CN114624352A - Method for detecting content of strychnine emplastrum and related substances and application - Google Patents
Method for detecting content of strychnine emplastrum and related substances and application Download PDFInfo
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- CN114624352A CN114624352A CN202110730629.1A CN202110730629A CN114624352A CN 114624352 A CN114624352 A CN 114624352A CN 202110730629 A CN202110730629 A CN 202110730629A CN 114624352 A CN114624352 A CN 114624352A
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- strychnine
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- emplastrum
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- QMGVPVSNSZLJIA-FVWCLLPLSA-N strychnine Chemical compound O([C@H]1CC(N([C@H]2[C@H]1[C@H]1C3)C=4C5=CC=CC=4)=O)CC=C1CN1[C@@H]3[C@]25CC1 QMGVPVSNSZLJIA-FVWCLLPLSA-N 0.000 title claims abstract description 140
- QMGVPVSNSZLJIA-UHFFFAOYSA-N Nux Vomica Natural products C1C2C3C4N(C=5C6=CC=CC=5)C(=O)CC3OCC=C2CN2C1C46CC2 QMGVPVSNSZLJIA-UHFFFAOYSA-N 0.000 title claims abstract description 76
- 241001279009 Strychnos toxifera Species 0.000 title claims abstract description 70
- 229960005453 strychnine Drugs 0.000 title claims abstract description 70
- 239000000126 substance Substances 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 239000011505 plaster Substances 0.000 claims abstract description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 61
- 238000000605 extraction Methods 0.000 claims description 51
- 239000000499 gel Substances 0.000 claims description 31
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 239000002904 solvent Substances 0.000 claims description 20
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 19
- 239000011259 mixed solution Substances 0.000 claims description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 13
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 239000012266 salt solution Substances 0.000 claims description 11
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 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 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 235000011054 acetic acid Nutrition 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 235000015165 citric acid Nutrition 0.000 claims description 4
- 150000007522 mineralic acids Chemical class 0.000 claims description 4
- 150000007524 organic acids Chemical class 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims 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 claims description 2
- -1 aromatic organic acid Chemical class 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 150000004671 saturated fatty acids Chemical class 0.000 claims description 2
- 239000001488 sodium phosphate Substances 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 2
- 239000011975 tartaric acid Substances 0.000 claims description 2
- 235000002906 tartaric acid Nutrition 0.000 claims description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 claims description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims 1
- 229910002651 NO3 Inorganic materials 0.000 claims 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims 1
- 238000005070 sampling Methods 0.000 claims 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 12
- 229930013930 alkaloid Natural products 0.000 abstract description 3
- 150000003797 alkaloid derivatives Chemical class 0.000 abstract description 2
- 238000005220 pharmaceutical analysis Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 16
- 239000012535 impurity Substances 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 235000002639 sodium chloride Nutrition 0.000 description 12
- 230000000694 effects Effects 0.000 description 8
- 238000011084 recovery Methods 0.000 description 7
- 238000009210 therapy by ultrasound Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- 244000107975 Strychnos nux-vomica Species 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 5
- 235000019799 monosodium phosphate Nutrition 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 5
- RRKTZKIUPZVBMF-IBTVXLQLSA-N brucine Chemical compound O([C@@H]1[C@H]([C@H]2C3)[C@@H]4N(C(C1)=O)C=1C=C(C(=CC=11)OC)OC)CC=C2CN2[C@@H]3[C@]41CC2 RRKTZKIUPZVBMF-IBTVXLQLSA-N 0.000 description 4
- RRKTZKIUPZVBMF-UHFFFAOYSA-N brucine Natural products C1=2C=C(OC)C(OC)=CC=2N(C(C2)=O)C3C(C4C5)C2OCC=C4CN2C5C31CC2 RRKTZKIUPZVBMF-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 201000008482 osteoarthritis Diseases 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 239000011241 protective layer Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 1
- AMBQHHVBBHTQBF-UHFFFAOYSA-N Loganin Natural products C12C(C)C(O)CC2C(C(=O)OC)=COC1OC1OC(CO)C(O)C(O)C1O AMBQHHVBBHTQBF-UHFFFAOYSA-N 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 150000001642 boronic acid derivatives Chemical class 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 235000011132 calcium sulphate Nutrition 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 150000004694 iodide salts Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- AMBQHHVBBHTQBF-UOUCRYGSSA-N loganin Chemical compound O([C@@H]1OC=C([C@H]2C[C@H](O)[C@H](C)[C@H]21)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O AMBQHHVBBHTQBF-UOUCRYGSSA-N 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- JESHZQPNPCJVNG-UHFFFAOYSA-L magnesium;sulfite Chemical compound [Mg+2].[O-]S([O-])=O JESHZQPNPCJVNG-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- JEUXZUSUYIHGNL-UHFFFAOYSA-N n,n-diethylethanamine;hydrate Chemical compound O.CCN(CC)CC JEUXZUSUYIHGNL-UHFFFAOYSA-N 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 150000002826 nitrites Chemical class 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 235000009518 sodium iodide Nutrition 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical class [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 150000004764 thiosulfuric acid derivatives Chemical class 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
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- 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)
Abstract
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the content of strychnine emplastrum and related substances. The detection of the content of strychnine in the strychnine gel emplastrum and the determination of related substances are important contents for the research of the medicine quality, and the premise of accurately determining the content is to completely extract the components to be detected. The invention comprises (1) a method for extracting strychnine from gel plaster; (2) a method for analyzing and detecting the strychnine content and related substances is high performance liquid chromatography. The invention provides technical support for the quality research of the strychnine gel emplastrum, and provides reference for the content of other alkaline alkaloid gel emplastrums and the research of related substances.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting the content of strychnine emplastrum and related substances.
Background
Nux vomica is bitter in property and warm; it is toxic. Has effects of dredging collaterals, relieving pain, resolving hard mass and relieving swelling. Can be used for treating traumatic injury, fracture, rheumatism, numbness, and paralysis. The main effective component of semen Strychni is total alkaloids including strychnine, loganin, and ecamine; wherein, the strychnine is the effective component of nux vomica for resisting inflammation, relieving pain and treating arthritis.
The brucine as the raw material medicine is obtained by extracting and separating from the processed nux vomica, the purity reaches more than 98 percent, and the strychnine hardly contains any toxic substance. The strychnine gel emplastrum has large drug-loading rate, accurate dosage, slow-release drug release and long-lasting drug effect. In the aspect of treating osteoarthritis, the toxicity of the bulk drug strychnine is greatly reduced and the drug effect is greatly improved compared with the traditional Chinese medicine nux vomica. The strychnine gel plaster has less blood invasion and high local medicine concentration in joints because of being an external medicine, thereby improving the local medicine effect and reducing the toxic and side effects of the whole body. Therefore, the strychnine gel emplastrum has important clinical value in the aspect of treating osteoarthritis.
At present, no report on content detection of strychnine gel plaster and related substance research exists. The detection of the content of strychnine in the strychnine gel emplastrum and the determination of related substances are important contents of medicine quality research, and the premise of accurately determining the content is to completely extract the components to be detected.
Extracting strychnine and related substances from the strychnine gel plaster needs to overcome the technical problem and find proper extraction conditions; meanwhile, the brucine and related substances cannot be completely separated by an HPLC mobile phase system for detecting the content of the brucine in Chinese pharmacopoeia. The content of strychnine emplastrum and a detection method of related substances are required to be developed to ensure the product quality.
Disclosure of Invention
The invention aims to provide a method for analyzing and detecting the content and related substances in strychnine gel emplastrum.
The invention also aims to provide the application of the analytical detection method of the content and related substances in the strychnine emplastrum in the aspect of controlling the quality of the strychnine emplastrum product.
The invention provides a method for analyzing and detecting the content of strychnine gel emplastrum and related substances, which comprises the following steps of (1) extracting strychnine from the gel emplastrum; (2) a method for analyzing and detecting strychnine content and related substances is high performance liquid chromatography.
First, the content measurement of strychnine emplastrum and the preparation of samples for the examination of related substances were performed. Extraction of strychnine and related impurities from gel plaster is a necessary condition for sample detection.
The method for extracting the strychnine from the gel plaster in the step (1) comprises the following steps:
the extraction research comprises an extraction solvent, the dosage of the solvent, the pH value of the solvent, an extraction mode, extraction time, extraction temperature and the like.
The extraction solvent considers the mixed solution of aqueous solution, inorganic salt solutions with different concentrations and different types and methanol.
The pH value range of the solution is 2.0-5.0, and the pH regulator is organic acid and inorganic acid;
the organic acid is selected from straight chain or branched chain saturated fatty acid, unsaturated fatty acid, and aromatic organic acid.
The organic acid is selected from formic acid, acetic acid, citric acid, tartaric acid and citric acid; the inorganic acid is selected from hydrochloric acid, phosphoric acid and sulfuric acid.
The extraction mode inspects the ultrasound and the stirring;
the extraction time is 5-20h by water bath stirring;
the extraction temperature range is 30-70 ℃.
Taking a proper amount of strychnine gel emplastrum, removing the protective layer, cutting into small pieces, sequentially putting into a 200-ml measuring flask filled with a proper amount of solvent for preventing adhesion, putting into a rotor, stirring at 30-70 ℃ for 5-20h, performing ultrasonic treatment for 0.5-3h, cooling, taking out the rotor, adding the solvent to dilute to a scale, shaking uniformly, and filtering with a 0.45-micrometer filter membrane. Making into solution containing brucine 0.2mg (related substance)/0.1 mg (content) per 1 ml.
The extraction solvent is a mixed solution of an inorganic salt solution and methanol;
the extraction solvent is preferably a mixed solution of 0.9-10% inorganic salt solution and methanol;
the extraction solvent is more preferably a mixture of 4-10% inorganic salt solution and methanol.
The inorganic salt in the extraction solvent is selected from: chlorides, iodides, phosphates, sulfates, nitrates, nitrites, borates, thiosulfates, sulfites, bisulfites, and the like. Such as sodium chloride, sodium iodide, sodium phosphate, calcium phosphate, sodium sulfate, magnesium sulfate, calcium sulfate, sodium nitrate, sodium nitrite, sodium borate, sodium thiosulfate, sodium sulfite, magnesium sulfite, sodium bisulfite, and the like.
The inorganic salt is more preferably sodium chloride, phosphate, sulfate; most preferred is sodium chloride.
The inorganic salt solution and the methanol mixed solution are mixed in any proportion, and the preferred proportion is 80: 20.
Research results show that the extraction solvent is inorganic salt aqueous solution and methanol at a ratio of 4:1, the pH value of the solvent is 2-5, the extraction mode comprises stirring for 5-16 hours, ultrasonic treatment for 0.5-1 hour, and the extraction effect is optimal when the extraction temperature is 40-60 ℃.
The method for analyzing and detecting the strychnine content and related substances in the step (2),
the mobile phase regulator researches acetic acid, triethylamine, phosphoric acid, sodium heptanesulfonate and sodium dihydrogen phosphate; the pH value of the mobile phase is 3.80-4.10.
A method for analyzing and detecting the strychnine content and related substances is high performance liquid chromatography. The liquid chromatography conditions used were as follows:
a chromatographic column: octadecylsilane bonded silica gel column, preferably Agilent ZORBAX SB-C18, 250mm × 4.6mm, 5 μm; kromasil C18, 250 mm. times.4.6 mm, 5 μm; ultimate LP-C18, 250mm × 4.6mm, 5 μm; waters Xbridge C18, 250mm × 4.6mm, 5 μm, etc.;
related substances mobile phase: equal-volume mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; preferably 18: 82. Adjusting the pH of the mobile phase to 3.90-4.10 by using 5% phosphoric acid;
content of mobile phase: equal volume of mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; preferably in a ratio of 21: 79. Adjusting the pH of the mobile phase to 3.80-4.10 by using 5% phosphoric acid;
the detection wavelength is 255-280 nm;
the flow rate is 0.5-2.0 ml/min;
the column temperature is 25-40 ℃;
the injection volume is 5-60 μ l.
The strychnine and related substances in the strychnine emplastrum provided by the invention are as follows:
drawings
FIG. 1 is a chromatogram obtained by using chromatographic conditions for measuring the content of semen Strychni preparata in the first part of Chinese pharmacopoeia 2020 edition;
FIG. 2 is a spectrum obtained using chromatographic conditions in the reference;
FIG. 3 is a graph obtained using chromatographic conditions established in this study-pH 3.90;
FIG. 4 is a graph, pH 4.00, obtained using chromatographic conditions established in this study;
FIG. 5 is a graph, pH 4.10, obtained using chromatographic conditions established in this study.
Detailed Description
Examples 1-19 determination of the content of strychnine in the plaster and preparation of samples for examination of the relevant substances are as follows:
example 1g of paste was taken and put into a 25ml measuring flask, 20ml of water solution (acetic acid to adjust pH to 2.0) was added and ultrasonic treatment was carried out for 3 hours, the mixture was stirred overnight (15 hours) in a 60 ℃ water bath (stirring speed is medium), ultrasonic treatment was carried out for 1 hour, and volume was fixed with methanol. The extraction recovery rate of strychnine is 99.7%.
The method can completely extract the strychnine in the gel plaster, but the ultrasonic treatment is carried out for 3 hours, and the extraction mode is too strong, so that the method is not suitable for daily sample inspection operation. There is therefore a need to optimize the extraction process.
Example 21 g of paste is taken and placed in a 25ml measuring flask, 20ml of 0.9% sodium chloride solution (formic acid to adjust the pH value to 2.0) is added, ultrasonic treatment is carried out for 1 hour, 5ml of methanol is added, water bath stirring is carried out at 60 ℃ overnight (15h), and the volume is determined by methanol. The extraction recovery rate of strychnine is measured to be 99.6%.
The test reduces the ultrasonic time, and the result has no obvious difference, which shows that the 0.9 percent sodium chloride solution is used for replacing water, thereby being beneficial to extracting the strychnine in the gel plaster.
Example 3-5 taking 1g of the paste, placing the paste in a 25ml measuring flask, adding 20ml of 0.9% sodium chloride solution (phosphoric acid is adjusted to different pH values), carrying out ultrasonic treatment for 1h, adding 5ml of methanol, stirring in a water bath at 60 ℃ overnight (15h), and adding methanol to fix the volume. The method comprises the following specific steps:
examples 6-14 gel patches 7 x 10cm (corresponding to 7g of paste) were weighed, cut into 3cm x 1.5cm pieces, placed in 200ml measuring flasks, charged to 200ml with different concentrations of inorganic salt solution (formic acid to adjust the pH to 3.0) -methanol (80:20), stirred in a water bath and sonicated for 0.5 h. The method comprises the following specific steps:
note: because the prescription process is mature, the sample in the experiment does not use the gel paste any more, but the formed gel plaster is adopted, the plaster is cut into pieces and then extracted, the extraction scale is enlarged, and the changes do not have obvious influence on the extraction.
Adjusting the pH value of the extraction solution to 3.0, increasing the concentration of inorganic salt, and inspecting the extraction effect. From the results of examples 6 to 8, 9, and 12 to 13, it was found that the increase in the concentration of the inorganic salt within a certain range indeed improves the extraction yield of strychnine, and the concentration of the inorganic salt was selected to be 4%.
Under the premise of keeping other conditions unchanged, the pH value of the extracting solution is continuously increased (the pH value is less than 7) (example 14), and the recovery rate of the strychnine is almost unchanged. The extract composition, i.e. 4% sodium chloride solution (formic acid adjusted to pH3.0) -methanol (80:20) was fixed and the effect of different extraction times on the extraction recovery was examined. From the results of examples 8 and 9, it can be seen that the extraction recovery rate RAD% was 0.7 with no significant change between the overnight stirring in water bath (15h) and the 5h extraction, and the extraction time was determined to be 5h for saving the time cost.
The composition and extraction time of the extract solution were fixed, i.e. the extract solution was 4% sodium chloride solution (pH adjusted to 3.0 with formic acid) -methanol (80:20), the extraction time was 5h, and the effect of different extraction temperatures on the extraction recovery rate was examined. As is clear from the results of examples 9 and 10, the extraction at 60 ℃ is not significantly different from that at 40 ℃.
The pH value (pH3.0), extraction time (5h) and extraction temperature (60 ℃) of the extract were fixed, and the extraction effect of the extraction solvent without methanol was examined. From the results of example 11, it can be seen that the solvent recovery without methanol is lower than that with methanol, and the optimal content sample preparation method is that used in examples 8 and 9.
In order to ensure that the extraction method is simultaneously suitable for preparation of related substance samples, the impurity extraction condition is researched.
Example 15 about 25mg of the raw material sample is taken, precisely weighed, placed in a 25mL measuring flask, dissolved in methanol and diluted to the scale, and shaken up; precisely measuring 5ml, placing in a 10ml measuring flask, and adding water to dilute to scale.
Examples 16-17 gel patches 7 x 10cm (corresponding to 7g of paste) were weighed, cut into 3cm x 1.5cm pieces, placed in a 200ml measuring flask, added with 4% sodium chloride solution (hydrochloric acid to adjust the pH to 5.0) -methanol (80:20) to 200ml, stirred in a water bath and sonicated for 0.5 h. The results are as follows:
| impurity name/RRT | Example 15 | Example 16 | Example 17 |
| 0.18 | / | 0.032 | 0.031 |
| |
/ | 0.650 | 0.725 |
| 0.46 | 0.097 | 0.089 | 0.088 |
| 0.60 | 0.032 | 0.030 | 0.030 |
| 0.69 | 0.043 | 0.044 | 0.044 |
| |
0.670 | 0.555 | 0.572 |
| 0.81 | / | 0.023 | 0.022 |
| |
0.073 | 0.086 | 0.073 |
| |
0.018 | 0.028 | 0.020 |
| Strychnine | 98.238 | 97.670 | 97.609 |
| |
0.050 | 0.047 | 0.047 |
| 1.54 | 0.022 | 0.024 | 0.021 |
| |
0.756 | 0.721 | 0.718 |
| Number of impurities | 9 | 12 | 12 |
Note: example 16 was stirred in a water bath for 5h and example 17 was stirred in a water bath overnight (15 h).
From the above results, it can be seen that: compared with the bulk drugs (example 15), in the gel plaster (examples 16-17), except unknown impurities and known impurities 8 of RRT0.18 and RRT0.81, all the other impurities are derived from the bulk drugs, the normalization result of each impurity has no obvious difference from the bulk drugs, and the result of prolonging the stirring time has no difference, which indicates that the sample is completely extracted.
Examples 18-19 methods of preparing samples of the release rates of strychnine emplastrum and the results of the tests are as follows:
taking the product, cutting into circular sample with diameter of 30mm, removing the protective layer to make the adhesive surface of the product face upward, placing in the release medium, respectively taking the release solution at different times, filtering, and measuring the subsequent filtrate.
The experiments show that the release degree of the strychnine gel plaster in a 2% sodium chloride solution is far greater than that of a pure water medium, and the addition of the sodium chloride also promotes the release of the strychnine from the gel plaster under the condition that the pH value is not adjusted by adding acid.
Examples 20-21 assay of strychnine-containing plasters and examination of related substances the following were screened:
example 20 the content of strychnos nux-vomica prepared in the first department of the Chinese pharmacopoeia 2020 edition was analyzed by octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 2.85 with 5% phosphoric acid) (21:79) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 10 ul. The chromatogram is shown in figure 1. :
the separation degree of each peak is poor, and the method is not suitable for detecting related substances, but can be used for measuring the content.
Example 21 the following chromatographic conditions were selected: bonding a silica gel column (4.6X 150mm, 5um) with octadecylsilane; the chromatographic column mobile phase A is acetonitrile, and the mobile phase B is 0.2% acetic acid and 0.2% triethylamine water solution; the flow rate is 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 10 ul. Gradient elution.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 5 | 95 |
| 20 | 21 | 79 |
| 25 | 60 | 40 |
| 35.01 | 10 | 90 |
| 35 | 10 | 90 |
The chromatogram is shown in FIG. 2.
Wherein, the impurity peak separation degrees of RT17.058min and RT17.375min are not enough.
Example 22 the selection of the use of an octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 3.90 with 5% phosphoric acid) (18:82) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 40 ul. The chromatogram is shown in FIG. 3.
Example 23 elected to use an octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 4.0 with 5% phosphoric acid) (18:82) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 40 ul. The chromatogram is shown in FIG. 4.
Example 24 selection of the use of an octadecylsilane bonded silica gel column (250 mm. times.4.6 mm, 5 μm); eluting with a mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L sodium dihydrogen phosphate (pH adjusted to 4.10 with 5% phosphoric acid) (18:82) at flow rate of 1 ml/min; the detection wavelength is 260nm, the column temperature is 30 ℃, and the injection volume is 40 ul. The chromatogram is shown in FIG. 5. The separation degree of each peak is good, and the method is applicable.
In summary, the method for extracting strychnine from the gel patch, preferably according to examples 8, 9, 10, 12, 13 and 14, shows that the addition of sodium chloride promotes the release of strychnine from the gel patch. The method for analyzing and detecting the content of the strychnine and related substances is better in examples 22, 23 and 24, wherein the separation degree between the strychnine and the related substances is good.
The analytical detection method of the content and related substances of the strychnine gel emplastrum is an important component of the quality research of the strychnine gel emplastrum, wherein the detection of the content and the related substances is the basis for setting the quality standard of the strychnine gel emplastrum, and the quality standard is a guideline for inspecting whether a preparation product is qualified or not. Therefore, the analytical detection method of the content of the strychnine gel emplastrum and related substances is a crucial factor in the quality of the whole preparation product. The research provides technical support for the quality research of the strychnine gel emplastrum, and simultaneously provides reference for the content research of other alkaline alkaloid gel emplastrums and the research of related substances.
Claims (10)
1. A method for analyzing and detecting the content and related substances in strychnine emplastrum is characterized in that,
comprises (1) extracting strychnine and related substances from gel plaster; (2) a method for analyzing and detecting the content of strychnine and related substances is high performance liquid chromatography.
2. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 1, is characterized by comprising the following steps: the extraction of strychnine from the gel plaster in the step (1) is as follows: stirring for 5-20h at 30-70 deg.C with extraction solvent, ultrasonic treating for 0.5-3h, and sampling; the extraction solvent is a mixed solution of an inorganic salt solution and methanol; the inorganic salt solution is adjusted by acid, and the pH value is 2.0-5.0.
3. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 1 or 2, which is characterized by comprising the following steps: the extraction solvent in the step (1) is a mixed solution of 0.9-10% of inorganic salt solution and methanol; the inorganic salt is selected from: chloride, iodide, phosphate, sulfate, nitrate, nitrite, borate, thiosulfate, sulfite, bisulfite.
4. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 3, is characterized by comprising the following steps: the extraction solvent in the step (1) is a mixed solution of 4-10% of inorganic salt solution and methanol, and the inorganic salt is preferably sodium chloride, phosphate and sulfate; the ratio of inorganic salt solution to methanol is preferably 80: 20.
5. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 1 or 2, which is characterized by comprising the following steps: the acid used for adjusting the pH value of the extraction solvent in the step (1) is selected from straight chain or branched chain saturated fatty acid, unsaturated fatty acid, aromatic organic acid and inorganic acid.
6. The method for analyzing and detecting the content and related substances in the strychnine emplastrum as claimed in claim 5, is characterized by comprising the following steps: the acid used for adjusting the pH value of the extraction solvent in the step (1), wherein the organic acid is selected from formic acid, acetic acid, citric acid, tartaric acid and citric acid; the inorganic acid is selected from hydrochloric acid, phosphoric acid and sulfuric acid.
7. The method for analyzing and detecting related substances in the strychnine emplastrum as claimed in claim 1, wherein the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel column;
related substances mobile phase: equal-volume mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; adjusting the pH of the mobile phase to 3.90-4.10 by using 5% phosphoric acid;
content of mobile phase: equal volume of mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 15:85-25: 75; adjusting the pH of the mobile phase to 3.80-4.10 by using 5% phosphoric acid;
the detection wavelength is 255-280 nm;
the flow rate is 0.5-2.0 ml/min;
the column temperature is 25-40 ℃;
the injection volume is 5-60 μ l.
8. The method for analyzing and detecting related substances in the strychnine emplastrum as claimed in claim 7, wherein the chromatographic conditions are as follows:
and (3) chromatographic column: agilent ZORBAX SB-C18, 250mm × 4.6mm, 5 μm; kromasil C18, 250mm × 4.6mm, 5 μm; ultimate LP-C18, 250mm × 4.6mm, 5 μm; waters Xbridge C18, 250 mm. times.4.6 mm, 5 μm;
related substances mobile phase: equal-volume mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 18: 82; adjusting the pH of the mobile phase to 3.90-4.10 by using 5% phosphoric acid;
content of mobile phase: equal volume of mixed solution of acetonitrile-0.01 mol/L sodium heptanesulfonate and 0.02mol/L potassium dihydrogen phosphate is 21: 79; adjusting the pH of the mobile phase to 3.80-4.10 by using 5% phosphoric acid;
the detection wavelength is 255-280 nm;
the flow rate is 0.5-2.0 ml/min;
the column temperature is 25-40 DEG C
The injection volume is 5-60 μ l.
9. The method for analyzing and detecting related substances in the strychnine emplastrum as claimed in claim 1 or 2, wherein the related substances in the strychnine emplastrum are:
10. use of the analytical detection method for the content and related substances in strychnine emplastrum as claimed in any one of claims 1-2 for controlling the quality of strychnine emplastrum products.
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| 蒋莹莹: "马钱子生物碱组分及其透皮制剂的研究", 《浙江工业大学硕士学位论文》, pages 30 - 46 * |
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