CN113248400A - Preparation method of folic acid intermediate impurity - Google Patents
Preparation method of folic acid intermediate impurity Download PDFInfo
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- CN113248400A CN113248400A CN202110540735.3A CN202110540735A CN113248400A CN 113248400 A CN113248400 A CN 113248400A CN 202110540735 A CN202110540735 A CN 202110540735A CN 113248400 A CN113248400 A CN 113248400A
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- mobile phase
- perborate
- folic acid
- molybdate
- acid intermediate
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- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 82
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 42
- 239000011724 folic acid Substances 0.000 title claims abstract description 42
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 229960000304 folic acid Drugs 0.000 title claims abstract description 39
- 239000012535 impurity Substances 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- MEFBJEMVZONFCJ-UHFFFAOYSA-N molybdate Chemical compound [O-][Mo]([O-])(=O)=O MEFBJEMVZONFCJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000003960 organic solvent Substances 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 44
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 23
- 238000001514 detection method Methods 0.000 claims description 22
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 19
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 14
- 229910052700 potassium Inorganic materials 0.000 claims description 14
- 239000011591 potassium Substances 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 13
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 13
- 229960001922 sodium perborate Drugs 0.000 claims description 13
- YKLJGMBLPUQQOI-UHFFFAOYSA-M sodium;oxidooxy(oxo)borane Chemical compound [Na+].[O-]OB=O YKLJGMBLPUQQOI-UHFFFAOYSA-M 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 235000015393 sodium molybdate Nutrition 0.000 claims description 11
- 239000011684 sodium molybdate Substances 0.000 claims description 11
- 239000013078 crystal Substances 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 238000010829 isocratic elution Methods 0.000 claims description 7
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 229910052744 lithium Inorganic materials 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 239000012071 phase Substances 0.000 description 54
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- 238000002425 crystallisation Methods 0.000 description 7
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- 239000000047 product Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
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- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 208000032170 Congenital Abnormalities Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 229930003761 Vitamin B9 Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
- C07C245/02—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides
- C07C245/06—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings
- C07C245/08—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a preparation method of folic acid intermediate impurities, which comprises the following steps: mixing N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate in organic solvent, and reacting at 20-80 deg.c.
Description
Technical Field
The invention relates to the technical field of cosmetics, and particularly relates to a preparation method of folic acid intermediate impurities.
Background
Folic acid, also known as vitamin B9, is a water-soluble vitamin that plays an important role in the growth, development, and metabolism of the human body. The activity of the folate metabolism enzyme is reduced due to the mutation of key genes participating in the folate metabolism pathway, so that the folate metabolism is disturbed, and the folate deficiency is caused. The demand of the pregnant woman for folic acid is 4 times higher than that of normal people, the lack of folic acid of the pregnant woman can cause serious consequences such as fetal birth defects, neural tube malformation and the like, and the risk of pregnancy-induced hypertension, spontaneous abortion and the like of the pregnant woman can be increased. The over-amount of folic acid affects the absorption of zinc element, which may lead to abnormal fetal development and increase the risk of fetal malformation. The reasonable use of folic acid has great influence on the maintenance of the physiological status of pregnant women and the normal development of fetuses. The risk type genes exist in certain proportion in Chinese population, so that different people have different folic acid utilization capacities, and folic acid supplementation needs individuation.
Although the mechanism of folic acid synthesis is composed of three fragments, although the mechanism is studied for many years, it is not clear whether the reaction is carried out in a synergistic manner or in a stepwise manner, wherein one possibility is that the first fragment is firstly cyclized with the second fragment and then nucleophilic substituted with the third fragment to generate folic acid:
based on this process reaction mechanism, it is presumed that (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid may be generated during the reaction, and the compound structure thereof is as follows:
(2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid is an azo compound, the structure of which is warned according to genotoxic impurities, and the compound is genotoxic impurities and needs to be quantitatively controlled strictly according to related guiding principles, but a synthetic method of the compound is not reported so far, and the synthesis is not easy to supposedly. In addition, the impurities are not commercially available at present, which adds a little difficulty to the research of related substances of folic acid raw materials.
Disclosure of Invention
In view of the above, it is necessary to provide a method for producing (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid, which has not been a problem of a definite synthetic method.
A preparation method of folic acid intermediate impurities comprises the following steps:
mixing N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate in organic solvent, and reacting at 20-80 deg.c.
In some embodiments, the perborate salt is selected from one or more of anhydrous sodium perborate, sodium perborate with crystal water, anhydrous potassium perborate, potassium perborate with crystal water, anhydrous lithium perborate, lithium perborate with crystal water.
In some of these embodiments, the molybdate is selected from one or more of sodium molybdate anhydrous, sodium molybdate with water of crystallization, potassium molybdate anhydrous and potassium molybdate with water of crystallization.
In some embodiments, the organic solvent is selected from one or both of formic acid and acetic acid.
In some embodiments, the molar ratio of perborate to molybdate is 5 to 15.
In some embodiments, the molar ratio of perborate to N-p-aminobenzoyl-L-glutamic acid is 0.1 to 5.
In some embodiments, the reaction temperature is 40-60 ℃, and the reaction time is 50-80 min.
In some of the embodiments, the method further comprises the steps of concentrating the reaction solution under reduced pressure after the reaction, and then dissolving the concentrated product with water and filtering.
In some embodiments, the method further comprises a step of detecting the filtered product, wherein the detecting step comprises:
the detection is carried out by adopting a high performance liquid chromatography, and the mobile phase consists of a mobile phase A and a mobile phase B, and the detection method is characterized in that the mobile phase A is a dipotassium hydrogen phosphate solution with the concentration, and the mobile phase B is methanol.
In some of the embodiments, the pH value of the dipotassium hydrogen phosphate solution is 2.0-3.0, and the concentration of the dipotassium hydrogen phosphate is 1 g/L-1.4 g/L.
In some of these embodiments, a gradient mobile phase elution is used, wherein the gradient of the mobile phase is set as follows, based on 100% of the volume of the mobile phase:
the volume ratio of the mobile phase A to the mobile phase B is kept between 90 and 99 and between 10 and 1 within 0 to 10min for isocratic elution;
within 10-35 min, gradually changing the volume ratio of the mobile phase A to the mobile phase B to (55-65): 45-35);
within 35-55 min, gradually changing the volume ratio of the mobile phase A to the mobile phase B to (65-75): 35-25);
the volume ratio of the mobile phase A to the mobile phase B is kept between 65 and 75 and between 35 and 25 for isocratic elution within 55 to 60 minutes;
within 60min-60.1min, the volume ratio of the mobile phase A to the mobile phase B gradually changes to (90-99): 10-1);
the volume ratio of the mobile phase A to the mobile phase B is kept (90-99) to (10-1) unchanged within 60.1-70 min.
According to the invention, the compound (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid is prepared by the reaction of N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate, and the compound with higher purity can be obtained by the method through analysis. And chromatographic analysis is carried out on the compound on the basis of the preparation method, so that the detection method of the compound is determined, and the feasibility of the method for confirming specificity (separation degree experiment of impurities and main components), precision, detection limit, accuracy and linear range is carried out. The invention fills the blank of industrial synthesis of the impurity compound in the folic acid intermediate, and lays a foundation for quantitative control of the impurity compound in the folic acid and research on related substances of folic acid raw materials.
Drawings
FIG. 1 is a mass spectrum of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid prepared according to one embodiment of the present invention;
FIG. 2 is an H spectrum of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid prepared according to one embodiment of the present invention;
FIG. 3 is a C spectrum of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid prepared according to one embodiment of the present invention;
FIG. 4 shows the specificity of the detection method for (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid according to an embodiment of the present invention;
FIG. 5 is a graph of the linear relationship of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid in accordance with one embodiment of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The embodiment of the invention provides a preparation method of folic acid intermediate impurities, which comprises the following steps:
mixing N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate in organic solvent, and reacting at 20-80 deg.c.
The chemical structural formula of the N-p-aminobenzoyl-L-glutamic acid is as follows:
the chemical structural formula of the compound (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid prepared by the reaction is as follows:
according to the invention, the compound (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid is prepared by the reaction of N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate, and the compound with higher purity can be obtained by the method through analysis. And chromatographic analysis is carried out on the compound on the basis of the preparation method, so that the detection method of the compound is determined, and the feasibility of the method for confirming specificity (separation degree experiment of impurities and main components), precision, detection limit, accuracy and linear range is carried out. The invention fills the blank of the industrial synthesis path of the impurity compound in the folic acid intermediate, and lays a foundation for the quantitative control of the impurity compound in the folic acid and the research of related substances of folic acid raw materials.
Preferably, the step of mixing N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate in an organic solvent may be: perborate and molybdate are mixed in an organic solvent, and then N-p-aminobenzoyl-L-glutamic acid is added to the mixture.
In some embodiments, the perborate salt is selected from one or more of anhydrous sodium perborate, sodium perborate with water of crystallization, anhydrous potassium perborate, potassium perborate with water of crystallization, anhydrous lithium perborate, lithium perborate with water of crystallization.
In some embodiments, the molybdate is selected from one or more of sodium molybdate anhydrous, sodium molybdate with water of crystallization, potassium molybdate anhydrous and potassium molybdate with water of crystallization.
In some embodiments, the organic solvent is selected from one or both of formic acid and acetic acid. Preferably, the organic solvent is acetic acid.
In some embodiments, the molar ratio of the perborate salt to the molybdate salt is 5 to 15. Specifically, the molar ratio of borate to molybdate may be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15: 1. Preferably, the molar ratio of the borate to the molybdate is 9 to 12, and more preferably 10 to 11.
In some embodiments, the molar ratio of perborate to N-p-aminobenzoyl-L-glutamic acid is 0.1 to 5. Specifically, the molar ratio of perborate to N-p-aminobenzoyl-L-glutamic acid may be 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5: 1. Preferably, the molar ratio of perborate to N-p-aminobenzoyl-L-glutamic acid is 0.5 to 2, more preferably 0.8 to 1.2.
Specifically, the reaction temperature may be 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃. Preferably, the temperature of the reaction is 40 to 60 ℃.
Specifically, the reaction time of the mixed N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate in the organic solvent may be 30min to 120min, specifically 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min and 120 min. Preferably, the reaction time is 50min to 80 min.
In some embodiments, the method further comprises the step of concentrating the reaction solution under reduced pressure after the above reaction, and then dissolving the concentrated product with water and filtering to obtain a white solid after filtration.
The method has the advantages of mild reaction conditions, cheap and easily-obtained starting materials for reaction, short reaction steps, simple post-treatment and high purity of obtained impurities.
In some embodiments, the method further comprises a step of detecting the product obtained by filtering, and verifying whether the product obtained by preparation contains the compound (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid and the content thereof.
The detection step of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid comprises:
detecting by adopting a high performance liquid chromatography, wherein the mobile phase consists of a mobile phase A and a mobile phase B, the mobile phase A is a dipotassium hydrogen phosphate solution with the concentration, and the mobile phase B is methanol.
In some embodiments, the dipotassium hydrogen phosphate solution has a pH of 2.0 to 3.0 and the dipotassium hydrogen phosphate has a concentration of 1g/L to 1.4 g/L.
In some embodiments, a gradient mobile phase elution is used, the gradient of the mobile phase being set as follows, based on 100% volume of the mobile phase:
the volume ratio of the mobile phase A to the mobile phase B is kept between 90 and 99 and between 10 and 1 within 0 to 10min for isocratic elution;
within 10-35 min, gradually changing the volume ratio of the mobile phase A to the mobile phase B to (55-65): 45-35);
within 35-55 min, gradually changing the volume ratio of the mobile phase A to the mobile phase B to (65-75): 35-25);
the volume ratio of the mobile phase A to the mobile phase B is kept between 65 and 75 and between 35 and 25 for isocratic elution within 55 to 60 minutes;
within 60min-60.1min, the volume ratio of the mobile phase A to the mobile phase B gradually changes to (90-99): 10-1);
the volume ratio of the mobile phase A to the mobile phase B is kept (90-99) to (10-1) unchanged within 60.1-70 min.
In the detection method, a silane-bonded silica gel is preferably used as a column, and for example, the column is phenyl-Hexyl.
Preferably, the length of the chromatographic column is 150 mm-160 mm, the diameter is 4 mm-5 mm, and the particle size of the filler is 2.5 μm-3.5 μm.
Preferably, the detection wavelength is 260nm to 270nm, preferably 264 nm.
In some embodiments, the flow rate is 0.6mL/mL to 0.8mL/mL, preferably 0.7 mL/mL.
In some embodiments, the column temperature is from 25 ℃ to 40 ℃, preferably 30 ℃.
In some embodiments, the sample solution is prepared by: the sample was dissolved in dipotassium hydrogen phosphate solution. The pH value of the dipotassium phosphate solution is 2-3, and preferably 2.5. The concentration of dipotassium hydrogen phosphate is 1 g/L-1.4 g/L, preferably 1.2 g/L.
In some embodiments, the sample size is 5 μ L to 50 μ L, and specifically may be 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L, 50 μ L. Preferably, it may be 10. mu.L.
The detection method is not only suitable for detecting the (2S,2'S) -2,2' - (4,4'- (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid prepared by the preparation method, but also suitable for detecting (2S,2' S) -2,2'- (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid in other folic acid related samples, such as folic acid samples or folic acid intermediate samples.
The following are specific examples.
The compound II in the following examples refers to (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Example 1: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 1.69g of sodium perborate and 0.24g of sodium molybdate, adding into a reaction bottle, adding 10ml of acetic acid, heating to 50 ℃, and stirring for dissolving; adding 3.99g of compound N-p-aminobenzoyl-L-glutamic acid into the reaction solution, and reacting for 1h at 50 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain the final product with purity of 95.3%.
Example 2: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 1.69g of sodium perborate and 0.48g of sodium molybdate, adding into a reaction bottle, adding 20ml of acetic acid, heating to 50 ℃, and stirring for dissolving; adding 6.81g of compound N-p-aminobenzoyl-L-glutamic acid into the reaction solution, and reacting for 1h at 50 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain the final product with purity of 96.0%.
Example 3: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 1.69g of potassium perborate and 0.24g of potassium molybdate, adding into a reaction bottle, adding 10ml of acetic acid, heating to 50 ℃, and stirring for dissolving; adding 3.99g of compound N-p-aminobenzoyl-L-glutamic acid into the reaction solution, and reacting for 50min at 50 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain the final product with purity of 95.3%.
Example 4: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 1.69g of sodium perborate and 0.24g of sodium molybdate, adding into a reaction bottle, adding 10ml of acetic acid, heating to 50 ℃, and stirring for dissolving; adding 3.99g of compound N-p-aminobenzoyl-L-glutamic acid into the reaction solution, and reacting for 40min at 80 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain the final product with purity of 94.8%.
Example 5: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 1.69g of sodium perborate and 0.24g of sodium molybdate, adding into a reaction bottle, adding 10ml of acetic acid, heating to 50 ℃, and stirring for dissolving; adding 3.99g of compound N-p-aminobenzoyl-L-glutamic acid into the reaction solution, and reacting for 1h at 100 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain the final product with purity of 91.1%.
Example 6: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 1.69g of sodium perborate and 1.69g of sodium molybdate, adding into a reaction bottle, adding 10ml of formic acid, heating to 50 ℃, and stirring for dissolving; adding 3.99g of compound N-p-aminobenzoyl-L-glutamic acid into the reaction solution, and reacting for 1h at 50 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain the final product with purity of 90.5%.
Example 7: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 16.9g of sodium perborate and 0.24g of sodium molybdate, adding into a reaction bottle, adding 10ml of acetic acid, heating to 50 ℃, and stirring for dissolving; adding 3.99g of compound N-p-aminobenzoyl-L-glutamic acid into the reaction solution, and reacting for 1h at 50 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain final product with purity of 87.9%.
Example 8: preparation of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid
Weighing 1.69g of sodium perborate and 0.24g of sodium molybdate, adding into a reaction bottle, adding 10ml of acetic acid, heating to 50 ℃, and stirring for dissolving; 133g of compound N-p-aminobenzoyl-L-glutamic acid is added into the reaction solution, and the reaction is carried out for 1h at 50 ℃; concentrating the reaction solution under reduced pressure, dissolving the residue with 40ml water under stirring for 20min, filtering to obtain white solid, drying, separating and purifying with preparative liquid phase, and lyophilizing to obtain the final product with purity of 85.4%.
The structural analysis of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid prepared in the examples of the present invention is shown in the following tables 1 and 2, fig. 1 to 3:
TABLE 1 Mass Spectrometry
TABLE 2 nuclear magnetism
Example 9 detection of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid in a sample
1. Chromatographic conditions
A chromatographic column: silane bonded silica gel is used as a filling agent; (phenyl-Hexyl C18, 4.6X 150mm, 3 μm)
Column temperature: 30 ℃;
detection wavelength: 264 nm;
flow rate: 0.7 ml/min;
sample introduction amount: 10 mu l of the mixture;
mobile phase A: 1.2g/L dipotassium hydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid);
mobile phase B: methanol;
gradient elution comprises the following steps: (1) the volume ratio of the mobile phase A to the mobile phase B is kept to be 95:5 isocratic elution within 0-10 minutes; (2) the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95:5 to 60:40 within 10-35 minutes; (3) the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 60:40 to 30:70 at a constant speed within 35-55 minutes; (4) the volume ratio of the mobile phase A to the mobile phase B is kept to be eluted at 30:70 isocratic within 55-60 minutes; (5) the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 95:5 at a constant speed within 60-60.1 minutes; (4) the volume ratio of mobile phase A to mobile phase B was kept constant at 95:5 for 60.1-70 minutes. The detailed elution procedure is shown in table 3 below.
TABLE 3 elution gradient conditions
2. Solution preparation
Control solution: an appropriate amount of a control (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid was dissolved in 1.2g/L dipotassium hydrogenphosphate solution (pH2.5) to prepare a solution containing about 0.5mg of Compound II per 1ml, as a control solution.
Test solution: taking a proper amount of the folic acid intermediate to be detected, adding 1.2g/L dipotassium hydrogen phosphate solution (pH2.5) for dissolving, and preparing a solution containing about 2mg folic acid intermediate in each 1ml to be used as a test solution.
3. Detection method verification
3.1 specificity
System applicability solution: a proper amount of the folic acid intermediate was added to a quantitative control solution, and 1.2g/L of a dipotassium hydrogenphosphate solution (pH2.5) was added to dissolve the folic acid intermediate to prepare a solution containing about 2mg of the folic acid intermediate and 0.75. mu.g of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid per 1ml, as a system suitability solution.
Sampling the system applicability solution, recording the chromatogram, and investigating the specificity of the method, wherein the results are shown in the following table 4 and fig. 4.
TABLE 4 specificity results
And (4) conclusion: the method can effectively separate the folic acid intermediate from the (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid, and has good specificity.
3.2 precision of sample introduction
The above control solution was sampled continuously for 6 needles, the chromatogram was recorded, and the precision of the sample injection was examined, the results are shown in Table 5 below.
TABLE 5 sample introduction precision results
And (4) conclusion: the determination method samples 6 times and the RSD percent of the peak area of the reference sample is less than 2.0; retention time RSD% is less than 1.0; the sample injection precision is good.
3.3 detection limit, quantitation limit
The detection limit and quantitation results for (2S,2'S) -2,2' - (4,4'- (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid are shown in table 6 below for a control solution of (2S,2' S) -2,2'- (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid that was gradually diluted and injected after the control solution was added.
TABLE 6 results of detection of the limiting limits
| Detecting items | Concentration (μ g/ml) | Corresponding to the concentration of the test sample% | S/N |
| Detection limit | 0.2525 | 0.0505 | 10.98 |
| Limit of quantification | 0.7575 | 0.1515 | 41.84 |
And (4) conclusion: the S/N values of the detection limit and the quantification limit are both larger than 10, and the detection requirement is met.
3.4 Linear relationship and Range
Linear solution: an appropriate amount of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid control was dissolved in 1.2g/L dipotassium hydrogen phosphate solution (ph2.5) to prepare linear solutions of each concentration, and the results of the chromatogram recording by sample injection were shown in table 7 and fig. 5.
TABLE 7 results of linear relationship
| Name (R) | Concentration (μ g/ml) | A | Corresponding to the concentration of the test sample% |
| Line-1 | 0.2525 | 3287 | 0.05％ |
| Line-2 | 0.505 | 6816 | 0.10％ |
| Line-3 | 5.05 | 56058 | 1％ |
| Line-4 | 10.1 | 111729 | 2％ |
| Line-5 | 15.15 | 169114 | 3％ |
| Line-6 | 37.875 | 435704 | 7.50％ |
| Line-7 | 252.5 | 2887422 | 50％ |
| Line-8 | 505 | 6325852 | 100％ |
| Line-9 | 757.5 | 9428526 | 150％ |
And (4) conclusion: the results show that the peak area and the concentration of the compound II are in a good linear relationship within the concentration range of 0.2525-757.5 mu g/ml.
3.5 degree of accuracy
An appropriate amount of the compound II is precisely weighed, and a solution of 1.2g/L dipotassium hydrogen phosphate (pH2.5) is used for preparing impurity mother liquor with a certain concentration. 9 parts of sample are precisely weighed, 50 percent, 100 percent and 150 percent of impurity mother liquor with limited amount of impurities are respectively added, and the sample is diluted to scale by 1.2g/L dipotassium hydrogen phosphate solution (pH2.5), wherein each concentration is in parallel with three parts. Each 10. mu.l of the solution was measured precisely and injected into a liquid chromatograph to measure the recovery rate, and the results are shown in Table 8 below.
TABLE 8 accuracy results
And (4) conclusion: the recovery rates of (2S,2' S) -2,2' - (4,4' - (hydrazino-1, 2-diyl) bis-benzoyl) diglutamic acid are all between 90% and 110%, and the recovery rate test requirements are met.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the patent protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.
Claims (10)
1. A preparation method of folic acid intermediate impurities is characterized by comprising the following steps:
mixing N-p-aminobenzoyl-L-glutamic acid, perborate and molybdate in organic solvent, and reacting at 20-80 deg.c.
2. The method of producing a folic acid intermediate impurity of claim 1, characterized in that the perborate salt is selected from one or more of anhydrous sodium perborate, sodium perborate with crystal water, anhydrous potassium perborate, potassium perborate with crystal water, anhydrous lithium perborate, lithium perborate with crystal water.
3. The method of producing folic acid intermediate impurities of claim 1 wherein said molybdate is selected from one or more of the group consisting of anhydrous sodium molybdate, sodium molybdate with crystal water, anhydrous potassium molybdate, and potassium molybdate with crystal water.
4. The method of producing folic acid intermediate impurities of claim 1, characterized in that said organic solvent is one or two selected from formic acid and acetic acid.
5. The method for preparing folic acid intermediate impurities as defined in claim 1, wherein the molar ratio of perborate to molybdate is 5-15, and the molar ratio of perborate to N-p-aminobenzoyl-L-glutamic acid is 0.1-5.
6. The method for preparing folic acid intermediate impurities according to claim 1, characterized in that the reaction temperature is 40-60 ℃ and the reaction time is 50-80 min.
7. The method of producing folic acid intermediate impurities according to claim 1, further comprising the step of, after said reaction, concentrating the reaction solution under reduced pressure, and then dissolving the concentrated product with water and filtering.
8. The method of claim 7, further comprising the step of detecting the filtered product, wherein the step of detecting comprises:
the detection is carried out by adopting a high performance liquid chromatography, and the mobile phase consists of a mobile phase A and a mobile phase B, and the detection method is characterized in that the mobile phase A is a dipotassium hydrogen phosphate solution with the concentration, and the mobile phase B is methanol.
9. The method for preparing folic acid intermediate impurities according to claim 8, wherein the pH value of the dipotassium phosphate solution is 2.0-3.0, and the concentration of the dipotassium phosphate is 1-1.4 g/L.
10. The method of claim 9, wherein a gradient mobile phase elution is used, wherein the gradient of the mobile phase is set as follows, based on 100% volume of the mobile phase:
the volume ratio of the mobile phase A to the mobile phase B is kept between 90 and 99 and between 10 and 1 within 0 to 10min for isocratic elution;
within 10-35 min, gradually changing the volume ratio of the mobile phase A to the mobile phase B to (55-65): 45-35);
within 35-55 min, gradually changing the volume ratio of the mobile phase A to the mobile phase B to (65-75): 35-25);
the volume ratio of the mobile phase A to the mobile phase B is kept between 65 and 75 and between 35 and 25 for isocratic elution within 55 to 60 minutes;
within 60min-60.1min, the volume ratio of the mobile phase A to the mobile phase B gradually changes to (90-99): 10-1);
the volume ratio of the mobile phase A to the mobile phase B is kept (90-99) to (10-1) unchanged within 60.1-70 min.
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