CN113670680B - Preparation method of acarbose impurity reference substance - Google Patents
Preparation method of acarbose impurity reference substance Download PDFInfo
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- CN113670680B CN113670680B CN202110741868.7A CN202110741868A CN113670680B CN 113670680 B CN113670680 B CN 113670680B CN 202110741868 A CN202110741868 A CN 202110741868A CN 113670680 B CN113670680 B CN 113670680B
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- 229960002632 acarbose Drugs 0.000 title claims abstract description 138
- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 title claims abstract description 138
- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 title claims abstract description 137
- 239000012535 impurity Substances 0.000 title claims abstract description 133
- 239000013558 reference substance Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 238000000926 separation method Methods 0.000 claims abstract description 27
- 238000000746 purification Methods 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 29
- BXWLVQXAFBWKSR-UHFFFAOYSA-N 2-methoxy-5-methylsulfonylbenzoic acid Chemical compound COC1=CC=C(S(C)(=O)=O)C=C1C(O)=O BXWLVQXAFBWKSR-UHFFFAOYSA-N 0.000 claims description 18
- 239000000945 filler Substances 0.000 claims description 18
- 238000007710 freezing Methods 0.000 claims description 14
- 230000008014 freezing Effects 0.000 claims description 14
- 238000012856 packing Methods 0.000 claims description 14
- 230000001105 regulatory effect Effects 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 8
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000004821 distillation Methods 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 4
- 239000004952 Polyamide Substances 0.000 claims 2
- 229920002647 polyamide Polymers 0.000 claims 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 abstract description 16
- 238000004811 liquid chromatography Methods 0.000 abstract description 11
- 238000001196 time-of-flight mass spectrum Methods 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 5
- 238000000862 absorption spectrum Methods 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 238000003908 quality control method Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 230000007812 deficiency Effects 0.000 abstract description 2
- 229910052799 carbon Inorganic materials 0.000 description 44
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 38
- 238000001228 spectrum Methods 0.000 description 33
- 229910052739 hydrogen Inorganic materials 0.000 description 23
- 239000001257 hydrogen Substances 0.000 description 19
- 238000005160 1H NMR spectroscopy Methods 0.000 description 14
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 13
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 12
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 11
- 238000005481 NMR spectroscopy Methods 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 238000011049 filling Methods 0.000 description 7
- 238000001819 mass spectrum Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000012790 confirmation Methods 0.000 description 6
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 125000005842 heteroatom Chemical group 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 3
- HCIFJKWFKHPOMC-IILQJVAWSA-N (1S,2S,3R,6S)-6-[[(2R,3S,4S,5R,6R)-6-[(2R,3S,4R,5R,6S)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(1R,4R,5S,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)cyclohex-2-en-1-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-methyloxan-3-yl]amino]-4-(hydroxymethyl)cyclohex-4-ene-1,2,3-triol Chemical compound C[C@H]1O[C@H](O[C@@H]2[C@@H](CO)O[C@H](O[C@H]3[C@H](O)[C@@H](O)[C@H](O)C=C3CO)[C@H](O)[C@H]2O)[C@H](O)[C@@H](O)[C@@H]1N[C@H]1C=C(CO)[C@@H](O)[C@H](O)[C@H]1O HCIFJKWFKHPOMC-IILQJVAWSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- UMFZJPRJFORIIJ-BGDITRTQSA-N (2R,3R,4R,5R)-4-[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-en-1-yl]amino]oxan-2-yl]oxy-2,3,5,6-tetrahydroxyhexanal Chemical compound C[C@H]1O[C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)[C@H](O)[C@@H](O)[C@@H]1N[C@@H]1[C@H](O)[C@@H](O)[C@H](O)C(CO)=C1 UMFZJPRJFORIIJ-BGDITRTQSA-N 0.000 description 1
- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 102400000471 Isomaltase Human genes 0.000 description 1
- 108010026867 Oligo-1,6-Glucosidase Proteins 0.000 description 1
- 101710184309 Probable sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 102400000472 Sucrase Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101710112652 Sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003888 alpha glucosidase inhibitor Substances 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 102000016679 alpha-Glucosidases Human genes 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- SNVLJLYUUXKWOJ-UHFFFAOYSA-N methylidenecarbene Chemical group C=[C] SNVLJLYUUXKWOJ-UHFFFAOYSA-N 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- 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
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N2001/2893—Preparing calibration standards
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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)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a preparation method of an acarbose impurity reference substance, and belongs to the technical field of medicine analysis. The method adopts various technologies such as preparation liquid chromatography, ultraviolet absorption spectrum, infrared absorption spectrum, time of flight mass spectrum (TOF-MS), nuclear magnetic resonance spectrum (NMR), freeze dryer, quantitative nuclear magnetic detection and the like to separate, purify, freeze dry, content detect, structure confirm and mark four impurities of acarbose impurities I, II, III and IV, and the four impurities can be used as impurity reference substances in quality detection of acarbose bulk drugs and preparations. The invention provides a separation and purification method of acarbose impurities, which is a general separation method of acarbose impurity reference substances, can be used for purifying and preparing other related substances of acarbose, and solves the problems of quality control of acarbose and impurity reference substance deficiency in impurity research at present.
Description
Technical Field
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a preparation method of an acarbose impurity reference substance.
Background
Acarbose (Acarbose) is O-4, 6-dideoxy-4- [1S,4R,5S,6S ] -4,5, 6-trihydroxy-3- (hydroxymethyl) -2-cyclohexene-1-amino ] -alpha-D-glucopyranosyl- (1- > 4) -O-alpha-D-glucopyranosyl- (1- > 4) -alpha-D-glucopyranose, is an alpha-glucosidase inhibitor, competitively inhibits glucoside hydrolase (maltase, isomaltase, glucoamylase and sucrase) in intestinal tracts, reduces decomposition of polysaccharide and sucrose into glucose, correspondingly slows down absorption of sugar, has the effect of reducing postprandial hyperglycemia, and achieves the effect of reducing blood glucose.
Acarbose is a raw material for oral preparations, and impurities contained in acarbose affect the use safety of products, and the impurities in samples are thoroughly researched and strictly limited. The existing methods and documents do not carry out detailed impurity preparation, and cannot provide an impurity reference substance preparation method which is necessary for acarbose quality control and impurity research.
Disclosure of Invention
Aiming at the problems, the invention provides a separation and purification method of acarbose impurities, which is used for carrying out separation and purification, freeze drying, structure confirmation and standardization by preparing liquid chromatography to obtain high-purity acarbose impurities I, II, III and IV, and is used for quality detection and research of acarbose bulk drugs and preparations.
The invention is realized by the following technical scheme:
the preparation method of the acarbose impurity reference substance comprises the following steps:
1) Taking acarbose impurity enrichment liquid, regulating the pH value to 3.0-6.0 by dilute hydrochloric acid, and concentrating by reduced pressure distillation until each milliliter of concentrated liquid contains 10-40mg of acarbose impurities;
2) Regulating the pH value of the concentrated solution in the step 1) to 7.0-10.0 by dilute ammonia water, and then filtering by a 0.45 mu m filter membrane to obtain an impurity concentrated solution;
3) Eluting the impurity concentrated solution twice in succession under the separation and purification condition of C 18 packing, and collecting fractions containing acarbose impurities;
4) Purifying the fraction collected in the step 3) under NH 2 filling separation and purification conditions, and collecting the fraction with the purity of more than 95%;
5) Mixing the fractions with the purity of more than 95% collected in the step 4), distilling under reduced pressure to remove methanol, adding water for dissolution, adjusting the pH value to 3.0-6.0 with dilute hydrochloric acid, and pre-freezing in a refrigerator;
6) Then freeze-drying the fraction after the pre-freezing treatment in the step 5) in a freeze dryer to obtain white crystalline powder, namely acarbose impurities; and finally, detecting the purity of the obtained acarbose impurity, and carrying out structure confirmation and standardization.
The preparation method of the acarbose impurity reference substance is characterized in that the acarbose impurities in the step 1) are respectively impurity I, impurity II, impurity III and impurity IV in acarbose of the second edition of the pharmacopoeia of the people's republic of China in 2020, and the structural formulas are respectively as follows:
the preparation method of the acarbose impurity reference substance is characterized in that the acarbose impurity reference substance is concentrated to 20-30mg of acarbose impurity in each milliliter of concentrated solution by reduced pressure distillation in the step 1).
The preparation method of the acarbose impurity reference substance is characterized in that the separation and purification conditions of the C 18 filling in the step 3) are as follows: chromatographic column: welch Xtimate C 18, 10 μm filler packing 200-400g; mobile phase: methanol-water solution=8-92 (V/V); flow rate: 40-70mL/min; sample injection volume: 10mL; detection wavelength: 210nm, wherein the methanol-water solution contains 0.1% NH 3.
The preparation method of the acarbose impurity reference substance is characterized in that the separation and purification conditions of NH 2 filling materials in the step 4) are as follows: chromatographic column: welch Ultimate XB-NH 2, 10 μm filler charge 150-350g; mobile phase: methanol; flow rate: 40-70mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
The preparation method of the acarbose impurity reference substance is characterized in that the flow rate is 50-60mL/min.
The preparation method of the acarbose impurity reference substance is characterized in that the acarbose enrichment liquid collected in the step 1) is 200-500ml in each time, and the volume of the acarbose enrichment liquid dissolved by adding water in the step 5) is 20-50ml.
The preparation method of the acarbose impurity reference substance is characterized in that the conditions of the freeze dryer in the step 6) are as follows: the freezing temperature is-70 ℃ to-80 ℃ under the pressure condition of 0.01 to 0.03 Mpa.
The preparation method of the acarbose impurity reference substance is characterized in that the freezing pressure is 0.02Mpa, and the freezing temperature is-76. DEG C
The method adopts various technologies such as preparation liquid chromatography, ultraviolet absorption spectrum, infrared absorption spectrum, time of flight mass spectrum (TOF-MS), nuclear magnetic resonance spectrum (NMR), freeze dryer and the like to separate and purify, freeze dry, detect the content, confirm and normalize the four impurities of acarbose impurities I, II, III and IV, and the four impurities can be used as impurity reference substances in quality detection of acarbose bulk drugs and preparations. The invention provides a separation and purification method of acarbose impurities, which is a general separation method of acarbose impurities, can be used for purifying and preparing other related substances of acarbose, and solves the problems of quality control of acarbose and impurity reference substance deficiency in impurity research at present. The method can rapidly prepare a large amount of impurities of high-purity acarbose, provides powerful technical support for defining the quality safety of acarbose products, guaranteeing the health of consumers and the like, and has important practical significance.
Drawings
FIG. 1 is an HPLC chromatogram of acarbose impurity I in example 1;
FIG. 2 is a time-of-flight mass spectrum of acarbose impurity I in example 1;
FIG. 3 is a nuclear magnetic resonance spectrum (H spectrum) of acarbose impurity I in example 1;
FIG. 4 is a nuclear magnetic resonance spectrum (C spectrum) of acarbose impurity I in example 1;
FIG. 5 is an HPLC chromatogram of acarbose impurity II in example 2;
FIG. 6 is a time-of-flight mass spectrum of acarbose impurity II in example 2;
FIG. 7 is a nuclear magnetic resonance spectrum (H spectrum) of acarbose impurity II in example 2;
FIG. 8 is a nuclear magnetic resonance spectrum (C spectrum) of acarbose impurity II in example 2;
FIG. 9 is an HPLC chromatogram of acarbose impurity III in example 3;
FIG. 10 is a time-of-flight mass spectrum of acarbose impurity III in example 3;
FIG. 11 is a nuclear magnetic resonance spectrum (H spectrum) of acarbose impurity III in example 3;
FIG. 12 is a nuclear magnetic resonance spectrum (C spectrum) of acarbose impurity III in example 3;
FIG. 13 is an HPLC chromatogram of acarbose impurity IV in example 4;
FIG. 14 is a time-of-flight mass spectrum of acarbose impurity IV in example 4;
FIG. 15 is a nuclear magnetic resonance spectrum (H spectrum) of acarbose impurity IV in example 4;
FIG. 16 is a nuclear magnetic resonance spectrum (C spectrum) of acarbose impurity IV in example 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings, and specific examples are given.
Example 1
1. Acarbose impurity I extraction
(1) Acarbose impurity I was enriched from acarbose samples by a LC silica gel pre-column with a particle size of 20-45. Mu.m. 300mL of acarbose impurity I enrichment solution is taken, the pH value is regulated to 3.0-6.0 by dilute hydrochloric acid, reduced pressure distillation and concentration are carried out to 30mg/mL, the pH value is regulated to 7.0-10.0 by dilute ammonia water, and finally the I impurity enrichment solution is obtained by filtering by a 0.45 mu m filter membrane.
(2) Eluting the I impurity concentrated solution twice in succession under the condition of separating and purifying by using C 18 packing. The separation and purification conditions of the C 18 filling material are as follows: instrument: DAC50 prepares a liquid chromatography system (beijing innovation general technology limited); chromatographic column: welch Xtimate C 18, 10 μm filler charge 300g; mobile phase: methanol-water (0.1% nh 3) solution = 8-92 (V/V); flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(3) And collecting fractions containing acarbose impurities I, purifying the fractions under the separation and purification conditions of NH 2 filling, and collecting fractions with the purity of more than 95%. The separation and purification conditions of the NH 2 filling material are as follows: instrument: DAC50 prepares a liquid chromatography system (beijing innovation general technology limited); chromatographic column: welch Ultimate XB-NH 2, 10 μm filler charge 250g; mobile phase: methanol; flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(4) Mixing acarbose impurity I fraction with purity greater than 95%, vacuum distilling to remove methanol, adding 20mL water for dissolving, regulating pH to 5.0-6.0 with diluted hydrochloric acid, and pre-freezing in refrigerator.
(5) And then freeze-drying is carried out in a freeze dryer at the temperature of minus 76 ℃ and the pressure of 0.02Mpa, so as to obtain white crystalline powder, namely the target product acarbose impurity I, and the product is preserved under the drying condition of 2-8 ℃.
2. Acarbose impurity I Structure confirmation
(1) The high resolution mass spectrum is shown in fig. 2, and the analysis and conclusion are as follows:
Sample molecular formula: c 25H43NO18 absolute molecular weight M:645.2480
[ M+H ] + Experimental values: 646.2573 Theoretical value of [ M+H ] +: 646.2553
As can be seen from the high-resolution mass spectrum of the acarbose impurity I, in the positive ion mode, the relative error between the experimental value of the molecular formula [ M+H ] + of the sample and the theoretical value of [ M+H ] + corresponding to the molecular formula C 25H43NO18 is 3.09ppm, so that the molecular formula of the acarbose impurity I is confirmed to be C 25H43NO18.
(2) The nuclear magnetic resonance spectrum, the nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the sample are shown in FIGS. 3 and 4.
1 The occurrence of proton signals in the H-NMR spectrum (FIG. 3) can be divided into 15 groups, and from the area integration of the hydrogen signal peaks, the sample contains at least 29 hydrogen atoms (without active hydrogen), including hydrogen signals on1 double bond (δ H 5.741-5.734, d, J=4.0Hz, 1H), 2 sugar end group hydrogen signals (δ H5.172-5.151,d,J＝3.5Hz,1H;δH 5.083-5.053, d, J=3.8Hz, 1H), 1 triplet methine signals (δ H 2.326-2.295, t, J=9.5Hz, 1H), 1 bimodal methyl signals (δ H 1.186.186-1.176, d, J=6.3Hz, 3H) and other methine and methylene signals.
13 C-NMR (FIG. 4) shows that the sample contains 34 groups of 25 pairs of signals of carbon, including 1 pair of double bond carbons (δ C140.820;δC 125.628), 1 quaternary carbon (δ C 103.794/100.138), 2 sugar end methines (δ C102.189/99.998;δC 101.711), 15 other methines (δC83.240/80.027;δC81.828/71.022;δC79.056;δC77.090/68.749;δC75.223/75.046;δC74.814;δC74.787;δC74.568;δC73.436;δC73.015;δC72.862/72.724;δC72.638;δC71.446;δC66.791;δC57.836)、4 methylenes (δ C65.635/64.239;δC65.251/64.549;δC63.464;δC 62.452) and 1 methyl group (δ C 19.184).
The nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the acarbose impurity I prove that C, H belongs to the molecular structural formula of the sample, and the structural formula is as follows:
3. Acarbose impurity I labeling
The HPLC purity of acarbose impurity I was 96.4%, see FIG. 1. The quantitative nuclear magnetic method is used for detecting the content of acarbose impurity I to be 90.5 percent and is used as an impurity reference substance.
Example 2
1. Acarbose impurity II extraction
(1) The acarbose impurity II is enriched from the acarbose sample by using an LC silica gel pre-column with the particle size of 20-45 mu m. 300mL of acarbose impurity II enrichment solution is taken, pH is regulated to 3.0-6.0 by dilute hydrochloric acid, reduced pressure distillation and concentration are carried out to 30mg/mL, pH is regulated to 7.0-10.0 by dilute ammonia water, and finally, the concentration solution is obtained by filtering by a 0.45 mu m filter membrane.
(2) Eluting the impurity II concentrated solution twice continuously under the separation and purification condition of C 18 filling, and collecting fraction containing acarbose impurity II. Separation and purification conditions of C 18 packing: instrument: DAC50 was used to prepare a liquid chromatography system (Jiangsu Hanbang technology Co., ltd.); chromatographic column: welch Xtimate C 18, 10 μm filler charge 300g; mobile phase: methanol-water (0.1% nh 3) solution = 8-92 (V/V); flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(3) And purifying the fraction under NH 2 packing separation and purification conditions, and collecting the fraction with the purity of more than 95%. NH 2 packing separation and purification conditions: instrument: DAC50 was used to prepare a liquid chromatography system (Jiangsu Hanbang technology Co., ltd.); chromatographic column: daisogel SP-120-APS-P,10 μm filler charge 300g; mobile phase: methanol; flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(4) Mixing acarbose impurity II fractions with purity of more than 95%, distilling under reduced pressure to remove methanol, adding 20mL of water for dissolution, adjusting pH to 5.0-6.0 with dilute hydrochloric acid, and pre-freezing in a refrigerator.
(5) Freeze-drying in a freeze dryer at-76 deg.C and 0.02Mpa to obtain white crystalline powder, which is the target product, acarbose impurity II, and storing at 2-8deg.C.
2. Acarbose impurity II Structure confirmation
(1) The high resolution mass spectrum is shown in fig. 6, and the analysis and conclusion are as follows:
Sample molecular formula: c 26H43NO17 absolute molecular weight M:641.2531
[ M+H ] + Experimental values: 642.2614 Theoretical value of [ M+H ] +: 642.2604
As can be seen from the high-resolution mass spectrum of the acarbose impurity B, in the positive ion mode, the relative error between the experimental value of the molecular formula [ M+H ] + of the sample and the theoretical value of [ M+H ] + corresponding to the molecular formula C 26H43NO17 is 1.55ppm, so that the molecular formula of the acarbose impurity B is confirmed to be C 26H43NO17.
(2) The nuclear magnetic resonance spectrum, the nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the sample are shown in FIGS. 7 and 8.
1 The proton signals of the 17 groups of samples, which appear in the H-NMR spectrum (FIG. 7), are found from their integrated areas to contain at least 29 hydrogen atoms (no active hydrogen). 1 The H-NMR spectrum shows proton signals on the 2 double bonds (δ H5.920,d,J＝3.7Hz,1H;δH 5.758, s, 1H) and 13 hydrogen proton signals on the 2 terminal groups (δ H5.471,d,J＝3.9Hz,1H;δH5.383,d,J＝3.7Hz,1H);1 H-NMR spectrum) in the high field region, including 4 overlapping proton signals (δH4.268-4.192,overlap,4H;δH3.964-3.932,overlap,2H;δH3.901-3.863,overlap,5H;δH3.684-3.623,overlap,3H),5 proton signals (δH 4.477,d,J＝7.5Hz,1H;δH 4.116,d,J＝6.8Hz,1H;δH4.046,br,1H;δH3.811,m,1H;δH3.481,dd,J＝9.9,8.7Hz,1H;δH2.941,br,1H),2 for the methylene group coupled to the carbon (δ H4.151,d,J＝13.5Hz,1H;δH 3.791, m, 1H), and 1 methyl group proton signals (δ H 1.406, d, j=6.1 hz, 3H).
13 C-NMR (FIG. 8) shows that the sample contains 24 carbon signals, the low field of the carbon spectrum shows 2 carbon signals, a quaternary carbon signal (delta C 136.321) and a tertiary carbon signal (delta C 127.518) on 1 double bond; the midfield portion exhibits 22 carbon signals, including 2 terminal tertiary carbon signals (δ C99.681;δC 98.206), 16 tertiary carbon signals associated with heteroatoms (δ C61.341;δC61.322;δC 60.548) for (δC78.640;δC77.220;δC75.371;δC75.214;δC73.303;δC72.456;δC72.176;δC71.303;δC71.020;δC70.747;δC70.718;δC70.007;δC 68.406;δC66.715;δC63.341;δC56.271),3 hydroxymethyl secondary carbon signals and 1 methyl carbon signal (δ C 17.351).
The nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the acarbose impurity II confirm that C, H belongs to the molecular structural formula of the sample, and the structural formula is as follows:
3. acarbose impurity II standardization
The HPLC purity of acarbose impurity ii was 94.0%, see fig. 5. The content of acarbose impurity II detected by the quantitative nuclear magnetic method is 86.7 percent, and the acarbose impurity II is used as an impurity reference substance.
Example 3
1. Acarbose impurity III extraction
(1) Acarbose impurity III was enriched from acarbose samples by LC silica gel pre-column with particle size of 20-45 μm. 300mL of acarbose impurity III enrichment solution is taken, pH is regulated to 3.0-6.0 by dilute hydrochloric acid, reduced pressure distillation and concentration are carried out to 30mg/mL, pH is regulated to 7.0-10.0 by dilute ammonia water, and finally, the concentration solution is obtained by filtering by a 0.45 mu m filter membrane.
(2) Eluting the concentrated solution under the separation and purification conditions of C 18 packing, and collecting fractions containing acarbose impurities III. Separation and purification conditions of C 18 packing: instrument: DAC50 was used to prepare a liquid chromatography system (Jiangsu Hanbang technology Co., ltd.); chromatographic column: welch Xtimate C 18, 10 μm filler charge 300g; mobile phase: methanol-water (0.1% nh 3) solution = 8-92 (V/V); flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(3) And purifying the fraction under NH 2 packing separation and purification conditions, and collecting the fraction with the purity of more than 95%. NH 2 packing separation and purification conditions: instrument: DAC50 was used to prepare a liquid chromatography system (Jiangsu Hanbang technology Co., ltd.); chromatographic column: daisogel SP-120-APS-P,10 μm filler charge 300g; mobile phase: methanol; flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(4) Mixing fractions of acarbose impurity III with purity of more than 95%, distilling under reduced pressure to remove methanol, adding 20mL of water for dissolution, adjusting pH to 5.0-6.0 with dilute hydrochloric acid, and pre-freezing in a refrigerator.
(5) Freeze-drying in a freeze dryer at-76 deg.C and 0.02Mpa to obtain white crystalline powder, which is the target product, acarbose impurity III, and storing at 2-8deg.C.
2. Acarbose impurity III Structure confirmation
(1) The high resolution mass spectrum is shown in fig. 10, and the analysis and conclusion are as follows:
Sample molecular formula: c 25H43NO18 absolute molecular weight M:645.2480
[ M+H ] + Experimental values: 646.2573 Theoretical value of [ M+H ] +: 646.2553
As can be seen from the high-resolution mass spectrum of the acarbose impurity III, in the positive ion mode, the relative error between the experimental value of the molecular formula [ M+H ] + of the sample and the theoretical value of [ M+H ] + corresponding to the molecular formula C 25H43NO18 is 3.05ppm, so that the molecular formula of the acarbose impurity III is confirmed to be C 25H43NO18.
(2) The nuclear magnetic resonance spectrum, the nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the sample are shown in FIGS. 11 and 12.
1 The proton signals of 13 groups of samples in the H-NMR spectrum (FIG. 11) show that the sample structure at least contains 13 groups of hydrogen nuclear spin systems with different chemical environments, and the integrated area of the sample structure at least contains 29 hydrogen atoms (methyl signals delta H1.962,s,3H).1 H-NMR spectrum without active hydrogen and residual additive acetic acid show proton signals on 1 group of double bonds in the low field region (12 groups of hydrogen proton signals in the high field region in the delta H5.909,m,1H);1 H-NMR spectrum, wherein the proton signals comprise 4 groups of overlapped proton signals (δH5.190,overlap,2H;δH4.096,overlap,2H;δH3.880-3.748,overlap,10H;δH3.697-3.632,overlap,4H),5 groups of methine proton signals (δH5.387,d,J＝3.9Hz,1H;δH4.017,dq,J＝12.5,6.2Hz,1H;δH3.942,m,1H;δH3.450,t,J＝9.5Hz,1H;δH2.897,t,J＝10.1Hz,1H),2 groups of proton signals (delta H4.235,d,J＝14.6Hz,1H;δH.205, d, J=14.6 Hz, 1H) and 1 group of methyl proton signals (delta H.395, d, J=6.2 Hz, 3H).
13 C-NMR (FIG. 12) shows that the samples contained 24 groups of 25 carbon signals (related carbon signals without residual additive acetic acid (CH 3COOH：δC22.500,δC 180.195)). The low field of the carbon spectrum exhibits 2 carbon signals, including 1 double bond quaternary carbon signal (delta C 143.025), 1 double bond tertiary carbon signal (delta C 118.923); the midfield portion exhibits 23 carbon signals, including 19 tertiary carbon signals (δC99.650;δC93.408;δC93.174;δC77.421;δC72.938;δC72.525;δC72.428;δC72.214;δC72.170;δC71.023;δC70.786;δC70.731;δC70.622;δC70.446;δC69.649;δC68.558;δC66.876;δC63.826;δC56.171) attached to heteroatoms and 3 secondary carbon signals (δ C61.326;δC 60.490 ×2) attached to heteroatoms and 1 primary carbon signal (δ C 17.314).
The nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the acarbose impurity III confirm that C, H belongs to the molecular structural formula of the sample, and the structural formula is as follows:
3. acarbose impurity III labelling
The HPLC purity of acarbose impurity iii was 98.5%, see fig. 9. The content of acarbose impurity III detected by a quantitative nuclear magnetic method is 89.4 percent, and the acarbose impurity III is used as an impurity reference substance.
Example 4
1. Acarbose impurity IV extraction
(1) The acarbose impurity IV is enriched from the acarbose sample by using an LC silica gel pre-column with the particle size of 20-45 mu m. 300mL of acarbose impurity IV enrichment solution is taken, the pH value is regulated to 3.0-6.0 by dilute hydrochloric acid, reduced pressure distillation and concentration are carried out to 30mg/mL, the pH value is regulated to 7.0-10.0 by dilute ammonia water, and finally the concentration solution is obtained by filtering by a 0.45 mu m filter membrane.
(2) Eluting the concentrated solution twice continuously under the separation and purification condition of C 18 packing, and collecting fractions containing acarbose impurity IV. Separation and purification conditions of C 18 packing: instrument: DAC50 was used to prepare a liquid chromatography system (Jiangsu Hanbang technology Co., ltd.); chromatographic column: welch Xtimate C 18, 10 μm filler charge 300g; mobile phase: methanol-water (0.1% nh 3) solution = 8-92 (V/V); flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(3) And (3) desalting the qualified fraction obtained by separating and purifying the C 18 filler under the following conditions, and collecting the fraction containing acarbose impurity IV and having the purity of more than 95%. Chromatographic conditions: instrument: DAC50 was used to prepare a liquid chromatography system (Jiangsu Hanbang technology Co., ltd.); chromatographic column: welch Xtimate C 18, 10 μm filler charge 300g; mobile phase: methanol-water solution=25-75 (V/V); flow rate: 50mL/min; sample injection volume: 10mL; detection wavelength: 210nm.
(4) Mixing acarbose impurity IV fractions with purity of more than 95%, vacuum distilling to remove the impurities, concentrating to 20mL, regulating pH to 5.0-6.0 with dilute hydrochloric acid, and pre-freezing in a refrigerator.
(5) And then freeze-drying is carried out in a freeze dryer at the temperature of minus 76 ℃ and the pressure of 0.02Mpa, so as to obtain white crystalline powder, namely the target product, acarbose impurity IV, and the product is preserved under the drying condition of 2-8 ℃.
2. Acarbose impurity IV structure confirmation
(1) The high resolution mass spectrum is shown in fig. 14, and the analysis and conclusion are as follows:
Sample molecular formula: c 19H33NO13 absolute molecular weight M:483.1952
[ M+H ] + Experimental values: 484.2015 Theoretical value of [ M+H ] +: 484.2025
As can be seen from the high-resolution mass spectrogram of the acarbose impurity D, in the positive ion mode, the relative error between the experimental value of the molecular formula [ M+H ] + of the sample and the theoretical value of [ M+H ] + corresponding to the molecular formula C 19H33NO13 is-2.07 ppm, so that the molecular formula of the acarbose impurity IV is confirmed to be C 19H33NO13.
(2) The nuclear magnetic resonance spectrum, the nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the sample are shown in FIGS. 15 and 16.
1 The occurrence of proton signals in the H-NMR spectrum (FIG. 15) can be divided into 14 groups, and the integration of the areas of the peaks of the hydrogen signals indicates that the sample contains at least 22 hydrogen atoms (no active hydrogen). 1 The low field part of the H-NMR spectrum sees the hydrogen signals on the 1 group double bond (δ H 5.941-5.932, d, J=5.0 Hz, 1H) and the 2 group sugar terminal methine hydrogen signals (5.359-5.350, d, J=3.4 Hz,1H;5.266-5.260, d, J=3.8 Hz, 1H); the mid-high field portion shares 11 sets of proton signal peaks including 3 overlapping sets of proton signals (δH 4.010-3.946,overlap,0.8H;δH3.811-3.754,overlap,3.6H;δH 3.654-3.565,overlap,4H),4 sets of methine signals (δH 4.083-4.072,d,J＝7.0Hz,1H;δH3.716-3.692,dd,J＝9.8,4.6Hz,1H;δH 3.581-3.565,d,J＝6.7Hz,1H;δH 2.526-2.495,t,J＝9.5Hz,1H),3 sets of carbon-coupled methylene signals (4.275-4.252, d, j=13.8 hz,1h; δ H 4.164-4.141,d,J＝13.8Hz,1H;δH 3.925.925-3.854, m,1 h) and 1 set of methyl signals (δ H 1.384-1.373, d, j=6.3 hz,3 h).
13 C-NMR (FIG. 16) showed that the sample contained 19 carbon signals, the low field of the carbon spectrum showed 4 carbon signals, including the carbon signal on 2 double bonds (delta C141.618,s;δC.338, d), the tertiary carbon signal on 2 sugar end groups (delta C102.492/102.373,d;δC.94.510, d). The middle-high field portion exhibited 15 carbon signals, including 11 tertiary carbon signals (δC79.945,d;δC 75.881,d;δC 75.542,d;δC75.358/75.254,d;δC73.882,d;δC 73.769,d;δC73.374,d;δC 72.625,d;δC 72.154,d;δC 67.568/67.540,d;δC58.595,d),2 methylene carbon signals (δ C64.213,t;δC 63.370/63.252, t) and 1 methyl carbon signal (δ C.19.932, q) on the six-membered cyclic sugar.
The nuclear magnetic resonance hydrogen spectrum (1 H-NMR) and the carbon spectrum (13 C-NMR) of the acarbose impurity IV confirm that C, H belongs to the molecular structural formula of the sample, and the structural formula is as follows:
3. Acarbose impurity IV labeling
The HPLC purity of acarbose impurity iv was 96.0%, see fig. 13. The content of acarbose impurity IV detected by a quantitative nuclear magnetic method is 92.0 percent, and the acarbose impurity IV is used as an impurity reference substance.
According to the invention, impurities are separated and prepared, so that an impurity monomer is obtained, a theoretical basis is provided for safe use of the medicine, effective data support is provided for improving the quality standard of acarbose, and effective guarantee is provided for clinical safe use of acarbose.
By using the technical scheme of the invention or under the inspired by the technical scheme of the invention, a similar technical scheme is designed by a person skilled in the art, so that the technical effects are achieved, and the technical effects fall into the protection scope of the invention.
Claims (6)
1. The preparation method of the acarbose impurity reference substance is characterized by comprising the following steps:
1) Taking acarbose impurity enrichment liquid, regulating the pH value to 3.0-6.0 by dilute hydrochloric acid, and concentrating by reduced pressure distillation until each milliliter of the enrichment liquid contains 10-40mg of acarbose impurities, thus obtaining acarbose impurity enrichment liquid;
2) Adjusting the pH value of the concentrated solution in the step 1) to 7.0-10.0 by using dilute ammonia water, and filtering the concentrated solution by using a filter membrane to obtain an impurity concentrated solution;
3) Eluting the impurity concentrated solution twice in reverse phase silica gel packing separation and purification condition, and collecting fraction containing acarbose impurity; the reverse silica gel filler is C 18 filler, and the separation and purification conditions are as follows: chromatographic column: welch Xtimate C 18, 10 μm filler packing 200-400g; mobile phase: the volume ratio of methanol to aqueous solution is 8:92; flow rate: 40-70mL/min; sample injection volume: 10mL; detection wavelength: 210 nm, wherein the methanol-water solution contains 0.1% NH 3;
4) Purifying the fraction collected in the step 3) under polyamide filler separation and purification conditions, and collecting the fraction with the purity of more than 95%;
5) Mixing the fractions with the purity of more than 95% collected in the step 4), distilling under reduced pressure to remove methanol, adding water for dissolution, adjusting the pH value to 3.0-6.0 by dilute hydrochloric acid, and pre-freezing in a refrigerator;
6) Then freeze-drying the fraction after the pre-freezing treatment in the step 5) in a freeze dryer to obtain white crystalline powder, namely acarbose impurities;
the acarbose impurities in the step 1) are respectively impurity I, impurity II, impurity III and impurity IV, and the structural formulas are respectively as follows:
；
Acarbose impurity I
；
Acarbose impurity II
；
Acarbose impurity III
；
Acarbose impurity IV.
2. The method for preparing acarbose impurity control according to claim 1, wherein the polyamide filler in the step 4) is NH 2 filler, and the separation and purification conditions are as follows: chromatographic column: welch Ultimate XB-NH 2, 10 μm filler charge 150-350g; mobile phase: methanol; flow rate: 40-70mL/min; sample injection volume: 10mL; detection wavelength: 210 nm.
3. The method for preparing acarbose impurity control according to claim 1, characterized in that the flow rate is 50-60mL/min.
4. The method for preparing acarbose impurity control according to claim 1, wherein the amount of acarbose enrichment liquid collected in step 1) each time is 200-500ml, and the volume dissolved by adding water in step 5) is 20-50ml.
5. The method for preparing acarbose impurity control according to claim 1, characterized in that the conditions of the freeze dryer in step 6) are: the freezing temperature is-70 ℃ to-80 ℃ under the pressure condition of 0.01-0.03 Mpa.
6. The method for preparing acarbose impurity control according to claim 5, wherein the freezing pressure is 0.02 Mpa and the freezing temperature is-76 ℃.
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