methanol water1Glucose with concentration of 6000ng/mL, trisaccharide with concentration of 4000ng/mL and ginsenoside Rb with concentration of 4000ng/mL1Ginsenoside Rd with the concentration of 4000ng/mL, pyroglutamic acid with the concentration of 2000ng/mL, rosmarinic acid with the concentration of 2000ng/mL, and notoginsenoside R with the concentration of 2000ng/mL1And mixed standard stock solution of other substances to be detected with the concentration of 1000 ng/mL; taking a proper amount of the mixed standard stock solution, preparing a series of mixed standard solutions with concentration by using 70% methanol according to dilution times of 2, 5, 10, 20, 50, 100, 200, 500 and 1000, and storing the mixed standard solutions in a refrigerator at 4 ℃ for later use;
preferably, step 3: the internal standard solution was prepared as follows: weighing L-lysine-4, 4,5,5-d4Accurately weighing appropriate amounts of (IS1), diazepam (IS2) and chloramphenicol (IS3), respectively adding 60-80% methanol water to prepare solutions with the concentration of 450-;
further preferably, step 3 is: preparation of internal standard solution: weighing L-lysine-4, 4,5,5-d4Accurately weighing appropriate amounts of (IS1), diazepam (IS2) and chloramphenicol (IS3), respectively adding 70% methanol water to prepare solutions with the concentration of 500 mug/mL as internal standard stock solutions, respectively accurately weighing appropriate amounts of the internal standard stock solutions, preparing IS reference substance solutions with the concentration of 100ng/mL by using 70% methanol water, and storing in a refrigerator at 4 ℃ for later use;
preferably, step 4: the test solution is prepared by the following steps: respectively and precisely measuring an equal series of mixed reference substance solutions or sample solutions with different concentrations and an IS reference substance solution to be mixed to prepare a series of mixed reference substance solutions or sample solutions containing an internal standard, storing the series of mixed reference substance solutions or sample solutions in a refrigerator at 4 ℃ for later use, wherein the series of mixed reference substance solutions are solutions with the dilution times of the internal standard solutions of 2, 4, 10, 20, 40, 100, 200, 400, 1000 and 2000, and the volume ratio of the mixed sample solution to the IS reference substance solution IS 1-10: 1-10;
further preferably, step 4: the test solution is prepared by the following steps: respectively and precisely measuring an equal series of mixed reference substance solutions or sample solutions with different concentrations and mixing with an IS reference substance solution to prepare a series of mixed reference substance solutions or sample solutions containing an internal standard, storing the series of mixed reference substance solutions or sample solutions in a refrigerator at 4 ℃ for later use, wherein the series of mixed reference substance solutions are solutions with the dilution times of the internal standard solutions of 2, 4, 10, 20, 40, 100, 200, 400, 1000 and 2000, and the sample solutions and the IS reference substance solutions are mixed in equal proportion;
preferably, step 5: injecting the sample solution into a chromatograph to obtain a chromatogram, and calculating the content of 31 components in the compound red sage root extract or related medicinal materials according to the chromatogram, wherein the content is calculated according to an internal standard method; wherein the chromatographic conditions are as follows:
reverse phase column using sub 2 μm C18 packed column, guard column using Waters in-line filter (2 μm), mobile phase consisting of 0.08-0.15% formic or acetic acid water (a1) and acetonitrile (B1), gradient program: 0min, 25% B; 0.5min, 25% B; 1.0min, 35% B; 5.0min, 60% B; 10.0min, 80% B; 15.0min, 80% B, flow rate of 0.1-0.12mL/min, column temperature of 25-45 deg.C, sample amount of 1-3 μ L;
the hydrophilic interaction chromatographic column uses a sub-2 μm hydrophilic chromatographic column, the guard column uses a Waters on-line filter (2 μm), the mobile phase consists of 0.08-0.15% formic or acetic acid water (a2) and acetonitrile (B2), and the gradient program is: 0min, 97% B; 10.0min, 50% B; 10.5min, 50% B; 11.0min, 97% B; 15.0min, 97% B; the flow rate is 0.35-0.45mL/min, and the column temperature is 25-45 ℃; the reversed phase chromatographic column and the hydrophilic chromatographic column are connected through a tee joint;
the mass spectrum conditions were as follows:
connecting with hydrophilic chromatographic column by using electrospray ionization (ESI) interface, setting the voltage of positive ion capillary tube to 5500V, and setting the voltage of negative ion capillary tube to-4500V; the ion source temperature was set at 550 ℃; high-purity nitrogen is used as the atomizing gas, the heating gas and the air curtain gas, the set pressure is 55 psi, 55 psi and 25psi respectively, and the object to be detected is quantitatively analyzed by using a positive and negative ion switching and time-course Multiple Reaction Monitoring (MRM) method.
The reverse phase chromatography column is preferably Shim-Pack XR-ODS III (1.6 μm, 2.0 mm. times.50 mm) or Waters Acquity
BEH C18(1.7μm，2.1×50mm)。
The hydrophilic chromatographic column is preferably an amide chromatographic column, and is more preferably a Waters Acquity
BEH Amide(1.7μm，2.1mm×100mm)。
Explanation: IS reference solution IS corresponding L-lysine-4, 4,5,5-d4(IS1), diazepam (IS2) and chloramphenicol (IS3) control solutions.
The method provided by the invention is obtained through a large number of screening experiments:
1. detecting 31 components in the compound salvia miltiorrhiza extract and the medicine, and respectively: amino acid, nucleotide, organic acid and polysaccharide belong to primary metabolites, and salvianolic acid, saponin and tanshinone belong to secondary metabolites.
The polarity is in the order from small to large: tanshinone, salvianolic acid, saponin, organic acid, nucleoside, amino acid, and sugar (not in absolute order, but in approximate order according to their properties, and may vary depending on the specificity of each substance).
Considering the availability of instruments and equipment and the difference of properties among substances, the most common methods for each substance are different, but the purpose of the experiment is to reveal the mass balance of the traditional Chinese medicine composition, and the results of respective determination are not comparable, so that simultaneous detection needs to be considered.
2. The method of the invention comprises the following steps: the reversed-phase hydrophilic tandem chromatography technology can be suitable for separating compounds with larger polarity difference, and the application of the reversed-phase hydrophilic tandem chromatography technology to compound salvia miltiorrhiza extracts or pseudo-ginseng and salvia miltiorrhiza is not reported at present.
The inventor is applied to the field of compound salvia miltiorrhiza extracts and related medicinal materials for the first time. The seven components selected in the experiment belong to main components (the mass ratio accounts for more than 50% of the total water content) in the compound salvia extract and are also common quality control indexes, wherein oligosaccharides, amino acids and nucleoside compounds belong to components with larger polarity, and if the components are measured independently, a hydrophilic interaction chromatogram is preferably selected, but the components do not retain with medium-polarity or non-polar substances in the hydrophilic interaction chromatogram and are not suitable for analysis. In contrast, the selected organic acids, phenolic acids, saponins and tanshinone compounds are preferably subjected to reversed phase chromatography. In view of time economy, reverse-phase hydrophilic tandem chromatography is required to achieve good separation of components with such complex polarity differences. The tandem mass spectrometry MRM mode is added so that the substance measurement does not interfere with each other.
3. Difficulty of reverse phase-hydrophilic detection method:
1) for the detection object: when the valve switching method is used for Chinese medicine detection, a valve switching method is mostly used, according to the peak time, a part suitable for reverse phase separation is separated by a reverse phase column, and an unsuitable part is switched to hydrophilic column separation, so that the reverse phase hydrophilic columns can only be combined but not connected in a full two-dimensional series manner, and the problem caused by the full two-dimensional series connection is that the reverse phase hydrophilic columns share the same mobile phase system. For example, when organic acids, saponins, oligosaccharides, amino acids and nucleosides are detected, experimental results show that the peak shapes of the substances in a newly established series system are not influenced and only have certain difference in peak-appearing time compared with the respective single and better chromatographic columns, and the substances belong to a normal phenomenon; for phenolic acid type components, there will be a slight tailing situation compared to the use of a reverse phase column alone, but this situation will be improved by the acid concentration of the additive in the mobile phase; for the tanshinone component, the most strongly retained in the reverse phase column, the last elution in the reverse phase column, but it needs to be in the case of a high proportion of organic phase in the hydrophilic column to elute, and actually according to the elution mode of the hydrophilic column, from the high organic phase to the organic phase: the aqueous phase/50: 50 ratio was subjected to gradient elution. The inventors found that the flow phase ratio did not meet the peak appearance condition of tanshinone by the time of the second dimension hydrophilic column after tanshinone eluted from the first dimension reverse phase column, and thus dispersion or no peak appearance may occur. Aiming at the situation, the inventor adopts a solution that according to the peak-appearing time, after ensuring that all other substances are normally appeared, the proportion of a mobile phase organic phase of a second-dimensional hydrophilic column is increased, so that tanshinone is normally reserved, and the test result is in line with expectation. To summarize: for non-polar substances, the method can be adopted for searching because of the discordance of the ratio of the first-dimension post-elution to the second-dimension post-operation mobile phase in the full two-dimension system.
2) The balance time is as follows: the system is a full two-dimensional serial connection and accurate quantification system. Therefore, the equilibrium time is longer than that of the single chromatographic column, otherwise, the peak time shifts, and the specific time is determined according to the situation.
3. When reversed-phase hydrophilic chromatography is combined, a sample (which cannot contain PEG, a surfactant and the like and cannot enter a mass spectrum) meeting the detection condition of the mass spectrum can be detected theoretically. The above discussion shows that the salvianolic acid and tanshinone have certain difficulty in using the method, and through a large number of experimental investigation and demonstration, a reversed phase-hydrophilic combined method suitable for three samples of compound salvia extract, salvia medicinal material and panax notoginseng medicinal material is finally determined. Other similar samples can be tested by the method after the method needs to be verified again.
The specific screening process is as follows:
firstly, the selection of chromatographic conditions, the establishment of direct tandem reverse phase chromatography and hydrophilic interaction chromatography systems must take into account solvent compatibility and the order of connection.
The weak elution solvent in reverse phase chromatography is the strong elution solvent in hydrophilic interaction chromatography and vice versa. The problem of solvent compatibility can be solved by an online dilution method, namely, the first chromatographic column is eluted at a low flow rate, and the second chromatographic column is eluted at a high flow rate (3-8 times), so that the mobile phase entering the second chromatographic column is compatible with a chromatographic system. Because two chromatographic columns are directly connected in series, the system pressure can be greatly improved. Hydrophilic interaction chromatography columns have a lower column pressure at high flow rates due to the use of a high proportion of organic phase for elution compared to reverse phase chromatography columns. Meanwhile, the ionization efficiency of the high-proportion organic phase is higher, which is beneficial to improving the sensitivity of the analysis method. Taking the above considerations together, experiments were performed using a combination of reversed-phase-hydrophilic interaction chromatography.
Second, column inspection
The specification of the chromatographic column and the optimal selection of the packing are controlled by fixing the factors of flow equality. The organic phase of the column is usually selected from acetonitrile and methanol, with the empirical use of acetonitrile and the column pressure being relatively low, as mentioned later. Whereas 0.1% formic acid is a general procedure for mobile phase additives, suitable for use in a process groping starting conditions.
The following mobile phases were used for the test experiments: phase A, 0.1% formic acid water; phase B, acetonitrile. Reverse phase chromatography the separation effect of different size C18 chromatography columns (column length 50mm and 100mm) on the analytes was examined. Since the first chromatographic column needs to be separated at a low flow rate, the flow rate is set to 0.1 mL/min. Chromatographic columns (50mm and 100mm) with two specifications can better separate phenolic acid, saponin and tanshinone compounds, but have poor retention on polar compounds such as amino acid, oligosaccharide and nucleoside. Under low flow rate, the separation of a 50mm chromatographic column can be completed within 8min, and the chromatographic peak does not generate peak broadening phenomenon; the separation of a 100mm chromatographic column needs to be carried out for 12min, and the chromatographic peak is seriously widened. Hydrophilic interaction chromatography two stationary phases, silica gel (HILIC) and Amide (Amide), were investigated at a flow rate of 0.4 mL/min. The silica gel fixation hardly retains other analytes except oligosaccharide, while the amide fixation retains oligosaccharide and saponin well, retains phenolic acid, nucleoside, amino acid and other model compounds, and does not retain tanshinone substances. The amide stationary phase exhibited good separation orthogonality with the C18 stationary phase, so a combination of C18 and amide columns was used.
Third, mobile phase System
Compared with a methanol water system, the system pressure of the acetonitrile water system is lower, and the separation performance of the hydrophilic interaction chromatography in the acetonitrile water system is better, so that the acetonitrile water is selected as a mobile phase. The selection of mobile phase additives included the addition of acid or the addition of a salt or acid salt combination, therefore, the inventors examined three mobile phase additive combinations, including a.0.1% formic acid, b.10mM ammonium formate, and c.0.1% formic acid-10 mM ammonium formate.
All additives were added in the aqueous phase. In both the reversed phase and hydrophilic separation modes, 0.1% formic acid significantly improved the peak profile of the phenolic compounds, while ammonium formate severely smeared the phenolic compounds. The three additives have little influence on the retention and response of oligosaccharide, saponin and tanshinone compounds. The addition of formic acid reduces the negative ion response but has an irreplaceable effect on improving the peak shape of the chromatographic peak. The separation effect of four formic acid addition levels, 0.00%, 0.02%, 0.05% and 0.10%, on the analyte was further examined in a direct tandem system to evaluate the compound response as peak area. When no formic acid is added, phenolic acid and tanshinone have low response, and the former has serious tailing; when 0.02% formic acid is added, the response of oligosaccharide, amino acid, nucleoside, saponin and tanshinone is improved, but the improvement of the phenolic acid peak shape is limited, and the quantitative requirement is difficult to meet; when 0.05% formic acid is added, the phenolic acid peak type is improved, the response of the salvianolic acid B, the salvianolic acid D and the lithospermic acid is obviously improved, but the danshensu cannot be detected; when 0.10% formic acid was added, the peak profile of phenolic acids was improved significantly, with all phenolic acid responses maximized, while saponin, pyroglutamic acid and adenine responses decreased (to a minimum of 28.6% of the response at the 0.02% addition level). By integrating the quantity and content information of the analytes in the sample, the method in a 0.10% formic acid water system can meet the analysis requirement.
Fourth, investigation of mobile phase mixing mode
The three-way connection and the length of the mixing line can affect the mixing effect of the mobile phase of the reversed phase chromatography and the hydrophilic interaction chromatography. As shown in FIG. 3, the three ways of connection are ABC.
Experimental results show that the C-mode chromatographic peak has a significant broadening, which may be a result of separation loss of the reversed-phase chromatography due to the vortex phenomenon occurring in the tee by the mobile phase in the convection junction. The A mode and the B mode can obtain good chromatographic peak patterns, and the response of the A mode is higher than that of the B mode, which is probably because the high-flow-rate mobile phase is connected into the tee joint in the vertical direction to obtain better mixing effect. The mixing effect is of great significance to the separation performance and stability of hydrophilic interaction chromatography. The mixing process should minimize diffusion of the analyte within the system and loss of resolution, thus increasing mixing efficiency by extending the line after mixing. The effect of metal tubing with an internal diameter of 0.01mm and lengths of 5, 10 and 15cm, respectively, on hydrophilic interaction chromatographic separation was examined. When the pipeline between the tee joint and the hydrophilic interaction chromatographic column is extended to 15cm, the peak type of each analyte is good, and the method can be stably repeated, which indicates that the two mobile phases are sufficiently mixed. From the above results, subsequent studies were conducted using the a mode and 15cm mixing line.
Fifthly, optimization of flow rate and gradient elution program
The problem of mobile phase compatibility is solved by adopting an online dilution method. The flow rate and ratio have a large influence on the chromatographic behavior and response of the analyte. First, a hydrophilic interaction chromatography flow rate of 0.50mL/min was set, and the chromatographic behavior and response of each analyte were examined at reverse phase chromatography flow rates of 0.06, 0.08, 0.10, 0.12, and 0.14mL/min (flow rate ratios of 1: 8.33, 1: 6.25, 1: 5.00, 1: 4.16, and 1: 3.57, respectively). As the reverse phase chromatography flow rate increased, the compound retention decreased while the response gradually increased. When the flow rate was increased to 0.12mL/min, the chromatographic peak response was substantially stabilized, so that reverse phase chromatographic separation was performed at this flow rate. Then, the flow rate of the hydrophilic interaction chromatography was examined, and three levels of 0.30, 0.40 and 0.50mL/min were set.
The results show that the flow rate has little effect on the chromatographic behavior of each compound, but that the chromatographic peak response increases slightly with increasing flow rate. When the flow rate was increased to 0.40mL/min, the chromatographic peak response was substantially stable. Therefore, the second dimension separation flow rate was set to 0.40 mL/min. The gradient elution procedure can significantly affect the chromatographic behavior of each compound. Since reverse phase chromatography separates at low flow rates, a higher proportion of the organic phase is required to separate the analyte in a short time. When the organic phase ratio of the initial mobile phase of the reversed phase chromatography is lower than 20%, each analyte has obvious peak broadening phenomenon, the chromatographic peak of the phenolic acid compound is trailing, and the elution time is long; when the initial mobile phase organic phase ratio increased to 25%, the improvement in chromatographic peak pattern was significant and all analytes could be eluted within 15 min. The hydrophilic interaction chromatography can further separate the reverse phase chromatography effluent, but the tanshinone compound cannot be eluted by the one-way gradient elution procedure. In the hydrophilic interaction chromatography, the tanshinone compound can be eluted in a high proportion of organic phases, but the dispersion phenomenon occurs in a low proportion of organic phases. In reversed phase chromatography, tanshinone compounds are eluted after 10min, and organic phase ratio of hydrophilic interaction chromatography is rapidly increased to 97% at 10.5min to ensure peak type of tanshinone.
Sixthly, optimization of mass spectrum conditions
1) Ion pair optimization
The amino acid, the nucleoside and the tanshinone have good response in a positive ion mode, and an obvious excimer ion peak can be detected. The response of the oligosaccharide, the organic acid, the phenolic acid and the saponin is higher than that of negative ions in a negative ion mode, and an obvious excimer ion peak can be detected. Pyroglutamic acid and adenine are respectively amino acid and nucleoside compound, but the response of the pyroglutamic acid and adenine is higher than that of positive ions in the negative ion mode, so that the negative ion mode is adopted to detect the two substances. And respectively removing the cluster voltage and the incident voltage to enable the peak response of the quasi-molecular ions of each compound to reach the maximum. And further carrying out sub-ion scanning on the quasi-molecular ion peak of each compound, and optimizing collision energy and collision chamber escape voltage to enable sub-ion response to be maximum. And selecting the ion pair with the highest response as a quantitative ion pair and selecting the ion pair with the second highest response as a qualitative ion pair.
2) Internal standard compound screening
The selection of the internal standard substance has important significance for ensuring the stability and reliability of the method. Because the variety of the measured substances is more, the difference of the structure and the physicochemical property of each compound is larger, and a multi-internal standard substance is adopted to establish an analysis method. Using L-lysine-4, 4,5,5-d in positive ion mode4As an internal standard substance for amino acids and nucleosides, diazepam asInternal standard substance of tanshinone, chloramphenicol was used as internal standard substance of all compounds in negative ion mode. L-lysine-4, 4,5,5-d4The structure of (a) is similar to the amino acid to be determined, and the retention behavior is similar to that of nucleoside; because the peak time of tanshinone is late, diazepam with similar polarity is used as an internal standard substance; the compounds to be detected in the negative ion mode all peak at 2-7min, and chloramphenicol (5.55min) can react with the ionization condition of the compounds with similar retention time. The three internal standard substances have high and stable response, sharp peak shape and good symmetry, and can meet the requirements of analysis and determination of each compound.
The method provided by the invention has the following advantages:
1. at present, no method can simultaneously carry out quantitative analysis on a plurality of compounds with greatly different chemical and physical properties and structures, such as oligosaccharide, amino acid, nucleoside, organic acid, phenolic acid, saponin, tanshinone and the like contained in the compound salvia miltiorrhiza dripping pill. The invention develops an accurate, reliable, simple, convenient and rapid determination method, can be used as a means for quantitatively analyzing various components of the compound red sage root dripping pill, and can simultaneously determine 7 types and 31 types of components in the compound red sage root extract. The method saves time cost and labor cost, is rapid and accurate, and can evaluate the quality of the preparation more comprehensively.
2. The Chinese medicinal composition is extremely complex and contains numerous primary metabolites and secondary metabolites. To clarify the composition of the chemical groups of Chinese herbs, it is not reasonable to measure only a single component or a single class of components. The process of analyzing by respectively measuring and combining a plurality of components by different methods not only wastes time and labor, but also has no comparability of the measurement results among the methods (the common methods for respective detection are shown in the second section of background technology).
3. The method established by the invention can cover a larger measurement range through one-time measurement, make up for the defects and lay a foundation for researching the mass balance of the traditional Chinese medicine.
Drawings
FIG. 1: structures of 31 analytes and 3 internal standard compounds;
FIG. 2: a schematic diagram of a direct tandem reversed phase-hydrophilic interaction liquid chromatography-mass spectrometry system;
FIG. 3: schematic diagram of three-way connection modes, wherein: the mobile phase 1 is a reversed phase chromatography mobile phase system; the mobile phase 2 is a hydrophilic interaction chromatography mobile phase system;
FIG. 4: the number of the extracted ion flow diagram of 31 analytes in the compound salvia miltiorrhiza extract is consistent with the number of the compound in the table 4.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Laboratory instruments and materials
1. Instrument for measuring the position of a moving object
Shimadzu ultra high performance liquid chromatograph (Shimadzu corporation, Japan) equipped with four high pressure pumps, an online degasser, an autosampler, and a column stability controller; qtrap 5500 triple quadrupole mass spectrometer (AB, USA); KQ-500DE ultrasonic instrument (ultrasonic instruments Co., Ltd., Kunshan city); XS105DU analytical balance (METTLER, Switzerland).
2. Reagent and medicine
HPLC grade and MS grade acetonitrile were purchased from Merck, germany. HPLC grade formic acid (> 95% purity) was purchased from Sigma, USA.
High purity deionized water was produced by a Milli-Q system (Milli-Q, USA).
Adenine, adenosine, uridine, succinic acid, glucose, sucrose, pyroglutamic acid, proline, valine, arginine, lysine, protocatechuic aldehyde, protocatechuic acid, tanshinol, rosmarinic acid, salvianolic acid B, and notoginsenoside R1Ginsenoside Rb1Ginsenoside Rd, ginsenoside Re, and ginsenoside Rg1Ginsenoside Rg2Ginsenoside Rh, ginsenoside Rh1Tanshinone I, tanshinone IIA, cryptotanshinone, diazepam (IS2) and chloramphenicol (IS3) were purchased from the national institute for drug and biological product assay;
salvianolic acid A, Salvianolic acid C, Salvianolic acid D and lithospermic acid were obtained from Tianjin Tech technologies, Inc.;
l-lysine-4, 4,5,5-d4(IS1) was purchased from Sigma, USA;
α -D-glucopyranose- (1 → 2) - β -D-fructofuranose- (1 → 1) - β -D-galactopyranose (trisaccharide) and O- α -D-galactopyranose- (1 → 6) -O- α -D-glucopyranose- (1 → 2) -O- α -D-fructofuranose (tetrasaccharide) were prepared by Tianshili modern Chinese medicine resources Co.
The structural formula of the analyte is shown in figure 1. All controls were more than 97% pure as calculated by peak area normalization using HPLC and ELDS.
Example 1: detection method
1. Preparation of Standard and internal Standard solutions
1.1 preparation of Standard series solutions
Weighing appropriate amount of standard substance of the substance to be detected, precisely weighing, and adding 70% methanol water to obtain solutions with concentration of 500 μ g/mL as stock solutions of the substances to be detected. Precisely measuring the stock solutions of the samples, and preparing tetrasaccharide (with concentration of about 70000ng/mL), sucrose (with concentration of about 50000ng/mL), salvianolic acid B (with concentration of about 12000ng/mL), and ginsenoside Rg with 70% methanol water1(concentration about 8000ng/mL), glucose (concentration about 6000ng/mL), trisaccharide, and ginsenoside Rb1Ginsenoside Rd (concentration about 4000ng/mL), pyroglutamic acid, rosmarinic acid and notoginsenoside R1(at a concentration of about 2000ng/mL) and the remainder of the test substance (at a concentration of about 1000 ng/mL). Taking a proper amount of the mixed standard stock solution, preparing a series of mixed standard solutions with concentration by using 70% methanol according to dilution times of 2, 5, 10, 20, 50, 100, 200, 500 and 1000, and storing the mixed standard solutions in a refrigerator at 4 ℃ for later use.
1.2 preparation of internal Standard solution
Weighing appropriate amount of IS1, IS2 and IS3, precisely weighing, and respectively adding 70% methanol water to prepare solutions with concentration of 500 μ g/mL as each internal standard stock solution. Respectively and precisely measuring appropriate amount of each internal standard stock solution, preparing IS reference substance solution with each internal standard concentration of 100ng/mL by using 70% methanol water, and storing in a refrigerator at 4 ℃ for later use.
Respectively and precisely measuring the same amount of series of mixed reference substance solutions with different concentrations and IS reference substance solutions, mixing to obtain series of mixed reference substance solutions (dilution times of 2, 4, 10, 20, 40, 100, 200, 400, 1000 and 2000) containing internal standard, and storing in refrigerator at 4 deg.C for use.
2. Chromatographic and mass spectral conditions
2.1 direct tandem reverse phase chromatography-hydrophilic interaction chromatography
The reversed phase column was used Shim-Pack XR-ODS III (1.6 μm, 2.0 mm. times.50 mm), and the guard column was used a Waters on-line filter (2 μm). The mobile phase consisted of 0.1% formic acid (a1) and acetonitrile (B1) with a gradient program: 0min, 25% B; 0.5min, 25% B; 1.0min, 35% B; 5.0min, 60% B; 10.0min, 80% B; 14.0min, 80% B. The flow rate is 0.12mL/min, the column temperature is 30 ℃, and the sample injection amount is2 mu L.
Hydrophilic interaction chromatography column Using Waters Acquity
BEH Amide (1.7 μm, 2.1 mm. times.100 mm), guard column using a Waters in-line filter (2 μm). The mobile phase consisted of 0.1% formic acid (a2) and acetonitrile (B2) with a gradient program: 0min, 97% B; 10.0min, 50% B; 10.5min, 50% B; 11.0min, 97% B; 14.0min, 97% B. The flow rate was 0.4mL/min, and the column temperature was 30 ℃. The reverse phase chromatography column and the hydrophilic interaction chromatography column are connected by a tee, see figure 2.
2.2 Mass Spectrometry conditions
An electrospray ionization (ESI) interface was used to interface to a hydrophilic interaction chromatography column. The voltage of the positive ion capillary is set to 5500V, and the voltage of the negative ion capillary is set to-4500V; the ion source temperature was set at 550 ℃; high purity nitrogen was used for the atomizing, heating and curtain gases, set at 55, 55 and 25psi, respectively. And quantitatively analyzing the analyte by using a positive and negative ion switching combined time-course multi-reaction monitoring (MRM) method, and respectively optimizing parameters of a declustering voltage (DP), an incident voltage (EP), Collision Energy (CE) and a collision chamber escape voltage (CXP). The quantitative and qualitative parameters are shown in table 1.
TABLE 1 Retention time and related Mass Spectrometry parameters for analytes and internal standards
*An internal standard compound;aquantifying ion pairs;band (5) qualitative ion pairs.
DP, declustering voltage; EP, incident voltage; CE, collision energy; CXP, collision cell escape voltage.
EXAMPLE 231 measurement of the content of ingredients
1. Preparation of sample test solution
Compound Saviae Miltiorrhizae radix extract S01-S05 (batches 20131205, 20141103, 20120201, 20150310 and 20150310 respectively) are provided by Tianshili pharmaceutical group, Inc.; saviae Miltiorrhizae radix material S06-S27 is collected from multiple provinces of China, and Notoginseng radix material S28-S52 is collected from Yunnan province, and pulverized in Tianshili research institute.
Respectively taking 0.10g of compound red sage root extract, pseudo-ginseng extract, red sage root powder and pseudo-ginseng powder, precisely weighing, adding 10mL of 70% methanol, carrying out ultrasonic treatment for 30min, centrifuging for 10min (16,000rpm), filtering through a 0.22 mu m filter membrane, respectively precisely weighing an equivalent sample solution and an IS reference solution, and mixing to prepare a sample test solution containing an internal standard for RP-HILIC-MS/MS analysis. All solutions were stored in a 4 ℃ refrigerator prior to analysis.
2. Validation of analytical methodology
2.1 method specificity
The 31 analytes were subjected to ionization and collision lysis under mass spectrometry conditions in 1.3. Taking the compound salvia miltiorrhiza extract as an example, the scanning mass spectrogram of the sub-ions of each analyte in different samples is observed. As shown in fig. 4, the sample matrix does not interfere with the determination of 31 analytes in the multiple reflection monitoring analysis window, indicating that the method specificity is good.
2.2 Standard Curve with lower quantitative Limit
The standard curve was obtained using a series of concentrations of mixed standard solutions, each concentration being subjected to a double sample analysis. The analyte concentration (ng/mL) is taken as the abscissa x, and the peak area ratio of the analyte to the internal standard substance is taken as the ordinateCoordinate y, with formaldehyde (w ═ 1/x)2) And performing regression calculation by a least square method to obtain a linear regression equation of the standard curve. The detection limit and the quantitation limit are the analyte concentrations at 3-fold and 10-fold signal-to-noise ratios, respectively. The internal standard compounds, regression equation, quantitation limit, and detection limit information are shown in table 2.
Internal standard compounds, regression equation, limit of quantitation (LOD) and limit of detection (LOQ) for 231 analytes
2.3 precision and sample stability Studies
Methodology studies were performed using compound salvia extracts, salvia miltiorrhiza and panax notoginseng. Precision was assessed using intra-and inter-day variability. The same sample was measured for 6 pins continuously for day precision and for 3 pins each for three consecutive days for day precision. Reproducibility was analyzed by preparing 6 samples in parallel. Stability the sample solutions were tested for analyte content at 4 ℃ for 0, 2, 4, 8, 10 and 12 h. All experiments were performed according to the standard curve of the day, and the measured concentration was calculated. Precision, reproducibility and stability results were expressed using RSD and are shown in Table 3.
TABLE 3 precision, repeatability and stability results of 31 analytes in Compound Salvia miltiorrhiza extract, Salvia miltiorrhiza and Panax notoginseng
Table 3 the results show: the precision RSD of the test sample in the day and the precision RSD in the day are respectively less than 4.87 percent and 5.41 percent, the repeatability RSD is less than 5.58 percent, and the stability RSD of the test sample is less than 4.90 percent.
2.4 accuracy survey
Accuracy was assessed using sample recovery. Known amounts of standards were added to the complex red sage extract, red sage and notoginseng, respectively, so that the analyte concentrations in the added samples were about 50%, 100% and 200% of the original concentrations, and 3 samples were prepared for analysis at each concentration. The recovery rate is calculated according to the formula: recovery (%) × (measured amount-original amount)/added amount) × 100%. The results are shown in Table 4.
Table 4 recovery (%) of 31 analytes in salvia miltiorrhiza, notoginseng and compound salvia miltiorrhiza extract (RSD,%, n ═ 3)
The recovery of the sample was 92.3-106.8% (RSD < 5.48%), 95.1-105.0% (RSD < 4.39%) and 95.6-106.4% (RSD < 5.34%) at the three addition levels of 50%, 100% and 200%, respectively.
3. Results of sample measurement
The content of 31 analytes in 5 batches of extract, 22 batches of salvia miltiorrhiza and 25 batches of panax notoginseng samples was determined using the established direct tandem reverse phase-hydrophilic interaction chromatography-mass spectrometry method, and the results are shown in table 5.
Table 5 the results show: 31 compounds are detected in the compound salvia miltiorrhiza extract, and the content of the detected substances accounts for 55.5-71.5% of the weight of the extract. S03 and S04 are salvia miltiorrhiza extracts, and no saponin compounds are detected; s05 is Notoginseng radix extract, and arginine, trisaccharide, tetrasaccharide, phenolic acid and tanshinone compounds are not detected. The compound Saviae Miltiorrhizae radix extract (S01 and S02) has high contents of oligosaccharide, saponin, phenolic acid and amino acid, respectively 40.3-46.6%, 11.3-11.9%, and 6.0-6.8% of the total weight of the extract. 20 compounds including 5 amino acids, 4 oligosaccharides, 1 nucleoside, 1 organic acid, 6 phenolic acids and 3 tanshinone are detected in Saviae Miltiorrhizae radix (S06-S27), and the content of the substances is 16.4-38.2% of the Saviae Miltiorrhizae radix. 18 compounds including 5 amino acids, 2 oligosaccharides, 3 nucleosides, 1 organic acid and 7 saponins are detected in the panax notoginseng (S28-S52), and the content of the substances is determined to be 8.6-35.9% of the weight of the panax notoginseng.
Claims (9)
1. A method for simultaneously determining 31 components in compound Saviae Miltiorrhizae radix extract or related medicinal materials adopts direct tandem-reversed phase-hydrophilic interaction chromatography-mass spectrometry combined method,
the method comprises the following steps:
step 1 preparation of sample solution: preparing compound Saviae Miltiorrhizae radix extract or related medicinal materials into methanol solution with methanol;
step 2 preparation of standard series solutions: preparing a methanol solution from 31 standard substances with methanol;
step 3 preparation of internal standard solution: taking an internal standard substance L-lysine-4, 4,5,5-d4Diazepam and chloramphenicol, respectively preparing methanol solutions with methanol;
step 4, preparation of a test solution: respectively mixing the solution obtained in the step 1 or the step 2 with the internal standard solution obtained in the step 3;
step 5, injecting the solution obtained in the step 4 into a direct tandem reverse phase-hydrophilic interaction liquid chromatography-mass spectrometry combined system to obtain an extracted ion flow diagram of 31 analytes, and calculating the content of 31 components in the compound salvia miltiorrhiza extract or related medicinal materials according to the extracted ion flow diagram;
wherein, the chromatographic conditions are as follows:
the reversed phase chromatographic column uses a chromatographic column with sub 2 mu m C18 packing, the protective column uses a Waters online filter with the particle size of 2 mu m, the mobile phase A1 is 10.08-0.15% formic acid or acetic acid water, the mobile phase B1 is acetonitrile, and the gradient program is as follows: 0min, 25% B1; 0.5min, 25% B1; 1.0min, 35% B1; 5.0min, 60% B1; 10.0min, 80% B1; 15.0min, 80% B1, flow rate of 0.1-0.12mL/min, column temperature of 25-45 deg.C, sample amount of 1-3 μ L;
the hydrophilic interaction chromatographic column adopts a sub-2 mu m hydrophilic chromatographic column, the protective column adopts a Waters online filter with the particle size of 2 mu m, the mobile phase A2 is 0.08-0.15% formic acid or acetic acid water, the mobile phase B2 is acetonitrile, and the gradient program is as follows: 0min, 97% B2; 10.0min, 50% B2; 10.5min, 50% B2; 11.0min, 97% B2; 15.0min, 97% B2; the flow rate is 0.35-0.45mL/min, and the column temperature is 25-45 ℃; the reversed phase chromatographic column and the hydrophilic chromatographic column are connected through a tee joint;
the reversed-phase hydrophilic columns share the same mobile phase system;
the mass spectrum conditions were as follows:
connecting an electrospray ionization interface with a hydrophilic interaction chromatographic column, wherein the voltage of a positive ion capillary is set to 5500V, and the voltage of a negative ion capillary is set to-4500V; the ion source temperature was set at 550 ℃; high-purity nitrogen is used as atomizing gas, heating gas and air curtain gas, the set pressure is 55 psi, 55 psi and 25psi respectively, and the object to be detected is quantitatively analyzed by using a positive and negative ion switching and time-course multi-reaction monitoring method;
wherein the related medicinal materials are selected from: the root of red-rooted salvia or pseudo-ginseng,
the 31 effective components are respectively: adenine, succinic acid, pyroglutamic acid, adenosine, uridine, tanshinol, valine, lysine, proline, protocatechualdehyde, glucose, sucrose, arginine, salvianolic acid D, notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, rosmarinic acid, trisaccharide, alkannic acid, salvianolic acid B, salvianolic acid A, ginsenoside Rb1, salvianolic acid C, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rd, tetrasaccharide, cryptotanshinone, tanshinone I, tanshinone IIA.
2. The method of claim 1, wherein the sample solution of step 1 is prepared by: respectively taking 0.10g of compound red sage root extract, pseudo-ginseng extract, red sage root powder or pseudo-ginseng powder, precisely weighing, adding 8-12mL of 60-80% methanol, carrying out ultrasonic treatment for 20-40min, centrifuging for 8-12min at 16,000rpm, filtering with a 0.22 mu m filter membrane, and storing all solutions in a refrigerator at 4 ℃ before analysis.
3. The method of claim 2, wherein the sample solution of step 1 is prepared by: respectively taking 0.10g of compound red sage root extract, pseudo-ginseng extract, red sage root powder or pseudo-ginseng powder, precisely weighing, adding 10mL of 70% methanol, carrying out ultrasonic treatment for 30min, centrifuging for 10min at 16,000rpm, filtering with a 0.22 mu m filter membrane, and storing all solutions in a refrigerator at 4 ℃ before analysis.
4. The method of claim 2, wherein the step 2 standard series solution is prepared by: weighing appropriate amount of standard substance of the object to be tested, precisely weighing, respectively adding 60-80% methanol water to prepare solution with concentration of 450-1Glucose with concentration of 6000ng/mL, trisaccharide with concentration of 4000ng/mL and ginsenoside Rb with concentration of 4000ng/mL1Ginsenoside Rd with the concentration of 4000ng/mL, pyroglutamic acid with the concentration of 2000ng/mL, rosmarinic acid with the concentration of 2000ng/mL, and notoginsenoside R with the concentration of 2000ng/mL1And mixed standard stock solution of other substances to be detected with the concentration of 1000 ng/mL; taking a proper amount of the mixed standard stock solution, preparing a series of mixed standard solutions with concentration by using 60-80% methanol according to the dilution times of 2, 5, 10, 20, 50, 100, 200, 500 and 1000, and storing the mixed standard solutions in a refrigerator at 4 ℃ for later use.
5. The method of claim 2, wherein the step 2 standard systemThe preparation method of the solution comprises the following steps: weighing appropriate amount of standard substance of the sample, precisely weighing, respectively adding 70% methanol water to obtain 500 μ g/mL solution as stock solution of each sample, precisely measuring appropriate amount of stock solution of each sample, respectively, preparing tetrasaccharide with concentration of 70000ng/mL, sucrose with concentration of 50000ng/mL, salvianolic acid B with concentration of 12000ng/mL, and ginsenoside Rg with concentration of 8000ng/mL with 70% methanol water1Glucose with concentration of 6000ng/mL, trisaccharide with concentration of 4000ng/mL and ginsenoside Rb with concentration of 4000ng/mL1Ginsenoside Rd with the concentration of 4000ng/mL, pyroglutamic acid with the concentration of 2000ng/mL, rosmarinic acid with the concentration of 2000ng/mL, and notoginsenoside R with the concentration of 2000ng/mL1And mixed standard stock solution of other substances to be detected with the concentration of 1000 ng/mL; taking a proper amount of the mixed standard stock solution, preparing a series of mixed standard solutions with concentration by using 70% methanol according to dilution times of 2, 5, 10, 20, 50, 100, 200, 500 and 1000, and storing the mixed standard solutions in a refrigerator at 4 ℃ for later use.
6. The method according to claim 2, wherein the step 3 internal standard solution is prepared by the following method: weighing L-lysine-4, 4,5,5-d4Precisely weighing appropriate amounts of diazepam and chloramphenicol, respectively adding 60-80% methanol water to prepare solutions with the concentration of 450-.
7. The method according to claim 6, wherein the preparation method of the internal standard solution in the step 3 is as follows: weighing L-lysine-4, 4,5,5-d4Respectively adding 70% methanol water to prepare solutions with the concentration of 500 mug/mL as internal standard stock solutions, respectively and precisely measuring the appropriate amount of each internal standard stock solution, preparing IS reference substance solutions with the concentration of 100ng/mL by using 70% methanol water, and storing the IS reference substance solutions for later use in a refrigerator at 4 ℃.
8. The method of claim 2, wherein the sample solution of step 4 is prepared by: respectively and precisely measuring an equal series of mixed reference substance solutions or sample solutions with different concentrations and mixing with an IS reference substance solution to prepare a series of mixed reference substance solutions or sample solutions containing an internal standard, storing the series of mixed reference substance solutions or sample solutions in a refrigerator at 4 ℃ for later use, wherein the series of mixed reference substance solutions are solutions with the dilution times of the internal standard solutions of 2, 4, 10, 20, 40, 100, 200, 400, 1000 and 2000, and the volume ratio of the mixed sample solution to the IS reference substance solution IS 1-10: 1-10.
9. The method of claim 8, wherein the sample solution of step 4 is prepared by: respectively and precisely measuring a series of equal-quantity mixed reference substance solutions with different concentrations or sample solutions and an IS reference substance solution to be mixed to prepare a series of mixed reference substance solutions or sample solutions containing internal standards, storing the series of mixed reference substance solutions or sample solutions in a refrigerator at 4 ℃ for later use, wherein the series of mixed reference substance solutions are solutions with the dilution times of the internal standard solutions of 2, 4, 10, 20, 40, 100, 200, 400, 1000 and 2000, and the sample solutions and the IS reference substance solutions are mixed in equal proportion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610969524.0A CN107991399B (en) | 2016-10-27 | 2016-10-27 | Method for simultaneously determining 31 components in compound red sage root extract or related medicinal materials |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610969524.0A CN107991399B (en) | 2016-10-27 | 2016-10-27 | Method for simultaneously determining 31 components in compound red sage root extract or related medicinal materials |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107991399A CN107991399A (en) | 2018-05-04 |
| CN107991399B true CN107991399B (en) | 2022-03-08 |
Family
ID=62028814
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610969524.0A Active CN107991399B (en) | 2016-10-27 | 2016-10-27 | Method for simultaneously determining 31 components in compound red sage root extract or related medicinal materials |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107991399B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918702A (en) * | 2018-05-21 | 2018-11-30 | 北京红太阳药业有限公司 | Method that is a kind of while measuring two active component contents in red seven preparations |
| CN109374779A (en) * | 2018-12-17 | 2019-02-22 | 杭州奕安济世生物药业有限公司 | A kind of rapid detection method of cane sugar content |
| CN109856267B (en) * | 2019-01-23 | 2022-02-18 | 牡丹江友搏药业有限责任公司 | Method for simultaneously determining contents of multiple components in Chinese medicinal preparation based on LC-MS technology |
| CN109900826A (en) * | 2019-03-26 | 2019-06-18 | 广州莱泰制药有限公司 | A kind of Fufang Danshen Pian Radix Notoginseng Content Test method |
| CN111812221B (en) * | 2020-05-29 | 2022-08-16 | 天津中医药大学 | Method for separating saponin component from Chinese medicinal materials of Panax |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012163B (en) * | 2006-11-03 | 2010-05-12 | 上海朗萨医药科技有限公司 | Method of preparing high purity Danshensu |
| EP2279251A2 (en) * | 2008-04-23 | 2011-02-02 | Danisco US Inc. | Isoprene synthase variants for improved microbial production of isoprene |
| CN101717384B (en) * | 2009-11-24 | 2011-10-12 | 正大青春宝药业有限公司 | Compound in salvia miltiorrhiza bungered sage root injection and application thereof in curing treating cardiovascular disease |
| NL2008662C2 (en) * | 2012-04-19 | 2013-10-23 | Univ Leiden | Electroextraction. |
| CN104749280A (en) * | 2015-03-30 | 2015-07-01 | 神威药业集团有限公司 | Quality control method for salvia miltiorrhiza injection |
| CN105510495A (en) * | 2015-10-22 | 2016-04-20 | 中国药科大学 | Method for revealing pharmaceutically active ingredients and combinations of pharmaceutically active ingredients |
-
2016
- 2016-10-27 CN CN201610969524.0A patent/CN107991399B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107991399A (en) | 2018-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107991399B (en) | Method for simultaneously determining 31 components in compound red sage root extract or related medicinal materials | |
| Yao et al. | Simultaneous quantitation of five Panax notoginseng saponins by multi heart-cutting two-dimensional liquid chromatography: Method development and application to the quality control of eight Notoginseng containing Chinese patent medicines | |
| Huang et al. | Fast separation of triterpenoid saponins using supercritical fluid chromatography coupled with single quadrupole mass spectrometry | |
| Kwon et al. | Determination of astragalin and astragaloside content in Radix Astragali using high-performance liquid chromatography coupled with pulsed amperometric detection | |
| He et al. | Rapid identification and quantitative analysis of the chemical constituents in Scutellaria indica L. by UHPLC–QTOF–MS and UHPLC–MS/MS | |
| Stavrianidi et al. | Combination of HPLC–MS and QAMS as a new analytical approach for determination of saponins in ginseng containing products | |
| CN103235050B (en) | Quality control method of panax notoginseng saponins injection | |
| Qian et al. | Rapid method for simultaneous determination of flavonoid, saponins and polyacetylenes in Folium Ginseng and Radix Ginseng by pressurized liquid extraction and high-performance liquid chromatography coupled with diode array detection and mass spectrometry | |
| Shi et al. | Simultaneous determination of nine ginsenosides in functional foods by high performance liquid chromatography with diode array detector detection | |
| CN103645257A (en) | Detection method for papaya decoction piece | |
| Chang et al. | Simultaneous determination of four phenolic acids and seven alkaloids in rat plasma after oral administration of traditional Chinese medicinal preparation Jinqi Jiangtang Tablet by LC-ESI–MS/MS | |
| Li et al. | Simultaneous determination of main phytoecdysones and triterpenoids in radix achyranthis bidentatae by high-performance liquid chromatography with diode array-evaporative light scattering detectors and mass spectrometry | |
| Xu et al. | Simultaneous quantitative assays of 15 ginsenosides from 119 batches of ginseng samples representing 12 traditional Chinese medicines by ultra-high performance liquid chromatography coupled with charged aerosol detector | |
| Xie et al. | Simultaneous determination of six main components in Bushen Huoxue prescription by HPLC-CAD | |
| Yu et al. | Simultaneous quantification of eight organic acid components in Artemisia capillaris Thunb (Yinchen) extract using high-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry | |
| Lee et al. | Quantification of isoflavonoids and triterpene saponins in Astragali Radix, the root of Astragalus membranaceus, via reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection | |
| CN103969346A (en) | Panax notoginseng saponin component analysis method | |
| Qu et al. | Simultaneous determination of eight active components in Chinese medicine ‘YIQING’capsule using high-performance liquid chromatography | |
| Liu et al. | Development and validation of liquid chromatography–tandem mass spectrometry method for simultaneous determination of six steroidal saponins in rat plasma and its application to a pharmacokinetics study | |
| Yu et al. | Simultaneously determination of five ginsenosides in rabbit plasma using solid-phase extraction and HPLC/MS technique after intravenous administration of ‘SHENMAI’injection | |
| CN108362783A (en) | A kind of detection method of 5 ingredients fingerprint of ginseng saponin F | |
| Zhang et al. | Trace analysis in complex mixtures using a high-component filtering strategy with liquid chromatography–mass spectrometry | |
| Yan et al. | Simultaneous determination of major bioactive components in Qingkailing injection by high-performance liquid chromatography with evaporative light scattering detection | |
| CN109828066B (en) | Method for determining chemical components in traditional Chinese medicine by liquid chromatography-mass spectrometry combined method and application | |
| Qin et al. | Ultrasonic-assisted liquid–liquid extraction and HILIC–ELSD analysis of ginsenoside Rb1, astragaloside IV and dulcitol in sugar-free “Fufangfufangteng Heji” |
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 |