CN106248860B - A kind of youngster's spleen is waken up the detection method of particle - Google Patents
A kind of youngster's spleen is waken up the detection method of particle Download PDFInfo
- Publication number
- CN106248860B CN106248860B CN201610887034.6A CN201610887034A CN106248860B CN 106248860 B CN106248860 B CN 106248860B CN 201610887034 A CN201610887034 A CN 201610887034A CN 106248860 B CN106248860 B CN 106248860B
- Authority
- CN
- China
- Prior art keywords
- solution
- methanol
- chromatography
- reference substance
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 52
- 239000002245 particle Substances 0.000 title claims abstract description 51
- 210000000952 spleen Anatomy 0.000 title claims abstract description 45
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 114
- 238000000034 method Methods 0.000 claims abstract description 65
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 claims abstract description 55
- 238000012360 testing method Methods 0.000 claims abstract description 49
- 235000009917 Crataegus X brevipes Nutrition 0.000 claims abstract description 45
- 235000013204 Crataegus X haemacarpa Nutrition 0.000 claims abstract description 45
- 235000009685 Crataegus X maligna Nutrition 0.000 claims abstract description 45
- 235000009444 Crataegus X rubrocarnea Nutrition 0.000 claims abstract description 45
- 235000009486 Crataegus bullatus Nutrition 0.000 claims abstract description 45
- 235000017181 Crataegus chrysocarpa Nutrition 0.000 claims abstract description 45
- 235000009682 Crataegus limnophila Nutrition 0.000 claims abstract description 45
- 235000004423 Crataegus monogyna Nutrition 0.000 claims abstract description 45
- 235000002313 Crataegus paludosa Nutrition 0.000 claims abstract description 45
- 235000009840 Crataegus x incaedua Nutrition 0.000 claims abstract description 45
- 238000003556 assay Methods 0.000 claims abstract description 32
- 241000287828 Gallus gallus Species 0.000 claims abstract description 30
- 239000012528 membrane Substances 0.000 claims abstract description 30
- 210000004317 gizzard Anatomy 0.000 claims abstract description 29
- 238000012850 discrimination method Methods 0.000 claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 18
- 235000002722 Dioscorea batatas Nutrition 0.000 claims abstract description 16
- 235000006536 Dioscorea esculenta Nutrition 0.000 claims abstract description 16
- 240000001811 Dioscorea oppositifolia Species 0.000 claims abstract description 16
- 235000003416 Dioscorea oppositifolia Nutrition 0.000 claims abstract description 16
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 claims abstract description 7
- 244000077995 Coix lacryma jobi Species 0.000 claims abstract description 5
- 240000008669 Hedera helix Species 0.000 claims abstract description 5
- 244000197580 Poria cocos Species 0.000 claims abstract description 5
- 235000008599 Poria cocos Nutrition 0.000 claims abstract description 5
- 210000000582 semen Anatomy 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 339
- 239000000243 solution Substances 0.000 claims description 173
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 135
- 239000013558 reference substance Substances 0.000 claims description 109
- 238000004587 chromatography analysis Methods 0.000 claims description 103
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 99
- 239000000463 material Substances 0.000 claims description 93
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 78
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 75
- 239000012085 test solution Substances 0.000 claims description 68
- 239000007788 liquid Substances 0.000 claims description 67
- 235000019441 ethanol Nutrition 0.000 claims description 58
- 239000000706 filtrate Substances 0.000 claims description 45
- 239000008187 granular material Substances 0.000 claims description 44
- 241001092040 Crataegus Species 0.000 claims description 43
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 42
- 239000000203 mixture Substances 0.000 claims description 42
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 40
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 38
- 238000002360 preparation method Methods 0.000 claims description 35
- 239000000741 silica gel Substances 0.000 claims description 33
- 229910002027 silica gel Inorganic materials 0.000 claims description 33
- 239000002904 solvent Substances 0.000 claims description 33
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 31
- 238000001914 filtration Methods 0.000 claims description 31
- 238000000605 extraction Methods 0.000 claims description 30
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 29
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 239000000047 product Substances 0.000 claims description 24
- 238000010992 reflux Methods 0.000 claims description 24
- 239000007921 spray Substances 0.000 claims description 23
- 239000000377 silicon dioxide Substances 0.000 claims description 21
- 239000000945 filler Substances 0.000 claims description 20
- 238000011161 development Methods 0.000 claims description 19
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 18
- 229960000583 acetic acid Drugs 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 16
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 14
- 235000019253 formic acid Nutrition 0.000 claims description 14
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 claims description 13
- 238000004811 liquid chromatography Methods 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 claims description 11
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 claims description 11
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 claims description 11
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 claims description 11
- 229940025878 hesperidin Drugs 0.000 claims description 11
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 claims description 11
- 229940096998 ursolic acid Drugs 0.000 claims description 11
- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 238000000527 sonication Methods 0.000 claims description 10
- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 9
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 230000007935 neutral effect Effects 0.000 claims description 8
- 238000011067 equilibration Methods 0.000 claims description 7
- 239000012467 final product Substances 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- 239000006071 cream Substances 0.000 claims description 4
- 238000004064 recycling Methods 0.000 claims description 4
- 239000012047 saturated solution Substances 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 3
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 3
- 238000007598 dipping method Methods 0.000 claims description 3
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 235000019640 taste Nutrition 0.000 claims description 3
- 241000446313 Lamella Species 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- 238000012545 processing Methods 0.000 claims 1
- 238000005303 weighing Methods 0.000 claims 1
- 239000002023 wood Substances 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 240000000171 Crataegus monogyna Species 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 80
- 239000003153 chemical reaction reagent Substances 0.000 description 16
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 238000010200 validation analysis Methods 0.000 description 12
- 238000011084 recovery Methods 0.000 description 11
- 239000012488 sample solution Substances 0.000 description 10
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 208000022531 anorexia Diseases 0.000 description 6
- 206010061428 decreased appetite Diseases 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- -1 biphosphate Ammonium salt Chemical class 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000003252 repetitive effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000002137 ultrasound extraction Methods 0.000 description 4
- 229930182470 glycoside Natural products 0.000 description 3
- 150000002338 glycosides Chemical class 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000008213 purified water Substances 0.000 description 3
- XHFLOLLMZOTPSM-UHFFFAOYSA-M sodium;hydrogen carbonate;hydrate Chemical compound [OH-].[Na+].OC(O)=O XHFLOLLMZOTPSM-UHFFFAOYSA-M 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 240000004307 Citrus medica Species 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 235000002710 Ilex cornuta Nutrition 0.000 description 2
- 241001310146 Ilex cornuta Species 0.000 description 2
- 235000010326 Osmanthus heterophyllus Nutrition 0.000 description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 2
- 150000001343 alkyl silanes Chemical group 0.000 description 2
- 229940121657 clinical drug Drugs 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 239000002304 perfume Substances 0.000 description 2
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 239000011975 tartaric acid Substances 0.000 description 2
- 235000002906 tartaric acid Nutrition 0.000 description 2
- 230000001550 time effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000000103 Anorexia Nervosa Diseases 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 201000009495 Hypotrichosis Diseases 0.000 description 1
- 208000015817 Infant Nutrition disease Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 208000019790 abdominal distention Diseases 0.000 description 1
- UGAPHEBNTGUMBB-UHFFFAOYSA-N acetic acid;ethyl acetate Chemical compound CC(O)=O.CCOC(C)=O UGAPHEBNTGUMBB-UHFFFAOYSA-N 0.000 description 1
- AOZUYISQWWJMJC-UHFFFAOYSA-N acetic acid;methanol;hydrate Chemical compound O.OC.CC(O)=O AOZUYISQWWJMJC-UHFFFAOYSA-N 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002996 emotional effect Effects 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000003676 hair loss Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 244000000053 intestinal parasite Species 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 208000007442 rickets Diseases 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
It wakes up the invention discloses a kind of youngster's spleen the detection method of particle, the drug is mainly prepared by hawthorn, malt, the membrane of a chicken's gizzard, Chinese yam, coix seed, Semen Lablab Album, dried orange peel and Poria cocos, and the detection method includes discrimination method and content assaying method；The discrimination method includes differentiating detection to the thin layer of hawthorn, dried orange peel, Chinese yam or the membrane of a chicken's gizzard；The content assaying method includes the assay to aurantiamarin or citric acid.The detection method is accurate, and high sensitivity is reproducible, and testing result is stablized, and can effectively detect youngster's spleen and wake up the quality of particle, and each detection standard can guarantee curative effect of medication.
Description
Technical field
It wakes up the present invention relates to a kind of youngster's spleen the detection method of particle, belongs to the field of drug technology；
Background technology
Baby anorexia is very common in children, and main symptom has vomiting, loss of appetite, diarrhea, constipation, abdominal distension, abdomen
Bitterly and have blood in stool.Children anorexia is the common and frequently-occurring disease of paediatrics, if effective prevention can not cause infant hypoevolutism, causes battalion
It nourishs one's nature anaemia, rickets, giving infant physical and mental health and causing to increase influences.There are Gastric gysrhythmias by apocleisis children simultaneously.
The cause of disease of baby anorexia is varied, and being summed up mainly has systemic disease, drug, weather, emotional factor
And improper feeding and anorexia nervosa, no matter baby anorexia caused by which kind of reason, the duration is longer can influence it is small
The normal growth of youngster's height, weight, merging is become thin, malnutritive, hypoimmunity, very then develops into infantile malnutrition due to digestive disturbances or intestinalparasites disease, shows as face Huang
Flesh is thin, hypotrichosis, tripe abdominal distention blue veins are exposed or the recessed such as boat of abdomen, seriously affects the growth and development of children, and easily triggers simultaneously
Send out disease.Western medicine has been avoided because the toxic side effects such as allergy, nausea, diarrhea, hepatic disorder is often caused increasingly to be tried one's best by people,
Baby anorexia first choice Chinese medicine is treated, when the traditional Chinese medical science tackles such disease, it is smaller that with obvious effects and adverse reaction often can be used
Drug.With youngster's spleen made of hawthorn, malt, the membrane of a chicken's gizzard, Chinese yam, coix seed, Semen Lablab Album, dried orange peel and Poria cocos wake up particle drug have
There is the effect of strengthening the spleen and stomach, promoting digestion and removing indigestion.Available for children anorexia, loose watery stool caused by spleen deficiency food stagnation, weak grade diseases of becoming thin.
In order to preferably control the quality of the product, it is ensured that clinical drug effect, the present invention provides the detection methods of this drug.
The content of the invention
It wakes up it is an object of the present invention to provide a kind of youngster's spleen the detection method of particle；The detection method is accurate, sensitivity
Height, reproducible, reliable results can effectively control the quality of the product, it is ensured that the clinical drug effect of drug.
In order to solve the above technical problems, the present invention adopts the following technical scheme that realization：
A kind of youngster's spleen is waken up the detection method of particle, main 300-340 parts of hawthorn, the wheat by calculating by weight of the particle
Bud
300-340 parts, 220-260 parts of the membrane of a chicken's gizzard, 220-260 parts of Chinese yam, 140-180 parts of coix seed, Semen Lablab Album 220-260
86-106 parts of part, 86-106 parts of dried orange peel and Poria cocos are made as follows：More than eight tastes, hawthorn, dried orange peel be ground into coarse powder, uses
65% ethanol as solvent, when dipping 24 is small after, carry out diacolation, collection is filtered liquid, and diacolation liquid recycling ethanol is concentrated into 55-65 DEG C of phase
It is spare to the thick paste that density is 1.35-1.40；The Six-elements medicinal material such as remaining malt adds water to cook three times, every time 1 it is small when, merge decoct
Liquid, filtration, relative density is 1.10 clear cream when filtrate is concentrated into 55-65 DEG C, and equivalent ethyl alcohol is added to make precipitation, takes supernatant, is returned
Ethyl alcohol is received, is concentrated into the thick paste that relative density is 1.35-1.40；With above-mentioned hawthorn, dried orange peel thick paste mixing, addition sucrose is appropriate,
Be made particle, it is dry to get；The detection method includes discrimination method and content assaying method；The discrimination method include pair
Hawthorn, dried orange peel, the thin layer of Chinese yam or the membrane of a chicken's gizzard differentiate detection；The content assaying method includes containing aurantiamarin or citric acid
It is fixed to measure.
Foregoing youngster's spleen is waken up the detection method of particle, and the discrimination method of the hawthorn is：It gets it filled composition granule, after finely ground, takes
4-12g adds 20-30ml ether, be heated to reflux 0.5-1.5 it is small when, filtration, filtrate volatilizes, and it is molten that residue adds methanol 0.5-1.5m1 to make
Solution, as test solution；Separately take hawthorn control medicinal material 0.1-0.3g, after finely ground, 4-12g is taken to add 20-30ml ether, is heated back
When stream 0.5-1.5 is small, filtration, filtrate volatilizes, and residue adds methanol 0.5-1.5m1 to make dissolving, and control medicinal material solution is made；Bear is taken again
Tartaric acid reference substance is appropriate, adds methanol that solution of every 0.5-1.5ml containing 0.05-0.15mg is made, as reference substance solution；In
The experiment of state pharmacopeia thin-layered chromatography, draws above-mentioned three a variety of each 5-10 μ l of solution, is put respectively on same silica gel g thin-layer plate, with
Toluene：Ethyl acetate：Formic acid=15-25: 2-6: 0.1-1.0 is solvent, is unfolded, and takes out, dries, spray molten with sulfuric acid ethyl alcohol
Liquid, the volume ratio of sulfuric acid and ethyl alcohol is 2-4 in ethanol solution of sulfuric acid:9-11；It is clear that spot development is heated at 105 DEG C, respectively
It puts and is inspected under daylight or 360-370nm ultraviolet lamps, in test sample chromatography, on position corresponding with control medicinal material chromatography, shown
The principal spot of same color or fluorescence principal spot；On position corresponding with reference substance chromatography, the spot or glimmering of same color is shown
Hot spot point.
Foregoing youngster's spleen is waken up the detection method of particle, and the discrimination method of the hawthorn is：It gets it filled composition granule, after finely ground, takes
8g adds 25ml ether, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds methanol 1m1 to make dissolving, as test solution；
Separately take hawthorn control medicinal material 0.2g, after finely ground, 8g is taken to add 25ml ether, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds
Methanol 1m1 makes dissolving, and control medicinal material solution is made；It takes ursolic acid reference substance appropriate again, adds methanol that every 1ml is made containing 0.1mg's
Solution, as reference substance solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned three a variety of each 5~10 μ l of solution, point
Other point is on same silica gel g thin-layer plate, with toluene：Ethyl acetate：Formic acid=20: be solvent at 4: 0.5 are unfolded, and are taken out, are dried in the air
It is dry, it sprays with ethanol solution of sulfuric acid, the volume ratio of sulfuric acid and ethyl alcohol is 3 in ethanol solution of sulfuric acid:10；Spot is heated at 105 DEG C
Colour developing is clear, puts inspected under daylight or 365nm ultraviolet lamps respectively, in test sample chromatography, corresponding with control medicinal material chromatography
On position, the principal spot of same color or fluorescence principal spot are shown；On position corresponding with reference substance chromatography, same color is shown
Spot or fluorescence spot.
Foregoing youngster's spleen is waken up the detection method of particle, and the discrimination method of the dried orange peel is：It gets it filled composition granule, after finely ground, takes
5-15g adds methanol 25-35ml, be heated to reflux 0.5-1.5 it is small when, filtration, filtrate volatilizes, and residue adds water 5-15ml to make dissolving, turns
It moves in separatory funnel, is extracted 2-3 times with ethyl acetate shaking, every time each 5-15ml, combined ethyl acetate extracting solution, in water-bath
It is evaporated, residue adds methanol 1-4ml to make dissolving, as test solution；Dried orange peel control medicinal material 0.5-1.5g separately is taken, is made in the same way of pair
According to medicinal material solution；It takes aurantiamarin reference substance appropriate again, adds methanol that saturated solution is made, as reference substance solution；According to middle traditional Chinese medicines
Allusion quotation thin-layered chromatography is tested, and draws above-mentioned each 2-5 μ l of three kinds of solution, puts use 0.1-1.0% sodium hydroxide solutions in same respectively
On the silica gel g thin-layer plate of preparation, with ethyl acetate：Methanol：Water=95-105: 15-19: 10-16 is solvent, is opened up to 1-6cm,
It takes out, dries, then with toluene：Ethyl acetate：Formic acid：The upper solution of water=15-25: 5-15: 0.5-1.5: 0.5-1.5 is exhibition
Agent is opened, opens up to 5-10cm, takes out, dry, spray with 1-10% alchlor ethanol solutions, it is ultraviolet for 360-370nm to be placed in wavelength
It is inspected under light lamp；In test sample chromatography, on position corresponding with control medicinal material chromatography, the fluorescence principal spot of same color is shown；
On position corresponding with reference substance chromatography, the fluorescence spot of same color is shown.
Foregoing youngster's spleen is waken up the detection method of particle, and the discrimination method of the membrane of a chicken's gizzard is：It gets it filled composition granule, it is finely ground, it takes
1-10g, adds water 25-35ml, steeped overnight, and aqueous solution adds water saturated n-butanol together in residue dislocation separatory funnel
Shaking extraction 2-4 times, each 15-25ml, divides and takes n-butanol liquid, be evaporated, residue adds proper amount of methanol to make dissolving, is added on 100-120
Mesh, 1-10g, internal diameter 10-15mm neutral alumina column on, with 5-15% methanol 90-110ml elute, collect eluent, steam
Dry, residue adds 0.5-1.5ml, 65-75% ethyl alcohol makes dissolving, as test solution；Separately take the membrane of a chicken's gizzard control medicinal material 0.5-
1.5g adds water 25-35ml, and when decoction 1-3 is small, aqueous solution is made in the same way of comparison medicine together in residue dislocation separatory funnel
Material solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws each 3~5 μ l of above two solution, put respectively in same silica G
On lamellae, with n-butanol：Glacial acetic acid：Water=5-15: 1-6: 0.5-1.5 is solvent, the lamellae 10- after pre-equilibration point sample
20 minutes, it is unfolded, takes out, dry, spray with ninhydrin solution, it is clear to be heated to spot development at 110 DEG C；In test sample chromatography,
On position corresponding with control medicinal material chromatography, the principal spot of same color is shown.
Foregoing youngster's spleen is waken up the detection method of particle, and the discrimination method of the membrane of a chicken's gizzard is：It gets it filled composition granule, it is finely ground, it takes
5g, adds water 30ml, steeped overnight, and aqueous solution is carried together in residue dislocation separatory funnel, adding water saturated n-butanol shaking
It takes 3 times, each 20ml, divides and take n-butanol liquid, be evaporated, residue adds proper amount of methanol to make dissolving, is added on 100-120 mesh, 5g, internal diameter
It on the neutral alumina column of 10-15mm, is eluted with 10% methanol 100ml, collects eluent, be evaporated, residue adds 1ml70% ethyl alcohol
Make dissolving, as test solution；The membrane of a chicken's gizzard control medicinal material 1g separately is taken, adds water 30ml, when decoction 2 is small, aqueous solution is together with residue
Together in dislocation separatory funnel, control medicinal material solution is made in the same way of；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned two
Kind of each 3~5 μ l of solution are put respectively on same silica gel g thin-layer plate, with n-butanol：Glacial acetic acid：Water=9: be solvent at 3: 1, in advance
Lamellae after balance point sample 15 minutes, is unfolded, and takes out, dries, spray with ninhydrin solution, spot development is heated at 110 DEG C
Clearly；In test sample chromatography, on position corresponding with control medicinal material chromatography, the principal spot of same color is shown.
Foregoing youngster's spleen is waken up the detection method of particle, and the content of hesperidin assay method is：
According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With methanol：Ice vinegar
Acid：Water=30-40: 1-8: 55-65 is mobile phase；Column temperature is 33-37 DEG C；Detection wavelength is 280-286nm；Number of theoretical plate is by orange
Skin glycosides peak, which calculates, should be not less than 3000；
The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol that every 0.5-1.5ml is made and contain
The solution of 0.005-0.015mg to get；
The preparation of test solution：It gets it filled composition granule content, mixing is finely ground, takes 0.2-0.6g, accurately weighed, puts tool
It fills in conical flask, precision adds in methanol 20-30ml, close plug, and weighed weight is ultrasonically treated, sonification power 200-300W,
Supersound process frequency is 30-40kHz, and sonication treatment time is 25-35 minutes, is let cool, then weighed weight, and less loss is supplied with methanol
Weight, shake up, filter, take subsequent filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product.
Foregoing youngster's spleen is waken up the detection method of particle, and the content of hesperidin assay method is：
According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With methanol：Ice vinegar
Acid：Water=35: be mobile phase at 4: 61；Column temperature is 35 DEG C；Detection wavelength is 283nm；Number of theoretical plate should not by the calculating of aurantiamarin peak
Less than 3000；
The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made containing 0.01mg
Solution to get；
The preparation of test solution：It gets it filled composition granule content, mixing is finely ground, takes 0.4g, accurately weighed, puts tool plug cone
In shape bottle, precision adds in methanol 25ml, close plug, and weighed weight is ultrasonically treated, sonification power 250W, is ultrasonically treated frequency
Rate is 35kHz, and sonication treatment time is 30 minutes, is let cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, and is filtered
Cross, take subsequent filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product.
Foregoing youngster's spleen is waken up the detection method of particle, and the citric acid content assaying method is：It is efficient according to Chinese Pharmacopoeia
Liquid chromatography for measuring：
Chromatographic condition and system suitability：It is filler with octadecylsilane chemically bonded silica；Number of theoretical plate presses Chinese holly
Rafter acid peak, which calculates, should be not less than 3000；
The preparation of reference substance solution：Take citric acid reference substance appropriate, it is accurately weighed, add water that every 0.5-1.5ml is made and contain
The solution of 0.1-0.6mg to get；
The preparation of test solution：It gets it filled composition granule content, finely ground, precision weighs 1-4g, puts in 20-30ml measuring bottles,
Precision adds in appropriate amount of water, is ultrasonically treated, sonification power 200-300W, and supersounds process frequency is 30-40kHz, it is ultrasonic at
It is 15-25 minutes to manage the time, lets cool, is diluted with water to scale and shakes up, and is filtered, and takes subsequent filtrate, crosses 0.35-0.55 μm of micropore filter
Film to get.
Measuring method：It is accurate respectively to draw reference substance solution and each 15-25 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed to get.
Foregoing youngster's spleen is waken up the detection method of particle, and the discrimination method of the hawthorn is：After composition granule of getting it filled is finely ground, 8g is taken
Add 25ml ether, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds methanol 1m1 to make dissolving, as test solution；Separately
Take hawthorn control medicinal material 0.2g, after finely ground, 8g is taken to add 25ml ether, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds first
Alcohol 1m1 makes dissolving, and control medicinal material solution is made；It takes ursolic acid reference substance appropriate again, adds methanol that every 1ml is made containing the molten of 0.1mg
Liquid, as reference substance solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned three a variety of each 5~10 μ l of solution, respectively
Point is on same silica gel g thin-layer plate, with toluene：Ethyl acetate：Formic acid=20: be solvent at 4: 0.5 are unfolded, and are taken out, are dried,
Spray is with ethanol solution of sulfuric acid, and the volume ratio of sulfuric acid and ethyl alcohol is 3 in ethanol solution of sulfuric acid:10；Spot development is heated at 105 DEG C
Clearly, put and inspected under daylight or 365nm ultraviolet lamps respectively, in test sample chromatography, in position corresponding with control medicinal material chromatography
On, show the principal spot of same color or fluorescence principal spot；On position corresponding with reference substance chromatography, the spot of same color is shown
Or fluorescence spot；
The discrimination method of the dried orange peel is：It gets it filled composition granule content, after finely ground, takes 10g, add methanol 30ml, heat back
Flow 1 it is small when, filtration, filtrate volatilizes, and residue adds water 10ml to make dissolving, is transferred in separatory funnel, with ethyl acetate shaking extraction 2
Secondary, each each 10ml, combined ethyl acetate extracting solution is evaporated in water-bath, and residue adds methanol 2ml to make dissolving, molten as test sample
Liquid；Dried orange peel control medicinal material 1g separately is taken, is made in the same way of control medicinal material solution；It takes aurantiamarin reference substance appropriate again, methanol is added to be made full
And solution, as reference substance solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned each 2~5 μ l of three kinds of solution, point
Other point in it is same with 0.5% sodium hydroxide solution prepare silica gel g thin-layer plate on, with ethyl acetate：Methanol：Water=100: 17:
13 be solvent, opens up to about 3cm, takes out, dry, then with toluene：Ethyl acetate：Formic acid：Water=20: 10: 1: 1 upper solution
It for solvent, opens up to 8cm, takes out, dry, spray with 5% alchlor ethanol solution, be placed in wavelength as under 365nm ultraviolet lamps
It inspects；In test sample chromatography, on position corresponding with control medicinal material chromatography, the fluorescence principal spot of same color is shown；With it is right
According on the corresponding position of product chromatography, the fluorescence spot of same color is shown；
The discrimination method of the membrane of a chicken's gizzard is：It gets it filled composition granule, it is finely ground, 5g is taken, adds water 30ml, steeped overnight, aqueous solution
Together with water saturated n-butanol shaking extraction 3 times, each 20ml in residue dislocation separatory funnel, is added, divide and take n-butanol liquid,
It is evaporated, residue adds proper amount of methanol to make dissolving, is added on the neutral alumina column of 100-120 mesh, 5g, internal diameter 10-15mm, with 10%
Methanol 100ml is eluted, and is collected eluent, is evaporated, residue adds 1ml70% ethyl alcohol to make dissolving, as test solution；Separately take in chicken
Golden control medicinal material 1g adds water 30ml, and when decoction 2 is small, aqueous solution is made in the same way of control together in residue dislocation separatory funnel
Medicinal material solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws each 3~5 μ l of above two solution, put respectively in same silica gel
On G lamellaes, with n-butanol：Glacial acetic acid：Water=9: be solvent at 3: 1, the lamellae after pre-equilibration point sample 15 minutes, expansion,
It takes out, dries, spray with ninhydrin solution, it is clear to be heated to spot development at 110 DEG C；In test sample chromatography, with control medicinal material
On the corresponding position of chromatography, the principal spot of same color is shown；
The content of hesperidin assay method is：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With methanol：Ice vinegar
Acid：Water=35: be mobile phase at 4: 61；Column temperature is 35 DEG C；Detection wavelength is 283nm；Number of theoretical plate should not by the calculating of aurantiamarin peak
Less than 3000；
The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made containing 0.01mg
Solution to get；
The preparation of test solution：It gets it filled composition granule content, mixing is finely ground, takes 0.4g, accurately weighed, puts tool plug cone
In shape bottle, precision adds in methanol 25ml, close plug, and weighed weight is ultrasonically treated, sonification power 250W, is ultrasonically treated frequency
Rate is 35kHz, and sonication treatment time is 30 minutes, is let cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, and is filtered
Cross, take subsequent filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product；The citric acid content assaying method is：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：
Chromatographic condition and system suitability：It is filler with octadecylsilane chemically bonded silica；With 5% biphosphate
Ammonium salt solution, is mobile phase with phosphoric acid slow-readjustment section pH to 3.0, and column temperature is 25 DEG C；Detection wavelength is 210nm；Number of theoretical plate presses citron
Sour peak, which calculates, should be not less than 3000；
The preparation of reference substance solution：Take citric acid reference substance appropriate, it is accurately weighed, add water that every 1ml is made containing the molten of 0.3mg
Liquid to get；
The preparation of test solution：It gets it filled composition granule content, finely ground, precision weighs 2.0g, puts in 25ml measuring bottles, accurate
Appropriate amount of water is added in, is ultrasonically treated, sonification power 250W, supersound process frequency is 35kHz, and sonication treatment time is 20 points
Clock is let cool, and is diluted with water to scale and is shaken up, and filtration takes subsequent filtrate, cross 0.45 μm of miillpore filter to get.
Measuring method：It is accurate respectively to draw reference substance solution and each 20 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product.
Inventor has carried out substantial amounts of experiment, is the research of detection method of the present invention below
Experimental example：Detection method research
First, sample
Sample：Youngster's spleen is waken up particle, is prepared as described in Example 1, is the particle of brown color；Gas micro-perfume, sweet and sour,
Slight bitter.Recording original project in WS-10441 (ZD-0441) -2002-2012Z, standard is：Character；Hawthorn, dried orange peel thin layer mirror
Not；It checks；Assay project is the measure of aurantiamarin in dried orange peel；According to quality criteria requirements are improved, intend to primary standard project
Revised and the side of adding in Chinese yam, the membrane of a chicken's gizzard thin layer differentiate, hawthorn assay.Through experimental study to this product quality standard
It proposes that increasing revision project has carried out methodology validation, increases revision verification Description Experimental according to as follows.
2nd, differentiate
1st, the thin layer of hawthorn differentiates：
Primary standard is recorded only by the use of ursolic acid as control in method, and specificity is not strong, therefore here, this discriminating is repaiied
It orders, adds hawthorn control material.Revision method is：The content under this product content uniformity item is taken, it is finely ground, about 8g is taken, is added diethyl ether
25ml, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds methanol 1m1 to make dissolving, as test solution.Separately take hawthorn
Control medicinal material 0.2g obtains control medicinal material solution with legal system.It takes ursolic acid reference substance appropriate again, adds methanol that every 1ml is made containing 0.1mg
Solution, as reference substance solution.It tests, draws above-mentioned according to thin-layered chromatography (four general rules 0502 of Chinese Pharmacopoeia version in 2015)
Three a variety of each 5~10 μ l of solution are put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (20: 4: 0.5)
For solvent, it is unfolded, takes out, dry, spray with ethanol solution of sulfuric acid (3 → 10), it is clear to be heated to spot development at 105 DEG C.Point
It does not put and is inspected under daylight or ultraviolet lamp (365nm), in test sample chromatography, on position corresponding with control medicinal material chromatography, shown
The principal spot of same color or fluorescence principal spot；On position corresponding with reference substance chromatography, the spot or glimmering of same color is shown
Hot spot point.On request, methodological study has been carried out to this discriminating, by methodology validation, the thin-layer identification method clear spot,
Separator well reappears, and negative noiseless.Methodology validation is as follows：
1.1 reagents and reagent
Youngster's spleen wakes up particle by the offer of Guizhou Run Sheng pharmacy industries Co., Ltd, i.e., is prepared by embodiment 1；Hawthorn comparison medicine
Material (121626-201402), ursolic acid reference substance (110742-201421) are provided by Nat'l Pharmaceutical ＆ Biological Products Control Institute；
Silica gel g thin-layer plate：The silica gel g thin-layer plate of subsidiary factory of Haiyang Chemical Plant, Qingdao production and self-control silica gel g thin-layer plate；Reagent：Toluene, second
Acetoacetic ester, formic acid are that analysis is pure；
1.2 specificity
Youngster's spleen is taken to wake up particle, hawthorn control medicinal material, ursolic acid reference substance and scarce hawthorn negative sample, with text the method
Test solution, control medicinal material solution, reference substance solution and negative test solution is made, point sample, expansion, colour developing, put respectively
Daylight or ultraviolet lamp (365nm) in test sample chromatography, on position corresponding with control medicinal material chromatography, show same color
Principal spot or fluorescence principal spot；On position corresponding with reference substance chromatography, the spot or fluorescence spot of same color are shown.And spot
Point separation is preferable, negative noiseless, is specifically shown in Fig. 1 and Fig. 2.
2nd, dried orange peel thin layer differentiates
After recording method extraction point sample due to primary standard, sample impurity is more, there is certain interference, this discriminating is extracted at this
Method further cleans, while adds reference substance point sample, and method is determined as：The content under this product content uniformity item is taken, is ground
Carefully, take about 10g, add methanol 30ml, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds water 10ml to make dissolving, is transferred to point
It in liquid funnel, is extracted 2 times, each each 10ml, combined ethyl acetate extracting solution, is evaporated in water-bath, residue with ethyl acetate shaking
Methanol 2ml is added to make dissolving, as test solution.Dried orange peel control medicinal material 1g separately is taken, is made in the same way of control medicinal material solution.Orange is taken again
Skin glycosides reference substance is appropriate, adds methanol that saturated solution is made, as reference substance solution.According to thin-layered chromatography (Chinese Pharmacopoeia 2015
Four general rules 0502 of version) experiment, above-mentioned each 2~5 μ l of three kinds of solution are drawn, are put respectively in same with 0.5% sodium hydroxide solution
On the silica gel g thin-layer plate of preparation, with acetate-methanol-water 100: 17: it is 13) solvent, opens up to about 3cm, take out, dry,
Again using the upper solution of toluene-ethyl acetate-formic acid-water (20: 10: 1: 1) as solvent, open up to about 8cm, take out, dry, spray
With 5% alchlor ethanol solution, put and inspected under ultraviolet lamp (365nm).In test sample chromatography, with control medicinal material chromatography phase
On the position answered, the fluorescence principal spot of same color is shown；On position corresponding with reference substance chromatography, the fluorescence of same color is shown
Spot.Methodological study is carried out to this discriminating, by methodology validation, the thin-layer identification method clear spot, separator well, weight
It is now good and negative noiseless.Methodology validation is as follows：
2.1 reagents and reagent
Youngster's spleen wakes up particle by the offer of Guizhou Run Sheng pharmacy industries Co., Ltd；Dried orange peel control medicinal material (120969-201109) and
Aurantiamarin reference substance (110721-201014) is provided by Nat'l Pharmaceutical ＆ Biological Products Control Institute；Lamellae：Qingdao Haiyang chemical industry
The silica gel g thin-layer plate of subsidiary factory of factory production immerses 0.5% sodium hydroxide solution, takes out, dries；Make 0.5% sodium hydroxide solution by oneself
The silica gel g thin-layer plate of preparation；Reagent：Toluene, ethyl acetate, formic acid, methanol are that analysis is pure；
2.2nd, specificity
Youngster's spleen is taken to wake up particle, dried orange peel control medicinal material, aurantiamarin reference substance and scarce dried orange peel negative sample, with text the method
Test solution, control medicinal material solution, reference substance solution and negative test solution is made, point sample, expansion, colour developing, put respectively
It is inspected under ultraviolet lamp (365nm), in test sample chromatography, the glimmering of same color is shown on position corresponding with control medicinal material chromatography
Light principal spot on position corresponding with reference substance chromatography, shows the fluorescence spot of same color.And spot separation is preferable, it is negative
It is noiseless, see Fig. 3.
2.3 Chinese yam thin layers differentiate
Through overtesting, method is determined as：It gets it filled composition granule, it is finely ground, about 5g is taken, add methylene chloride 50ml, and it is small to be heated to reflux 2
When, filtration takes filtrate to put in separatory funnel, is washed with water 3 times, each 50ml, divides and takes dichloromethane solution, is evaporated, residue adds 1ml
Dichloromethane makes dissolving, as test solution.Chinese yam control medicinal material 0.5g separately is taken, is made in the same way of control medicinal material solution.According to thin
Layer chromatography (four general rules 0502 of Chinese Pharmacopoeia version in 2015) test, draw each 5~10 μ l of above two solution, put respectively in
On same silica gel g thin-layer plate, with chloroform-methanol-water (16: 3: 0.1) for solvent, the lamellae 15 after pre-equilibration point sample
Minute, it is unfolded, takes out, dry, spray with phosphomolybdic acid test solution, it is clear to be heated to spot development at 110 DEG C.In test sample chromatography,
On position corresponding with control medicinal material chromatography, the principal spot of same color is shown.By pharmacopoeial requirements, methodology has been carried out to this discriminating
It investigates, by methodology validation, the thin-layer identification method clear spot, separator well reappears, and negative noiseless.Methodology
Verification is as follows：
2.3.1, reagent and reagent
Youngster's spleen wake up particle Guizhou Run Sheng pharmacy industries Co., Ltd offer；Chinese yam control medicinal material (121137-200402) is in
State's drug biological products assay institute provides；Silica gel thin-layer plate：The silica gel g thin-layer plate of subsidiary factory of Haiyang Chemical Plant, Qingdao production and self-control
Silica gel g thin-layer plate reagent：Dichloromethane, chloroform, methanol are that analysis is pure, and water is super purified water；
2.3.2, specificity
Youngster's spleen is taken to wake up particle, Chinese yam control medicinal material and scarce Chinese yam negative sample, it is molten that test sample is made with text the method
Liquid, control medicinal material solution, reference substance solution and negative test solution, point sample, expansion, spray are with phosphomolybdic acid test solution respectively, 110
It is clear DEG C to be heated to spot development, in test sample chromatography, the master of same color is shown on position corresponding with control medicinal material chromatography
Spot, and spot separation is preferable, negative noiseless, the result is shown in Fig. 4.
The thin layer of 2.4 thes membrane of a chicken's gizzard differentiates
Through overtesting, method is determined as：It gets it filled composition granule, it is finely ground, about 5g is taken, adds water 30ml, steeped overnight, aqueous solution connects
With water saturated n-butanol shaking extraction 3 times, each 20ml in residue together dislocation separatory funnel, is added, divide and take n-butanol liquid, steam
Dry, residue adds proper amount of methanol to make dissolving, is added on neutral alumina column (100-120 mesh, 5g, internal diameter 10-15mm), with 10% first
Alcohol 100ml is eluted, and is collected eluent, is evaporated, residue adds 1ml70% ethyl alcohol to make dissolving, as test solution.Separately take the membrane of a chicken's gizzard
Control medicinal material 1g adds water 30ml, and when decoction 2 is small, aqueous solution is made in the same way of comparison medicine together in residue dislocation separatory funnel
Material solution.It is tested according to thin-layered chromatography (four general rules 0502 of Chinese Pharmacopoeia version in 2015), draws each 3~5 μ of above two solution
L is put respectively on same silica gel g thin-layer plate, with n-butanol-glacial acetic acid-water (9:3:1) it is solvent, after pre-equilibration point sample
Lamellae 15 minutes is unfolded, and takes out, dries, spray with ninhydrin solution, it is clear to be heated to spot development at 110 DEG C.Test sample color
In spectrum, on position corresponding with control medicinal material chromatography, the spot of same color is shown.By pharmacopoeial requirements, this discriminating is carried out
Methodological study, by methodology validation, the thin-layer identification method clear spot, separator well reappears, and negative noiseless.
Methodology validation is as follows：
2.4.1 reagent and reagent
Youngster's spleen wakes up particle by the offer of Guizhou Run Sheng pharmacy industries Co., Ltd；The membrane of a chicken's gizzard control medicinal material (1153-200001) by
Nat'l Pharmaceutical ＆ Biological Products Control Institute provides；Silica gel g thin-layer plate：Subsidiary factory of Haiyang Chemical Plant, Qingdao production silica gel g thin-layer plate and
Make silica gel g thin-layer plate by oneself；Reagent：Methanol, ethyl alcohol, n-butanol are that analysis is pure, and water is super purified water；
2.4.2 specificity
Youngster's spleen is taken to wake up particle, the membrane of a chicken's gizzard control medicinal material and scarce the membrane of a chicken's gizzard negative sample, is made with text the method for examination
Product solution, control medicinal material solution, reference substance solution and negative test solution, point sample, expansion, spray be with ninhydrin solution respectively,
It is clear that spot development is heated at 110 DEG C, in test sample chromatography, same color is shown on position corresponding with control medicinal material chromatography
Principal spot, and spot separation is preferable, negative noiseless, sees Fig. 5.
2nd, assay
1 content of hesperidin measures
Dried orange peel：Dried orange peel assay is that primary standard is recorded, and has no revision herein, as requested, primary standard is recorded old
Skin assay has carried out methodology validation, and methodology validation is as follows：
1.1 instruments and reagent
High performance liquid chromatograph：Shimadzu liquid chromatograph；Youngster's spleen is waken up particle：Guizhou Runsheng Pharmaceutical Co., Ltd.；Aurantiamarin
Reference substance is provided by Nat'l Pharmaceutical ＆ Biological Products Control Institute, lot number 110721-201014；Reagent：Methanol is chromatography alcohol, water is
Double distilled water.
1.2 assay method
1.2.1 chromatography condition：
(1) chromatographic column：With reference to primary standard and version pharmacopeia dried orange peel assay in 2010, octadecylsilane bonded silica is selected
Glue is investigated for the pillar of filler.The result shows that octadecylsilane chemically bonded silica is used to separate effect for the pillar of filler
Fruit is preferable.
(2) mobile phase：With reference to primary standard, methanol-glacial acetic acid-water (35: 4: 61) is used as mobile phase, ingredient orange peel to be measured
Glycosides and other impurities can obtain preferable separation.
(3) Detection wavelength：It is measured with reference to primary standard and version pharmacopeia content of hesperidin in 2010, Detection wavelength is set to 283nm.
(4) theoretical cam curve：According to different pillar testing results, referring concurrently to version pharmacopeia dried orange peel assay in 2010,
Tentative theoretical cam curve is calculated by aurantiamarin peak, should be not less than 3000.
1.2.2 the preparation of test solution
It gets it filled composition granule, mixing is finely ground, takes about 0.4g, accurately weighed, puts in conical flask with cover, and precision adds in methanol
25ml, close plug, weighed weight is ultrasonically treated (power 250W, frequency 35kHz) 30 minutes, lets cool, then weighed weight, uses methanol
Supply less loss weight, shake up, filter, take subsequent filtrate to get.
1.2.3, the preparation of reference substance solution
Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol be made solution of every 1ml containing 0.01mg to get.
1.3 method validation
1.3.1 it is linear to investigate
Accurate aurantiamarin reference substance solution (0.031906mg/ml) 1ml, 2ml, 3ml, 5ml, 6ml, the 10ml that draw is arrived respectively
In 10ml volumetric flasks, methanol is added to divide to scale and take 10 μ l sample introductions, chromatography is recorded, using peak area as ordinate, with sample size (μ g)
It maps for abscissa, calculating regression equation is：A=1847322.47C+3897.83801 (r=0.9999), regression equation fitting
The linear equation for crossing origin is：A=1866179.663C；Seeing Fig. 6 and table 1., sample introduction is analyzed by a test solution, gained peak face
Integration does not substitute into the calculating of upper two formula, relative deviation 0.32%, therefore it is believed that standard curve crosses origin, regression equation intercept is
Zero.Thus one point external standard method calculating can be used in assay.Aurantiamarin linear relationship between the μ g of 0.032 μ g~0.319 is good.
1 aurantiamarin linear relationship of table is investigated
1.3.2, precision test
Reference substance solution 0.011867mg/ml is taken, repeats sample introduction 6 times, chromatography is recorded, the results are shown in Table 2 and precision collection of illustrative plates,
RSD is 0.41%, illustrates there is good precision.
2 precision test of table
1.3.3, repetitive test
A sample is taken to prepare 6 parts of test solutions as described in text, respectively sample introduction, records chromatography, calculated, the results are shown in Table 3,
Repeated collection of illustrative plates, RSD 0.58% illustrate there is good repeatability.
3 repetitive test of table
1.3.4, recovery test
It is recycled using sample-adding, precision draws the sample 0.2g (0.5789mg/g) for having measured content, adds aurantiamarin pair
Appropriate according to product, chromatographic condition measures as described in text, calculates the rate of recovery, the results are shown in Table 4, rate of recovery collection of illustrative plates, the recycling of aurantiamarin
Rate is 97.81%, RSD 0.75%, illustrates that this law has the good rate of recovery.
4 recovery test of table
1.3.5, specificity
Take that youngster's spleen wakes up particle and negative sample prepares sample solution and negative sample solution by test solution preparation method,
By above-mentioned chromatographic condition, reference substance solution, sample solution, negative sample solution injection liquid chromatograph, sample solution are taken respectively
Chromatography is equipped with corresponding chromatographic peak in reference substance solution chromatography corresponding positions, and negative noiseless, and the result is shown in Fig. 8, Fig. 9 and Figure 10.
1.3.6, durability
1.3.6.1 the selection of sample extraction solvent
It gets it filled composition granule, it is finely ground, take 5 parts, every part of about 0.4g, it is accurately weighed, it puts in conical flask with cover, it is accurate respectively to add in
Ethyl alcohol, methanol, 60% methanol, 70% methanol, each 25ml of 80% methanol, weighed weight are ultrasonically treated 30 minutes, let cool, then claim
Determine weight, supply the weight of less loss with ethyl alcohol, methanol, 60% methanol, 70% methanol, 80% methanol respectively, shake up, filter, take
Subsequent filtrate is as test solution.Each 10 μ l injections liquid chromatograph of above-mentioned solution is taken respectively, is calculated, be the results are shown in Table 5, illustrates this
Product using methanol as Extraction solvent, extract more complete by content.
The selection of 5 Extraction solvent of table
1.3.6.2, the selection of sample extraction method
Get it filled composition granule, it is finely ground, take 2 parts, every part of about 0.4g, accurately weighed, the 1st part is put in conical flask with cover, precision plus
Enter methanol 25ml, be ultrasonically treated 30 minutes, another refluxing extraction 30 minutes, take out, let cool, less loss weight is supplied with methanol,
It shakes up, filters；Above-mentioned 2 kinds of solution, 10 μ l injections liquid chromatograph is taken respectively, is calculated, the results are shown in Table 6, and refluxing extraction content surpasses
Sound extracts content without essential difference, therefore selects ultrasonic extraction.
The selection of 6 extracting method of table
1.3.6.3, sample extraction time effects
The content drug granule under content uniformity item is taken, it is finely ground, take 3 parts, every part of about 0.4g, it is accurately weighed, it puts respectively
In conical flask with cover, precision add in methanol 25ml, weighed weight, respectively ultrasonic extraction 10,15,30,40 minutes, take out, let cool,
Less loss weight is supplied with methanol, is shaken up, is filtered, 10 μ l subsequent filtrates is taken to inject liquid chromatograph respectively, content is calculated, the results are shown in Table
7, ultrasonic extraction just extraction completely in 30 minutes, therefore the selective extraction time is 30 minutes.
7 extraction time of table influences
Extracting method is determined as:It gets it filled composition granule, mixing is finely ground, takes about 0.4g, accurately weighed, puts in conical flask with cover,
Precision adds in methanol 25ml, close plug, and weighed weight is ultrasonically treated (power 250W, frequency 35kHz) 30 minutes, lets cool, then weighed
Weight is supplied less loss weight with methanol, is shaken up, filtration, take subsequent filtrate to get.
1.3.6.4, the influence of different brands pillar
Pillar of the different brands octadecylsilane chemically bonded silica for filler is used, youngster's spleen is determined respectively and wakes up in particle
The content of aurantiamarin investigates the influence of the chromatographic column of different brands.Assay the results are shown in Table 8.The result shows that different brands ten
Eight alkyl silane bonded silica gels for filler chromatographic column on assay without influence.
The measurement result of 8 different brands chromatographic column of table
1.3.6.5, sample stability experiment
Youngster's spleen is taken to wake up particle, is prepared by test solution preparation method, aurantiamarin peak area is measured by above-mentioned chromatographic condition,
It the results are shown in Table 9.
9 sample stability of table is tested
1.3.7 ten batches of content samples measure：As a result table 10；
10 10 batches of sample assay results of table
According to primary standard content limit and 10 batches of sample measurements, this product dried orange peel assay limit is：Every bag of this product contains
Dried orange peel is with aurantiamarin (C28H34O15) meter, 0.62mg must not be less than.
2nd, hawthorn assay
2.1st, instrument and reagent
Liquid chromatograph：Waters e2695 liquid chromatographs；Youngster's spleen is waken up particle：Guizhou Runsheng Pharmaceutical Co., Ltd.；Chinese holly
Rafter acid reference substance (100396-200301) is provided by Nat'l Pharmaceutical ＆ Biological Products Control Institute；Reagent：Ammonium dihydrogen phosphate, phosphoric acid are
Analysis level, water are super purified water.
2.2nd, assay method
2.2.1, chromatography condition
(1) chromatographic column：Octadecylsilane chemically bonded silica is selected to be investigated for the pillar of filler.The result shows that using
Octadecylsilane chemically bonded silica is preferable for the pillar separating effect of filler.
(2) mobile phase：Different mobile phases are compared, using 5% ammonium dihydrogen phosphate (with phosphoric acid slow-readjustment section pH extremely
3.0) it is mobile phase, ingredient citric acid to be measured and other impurities can obtain preferable separation.
(3) Detection wavelength：According to the UV absorption wavelength of citric acid reference substance, Detection wavelength is set to 210nm.
(4) theoretical cam curve：According to different pillar testing results, fix tentatively theoretical cam curve and calculated by citric acid, it should not be low
In 3000.
2.2.2, the preparation of test solution
It gets it filled composition granule, finely ground, precision weighs about 2.0g, puts in 25ml measuring bottles, and precision adds in appropriate amount of water, is ultrasonically treated
(power 250W, frequency 35kHz) 20 minutes, lets cool, is diluted with water to scale, shake up, filtration, takes subsequent filtrate, and 0.45 μm excessively micro-
Hole filter membrane to get.
2.2.3, the preparation of reference substance solution
Take citric acid reference substance appropriate, it is accurately weighed, add water be made solution of every 1ml containing 0.5mg to get.
2.3rd, method validation
2.3.1, linearly investigate precision and weigh citric acid reference substance 0.2010g and obtain A liquid (2.01mg/ml) to 10ml measuring bottles；
1mlA liquid to 2ml measuring bottles is taken to obtain B liquid (1.005mg/ml)；2mlA liquid to 5ml measuring bottles is taken to obtain C liquid (0.804mg/ml)；It takes
1.5mlA liquid to 5ml measuring bottles obtain D liquid (0.603mg/ml)；1ml B liquid to 2ml measuring bottles is taken to obtain E liquid (0.402mg/ml)；Take 1ml
E liquid to 2ml measuring bottles obtain F liquid (0.201mg/ml)；1ml F liquid to 2ml measuring bottles is taken to obtain G liquid (0.1005mg/ml), B, C, D, E, F,
G points take 20 μ l sample introductions.Chromatography is recorded, using peak area as ordinate, is mapped with sample size (μ g) for abscissa, calculates regression equation
For：A=146399.4943C-65747.9151 (r=0.9998), the linear equation that regression equation was fitted origin are：A=
141811.156C；By a test solution, sample introduction is analyzed, and gained peak area substitutes into the calculating of two formulas respectively, and relative deviation is
3.27%, therefore it is believed that standard curve crosses origin, regression equation no intercept.Thus one point external standard method meter can be used in assay
It calculates.Citric acid linear relationship between the μ g of 2.02 μ g~20.1 is good.Concrete outcome is shown in Table 11 and Fig. 7.
11 citric acid linear relationship of table investigates (n=2)
2.3.2, precision test
Same citric acid reference substance solution is taken, repeats sample introduction 6 times, chromatography is recorded, the results are shown in Table 12 and precision collection of illustrative plates,
RSD is 0.19%, illustrates there is good precision.
12 precision test of table
2.3.3 repetitive test
A sample is taken to prepare 6 parts of test solutions as described in text, respectively sample introduction, record chromatography, calculated, the results are shown in Table
13, RSD 1.01%, illustrate there is good repeatability.
13 repetitive test of table
2.3.4 recovery test
It is recycled using sample-adding, precision draws the sample 1g (4.007mg/g) for having measured content, adds citric acid reference substance
In right amount, chromatographic condition measures as described in text, calculates the rate of recovery, the results are shown in Table 14, rate of recovery collection of illustrative plates, and the rate of recovery of citric acid is
102.93%, RSD 0.92% illustrates that this law has the good rate of recovery.
14 recovery test of table
2.3.5, specificity
Take that youngster's spleen wakes up particle and negative sample prepares sample solution and negative sample solution by test solution preparation method,
By above-mentioned chromatographic condition, reference substance solution, sample solution, negative sample solution injection liquid chromatograph, sample solution are taken respectively
Chromatography is equipped with corresponding chromatographic peak, and negative noiseless, the result is shown in Figure 11, Figure 12 and figure in reference substance solution chromatography corresponding positions
13。
2.3.6, durability,
2.3.6.1, the selection of sample extraction solvent
It gets it filled composition granule, it is finely ground, take 4 parts, every part of about 2g, it is accurately weighed, it puts in 25ml measuring bottles, is soluble according to organic acid
Water, methanol, the property of ethyl alcohol, therefore accurate addition suitable quantity of water, 1% sodium bicarbonate water, methanol, ethyl alcohol respectively, weighed weight, ultrasound
Handle 1 it is small when, let cool, be diluted to scale with water, 1% sodium bicarbonate water, methanol, ethyl alcohol respectively, shake up, filter, take subsequent filtrate,
0.45 μm of miillpore filter is crossed, as test solution.Take each 20 μ l of above-mentioned solution injection liquid chromatographs respectively, by methanol,
Chromatogram separating effect that ethyl alcohol extracts is poor can not to calculate content, therefore water and 1% sodium bicarbonate water are compared, and count
It calculates, the results are shown in Table 15, this product using water as Extraction solvent, extract more complete by content.
The selection of 15 Extraction solvent of table
2.3.6.2, the selection of sample extraction method
It gets it filled composition granule, it is finely ground, take 3 parts, every part of about 2g, it is accurately weighed, it is accurate respectively to add in 25ml water, respectively by returning
Stream, ultrasound, soak extraction, weighed weight when reflux 1 is small, when ultrasound 1 is small, when immersion 1 is small, lets cool, supply less loss with water respectively
Weight, filtration take subsequent filtrate, 0.45 μm of miillpore filter are crossed, as test solution.Each 20 μ l injections liquid of above-mentioned solution is taken respectively
Chromatography calculates, and the results are shown in Table 16, the content that three kinds of reflux, ultrasound, immersion extracting methods are surveyed is not much different, therefore selects
Easy, the preferable ultrasonic extraction of effect is as extracting method.
The selection of 16 extracting method of table
2.3.6.3, sample extraction time effects
It gets it filled composition granule, it is finely ground, take 4 parts, every part of about 2.0g, it is accurately weighed, it puts respectively in 25ml measuring bottles, precision adds in water
In right amount, it is 10,20,40,60 minutes ultrasonic respectively, it lets cool, is diluted with water to scale, shake up, filter, take subsequent filtrate, cross 0.45 μm
Miillpore filter, as test solution.Each 20 μ l injections liquid chromatograph of above-mentioned solution is taken respectively, is calculated, be the results are shown in Table 17,
10th, the content measured by 20,40,60 minutes is not much different, and in order to ensure that extraction is complete, therefore the selective extraction time is 20 minutes.
17 extraction time of table influences
Extracting method is determined as:It gets it filled composition granule, finely ground, precision weighs about 2.0g, puts in 25ml measuring bottles, and precision adds in water
In right amount, it is ultrasonically treated (power 250W, frequency 30kHz) 20 minutes, lets cool, be diluted with water to scale, shake up, filter, take continuous filter
Liquid, cross 0.45 μm of miillpore filter to get.
2.3.6.4, the influence of different brands pillar
Pillar of the different brands octadecylsilane chemically bonded silica for filler is used, youngster's spleen is determined respectively and wakes up in particle
The content of citric acid investigates the influence of the chromatographic column of different brands.Assay the results are shown in Table 18.The result shows that different brands ten
Eight alkyl silane bonded silica gels for filler chromatographic column on assay without influence.
The measurement result of 18 different brands chromatographic column of table
2.3.6.5, sample stability experiment
Youngster's spleen is taken to wake up particle, is prepared by test solution preparation method, citric acid peak area is measured by above-mentioned chromatographic condition,
It the results are shown in Table 19.
19 sample stability of table is tested
2.4.10 batch content sample measures：
It the results are shown in Table 20.
20 10 batches of sample assay results of table
According to 10 batches of sample measurements, this product hawthorn assay limit is：Every bag of this product is containing hawthorn with citric acid
(C6H8O7) meter, 6.0mg must not be less than.
The beneficial effects of the present invention are：It wakes up the present invention provides a kind of youngster's spleen the detection method of particle, including with effective
Ingredient aurantiamarin and citric acid are index, using the content of high effective liquid chromatography for measuring aurantiamarin and citric acid；And drug
In to the thin-layer identification method of hawthorn, dried orange peel, Chinese yam and the membrane of a chicken's gizzard；The detection method is accurate, and high sensitivity is reproducible,
Testing result is stablized, and youngster's spleen can effectively be controlled to wake up the quality of particle, be both more advantageous to manufacturer and supervisory and management department to production
The monitoring of quality, or the treatment of medical department and patient, which provide, preferably to be ensured.
Description of the drawings
When Fig. 1 is that hawthorn thin-layer differentiates, when specificity is tested, figure is inspected under daylight；1-20151001,2-20151002,
3-20151003,4- negative sample, 5- hawthorn control medicinal materials, 6- ursolic acid reference substances；
When Fig. 2 is that hawthorn thin-layer differentiates, when specificity is tested, figure is inspected under ultraviolet lamp (365nm)；1-20151001,
2-20151002,3-20151003,4- negative sample, 5- hawthorn control medicinal materials, 6- ursolic acid reference substances；
When Fig. 3 is that hawthorn thin-layer differentiates, when specificity is tested, figure is inspected under daylight；1-20151001,2-20151002,
3-20151003,4- negative sample, 5- dried orange peel control medicinal materials, 6- aurantiamarin reference substances；
When Fig. 4 is that Chinese yam thin layer differentiates；When specificity is tested, result figure, 1-20151001,2- are inspected under daylight
20151002,3-20151003,4- Chinese yam control medicinal material, 5- negative samples；
When Fig. 5 is that the membrane of a chicken's gizzard thin layer differentiates, when specificity is tested, result figure is inspected under daylight；1-20151001,2-
20151002,3-20151003,4- the membrane of a chicken's gizzard control medicinal material, 5- negative samples；
Fig. 6 is aurantiamarin canonical plotting, and X-axis is sample size (μ g), Y-axis is peak area；
Fig. 7 is citric acid canonical plotting, and X-axis is sample size (μ g), Y-axis is peak area；
When Fig. 8 is that content of hesperidin measures, aurantiamarin reference substance specificity lab diagram；
When Fig. 9 is that content of hesperidin measures, aurantiamarin test sample specificity lab diagram；
When Figure 10 content of hesperidin measures, aurantiamarin negative sample specificity lab diagram；
When Figure 11 is citric acid assay, citric acid reference substance specificity lab diagram；
When Figure 12 is citric acid assay, citric acid test sample specificity lab diagram；
When Figure 13 is citric acid assay, citric acid negative sample specificity lab diagram.
With reference to embodiment, the present invention is further illustrated
Specific embodiment
Embodiment：The detection method of granule
Granular formulations：Hawthorn 320g, malt 320g, the membrane of a chicken's gizzard 240g, Chinese yam 240g, coix seed 160g, Semen Lablab Album
240g, dried orange peel 96g and Poria cocos 96g.
Technique：More than eight tastes, hawthorn, dried orange peel be ground into coarse powder, with 65% ethanol as solvent, when dipping 24 is small after, carry out
Diacolation collects liquid of filtering, and diacolation liquid recycling ethanol, relative density is the thick paste of 1.35-1.40 when being concentrated into 55-65 DEG C, spare；Remaining
The Six-elements medicinal material such as malt adds water to cook three times, every time 1 it is small when, collecting decoction, filtration, filtrate is concentrated into relative density as 1.10
The clear cream of (60 DEG C), adds equivalent ethyl alcohol to make precipitation, takes supernatant, recycles ethyl alcohol, and it is the thick of 1.35-1.40 to be concentrated into relative density
Cream；With above-mentioned hawthorn, dried orange peel thick paste mixing, it is appropriate to add in sucrose, is made particle, it is dry to get.
Usage and dosage：Warm boiled water.1~5 year old once a bag, 3 times a day；5 years old or more once 2 bags, 3 times a day；10
Days as a treatment course.
Specification：Per packed 2.5g.
Detection method：
Character：Drug is the particle of brown color；Gas micro-perfume, sweet and sour, slight bitter.
Differentiate：
(1) get it filled composition granule content, after finely ground, 8g taken to add 25ml ether, be heated to reflux 1 it is small when, filtration, filtrate is waved
Dry, residue adds methanol 1m1 to make dissolving, as test solution；Hawthorn control medicinal material 0.2g separately is taken, after finely ground, 8g is taken to add 25ml
Ether, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds methanol 1m1 to make dissolving, and control medicinal material solution is made；Bear is taken again
Tartaric acid reference substance is appropriate, adds methanol that solution of every 1ml containing 0.1mg is made, as reference substance solution；According to Chinese Pharmacopoeia thin layer color
Spectrometry is tested, and draws above-mentioned three a variety of each 5~10 μ l of solution, is put respectively on same silica gel g thin-layer plate, with toluene：Acetic acid second
Ester：Formic acid=20: be solvent at 4: 0.5 are unfolded, and are taken out, are dried, spray with ethanol solution of sulfuric acid (3 → 10), in 105 DEG C of heating
It is clear to spot development, put inspected under daylight or 365nm ultraviolet lamps respectively, in test sample chromatography, with control medicinal material chromatography
On corresponding position, the principal spot of same color or fluorescence principal spot are shown；On position corresponding with reference substance chromatography, show identical
The spot or fluorescence spot of color.
(2) get it filled composition granule content, after finely ground, take 10g, add methanol 30ml, be heated to reflux 1 it is small when, filtration, filtrate is waved
Dry, residue adds water 10ml to make dissolving, is transferred in separatory funnel, and with ethyl acetate shaking extraction 2 times, each each 10ml merges
Acetic acid ethyl acetate extract is evaporated in water-bath, and residue adds methanol 2ml to make dissolving, as test solution；Separately take dried orange peel control medicinal material
1g is made in the same way of control medicinal material solution；It takes aurantiamarin reference substance appropriate again, adds methanol that saturated solution is made, it is molten as reference substance
Liquid；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned each 2~5 μ l of three kinds of solution, put respectively in same with 0.5% hydrogen
On silica gel g thin-layer plate prepared by sodium hydroxide solution, with ethyl acetate：Methanol：Water=100: be solvent at 17: 13, Zhan Zhiyue
3cm takes out, dries, then with toluene：Ethyl acetate：Formic acid：Water=20: 10: 1: 1 upper solution is solvent, is opened up to 8cm,
It takes out, dries, spray with 5% alchlor ethanol solution, be placed in wavelength to be inspected under 365nm ultraviolet lamps；In test sample chromatography,
On position corresponding with control medicinal material chromatography, the fluorescence principal spot of same color is shown；In position corresponding with reference substance chromatography
On, the fluorescence spot of aobvious same color.
(3) get it filled composition granule content, it is finely ground, 5g is taken, adds water 30ml, steeped overnight, aqueous solution is moved together with residue
It puts in separatory funnel, adds water saturated n-butanol shaking extraction 3 times, each 20ml, divide and take n-butanol liquid, be evaporated, residue adds suitable
Amount methanol makes dissolving, is added on the neutral alumina column of 100-120 mesh, 5g, internal diameter 10-15mm, is washed with 10% methanol 100ml
It is de-, eluent is collected, is evaporated, residue adds 1ml70% ethyl alcohol to make dissolving, as test solution；Separately take the membrane of a chicken's gizzard control medicinal material
1g adds water 30ml, and when decoction 2 is small, aqueous solution is made in the same way of control medicinal material solution together in residue dislocation separatory funnel；
It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws each 3~5 μ l of above two solution, put respectively in same silica gel g thin-layer plate
On, with n-butanol：Glacial acetic acid：Water=9: be solvent at 3: 1, the lamellae after pre-equilibration point sample 15 minutes are unfolded, and take out, dry in the air
It is dry, it sprays with ninhydrin solution, it is clear to be heated to spot development at 110 DEG C；In test sample chromatography, corresponding to control medicinal material chromatography
Position on, show same color principal spot.
Should meet (《Chinese Pharmacopoeia》The four annex I C of version in 2015) every regulation related under granule item.
Assay：
(1) aurantiamarin：
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With methanol：Ice vinegar
Acid：Water=35: be mobile phase at 4: 61；Column temperature is 35 DEG C；Detection wavelength is 283nm；Number of theoretical plate should not by the calculating of aurantiamarin peak
Less than 3000；
The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made containing 0.01mg
Solution to get；
The preparation of test solution：It gets it filled composition granule content, mixing is finely ground, takes 0.4g, accurately weighed, puts tool plug cone
In shape bottle, precision adds in methanol 25ml, close plug, and weighed weight is ultrasonically treated, sonification power 250W, is ultrasonically treated frequency
Rate is 35kHz, and sonication treatment time is 30 minutes, is let cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, and is filtered
Cross, take subsequent filtrate to get；
Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product.
Every bag of this product is containing dried orange peel with aurantiamarin (C28H34O15) meter, 0.62mg must not be less than.
(2) citric acid：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：
Chromatographic condition and system suitability：It is filler with octadecylsilane chemically bonded silica；With 5% biphosphate
Ammonium salt solution (using phosphoric acid slow-readjustment section pH to 3.0) is mobile phase, and column temperature is 25 DEG C；Detection wavelength is 210nm；Number of theoretical plate presses citron
Sour peak, which calculates, should be not less than 3000；
The preparation of reference substance solution：Take citric acid reference substance appropriate, it is accurately weighed, add water that every 1ml is made containing the molten of 0.3mg
Liquid to get；
The preparation of test solution：It gets it filled composition granule content, finely ground, precision weighs 2.0g, puts in 25ml measuring bottles, accurate
Appropriate amount of water is added in, is ultrasonically treated, sonification power 250W, supersound process frequency is 35kHz, and sonication treatment time is 20 points
Clock is let cool, and is diluted with water to scale and is shaken up, and filtration takes subsequent filtrate, cross 0.45 μm of miillpore filter to get.
Measuring method：It is accurate respectively to draw reference substance solution and each 20 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product.
Every bag of this product is containing hawthorn with citric acid (C6H8O7) meter, 6.0mg must not be less than.
Claims (7)
- The detection method of particle 1. a kind of youngster's spleen is waken up, the particle by calculate by weight 300-340 parts of hawthorn, malt 300-340 parts, 220-260 parts of the membrane of a chicken's gizzard, 220-260 parts of Chinese yam, 140-180 parts of coix seed, 220-260 parts of Semen Lablab Album, dried orange peel 86-106 parts are made as follows with 86-106 parts of Poria cocos：More than eight tastes, hawthorn, dried orange peel be ground into coarse powder, is made with 65% ethyl alcohol Solvent, when dipping 24 is small after, carry out diacolation, collection is filtered liquid, and diacolation liquid recycling ethanol, relative density is when being concentrated into 55-65 DEG C 1.35-1.40 thick paste, it is spare；The Six-elements medicinal material such as remaining malt adds water to cook three times, every time 1 it is small when, collecting decoction, filtration, Relative density is 1.10 clear cream when filtrate is concentrated into 55-65 DEG C, and equivalent ethyl alcohol is added to make precipitation, takes supernatant, recycles ethyl alcohol, dense It is reduced to the thick paste that relative density is 1.35-1.40；With above-mentioned hawthorn, dried orange peel thick paste mixing, addition sucrose is appropriate, and particle is made, It is dry to get；It is characterized in that：The detection method includes discrimination method and content assaying method；The discrimination method includes Detection is differentiated to the thin layer of hawthorn, dried orange peel and the membrane of a chicken's gizzard；The content assaying method includes the content to aurantiamarin and citric acid It measures；The discrimination method of the hawthorn is：It gets it filled composition granule, after finely ground, 4-12g is taken to add 20-30ml ether, is heated to reflux When 0.5-1.5 is small, filtration, filtrate volatilizes, and residue adds methanol 0.5-1.5m1 to make dissolving, as test solution；Separately take hawthorn pair According to medicinal material 0.1-0.3g, after finely ground, 4-12g is taken to add 20-30ml ether, be heated to reflux 0.5-1.5 it is small when, filtration, filtrate volatilizes, Residue adds methanol 0.5-1.5m1 to make dissolving, and control medicinal material solution is made；It takes ursolic acid reference substance appropriate again, methanol is added to be made often Solution of the 0.5-1.5ml containing 0.05-0.15mg, as reference substance solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, in absorption Each 5-10 μ l of three kinds of solution are stated, are put respectively on same silica gel g thin-layer plate, with toluene：Ethyl acetate：Formic acid=15-25: 2-6: 0.1-1.0 is solvent, is unfolded, and takes out, dries, and is sprayed with ethanol solution of sulfuric acid, the body of sulfuric acid and ethyl alcohol in ethanol solution of sulfuric acid Product is than being 2-4:9-11；It is clear that spot development is heated at 105 DEG C, puts inspected under daylight or 360-370nm ultraviolet lamps respectively, In test sample chromatography, on position corresponding with control medicinal material chromatography, the principal spot of same color or fluorescence principal spot are shown；With On the corresponding position of reference substance chromatography, the spot or fluorescence spot of same color are shown；The discrimination method of the dried orange peel is：It gets it filled object Particle after finely ground, takes 5-15g, adds methanol 25-35ml, be heated to reflux 0.5-1.5 it is small when, filtration, filtrate volatilizes, and residue adds water 5-15ml makes dissolving, is transferred in separatory funnel, and with ethyl acetate shaking extraction 2-3 times, each each 5-15ml merges acetic acid second Ester extracting solution is evaporated in water-bath, and residue adds methanol 1-4ml to make dissolving, as test solution；Separately take dried orange peel control medicinal material 0.5- 1.5g is made in the same way of control medicinal material solution；It takes aurantiamarin reference substance appropriate again, adds methanol that saturated solution is made, as reference substance Solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned each 2-5 μ l of three kinds of solution, put use 0.1- in same respectively On silica gel g thin-layer plate prepared by 1.0% sodium hydroxide solution, with ethyl acetate：Methanol：Water=95-105: 15-19: 10-16 is exhibition Agent is opened, is opened up to 1-6cm, is taken out, dry, then with toluene：Ethyl acetate：Formic acid：Water=15-25: 5-15: 0.5-1.5: 0.5-1.5 Upper solution for solvent, open up to 5-10cm, take out, dry, spray with 1-10% alchlor ethanol solutions, being placed in wavelength is It is inspected under 360-370nm ultraviolet lamps；In test sample chromatography, on position corresponding with control medicinal material chromatography, same color is shown Fluorescence principal spot；On position corresponding with reference substance chromatography, the fluorescence spot of same color is shown；The discriminating of the membrane of a chicken's gizzard Method is：It gets it filled composition granule, it is finely ground, 1-10g is taken, adds water 25-35ml, steeped overnight, aqueous solution is together with residue dislocation point In liquid funnel, add water saturated n-butanol shaking extraction 2-4 times, each 15-25ml, divide and take n-butanol liquid, be evaporated, residue adds suitable Amount methanol makes dissolving, is added on the neutral alumina column of 100-120 mesh, 1-10g, internal diameter 10-15mm, with 5-15% methanol 90- 110ml is eluted, and is collected eluent, is evaporated, residue adds 0.5-1.5ml 65-75% ethyl alcohol to make dissolving, as test solution；Separately The membrane of a chicken's gizzard control medicinal material 0.5-1.5g is taken, adds water 25-35ml, when decoction 1-3 is small, aqueous solution is together with residue dislocation liquid separation In funnel, control medicinal material solution is made in the same way of；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above two solution each 3~5 μ l are put respectively on same silica gel g thin-layer plate, with n-butanol：Glacial acetic acid：Water=5-15: 1-6: 0.5-1.5 is solvent, pre- flat Lamellae after weighing apparatus point sample 10-20 minutes, is unfolded, and takes out, dries, spray with ninhydrin solution, spot development is heated at 110 DEG C Clearly；In test sample chromatography, on position corresponding with control medicinal material chromatography, the principal spot of same color is shown.
- The detection method of particle 2. youngster's spleen as described in claim 1 is waken up, it is characterised in that：The discrimination method of the hawthorn is： Get it filled composition granule, after finely ground, 8g taken to add 25ml ether, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds methanol 1m1 to make Dissolving, as test solution；Separately take hawthorn control medicinal material 0.2g, after finely ground, 8g is taken to add 25ml ether, be heated to reflux 1 it is small when, Filtration, filtrate volatilize, and residue adds methanol 1m1 to make dissolving, and control medicinal material solution is made；It takes ursolic acid reference substance appropriate again, adds first Solution of every 1ml containing 0.1mg is made in alcohol, as reference substance solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned three Kind of each 5~10 μ l of solution are put respectively on same silica gel g thin-layer plate, with toluene：Ethyl acetate：Formic acid=20: be expansion at 4: 0.5 Agent is unfolded, and takes out, dries, and sprays with ethanol solution of sulfuric acid, and the volume ratio of sulfuric acid and ethyl alcohol is 3 in ethanol solution of sulfuric acid:10； 105 DEG C to be heated to spot development clear, puts inspected under daylight or 365nm ultraviolet lamps respectively, in test sample chromatography, with compareing On the corresponding position of medicinal material chromatography, the principal spot of same color or fluorescence principal spot are shown；In position corresponding with reference substance chromatography On, the spot or fluorescence spot of aobvious same color.
- The detection method of particle 3. youngster's spleen as described in claim 1 is waken up, it is characterised in that：The discrimination method of the membrane of a chicken's gizzard For：Get it filled composition granule, it is finely ground, take 5g, add water 30ml, steeped overnight, aqueous solution together in residue dislocation separatory funnel, Adding water saturated n-butanol shaking extraction 3 times, each 20ml, divide and take n-butanol liquid, be evaporated, residue adds proper amount of methanol to make dissolving, It is added on the neutral alumina column of 100-120 mesh, 5g, internal diameter 10-15mm, is eluted with 10% methanol 100ml, collect eluent, It is evaporated, residue adds 1ml70% ethyl alcohol to make dissolving, as test solution；The membrane of a chicken's gizzard control medicinal material 1g separately is taken, adds water 30ml, is decocted Boil 2 it is small when, aqueous solution is made in the same way of control medicinal material solution together in residue dislocation separatory funnel；It is thin according to Chinese Pharmacopoeia Layer chromatography is tested, and draws each 3~5 μ l of above two solution, is put respectively on same silica gel g thin-layer plate, with n-butanol：Ice vinegar Acid：Water=9: be solvent at 3: 1, the lamellae after pre-equilibration point sample 15 minutes are unfolded, and take out, dry, spray with ninhydrin solution, It is clear that spot development is heated at 110 DEG C；In test sample chromatography, on position corresponding with control medicinal material chromatography, identical face is shown The principal spot of color.
- The detection method of particle 4. youngster's spleen as described in claim 1 is waken up, it is characterised in that：The content of hesperidin assay method For：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With methanol：Glacial acetic acid：Water= 30-40: 1-8: 55-65 is mobile phase；Column temperature is 33-37 DEG C；Detection wavelength is 280-286nm；Number of theoretical plate presses aurantiamarin peak 3000 should be not less than by calculating；The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol that every 0.5-1.5ml is made and contain The solution of 0.005-0.015mg to get；The preparation of test solution：It gets it filled composition granule content, mixing is finely ground, takes 0.2-0.6g, accurately weighed, puts tool plug cone In shape bottle, precision adds in methanol 20-30ml, close plug, and weighed weight is ultrasonically treated, sonification power 200-300W, ultrasound Processing frequency is 30-40kHz, and sonication treatment time is 25-35 minutes, is let cool, then weighed weight, and the weight of less loss is supplied with methanol Amount, shake up, filter, take subsequent filtrate to get；Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
- The detection method of particle 5. youngster's spleen as claimed in claim 4 is waken up, it is characterised in that：The content of hesperidin assay method For：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With methanol：Glacial acetic acid：Water= Be mobile phase at 35: 4: 61；Column temperature is 35 DEG C；Detection wavelength is 283nm；Number of theoretical plate should be not less than by the calculating of aurantiamarin peak 3000；The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made containing the molten of 0.01mg Liquid to get；The preparation of test solution：It gets it filled composition granule content, mixing is finely ground, takes 0.4g, accurately weighed, puts conical flask with cover In, precision adds in methanol 25ml, close plug, and weighed weight is ultrasonically treated, sonification power 250W, is ultrasonically treated frequency and is 35kHz, sonication treatment time are 30 minutes, are let cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, and filter, take Subsequent filtrate to get；Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
- The detection method of particle 6. youngster's spleen as described in claim 1 is waken up, it is characterised in that：The citric acid content assaying method For：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：Chromatographic condition and system suitability：It is filler with octadecylsilane chemically bonded silica；Number of theoretical plate presses citric acid Peak, which calculates, should be not less than 3000；The preparation of reference substance solution：Take citric acid reference substance appropriate, it is accurately weighed, add water that every 0.5-1.5ml is made containing 0.1- The solution of 0.6mg to get；The preparation of test solution：It gets it filled composition granule content, finely ground, precision weighs 1-4g, puts in 20-30ml measuring bottles, accurate Appropriate amount of water is added in, is ultrasonically treated, sonification power 200-300W, supersounds process frequency is 30-40kHz, during supersound process Between for 15-25 minute, let cool, be diluted with water to scale and shake up, filter, take subsequent filtrate, 0.35-0.55 μm of miillpore filter of mistake, i.e., ；Measuring method：It is accurate respectively to draw reference substance solution and each 15-25 μ l of test solution, liquid chromatograph is injected, is measured, i.e., .
- The detection method of particle 7. youngster's spleen as described in claim 1 is waken up, it is characterised in that：The discrimination method of the hawthorn is：After composition granule of getting it filled is finely ground, 8g is taken to add 25ml ether, be heated to reflux 1 it is small when, filtration, Filtrate volatilizes, and residue adds methanol 1m1 to make dissolving, as test solution；Hawthorn control medicinal material 0.2g separately is taken, after finely ground, takes 8g Add 25ml ether, be heated to reflux 1 it is small when, filtration, filtrate volatilizes, and residue adds methanol 1m1 to make dissolving, and control medicinal material solution is made； It takes ursolic acid reference substance appropriate again, adds methanol that solution of every 1ml containing 0.1mg is made, as reference substance solution；According to Chinese Pharmacopoeia Thin-layered chromatography is tested, and draws above-mentioned each 5~10 μ l of three kinds of solution, is put respectively on same silica gel g thin-layer plate, with toluene：Second Acetoacetic ester：Formic acid=20: be solvent at 4: 0.5 are unfolded, and are taken out, are dried, and are sprayed with ethanol solution of sulfuric acid, sulphur in ethanol solution of sulfuric acid The volume ratio of acid and ethyl alcohol is 3:10；It is clear that spot development is heated at 105 DEG C, is put respectively under daylight or 365nm ultraviolet lamps It inspects, in test sample chromatography, on position corresponding with control medicinal material chromatography, shows the principal spot of same color or the main spot of fluorescence Point；On position corresponding with reference substance chromatography, the spot or fluorescence spot of same color are shown；The discrimination method of the dried orange peel is：It gets it filled composition granule content, after finely ground, takes 10g, add methanol 30ml, be heated to reflux 1 Hour, filtration, filtrate volatilizes, and residue adds water 10ml to make dissolving, is transferred in separatory funnel, is extracted 2 times with ethyl acetate shaking, Each each 10ml, combined ethyl acetate extracting solution are evaporated in water-bath, and residue adds methanol 2ml to make dissolving, as test solution； Dried orange peel control medicinal material 1g separately is taken, is made in the same way of control medicinal material solution；It takes aurantiamarin reference substance appropriate again, adds methanol that saturation is made molten Liquid, as reference substance solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws above-mentioned each 2~5 μ l of three kinds of solution, respectively point In it is same with 0.5% sodium hydroxide solution prepare silica gel g thin-layer plate on, with ethyl acetate：Methanol：Water=100: be exhibition at 17: 13 Agent is opened, is opened up to about 3cm, is taken out, dry, then with toluene：Ethyl acetate：Formic acid：Water=20: 10: 1: 1 upper solution is expansion Agent opens up to 8cm, takes out, dry, spray with 5% alchlor ethanol solution, is placed in wavelength to be inspected under 365nm ultraviolet lamps；For In test product chromatography, on position corresponding with control medicinal material chromatography, the fluorescence principal spot of same color is shown；With reference substance chromatography On corresponding position, the fluorescence spot of same color is shown；The discrimination method of the membrane of a chicken's gizzard is：Get it filled composition granule, it is finely ground, take 5g, add water 30ml, steeped overnight, aqueous solution together with Residue in dislocation separatory funnel, adds water saturated n-butanol shaking extraction 3 times, each 20ml, divides and take n-butanol liquid, steam together Dry, residue adds proper amount of methanol to make dissolving, is added on the neutral alumina column of 100-120 mesh, 5g, internal diameter 10-15mm, with 10% first Alcohol 100ml is eluted, and is collected eluent, is evaporated, residue adds 1ml70% ethyl alcohol to make dissolving, as test solution；Separately take the membrane of a chicken's gizzard Control medicinal material 1g adds water 30ml, and when decoction 2 is small, aqueous solution is made in the same way of control together in residue dislocation separatory funnel Medicinal material solution；It is tested according to Chinese Pharmacopoeia thin-layered chromatography, draws each 3~5 μ l of above two solution, put respectively in same silica gel On G lamellaes, with n-butanol：Glacial acetic acid：Water=9: be solvent at 3: 1, the lamellae after pre-equilibration point sample 15 minutes, expansion take Go out, dry, spray with ninhydrin solution, it is clear to be heated to spot development at 110 DEG C；In test sample chromatography, with comparison medicine wood color It composes on corresponding position, shows the principal spot of same color；The content of hesperidin assay method is：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With methanol：Glacial acetic acid：Water= Be mobile phase at 35: 4: 61；Column temperature is 35 DEG C；Detection wavelength is 283nm；Number of theoretical plate should be not less than by the calculating of aurantiamarin peak 3000；The preparation of reference substance solution：Take aurantiamarin reference substance appropriate, it is accurately weighed, add methanol that every 1ml is made containing the molten of 0.01mg Liquid to get；The preparation of test solution：It gets it filled composition granule content, mixing is finely ground, takes 0.4g, accurately weighed, puts conical flask with cover In, precision adds in methanol 25ml, close plug, and weighed weight is ultrasonically treated, sonification power 250W, is ultrasonically treated frequency and is 35kHz, sonication treatment time are 30 minutes, are let cool, then weighed weight, and the weight of less loss is supplied with methanol, is shaken up, and filter, take Subsequent filtrate to get；Measuring method：It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get；The citric acid content assaying method is：According to Chinese Pharmacopoeia high effective liquid chromatography for measuring：Chromatographic condition and system suitability：It is filler with octadecylsilane chemically bonded silica；It is molten with 5% ammonium dihydrogen phosphate Liquid, is mobile phase with phosphoric acid slow-readjustment section pH to 3.0, and column temperature is 25 DEG C；Detection wavelength is 210nm；Number of theoretical plate presses citric acid peak 3000 should be not less than by calculating；The preparation of reference substance solution：Take citric acid reference substance appropriate, it is accurately weighed, add water that solution of every 1ml containing 0.3mg is made, To obtain the final product；The preparation of test solution：It gets it filled composition granule content, finely ground, precision weighs 2.0g, puts in 25ml measuring bottles, and precision adds in Appropriate amount of water is ultrasonically treated, sonification power 250W, and supersound process frequency is 35kHz, and sonication treatment time is 20 minutes, Let cool, be diluted with water to scale and shake up, filter, take subsequent filtrate, cross 0.45 μm of miillpore filter to get；Measuring method：It is accurate respectively to draw reference substance solution and each 20 μ l of test solution, inject liquid chromatograph, measure to get.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610887034.6A CN106248860B (en) | 2016-10-11 | 2016-10-11 | A kind of youngster's spleen is waken up the detection method of particle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610887034.6A CN106248860B (en) | 2016-10-11 | 2016-10-11 | A kind of youngster's spleen is waken up the detection method of particle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106248860A CN106248860A (en) | 2016-12-21 |
| CN106248860B true CN106248860B (en) | 2018-05-29 |
Family
ID=57611409
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610887034.6A Active CN106248860B (en) | 2016-10-11 | 2016-10-11 | A kind of youngster's spleen is waken up the detection method of particle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106248860B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110470783B (en) * | 2019-08-05 | 2021-07-16 | 中南大学湘雅二医院 | Quality detection method of Shuanghuang lipid-lowering granules |
| CN114137117A (en) * | 2021-11-29 | 2022-03-04 | 上海方予健康医药科技有限公司 | Method for determining content of citric acid or citrate in preparation |
| CN114522211A (en) * | 2021-12-29 | 2022-05-24 | 江苏七○七天然制药有限公司 | Preparation and detection method of infant spleen-tonifying and digestion-promoting granules |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101444604A (en) * | 2009-01-06 | 2009-06-03 | 贵州本草堂药业有限公司 | Chinese medicine granular formulation to therapy children anorexia and preparation method thereof and detection method |
| CN102114155A (en) * | 2010-01-06 | 2011-07-06 | 山东明仁福瑞达制药有限公司 | Serial Chinese medicinal preparation for treating phaseolus cold and preparation process and quality control method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101654989B1 (en) * | 2014-11-07 | 2016-09-07 | 건국대학교 산학협력단 | A method for identifying vinegar and a composition therefor |
-
2016
- 2016-10-11 CN CN201610887034.6A patent/CN106248860B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101444604A (en) * | 2009-01-06 | 2009-06-03 | 贵州本草堂药业有限公司 | Chinese medicine granular formulation to therapy children anorexia and preparation method thereof and detection method |
| CN102114155A (en) * | 2010-01-06 | 2011-07-06 | 山东明仁福瑞达制药有限公司 | Serial Chinese medicinal preparation for treating phaseolus cold and preparation process and quality control method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 运脾颗粒的质量标准研究;童鑫等;《中国中医药信息杂志》;20111031;第18卷(第10期);第2.1.4节 * |
| 高效液相色谱法测定山楂中枸橼酸的含量;杨滨等;《中国药学杂志》;20040930;第39卷(第9期);第2节，第3节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106248860A (en) | 2016-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105259295B (en) | Quality detection method for ginseng, cassia twig and poria cocos oral solution | |
| CN107478762B (en) | The discriminating of children's compound the membrane of a chicken's gizzard chewable tablets and content assaying method | |
| CN102590433B (en) | A kind of quality determining method of the smooth preparation of liver | |
| CN102854281B (en) | Detection method of sugar-free strong loquat syrup | |
| CN106248860B (en) | A kind of youngster's spleen is waken up the detection method of particle | |
| CN104306500B (en) | Method for detecting medicine for treating irritable bowel syndrome | |
| CN105301168B (en) | The detection method of dredging collateral resolving sputum capsule | |
| CN107315061B (en) | A kind of detection method of alizarin root of Dahurian angelica Chinese materia medica preparation that treating uterus bleeding | |
| CN108760945A (en) | A kind of detection method of Qijiaoshenbai capsule | |
| CN102078403A (en) | Detection method of Chinese traditional medicine preparation of oral liquid for treating infant cough with lung heat | |
| CN106442843A (en) | Quality check method of children's granules for clearing heat from throat | |
| CN109692291A (en) | Lung power cough mixture medicinal extract and application thereof and method of quality control | |
| CN106153812A (en) | A kind of detection method of the Chinese medicine preparation treating endometriosis | |
| CN102068627A (en) | Quality control method for Chinese medicine preparation Xinnaojing tabelets | |
| CN104155383B (en) | The detection method of blue or green Pu granule | |
| CN103163272B (en) | Quality control method of infant spleen tonifying medicament | |
| CN106680414A (en) | Detection method of compound ardisia japonica tablet | |
| CN104069200A (en) | "Sanhuang" heart-fire removing decoction formula granules and preparing method and detection method thereof | |
| CN103230448A (en) | Coptis and colla corii asini decoction compound formula granule, as well as preparation method and detection method thereof | |
| CN110292617A (en) | Lung power cough capsule medicinal extract and application thereof and method of quality control | |
| CN108653550A (en) | A kind of fiber crops a kind of reed mentioned in ancient books disappears cough drug and preparation method thereof and detection method | |
| CN104873686A (en) | Quality detection method for ginseng antler brain-boosting capsule | |
| CN108037200A (en) | A kind of quality determining method of Zishenningshenwan | |
| CN108614065A (en) | A kind of method of quality control of mountain chrysanthemum Jiangya Granula | |
| CN102068599B (en) | Detection method for phlegm eliminating and asthma relieving cough syrup |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A detection method for Erpixing granules Effective date of registration: 20231106 Granted publication date: 20180529 Pledgee: Agricultural Bank of China Limited Guiyang Jiaxiu sub branch Pledgor: GUIZHOU RUNSHENG PHARMACY CO.,LTD. Registration number: Y2023520000063 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |