CN109096367B - Separation and purification method of impurity G generated in reduced glutathione fermentation process and application thereof - Google Patents
Separation and purification method of impurity G generated in reduced glutathione fermentation process and application thereof Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 89
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 71
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0819—Tripeptides with the first amino acid being acidic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
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Abstract
The invention discloses a method for separating and purifying an impurity G, namely L-glutamic acid-L-cysteine-alpha-alanine, generated in a reduced glutathione fermentation process, which comprises five steps of sample concentration, sample preparation, first purification and separation, second purification and separation and purity detection. The invention also discloses application of the impurity G as an impurity reference substance in detection of reduced glutathione-related substances. The method has the advantages that unknown fermentation impurities in the fermentation process of the reduced glutathione are positioned, determined, separated and purified, and the application of the impurities as impurity reference substances in the detection of related substances of the reduced glutathione is utilized, so that the medicine quality and the clinical medication safety of the existing reduced glutathione are improved.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a separation and purification method of an impurity G generated in a reduced glutathione fermentation process, namely a separation and purification method of L-glutamic acid-L-cysteine-alpha-alanine, and application of the separation and purification method as an impurity reference substance in detection of reduced glutathione related substances.
Background
Glutathione has two forms, namely, reduced form (G-SH) and oxidized form (G-S-S-G), wherein reduced glutathione is the majority of glutathione under physiological conditions, and glutathione reductase can catalyze interconversion between the two forms. The coenzyme of the enzyme is NADPH provided by the pentose phosphate shunt metabolism. Reduced glutathione is present in almost every cell of the body. The reduced glutathione can help to maintain the functions of a normal immune system and has the functions of antioxidation and integrated detoxification, and the sulfhydryl on the cysteine is an active group (so the sulfhydryl is often abbreviated as G-SH) of the reduced glutathione and is easy to be combined with certain medicines (such as paracetamol), toxins (such as free radicals, iodoacetic acid, mustard gas, lead, mercury, arsenic and other heavy metals) and the like, so the reduced glutathione has the functions of integrated detoxification. Reduced glutathione (especially glutathione in liver cells) can participate in biotransformation, so that harmful toxic substances in the organism are transformed into harmless substances and excreted out of the body, therefore, the glutathione has broad-spectrum detoxification function, can be used for medicines, can be used as a base material of functional foods, and is widely applied to the functional foods for delaying senility, enhancing immunity, resisting tumors and the like.
According to the technical guidelines for the study of impurities in chemical drugs, any substance that affects the purity of a drug is collectively referred to as an impurity. The research on impurities is an important content of drug development. Reduced glutathione is produced by biological fermentation, other fermentation impurities can be produced in the fermentation process, and the impurities can not be removed in the purification process, and can be introduced into the final finished product of the raw material medicine, thereby influencing the quality of the product. Therefore, the detection and control of impurities in the reduced glutathione bulk drug and the preparation are regarded as important guarantees for ensuring the drug quality and the clinical medication safety. Therefore, it is necessary to intensively study impurities of reduced glutathione.
In the existing research on fermentation impurities of reduced glutathione, 4 known impurities (a-D) and 1 unknown impurity are specified under the examination item of impurities of reduced glutathione in european pharmacopoeia 9.0, and the known impurities have the following structures:
in the Chongqing Yaoyou patent "a separation and purification method of reduced glutathione degradation product F", a known impurity F is reported, which is an oxidation product of reduced glutathione and has the following structure:
according to the data of the inquired literature, K lapheck S finds that homologues of glutathione exist in plants in 1988, for example, leguminous plants can synthesize high glutathione, namely glycine at the hydroxyl terminal of reduced glutathione is replaced by beta-alanine to form gamma-glutamic acid-cysteine-beta-alanine; in contrast, the fermentation produced glycine at the hydroxyl terminus of reduced glutathione was replaced with alpha-alanine.
In the research process of reduced glutathione related substances, the existence of an unknown impurity is always found in the fermentation process, the purification process and the finished product except for the above-found impurities.
The common impurity detection methods include a self-control method, an internal standard method, an external standard method and the like, and the high performance liquid chromatography self-control method adopting a correction factor in the import registration standard of the reduced glutathione for injection is used for detection. The self-contrast method is that on the premise that all components are peaked, all impurities are separated from the main component, and all impurities are the same as the response factors of the main component, the detection result can completely and truly indicate the purity of the substance, and the correction factors are added to overcome the errors of the impurities different from the response factors of the main component, so that the result is more accurate. The method for detecting the impurity content by adopting the known impurity reference substance method is not influenced by inconsistent response with the main component, and can accurately position the impurity and accurately determine the impurity content.
Therefore, the development of the separation and purification of the unknown impurities and the application of the unknown impurities as impurity reference substances in the detection of reduced glutathione related substances are important guarantee means for ensuring the quality of reduced glutathione medicines and the safety of clinical medication.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a method for separating and purifying impurity G generated in the reduced glutathione fermentation process, namely a method for separating and purifying L-glutamic acid-L-cysteine-alpha-alanine generated in the reduced glutathione fermentation process.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a method for separating and purifying impurity G generated in the process of reduced glutathione fermentation comprises the following steps:
step (1), concentrating a sample: firstly, carrying out rotary evaporation and vacuum concentration on reduced glutathione crystallization mother liquor to obtain a first solid;
step (2) sample preparation: dissolving the first solid obtained in the step (1) by using deionized water to prepare a first solution;
step (3), first purification and separation: performing gradient elution on the first solution prepared in the step (2) by using a cation exchange column after pH adjustment, monitoring by using high performance liquid chromatography in the elution process, and combining the first eluents according to the detection result;
and (4) second purification and separation: performing vacuum concentration on the first eluent obtained in the step (3) to obtain a second solid, dissolving the second solid with deionized water to obtain a second solution, performing second separation by using a high performance liquid preparative chromatography, performing separation according to an online chromatogram, positioning by using a high performance liquid, determining a collecting solution containing an impurity G, combining the collecting solutions, and performing freeze drying treatment to obtain a white solid, namely the impurity G;
and (5) purity detection: after the impurity G obtained in the step (4) is pretreated, a high performance liquid chromatograph is used to perform purity analysis, and the chromatographic column and the mobile phase used in the high performance liquid chromatograph are the same as those used in the step (4) and the high performance liquid chromatography is used for positioning.
In a preferred embodiment of the present invention, in step (2), 250mg of the first solid is contained per 1ml of the first solution.
In a preferred embodiment of the present invention, in step (3), the pH of the pH-adjusted cation exchange column is adjusted to 6 with NaOH at a concentration of 20% by mass before loading.
In a preferred embodiment of the present invention, in the step (3), the diameter of the cation exchange column after pH adjustment is 5-10cm, and the height thereof is 50-100 cm; the height is preferably 80 cm.
In a preferred embodiment of the present invention, in step (3), the filler in the cation exchange column after pH adjustment is any one or a mixture of two or more of D001x7 strong acid cation exchange resin, D151 macroporous weak acid acrylic cation exchange resin, D152D151 macroporous weak acid acrylic cation exchange resin, and CD-180 macroporous weak acid cation exchange resin.
In a most preferred embodiment of the present invention, in step (3), the packing material in the pH-adjusted cation exchange column is D001x7 strong acid cation exchange resin with a corresponding particle size range of 0.315 to 1.25 mm; the diameter of the cation exchange column after pH adjustment is 6 cm.
In a preferred embodiment of the invention, in the step (3), gradient elution is adopted in the elution process, wherein the gradient elution step is that water is firstly used for elution, the flow rate is 15-20 ml per minute, and the elution time is 30-45 minutes; then, eluting with 1.8 percent acetic acid aqueous solution by volume percentage concentration, wherein the flow rate is 15-20 ml per minute, and the elution time is 20-30 minutes; respectively eluting with trifluoroacetic acid with the volume percentage concentration of 0.5% and trifluoroacetic acid with the volume percentage concentration of 1%, wherein the flow rate of the trifluoroacetic acid with the volume percentage concentration of 0.5% is 10-15 ml per minute, and the elution time is 20-30 minutes; the flow rate of the trifluoroacetic acid with the volume percentage concentration of 1% is 10-15 ml per minute, and the elution time is 20-30 minutes.
In a preferred embodiment of the present invention, in the step (3), the monitoring is performed by high performance liquid chromatography, and the mobile phase used in the high performance liquid chromatography comprises any one of or a mixture of a buffer solution of sodium heptanesulfonate and a buffer solution of phosphate.
In a preferred embodiment of the present invention, in the step (3), the monitoring is performed by high performance liquid chromatography, wherein the high performance liquid chromatography uses a C18 column with a diameter of 4.6mm, a length of 150mm and a particle size of 3um or 3.5 um.
In a preferred embodiment of the present invention, in step (3), 200mg of the second solid is contained per 1ml of the second solution.
In a preferred embodiment of the present invention, in the step (4), the mobile phase used in the second separation by high performance liquid chromatography is 0.1% trifluoroacetic acid aqueous solution by mass, and the flow rate is 10 ml/min.
In a preferred embodiment of the present invention, in the step (4), the chromatographic column used in the second separation by high performance liquid preparative chromatography is a C18 column having a diameter of 19mm, a length of 210mm and a particle size of 10 um.
In a preferred embodiment of the present invention, in the step (4), the detection wavelength used in the second separation by high performance liquid preparative chromatography is 210 nm.
In a preferred embodiment of the present invention, in the step (4), the mobile phase used in the positioning with the high performance liquid phase comprises any one of a buffer solution of sodium heptanesulfonate and a buffer solution of phosphate or a mixture of the two.
In a preferred embodiment of the present invention, in the step (4), the chromatographic column used in the positioning with the high performance liquid phase is a C18 column with a diameter of 4.6mm, a length of 150mm and a particle size of 3um or 3.5 um.
In a preferred embodiment of the present invention, in the step (5), the impurity G is pretreated by dissolving the impurity G obtained in the step (4) with purified water to prepare a sample of 1 mg/ml.
In a preferred embodiment of the present invention, in steps (4) and (5), the impurity G is L-glutamic acid-L-cysteine-alpha-alanine.
The invention also aims to provide application of impurity G generated in the process of reduced glutathione fermentation as an impurity reference substance in reduced glutathione related substance detection, which is characterized in that the HPLC purity of the impurity G obtained by separation and purification is more than 98 percent, and the requirement of the impurity G for the application of the impurity reference substance can be met.
The main innovation points of the invention are as follows:
unknown fermentation impurities in the fermentation process of the reduced glutathione are positioned, determined, separated and purified, and the application of the impurities as impurity reference substances in the detection of related substances of the reduced glutathione is utilized, so that the medicine quality and the clinical medication safety of the existing reduced glutathione are improved.
Drawings
FIG. 1 is a high performance liquid chromatography detection spectrum of impurity G in the eluted fraction obtained by the first separation and purification.
FIG. 2 is a high performance liquid chromatography purity detection spectrum of impurity G after the second separation and purification.
FIG. 3 is a MS-ESI chart of mass spectrum information of impurity G.
FIG. 4 nuclear magnetic hydrogen spectrum of impurity G1H-NMR chart.
FIG. 5 is a high performance liquid chromatogram of the related substances of the purchased reduced glutathione sample solution.
FIG. 6 is a high performance liquid chromatogram of the relevant substances in a self-developed reduced glutathione sample solution.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the invention thereto.
Example 1
Separation and purification method of reduced glutathione impurity G
(1) Concentration: 1L of reduced glutathione crystallization mother liquor was taken, concentrated in vacuo using a rotary evaporator to a white solid, and weighed about 35 g.
(2) Sample preparation: dissolving the white solid obtained in the step (1) with deionized water to prepare 20ml of a solution containing about 250mg of the white solid per 1 ml.
(3) First purification and separation: d001x7 strong acid cation exchange column, particle size range 0.315-1.25mm, diameter 6cm, height 80 cm. Before loading, the pH value is adjusted to 6 by using NaOH with the mass percent concentration of 20 percent, elution is carried out by using water with the flow rate of about 20 milliliters per minute, and then elution is carried out by using acetic acid water solution with the mass percent concentration of 1.8 percent. Then, the mixture is eluted by trifluoroacetic acid aqueous solution with the mass percent concentration of 0.5 percent and trifluoroacetic acid aqueous solution with the mass percent concentration of 1 percent respectively. Monitoring by high performance liquid chromatography (see figure 1), wherein the mobile phase is buffer solution of sodium heptanesulfonate and phosphate, and the eluent is combined according to the detection result.
The conditions of the high performance liquid chromatography are as follows: the chromatographic column is an Inertsil ODS-HL column (4.6mm x 150mm, 3um), the mobile phase is phosphate buffer solution (6.8 g of potassium dihydrogen phosphate and 2.2g of sodium heptanesulfonate are dissolved by adding 1000ml of water, the pH value is adjusted to 2.50 +/-0.02) by using phosphoric acid) -acetonitrile (90:10,), and the flow rate is 1.0 ml/min; the detection wavelength is 210nm, and the column temperature is 30 ℃.
(4) And (3) second purification and separation: concentrating the eluate obtained in step (3) in vacuum to obtain a solid, to obtain 326 mg; dissolving with deionized water to obtain 1ml of solution containing about 200mg of solids, performing secondary separation by using a high performance liquid preparative chromatography, wherein a mobile phase is a trifluoroacetic acid aqueous solution with the mass percentage concentration of 0.1%, a chromatographic column is C18, the diameter of the chromatographic column is 19mm, the length of the chromatographic column is 210mm, the particle size of the chromatographic column is 10um, the flow rate of the chromatographic column is 10ml/min, the detection wavelength of the chromatographic column is 210nm, collecting according to an online chromatogram, and positioning by using the high performance liquid, and the chromatographic conditions are as follows: the chromatographic column is an Inertsil ODS-HL column (4.6mm x 150mm, 3um), the mobile phase is phosphate buffer solution (6.8 g of potassium dihydrogen phosphate and 2.2g of sodium heptanesulfonate are dissolved by adding 1000ml of water, the pH value is adjusted to 2.50 +/-0.02) by using phosphoric acid) -acetonitrile (90:10,), and the flow rate is 1.0 ml/min; detecting the wavelength of 210nm, column temperature of 30 deg.C, determining the collected liquid containing impurity G, mixing the collected liquids, and freeze drying to obtain white solid 125mg, i.e. impurity G.
(5) And (3) purity detection: dissolving the white solid obtained in the step (4) by using purified water to prepare a sample injection high performance liquid chromatograph with the concentration of 1mg/ml, wherein the chromatographic conditions are as follows: the chromatographic column is an Inertsil ODS-HL column (4.6mm x 150mm, 3um), the mobile phase is phosphate buffer solution (6.8 g of potassium dihydrogen phosphate and 2.2g of sodium heptanesulfonate are dissolved by adding 1000ml of water, the pH value is adjusted to 2.50 +/-0.02) by using phosphoric acid) -acetonitrile (90:10,), and the flow rate is 1.0 ml/min; the detection wavelength was 210nm, the column temperature was 30 ℃, and purity analysis was performed, and the purity analysis result was obtained (see fig. 2).
And (3) confirming structural information through mass spectrum and nuclear magnetic hydrogen spectrum: referring to FIG. 3, the mass spectrum information MS-ESI is called for C11H19N3O6S,322.3(M + H), found 321.3; referring to FIG. 4, nuclear magnetic hydrogen spectrum1H-NMR(400Hz,D2O)δppm:4.40-4.42(1H,m,6-H),4.25-4.29(1H,m,9-H),3.86-3.89(1H,m,2-H),2.762.87(2H, m,7-H),2.42-2.49(2H, m,4H),2.07-2.13(2H, m,3-H),1.32-1.34(3H, m,10-H) (see FIG. 4).
Example 2
High performance liquid chromatography for determining content of impurity G in reduced glutathione
Chromatographic conditions are as follows: the chromatographic column is an Inertsil ODS-HL column (4.6mm x 150mm, 3um), the mobile phase is phosphate buffer solution (6.8 g of potassium dihydrogen phosphate and 2.2g of sodium heptanesulfonate are dissolved by adding 1000ml of water, the pH value is adjusted to 2.50 +/-0.02) by using phosphoric acid) -acetonitrile (90:10,), and the flow rate is 1.0 ml/min; the detection wavelength is 210nm, and the column temperature is 30 ℃.
Weighing about 25g each of purchased (Shandong Jincheng) and self-grinding reduced glutathione, precisely weighing, putting into a 50ml volumetric flask (newly prepared), adding mobile phase to dissolve and dilute to scale, shaking up to obtain sample solution; precisely weighing a proper amount of impurity G reference substance, dissolving and diluting the impurity G reference substance into a solution of 0.5mg/ml by using a solvent to serve as a reference substance solution; 10ul of each of the test solution and the reference solution is taken, injected into a liquid chromatograph, and the chromatogram is recorded until the retention time of the main component peak is 3 times.
FIG. 5 is a test pattern of substances related to purchased reduced glutathione, and FIG. 6 is a test pattern of substances related to self-developed reduced glutathione, wherein the retention time of impurity G is 11.784 min. As can be seen from the figure, the impurity G does not interfere with the main peak and other impurities, so the method can control the quality of the content of the impurity G in the reduced glutathione by a reference external standard method.
Claims (16)
1. A method for separating and purifying impurity G generated in the process of reduced glutathione fermentation is characterized by comprising the following steps:
step (1), concentrating a sample: firstly, carrying out rotary evaporation and vacuum concentration on reduced glutathione crystallization mother liquor to obtain a first solid;
step (2) sample preparation: dissolving the first solid obtained in the step (1) by using deionized water to prepare a first solution;
step (3), first purification and separation: performing gradient elution on the first solution prepared in the step (2) by using a cation exchange column after pH adjustment, monitoring by using high performance liquid chromatography in the elution process, and combining the first eluents according to the detection result;
and (4) second purification and separation: performing vacuum concentration on the first eluent obtained in the step (3) to obtain a second solid, dissolving the second solid with deionized water to obtain a second solution, performing second separation by using a high performance liquid preparative chromatography, performing separation according to an online chromatogram, positioning by using a high performance liquid, determining a collecting solution containing an impurity G, combining the collecting solutions, and performing freeze drying treatment to obtain a white solid, namely the impurity G;
and (5) purity detection: after the impurity G obtained in the step (4) is pretreated, a high performance liquid chromatograph is used for purity analysis, and a chromatographic column and a mobile phase used by the high performance liquid chromatograph are the same as those used in the step (4) and positioned by the high performance liquid chromatograph;
in the step (3), the filler in the cation exchange column after pH adjustment is any one or a mixture of more than two of D001x7 strong acid cation exchange resin, D151 macroporous weak acid acrylic acid cation exchange resin, D152D151 macroporous weak acid acrylic acid cation exchange resin and CD-180 macroporous weak acid cation exchange resin;
in the steps (4) and (5), the impurity G is L-glutamic acid-L-cysteine-alpha-alanine.
2. The method of claim 1, wherein the step (2) comprises the step of separating and purifying the impurity G produced during the reduced glutathione fermentation process, wherein the first solid content is 250mg per 1ml of the first solution.
3. The method of claim 1, wherein in step (3), the pH of the pH-adjusted cation exchange column is adjusted to pH 6 with NaOH at a concentration of 20% by mass before loading.
4. The method for separating and purifying impurity G produced in reduced glutathione fermentation according to claim 1, wherein in the step (3), the diameter of the cation exchange column after pH adjustment is 5-10cm, and the height is 50-100 cm.
5. The method for separating and purifying impurity G produced in reduced glutathione fermentation process of claim 1, wherein in the step (3), the packing material of the cation exchange column after pH adjustment is D001x7 strong acid cation exchange resin with corresponding particle size range of 0.315-1.25 mm; the diameter of the cation exchange column after pH adjustment is 6 cm.
6. The method for separating and purifying the impurity G generated in the reduced glutathione fermentation process in the step (3), wherein in the step (3), gradient elution is adopted, and the gradient elution step comprises the steps of eluting with water firstly, wherein the flow rate is 15-20 ml per minute, and the elution time is 30-45 minutes; then, eluting with 1.8 percent acetic acid aqueous solution by volume percentage concentration, wherein the flow rate is 15-20 ml per minute, and the elution time is 20-30 minutes; respectively eluting with trifluoroacetic acid with the volume percentage concentration of 0.5% and trifluoroacetic acid with the volume percentage concentration of 1%, wherein the flow rate of the trifluoroacetic acid with the volume percentage concentration of 0.5% is 10-15 ml per minute, and the elution time is 20-30 minutes; the flow rate of the trifluoroacetic acid with the volume percentage concentration of 1% is 10-15 ml per minute, and the elution time is 20-30 minutes.
7. The method according to claim 1, wherein in the step (3), the monitoring is performed by high performance liquid chromatography, and the mobile phase used in the high performance liquid chromatography comprises any one or a mixture of a buffer solution of sodium heptanesulfonate and a buffer solution of phosphate.
8. The method of claim 1, wherein the step (3) of separating and purifying the impurity G produced in the reduced glutathione fermentation process is carried out by using high performance liquid chromatography, wherein the high performance liquid chromatography uses a C18 column with a diameter of 4.6mm, a length of 150mm and a particle size of 3um or 3.5 um.
9. The method of claim 1, wherein the second solid is present in an amount of 200mg per 1ml of the second solution in step (3).
10. The method of claim 1, wherein in the step (4), the mobile phase used in the second separation by HPLC is 0.1% trifluoroacetic acid in water at a flow rate of 10 ml/min.
11. The method of claim 1, wherein the column used in the second separation by HPLC (high performance liquid chromatography) in the step (4) is a C18 column with a diameter of 19mm, a length of 210mm and a particle size of 10 μm.
12. The method of claim 1, wherein the detection wavelength used in the second separation by HPLC is 210nm in step (4).
13. The method for separating and purifying the impurity G generated in the reduced glutathione fermentation process of claim 1, wherein the mobile phase used in the positioning by using the high performance liquid phase in the step (4) comprises any one or a mixture of a buffer solution of sodium heptanesulfonate and a buffer solution of phosphate.
14. The method for separating and purifying impurity G produced in reduced glutathione fermentation according to claim 1, wherein in the step (4), the chromatographic column used for positioning by using the high performance liquid is a C18 column, the diameter is 4.6mm, the length is 150mm, and the particle size is 3um or 3.5 um.
15. The method of claim 1, wherein the pre-treatment of the impurity G in the step (5) is to dissolve the impurity G obtained in the step (4) with purified water to prepare a sample of 1 mg/ml.
16. The application of the impurity G generated in the fermentation process of the reduced glutathione as an impurity reference substance in the detection of the reduced glutathione-related substance is characterized in that the impurity G obtained by separation and purification has the HPLC purity of more than 98 percent and can meet the requirement of the impurity G for the application of the impurity reference substance, and when the detection of the reduced glutathione-related substance is carried out, the impurity G is used as the impurity reference substance, and the content of the impurity G in the reduced glutathione is measured by adopting a high performance liquid chromatography; the impurity G is L-glutamic acid-L-cysteine-alpha-alanine.
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| JPS6127999A (en) * | 1984-07-16 | 1986-02-07 | Kanegafuchi Chem Ind Co Ltd | Method for purifying glutathione |
| CN106226445A (en) * | 2016-01-30 | 2016-12-14 | 重庆药友制药有限责任公司 | A kind of isolation and purification method of reduced glutathion catabolite F |
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| JPS6127999A (en) * | 1984-07-16 | 1986-02-07 | Kanegafuchi Chem Ind Co Ltd | Method for purifying glutathione |
| CN106226445A (en) * | 2016-01-30 | 2016-12-14 | 重庆药友制药有限责任公司 | A kind of isolation and purification method of reduced glutathion catabolite F |
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Denomination of invention: A method for separating and purifying impurity G generated during the fermentation process of reduced glutathione and its application Granted publication date: 20210813 Pledgee: Agricultural Bank of China Limited Shanghai Yangtze River Delta Integrated Demonstration Zone Sub branch Pledgor: SHANGHAI QINGPING PHARMACEUTICAL Co.,Ltd. Registration number: Y2024310000191 |
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