CN112924566B - Method for simultaneously detecting glycine and serine in enzymatic reaction liquid - Google Patents
Method for simultaneously detecting glycine and serine in enzymatic reaction liquid Download PDFInfo
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- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 title claims abstract description 177
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 title claims abstract description 90
- 239000004471 Glycine Substances 0.000 title claims abstract description 88
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 62
- 238000006911 enzymatic reaction Methods 0.000 title claims abstract description 33
- 239000012295 chemical reaction liquid Substances 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 63
- 238000010521 absorption reaction Methods 0.000 claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 18
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 239000013558 reference substance Substances 0.000 claims description 53
- 239000000243 solution Substances 0.000 claims description 42
- 239000000523 sample Substances 0.000 claims description 40
- 239000011550 stock solution Substances 0.000 claims description 26
- 239000012488 sample solution Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000007865 diluting Methods 0.000 claims description 16
- 239000012153 distilled water Substances 0.000 claims description 15
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 14
- 239000007924 injection Substances 0.000 claims description 13
- 238000002347 injection Methods 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- VYCBPGNEBZTCTF-UHFFFAOYSA-N [Na].CCCCCCCCCC Chemical compound [Na].CCCCCCCCCC VYCBPGNEBZTCTF-UHFFFAOYSA-N 0.000 claims description 12
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 238000010812 external standard method Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- 239000003002 pH adjusting agent Substances 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000001121 post-column derivatisation Methods 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 229960001153 serine Drugs 0.000 description 78
- 238000001212 derivatisation Methods 0.000 description 8
- 238000003860 storage Methods 0.000 description 8
- 238000005259 measurement Methods 0.000 description 7
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000002211 ultraviolet spectrum Methods 0.000 description 3
- 235000013365 dairy product Nutrition 0.000 description 2
- SBNUEVPRRHCPNQ-UHFFFAOYSA-N decane-1-sulfonic acid;sodium Chemical compound [Na].CCCCCCCCCCS(O)(=O)=O SBNUEVPRRHCPNQ-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229940107700 pyruvic acid Drugs 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- YPWSLBHSMIKTPR-UHFFFAOYSA-N Cystathionine Natural products OC(=O)C(N)CCSSCC(N)C(O)=O YPWSLBHSMIKTPR-UHFFFAOYSA-N 0.000 description 1
- ILRYLPWNYFXEMH-UHFFFAOYSA-N D-cystathionine Natural products OC(=O)C(N)CCSCC(N)C(O)=O ILRYLPWNYFXEMH-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 239000004909 Moisturizer Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000010836 blood and blood product Substances 0.000 description 1
- 229940125691 blood product Drugs 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 1
- 229940097068 glyphosate Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000037257 muscle growth Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003354 serine derivatives Chemical class 0.000 description 1
- AIMUHNZKNFEZSN-UHFFFAOYSA-M sodium;decane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCS([O-])(=O)=O AIMUHNZKNFEZSN-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 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
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- 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)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to the field of biological analysis, in particular to a method for simultaneously detecting glycine and serine in enzymatic reaction liquid. The invention adopts high performance liquid chromatography, injects enzymatic reaction liquid into the high performance liquid chromatograph, adopts C18 column separation and adopts an ultraviolet absorption detector for detection. The invention realizes simultaneous detection of glycine and serine content in enzymatic reaction liquid, can provide reliable data support for real-time monitoring of serine production, and the detection sample does not need pre-column or post-column derivatization treatment, so that the detection time is greatly shortened, and the detection result is more accurate.
Description
Technical Field
The invention relates to the field of biological analysis, in particular to a method for simultaneously detecting glycine and serine in enzymatic reaction liquid.
Background
Serine (Ser) is also known as beta-hydroxy alanine, which has the chemical formula C 3 H 7 NO 3 The method comprises the steps of carrying out a first treatment on the surface of the Glycine (Gly) is also known as Glycine, and has the chemical formula C 2 H 5 NO 2 . They are all non-essential amino acids, and are widely used in the fields of cosmetics, medicines, foods, etc., and play a key role. For example, the number of the cells to be processed,serine plays an important role in the metabolism of fat and fatty acids and in muscle growth. It also contributes to the production of immune hemoglobin and antibodies and maintains the health of the immune system. Serine is also a nutritional supplement, serine derivative raw material, eye drop pH regulator and natural moisturizer. Glycine is an important intermediate of organic chemistry, mainly for the synthesis of glyphosate. Glycine has the functions of regulating food flavor, inhibiting the propagation of escherichia coli, inhibiting the oxidization of dairy products and the like. With the wide application of the special functions of serine and glycine, the market demand of the serine and glycine is increasing year by year.
At present, amino acid is mainly produced by adopting methods such as chemical synthesis, extraction, enzymatic method, fermentation and the like. The enzymatic method is widely used because it can obtain a product with a single structure and high purity. The serine is produced by adopting the method, a method suitable for serine and glycine separation and quantitative detection needs to be established, and the method needs to have the characteristics of accuracy, rapidness, simplicity and the like.
In the related art, the literature "the contents of L-serine and glycine in fermentation broth are determined by paper chromatography-spectrophotometry" (Lu Fa, zhang Weiguo, food and fermentation industry, 2005 3), which mainly includes centrifugation, spotting, blow-drying, color development, elution, etc., and the detection time is long. The method needs to be provided with the color reagent, optimizes a plurality of experimental factors, has large experimental error and inaccurate detection result. The method can only indirectly reflect the content of glycine and serine in the fermentation liquor.
At present, high performance liquid chromatography and an automatic amino acid analyzer are also adopted to detect serine and glycine respectively. For example, patent ZL201510024181.6, entitled high performance liquid chromatography of serine, adopts derivatization reagent to derivatize detection sample, and then indirectly measures serine content by high performance liquid chromatography. The method has complicated derivatization steps and inconvenient operation. In addition, derivatization produces a number of byproducts that make the chromatograms difficult to isolate, thereby affecting the accuracy of the serine quantitative analysis. And the derivative reagent has various types, is difficult to screen, has high price and high detection cost, and is not suitable for detection of multiple samples. The patent ZL201310591734.7 is named as a method for quantitatively detecting serine, adopts cystathionine lyase (CBL) to hydrolyze serine to generate pyruvic acid, and indirectly detects the serine content in a sample by measuring the pyruvic acid content. The method also has the problems of troublesome sample treatment, complicated experimental operation, inaccurate experimental results and the like. The glycine content is measured indirectly by a derivatization method in the literature 'the content of glycine in blood products is measured by a post-column derivatization HPLC method and the content of glycine in dairy products is measured by a pre-column derivatization high performance liquid chromatography method', and patent application CN201710050705.8 (the name of the invention is a method for detecting glycine and impurities thereof by high performance liquid chromatography). While the detector used in patent application CN201811635412.7 (entitled new glycine content detection method) is ELSD detector, the method requires high requirements for chromatographic detection instrument.
The method can only be established at a laboratory level for detecting the conventional samples, and is not suitable for enterprises with more samples and large workload. Moreover, only the content of one amino acid can be detected by the high-precision method among the above methods. Therefore, there is an urgent need to establish an accurate and convenient method for simultaneously determining the glycine and serine contents in the enzymatic reaction solution.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting glycine and serine in an enzymatic reaction solution.
In order to achieve the aim of the invention, the technical scheme adopted is as follows:
the invention provides a method for simultaneously detecting glycine and serine in enzymatic reaction liquid, which adopts high performance liquid chromatography, injects the enzymatic reaction liquid into a high performance liquid chromatograph, adopts C18 column separation and adopts an ultraviolet absorption detector for detection;
the mobile phase A of the high performance liquid chromatography is a mixed solution of 0.01-0.1 mol/L dipotassium hydrogen phosphate and 0.001-0.01 mol/L decane sodium sulfonate, and the pH value is 1-5; the mobile phase B of the high performance liquid chromatography is absolute ethyl alcohol.
Alternatively, the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L decane sulfonic acid sodium.
Optionally, the pH of mobile phase a is 2.3.
Optionally, the volume ratio of the mobile phase a to the mobile phase B is 1000: 50-1000: 90, preferably 1000:70.
optionally, the mobile phase a further contains a pH regulator, and the pH regulator is preferably at least one of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid and acetic acid.
Optionally, the chromatographic conditions of the high performance liquid chromatography are: the column temperature is 20-40 ℃, the detection wavelength is 190-230 nm, the sample flow rate is 0.8-1.5 mL/min, and the sample injection amount is 5-20 mu L.
Optionally, the chromatographic conditions of the high performance liquid chromatography are: the column temperature is 25-35 ℃, preferably 30 ℃, the detection wavelength is 200-220 nm, preferably 210nm, the sample flow rate is 0.8-1 mL/min, preferably 1mL/min, and the sample injection amount is 8-15 mu L, preferably 10 mu L.
Optionally, the method at least comprises the following steps:
(1) Preparing a mixed reference substance solution: the concentration of glycine reference substance in the mixed reference substance solution is 0.1-5 g/L, and the concentration of serine reference substance is 0.1-5 g/L;
(2) Preparing a test sample solution: diluting the enzymatic reaction solution to be tested to a concentration of 0.1-5 g/L to obtain a sample solution;
(3) And (3) detection: and respectively taking the mixed reference substance solution and the sample solution, directly injecting the mixed reference substance solution and the sample solution into a high performance liquid chromatograph, adopting a C18 column for separation, detecting a glycine absorption peak and a serine absorption peak which are separated by an ultraviolet absorption detector, and calculating the content of glycine and serine in the enzymatic reaction liquid to be detected by an external standard method.
Optionally, in step (1), preparing a glycine reference substance storage solution and a serine reference substance storage solution respectively, and mixing the glycine reference substance storage solution and the serine reference substance storage solution respectively before detection, and diluting with distilled water to obtain the mixed reference substance solution;
the preparation method of the glycine control stock solution preferably comprises the following steps: weighing glycine reference substance, and diluting with distilled water to obtain glycine reference substance stock solution with concentration of 1-10 g/L;
the preparation method of the serine reference substance stock solution preferably comprises the following steps: weighing serine reference substance, and diluting with distilled water to obtain serine reference substance stock solution with concentration of 1-10 g/L.
Optionally, in the step (2), the enzymatic reaction solution to be tested is diluted and then filtered, and the filtration is performed by adopting a 0.45 μm water-based filter membrane.
The invention has at least the following beneficial effects:
the invention realizes the simultaneous detection of glycine and serine content in the enzymatic reaction liquid, can provide reliable data support for real-time monitoring of serine production, and has the following technical effects:
(1) The invention can simultaneously detect the glycine and serine content in the enzymatic reaction liquid.
(2) The detection sample does not need to be subjected to pre-column or post-column derivatization treatment, so that the detection time is greatly shortened, and the detection result is more accurate.
(3) The method can directly take the detection sample, can be used for on-machine detection after treatment and filtration by deionized water, has simple detection sample treatment steps and reduces the experimental operation error rate.
(4) The chromatographic column is a common C18 column, the detector is an ultraviolet detector, the chromatographic condition is simple, the detection cost is low, the operation is simple and convenient, the chromatographic column is suitable for enterprises with more sample detection and large workload, and the economic burden of the enterprises can be reduced.
(5) The chromatographic detection principle is more scientific, the detection result is more convincing, and the method has guiding significance for the industrial production of serine by an enzyme method.
Drawings
FIG. 1 is a graph showing the detection of the mixed control solution in example 1;
FIG. 2 is a graph showing the detection pattern of the enzymatic reaction solution in example 2;
FIG. 3 is a graph showing the peak area and linearity of glycine concentration in example 3;
FIG. 4 is a linear graph of serine concentration peak area and serine in example 4;
FIG. 5 is a detection chart of mobile phase D1 detection of mixed control solution in example 6;
FIG. 6 is a detection pattern of mobile phase D2 detection of mixed control solution in example 6.
The invention will be further illustrated with reference to specific examples. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present invention.
Detailed Description
The embodiment of the invention provides a method for simultaneously detecting glycine and serine in enzymatic reaction liquid. Specifically, the method adopts high performance liquid chromatography, enzymatic reaction liquid containing glycine and serine is injected into the high performance liquid chromatograph, C18 column separation is adopted, and an ultraviolet absorption detector is used for detecting the separated glycine absorption peak and serine absorption peak. Because glycine and serine have poor absorption in the ultraviolet spectrum, the prior art can only convert glycine or serine into other substances with absorption in the ultraviolet spectrum by using a derivatization method and an enzymolysis method, or detect the substances by using other detectors. Through intensive research, the embodiment of the invention does not need other processing steps, so that glycine and serine have obvious and separated absorption peaks on an ultraviolet spectrum at the same time, thereby realizing the invention.
The detection method of the embodiment of the invention overcomes the defects brought by indirect detection by adopting a derivatization method in the prior art, realizes simultaneous detection of glycine and serine content in enzymatic reaction liquid by utilizing a common C18 column, does not need an expensive chromatographic column, does not need an expensive detection device, and can be realized by adopting an ultraviolet detector. The detection method provided by the embodiment of the invention has simple operation steps, and can be used for detection only by adjusting the concentration of the enzymatic reaction liquid. Therefore, the method has the characteristics of direct, quick and accurate detection, is suitable for industrial production with a large number of detection samples and large workload, and is convenient to popularize and apply in the actual production process.
Specifically, the mobile phase A selected in the embodiment of the invention is a mixed solution of 0.01-0.1 mol/L dipotassium hydrogen phosphate and 0.001-0.01 mol/L decane sodium sulfonate, and the pH value is 1-5, and the separation degree can be influenced if the pH value is too high or too low; mobile phase B is absolute ethanol. The mobile phase of the embodiment of the invention not only ensures that glycine and serine can have good absorption peaks on an ultraviolet absorption detector, but also has good separation degree of glycine absorption peaks and serine absorption peaks, thereby realizing simultaneous detection of glycine and serine.
Further alternatively, mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decanesulfonate.
Further alternatively, mobile phase a has a pH of 2.3.
Further optionally, the volume ratio of mobile phase a to mobile phase B is 1000: 50-1000: 90, preferably 1000:70, mixing the mobile phase A and the mobile phase B according to a proportion to obtain the mobile phase. If the volume ratio of mobile phase A to mobile phase B is too large or too small, it will have a certain effect on the degree of separation of the two absorption peaks.
Further optionally, the mobile phase a further contains a pH adjustor, and the pH of the mobile phase a is adjusted within the scope of the embodiment of the present invention, where the pH adjustor is preferably at least one of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and acetic acid.
The C18 column used in the embodiment of the present invention may be a common commercial C18 column, for example: siemens Syncronis C18 column, epidert Hypersil ODS 2C 18 column, siemens BDS Hypersil TM C column, etc
The chromatographic conditions of the high performance liquid chromatography of the embodiment of the invention are as follows:
the column temperature is 20 to 40 ℃, preferably 25 to 35 ℃, and more preferably 30 ℃.
The detection wavelength is 190 to 230nm, preferably 200 to 220nm, and more preferably 210nm.
The flow rate of the sample is 0.8-1.5 mL/min, if the flow rate of the sample is too fast, the peak can be overlapped with other peaks (such as solvent peaks) too early, and if the flow rate of the sample is too slow, the analysis time can be prolonged, and the working efficiency is reduced; preferably, the sample flow rate is 0.8 to 1mL/min, more preferably 1mL/min.
The sample injection amount is 5-20 mu L, if the sample injection amount is too large, the instrument load can be caused, the tailing phenomenon can also occur on the absorption peak, and the separation degree between the glycine absorption peak and the serine absorption peak is influenced; if the sample injection amount is too small, the measurement is inaccurate; preferably, the amount of sample introduced is 8 to 15. Mu.L, more preferably 10. Mu.L.
Optionally, the method of the embodiment of the invention at least comprises the following steps:
(1) Preparing a mixed reference substance solution: the concentration of glycine reference substance in the mixed reference substance solution is 0.1-5 g/L, and the concentration of serine reference substance is 0.1-5 g/L;
(2) Preparing a test sample solution: diluting the enzymatic reaction solution to be tested to a concentration of 0.1-5 g/L to obtain a sample solution;
(3) And (3) detection: and respectively taking a mixed reference substance solution and a sample solution, directly adding the mixed reference substance solution and the sample solution into a high performance liquid chromatograph, adopting a C18 column for separation, detecting a glycine absorption peak and a serine absorption peak which are separated by an ultraviolet absorption detector, and calculating the content of glycine and serine in the enzymatic reaction solution to be detected by an external standard method.
According to the research of the embodiment of the invention, when the concentration range of the reference substance and the solution of the test substance is between 0.1 and 5g/L, the peak areas of glycine and serine at each concentration show good linear relation with the concentration. The content of serine and glycine can be calculated by an external standard method within the linear range. Wherein the concentration of the sample solution can be obtained by simple calculation according to the addition amount of the raw materials.
Optionally, in step (1), preparing a glycine reference substance stock solution and a serine reference substance stock solution respectively, and mixing the glycine reference substance stock solution and the serine reference substance stock solution respectively before detection, and diluting with distilled water to obtain a mixed reference substance solution.
Specifically, the preparation method of the glycine control stock solution comprises the following steps: weighing glycine reference substance, and diluting with distilled water to obtain glycine reference substance stock solution with concentration of 1-10 g/L;
the preparation method of the serine reference substance stock solution comprises the following steps: weighing serine reference substance, and diluting with distilled water to obtain serine reference substance stock solution with concentration of 1-10 g/L.
Optionally, in the step (2), the enzymatic reaction solution to be tested is diluted and then filtered, and the filtration is performed by adopting a 0.45 μm water-based filter membrane. The embodiment of the invention can detect the enzymatic reaction liquid by simply filtering without pretreatment such as derivatization and enzymolysis.
The following examples are provided to further illustrate and explain the principles of the present invention.
Example 1
The apparatus and the detection conditions adopted in this embodiment are as follows:
instrument: dionex Ultimate 3000 high performance liquid chromatograph; a VWD ultraviolet detector;
chromatographic column: siemens Syncronis C18 (5 μm, 4.6X1250 mm);
column temperature: 30 ℃;
wavelength: 210nm;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L decane sodium sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a to mobile phase B: 1000:70.
the specific operation steps of the standard glycine and serine content measurement are as follows:
1. preparing a mixed reference substance solution:
glycine control stock solution: precisely weighing 1g of glycine reference substance, and dissolving and diluting with distilled water to obtain glycine reference stock solution with concentration of 10 g/L;
serine control stock solution: accurately weighing 1g of serine reference substance, and dissolving and diluting with distilled water to obtain serine reference stock solution with concentration of 10 g/L;
2. mixing the reference substance solution: and respectively taking a proper amount of glycine reference substance storage solution and serine reference substance storage solution before use, mixing the glycine reference substance storage solution and the serine reference substance storage solution, and then diluting with distilled water to obtain mixed reference substance solutions with glycine and serine concentrations of 0.5g/L respectively.
3. Precisely sucking 10 μl of the mixed reference solution, injecting into liquid chromatograph for measurement, and recording chromatogram. The experimental results are shown in FIG. 1.
Example 2
The apparatus and the detection conditions adopted in this embodiment are as follows:
instrument: dionex Ultimate 3000 high performance liquid chromatograph; a VWD ultraviolet detector;
chromatographic column: siemens Syncronis C18 (5 μm, 4.6X1250 mm);
column temperature: 30 ℃;
wavelength: 210nm;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L decane sodium sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a to mobile phase B: 1000:70.
the specific operation steps of the determination of the glycine and serine content in the enzymatic reaction liquid are as follows:
1. taking 1mL of the enzymatic reaction solution in a 10mL volumetric flask, fixing the volume by distilled water, and shaking uniformly to obtain the product.
2. The above solution was filtered through a 0.45 μm aqueous filter membrane, and the filtrate was used as a sample solution.
3. Taking 10 mu L of sample test solution, injecting into a liquid chromatograph for measurement, and recording a chromatogram. The experimental results are shown in FIG. 2. The concentration of the sample test solution can be calculated by using the data of fig. 1 and 2 and adopting an external standard method.
Example 3
The apparatus and the detection conditions adopted in this embodiment are as follows:
instrument: dionex Ultimate 3000 high performance liquid chromatograph; a VWD ultraviolet detector;
chromatographic column: siemens Syncronis C18 (5 μm, 4.6X1250 mm);
column temperature: 30 ℃;
wavelength: 210nm;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L decane sodium sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a to mobile phase B: 1000:70.
the specific operation steps of the glycine linearity test are as follows:
1. and (3) taking a proper amount of glycine reference substance stock solution, and diluting with distilled water to obtain sample solutions with the concentration of 0.1g/L, 0.5g/L, 1g/L, 2.5g/L and 5 g/L.
2. Taking 10 mu L of sample solution, injecting into a liquid chromatograph for measurement, recording a chromatogram, taking the glycine peak area as an ordinate Y and the glycine concentration as an abscissa X, and performing linear regression to obtain a linear equation correlation coefficient R 2 =1. The peak areas of the concentrations and glycine linearity are shown in FIG. 3. As can be seen from the glycine linear graph of fig. 3: glycine has good linearity in the concentration range of 0.1-5 g/L.
Example 4
The apparatus and the detection conditions adopted in this embodiment are as follows:
instrument: dionex Ultimate 3000 high performance liquid chromatograph; a VWD ultraviolet detector;
chromatographic column: siemens Syncronis C18 (5 μm, 4.6X1250 mm);
column temperature: 30 ℃;
wavelength: 210nm;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L decane sodium sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a to mobile phase B: 1000:70.
the specific procedure for the serine linearity test is as follows:
1. and (3) taking a proper amount of serine control stock solution, and diluting with distilled water to obtain sample solutions with the concentration of 0.1g/L, 0.5g/L, 1g/L, 2.5g/L and 5 g/L.
2. Taking 10 mu L of sample solution, injecting into a liquid chromatograph for measurement, recording a chromatogram, taking serine peak area as an ordinate Y and glycine concentration as an abscissa X, and performing linear regression to obtain a linear equation correlation coefficient R 2 =1. The peak area and serine linearity diagrams for each concentration are shown in FIG. 4. From the serine linear diagram of fig. 4, it can be seen that: serine has good linearity in the concentration range of 0.1-5 g/L.
Example 5
In this example, the durability of the method was examined by changing any one of chromatographic conditions such as column temperature, wavelength, sample injection amount, flow rate, column chromatography, mobile phase volume ratio, and the like.
The instrument and the detection conditions adopted in the embodiment are as follows:
instrument: dionex Ultimate 3000 high performance liquid chromatograph; a VWD ultraviolet detector;
chromatographic column:
airit Hypersil ODS 2C 18 (5 μm, 4.6X1250 mm),
B Siemens Syncronis C18 (5 μm, 4.6X1250 mm),
C Sieimer's flight BDS Hypersil TM C (5 μm, 4.6X1250 mm);
(other chromatographic conditions were the same as in example 1 when the column was changed).
Column temperature: 20 ℃, 30 ℃ and 40 ℃; (other chromatographic conditions were the same as in example 1 when the column temperature was changed).
Wavelength: 190nm, 210nm, 230nm; (other chromatographic conditions were the same as in example 1 when the wavelength was changed).
Sample injection amount: 5. Mu.L, 10. Mu.L, 20. Mu.L; (other chromatographic conditions were the same as in example 1 when the amount of sample introduced was changed).
Flow rate: 0.8mL/min, 1.0mL/min, 1.5mL/min; (other chromatographic conditions were the same as in example 1 when the flow rate was changed).
Mobile phase:
the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L decane sodium sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
the F mobile phase A is a mixed solution of 0.01mol/L dipotassium hydrogen phosphate and 0.001mol/L decane sodium sulfonate, the pH value is 2.0, and the mobile phase B is absolute ethyl alcohol;
the G mobile phase A is a mixed solution of 0.1mol/L dipotassium hydrogen phosphate and 0.01mol/L decane sodium sulfonate, the pH value is 2.5, and the mobile phase B is absolute ethyl alcohol;
(other chromatographic conditions were the same as in example 1 when the mobile phase composition was changed).
Volume ratio of mobile phase a to mobile phase B: 1000: 50. 1000:70. 1000:90; (other chromatographic conditions were the same as in example 1 when the volume ratio of mobile phase A and mobile phase B was changed).
A mixed control was prepared with glycine and serine concentrations of 2.5g/L, respectively, and tested according to the procedure of example 1. A sample having a theoretical glycine content of 1.0g/L and a theoretical serine concentration of 1.2g/L was prepared and tested according to the procedure of example 2. The results of the glycine and serine content measurements under different chromatographic conditions are recorded in Table 1.
TABLE 1
As can be seen from Table 1, the chromatographic conditions are within the scope of the examples of the present invention, and the accuracy of the assay can be maintained. The average value of the serine content of the sample is 1.13g/L, the detection result is very close to the theoretical serine content (1.2 g/L), and the RSD value of serine is less than 2%. The average value of the glycine content of the sample is 0.95g/L, the detection result is very close to the theoretical glycine content (1 g/L), and the RSD value of glycine is less than 2%. In conclusion, the method provided by the embodiment of the invention has good precision and accuracy.
Example 6
The instrument and the detection conditions adopted in the embodiment are as follows:
instrument: dionex Ultimate 3000 high performance liquid chromatograph; a VWD ultraviolet detector;
chromatographic column: siemens Syncronis C18 (5 μm, 4.6X1250 mm);
column temperature: 30 ℃;
wavelength: 210nm;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
the mobile phase employs two conventional mobile phase conditions:
d1:10% aqueous methanol;
d2:0.05mol/L potassium dihydrogen phosphate buffer solution;
d3: mobile phase A is 0.05mol/L dipotassium hydrogen phosphate, pH value is 2.3, mobile phase B is absolute ethyl alcohol, and mobile phase volume ratio is 1000:70;
d4: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.02mol/L decane sodium sulfonate, the pH value is 2.3, the mobile phase B is absolute ethyl alcohol, and the volume ratio of the mobile phase is 1000:70;
the specific procedure is as in example 5, with a theoretical glycine content of 1.0g/L and a theoretical serine concentration of 1.2 g/L.
The high performance liquid chromatograms obtained by detecting the mobile phases D1 and D2 are respectively shown in fig. 5 and 6, and in the mobile phases of the two conventional chromatographic conditions, glycine and serine can not be separated, and only one liquid chromatographic peak exists on the chromatogram.
The mobile phases D3 and D4 were used for detection, and could not be detected. When the decane sodium sulfonate is not added in the mobile phase, the separation time of the sample on the chromatographic column is short, the retention time of the detection sample is influenced, and therefore, the effective experimental result cannot be qualitatively and quantitatively detected. When the addition amount of the decane sulfonic acid sodium is too large, the separation effect of the chromatographic column is seriously affected, and no sample can be detected.
While the preferred embodiment has been described, it is not intended to limit the scope of the claims, and any person skilled in the art can make several possible variations and modifications without departing from the spirit of the invention, so the scope of the invention shall be defined by the claims.
Claims (18)
1. A method for simultaneously detecting glycine and serine in enzymatic reaction liquid is characterized in that the method adopts high performance liquid chromatography, the enzymatic reaction liquid is injected into the high performance liquid chromatograph, C18 column separation is adopted, and an ultraviolet absorption detector is adopted for detection;
the mobile phase A of the high performance liquid chromatography is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L decane sodium sulfonate, and the pH value is 2.3; the mobile phase B of the high performance liquid chromatography is absolute ethyl alcohol;
the volume ratio of the mobile phase A to the mobile phase B is 1000: 50-1000: 90.
2. the method according to claim 1, wherein the volume ratio of mobile phase a to mobile phase B is 1000:70.
3. the method of claim 1, wherein the mobile phase a further comprises a pH adjuster.
4. A method according to claim 3, wherein the pH modifier is selected from at least one of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid.
5. The method of claim 1, wherein the chromatographic conditions of the high performance liquid chromatography are: the column temperature is 20-40 ℃, the detection wavelength is 190-230 nm, the flow rate is 0.8-1.5 mL/min, and the sample injection amount is 5-20 mu L.
6. The method of claim 1, wherein the column temperature is 25 to 35 ℃.
7. The method of claim 1, wherein the column temperature is 30 ℃.
8. The method according to claim 1, wherein the detection wavelength is 200-220 nm.
9. The method of claim 1, wherein the detection wavelength is 210nm.
10. The method of claim 1, wherein the sample flow rate is 0.8 to 1mL/min.
11. The method of claim 1, wherein the sample flow rate is 1mL/min.
12. The method of claim 1, wherein the sample is introduced in an amount of 8 to 15 μl.
13. The method of claim 1, wherein the sample is introduced at a volume of 10 μl.
14. Method according to any one of claims 1-13, characterized in that the method comprises at least the following steps:
(1) Preparing a mixed reference substance solution: the concentration of glycine reference substance in the mixed reference substance solution is 0.1-5 g/L, and the concentration of serine reference substance is 0.1-5 g/L;
(2) Preparing a test sample solution: diluting the enzymatic reaction solution to be tested to a concentration of 0.1-5 g/L to obtain a sample solution;
(3) And (3) detection: and respectively taking the mixed reference substance solution and the sample solution, injecting the mixed reference substance solution and the sample solution into a high performance liquid chromatograph, adopting a C18 column for separation, detecting a glycine absorption peak and a serine absorption peak which are separated by an ultraviolet detector, and calculating the content of glycine and serine in the enzymatic reaction liquid to be detected by an external standard method.
15. The method according to claim 14, wherein in step (1), a glycine control stock solution and a serine control stock solution are prepared, respectively, and before detection, the glycine control stock solution and the serine control stock solution are taken and mixed, and diluted with distilled water, to obtain the mixed control solution.
16. The method of claim 14, wherein the glycine control stock solution is formulated by: weighing glycine reference substance, and diluting with distilled water to obtain glycine reference substance stock solution with concentration of 1-10 g/L.
17. The method of claim 14, wherein the serine control stock solution is formulated by: weighing serine reference substance, and diluting with distilled water to obtain serine reference substance stock solution with concentration of 1-10 g/L.
18. The method according to claim 14, wherein in the step (2), the enzymatic reaction solution to be measured is diluted and then filtered by using a 0.45 μm aqueous filter membrane.
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