CN109721632B - High-purity ganglioside GM1 and preparation method thereof - Google Patents
High-purity ganglioside GM1 and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 39
- QPJBWNIQKHGLAU-IQZHVAEDSA-N ganglioside GM1 Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 QPJBWNIQKHGLAU-IQZHVAEDSA-N 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 53
- 238000000746 purification Methods 0.000 claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000011068 loading method Methods 0.000 claims abstract description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 44
- 150000002270 gangliosides Chemical class 0.000 claims description 32
- 239000012043 crude product Substances 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000000945 filler Substances 0.000 claims description 26
- 239000000872 buffer Substances 0.000 claims description 25
- 239000007864 aqueous solution Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 23
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 22
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical group [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 20
- 238000002347 injection Methods 0.000 claims description 20
- 239000007924 injection Substances 0.000 claims description 20
- 239000002245 particle Substances 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 18
- 238000001704 evaporation Methods 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 12
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 12
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 10
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 8
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 8
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 8
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 8
- 230000005526 G1 to G0 transition Effects 0.000 claims description 7
- 239000012045 crude solution Substances 0.000 claims description 7
- 230000002209 hydrophobic effect Effects 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 239000008351 acetate buffer Substances 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 4
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 4
- 239000007974 sodium acetate buffer Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 230000006835 compression Effects 0.000 claims description 3
- 238000007906 compression Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000006012 monoammonium phosphate Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 238000000825 ultraviolet detection Methods 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 2
- 239000004254 Ammonium phosphate Substances 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 2
- 239000001099 ammonium carbonate Substances 0.000 claims description 2
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 2
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 2
- 238000011097 chromatography purification Methods 0.000 claims description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 2
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 235000011056 potassium acetate Nutrition 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- FVSKHRXBFJPNKK-UHFFFAOYSA-N propionitrile Chemical compound CCC#N FVSKHRXBFJPNKK-UHFFFAOYSA-N 0.000 claims description 2
- 238000005086 pumping Methods 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- 239000001488 sodium phosphate Substances 0.000 claims description 2
- 235000011008 sodium phosphates Nutrition 0.000 claims description 2
- 238000010025 steaming Methods 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical group [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract description 2
- 239000008363 phosphate buffer Substances 0.000 description 23
- 239000000523 sample Substances 0.000 description 23
- 239000000047 product Substances 0.000 description 22
- 150000003839 salts Chemical class 0.000 description 21
- 239000011148 porous material Substances 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 14
- 150000007960 acetonitrile Chemical class 0.000 description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 239000012267 brine Substances 0.000 description 4
- 239000000337 buffer salt Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108091006112 ATPases Proteins 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 206010001497 Agitation Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 229930182565 Australin Natural products 0.000 description 1
- 239000005740 Boscalid Substances 0.000 description 1
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- WYEMLYFITZORAB-UHFFFAOYSA-N boscalid Chemical compound C1=CC(Cl)=CC=C1C1=CC=CC=C1NC(=O)C1=CC=CN=C1Cl WYEMLYFITZORAB-UHFFFAOYSA-N 0.000 description 1
- 229940118790 boscalid Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 206010008129 cerebral palsy Diseases 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 150000002339 glycosphingolipids Chemical class 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Abstract
The invention belongs to the technical field of biological medicine, and particularly relates to a high-purity ganglioside GM1 single component (GM 1A, GM 1B), a mixed component and a preparation method thereof. The separation and purification method provided by the invention adopts a new mobile phase, the ganglioside GM1 and main components thereof are well separated, the problems of insufficient separation degree, more impurities and difficult separation are effectively solved, the cycle time is short, the sample loading amount is high, and the industrial mass production is easy to realize.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a high-purity ganglioside GM1 single component, a mixed component and a preparation method thereof.
Background
Gangliosides (gangliosides) are the most complex glycosphingolipids, widely distributed on the cell membrane of vertebrates, with the highest content in the central nervous system. It is composed of oligosaccharide chains with sialic acid and ceramide, and gangliosides can be classified according to the number of sialic acid and oligosaccharide glycosyl groups and the difference of sialic acid connecting sites. The ganglioside with higher content is GM1, GDla, GDlb, GD3 and GTlb.
GM1 is prepared by reacting Na + ，K + "Compensation" of ATPase Activity to function in Na + ，K + ATPases play an important role in maintaining membrane stability and excitability, and are important for the normal function of the cells. The function of GM1 can be broadly summarized as follows: 1. promoting normal development of nerve cells and brain tissues, and preventing and treating cerebral palsy and other diseases; 2. repairing damaged nerve and brain tissue, and preventing and treating cerebral apoplexy; 3. enhancing memory function; 4. delay the aging of nerve cells, and prevent and treat diseases such as Parkinson, senile dementia, etc. Its role is of concern in the medical field. The existing monosialotetrahexosyl ganglioside (GM 1 for short) preparation in China mainly comprises the following components: boscalid, shen Jie, benevolence australia, celebrity, australin, brain bond, etc.
Column chromatography is currently a very efficient method for preparing and separating and purifying GM1. Pan Ying et al used centrifugation liquid chromatography, methanol/chloroform/water elution to purify gangliosides, which took longer time, required 10 hours for one purification, and had low solvent consumption, low purity and poor reproducibility [ Pan Ying et al, chinese Biochemical Journal, vol.10, no.4, aug.,1994 ]; guo et al used low pressure silica gel column chromatography, methanol/chloroform/water elution to purify gangliosides, which required two column separations, was relatively complex and was only suitable for the preparation of mg-grade pure products, and was not suitable for scale-up production [ Guo, et al, chinese Journal of Chromatography, vol.10, no.3,1992 ]; huang Ru et al extract high purity gangliosides from pig brain by a combination of centrifugal liquid chromatography and gel chromatography, which is cumbersome, requires a large amount of solvents, has a long period of time, and is not suitable for mass production [ Huang Ru, et al, prog. Bioehem. Biophys, vol.21, no.5,1994 ]. The purification steps disclosed in the patent CN103524572B are desalting, silica gel chromatography, reversed phase chromatography, concentration, acetone recrystallization and freeze-drying, so that the GM1 with high purity can be obtained, but the purification process is complicated, the recovery rate is low, the time is long, and the purification method is not suitable for mass production; the purification steps disclosed in the patent CN1353112A are ion exchange column chromatography and reversed phase column chromatography, and the problems of complicated operation and long production period exist. The above methods have drawbacks in terms of purity and yield of GM1, and effectiveness in terms of cost, efficiency and application to industrial scale.
Disclosure of Invention
The first aspect of the present invention provides a high purity ganglioside GM1, comprising one or both of GM1A, GM 1B; wherein the high purity means that the total HPLC content of GM1A, GM B is 95% or more, preferably 96% or more, 97% or more, or 98% or more, more preferably 99% or more; the GM1A and GM1B each have the following chemical structure:
in one embodiment of the present invention, the high purity GM1 according to the first aspect of the present invention is characterized in that GM1 is a mixture of GM1A and GM 1B; preferably, the molar ratio of GM1A to GM1B is 1:0.8 to 1.2, more preferably 1:0.9 to 1.1.
In a second aspect, the present invention provides a process for preparing the high purity ganglioside GM1 according to the first aspect of the present invention, comprising the steps of:
1) Mobile phase configuration: mixing an organic solvent and a buffer solution, and regulating the pH value to 2.5-5.5, preferably 3.0-5.0 by using an aqueous NaOH solution or an aqueous phosphoric acid solution to obtain a mobile phase; wherein the volume ratio of the organic solvent to the buffer solution is 0.5-8: 1, preferably 1 to 5:1, more preferably 1 to 4:1; the organic solvent is selected from methanol, ethanol, isopropanol, acetonitrile, propionitrile and tetrahydrofuran; the molar concentration of the buffer solution is 1-1000 mmol/L, preferably 10-200 mmol/L, 20-80 mmol/L, 30-60 mmol/L, 300-400 mmol/L, more preferably 10mmol/L, 30mmol/L or 50mmol/L; the buffer solution is selected from phosphate buffer solution, acetate buffer solution and bicarbonate buffer solution; the phosphate is selected from sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium phosphate, diammonium hydrogen phosphate, monoammonium phosphate and triethylamine phosphate; preferably, the acetate buffer is selected from the group consisting of sodium acetate buffer, ammonium acetate buffer, potassium acetate buffer; the bicarbonate buffer is selected from sodium bicarbonate buffer, potassium bicarbonate buffer and ammonium bicarbonate buffer; the NaOH aqueous solution and the phosphoric acid aqueous solution are conventional concentrations used by those skilled in the art for adjusting the pH value of the solution, and the molar concentration is preferably 0.5-2.0 mol/L, more preferably 1.0mol/L;
2) Sample dissolution: dissolving the GM1 crude product fully by using a proper amount of mobile phase obtained by the method in the step 1), and filtering to obtain a crude product solution; wherein the concentration of the resulting crude solution is 10 to 1000mg/ml, preferably 20 to 100mg/ml, more preferably 60 to 80mg/ml.
3) Loading and eluting: pumping the crude product solution obtained in the step 2) into a dynamic axial compression preparation column filled with hydrophobic filler by using a sample pump, eluting by using a mobile phase prepared by the method of the step 1), collecting components GM1A and GM1B at corresponding eluting time and stage, respectively evaporating the solvent to obtain corresponding single-component samples, or mixing the collected eluents and steaming to obtain high-purity GM1.
Preferably, the hydrophobic filler in the step 3) is octadecyl bonded silica gel (called "bonded filler" for short), and the carbon number of the stationary phase is 1-50 n-chain alkyl, preferably the carbon number of the stationary phase is 1-30; the particle size of the stationary phase is 1-200 mu m, preferably 5-100 mu m; the aperture isSpecific surface area of 100-500 m 2 /g；
Preferably, the crude GM1 in the crude solution in step 3) is 0.1% to 5.0% by weight, more preferably 0.2% to 0.4% by weight of hydrophobic filler;
preferably, the preparation column of step 3) is a reverse phase preparation column;
preferably, in step 3) the flow rate of the mobile phase in the chromatographic purification is 0.2-2 mm/s, the temperature is 10-60 ℃, and the ultraviolet detection wavelength is 200-220 nm, preferably 205nm;
preferably, the inner diameter of the preparation column in the step 3) is 20 mm-1600 mm, and the height of the preparation column bed is 100 mm-1000 mm.
The invention relates to a method for separating and preparing gangliosides, which is characterized by comprising the following steps: separating ganglioside crude product solution by single needle column and collecting in segments; or a multi-needle batch continuous mode is adopted for column feeding and a separated continuous sample injection purification mode, and each batch of eluent is collected in a segmented mode.
In the invention, the reagents used are all conventional reagents and can be obtained through commercial purchase, and the raw materials used are all conventional chemical raw materials and can be obtained through commercial purchase or prepared according to published prior documents. Unless otherwise specified, all operations of the present invention are carried out at room temperature, which has the meaning usual in the art, preferably from 20 to 35℃and preferably from 20 to 25 ℃. In addition, the person skilled in the art can select the preparation columns with different specifications according to the weight of the crude GM1.
The invention has the following advantages: 1. high degree of separation: aiming at the problems encountered in the current ganglioside GM1 separation and purification, the invention provides a separation and purification method which adopts a new mobile phase, so that ganglioside GM1 and main components thereof are better separated, and the problems of insufficient separation degree, more impurities and difficult separation are effectively solved. 2. High efficiency: the cycle time is short, and the production efficiency is better improved. 3. The sample loading amount is large: is easy to realize industrialized mass production. 4. High repeatability: the experimental operation is simple and controllable, the automation is easy to realize, the process is stable, and the repeatability is good.
Drawings
FIG. 1 is an HPLC chart of the crude ganglioside GM1 product obtained in preparation example 1;
FIG. 2 is an HPLC plot of the ganglioside GM1 product obtained in example 1.
Detailed Description
The foregoing is further elaborated by the following specific embodiments, which are not to be construed as limiting the claimed subject matter. All technical schemes realized based on the above content of the invention belong to the scope of the invention. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. In the embodiment of the invention, if not specified, the reverse phase chromatographic column refers to a dynamic axial compression preparation column, and the filler is octadecyl bonded silica gel.
Preparation example 1: preparation of ganglioside GM1 crude product
20kg of clean fresh pig brain tissue is homogenized by a homogenizer, 10 times of chloroform/methanol mixed solution (1:1, V/V) is added, stirring and extracting are carried out overnight, supernatant is taken, spin-evaporating and concentrating are carried out at 40 ℃ until the volume is 1/4 of the original volume, the supernatant is placed in a-20 refrigerator overnight, filtering is carried out, the mixed solution (1:1, V/V) of NaCl aqueous solution (5% of mass fraction) and methanol is added according to the volume of 1/5 of filtrate for 3 times of extraction, the supernatant is combined, the temperature is 4 ℃ lower than Wen Xijing after concentration, and ganglioside total lipid is obtained after filtering.
Taking 100g of 500-600 mesh amorphous silica gel, homogenizing 200ml of isopropanol, loading into a column, and balancing by using chloroform/methanol/water=50/50/8 (abbreviated as A solution); 30g of ganglioside total lipid is added into 10 times volume of A solution to be completely dissolved, the mixture is put on a column, the A solution is eluted at the flow rate of 80ml/min, one bottle is collected every 250ml, the eluate containing ganglioside is collected through HPTLC detection, the eluate is evaporated and dried under reduced pressure in a rotary evaporation way, the ganglioside crude product is obtained, the total HPLC purity of GM1A and GM1B is 86%, and the HPLC diagram is shown in figure 1.
Example 1
600mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 10mL of methanol-10 mM ammonium dihydrogen phosphate buffer saline solution (pH=2.5), and the sample injection amount is 10mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 10 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 0.6mm/s; the mobile phase was methanol-10 mM ammonium dihydrogen phosphate buffer salt (ph=2.5), wherein the volume ratio of methanol/buffer saline solution was 60/40; an ultraviolet detector for detecting the wavelength of 205nm and collecting sample eluent in stages; the HPLC purities of GM1A, GM1B were determined to be 99.80% and 99.20%, respectively; the eluents were combined and the solvent was distilled off, and the HPLC profile of the resulting product was shown in fig. 2.
Example 2
1000mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 17mL of methanol-1Sample injection amount 17mL in 0mM monoammonium phosphate buffer saline solution (ph=2.5); reversed-phase chromatography column (column size 50X 250mm, particle diameter 20 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 0.9mm/s; the mobile phase was methanol-10 mM ammonium dihydrogen phosphate buffer salt (ph=2.5), wherein the volume ratio of methanol/buffer saline solution was 60/40; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.5%, and the molar ratio of GM1A to GM1B is 1:1.06; the HPLC profile is substantially identical to fig. 2.
Example 3
800mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 10mL of ethanol-10 mM triethylamine phosphate buffer saline solution (pH=3.0), and the sample injection amount is 1mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 15 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 0.9mm/s; the mobile phase is ethanol-10 mM triethylamine phosphate buffer salt (ph=3.0), wherein the volume ratio of ethanol/buffer saline solution is 50/50; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.2%, and the molar ratio of GM1A to GM1B is 1:0.93; the HPLC profile is substantially identical to fig. 2.
Example 4
1500mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-10 mM ammonium dihydrogen phosphate buffer saline solution (pH=2.5), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 10 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 0.4mm/s; the mobile phase was acetonitrile-10 mM ammonium dihydrogen phosphate buffer salt (ph=2.5), wherein the volume ratio of acetonitrile/buffer salt aqueous solution was 80/20;the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, and the HPLC total purity of GM1A and GM1B is 99.6% according to the measurement; the molar ratio of GM1A to GM1B is 1:0.85; the HPLC profile is substantially identical to fig. 2.
Example 5
600mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-50 mM disodium hydrogen phosphate buffer salt aqueous solution (pH=5.0), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 30 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 1.0mm/s; the mobile phase was acetonitrile-10 mM disodium hydrogen phosphate buffer salt (ph=5.0), wherein the volume ratio of acetonitrile/buffer salt aqueous solution was 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, and the HPLC total purity of GM1A and GM1B is 99.3 percent according to the measurement; the molar ratio of GM1A to GM1B is 1:1, a step of; the HPLC profile is substantially identical to fig. 2.
Example 6
300mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-50 mM sodium bicarbonate buffer saline solution (pH=5.5), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 10 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 1.0mm/s; the mobile phase was acetonitrile-50 mM sodium bicarbonate buffer salt (ph=5.5), wherein the volume ratio of acetonitrile/buffer brine solution was 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.7%, and the molar ratio of GM1A to GM1B is 1:0.9; the HPLC profile is substantially identical to fig. 2.
Example 7
300mg of ganglioside crude product obtained by the method of preparation example 1 was dissolved in 15mL of acetonitrile-50 mM sodium bicarbonate buffer saline solutionIn (ph=5.5), the sample injection amount was 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 60 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 1.0mm/s; the mobile phase was acetonitrile-50 mM sodium bicarbonate buffer salt (ph=5.5), wherein the volume ratio of acetonitrile/buffer brine solution was 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.5%, and the molar ratio of GM1A to GM1B is 1:0.98; the HPLC profile is substantially identical to fig. 2.
Example 8
300mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-50 mM sodium bicarbonate buffer saline solution (pH=5.5), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 15 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 0.6mm/s; the mobile phase was acetonitrile-50 mM sodium bicarbonate buffer salt (ph=5.5), wherein the volume ratio of acetonitrile/buffer brine solution was 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.2%, and the molar ratio of GM1A to GM1B is 1:1.1; the HPLC profile is substantially identical to fig. 2.
Example 9
300mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-50 mM sodium bicarbonate buffer saline solution (pH=5.5), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 10 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 0.7mm/s; the mobile phase was acetonitrile-50 mM sodium bicarbonate buffer salt (ph=5.5), wherein the volume ratio of acetonitrile/buffer brine solution was 80/20; ultraviolet detector with detection wavelength of 205nm and collecting in stagesThe eluents of samples GM1A and GM1B are combined and evaporated to dryness to obtain the product of GM1, and the HPLC total purity of GM1A and GM1B is 99.8%, and the molar ratio of GM1A to GM1B is 1:1.05; the HPLC profile is substantially identical to fig. 2.
Example 10
300mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-50 mM sodium dihydrogen phosphate buffer salt aqueous solution (pH=5.0), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 15 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 1.2mm/s; the mobile phase is acetonitrile-50 mM sodium dihydrogen phosphate buffer salt (ph=5.0), wherein the volume ratio of acetonitrile/buffer salt aqueous solution is 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.6%, and the molar ratio of GM1A to GM1B is 1:1, a step of; the HPLC profile is substantially identical to fig. 2.
Example 11
1000mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-50 mM sodium acetate buffer saline solution (pH=4.6), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 15 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 1.0mm/s; the mobile phase was acetonitrile-50 mM sodium acetate buffer salt (ph=5.5), wherein the volume ratio of acetonitrile/buffer salt aqueous solution was 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.5%, and the molar ratio of GM1A to GM1B is 1:1.15; the HPLC profile is substantially identical to fig. 2.
Example 12
300mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-30 mM disodium hydrogen phosphate buffer salt aqueous solution (pH=4.0), and the sample injection amount is 15mL; using opposite phase coloursSpectrum column (column specification 50X 250mm, particle size 10 μm, pore size)The mass of the bonding filler is 300 g), and the flow rate is 1.0mm/s; the mobile phase was acetonitrile-30 mM disodium hydrogen phosphate buffer salt (ph=4.0), wherein the volume ratio of acetonitrile/buffer salt aqueous solution was 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, eluent of samples GM1A and GM1B are collected in stages, and are combined and evaporated to dryness to obtain a product of GM1, and the product with the HPLC total purity of GM1A and GM1B of 99.8 percent is determined, and the molar ratio of GM1A to GM1B is 1:0.98; the HPLC profile is substantially identical to fig. 2.
Example 13
900mg of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 15mL of acetonitrile-30 mM disodium hydrogen phosphate buffer salt aqueous solution (pH=4.0), and the sample injection amount is 15mL; reversed-phase chromatography column (column size 50X 250mm, particle diameter 12 μm, pore diameter)The mass of the bonding filler is 300 g), and the flow rate is 1.2mm/s; the mobile phase was acetonitrile-30 mM disodium hydrogen phosphate buffer salt (ph=4.0), wherein the volume ratio of acetonitrile/buffer salt aqueous solution was 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.7%, and the molar ratio of GM1A to GM1B is 1:0.99; the HPLC profile is substantially identical to fig. 2.
Example 14
8.1g of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 300mL of acetonitrile-30 mM sodium dihydrogen phosphate buffer salt aqueous solution (pH=4.0), and the sample injection amount is 300mL; using a reversed-phase chromatographic column (column specification 150X 300mm, particle size 12 μm, pore size)2700g of bonding filler mass) and flow rate of 1.2mm/s; the mobile phase is acetonitrile-30 mM sodium dihydrogen phosphate buffer salt (ph=4.0), wherein the volume ratio of acetonitrile/buffer salt aqueous solution is 80/20; ultraviolet detector for detecting wavelength of 205nm and collecting samples GM1A and GM in stagesThe eluents of 1B are combined and evaporated to dryness to obtain a GM1 product, and the HPLC total purity of GM1A and GM1B is 99.3 percent, and the molar ratio of GM1A to GM1B is 1:1.05; the HPLC profile is substantially identical to fig. 2.
Example 15
8.1g of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 300mL of acetonitrile-30 mM sodium dihydrogen phosphate buffer salt aqueous solution (pH=4.0), and the sample injection amount is 300mL; using a reversed-phase chromatographic column (column specification 150X 300mm, particle size 15 μm, pore size)2700g of bonding filler mass) and flow rate of 1.2mm/s; the mobile phase is acetonitrile-30 mM sodium dihydrogen phosphate buffer salt (ph=4.0), wherein the volume ratio of acetonitrile/buffer salt aqueous solution is 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 99.1%, and the molar ratio of GM1A to GM1B is 1:1, a step of; the HPLC profile is substantially identical to fig. 2.
Example 16
270g of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 16L of acetonitrile-30 mM sodium dihydrogen phosphate buffer salt aqueous solution (pH=4.0), and the sample injection amount is 16L; using a reversed-phase chromatographic column (column specification 1000X 300mm, particle size 15 μm, pore size)The mass of the bonding filler is 135 kg), and the flow rate is 1.2mm/s; the mobile phase is acetonitrile-30 mM sodium dihydrogen phosphate buffer salt (ph=4.0), wherein the volume ratio of acetonitrile/buffer salt aqueous solution is 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a GM1 product, wherein the HPLC total purity of the GM1A and the GM1B is 99.3%, and the molar ratio of GGM1A to GM1B is 1:0.93; the HPLC profile is substantially identical to fig. 2.
Example 17
270g of ganglioside crude product obtained by the method of preparation example 1 is dissolved in 16L of acetonitrile-50 mM sodium dihydrogen phosphate buffer salt aqueous solution (pH=4.0), and the sample injection amount is 16L; using reversed phase chromatography columns(column size 1000X 300mm, particle size 10 μm, pore size)The mass of the bonding filler is 135 kg), and 1.0mm/s; the mobile phase is acetonitrile-30 mM sodium dihydrogen phosphate buffer salt (pH=4.0, no), wherein the volume ratio of acetonitrile/buffer salt water solution is 80/20; the ultraviolet detector is used for detecting the wavelength of 205nm, collecting the eluates of samples GM1A and GM1B in stages, combining and evaporating to dryness to obtain a product of GM1, wherein the HPLC total purity of GM1A and GM1B is 98.6%, and the molar ratio of GM1A to GM1B is 1:1, a step of; the HPLC profile is substantially identical to fig. 2.
Detection methods the HPLC content of the products obtained in examples 1-17 and preparation 1 of the present invention were determined as follows:
instrument and conditions
High performance liquid chromatograph: waterse2695-2489
Chromatographic column: c18 (Waters, 4.6X105 mm,3.5 μm)
Mobile phase a:0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (30:70), ph=7.0 with triethylamine;
mobile phase B acetonitrile
The gradient elution procedure was used:
flow rate: 1.0ml/min, detection wavelength 205nm, sample injection volume: column temperature 10 μl: 40 ℃.
The experimental steps are as follows:
taking a proper amount of ganglioside GM1 and related substances, dissolving a sample with an aqueous solution to prepare a sample solution containing about 1.0mg/ml ganglioside GM1. Performing high performance liquid chromatography under the above test conditions, and recording chromatogram, wherein the HPLC chromatogram is shown in figure 2; the content was calculated as peak area by the external standard method.
Claims (28)
1. A high purity ganglioside GM1, wherein GM1 is a mixture of GM1A and GM 1B; the molar ratio of GM1A to GM1B is 1: 0.8-1.2; wherein the high purity means that the HPLC total content of GM1A, GM1B is above 98%; the GM1A and GM1B each have the following chemical structure:
。
2. the high purity ganglioside GM1 according to claim 1, wherein the high purity means that the total HPLC content of GM1A, GM B is 99% or more.
3. The high purity ganglioside GM1 according to claim 1, wherein the molar ratio of GM1A to GM1B is 1:0.9 to 1.1.
4. A process for preparing the high purity ganglioside GM1 according to any one of claims 1-3, comprising the steps of:
1) Mobile phase configuration: mixing an organic solvent and a buffer solution, and regulating the pH value to 2.5-5.5 by using an aqueous NaOH solution or an aqueous phosphoric acid solution to obtain a mobile phase;
2) Sample dissolution: dissolving the GM1 crude product fully by using a proper amount of mobile phase obtained by the method in the step 1), and filtering to obtain a crude product solution;
3) Loading and eluting: pumping the crude product solution obtained in the step 2) into a dynamic axial compression preparation column filled with hydrophobic filler by using a sample pump, eluting by using a mobile phase prepared by the method of the step 1), collecting components GM1A and GM1B at corresponding eluting time and stage, respectively evaporating the solvent to obtain corresponding single-component samples, or mixing the collected eluents and steaming to obtain high-purity GM1.
5. The method of claim 4, wherein the mobile phase is adjusted to a pH of 3.0-5.0 in step 1).
6. The method of claim 4, wherein the volume ratio of the organic solvent to the buffer solution in step 1) is 0.5-8: 1, a step of; the organic solvent is selected from methanol, ethanol, isopropanol, acetonitrile, propionitrile and tetrahydrofuran; the molar concentration of the buffer solution is 1-1000 mmol/L; the buffer solution is selected from phosphate buffer solution, acetate buffer solution and bicarbonate buffer solution; the phosphate is selected from sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium phosphate, diammonium hydrogen phosphate, monoammonium phosphate and triethylamine phosphate; the acetate buffer is selected from sodium acetate buffer, ammonium acetate buffer and potassium acetate buffer; the bicarbonate buffer is selected from sodium bicarbonate buffer, potassium bicarbonate buffer and ammonium bicarbonate buffer; the molar concentration of the NaOH aqueous solution and the phosphoric acid aqueous solution is 0.5-2.0 mol/L.
7. The method of claim 4, wherein the volume ratio of the organic solvent to the buffer solution in step 1) is 1-5: 1.
8. the method of claim 4, wherein the volume ratio of the organic solvent to the buffer solution in step 1) is 1-4: 1.
9. the method according to claim 4, wherein the molar concentration of the buffer solution in step 1) is 10-200 mmol/L.
10. The method according to claim 4, wherein the molar concentration of the buffer solution in step 1) is 20-80 mmol/L.
11. The method according to claim 4, wherein the molar concentration of the buffer solution in step 1) is 30-60 mmol/L.
12. The method according to claim 4, wherein the molar concentration of the buffer solution in step 1) is 300-400 mmol/L.
13. The method according to claim 4, wherein the molar concentration of the buffer in step 1) is 10 mmol/L.
14. The method according to claim 4, wherein the molar concentration of the buffer in step 1) is 30 mmol/L.
15. The method according to claim 4, wherein the molar concentration of the buffer in step 1) is 50 mmol/L.
16. The method according to claim 4, wherein the molar concentration of the phosphoric acid aqueous solution in step 1) is 1.0 mol/L.
17. The process according to claim 4, wherein the crude solution obtained in step 2) has a concentration of 10 to 1000mg/ml.
18. The method according to claim 4, wherein the concentration of the crude solution obtained in step 2) is 20-100 mg/ml.
19. The method according to claim 4, wherein the concentration of the crude solution obtained in step 2) is 60-80 mg/ml.
20. The method according to claim 4, wherein the hydrophobic filler in the step 3) is octadecyl bonded silica gel, and the stationary phase has a carbon number of 1-50 n-chain alkyl groups; the particle size of the stationary phase is 1-200 mu m; the aperture is 60-300A; specific surface area of
100~500m 2 /g。
21. The method according to claim 4, wherein the stationary phase in step 3) has 1 to 30 carbon atoms.
22. The method of claim 4, wherein the stationary phase in step 3) has a particle size of 5-100 μm.
23. The method according to claim 4, wherein the weight of the GM1 crude product in the crude product solution in step 3) is 0.1% -5.0% of the weight of the hydrophobic filler; the preparation column in the step 3) is a reverse phase preparation column.
24. The method according to claim 4, wherein the crude GM1 in the crude solution in step 3) is 0.2% -0.4% by weight of hydrophobic filler.
25. The method according to claim 4, wherein in the step 3), the flow rate of the mobile phase in the chromatographic purification is 0.2-2 mm/s, and the temperature is 10-60 DEG C o And C, the ultraviolet detection wavelength is 200nm-220nm.
26. The method according to claim 4, wherein the ultraviolet detection wavelength in step 3) is 205nm.
27. The method according to claim 4, wherein the inner diameter of the preparation column in the step 3) is 20mm to 160 mm, and the height of the preparation column bed is 100mm to 1000mm.
28. The method of claim 4, wherein the ganglioside crude solution is separated by single needle column separation and collected in stages; or a multi-needle batch continuous mode is adopted for column feeding and a separated continuous sample injection purification mode, and each batch of eluent is collected in a segmented mode.
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