CN114544786A - Chromatographic separation method of organic amine alkaloid in relative ephedra by utilizing fluorine-containing fixation - Google Patents
Chromatographic separation method of organic amine alkaloid in relative ephedra by utilizing fluorine-containing fixation Download PDFInfo
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- 241000218671 Ephedra Species 0.000 title claims abstract description 29
- 229930013930 alkaloid Natural products 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 27
- 229910052731 fluorine Inorganic materials 0.000 title claims abstract description 21
- 239000011737 fluorine Substances 0.000 title claims abstract description 21
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 238000013375 chromatographic separation Methods 0.000 title claims abstract description 13
- -1 amine alkaloid Chemical class 0.000 title claims description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 39
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 10
- 150000001412 amines Chemical class 0.000 claims abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- KWGRBVOPPLSCSI-WPRPVWTQSA-N (-)-ephedrine Chemical compound CN[C@@H](C)[C@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WPRPVWTQSA-N 0.000 claims description 20
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 claims description 19
- 239000000523 sample Substances 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 229960002179 ephedrine Drugs 0.000 claims description 11
- 239000000741 silica gel Substances 0.000 claims description 11
- 229910002027 silica gel Inorganic materials 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 239000003643 water by type Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 6
- 229960003908 pseudoephedrine Drugs 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- 238000010829 isocratic elution Methods 0.000 claims description 5
- KWGRBVOPPLSCSI-WCBMZHEXSA-N pseudoephedrine Chemical compound CN[C@@H](C)[C@@H](O)C1=CC=CC=C1 KWGRBVOPPLSCSI-WCBMZHEXSA-N 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 4
- FMCGSUUBYTWNDP-ONGXEEELSA-N (1R,2S)-2-(dimethylamino)-1-phenyl-1-propanol Chemical compound CN(C)[C@@H](C)[C@H](O)C1=CC=CC=C1 FMCGSUUBYTWNDP-ONGXEEELSA-N 0.000 claims description 3
- FMCGSUUBYTWNDP-UHFFFAOYSA-N N-Methylephedrine Natural products CN(C)C(C)C(O)C1=CC=CC=C1 FMCGSUUBYTWNDP-UHFFFAOYSA-N 0.000 claims description 3
- 239000004695 Polyether sulfone Substances 0.000 claims description 3
- 229960002221 methylephedrine Drugs 0.000 claims description 3
- 229920006393 polyether sulfone Polymers 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- PISDRBMXQBSCIP-UHFFFAOYSA-N trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane Chemical compound FC(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)CC[Si](Cl)(Cl)Cl PISDRBMXQBSCIP-UHFFFAOYSA-N 0.000 claims description 3
- 239000013076 target substance Substances 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims 2
- 239000000460 chlorine Substances 0.000 claims 2
- 239000000758 substrate Substances 0.000 claims 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 1
- 125000001309 chloro group Chemical group Cl* 0.000 claims 1
- 239000002131 composite material Substances 0.000 claims 1
- 239000007822 coupling agent Substances 0.000 claims 1
- 125000005007 perfluorooctyl group Chemical group FC(C(C(C(C(C(C(C(F)(F)F)(F)F)(F)F)(F)F)(F)F)(F)F)(F)F)(F)* 0.000 claims 1
- SBEQWOXEGHQIMW-UHFFFAOYSA-N silicon Chemical compound [Si].[Si] SBEQWOXEGHQIMW-UHFFFAOYSA-N 0.000 claims 1
- 239000010703 silicon Substances 0.000 claims 1
- 229910052710 silicon Inorganic materials 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 8
- 150000003797 alkaloid derivatives Chemical class 0.000 abstract description 5
- 238000002137 ultrasound extraction Methods 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 4
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 abstract description 3
- 239000012528 membrane Substances 0.000 abstract description 2
- 238000002156 mixing Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 18
- 239000012071 phase Substances 0.000 description 11
- 239000012085 test solution Substances 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 7
- 229960001866 silicon dioxide Drugs 0.000 description 7
- 239000000126 substance Substances 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 229960002534 ephedrine hydrochloride Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012088 reference solution Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241001465251 Ephedra sinica Species 0.000 description 2
- BALXUFOVQVENIU-GNAZCLTHSA-N Ephedrine hydrochloride Chemical compound Cl.CN[C@@H](C)[C@H](O)C1=CC=CC=C1 BALXUFOVQVENIU-GNAZCLTHSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- NTCYWJCEOILKNG-ROLPUNSJSA-N [(1r,2s)-1-hydroxy-1-phenylpropan-2-yl]-dimethylazanium;chloride Chemical compound Cl.CN(C)[C@@H](C)[C@H](O)C1=CC=CC=C1 NTCYWJCEOILKNG-ROLPUNSJSA-N 0.000 description 2
- JQVDAXLFBXTEQA-UHFFFAOYSA-N dibutylamine Chemical compound CCCCNCCCC JQVDAXLFBXTEQA-UHFFFAOYSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 229940051020 methylephedrine hydrochloride Drugs 0.000 description 2
- 125000005010 perfluoroalkyl group Chemical group 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 229960003447 pseudoephedrine hydrochloride Drugs 0.000 description 2
- BALXUFOVQVENIU-KXNXZCPBSA-N pseudoephedrine hydrochloride Chemical compound [H+].[Cl-].CN[C@@H](C)[C@@H](O)C1=CC=CC=C1 BALXUFOVQVENIU-KXNXZCPBSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- LYUQWQRTDLVQGA-UHFFFAOYSA-N 3-phenylpropylamine Chemical class NCCCC1=CC=CC=C1 LYUQWQRTDLVQGA-UHFFFAOYSA-N 0.000 description 1
- 241000218670 Ephedraceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- LRCFXGAMWKDGLA-UHFFFAOYSA-N dioxosilane;hydrate Chemical compound O.O=[Si]=O LRCFXGAMWKDGLA-UHFFFAOYSA-N 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229960001252 methamphetamine Drugs 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 235000009048 phenolic acids Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000002040 relaxant effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229960004029 silicic acid Drugs 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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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
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a chromatographic separation method of organic amine alkaloids in ephedra by utilizing fluorine-containing fixed phase, which is a rapid chromatographic separation method for establishing high performance liquid chromatography for 5 organic amine alkaloids in ephedra, including 2 pairs of isomers, on the basis of a novel fluorine-containing chromatographic column material. The invention comprises the following steps: 1) adding acetic acid/water into herba Ephedrae sample, performing ultrasonic extraction for two times, and mixing the supernatants; 2) the combined supernatant is processed by a water membrane; 3) the solution after passing through the water film is analyzed by high performance liquid chromatography. The novel fluorine-containing stationary phase material utilized in the invention can well retain alkaloids in natural plants. Based on the characteristics, the problems that the alkaloid in the ephedra is weak to be reserved on a conventional reversed phase chromatographic column, and the structure is similar and is difficult to distinguish are solved. The method established by the invention has the characteristics of rapidness, simplicity, accurate and reliable result, good stability and the like, and is suitable for chromatographic separation of 5 organic amine alkaloids in the ephedra.
Description
Technical Field
The invention establishes a rapid chromatographic separation method of high performance liquid chromatography for 5 organic amine alkaloids in ephedra, including 2 pairs of isomers, based on novel fluorine-containing chromatographic column materials. The invention comprises the following steps: 1) adding acetic acid/water into herba Ephedrae sample, performing ultrasonic extraction for two times, and mixing the supernatants; 2) the combined supernatant is processed by a water membrane; 3) and analyzing the solution after passing through the water film by adopting high performance liquid chromatography. The novel fluorine-containing stationary phase material utilized by the invention can well retain alkaloids in natural plants. Based on the characteristics, the problems that the alkaloid in the ephedra is weak to be reserved on a conventional reversed phase chromatographic column, and the structure is similar and is difficult to distinguish are solved. The method established by the invention has the characteristics of rapidness, simplicity, accurate and reliable result, good stability and the like, and is suitable for chromatographic separation of 5 organic amine alkaloids in the ephedra herb.
Technical Field
The herba Ephedrae is perennial herb-like shrubbery plant of Ephedra of Ephedraceae, is a common Chinese medicinal herb, has long medicinal history, and has effects of inducing sweat, relieving exterior syndrome, inducing diuresis, relieving swelling, dispersing lung qi, relieving asthma, exciting central nervous system, relaxing bronchial smooth muscle, suppressing immunity, resisting oxidation, resisting blood coagulation, resisting virus, and resisting cancer. The chemical components in the ephedra are complex, contain alkaloids, flavonoids, volatile oil, organic phenolic acids, lignin and other chemical components, and have rich pharmacological action. The alkaloid is the main effective component in herba Ephedrae, mainly including ephedrine and pseudoephedrine, and secondly including demethyl ephedrine, demethyl pseudoephedrine and methyl ephedrine. The benzene ring side chain of the alkaloid in the ephedra contains nitrogen atoms, belongs to organic amine alkaloid, and has larger polarity, so the retention on a conventional reversed phase chromatographic column is weak; and because the relative molecular mass and chemical structure of several alkaloids in the ephedra are similar, only some differences exist on nitrogen-containing side chains, so the alkaloids are not easy to separate on a chromatographic column. The difference in structure results in a difference in pharmacological activity, and thus different ephedrine has different medicinal values. In addition, demethyl ephedrine and demethyl pseudoephedrine are also the main raw materials for preparing phenylpropylamines such as methamphetamine and the like synthetic drugs, so that the realization of the rapid chromatographic separation of the alkaloids in the ephedra has important significance.
The chromatographic quantitative method for ephedra in pharmacopoeia at present uses polar ether to connect the stationary phase of phenyl bonded silica gel and methanol-0.092% phosphoric acid solution (containing 0.04% triethylamine and 0.02% di-n-butylamine) so as to solve the problem of weak retention of ephedrine and only separate two alkaloids of ephedrine and pseudoephedrine.
The method comprises the steps of carrying out simple pretreatment on the ephedra medicinal material to obtain a test solution, and then realizing the rapid chromatographic separation of 5 organic amine alkaloids in the ephedra, including 2 pairs of isomers, by using a novel fluorine-containing chromatographic column-based high performance liquid chromatography method. The process is simple, the time consumption is low, and the separation of the target object can be quickly realized.
Disclosure of Invention
The invention relates to a rapid chromatographic separation method for establishing high performance liquid chromatography for 5 organic amine alkaloids in ephedra medicinal materials on the basis of a fluorine-containing chromatographic column.
In order to achieve the purpose, the invention adopts the technical scheme that:
1) preparation of a solution to be tested: preparation of a solution to be tested: weighing 0.5g of ephedra sample, adding 10mL of 1% acetic acid/water into the ephedra sample, carrying out ultrasonic extraction for 30min, carrying out centrifugation at 5000 rpm, collecting supernatant, adding 10mL of 1% acetic acid/water into residue again, repeating the above process, combining the two supernatants, and passing the supernatant through 0.45 mu m of polyethersulfone resin film to obtain a test solution.
2) Preparation of control solutions: weighing ephedrine hydrochloride, pseudoephedrine hydrochloride and 1mg of methylephedrine hydrochloride, respectively dissolving in 1mL of methanol to obtain 3 single targets with concentration of 1 mg/mL; then respectively measuring 40 mu L of each single standard, putting the single standard into the same centrifuge tube, adding methanol until the total volume is 1mL to obtain a reference solution, wherein the concentration of each standard is 40 mu g/mL.
3) And (3) measuring the sample to be measured and the reference substance solution by adopting high performance liquid chromatography, and separating 5 target substances at the same time.
High performance liquid chromatography conditions
The instrument comprises the following steps: the HPLC is Waters e2695 HPLC
A chromatographic column: fluorine-containing chromatographic column (4.6X 150mm, filler particle size 5 μm)
Mobile phase: a, ammonium formate solution (200mM, pH 3); b, acetonitrile; d, water;
isocratic elution: a: b: d is 3: 92: 5;
flow rate: 1mL/min
Column temperature: 30 deg.C
Detection wavelength: 210nm
The extraction solvent in the step 1) is acetic acid/water, acetic acid/methanol, phosphoric acid/water and the like.
The proportion of acid in the solution extracted in the step 1) is 1-10%.
The volume of the extractant in the step 1) is 5-20 mL.
The ultrasonic extraction time in the step 1) is 20-40 min.
The ultrasonic temperature in the step 1) is 20-40 ℃.
The high performance liquid chromatography column in the step 3) has the particle size of 3-5 mu m.
The column temperature in the step 3) is 30-45 ℃.
The salt of the mobile phase in the step 3) is ammonium formate or phosphate.
And the pH value of the ammonium formate in the step 3) is 3-6.
The proportion of the ammonium formate in the step 3) is 2-10 mM.
The mobile phase in the step 3) is acetonitrile or methanol and water.
The proportion of the organic phase in the step 3) is 80-95%.
The invention has the following advantages:
the novel fluorine-containing stationary phase material utilized in the invention can well retain alkaloids in natural plants. Based on the characteristics, the problems that the alkaloid in the ephedra is weak to be reserved on a conventional reversed phase chromatographic column, and the structure is similar and is difficult to distinguish are solved. The method established by the invention has the characteristics of rapidness, simplicity, accurate and reliable result, good stability and the like, and is suitable for chromatographic separation of 5 organic amine alkaloids in the ephedra herb.
Drawings
FIG. 1 is a comparison of a sample and a standard sample of Ephedra sinica Stapf;
FIG. 2 is the chemical structure of five compounds;
FIG. 3 is a graph comparing the amounts of five compounds in the samples of examples 1-3.
Detailed Description
Example 1
Fluorine-containing stationary phase
1) The mixed mode chromatographic stationary phase contains perfluoroalkyl chain in the bonded phase. The structural formula is as follows:
wherein the Silica is Silica gel. Each gram of silica gel contains 1.2mmol of perfluoroalkyl chain;
the preparation process of the chromatographic stationary phase comprises the following steps:
adding 10g of silica gel into a 250mL flask, adding the silica gel into 100mL of hydrochloric acid solution with the concentration of 38 wt%, heating, refluxing and stirring for 2 hours, filtering, washing with water until the pH value is 6-7, and drying the obtained solid in a drying oven at 160 ℃ to constant weight. Placing the obtained dry silica gel in an air atmosphere with the humidity of 50% for 24 hours to ensure that the water absorption weight of the silica gel is increased by 3%;
under the protection of nitrogen, 10.3g of hydrated silica gel is added into a 250mL flask, 100mL of dimethylbenzene is added and uniformly stirred, then 5mL of 1H,1H,2H, 2H-perfluorooctyl trichlorosilane is dropwise added to react for 3 hours at 30 ℃, the mixture is filtered and washed by dichloromethane, methanol water, methanol and tetrahydrofuran in sequence, and the obtained solid is dried for 24 hours in a drying oven at 80 ℃ to obtain the chromatographic stationary phase.
The fixed phase prepared above was used as a packing to pack an FC8 column for use in the following examples;
(II) sample treatment Process
1) Preparation of a solution to be tested: weighing 0.5g of ephedra sample, adding 10mL of 1% acetic acid/water with volume concentration for ultrasonic extraction for 30min, centrifuging at 5000 r/min, collecting supernatant, adding 10mL of 1% acetic acid/water with volume concentration again into residue, repeating the above process, combining the two supernatants, and passing the supernatant through a polyethersulfone resin film (Jinteng, with pore diameter of 0.45 μm and thickness of 50mm) with pore diameter of 0.45 μm.
2) Preparation of control solutions: weighing ephedrine hydrochloride, pseudoephedrine hydrochloride and 1mg of methylephedrine hydrochloride, respectively dissolving in 1mL of methanol to obtain 3 single targets with concentration of 1 mg/mL; then, 40. mu.L of each single label is measured and placed in the same centrifuge tube, and methanol is added until the total volume is 1mL, and the concentration of each substance is 40. mu.g/mL.
(III) high performance liquid chromatography method
1) High performance liquid chromatography analysis method
The high performance liquid chromatograph is Waters e2695 HPLC, and comprises a quaternary solvent manager, an automatic sample injector, a column incubator and an Empower chromatographic workstation; the 3 kinds of ephedrine are standard samples of Qiyun biotechnology limited company in Guangzhou city. Acetonitrile is chromatographically pure; the water is Mill-Q ultrapure water.
The instrument comprises the following steps: the HPLC is Waters e2695 HPLC
A chromatographic column: fluorine-containing chromatographic column (4.6X 150mm, filler particle size 5 μm)
Mobile phase: a, ammonium formate solution (200mM, pH 3); b, acetonitrile; d, water;
isocratic elution volume ratio: a: b: d is 3: 92: 5;
flow rate: 1mL/min
Column temperature: 30 deg.C
Detection wavelength: 210nm
2) Comparison of herba Ephedrae test solution and control solution
Processing herba Ephedrae and reference substance according to 1) and 2) in the sample processing procedure, wherein herba Ephedrae is available under the conditions of lot number 770190901, Beijing hongji, and is marked as A. Respectively obtaining herba Ephedrae test solution and control solution, and analyzing by high performance liquid chromatography of 1) in (III), the result is shown in FIG. 1. The structures of 5 alkaloids are shown in FIG. 2. The degrees of separation (degrees of separation) of two adjacent peaks of the five compounds are shown in table 1, and table 1 shows the degrees of separation of the five compounds.
The comparison results of different batches of ephedra herb materials are shown in figure 3.
TABLE 15 degrees of separation of the Compounds
| Name of Compound | Demethylation ephedrine | Demethylated pseudoephedrine | Ephedrine hydrochloride | Pseudoephedrine | Methylephedrine |
| Degree of separation | 1.6 | 2.9 | 2.0 | 1.6 |
Example 2
The ephedra herb in section (2) of the third section (III) of the example 1) is changed into a sample B, the batch number of the herb is MH16090301, the source of the ephedra herb is shenggui baicao, and the rest is the same as the example 1.
(III) high performance liquid chromatography method
1) High performance liquid chromatography analysis method
The high performance liquid chromatograph is Waters e2695 HPLC, and comprises a quaternary solvent manager, an automatic sample injector, a column incubator and an Empower chromatographic workstation; the 3 kinds of ephedrine are standard samples of Qiyun biotechnology limited company in Guangzhou city. Acetonitrile is chromatographically pure; the water is Mill-Q ultrapure water.
The instrument comprises the following steps: the HPLC is Waters e2695 HPLC
A chromatographic column: fluorine-containing chromatographic column (4.6X 150mm, filler particle size 5 μm)
Mobile phase: a, ammonium formate solution (200mM, pH 3); b, acetonitrile; d, water;
isocratic elution: a: b: d is 3: 92: 5;
flow rate: 1mL/min
Column temperature: 30 deg.C
Detection wavelength: 210nm
2) Comparison of herba Ephedrae test solution and control solution
Processing herba Ephedrae and reference substance according to 1) and 2) in (II), wherein herba Ephedrae is under batch number MH16090301, and is derived from Herbaea paniculata and marked as B. Respectively obtaining herba Ephedrae test solution and reference solution, analyzing by high performance liquid chromatography of 1) in (III), and comparing the results of herba Ephedrae with different batches as shown in FIG. 3.
Example 3
The ephedra herb in section (2) of the third section of the example 1 is changed into a sample C, the batch number of the herb is MH200330, the source is Kyushutong, and the rest is the same as the example 1.
(III) high performance liquid chromatography method
1) High performance liquid chromatography analysis method
The high performance liquid chromatograph is Waters e2695 HPLC, and comprises a quaternary solvent manager, an automatic sample injector, a column incubator and an Empower chromatographic workstation; the 3 kinds of ephedrine are standard samples of Qiyun biotechnology limited company in Guangzhou city. Acetonitrile is chromatographically pure; the water was Mill-Q ultrapure water.
The instrument comprises the following steps: the HPLC is Waters e2695 HPLC
A chromatographic column: fluorine-containing chromatographic column (4.6X 150mm, filler particle size 5 μm)
Mobile phase: a, ammonium formate solution (200mM, pH 3); b, acetonitrile; d, water;
isocratic elution: a: b: d is 3: 92: 5;
flow rate: 1mL/min
Column temperature: 30 deg.C
Detection wavelength: 210nm
2) Comparison of herba Ephedrae test solution and control solution
Processing herba Ephedrae and reference substance according to 1) and 2) in (II), wherein herba Ephedrae has batch number MH200330, and is from Kyushutong, and is marked as C. Respectively obtaining herba Ephedrae test solution and reference solution, analyzing by high performance liquid chromatography of 1) in (III), and comparing the results of herba Ephedrae with different batches as shown in FIG. 3.
Example 4
The HPLC method in section (1) of section (third) of example 1 is used to separate and purify the sample solution of Ephedra sinica Stapf, and fractions are collected by chromatographic peak to obtain single compounds.
Claims (5)
1. The chromatographic separation method of organic amine alkaloid in the fluorine-containing fixed relative ephedra is characterized by comprising the following steps: is a high performance liquid chromatography method based on a fluorine-containing stationary phase chromatographic column; the method comprises the following steps:
1) preparation of a solution to be tested: weighing 0.5g of ephedra sample, adding 5-20 mL of extracting solution for extraction, centrifuging, and collecting supernatant; adding 5-20 mL of extracting solution with volume concentration into the residues again, centrifuging, collecting supernate, combining the collected supernate, and then passing the supernate through a polyethersulfone resin film with the aperture of 0.45 mu m to obtain a sample solution to be detected;
2) the high performance liquid chromatography separation of a sample solution to be detected is carried out by adopting a chromatographic column of a stationary phase containing a fluorine bonding phase, and simultaneously 5 target substances are separated, wherein the separation comprises 2 pairs of isomer rapid chromatographic separation, and the 5 organic amine alkaloids are demethylephedrine, demethylpseudoephedrine, ephedrine, pseudoephedrine and methylephedrine respectively.
2. The method of claim 1, wherein:
high performance liquid chromatography conditions
The instrument comprises the following steps: the HPLC is Waters e2695 HPLC
A chromatographic column: fluorine-containing chromatographic column (4.6X 150mm, filler particle diameter 3 ~ 5 μm)
Mobile phase: a, ammonium formate solution (100-200 mM, pH 3-6); b, acetonitrile; d, water;
isocratic elution (volume ratio): a: b: d is 1-5: 95-80: 4-19;
flow rate: 0.5 to 1mL/min
Column temperature: 30-45 DEG C
Detection wavelength: 210 nm.
3. The method of claim 1, wherein: the high performance liquid chromatography column is a fluorine-containing bonding phase;
the specific phase of the fluorine-containing bonding phase is silica gel as a substrate, and the bonding phase on the surface of the substrate contains a perfluorooctyl chain; the silicon-silicon composite material is obtained by bonding silicon and silica gel surface after chlorine removal by using a 1H,1H,2H, 2H-perfluoro octyl trichlorosilane coupling agent, and has the following structure:
wherein X is chlorine, and m is 0-20; and each gram of silica gel contains 0.1-10 mmol of 1H,1H,2H, 2H-perfluorooctyl trichlorosilane group.
4. The method of claim 1, wherein: the extractive solution can be acetic acid/water, acetic acid/methanol, phosphoric acid/water; the volume ratio of acetic acid/water to acetic acid in the acetic acid/methanol extracting solution is 1-10%; the volume ratio of phosphoric acid/phosphoric acid in water is 1-10%; the volume of the extract is preferably 10 mL.
5. The method of claim 1, wherein: the extraction time is 20-40 min; the extraction temperature is 20-40 ℃.
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| CN115969905A (en) * | 2022-12-15 | 2023-04-18 | 赣江中药创新中心 | Novel online two-dimensional orthogonal separation method for coptis isoquinoline alkaloid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103512998A (en) * | 2013-09-10 | 2014-01-15 | 张文臣 | Chinese ephedra medicinal material, and content determination method of three alkaloids in preparation thereof |
-
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|---|---|---|---|---|
| CN103512998A (en) * | 2013-09-10 | 2014-01-15 | 张文臣 | Chinese ephedra medicinal material, and content determination method of three alkaloids in preparation thereof |
Non-Patent Citations (4)
| Title |
|---|
| DAVID S. BELL等: "Rational method development strategies on a fluorinated liquid chromatography stationary phase: Mobile phase ion concentration and temperature effects on the separation of ephedrine alkaloids", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| FEDERICA PELLATI等: "Determination of ephedrine alkaloids in Ephedra natural products using HPLC on a pentafluorophenylpropyl stationary phase", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| JOSEPH J. PESEK等: "Evaluation of the dual retention properties of stationary phases based on silica hydride: Perfluorinated bonded material", 《JOURNAL OF SEPARATION SCIENCE》 * |
| 史丽颖等: "麻黄中生物碱类成分富集新方法及化学成分分析", 《中国实验方剂学杂志》 * |
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|---|---|---|---|---|
| CN115969905A (en) * | 2022-12-15 | 2023-04-18 | 赣江中药创新中心 | Novel online two-dimensional orthogonal separation method for coptis isoquinoline alkaloid |
| CN115969905B (en) * | 2022-12-15 | 2024-02-09 | 赣江中药创新中心 | Online two-dimensional orthogonal separation method for coptis isoquinoline alkaloid |
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