CN1772013A - Quality control method for ginkgo-dipyridamine injection - Google Patents
Quality control method for ginkgo-dipyridamine injection Download PDFInfo
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- CN1772013A CN1772013A CN 200510200658 CN200510200658A CN1772013A CN 1772013 A CN1772013 A CN 1772013A CN 200510200658 CN200510200658 CN 200510200658 CN 200510200658 A CN200510200658 A CN 200510200658A CN 1772013 A CN1772013 A CN 1772013A
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- 229960002768 dipyridamole Drugs 0.000 title claims abstract description 110
- 238000003908 quality control method Methods 0.000 title claims abstract description 40
- 239000007924 injection Substances 0.000 title claims description 253
- 238000002347 injection Methods 0.000 title claims description 253
- 239000013558 reference substance Substances 0.000 claims abstract description 336
- MOLPUWBMSBJXER-YDGSQGCISA-N bilobalide Chemical compound O([C@H]1OC2=O)C(=O)[C@H](O)[C@@]11[C@@](C(C)(C)C)(O)C[C@H]3[C@@]21CC(=O)O3 MOLPUWBMSBJXER-YDGSQGCISA-N 0.000 claims abstract description 168
- 239000000284 extract Substances 0.000 claims abstract description 142
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims abstract description 139
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims abstract description 136
- 239000000843 powder Substances 0.000 claims abstract description 123
- IZEKFCXSFNUWAM-UHFFFAOYSA-N dipyridamole Chemical compound C=12N=C(N(CCO)CCO)N=C(N3CCCCC3)C2=NC(N(CCO)CCO)=NC=1N1CCCCC1 IZEKFCXSFNUWAM-UHFFFAOYSA-N 0.000 claims abstract description 81
- GQODBWLKUWYOFX-UHFFFAOYSA-N Isorhamnetin Natural products C1=C(O)C(C)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 GQODBWLKUWYOFX-UHFFFAOYSA-N 0.000 claims abstract description 75
- IZQSVPBOUDKVDZ-UHFFFAOYSA-N isorhamnetin Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 IZQSVPBOUDKVDZ-UHFFFAOYSA-N 0.000 claims abstract description 75
- 235000008800 isorhamnetin Nutrition 0.000 claims abstract description 75
- -1 flavone compound Chemical class 0.000 claims abstract description 70
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims abstract description 63
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims abstract description 63
- 235000005875 quercetin Nutrition 0.000 claims abstract description 63
- 229960001285 quercetin Drugs 0.000 claims abstract description 63
- 235000007586 terpenes Nutrition 0.000 claims abstract description 63
- 238000007689 inspection Methods 0.000 claims abstract description 44
- 229930003944 flavone Natural products 0.000 claims abstract description 8
- 235000011949 flavones Nutrition 0.000 claims abstract description 8
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 claims abstract description 3
- FOGVNFMUZXDMTR-UHFFFAOYSA-N [Mg].Cl Chemical compound [Mg].Cl FOGVNFMUZXDMTR-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 3
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 624
- 239000000243 solution Substances 0.000 claims description 518
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 384
- 239000007788 liquid Substances 0.000 claims description 268
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 245
- 229940090044 injection Drugs 0.000 claims description 243
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 213
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 207
- 238000012360 testing method Methods 0.000 claims description 198
- 235000008100 Ginkgo biloba Nutrition 0.000 claims description 169
- 241000218628 Ginkgo Species 0.000 claims description 168
- 235000011201 Ginkgo Nutrition 0.000 claims description 168
- 239000008176 lyophilized powder Substances 0.000 claims description 167
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 claims description 146
- 238000000034 method Methods 0.000 claims description 128
- 238000002360 preparation method Methods 0.000 claims description 126
- 239000003978 infusion fluid Substances 0.000 claims description 124
- 238000004108 freeze drying Methods 0.000 claims description 111
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 105
- 238000005406 washing Methods 0.000 claims description 105
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 94
- 239000000047 product Substances 0.000 claims description 85
- 229930003935 flavonoid Natural products 0.000 claims description 81
- 150000002215 flavonoids Chemical class 0.000 claims description 81
- 235000017173 flavonoids Nutrition 0.000 claims description 81
- 239000011259 mixed solution Substances 0.000 claims description 75
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 claims description 73
- 235000008777 kaempferol Nutrition 0.000 claims description 73
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 claims description 73
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 claims description 69
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 69
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 68
- 239000001632 sodium acetate Substances 0.000 claims description 68
- 235000017281 sodium acetate Nutrition 0.000 claims description 68
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 61
- 239000012528 membrane Substances 0.000 claims description 60
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 54
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 54
- 238000005303 weighing Methods 0.000 claims description 49
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 48
- 238000010992 reflux Methods 0.000 claims description 45
- 239000012071 phase Substances 0.000 claims description 44
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 42
- 230000033228 biological regulation Effects 0.000 claims description 39
- 229910001385 heavy metal Inorganic materials 0.000 claims description 39
- 239000002904 solvent Substances 0.000 claims description 39
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 33
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 32
- 238000000605 extraction Methods 0.000 claims description 31
- 238000009835 boiling Methods 0.000 claims description 28
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 27
- 229960004756 ethanol Drugs 0.000 claims description 27
- 238000011003 system suitability test Methods 0.000 claims description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 26
- 239000000945 filler Substances 0.000 claims description 26
- 238000004811 liquid chromatography Methods 0.000 claims description 26
- 239000002351 wastewater Substances 0.000 claims description 26
- 238000003556 assay Methods 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 23
- 239000000377 silicon dioxide Substances 0.000 claims description 23
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 22
- 239000007921 spray Substances 0.000 claims description 22
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 20
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 20
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 20
- 238000010438 heat treatment Methods 0.000 claims description 19
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 18
- 238000012850 discrimination method Methods 0.000 claims description 17
- 229960000583 acetic acid Drugs 0.000 claims description 16
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 16
- 238000010790 dilution Methods 0.000 claims description 16
- 239000012895 dilution Substances 0.000 claims description 16
- 239000012362 glacial acetic acid Substances 0.000 claims description 16
- 239000000741 silica gel Substances 0.000 claims description 15
- 229910002027 silica gel Inorganic materials 0.000 claims description 15
- 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 13
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 13
- 239000004952 Polyamide Substances 0.000 claims description 13
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 13
- 230000037396 body weight Effects 0.000 claims description 13
- 239000003085 diluting agent Substances 0.000 claims description 13
- YWEUIGNSBFLMFL-UHFFFAOYSA-N diphosphonate Chemical compound O=P(=O)OP(=O)=O YWEUIGNSBFLMFL-UHFFFAOYSA-N 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 13
- 238000000105 evaporative light scattering detection Methods 0.000 claims description 13
- 230000036512 infertility Effects 0.000 claims description 13
- 239000013618 particulate matter Substances 0.000 claims description 13
- DLYUQMMRRRQYAE-UHFFFAOYSA-N phosphorus pentoxide Inorganic materials O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 claims description 13
- 229920002647 polyamide Polymers 0.000 claims description 13
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 13
- 239000002510 pyrogen Substances 0.000 claims description 13
- 238000011046 pyrogen test Methods 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 13
- 238000010998 test method Methods 0.000 claims description 13
- 239000008215 water for injection Substances 0.000 claims description 13
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 12
- 239000000853 adhesive Substances 0.000 claims description 12
- 230000001070 adhesive effect Effects 0.000 claims description 12
- 230000001476 alcoholic effect Effects 0.000 claims description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 12
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 11
- 229920003123 carboxymethyl cellulose sodium Polymers 0.000 claims description 11
- 229940063834 carboxymethylcellulose sodium Drugs 0.000 claims description 11
- 229940093181 glucose injection Drugs 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000007791 liquid phase Substances 0.000 claims description 6
- 239000001117 sulphuric acid Substances 0.000 claims description 6
- 235000011149 sulphuric acid Nutrition 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 238000005374 membrane filtration Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 3
- HPKPFIHCMIKXMU-OUUBHVDSSA-N [(2r,3r,4s,5r,6s)-3,4,5-triacetyloxy-6-phenoxyoxan-2-yl]methyl acetate Chemical compound CC(=O)O[C@@H]1[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](COC(=O)C)O[C@H]1OC1=CC=CC=C1 HPKPFIHCMIKXMU-OUUBHVDSSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 244000194101 Ginkgo biloba Species 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 229930063422 Bilobalide A Natural products 0.000 abstract 4
- PUPKKEQDLNREIM-UHFFFAOYSA-N Kaempferitin Natural products OC1C(O)C(O)C(C)OC1OC1=CC(O)=C2C(=O)C(OC3C(C(O)C(O)C(C)O3)O)=C(C=3C=CC(O)=CC=3)OC2=C1 PUPKKEQDLNREIM-UHFFFAOYSA-N 0.000 abstract 2
- SOSLMHZOJATCCP-PADPQNGGSA-N afzelin Natural products O([C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](C)O1)C1=C(c2ccc(O)cc2)Oc2c(c(O)cc(O)c2)C1=O SOSLMHZOJATCCP-PADPQNGGSA-N 0.000 abstract 2
- SQOJOAFXDQDRGF-ZMVGXLHTSA-N ginkgolide b Chemical compound O[C@H]([C@]12[C@H](C(C)(C)C)C[C@H]3OC4=O)C(=O)O[C@H]2O[C@]24[C@@]13[C@H](O)[C@@H]1OC(=O)[C@@H](C)[C@]21O SQOJOAFXDQDRGF-ZMVGXLHTSA-N 0.000 abstract 2
- ZMGSKTZDVIZXJS-UHFFFAOYSA-N kaempferitrin Natural products CC1OC(OC2C(Oc3cc(OC4OC(C)C(O)C(O)C4O)cc(O)c3C2=O)c5ccc(O)cc5)C(O)C(O)C1O ZMGSKTZDVIZXJS-UHFFFAOYSA-N 0.000 abstract 2
- PUPKKEQDLNREIM-QNSQPKOQSA-N kaempferol 3,7-di-O-alpha-L-rhamnoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=CC(O)=C2C(=O)C(O[C@H]3[C@@H]([C@H](O)[C@@H](O)[C@H](C)O3)O)=C(C=3C=CC(O)=CC=3)OC2=C1 PUPKKEQDLNREIM-QNSQPKOQSA-N 0.000 abstract 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 6
- 239000009429 Ginkgo biloba extract Substances 0.000 description 5
- 150000001343 alkyl silanes Chemical group 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 150000002213 flavones Chemical class 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 229930182470 glycoside Natural products 0.000 description 5
- 238000012417 linear regression Methods 0.000 description 5
- 230000002349 favourable effect Effects 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 3
- 230000002526 effect on cardiovascular system Effects 0.000 description 3
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 description 1
- SQGLUEWZRKIEGS-UHFFFAOYSA-N Ginkgetin Natural products C1=CC(OC)=CC=C1C1=CC(=O)C2=C(O)C=C(OC)C(C=3C(=CC=C(C=3)C=3OC4=CC(O)=CC(O)=C4C(=O)C=3)O)=C2O1 SQGLUEWZRKIEGS-UHFFFAOYSA-N 0.000 description 1
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- AIFCFBUSLAEIBR-UHFFFAOYSA-N ginkgetin Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C(C=1)=CC=C(OC)C=1C1=C(O)C=C(O)C(C(C=2)=O)=C1OC=2C1=CC=C(O)C=C1 AIFCFBUSLAEIBR-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The quality control method includes mainly character inspection, and identification and/or content determination. The identification includes the specific reaction between flavone compound and magnesium hydrochloride powder, characteristic indentification of dipyridamine, thin layer identification with reference substances of various gingko leaf extract components quercetin, kaempferitrin, isorhamnetin, bilobalide A, bilobalide B, bilobalide C, etc. The content determination includes the determination of dipyridamine, quercetin, kaempferitrin, isorhamnetin, bilobalide A, bilobalide B and bilobalide C via comparison with corresponding reference substances. Compared with available technology, the present invention has increased thin layer identification and terpene lactone content determination, and thus raised quality control standard and ensured clinical curative effect.
Description
Technical field: the present invention relates to a kind of method of quality control of ginkgo-dipyridamine injection, belong to the technical field of medicine being carried out quality control.
Background technology: the harm that diseases of cardiovascular and cerebrovascular systems causes the mankind has made its mortality rate be in first.Through a large amount of clinical trials, prove that this product can effectively adjust antiotasis by " Ginkgo Leaf Extract and Dipyridamole Injection " of the applicant development, remove free radical, improve hemorheology, the antagonism platelet activating factor alleviates neuronal damage, is the good medicine of treatment cardiovascular and cerebrovascular disease.But discover through us, existing " Ginkgo Leaf Extract and Dipyridamole Injection " exists quality control standard simple, and the uppity shortcoming of product quality is in process of producing product, the quality of product can not be effectively controlled with this method of quality control, thereby the clinical efficacy of ginkgo-dipyridamine injection will be influenced.
Summary of the invention: the objective of the invention is to: a kind of method of quality control of ginkgo-dipyridamine injection is provided, and this ejection preparation comprises injection, lyophilized powder and infusion solutions.It is simple to the present invention is directed to existing " Ginkgo Leaf Extract and Dipyridamole Injection " quality control standard, the uppity shortcoming of product quality, method of quality control to this preparation is studied, increased with Folium Ginkgo reference extract, Quercetin, kaempferol, isorhamnetin reference substance and bilobalide, ginkalide A, ginkalide B, ginkalide C reference substance is the thin layer discrimination method of contrast, also increased the content assaying method of obedient class lactone, improve the quality control standard of ginkgo-dipyridamine injection, thereby guaranteed the clinical efficacy of this preparation.
Ginkgo-dipyridamine injection described in the present invention is the ejection preparation that Folium Ginkgo extract and dipyridamole are mixed and made into, and can be added with adjuvant in this ejection preparation, also can not add adjuvant.
Described method of quality control mainly comprises character, inspection, and in the discriminating, assay one or all; The feature of wherein differentiating the specific reaction comprise flavone compound and hydrochloric acid magnesium powder, dipyridamole differentiates, be thin layer discriminating part or all of that contrast was differentiated and be to the thin layer of contrast with ginkalide A reference substance, ginkalide B reference substance, ginkalide C reference substance and bilobalide reference substance with Folium Ginkgo reference extract and Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance; Assay comprises that with dipyridamole reference substance, Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance, bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance be the content assaying method of contrast.
The thin layer of Flavonoid substances differentiates that be is contrast with Folium Ginkgo reference extract and Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance in the Folium Ginkgo extract, and with dichloromethane: glacial acetic acid: 50～99% ethanol=1～10: 1～6: 0.2～5 is the thin layer discrimination method of developing solvent.
The thin layer discrimination method of terpene lactone is to be contrast with ginkalide A reference substance, ginkalide B reference substance, ginkalide C reference substance and bilobalide reference substance in the Folium Ginkgo extract, and with toluene: ethyl acetate: acetone: dehydrated alcohol=5～20: 2～10: 2～10: 0.5～5 is the thin layer discrimination method of developing solvent.
Discrimination method in the described method of quality control comprises the part or all of of following project:
(1) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is dissolved in water, taking liquid 1-10ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement.
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is dissolved in water, and taking liquid 1-10ml adds ethanol dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear.
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.1-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly,, shake up with the methanol dilution, get filtrate 20-100ml, add water 15-100ml mixing, put and steam in the water-bath to 10-50ml, put coldly,, get ether extracted liquid with ether extraction 1-5 time, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds dissolve with methanol, as need testing solution.Other gets Folium Ginkgo reference extract 20-100mg, add methanol: 5～50% hydrochloric acid=1～8: 0.5～5 mixed solution 10-60ml water-bath reflux, extract,, add water 10-50ml mixing, put and steam in the water-bath, put cold to 5-50ml, with ether extraction 1-5 time, get ether extracted liquid, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds methanol 0.5-5ml makes dissolving, makes Folium Ginkgo reference extract solution.Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add dissolve with methanol.Draw each 5-20 μ l of above-mentioned need testing solution, reference extract solution and reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 50～99% ethanol=1～10: 1～6: 0.2～5 is developing solvent, launch, take out, dry, spray is with 0.1～20% aluminum chloride alcoholic solution, in the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder add warm water makes dissolving, adds the 0.5-10% hydrochloric acid solution, extract 2-10 time with the ethyl acetate jolting, merge extractive liquid, is with the washing of 1-20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing 1-10 time, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution is got 1-10ml, evaporate to dryness, add the ethyl acetate dissolving, put in the separatory funnel, add 0.1～20% hydrochloric acid solution washing 1-10 time, get ethyl acetate liquid, evaporate to dryness, residue add acetone 1-5ml dissolving, as need testing solution; Take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, add dissolve with methanol, promptly get reference substance solution; Draw each 5-20 μ l of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 0.1～20% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=5～20: 2～10: 2～10: 0.5～5 is developing solvent, launch, take out, dry, spray is with acetic anhydride, heating is put coldly, puts under the ultra-violet lamp and inspects.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
Concrete discrimination method comprises the part or all of of following project:
(1) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, taking liquid 2ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement.
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, and taking liquid 2ml adds ethanol 10ml dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear.
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale, shake up with methanol, filter with microporous filter membrane 0.45 μ m, get 40ml, add water 30ml, mixing, put and steam in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Folium Ginkgo reference extract 50mg, adds the mixed solution 25ml of methanol or acetonitrile-25% hydrochloric acid (4: 1), water-bath reflux, extract, 1 hour, add water 30ml, mixing is put and is steamed in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, makes Folium Ginkgo reference extract solution.Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add methanol and make the reference substance solution that every 1ml contains 0.5mg.Draw above-mentioned need testing solution, reference extract solution and each 5ul of reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 95% ethanol=5: 3: 1 is developing solvent, launch, exhibition is taken out apart from about 5～6 centimetres, dry, spray is with 3% aluminum chloride alcoholic solution.In the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder 200mg add warm water 10ml makes dissolving, adds 2 of 2% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml divides the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, add acetone to scale, shake up, filter with 0.45 μ m microporous filter membrane, get 2ml, evaporate to dryness adds ethyl acetate 20ml dissolving, puts in the separatory funnel, add 5% hydrochloric acid solution washing 3 times, each 30ml gets ethyl acetate liquid, evaporate to dryness, residue adds acetone 1ml makes dissolving, as need testing solution.It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, adds methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly gets reference substance solution.Draw each 10ul of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 4% sodium acetate, (10: 5: 5: 1) be developing solvent, expansion was opened up apart from about 8～10cm with toluene-ethyl acetate-acetone-dehydrated alcohol, take out, dry, spray is with acetic anhydride, 140～160 ℃ of heating 30 minutes, put coldly, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
Inspection in the described method of quality control comprises:
(1) clarity: get clean tool plug nessler colorimetric tube, lyophilized powder adds filterable in advance water for injection dissolving, by " clarity test detailed rules and regulations and criterion " regulation inspection about the injection clarity test, and should be up to specification.
(2) pH value; Get this product content, 1 content of lyophilized powder adds 1～10ml water dissolution, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～6.
(3) loss on drying: get the lyophilized powder content, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get this product content, the lyophilized powder precision adds water makes dissolving, gets a little, puts in the crucible, and water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get this product content, lyophilized powder makes dissolving with 0.9% aseptic sodium chloride solution, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get this product content, lyophilized powder checks that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 0.1～50ml by rabbit body weight 1kg, should be up to specification in every ratio obtain solution that adds 5～20% glucose injections, 5～20ml.
(7) particulate matter: get this product content, lyophilized powder is made blank with the 10-200ml dissolving of purifying waste water of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item.
The content assaying method of dipyridamole is with methanol or acetonitrile: 0.05～10% sodium dihydrogen phosphate=1～9: 9～1 is the high-efficient liquid phase determining method of mobile phase.
The content assaying method of total flavonoids is with methanol or acetonitrile: 0.05～10% phosphoric acid solution=1～9: 9～1 is the high-efficient liquid phase determining method of mobile phase.
The content assaying method of terpene lactone is with normal propyl alcohol: oxolane: water=0.1～10: 3～50: 20～150 is the high-efficient liquid phase determining method of mobile phase.
Content assaying method in the described method of quality control comprises the part or all of of following project:
(1) dipyridamole: adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, with methanol or acetonitrile: 0.05～10% sodium dihydrogen phosphate=1～9: 9～1 is mobile phase, 0.05～10% sodium dihydrogen phosphate is used phosphoric acid solution 1 → 3 adjust pH to 2～7 in advance, the detection wavelength is 200～500nm; Precision takes by weighing the dipyridamole reference substance, uses dissolve with methanol, shakes up, and promptly gets reference substance solution; Get the lyophilized powder content, with 20～100% dissolve with methanol solution, the microporous filter membrane that 0.65 μ m reaches less than 0.65 μ m filters, promptly.High-capacity injection can be directly as need testing solution; Respectively accurate above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, and the record chromatogram calculates, and lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml.
(2) total flavonoids:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with methanol or acetonitrile: 0.05～10% phosphoric acid solution=1～9: 9～1 is mobile phase; The detection wavelength is 100～500nm; Accurate respectively Quercetin reference substance, kaempferol reference substance, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight adds dissolve with methanol, promptly gets reference substance solution.Get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.1-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, use smaller or equal to the filter membrane filtration of 0.65 μ m and get lyophilized powder, add methanol: 5～50% hydrochloric acid=1～10: 0.1～5 mixed solution, put reflux on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, filter with 0.65 μ m and less than 0.65 μ m filter membrane, promptly.High-capacity injection adds sulphuric acid liquid 5 → 100, methanol, and water-bath refluxes, and puts coldly, adds dissolve with methanol, with 0.65 μ m and less than the filtration of 0.65 μ m filter membrane, promptly gets need testing solution.Accurate respectively above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, measures, and calculates the content of Quercetin, kaempferol and isorhamnetin respectively, is converted into the content of total flavonoids with following formula.Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
(3) terpene lactone:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with normal propyl alcohol: oxolane: water=0.1～10: 3～50: 20～150 is mobile phase, detects with evaporative light scattering detector; It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance reference substance respectively, adds dissolve with methanol, promptly gets reference substance solution; Get the lyophilized powder content, accurate claim surely, add warm water and make dissolving, add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, wash with 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, promptly.Get high-capacity injection add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, with the washing of 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, and promptly gets need testing solution; Accurate respectively reference substance solution, the need testing solution drawn injects chromatograph of liquid, measures, and calculates bilobalide C respectively with external standard two-point method logarithmic equation
15H
18O
8, ginkalide A C
20H
24O
9, ginkalide B C
20H
24O
10With ginkalide C C
20H
24O
11Content, promptly.Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
Concrete content assaying method comprises the part or all of of following project:
(1) dipyridamole is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With methanol-0.1% sodium dihydrogen phosphate (using phosphoric acid solution (1 → 3) adjust pH to 4.6 in advance) (75: 25) is mobile phase; The detection wavelength is 288nm.Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000.
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly.
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 80% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 80% methanol solution to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates.
Lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml.
(2) total flavonoids is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-0.4% phosphoric acid solution (52: 48) is a mobile phase; The detection wavelength is 360nm.Number of theoretical plate calculates by the Quercetin peak should be not less than 2500.The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5.
Accurate respectively Quercetin reference substance, kaempferol v, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly.
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, the mixed solution 25ml that adds methanol-25% hydrochloric acid (4: 1), put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane (0.45 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml.
(3) terpene lactone is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With normal propyl alcohol-oxolane-water (1: 15: 84) is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the bilobalide peak should be not less than 2500.The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5.
The preparation of reference substance solution respectively precision to take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly.
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 2% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shakes up, filter (0.45 μ m) with microporous filter membrane, promptly.
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate bilobalide (C respectively with external standard two-point method logarithmic equation
15H
18O
8), ginkalide A (C
20H
24O
9), ginkalide B (C
20H
24O
10) and ginkalide C (C
20H
24O
11) content.
Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
Method of quality control of the present invention comprises:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and lyophilized powder is dissolved in water, taking liquid 1-10ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement.
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is dissolved in water, and taking liquid 1-10ml adds ethanol dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear.
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.1-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly,, shake up with the methanol dilution, get filtrate 20-100ml, add water 15-100ml mixing, put and steam in the water-bath to 10-50ml, put coldly,, get ether extracted liquid with ether extraction 1-5 time, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds dissolve with methanol, as need testing solution.Other gets Folium Ginkgo reference extract 20-100mg, add methanol: 5～50% hydrochloric acid=1～8: 0.5～5 mixed solution 10-60ml water-bath reflux, extract,, add water 10-50ml mixing, put and steam in the water-bath, put cold to 5-50ml, with ether extraction 1-5 time, get ether extracted liquid, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds methanol 0.5-5ml makes dissolving, makes Folium Ginkgo reference extract solution.Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add dissolve with methanol.Draw each 5-20 μ l of above-mentioned need testing solution, reference extract solution and reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 50～99% ethanol=1～10: 1～6: 0.2～5 is developing solvent, launch, take out, dry, spray is with 0.1～20% aluminum chloride alcoholic solution, in the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder add warm water makes dissolving, adds the 0.5-10% hydrochloric acid solution, extract 2-10 time with the ethyl acetate jolting, merge extractive liquid, is with the washing of 1-20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing 1-10 time, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution is got 1-10ml, evaporate to dryness, add the ethyl acetate dissolving, put in the separatory funnel, add 0.1～20% hydrochloric acid solution washing 1-10 time, get ethyl acetate liquid, evaporate to dryness, residue add acetone 1-5ml dissolving, as need testing solution; Take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, add dissolve with methanol, promptly get reference substance solution; Draw each 5-20 μ l of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 0.1～20% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=5～20: 2～10: 2～10: 0.5～5 is developing solvent, launch, take out, dry, spray is with acetic anhydride, heating is put coldly, puts under the ultra-violet lamp and inspects.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
Check: (1) clarity: get clean tool plug nessler colorimetric tube, lyophilized powder adds filterable in advance water for injection dissolving, by " clarity test detailed rules and regulations and criterion " regulation inspection about the injection clarity test, and should be up to specification.
(2) pH value: get this product content, 1 content of lyophilized powder adds 1～10ml water dissolution, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～6.
(3) loss on drying: get the lyophilized powder content, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get this product content, the lyophilized powder precision adds water makes dissolving, gets a little, puts in the crucible, and water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get this product content, lyophilized powder makes dissolving with 0.9% aseptic sodium chloride solution, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get this product content, lyophilized powder checks that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 0.1～50ml by rabbit body weight 1kg, should be up to specification in every ratio obtain solution that adds 5～20% glucose injections, 5～20ml.
(7) particulate matter: get this product content, lyophilized powder is made blank with the 10-200ml dissolving of purifying waste water of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item.
Assay:
(1) dipyridamole: adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, with methanol or acetonitrile: 0.05～10% sodium dihydrogen phosphate=1～9: 9～1 is mobile phase, 0.05～10% sodium dihydrogen phosphate is used phosphoric acid solution 1 → 3 adjust pH to 2～7 in advance, the detection wavelength is 200～500nm; Precision takes by weighing the dipyridamole reference substance, uses dissolve with methanol, shakes up, and promptly gets reference substance solution; Get the lyophilized powder content, with 20～100% dissolve with methanol solution, the microporous filter membrane that 0.65 μ m reaches less than 0.65 μ m filters, promptly.High-capacity injection can be directly as need testing solution; Respectively accurate above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, and the record chromatogram calculates, and lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml.
(2) total flavonoids:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with methanol or acetonitrile: 0.05～10% phosphoric acid solution=1～9: 9～1 is mobile phase; The detection wavelength is 100～500nm; Accurate respectively Quercetin reference substance, kaempferol reference substance, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight adds dissolve with methanol, promptly gets reference substance solution.Get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.1-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, use smaller or equal to the filter membrane filtration of 0.65 μ m and get lyophilized powder, add methanol: 5～50% hydrochloric acid=1～10: 0.1～5 mixed solution, put reflux on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, filter with 0.65 μ m and less than 0.65 μ m filter membrane, promptly.High-capacity injection adds sulphuric acid liquid 5 → 100, methanol, and water-bath refluxes, and puts coldly, adds dissolve with methanol, with 0.65 μ m and less than the filtration of 0.65 μ m filter membrane, promptly gets need testing solution.Accurate respectively above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, measures, and calculates the content of Quercetin, kaempferol and isorhamnetin respectively, is converted into the content of total flavonoids with following formula.Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
(3) terpene lactone:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with normal propyl alcohol: oxolane: water=0.1～10: 3～50: 20～150 is mobile phase, detects with evaporative light scattering detector; It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance reference substance respectively, adds dissolve with methanol, promptly gets reference substance solution; Get the lyophilized powder content, accurate claim surely, add warm water and make dissolving, add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, wash with 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, promptly.Get high-capacity injection add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, with the washing of 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, and promptly gets need testing solution; Accurate respectively reference substance solution, the need testing solution drawn injects chromatograph of liquid, measures, and calculates bilobalide C respectively with external standard two-point method logarithmic equation
15H
18O
8, ginkalide A C
20H
24O
9, ginkalide B C
20H
24O
10With ginkalide C C
20H
24O
11Content, promptly.Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
Find after deliberation, adopt the quality of the easier control ginkgo-dipyridamine injection of following method of quality control, be more conducive to guarantee the clinical efficacy of this preparation.So described method of quality control also can comprise:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and the content that lyophilized powder is adds water 5ml and makes dissolving, taking liquid 2ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement.
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, and taking liquid 2ml adds ethanol 10ml dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear.
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale, shake up with methanol, filter with microporous filter membrane 0.45 μ m, get 40ml, add water 30ml, mixing, put and steam in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Folium Ginkgo reference extract 50mg, adds the mixed solution 25ml of methanol or acetonitrile-25% hydrochloric acid (4: 1), water-bath reflux, extract, 1 hour, add water 30ml, mixing is put and is steamed in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, makes Folium Ginkgo reference extract solution.Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add methanol and make the reference substance solution that every 1ml contains 0.5mg.Draw above-mentioned need testing solution, reference extract solution and each 5ul of reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 95% ethanol=5: 3: 1 is developing solvent, launch, exhibition is taken out apart from about 5～6 centimetres, dry, spray is with 3% aluminum chloride alcoholic solution.In the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder 200mg add warm water 10ml makes dissolving, adds 2 of 2% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml divides the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, add acetone to scale, shake up, filter with 0.45 μ m microporous filter membrane, get 2ml, evaporate to dryness adds ethyl acetate 20ml dissolving, puts in the separatory funnel, add 5% hydrochloric acid solution washing 3 times, each 30ml gets ethyl acetate liquid, evaporate to dryness, residue adds acetone 1ml makes dissolving, as need testing solution.It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, adds methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly gets reference substance solution.Draw each 10ul of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 4% sodium acetate, (10: 5: 5: 1) be developing solvent, expansion was opened up apart from about 8～10cm with toluene-ethyl acetate-acetone-dehydrated alcohol, take out, dry, spray is with acetic anhydride, 140～160 ℃ of heating 30 minutes, put coldly, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 5ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2.5～4.5.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 10% glucose injection 10ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 3ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 80ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet relevant every regulation under the Chinese Pharmacopoeia appendix injection item.
Assay:
(1) dipyridamole is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With methanol-0.1% sodium dihydrogen phosphate (using phosphoric acid solution (1 → 3) adjust pH to 4.6 in advance) (75: 25) is mobile phase; The detection wavelength is 288nm.Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000.
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly.
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 80% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 80% methanol solution to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates.
Lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml.
(2) total flavonoids is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol-0.4% phosphoric acid solution (52: 48) is a mobile phase; The detection wavelength is 360nm.Number of theoretical plate calculates by the Quercetin peak should be not less than 2500.The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5.
Accurate respectively Quercetin reference substance, kaempferol v, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly.
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, the mixed solution 25ml that adds methanol-25% hydrochloric acid (4: 1), put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane (0.45 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula.
Total flavonoids content=(quercetin content+kaempferol content ten isorhamnetin content) * 2.51
Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml.
(3) terpene lactone is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With normal propyl alcohol-oxolane-water (1: 15: 84) is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the bilobalide peak should be not less than 2500.The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5.
The preparation of reference substance solution respectively precision to take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly.
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 2% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shakes up, filter (0.45 μ m) with microporous filter membrane, promptly.
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate bilobalide (C respectively with external standard two-point method logarithmic equation
15H
18O
8), ginkalide A (C
20H
24O
9), ginkalide B (C
20H
24O
10) and ginkalide C (C
20H
24O
11) content.
Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
This method of quality control is compared with " Ginkgo Leaf Extract and Dipyridamole Injection " quality standard, increased the thin layer of terpene lactone has been differentiated and to the content assaying method of terpene lactone, because effective ingredient comprises Ginkgo total flavones and bilobalide in the Folium Ginkgo extract, ginkgetin has blood vessel dilating, blood flow increasing, improves cerebral arteries and the end effect of blood flow slightly; Bilobalide is strong platelet activating factor antagonist, but specificity antagonism paf receptor suppresses platelet aggregation, vascular endothelial injury that PAF causes, effects such as microthrombusis and lipid metabolic disorder.So Ginkgo total flavones and bilobalide are the main effective ingredient of extract of ginkgo biloba for treating cardiovascular and cerebrovascular disease.The present invention controls the curative effect that can guarantee Folium Ginkgo extract to the content of bilobalide.
Wherein Ginkgo total flavones mainly comprises Quercetin, kaempferol and isorhamnetin, so among the present invention with Quercetin, kaempferol and isorhamnetin reference substance be Ginkgo total flavones in this preparation of control test than only being that reference substance detects in the primary standard with the Quercetin, its testing result is more accurate.
In the content assaying method of Ginkgo total flavones, in the primary standard in the preparation method of need testing solution, medicinal liquid is with the mixed solution water-bath reflux, extract, of sulphuric acid liquid and methanol; Adopt the mixed solution of methanol and hydrochloric acid to extract medicinal liquid in the existing standard, the preparation need testing solution, extract drugs is more complete, and testing result is more accurate.
Terpene lactone mainly comprises bilobalide, ginkalide A, ginkalide B and ginkalide C, is terpene lactone in this ejection preparation of control test with above-mentioned reference substance among the present invention, and its result is accurate.
For reasonability of verifying content assaying method and discrimination method among the present invention etc., carried out following experimentation.
One, total flavonoids assay
1, need testing solution extracts the investigation of solvent:
Extracting method: get ginkgo-dipyridamine injection, add to extract solvent, put reflux, extract, in the water-bath, put coldly, move in the measuring bottle,, the results are shown in following table with the methanol dilution:
| Extract solvent | Sulphuric acid liquid (5 → 100) and methanol mixed solution | 25% hydrochloric acid and methanol mixed solution |
| Content (%) | 18.21 | 21.98 |
| RSD(％) | 1.21 | 0.73 |
As seen from the above table, select for use 25% hydrochloric acid and methanol mixed solution as extracting solution, total flavonoids extracts fully, and relative standard deviation is less, is solvent so the present invention selects 25% hydrochloric acid and methanol mixed solution for use.
2, stability test is investigated: the stability test of Quercetin, kaempferol, isorhamnetin in the need testing solution: the preparation method by test liquid under the assay item in the quality standard draft text prepares test liquid, respectively at measuring Quercetin, kaempferol, isorhamnetin peak area in 0,1,2,4,8 hour, wherein the Quercetin average peak area is 923415, RSD is 1.39%, the kaempferol average peak area is 707139, and RSD is 1.33%; The isorhamnetin average peak area is 218444, and RSD is 1.00%.The result shows that test liquid is good at 8 hours internal stabilities.
Two, terpene lactone contents is measured
1, methodological study:
The system suitability test: the negative need testing solution of getting ginkalide A, ginkalide B, ginkalide C, bilobalide reference substance mixed solution, " ginkgo-dipyridamole for injection " need testing solution and scarce Folium Ginkgo extract respectively injects chromatograph of liquid respectively, the record chromatograph.As seen from the figure, the retention time (t of ginkalide C, bilobalide, ginkalide A, ginkalide B
R) be about 10 minutes respectively, 12 minutes, 14 minutes, 19 minutes, ginkalide C, bilobalide, ginkalide A, ginkalide B peak separate fully (separating degree＞1.5) with impurity peaks, negative sample is noiseless.Theoretical cam curve is calculated with the bilobalide peak, is not less than 2500.Illustrate that this method system suitability is good.
Linear relationship is investigated: respectively variable concentrations bilobalide, ginkalide A, B, C reference substance solution 10 μ l are injected chromatograph of liquid, natural logrithm (lnA) with peak area A (μ Vs) is a vertical coordinate, the natural logrithm (lnX) of mass number X (μ g) is an abscissa, carries out linear regression and calculates.
The ginkalide C equation of linear regression is: lnA=1.43422 * lnX+10.6386, r=0.99994;
The bilobalide equation of linear regression is: lnA=1.33793 * lnX+10.8845, r=0.9995;
The ginkalide A equation of linear regression is: lnA=1.35288 * lnX+10.7326, r=0.9999;
The ginkalide B equation of linear regression is: lnA=1.36827 * lnX+10.2757, r=0.9999.
Precision test: press in the quality standard draft text terpene lactone chromatographic condition under the assay item, accurate bilobalide, ginkalide A, B, each 10 μ l of C reference substance solution of drawing inject chromatograph of liquid, repeat sample introduction 5 times, the mensuration peak area.Ginkalide C peak area meansigma methods is 494183, and RSD is 1.28%; Bilobalide peak area meansigma methods is 456146, and RSD is 1.53%; Ginkalide A peak area meansigma methods is 957259, and RSD is 1.16%; Ginkalide B peak area meansigma methods is 87616, and RSD is 0.47%.The result as seen, this method precision is good.
The repeatability test: prepare 5 parts of test liquids respectively by test liquid preparation method under the terpene lactone contents mensuration item in the quality standard draft text, sample introduction is measured peak area, calculates content.Wherein the average content of ginkalide C is 1.457%, and RSD is 2.58%; The average content of bilobalide is 1.434%, and RSD is 2.15%; The average content of ginkalide A is 2.475%, and RSD is 1.88%; The average content of ginkalide B is 0.554%, and RSD is 2.30%.The result shows that this test repeatability is good.
Stability test: the stability test of ginkalide C, bilobalide, ginkalide A, ginkalide B in the need testing solution: prepare test liquid by test liquid preparation method under the terpene lactone contents mensuration item in the quality standard draft text, respectively at measuring ginkalide C, bilobalide, ginkalide A, ginkalide B peak area in 0,1,2,4,8 hour, wherein the ginkalide C average peak area is 555101, and RSD is 0.88%; The bilobalide average peak area is 567493, and RSD is 2.06%; The ginkalide A average peak area is 1036506, and RSD is 0.79%; The ginkalide B average peak area is 93139, and RSD is 1.70%.The result shows that need testing solution is good at 8 hours internal stabilities.
Three, dipyridamole content assaying method research
Under existing dipyridamole chromatographic condition, we find that this preparation all has absorption at 200-500nm, but at 288nm maximum absorption band are arranged, so the content assaying method of dipyridamole in this preparation, the optimum detection wavelength is 288nm in the chromatographic condition, and its testing result is more accurate.
Four, during the thin layer of Flavonoid substances is differentiated in the Folium Ginkgo extract, with Folium Ginkgo extract in Folium Ginkgo reference extract and Quercetin, kaempferol, the isorhamnetin reference substance discriminating side, being chosen as of its thin layer discrimination method:
Need testing solution preparation method 1: get ginkgo-dipyridamine injection, add butanol solution, in water-bath warm macerating 15-30 minute, jolting constantly, put coldly, filter, filtrate adds 0.025mol/L hydrochloric acid solution washing 2 times, discard hydrochloric acid solution, n-butanol layer adds water washing, divides and gets n-butanol layer, evaporate to dryness, residue adds ethanol 1ml makes dissolving, as need testing solution.
Need testing solution preparation method 2: get [assay] item need testing solution 40ml of total flavonoids down, add water 30ml, mixing, put and steam in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.
Developing solvent is selected: be developing solvent with the mixed solution of ethyl acetate, butanone, methanol and water and dichloromethane, glacial acetic acid in 95% alcoholic acid mixed solution respectively, put respectively in the sodium carboxymethyl cellulose that contains 4% sodium acetate be on the silica gel g thin-layer plate of adhesive with polyamide film on.
Result: the preparation method of test sample selecting method 2, adopt polyamide film, with dichloromethane: glacial acetic acid: 50～99% ethanol=1～10: 1～6: 0.2～5 is that the unfolded separating degree of developing solvent is good, the speckle colour developing is clear, negative control is noiseless, the method favorable reproducibility, best developing solvent is: dichloromethane: glacial acetic acid: 95% ethanol=5: 3: 1.
Five, the thin layer of terpene lactone research in the Folium Ginkgo extract: with Folium Ginkgo extract in ginkalide A, B, C and the bilobalide reference substance discriminating side.
Need testing solution preparation method 1: get need testing solution under [assay] assay item, as need testing solution.
Need testing solution preparation method 2: get ginkgo-dipyridamine injection, add 2% hydrochloric acid solution 1-5 and drip, use ethyl acetate extraction, merge ethyl acetate extraction liquid, add the washing of 0.025mol/L hydrochloric acid solution, get the ethyl acetate layer, evaporate to dryness, residue add acetone 1ml makes dissolving, as need testing solution.
Need testing solution preparation method 3: get the need testing solution 2ml of terpene lactone under the assay item, evaporate to dryness adds ethyl acetate 20ml dissolving, put in the separatory funnel, add the washing of 5% hydrochloric acid solution, get ethyl acetate liquid, evaporate to dryness, residue add acetone 1ml makes dissolving, as need testing solution.
Developing solvent is selected: respectively with the mixed solution of toluene, ethyl acetate, acetone and dehydrated alcohol; The mixed solution of toluene, ethyl acetate, acetone and methanol is developing solvent.
Result: the preparation method of test sample selecting method 3, with toluene: ethyl acetate: acetone: dehydrated alcohol=5～20: 2～10: 2～10: the separating degree of 0.5～5 developing solvent is good, the speckle colour developing is clear, negative control is noiseless, method favorable reproducibility, best developing solvent are toluene: ethyl acetate: acetone: dehydrated alcohol=10: 5: 5: 1.
Above experimental result shows, among the present invention, in the content assaying method and discrimination method of ginkgo-dipyridamine injection, the solvent of need testing solution is selected and is prepared rationally, and precision height, favorable reproducibility, good stability, therefore the inventive method is reasonable, feasible, is control ginkgo-dipyridamine injection quality method preferably.
Compared with the prior art, it is simple to the present invention is directed to existing " Ginkgo Leaf Extract and Dipyridamole Injection " quality control standard, the uppity shortcoming of product quality, method of quality control to this preparation is studied, increased with Folium Ginkgo reference extract, Quercetin, kaempferol, isorhamnetin reference substance and bilobalide, ginkalide A, ginkalide B, ginkalide C reference substance is the thin layer discrimination method of contrast, and increased the content assaying method of terpene lactone, improve the quality control standard of ginkgo-dipyridamine injection, thereby guaranteed the clinical efficacy of this preparation.Adopt the quality of the inventive method control ginkgo-dipyridamine injection, the discrimination method specificity is strong, the content assaying method favorable reproducibility, and used discrimination method and assay project can be guaranteed the curative effect of medicine, have reached the goal of the invention of effective control drug quality.
The specific embodiment:
Embodiments of the invention 1:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and the content that lyophilized powder is adds water 5ml and makes dissolving, taking liquid 2ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement.
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, and taking liquid 2ml adds ethanol 10ml dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear.
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale, shake up with methanol, filter with microporous filter membrane 0.45 μ m, get 40ml, add water 30ml, mixing, put and steam in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Folium Ginkgo reference extract 50mg, adds methanol or acetonitrile: the mixed solution 25ml of 25% hydrochloric acid=4: 1, water-bath reflux, extract, 1 hour, add water 30ml, mixing is put and is steamed in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, makes Folium Ginkgo reference extract solution.Get Quercetin, kaempferol, isorhamnetin reference substance more respectively, add methanol and make the reference substance solution that every 1ml contains 0.5mg.Draw above-mentioned need testing solution, reference extract solution and each 5ul of reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 95% ethanol=5: 3: 1 is developing solvent, launch, exhibition is taken out apart from about 5～6 centimetres, dry, spray is with 3% aluminum chloride alcoholic solution.In the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder 200mg add warm water 10ml makes dissolving, adds 2 of 2% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml divides the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, add acetone to scale, shake up, filter with 0.45 μ m microporous filter membrane, get 2ml, evaporate to dryness adds ethyl acetate 20ml dissolving, puts in the separatory funnel, add 5% hydrochloric acid solution washing 3 times, each 30ml gets ethyl acetate liquid, evaporate to dryness, residue adds acetone 1ml makes dissolving, as need testing solution.It is an amount of that precision takes by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance respectively, adds methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly gets reference substance solution.Draw each 10ul of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 4% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=10: 5: 5: 1 is developing solvent, launches, and exhibition is apart from about 8～10cm, take out, dry, spray is with acetic anhydride, 140～160 ℃ of heating 30 minutes, put coldly, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 5ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2.5～4.5.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 10% glucose injection 10ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 3ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 80ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet relevant every regulation under the Chinese Pharmacopoeia appendix injection item.
Assay:
(1) dipyridamole is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With methanol: 0.1% sodium dihydrogen phosphate (using phosphoric acid solution (1 → 3) adjust pH to 4.6 in advance)=75: 25 is a mobile phase; The detection wavelength is 288nm.Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000.
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly.
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 80% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 80% methanol solution to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates, promptly.Every of lyophilized powder contains dipyridamole and should be 0.5～20mg in this preparation; The every 1ml of high-capacity injection contains dipyridamole and should be 0.0005～0.3mg.
(2) total flavonoids is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol: 0.4% phosphoric acid solution=52: 48 is a mobile phase; The detection wavelength is 360nm.Number of theoretical plate calculates by the Quercetin peak should be not less than 2500.The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5.
Accurate respectively Quercetin, kaempferol, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly.
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, add methanol: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane (0.45 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula.Every of lyophilized powder contains total flavonoids and should be 1～30mg in this preparation; The every 1ml of high-capacity injection contains total flavonoids and should be 0.001～0.6mg.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
(3) terpene lactone is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With normal propyl alcohol: oxolane: water=1: 15: 84 is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the bilobalide peak should be not less than 2500.The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5.
The preparation of reference substance solution respectively precision to take by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly.
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 2% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shakes up, filter (0.45 μ m) with microporous filter membrane, promptly.
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate bilobalide (C respectively with external standard two-point method logarithmic equation
15H
18O
8), ginkalide A (C
20H
24O
9), ginkalide B (C
20H
24O
10) and ginkalide C (C
20H
24O
11) content, promptly.Every of lyophilized powder contains terpene lactone and should be 0.1～20mg in this preparation; The every 1ml of high-capacity injection contains the terpene lactone glycosides and should be 0.0004～0.2mg.
Embodiments of the invention 2:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add the acetonitrile of 0.5 times of amount: the mixed solution of 10% hydrochloric acid=10: 0.1, put on the boiling water bath reflux 1 hour, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale, shake up with methanol, filter with microporous filter membrane 0.45 μ m, get 20ml, add water 15ml, mixing, put and steam in the water-bath to about 10ml, put cold, with ether extraction 1 time, each 20ml, get ether extracted liquid, add water washing 1 time, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Folium Ginkgo reference extract 20mg, adds acetonitrile: the mixed solution 10ml of 5% hydrochloric acid=8: 0.5, water-bath reflux, extract, 1 hour, add water 10ml, mixing is put and is steamed in the water-bath to about 5ml, put cold, with ether extraction 1 time, each 20ml, get ether extracted liquid, add water washing 1 time, each 20ml, ether solution evaporate to dryness, residue adds methanol 0.5ml makes dissolving, makes Folium Ginkgo reference extract solution.Get Quercetin, kaempferol, isorhamnetin reference substance more respectively, add methanol and make the reference substance solution that every 1ml contains 0.5mg.Draw above-mentioned need testing solution, reference extract solution and each 10ul of reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 50% ethanol=10: 1: 0.2 is developing solvent, launch, exhibition is apart from about 5～6 centimetres, take out, dry, spray is with 0.1% aluminum chloride alcoholic solution.In the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 1ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～4.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 5% glucose injection 5ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 50ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 10ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet relevant every regulation under two appendix injections of Chinese Pharmacopoeia version in 2000 item.
Assay:
(1) dipyridamole is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with tetraalkyl silane group silica gel; With acetonitrile: 0.05% sodium dihydrogen phosphate (using phosphoric acid solution (1 → 3) adjust pH to 2 in advance)=10: 90 is a mobile phase; The detection wavelength is 200nm.Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000.
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly.
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 20% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 20% methanol solution to scale, shakes up, and filters with microporous filter membrane (0.65 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates, promptly.Every of lyophilized powder contains dipyridamole and should be 0.5～20mg in this preparation; The every 1ml of high-capacity injection contains dipyridamole and should be 0.0005～0.3mg.
(2) total flavonoids is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with tetraalkyl silane group silica gel; Acetonitrile: 0.05% phosphoric acid solution=10; 90 is mobile phase; The detection wavelength is 100nm.Number of theoretical plate calculates by the Quercetin peak should be not less than 2500.The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5.
Accurate respectively Quercetin, kaempferol, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly.
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, the acetonitrile that adds 0.5 times of amount: the mixed solution of 10% hydrochloric acid=1: 5, put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane (0.65 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula.Every of lyophilized powder contains total flavonoids and should be 1～30mg in this preparation; The every 1ml of high-capacity injection contains total flavonoids and should be 0.001～0.6mg.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
Embodiments of the invention 3:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder 200mg add warm water 10ml makes dissolving, adds 2 of 0.5% hydrochloric acid solutions, extract 2 (15ml with the ethyl acetate jolting, 10ml), merge extractive liquid, washs with 1% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 1 time, each 20ml divides the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, add acetone to scale, shake up, filter with 0.45 μ m microporous filter membrane, get 1ml, evaporate to dryness adds ethyl acetate 20ml dissolving, puts in the separatory funnel, add 0.1% hydrochloric acid solution washing 1 time, each 30ml gets ethyl acetate liquid, evaporate to dryness, residue adds acetone 3ml makes dissolving, as need testing solution.It is an amount of that precision takes by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance respectively, adds methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly gets reference substance solution.Draw each 5ul of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 0.1% sodium acetate, (5: 10: 10: 5) be developing solvent, expansion was opened up apart from about 8～10cm with toluene-ethyl acetate-acetone-dehydrated alcohol, take out, dry, spray is with acetic anhydride, 140～160 ℃ of heating 30 minutes, put coldly, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
Check; (1) clarity; In super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 10ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 4～6.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia version heavy metal in 2000 inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 20% glucose injection 20ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 0.1ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 200ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item.
Assay:
(1) total flavonoids is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with eight alkyl silane bonded silica gels; Methanol: 10% phosphoric acid solution=90: 10 is a mobile phase; The detection wavelength is 500nm.Number of theoretical plate calculates by the Quercetin peak should be not less than 2500.The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5.
Accurate respectively Quercetin, kaempferol, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly.
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, the methanol that adds 10 times of amounts: the mixed solution of 50% hydrochloric acid=10: 0.1, put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane (0.55 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula.Every of lyophilized powder contains total flavonoids and should be 1～30mg in this preparation; The every 1ml of high-capacity injection contains total flavonoids and should be 0.001～0.6mg.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
(2) terpene lactone is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with tetraalkyl silane group silica gel; With normal propyl alcohol: oxolane: water=0.1: 3: 150 is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the bilobalide peak should be not less than 2500.The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5.
The preparation of reference substance solution respectively precision to take by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly.
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 0.5% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 0.5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shakes up, filter (0.65 μ m) with microporous filter membrane, promptly
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate bilobalide (C respectively with external standard two-point method logarithmic equation
15H
18O
8), ginkalide A (C
20H
24O
9), ginkalide B (C
20H
24O
10) and ginkalide C (C
20H
24O
11) content, promptly.Every of lyophilized powder contains terpene lactone and should be 0.1～20mg in this preparation; The every 1ml of high-capacity injection contains the terpene lactone glycosides and should be 0.0004～0.2mg.
Embodiments of the invention 4:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and the content that lyophilized powder is adds water 5ml and makes dissolving, taking liquid 1ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement.
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, and taking liquid 1ml adds ethanol 10ml dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear.
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 8ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～5.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 15% glucose injection 15ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 10ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 100ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item.
Assay:
(1) dipyridamole is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with eight alkyl silane bonded silica gels; With methanol: 10% sodium dihydrogen phosphate (using phosphoric acid solution (1 → 3) adjust pH to 7 in advance)=90: 10 is a mobile phase; The detection wavelength is 500nm.Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000.
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly.
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 100% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 100% methanol solution to scale, shakes up, and filters with microporous filter membrane (0.55 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates, promptly.Every of lyophilized powder contains dipyridamole and should be 0.5～20mg in this preparation; The every 1ml of high-capacity injection contains dipyridamole and should be 0.0005～0.3mg.
(2) terpene lactone is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with eight alkyl silane bonded silica gels; With normal propyl alcohol: oxolane: water=10: 3: 20 is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the bilobalide peak should be not less than 2500.The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5.
The preparation of reference substance solution respectively precision to take by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly.
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 10% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 20% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shakes up, filter (0.55 μ m) with microporous filter membrane, promptly
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate bilobalide (C respectively with external standard two-point method logarithmic equation
15H
18O
8), ginkalide A (C
20H
24O
9), ginkalide B (C
20H
24O
10) and ginkalide C (C
20H
24O
11) content, promptly.Every of lyophilized powder contains terpene lactone and should be 0.1～20mg in this preparation; The every 1ml of high-capacity injection contains the terpene lactone glycosides and should be 0.0004～0.2mg.
Embodiments of the invention 5:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and the content that lyophilized powder is adds water 5ml and makes dissolving, taking liquid 10ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement.
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add the methanol of 10 times of amounts: the mixed solution of 50% hydrochloric acid=1: 5, put on the boiling water bath reflux 1 hour, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale, shake up with methanol, filter with microporous filter membrane 0.45 μ m, get 100ml, add water 100ml, mixing, put and steam in the water-bath to about 50ml, put cold, with ether extraction 5 times, each 20ml, get ether extracted liquid, add water washing 5 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Other gets Folium Ginkgo reference extract 100mg, adds methanol: the mixed solution 60ml of 50% hydrochloric acid=1: 5, water-bath reflux, extract, 1 hour, add water 50ml, mixing is put and is steamed in the water-bath to about 50ml, put cold, with ether extraction 5 times, each 20ml, get ether extracted liquid, add water washing 5 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 5ml makes dissolving, makes Folium Ginkgo reference extract solution.Get Quercetin, kaempferol, isorhamnetin reference substance more respectively, add methanol and make the reference substance solution that every 1ml contains 0.5mg.Draw above-mentioned need testing solution, reference extract solution and each 20ul of reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 99% ethanol=1: 6: 5 is developing solvent, launch, exhibition is apart from about 5～6 centimetres, take out, dry, spray is with 20% aluminum chloride alcoholic solution.In the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively.
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 3ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～6.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 8% glucose injection 8ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 30ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 150ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item.
Assay:
Total flavonoids is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Acetonitrile: 4% phosphoric acid solution=40: 60 is a mobile phase; The detection wavelength is 200nm.Number of theoretical plate calculates by the Quercetin peak should be not less than 2500.The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5.
Accurate respectively Quercetin, kaempferol, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly.
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, add acetonitrile: the mixed solution 25ml of 40% hydrochloric acid=10: 1, put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane (0.35 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula.Every of lyophilized powder contains total flavonoids and should be 1～30mg in this preparation; The every 1ml of high-capacity injection contains total flavonoids and should be 0.001～0.6mg.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
Embodiments of the invention 6:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and the content that lyophilized powder is adds water 5ml and makes dissolving, and taking liquid 10ml adds ethanol 10ml dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear.
(2) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder 200mg add warm water 10ml makes dissolving, adds 2 of 10% hydrochloric acid solutions, extract 10 times each 10ml, merge extractive liquid, with the ethyl acetate jolting, with 20% sodium acetate solution 20ml washing, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 10 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shake up, filter with 0.45 μ m microporous filter membrane, get 10ml, evaporate to dryness, add ethyl acetate 20ml dissolving, put in the separatory funnel, add 20% hydrochloric acid solution washing 10 times, each 30ml, get ethyl acetate liquid, evaporate to dryness, residue add acetone 5ml makes dissolving, as need testing solution.It is an amount of that precision takes by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance respectively, adds methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly gets reference substance solution.Draw each 20ul of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 20% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=20: 2: 2: 0.5 is developing solvent, launches, and exhibition is apart from about 8～10cm, take out, dry, spray is with acetic anhydride, 140～160 ℃ of heating 30 minutes, put coldly, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 5ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 3～5.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 12% glucose injection 12ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 10ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 180ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet relevant every regulation under two appendix injections of Chinese Pharmacopoeia version in 2000 item.
Assay:
Terpene lactone is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With normal propyl alcohol: oxolane: water=0.1: 50: 20 is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the bilobalide peak should be not less than 2500.The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5.
The preparation of reference substance solution respectively precision to take by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly.
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 5% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 10% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shakes up, filter (0.35 μ m) with microporous filter membrane, promptly
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate bilobalide (C respectively with external standard two-point method logarithmic equation
15H
18O
8), ginkalide A (C
20H
24O
9), ginkalide B (C
20H
24O
10) and ginkalide C (C
20H
24O
11) content, promptly.Every of lyophilized powder contains terpene lactone and should be 0.1～20mg in this preparation; The every 1ml of high-capacity injection contains the terpene lactone glycosides and should be 0.0004～0.2mg.
Embodiments of the invention 7:
Character:
For injection: product is yellow to pale brown color clear liquid.
For lyophilized preparation: product is faint yellow to the loose block of pale brown color.
For infusion solutions: product is faint yellow to pale brown color clear liquid.
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, by the regulation inspection under " clarity test detailed rules and regulations and criterion " injectable sterile powder item, should be up to specification.
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 5ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～6.
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%.
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths.
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification.
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 10% glucose injection 10ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 5ml by rabbit body weight 1kg, should be up to specification.
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 50ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification.
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item.
Assay:
(1) dipyridamole is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With acetonitrile: 5% sodium dihydrogen phosphate (using phosphoric acid solution (1 → 3) adjust pH to 6 in advance)=30: 70 is a mobile phase; The detection wavelength is 360nm.Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000.
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly.
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 50% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 50% methanol solution to scale, shakes up, and filters with microporous filter membrane (0.35 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates, promptly.Every of lyophilized powder contains dipyridamole and should be 0.5～20mg in this preparation; The every 1ml of high-capacity injection contains dipyridamole and should be 0.0005～0.3mg.
(2) total flavonoids is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with eight alkyl silane bonded silica gels; Methanol: 7% phosphoric acid solution=80: 20 is a mobile phase; The detection wavelength is 440nm.Number of theoretical plate calculates by the Quercetin peak should be not less than 2500.The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5.
Accurate respectively Quercetin, kaempferol, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly.
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, add methanol: the mixed solution 25ml of 15% hydrochloric acid=1: 4, put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane (0.45 μ m), promptly.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula.Every of lyophilized powder contains total flavonoids and should be 1～30mg in this preparation; The every 1ml of high-capacity injection contains total flavonoids and should be 0.001～0.6mg.
Total flavonoids content=(quercetin content+kaempferol content+isorhamnetin content) * 2.51
(3) terpene lactone is according to high effective liquid chromatography for measuring.
Chromatographic condition and system suitability test are filler with eight alkyl silane bonded silica gels; With normal propyl alcohol: oxolane: water=5: 30: 100 is mobile phase; Detect with evaporative light scattering detector.Number of theoretical plate calculates by the bilobalide peak should be not less than 2500.The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5.
The preparation of reference substance solution respectively precision to take by weighing bilobalide, ginkalide A, ginkalide B and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly.
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 8% hydrochloric acid solutions, extract 5 (15ml with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml), merge extractive liquid, washs with 15% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml, divide the water intaking washing liquid, add ethyl acetate 10ml and extract, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shakes up, filter (0.45 μ m) with microporous filter membrane, promptly.
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate bilobalide (C respectively with external standard two-point method logarithmic equation
15H
18O
8), ginkalide A (C
20H
24O
9), ginkalide B (C
20H
24O
10) and ginkalide C (C
20H
24O
11) content, promptly.Every of lyophilized powder contains terpene lactone and should be 0.1～20mg in this preparation; The every 1ml of high-capacity injection contains the terpene lactone glycosides and should be 0.0004～0.2mg.
Claims (13)
1. the method for quality control of a ginkgo-dipyridamine injection, described ejection preparation comprises injection, lyophilized powder and infusion solutions, it is characterized in that: described method of quality control mainly comprises character, inspection, and in the discriminating, assay one or all; The feature of wherein differentiating the specific reaction comprise flavone compound and hydrochloric acid magnesium powder, dipyridamole differentiates, be thin layer discriminating part or all of that contrast was differentiated and be to the thin layer of contrast with ginkalide A reference substance, ginkalide B reference substance, ginkalide C reference substance and bilobalide reference substance with Folium Ginkgo reference extract and Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance; Assay comprises that with dipyridamole reference substance, Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance, bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance be the content assaying method of contrast.
2. according to the method for quality control of the described ginkgo-dipyridamine injection of claim 1, it is characterized in that: the thin layer of Flavonoid substances differentiates that be is contrast with Folium Ginkgo reference extract and Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance in the Folium Ginkgo extract, and with dichloromethane: glacial acetic acid: 50～99% ethanol=1～10: 1～6: 0.2～5 is the thin layer discrimination method of developing solvent.
3. according to the method for quality control of the described ginkgo-dipyridamine injection of claim 1, it is characterized in that: the thin layer discrimination method of terpene lactone is to be contrast with ginkalide A reference substance, ginkalide B reference substance, ginkalide C reference substance and bilobalide reference substance in the Folium Ginkgo extract, and with toluene: ethyl acetate: acetone: dehydrated alcohol=5～20: 2～10: 2～10: 0.5～5 is the thin layer discrimination method of developing solvent.
4. according to the method for quality control of claim 1,2 or 3 described ginkgo-dipyridamine injections, it is characterized in that: discrimination method comprises the part or all of of following project:
(1) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is dissolved in water, taking liquid 1-10ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement;
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is dissolved in water, and taking liquid 1-10ml adds ethanol dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear;
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.1-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly,, shake up with the methanol dilution, get filtrate 20-100ml, add water 15-100ml mixing, put and steam in the water-bath to 10-50ml, put coldly,, get ether extracted liquid with ether extraction 1-5 time, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Other gets Folium Ginkgo reference extract 20-100mg, add methanol: 5～50% hydrochloric acid=1～8: 0.5～5 mixed solution 10-60ml water-bath reflux, extract,, add water 10-50ml mixing, put and steam in the water-bath, put cold to 5-50ml, with ether extraction 1-5 time, get ether extracted liquid, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds methanol 0.5-5ml makes dissolving, makes Folium Ginkgo reference extract solution; Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add dissolve with methanol; Draw each 5-20 μ l of above-mentioned need testing solution, reference extract solution and reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 50～99% ethanol=1～10: 1～6: 0.2～5 is developing solvent, launch, take out, dry, spray is with 0.1～20% aluminum chloride alcoholic solution, in the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively;
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder add warm water makes dissolving, adds the 0.5-10% hydrochloric acid solution, extract 2-10 time with the ethyl acetate jolting, merge extractive liquid, is with the washing of 1-20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing 1-10 time, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution is got 1-10ml, evaporate to dryness, add the ethyl acetate dissolving, put in the separatory funnel, add 0.1～20% hydrochloric acid solution washing 1-10 time, get ethyl acetate liquid, evaporate to dryness, residue add acetone 1-5ml dissolving, as need testing solution; Take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, add dissolve with methanol, promptly get reference substance solution; Draw each 5-20 μ l of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 0.1～20% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=5～20: 2～10: 2～10: 0.5～5 is developing solvent, launch, take out, dry, spray is with acetic anhydride, heating is put coldly, puts under the ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
5. according to the method for quality control of the described ginkgo-dipyridamine injection of claim 4, it is characterized in that: discrimination method comprises the part or all of of following project:
(1) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, taking liquid 2ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement;
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, and taking liquid 2ml adds ethanol 10ml dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear;
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale, shake up with methanol, filter with microporous filter membrane 0.45 μ m, get 40ml, add water 30ml, mixing, put and steam in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Other gets Folium Ginkgo reference extract 50mg, adds methanol or acetonitrile: 4: 1 mixed solution 25ml of 25% hydrochloric acid, water-bath reflux, extract, 1 hour, add water 30ml, mixing is put and is steamed in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, makes Folium Ginkgo reference extract solution; Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add methanol and make the reference substance solution that every 1ml contains 0.5mg; Draw above-mentioned need testing solution, reference extract solution and each 5ul of reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 95% ethanol=5: 3: 1 is developing solvent, launch, exhibition is taken out apart from about 5～6 centimetres, dry, spray is with 3% aluminum chloride alcoholic solution; In the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively;
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder 200mg add warm water 10ml makes dissolving, adds 2 of 2% hydrochloric acid solutions, extract 5 times with the ethyl acetate jolting, add 15ml respectively, 10ml, 10ml, 10ml, 10ml, merge extractive liquid,, wash with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml divides the water intaking washing liquid, adds ethyl acetate 10ml and extracts, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shake up, filter with 0.45 μ m microporous filter membrane, get 2ml, evaporate to dryness, add ethyl acetate 20ml dissolving, put in the separatory funnel, add 5% hydrochloric acid solution washing 3 times, each 30ml, get ethyl acetate liquid, evaporate to dryness, residue add acetone 1ml makes dissolving, as need testing solution; It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, adds methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly gets reference substance solution; Draw each 10ul of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 4% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=10: 5: 5: 1 is developing solvent, launches, and exhibition is apart from about 8～10cm, take out, dry, spray is with acetic anhydride, 140～160 ℃ of heating 30 minutes, put coldly, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively.
6. according to the method for quality control of the described ginkgo-dipyridamine injection of claim 1, it is characterized in that: the inspection in the described method of quality control comprises:
(1) clarity: get clean tool plug nessler colorimetric tube, lyophilized powder adds filterable in advance water for injection dissolving, presses the regulation inspection about the injection clarity test of clarity test detailed rules and regulations and criterion, should be up to specification;
(2) pH value: get this product content, 1 content of lyophilized powder adds 1～10ml water dissolution, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～6;
(3) loss on drying: get the lyophilized powder content, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%;
(4) heavy metal: get this product content, the lyophilized powder precision adds water makes dissolving, gets a little, puts in the crucible, and water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
(5) aseptic: get this product content, lyophilized powder makes dissolving with 0.9% aseptic sodium chloride solution, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification;
(6) pyrogen: get this product content, lyophilized powder checks that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 0.1～50ml by rabbit body weight 1kg, should be up to specification in every ratio obtain solution that adds 5～20% glucose injections, 5～20ml;
(7) particulate matter: get this product content, lyophilized powder is made blank with the 10-200ml dissolving of purifying waste water of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification;
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item.
7. according to the method for quality control of the described ginkgo-dipyridamine injection of claim 1, it is characterized in that: the content assaying method of dipyridamole is with methanol or acetonitrile: 0.05～10% sodium dihydrogen phosphate=1～9: 9～1 is the high-efficient liquid phase determining method of mobile phase.
8. according to the method for quality control of the described ginkgo-dipyridamine injection of claim 1, it is characterized in that: the content assaying method of total flavonoids is with methanol or acetonitrile: 0.05～10% phosphoric acid solution=1～9: 9～1 is the high-efficient liquid phase determining method of mobile phase.
9. according to the method for quality control of the described ginkgo-dipyridamine injection of claim 1, it is characterized in that: the content assaying method of terpene lactone is with normal propyl alcohol: oxolane: water=0.1～10: 3～50: 20～150 is the high-efficient liquid phase determining method of mobile phase.
10. according to the method for quality control of claim 1,7,8 or 9 described ginkgo-dipyridamine injections, it is characterized in that: content assaying method comprises:
(1) dipyridamole: adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, with methanol or acetonitrile: 0.05～10% sodium dihydrogen phosphate=1～9: 9～1 is mobile phase, 0.05～10% sodium dihydrogen phosphate is used phosphoric acid solution 1 → 3 adjust pH to 2～7 in advance, the detection wavelength is 200～500nm; Precision takes by weighing the dipyridamole reference substance, uses dissolve with methanol, shakes up, and promptly gets reference substance solution; Get the lyophilized powder content, with 20～100% dissolve with methanol solution, the microporous filter membrane that 0.65 μ m reaches less than 0.65 μ m filters, promptly; High-capacity injection can be directly as need testing solution; Respectively accurate above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, and the record chromatogram calculates, and lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml;
(2) total flavonoids:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with methanol or acetonitrile: 0.05～10% phosphoric acid solution=1～9: 9～1 is mobile phase; The detection wavelength is 100～500nm; Accurate respectively Quercetin reference substance, kaempferol reference substance, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight adds dissolve with methanol, promptly gets reference substance solution; Get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.1-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, use smaller or equal to the filter membrane filtration of 0.65 μ m and get lyophilized powder, add methanol: 5～50% hydrochloric acid=1～10: 0.1～5 mixed solution, put reflux on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, filter with 0.65 μ m and less than 0.65 μ m filter membrane, promptly; High-capacity injection adds sulphuric acid liquid 5 → 100, methanol, and water-bath refluxes, and puts coldly, adds dissolve with methanol, with 0.65 μ m and less than the filtration of 0.65 μ m filter membrane, promptly gets need testing solution; Accurate respectively above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, measures, and calculates the content of Quercetin, kaempferol and isorhamnetin respectively, is converted into the content of total flavonoids with following formula; Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml;
Total flavonoids content=quercetin content * 2.51+ kaempferol content * 2.51+ isorhamnetin content * 2.51
(3) terpene lactone:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with normal propyl alcohol: oxolane: water=0.1～10: 3～50: 20～150 is mobile phase, detects with evaporative light scattering detector; It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance reference substance respectively, adds dissolve with methanol, promptly gets reference substance solution; Get the lyophilized powder content, accurate claim surely, add warm water and make dissolving, add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, wash with 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, promptly; Get high-capacity injection add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, with the washing of 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, and promptly gets need testing solution; Accurate respectively reference substance solution, the need testing solution drawn, inject chromatograph of liquid, measure, calculate the content of bilobalide C15H1808, ginkalide A C20H2409, ginkalide B C20H24010 and ginkalide C C20H24011 respectively with external standard two-point method logarithmic equation, promptly; Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
11. according to the method for quality control of claim 1,7,8 or 9 described ginkgo-dipyridamine injections, it is characterized in that: content assaying method comprises:
(1) dipyridamole is according to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With methanol-0.1% sodium dihydrogen phosphate=75: 25 was mobile phase, and 0.1% sodium dihydrogen phosphate is used phosphoric acid solution 1 → 3 adjust pH to 4.6 in advance; The detection wavelength is 288nm; Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000;
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly;
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 80% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 80% methanol solution to scale, shakes up, and filters with microporous filter membrane 0.45 μ m, promptly;
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates;
Lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml;
(2) total flavonoids is according to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol: 0.4% phosphoric acid solution=52: 48 is a mobile phase; The detection wavelength is 360nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 2500; The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5;
Accurate respectively Quercetin reference substance, kaempferol v, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly;
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, add methanol: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane 0.45 μ m, promptly;
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula;
Total flavonoids content=quercetin content * 2.51+ kaempferol content * 2.51+ isorhamnetin content * 2.51
Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml;
(3) terpene lactone is according to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With normal propyl alcohol: oxolane: water=1: 15: 84 is mobile phase; Detect with evaporative light scattering detector; Number of theoretical plate calculates by the bilobalide peak should be not less than 2500; The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5;
The preparation of reference substance solution respectively precision to take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly;
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 2% hydrochloric acid solutions, extract 5 times, add 15ml respectively with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml, merge extractive liquid,, with 5% sodium acetate solution 20ml washing, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml divides the water intaking washing liquid, adding ethyl acetate 10ml extracts, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, add acetone to scale, shake up, filter 0.45 μ m with microporous filter membrane, promptly;
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate the content of bilobalide, ginkalide A, ginkalide B and ginkalide C respectively with external standard two-point method logarithmic equation;
Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
12. the method for quality control according to the described ginkgo-dipyridamine injection of claim 1 is characterized in that: the character in the described method of quality control is:
Character:
For injection: product is yellow to pale brown color clear liquid;
For lyophilized preparation: product is faint yellow to the loose block of pale brown color;
For infusion solutions: product is faint yellow to pale brown color clear liquid;
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and lyophilized powder is dissolved in water, taking liquid 1-10ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement;
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is dissolved in water, and taking liquid 1-10ml adds ethanol dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear;
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.2-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly,, shake up with the methanol dilution, get filtrate 20-100ml, add water 15-100ml mixing, put and steam in the water-bath to 10-50ml, put coldly,, get ether extracted liquid with ether extraction 1-5 time, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds dissolve with methanol, as need testing solution; Other gets Folium Ginkgo reference extract 20-100mg, add methanol: 5～50% hydrochloric acid=1～8: 0.5～5 mixed solution 10-60ml water-bath reflux, extract,, add water 10-50ml mixing, put and steam in the water-bath, put cold to 5-50ml, with ether extraction 1-5 time, get ether extracted liquid, add water washing 1-5 time, the ether solution evaporate to dryness, residue adds methanol 0.5-5ml makes dissolving, makes Folium Ginkgo reference extract solution; Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add dissolve with methanol; Draw each 5-20 μ l of above-mentioned need testing solution, reference extract solution and reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 50～99% ethanol=1～10: 1～6: 0.2～5 is developing solvent, launch, take out, dry, spray is with 0.1～20% aluminum chloride alcoholic solution, in the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively;
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder add warm water makes dissolving, adds the 0.5-10% hydrochloric acid solution, extract 2-10 time with the ethyl acetate jolting, merge extractive liquid, is with the washing of 1-20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing 1-10 time, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution is got 1-10ml, evaporate to dryness, add the ethyl acetate dissolving, put in the separatory funnel, add 0.1～20% hydrochloric acid solution washing 1-10 time, get ethyl acetate liquid, evaporate to dryness, residue add acetone 1-5ml dissolving, as need testing solution; Take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, add dissolve with methanol, promptly get reference substance solution; Draw each 5-20 μ l of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 0.1～20% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=5～20: 2～10: 2～10: 0.5～5 is developing solvent, launch, take out, dry, spray is with acetic anhydride, heating is put coldly, puts under the ultra-violet lamp and inspects; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively;
Check: (1) clarity: get clean tool plug nessler colorimetric tube, lyophilized powder adds filterable in advance water for injection dissolving, presses the regulation inspection about the injection clarity test of clarity test detailed rules and regulations and criterion, should be up to specification;
(2) pH value: get this product content, 1 content of lyophilized powder adds 1～10ml water dissolution, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2～6;
(3) loss on drying: get the lyophilized powder content, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%;
(4) heavy metal: get this product content, the lyophilized powder precision adds water makes dissolving, gets a little, puts in the crucible, and water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
(5) aseptic: get this product content, lyophilized powder makes dissolving with 0.9% aseptic sodium chloride solution, checks according to Chinese Pharmacopoeia sterility test method, should be up to specification;
(6) pyrogen: get this product content, lyophilized powder checks that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 0.1～50ml by rabbit body weight 1kg, should be up to specification in every ratio obtain solution that adds 5～20% glucose injections, 5～20ml;
(7) particulate matter: get this product content, lyophilized powder is made blank with the 10-200ml dissolving of purifying waste water of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification;
(8) other: should meet every regulation relevant under the Chinese Pharmacopoeia injection item;
Assay:
(1) dipyridamole: adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, with methanol or acetonitrile: 0.05～10% sodium dihydrogen phosphate=1～9: 9～1 is mobile phase, 0.05～10% sodium dihydrogen phosphate is used phosphoric acid solution 1 → 3 adjust pH to 2～7 in advance, the detection wavelength is 200～500nm; Precision takes by weighing the dipyridamole reference substance, uses dissolve with methanol, shakes up, and promptly gets reference substance solution; Get the lyophilized powder content, with 20～100% dissolve with methanol solution, the microporous filter membrane that 0.65 μ m reaches less than 0.65 μ m filters, promptly; High-capacity injection can be directly as need testing solution; Respectively accurate above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, and the record chromatogram calculates, and lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml;
(2) total flavonoids:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with methanol or acetonitrile: 0.05～10% phosphoric acid solution=1～9: 9～1 is mobile phase; The detection wavelength is 100～500nm; Accurate respectively Quercetin reference substance, kaempferol reference substance, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight adds dissolve with methanol, promptly gets reference substance solution; Get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile that 0.5-10 doubly measures: the mixed solution of 10-50% hydrochloric acid=1-10: 0.1-5, put heating and refluxing extraction on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, use smaller or equal to the filter membrane filtration of 0.65 μ m and get lyophilized powder, add methanol: 5～50% hydrochloric acid=1～10: 0.1～5 mixed solution, put reflux on the boiling water bath, be cooled to room temperature rapidly, dilute with methanol, shake up, filter with 0.65 μ m and less than 0.65 μ m filter membrane, promptly; High-capacity injection adds sulphuric acid liquid 5 → 100, methanol, and water-bath refluxes, and puts coldly, adds dissolve with methanol, with 0.65 μ m and less than the filtration of 0.65 μ m filter membrane, promptly gets need testing solution; Accurate respectively above-mentioned reference substance solution and the need testing solution drawn injects chromatograph of liquid, measures, and calculates the content of Quercetin, kaempferol and isorhamnetin respectively, is converted into the content of total flavonoids with following formula; Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml;
Total flavonoids content=quercetin content * 2.51+ kaempferol content * 2.51+ isorhamnetin content * 2.51
(3) terpene lactone:
Adopt high performance liquid chromatography, chromatographic column is C18 or C4 or C8 post, and with normal propyl alcohol: oxolane: water=0.1～10: 3～50: 20～150 is mobile phase, detects with evaporative light scattering detector; It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance reference substance respectively, adds dissolve with methanol, promptly gets reference substance solution; Get the lyophilized powder content, accurate claim surely, add warm water and make dissolving, add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, wash with 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, promptly; Get high-capacity injection add 0.5～10% hydrochloric acid solution a little, extract with the ethyl acetate jolting, get extracting solution, with the washing of 0.5～20% sodium acetate solution, divide and get sodium acetate liquid, add ethyl acetate extraction, merge ethyl acetate extraction liquid, add water washing, divide the water intaking washing liquid, add ethyl acetate extraction, merge ethyl acetate liquid, evaporate to dryness, the residue acetone solution, shake up, 0.65 μ m reaches less than 0.65 μ m microporous filter membrane and filters, and promptly gets need testing solution; Accurate respectively reference substance solution, the need testing solution drawn, inject chromatograph of liquid, measure, calculate the content of bilobalide C15H1808, ginkalide A C20H24O9, ginkalide B C20H24O10 and ginkalide C C20H24O11 respectively with external standard two-point method logarithmic equation, promptly; Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
13. the method for quality control according to the described ginkgo-dipyridamine injection of claim 12 is characterized in that: the character in the described method of quality control is:
For injection: product is yellow to pale brown color clear liquid;
For lyophilized preparation: product is faint yellow to the loose block of pale brown color;
For infusion solutions: product is faint yellow to pale brown color clear liquid;
Differentiate: (1) gets ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, and the content that lyophilized powder is adds water 5ml and makes dissolving, taking liquid 2ml, add magnesium powder a little, add concentrated hydrochloric acid number droplet, fade in redness after the placement;
(2) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the content that lyophilized powder is adds water 5ml and makes dissolving, and taking liquid 2ml adds ethanol 10ml dilution, promptly shows green fluorescence, add dilute hydrochloric acid after fluorescence disappear;
(3) get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, add methanol or acetonitrile: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale, shake up with methanol, filter with microporous filter membrane 0.45 μ m, get 40ml, add water 30ml, mixing, put and steam in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Other gets Folium Ginkgo reference extract 50mg, adds methanol or acetonitrile: 4: 1 mixed solution 25ml of 25% hydrochloric acid, water-bath reflux, extract, 1 hour, add water 30ml, mixing is put and is steamed in the water-bath to about 20ml, put cold, with ether extraction 3 times, each 20ml, get ether extracted liquid, add water washing 3 times, each 20ml, ether solution evaporate to dryness, residue adds methanol 1ml makes dissolving, makes Folium Ginkgo reference extract solution; Get Quercetin reference substance, kaempferol reference substance, isorhamnetin reference substance more respectively, add methanol and make the reference substance solution that every 1ml contains 0.5mg; Draw above-mentioned need testing solution, reference extract solution and each 5ul of reference substance solution, put respectively on same polyamide film, with dichloromethane: glacial acetic acid: 95% ethanol=5: 3: 1 is developing solvent, launch, exhibition is taken out apart from about 5～6 centimetres, dry, spray is with 3% aluminum chloride alcoholic solution; In the test sample chromatograph, with Folium Ginkgo reference extract and the corresponding position of reference substance chromatograph on, show the speckle of same color respectively;
(4) get the ginkgo Damo freeze-drying powder respectively, injection or infusion solutions, lyophilized powder 200mg add warm water 10ml makes dissolving, adds 2 of 2% hydrochloric acid solutions, extract 5 times with the ethyl acetate jolting, add 15ml respectively, 10ml, 10ml, 10ml, 10ml, merge extractive liquid,, wash with 5% sodium acetate solution 20ml, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml divides the water intaking washing liquid, adds ethyl acetate 10ml and extracts, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, adds acetone to scale, shake up, filter with 0.45 μ m microporous filter membrane, get 2ml, evaporate to dryness, add ethyl acetate 20ml dissolving, put in the separatory funnel, add 5% hydrochloric acid solution washing 3 times, each 30ml, get ethyl acetate liquid, evaporate to dryness, residue add acetone 1ml makes dissolving, as need testing solution; It is an amount of that precision takes by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance respectively, adds methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly gets reference substance solution; Draw each 10ul of above-mentioned need testing solution and reference substance solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the carboxymethylcellulose sodium solution that contains 4% sodium acetate, with toluene: ethyl acetate: acetone: dehydrated alcohol=10: 5: 5: 1 is developing solvent, launches, and exhibition is apart from about 8～10cm, take out, dry, spray is with acetic anhydride, 140～160 ℃ of heating 30 minutes, put coldly, put under the ultra-violet lamp 365nm and inspect; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color respectively;
Check: (1) clarity: in super-clean bench, operate, get 5 of clean tool plug nessler colorimetric tubes, be respectively charged into ginkgo Damo freeze-drying powder, injection or infusion solutions, lyophilized powder adds filterable in advance water for injection 5ml dissolving, press the regulation inspection under clarity test detailed rules and regulations and the criterion injectable sterile powder item, should be up to specification;
(2) pH value: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder adds water 5ml makes dissolving, measures according to Chinese Pharmacopoeia pH value algoscopy, should be 2.5～4.5;
(3) loss on drying: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the accurate title, decide, and measures according to the Chinese Pharmacopoeia dry weightless mensuration, must not cross 5.0%;
(4) heavy metal: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, the lyophilized powder precision adds water 5ml makes dissolving, and precision is measured 2ml, put in the crucible, water bath method according to the second method inspection of Chinese Pharmacopoeia heavy metal inspection technique, contains heavy metal and must not cross 5/1000000ths;
(5) aseptic: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is got 10 contents, makes dissolving with 0.9% aseptic sodium chloride solution 50ml, checks according to Chinese Pharmacopoeia sterility test method method, should be up to specification;
(6) pyrogen: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, lyophilized powder is in every ratio obtain solution that adds 10% glucose injection 10ml, check that according to the Chinese Pharmacopoeia pyrogen test dosage is slowly injected diluent 3ml by rabbit body weight 1kg, should be up to specification;
(7) particulate matter: get ginkgo Damo freeze-drying powder, injection or infusion solutions respectively, 1 content of lyophilized powder with the 80ml dissolving of purifying waste water, is made blank of purifying waste water, check according to the Chinese Pharmacopoeia microscopic counting, should be up to specification;
(8) other: should meet relevant every regulation under the Chinese Pharmacopoeia appendix injection item;
Assay:
(1) dipyridamole is according to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With methanol-0.1% sodium dihydrogen phosphate=75: 25 was mobile phase, and 0.1% sodium dihydrogen phosphate is used phosphoric acid solution 1 → 3 adjust pH to 4.6 in advance; The detection wavelength is 288nm; Number of theoretical plate calculates by the dipyridamole peak should be not less than 2000;
The preparation precision of reference substance solution takes by weighing dipyridamole reference substance 15mg, puts in the 10ml measuring bottle, with dissolve with methanol and be diluted to scale, shakes up; Precision is measured 1ml, puts in the 50ml measuring bottle, adds methanol to scale, shakes up, and makes the solution that every 1ml contains 30 μ g, promptly;
The about 48mg of this product content is got in the preparation of need testing solution, accurate claims surely, puts in the 10ml measuring bottle, with 80% dissolve with methanol solution and be diluted to scale, shakes up; Precision is measured 2ml, puts in the 25ml measuring bottle, adds 80% methanol solution to scale, shakes up, and filters with microporous filter membrane 0.45 μ m, promptly;
Accurate respectively above-mentioned reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, and the record chromatogram calculates;
Lyophilized powder contains dipyridamole and should be 3-20% in this preparation; Injection contains dipyridamole and should be 0.1-0.8mg/ml; High-capacity injection contains dipyridamole and should be 0.002～0.05mg/ml;
(2) total flavonoids is according to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; Methanol: 0.4% phosphoric acid solution=52: 48 is a mobile phase; The detection wavelength is 360nm; Number of theoretical plate calculates by the Quercetin peak should be not less than 2500; The separating degree at kaempferol peak and isorhamnetin peak should be greater than 1.5;
Accurate respectively Quercetin reference substance, kaempferol v, the isorhamnetin reference substance that takes by weighing through the phosphorus pentoxide dried overnight of the preparation of reference substance solution adds methanol and makes the mixed solution that every 1ml contains 30 μ g, 25 μ g, 10 μ g, promptly;
The about 35mg of this product is got in the preparation of need testing solution, the accurate title, decide, add methanol: the mixed solution 25ml of 25% hydrochloric acid=4: 1, put on the boiling water bath reflux 1 hour, and be cooled to room temperature rapidly, be transferred in the 50ml measuring bottle, be diluted to scale with methanol, shake up, filter with microporous filter membrane 0.45 μ m, promptly;
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and calculate the content of Quercetin, kaempferol and isorhamnetin respectively, are converted into the content of total flavonoids with following formula;
Total flavonoids content=quercetin content * 2.51+ kaempferol content * 2.51+ isorhamnetin content * 2.51
Lyophilized powder contains total flavonoids and should be 8-35% in this preparation; Injection contains total flavonoids and should be 0.5-1.5mg/ml; High-capacity injection contains total flavonoids and should be 0.008～0.08mg/ml;
(3) terpene lactone is according to high effective liquid chromatography for measuring;
Chromatographic condition and system suitability test are filler with octadecylsilane chemically bonded silica; With normal propyl alcohol: oxolane: water=1: 15: 84 is mobile phase; Detect with evaporative light scattering detector; Number of theoretical plate calculates by the bilobalide peak should be not less than 2500; The separating degree at bilobalide peak and ginkalide C peak should be greater than 1.5;
The preparation of reference substance solution respectively precision to take by weighing bilobalide reference substance, ginkalide A reference substance, ginkalide B reference substance and ginkalide C reference substance an amount of, add methanol and make the mixed solution that every 1ml contains 0.7mg, 1.2mg, 0.35mg, 0.7mg respectively, promptly;
The about 200mg of this product content is got in the preparation of need testing solution, and accurate the title decides, and adds warm water 10ml and makes dissolving, add 2 of 2% hydrochloric acid solutions, extract 5 times, add 15ml respectively with the ethyl acetate jolting, 10ml, 10ml, 10ml, 10ml, merge extractive liquid,, with 5% sodium acetate solution 20ml washing, divide and get sodium acetate liquid, add ethyl acetate 10ml and extract, merge ethyl acetate extraction liquid, add water washing 2 times, each 20ml divides the water intaking washing liquid, adding ethyl acetate 10ml extracts, merge ethyl acetate liquid, evaporate to dryness, residue is with acetone solution and be transferred in the 5ml measuring bottle, add acetone to scale, shake up, filter 0.45 μ m with microporous filter membrane, promptly;
Accurate respectively reference substance solution 5 μ l, the 10 μ l of drawing of algoscopy, need testing solution 10 μ l inject chromatograph of liquid, measure, and calculate the content of bilobalide, ginkalide A, ginkalide B and ginkalide C respectively with external standard two-point method logarithmic equation;
Lyophilized powder contains terpene lactone and should be 2-10% in this preparation; Injection contains terpene lactone and should be 0.1-0.8mg/ml; High-capacity injection contains terpene lactone and should be 0.002～0.03mg/ml.
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| CN101869593A (en) * | 2010-06-29 | 2010-10-27 | 广东逸舒制药有限公司 | Detection method of pill capable of dissipating phlegm and stopping coughing |
| CN101936958A (en) * | 2009-07-01 | 2011-01-05 | 秦引林 | Method for detecting and analyzing methanesulfonic amine ginkgolide B by using high-performance liquid-evaporative light |
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| CN103239487A (en) * | 2012-04-23 | 2013-08-14 | 成都百裕科技制药有限公司 | Bilobalide injection and content determination method |
| CN105372338A (en) * | 2014-08-08 | 2016-03-02 | 南京中科药业有限公司 | Compound yinlingtong capsule quality detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101936958A (en) * | 2009-07-01 | 2011-01-05 | 秦引林 | Method for detecting and analyzing methanesulfonic amine ginkgolide B by using high-performance liquid-evaporative light |
| CN101869593A (en) * | 2010-06-29 | 2010-10-27 | 广东逸舒制药有限公司 | Detection method of pill capable of dissipating phlegm and stopping coughing |
| CN101869593B (en) * | 2010-06-29 | 2014-07-02 | 广东逸舒制药有限公司 | Detection method of pill capable of dissipating phlegm and stopping coughing |
| CN103239487A (en) * | 2012-04-23 | 2013-08-14 | 成都百裕科技制药有限公司 | Bilobalide injection and content determination method |
| CN102707008A (en) * | 2012-06-15 | 2012-10-03 | 湖南麓山天然植物制药有限公司 | Detection method of gynostemma total saponin granules |
| CN105372338A (en) * | 2014-08-08 | 2016-03-02 | 南京中科药业有限公司 | Compound yinlingtong capsule quality detection method |
| CN106248856A (en) * | 2016-06-12 | 2016-12-21 | 杭州康恩贝制药有限公司 | The discrimination method of effective ingredient in a kind of gingko leaf preparation |
| CN111983077A (en) * | 2020-08-18 | 2020-11-24 | 贵州益佰制药股份有限公司 | Detection method of ginkgo dipyridamole injection preparation |
| CN111983077B (en) * | 2020-08-18 | 2022-08-30 | 贵州益佰制药股份有限公司 | Detection method of ginkgo dipyridamole injection preparation |
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