CN112924607A - Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid - Google Patents
Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid Download PDFInfo
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- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 title claims abstract description 160
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 title claims abstract description 152
- 229960002989 glutamic acid Drugs 0.000 title claims abstract description 78
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- 229940009533 alpha-ketoglutaric acid Drugs 0.000 title claims abstract description 77
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- AIMUHNZKNFEZSN-UHFFFAOYSA-M sodium;decane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCS([O-])(=O)=O AIMUHNZKNFEZSN-UHFFFAOYSA-M 0.000 claims description 20
- 239000011550 stock solution Substances 0.000 claims description 20
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 19
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- 238000002347 injection Methods 0.000 claims description 8
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- 238000004519 manufacturing process Methods 0.000 abstract description 6
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- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
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- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010010075 Coma hepatic Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
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- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
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- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- JFCQEDHGNNZCLN-UHFFFAOYSA-N glutaric acid Chemical class OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
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- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
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- 229960003104 ornithine Drugs 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
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- 239000002243 precursor Substances 0.000 description 1
- 230000003658 preventing hair loss Effects 0.000 description 1
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8818—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving amino acids
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The invention relates to the field of biological analysis, in particular to a method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction liquid. The invention adopts high performance liquid chromatography, the enzymatic reaction liquid is injected into a high performance liquid chromatograph, C18 column separation is adopted, and an ultraviolet absorption detector is used for detection. The invention realizes the simultaneous detection of the contents of alpha-ketoglutaric acid and L-glutamic acid in the enzymatic reaction liquid, can provide reliable data support for the real-time monitoring of the production of the alpha-ketoglutaric acid, does not need pre-column or post-column derivatization treatment for a detection sample, greatly shortens the detection time and has more accurate detection result.
Description
Technical Field
The invention relates to the field of biological analysis, in particular to a method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction liquid.
Background
Ketoglutaric acid (Ketoglutaric acid) is two keto-group-bearing derivatives of glutaric acid, and is classified into two types, alpha-type and beta-type. Alpha-ketoglutarate is mostly referred to as the alpha form. Alpha-ketoglutarate is one of key intermediates of basic metabolism tricarboxylic acid cycle in a life system, regulates a plurality of basic metabolic processes, and can provide a carbon skeleton for biosynthesis of L-glutamic acid, glutamine, ornithine and the like. The research finds that the alpha-ketoglutaric acid serving as a precursor of glutamine (Gln) can provide energy for gastrointestinal tract cells, promote the proliferation of the gastrointestinal tract cells and synergistically maintain the gastrointestinal tract health. It is also a pharmaceutical preparation, a dietary supplement and a special medicinal food and feed additive.
L-glutamic acid (L-Glu) is an acidic amino acid and has the chemical name of alpha-aminoglutaric acid. L-glutamic acid plays a key role in protein metabolism in organisms, and it also has various physiological functions. For example, in medicine, it can improve children's intelligence development, and can be used as a medicine to participate in the metabolism of protein and sugar in brain, and combine with ammonia in vivo to form non-toxic glutamine, so as to reduce blood ammonia content, and thus alleviate hepatic coma. In the food industry, the raw materials for producing monosodium glutamate, spices, nutritional supplements, biochemical reagents and the like. Meanwhile, L-glutamic acid is also a hair restoration agent for preventing hair loss. L-glutamic acid is used for skin, and can effectively treat wrinkles.
Both have important application prospects in various fields, and the market demand is very large.
The chemical synthesis method is adopted to synthesize the alpha-ketoglutaric acid, but the alpha-ketoglutaric acid is gradually eliminated due to a series of problems of environmental pollution and the like. The enzyme method can obtain a product with a single structure and high purity, and is widely applied. The production and preparation of alpha-ketoglutaric acid by fermentation method has become a research hotspot. The method for producing the L-glutamic acid needs to establish a method suitable for separating and quantitatively detecting the L-glutamic acid and the alpha-ketoglutaric acid, and the method needs to have the characteristics of accuracy, rapidness, simplicity and the like.
The prior art does not report that the content of L-glutamic acid and alpha-ketoglutaric acid in fermentation liquor is simultaneously measured by adopting a high performance liquid chromatography. At present, the content of alpha-ketoglutaric acid is determined by acid-base titration, spectrophotometry (Lipuidization factor analysis-spectrophotometry in the determination of alpha-ketoglutaric acid and pyruvic acid in the analysis of mixture components, analytical chemistry, 1987(10):29-34) and HPLC (Maoying, Bizhou, HPLC for determining the content of alpha-ketoglutaric acid, Chinese medicine, 2008,22(10):895 and 896). However, the specificity of the acid-base titration method is poor, and the measured result cannot correctly reflect the content of the alpha-ketoglutaric acid in the sample and is gradually eliminated. The spectrophotometry has poor detection result accuracy, low precision and long detection time, and only can detect the content of the alpha-ketoglutaric acid in the fermentation liquor. The established HPLC method has good detection result accuracy, but cannot simultaneously detect the contents of L-glutamic acid and alpha-ketoglutaric acid in fermentation liquor. Patent ZL201610395430.7 (entitled electrochemical biosensor for detecting α -ketoglutaric acid, preparation method and application thereof) applies electrochemical principles to detect α -ketoglutaric acid content, which is difficult to implement in practical applications.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction liquid. The invention establishes an accurate and convenient method for simultaneously determining the contents of alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction solution, and can meet the requirements of production enterprises with more detection samples and large workload.
In order to realize the purpose of the invention, the technical scheme is as follows:
the invention provides a method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction solution, which adopts a high performance liquid chromatography to inject the enzymatic reaction solution into a high performance liquid chromatograph, adopts a C18 column for 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 sodium decane sulfonate, and the pH value is 1-5; and the mobile phase B of the high performance liquid chromatography is absolute ethyl alcohol.
Optionally, the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate.
Optionally, the pH of the 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 as follows: 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 muL.
Optionally, the chromatographic conditions of the high performance liquid chromatography are as follows: the column temperature is 25-35 ℃, preferably 30 ℃, the detection wavelength is 200-220 nm, preferably 215nm, the sample flow rate is 0.8-1 mL/min, preferably 1mL/min, and the sample injection amount is 8-15 muL, preferably 10 muL.
Optionally, the method at least comprises the following steps:
(1) preparing a mixed reference solution: the concentration of the alpha-ketoglutaric acid reference substance in the mixed reference substance solution is 0.2-1.83 g/L, L-the concentration of the glutamic acid reference substance is 1.2-10.79 g/L;
(2) preparing a test solution: diluting an enzymatic reaction solution to be detected to the concentration of 0.1-5 g/L to serve as a test solution;
(3) and (3) detection: and (3) respectively taking the mixed reference solution and the test solution, directly injecting into a high performance liquid chromatograph, separating by adopting a C18 column, detecting the separated alpha-ketoglutaric acid absorption peak and L-glutamic acid absorption peak by using an ultraviolet absorption detector, and calculating by using an external standard method to obtain the contents of alpha-ketoglutaric acid and L-glutamic acid in the enzymatic reaction liquid to be detected.
Optionally, in the step (1), preparing a mixed stock solution containing both a ketoglutaric acid reference substance and an L-glutamic acid reference substance, and diluting the mixed stock solution with distilled water before detection to obtain a mixed reference substance solution;
the preparation method of the mixed stock solution is preferably as follows: weighing alpha-ketoglutaric acid reference substance and L-glutamic acid reference substance in the same volumetric flask respectively, adding acid for dissolving, and diluting with distilled water to obtain mixed stock solution of alpha-ketoglutaric acid with concentration of 2g/L and L-glutamic acid with concentration of 12g/L respectively.
Optionally, in the step (2), the enzymatic reaction solution to be detected is diluted and then filtered, and the filtering is performed by using a 0.45 μm water system filter membrane.
The invention has at least the following beneficial effects:
the invention realizes the simultaneous detection of the contents of alpha-ketoglutaric acid and L-glutamic acid in the enzymatic reaction liquid, can provide reliable data support for the real-time monitoring of the production of the L-glutamic acid, and has the following technical effects:
(1) the invention can simultaneously detect the contents of alpha-ketoglutaric acid and L-glutamic acid 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 detection sample can be directly taken and can be detected on the machine after being treated and filtered by deionized water, the treatment steps of the detection sample are simple, and the error rate of experimental operation is reduced.
(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 method is suitable for enterprises with more samples 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 the alpha-ketoglutaric acid and the L-glutamic acid by the enzyme method.
Drawings
FIG. 1 is a detection spectrum of a mixed control solution in example 1;
FIG. 2 is a detection map of the enzymatic reaction solution in example 2;
FIG. 3 is a graph showing the peak area of each concentration of α -ketoglutarate and the linear α -ketoglutarate in example 3;
FIG. 4 is a graph showing the peak area of L-glutamic acid concentration and the linearity of L-glutamic acid in example 4;
FIG. 5 is a detection map of mobile phase D3 in example 6;
FIG. 6 is a detection map of mobile phase D4 in example 6.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only 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 alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction liquid. Specifically, the method adopts high performance liquid chromatography, the enzymatic reaction solution containing alpha-ketoglutaric acid and L-glutamic acid is injected into a high performance liquid chromatograph, C18 column separation is adopted, and an ultraviolet absorption detector is utilized to detect the separated alpha-ketoglutaric acid absorption peak and L-glutamic acid absorption peak. Because alpha-ketoglutaric acid and L-glutamic acid have poor absorption in the ultraviolet spectrum, in the prior art, only a derivation method can be adopted to convert alpha-ketoglutaric acid or L-glutamic acid into other substances with absorption in the ultraviolet spectrum, or other detectors are adopted to detect. The embodiment of the invention realizes the invention by keen research and without other processing steps, so that the alpha-ketoglutaric acid and the L-glutamic acid have obvious and separated absorption peaks on the ultraviolet spectrum simultaneously.
The detection method of the embodiment of the invention overcomes the defects brought by indirect detection by a derivation method in the prior art, realizes simultaneous detection of the contents of alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction solution 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 is simple in operation steps, and can carry out detection only by adjusting the concentration of the enzymatic reaction solution. Therefore, the method has the characteristics of direct, rapid 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 sodium decane sulfonate, the pH value is 1-5, and if the pH value is too high or too low, the separation degree is affected; the mobile phase B is absolute ethyl alcohol. The mobile phase of the embodiment of the invention not only enables the alpha-ketoglutaric acid and the L-glutamic acid to have good absorption peaks on an ultraviolet absorption detector, but also enables the alpha-ketoglutaric acid absorption peaks and the L-glutamic acid absorption peaks to have good separation degrees, thereby realizing the simultaneous detection of the alpha-ketoglutaric acid and the L-glutamic acid.
Further alternatively, the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate.
Further optionally, the pH of mobile phase a is 2.3.
Further optionally, the volume ratio of mobile phase a to mobile phase B is 1000: 50-1000: 90, preferably 1000: and 70, mixing the mobile phase A and the mobile phase B according to the proportion to obtain the mobile phase. If the volume ratio of the mobile phase A to the mobile phase B is too large or too small, the separation degree of the two absorption peaks is affected.
Further optionally, the mobile phase a further contains a pH adjusting agent, and the pH of the mobile phase a is adjusted to be within the range of the embodiment of the present invention, and the pH adjusting agent 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 invention can be a common commercial C18 column, such as: a Saimer fly Syncronis C18 column, an Elite Hypersil ODS 2C 18 column, a Saimer fly BDS Hypersil TM C18 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 190-230 nm, preferably 200-220 nm, and more preferably 215 nm.
The flow rate of the sample is 0.8-1.5 mL/min, if the flow rate of the sample is too high, the peak is too early and can coincide with other peaks (such as solvent peaks), and if the flow rate of the sample is too low, the analysis time can be prolonged, and the working efficiency is reduced; preferably, the sample flow rate is 0.8-1 mL/min, more preferably 1 mL/min.
The sample injection amount is 5-20 mu L, if the sample injection amount is too large, instrument load can be caused, the absorption peak can also be subjected to tailing phenomenon, and the separation degree between the alpha-ketoglutaric acid absorption peak and the L-glutamic acid absorption peak is influenced; if the sample injection amount is too small, the measurement is inaccurate; preferably, the sample injection amount is 8-15 μ L, and more preferably 10 μ L.
Optionally, the method of the embodiment of the present invention at least includes the following steps:
(1) preparing a mixed reference solution: the concentration of the alpha-ketoglutaric acid reference substance in the mixed reference substance solution is 0.2-1.83 g/L, L-the concentration of the glutamic acid reference substance is 1.2-10.79 g/L;
(2) preparing a test solution: diluting an enzymatic reaction solution to be detected to the concentration of 0.1-5 g/L to serve as a test solution;
(3) and (3) detection: and respectively taking the mixed reference solution and the test solution, directly adding the mixed reference solution and the test solution into a high performance liquid chromatograph, separating by adopting a C18 column, detecting the separated alpha-ketoglutaric acid absorption peak and L-glutamic acid absorption peak by using an ultraviolet absorption detector, and calculating by using an external standard method to obtain the contents of alpha-ketoglutaric acid and L-glutamic acid in the enzymatic reaction solution to be detected.
The research of the embodiment of the invention finds that when the concentration ranges of the alpha-ketoglutaric acid reference substance and the alpha-ketoglutaric acid test solution are between 0.2g/L and 1.83g/L, the alpha-ketoglutaric acid presents a good linear relationship. When the concentration ranges of the L-glutamic acid reference substance and the L-glutamic acid test solution are between 1.2g/L and 10.79g/L, the L-glutamic acid presents a good linear relation. The contents of L-glutamic acid and alpha-ketoglutaric acid can be calculated in the linear range by adopting an external standard method. Wherein the concentration of the sample solution can be obtained by simple calculation according to the addition of the raw materials.
Optionally, in step (1), a mixed stock solution containing both a ketoglutaric acid reference substance and an L-glutamic acid reference substance is prepared, and the mixed stock solution is diluted with distilled water before detection to obtain a mixed reference substance solution.
The method for preparing the mixed stock solution is preferably: weighing alpha-ketoglutaric acid reference substance and L-glutamic acid reference substance in the same volumetric flask respectively, adding acid for dissolving, and diluting with distilled water to obtain mixed stock solution of alpha-ketoglutaric acid with concentration of 2g/L and L-glutamic acid with concentration of 12g/L respectively. The acid used can be hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, etc., preferably hydrochloric acid.
Optionally, in the step (2), the enzymatic reaction solution to be detected is diluted and then filtered, and the filtering is performed by using a 0.45 μm water system filter membrane. In the embodiment of the present invention, the enzyme reaction solution is not required to be pretreated by, for example, a derivatization method, and can be detected by simple filtration.
The contents of the embodiments of the present invention are further explained and illustrated by the following examples.
Example 1
The instrumentation and detection conditions employed in this example were as follows:
the instrument comprises the following steps: a Dionex Ultimate 3000 hplc; a VWD ultraviolet detector;
a chromatographic column: saimeifei Syncronis C18(5 μm, 4.6X 250 mm);
column temperature: 30 ℃;
wavelength: 215 nm;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a and mobile phase B: 1000: 70.
the specific operation steps for measuring the contents of the standard products of alpha-ketoglutaric acid and L-glutamic acid are as follows:
1. preparing a reference substance mixed stock solution: accurately weighing 0.2g of alpha-ketoglutaric acid reference substance and 1.2g of L-glutamic acid reference substance in the same volumetric flask, adding 10mL of 1mol/L hydrochloric acid for dissolving, and dissolving and diluting with distilled water to obtain 2g/L alpha-ketoglutaric acid reference stock solution and 12 g/L-glutamic acid reference stock solution.
2. Preparing mixed reference substance solutions with different concentrations: before use, 0.1mL of the stock solution of the above control is taken out and put into a 10mL volumetric flask, and the stock solution is diluted with distilled water to obtain a mixed control solution of alpha-ketoglutaric acid and L-glutamic acid with the concentrations of 0.2g/L and 1.2g/L respectively.
3. And (3) detection: precisely sucking 10 μ L of the mixed reference solution, injecting into a liquid chromatograph for measurement, and recording chromatogram. The results of the experiment are shown in FIG. 1.
Example 2
The instrumentation and detection conditions employed in this example were as follows:
the instrument comprises the following steps: a Dionex Ultimate 3000 hplc; a VWD ultraviolet detector;
a chromatographic column: saimeifei Syncronis C18(5 μm, 4.6X 250 mm);
column temperature: 30 ℃;
wavelength: 215 nm;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a and mobile phase B: 1000: 70.
the specific operation steps for measuring the content of alpha-ketoglutaric acid and L-glutamic acid in the enzymatic reaction liquid are as follows:
1. preparing a test solution: and (3) putting 1mL of the enzymatic reaction solution into a 10mL volumetric flask, diluting to constant volume with distilled water, and shaking up to obtain the enzyme-linked immunosorbent assay. The solution was filtered through a 0.45 μm aqueous membrane, and the filtrate was used as a sample test solution.
2. And (3) detection: sampling 10 μ L of sample solution, injecting into liquid chromatograph, measuring, and recording chromatogram. The results of the experiment 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 instrumentation and detection conditions employed in this example were as follows:
the instrument comprises the following steps: a Dionex Ultimate 3000 hplc; a VWD ultraviolet detector;
a chromatographic column: saimeifei Syncronis C18(5 μm, 4.6X 250 mm);
column temperature: 30 ℃;
wavelength: 215 nm;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a and mobile phase B: 1000: 70.
the specific operation steps of the alpha-ketoglutaric acid linear test are as follows:
1. preparing a test solution: before use, 0.1mL, 0.3mL, 0.5mL, 0.7mL and 0.9mL of control mixed stock solutions of alpha-ketoglutaric acid and L-glutamic acid are respectively taken and put into a 10mL volumetric flask, and diluted by distilled water to obtain test solution with the concentration of 0.20g/L, 0.61g/L, 1.02g/L, 1.42g/L and 1.83 g/L.
2. And (6) detecting. And (3) taking 10 mu L of test solution, injecting the test solution into a liquid chromatograph for measurement, and recording a chromatogram. Taking the peak area of alpha-ketoglutaric acid as ordinate Y and the concentration of alpha-ketoglutaric acid as abscissa X to perform linear regression to obtain correlation coefficient R of linear equation 21. The area of each concentration peak and the linearity of α -ketoglutarate are shown in FIG. 3.
From the α -ketoglutarate line plot of FIG. 3, it can be seen that: alpha-ketoglutaric acid has good linearity in the concentration range of 0.2-1.83 g/L.
Example 4
The instrumentation and detection conditions employed in this example were as follows:
the instrument comprises the following steps: a Dionex Ultimate 3000 hplc; a VWD ultraviolet detector;
a chromatographic column: saimeifei Syncronis C18(5 μm, 4.6X 250 mm);
column temperature: 30 ℃;
wavelength: 215 nm;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
mobile phase: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
volume ratio of mobile phase a and mobile phase B: 1000: 70.
the specific operation steps of the L-glutamic acid linear test are as follows:
1. preparing a test solution: before use, 0.1mL, 0.3mL, 0.5mL, 0.7mL and 0.9mL of the mixed stock solutions of the alpha-ketoglutaric acid and the L-glutamic acid reference substances are respectively taken and put into a 10mL volumetric flask, and the mixed stock solutions are diluted by distilled water to obtain test solution with the concentration of 1.2g/L, 3.60g/L, 6g/L, 8.39g/L and 10.79 g/L.
2. And (3) detection: taking 10 μ L of sample solution, injecting into liquid chromatograph, measuring, and recording chromatogram to obtainTaking the peak area of L-glutamic acid as ordinate Y and the concentration of alpha-ketoglutaric acid as abscissa X, performing linear regression to obtain correlation coefficient R of linear equation 21. The peak area and L-glutamic acid linearity at each concentration are shown in FIG. 4.
From the L-glutamic acid line graph of FIG. 4, it can be seen that: the L-glutamic acid has good linearity within the concentration range of 1.2-10.79 g/L.
Example 5
In this example, the durability of the method was examined by changing any chromatographic conditions such as column temperature, wavelength, sample volume, flow rate, column, and mobile phase volume ratio.
The present embodiment adopts the following instruments and devices and detection conditions:
the instrument comprises the following steps: a Dionex Ultimate 3000 hplc; a VWD ultraviolet detector;
a chromatographic column:
aylett Hypersil ODS 2C 18(5 μm, 4.6X 250mm),
B Silmer fly Syncronis C18(5 μm, 4.6 × 250mm),
C Saimer fly BDS Hypersil TM C18(5 μm, 4.6X 250 mm);
(when the column was changed, other chromatographic conditions were the same as in example 1).
Column temperature: 20 ℃, 30 ℃ and 40 ℃; (when the column temperature was changed, other chromatographic conditions were the same as in example 1).
Wavelength: 190nm, 215nm, 230 nm; (other chromatographic conditions were the same as in example 1 when the wavelength was changed).
Sample introduction amount: 5. mu.L, 10. mu.L, 20. mu.L; (when the amount of sample was changed, other chromatographic conditions were the same as in example 1).
Flow rate: 0.8mL/min, 1.0mL/min, 1.5 mL/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 sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
the mobile phase A is a mixed solution of 0.1mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
g, the mobile phase A is a mixed solution of 0.01mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
h, the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.001mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.01mol/L sodium decane sulfonate, the pH value is 2.3, and the mobile phase B is absolute ethyl alcohol;
(when the mobile phase composition was changed, other chromatographic conditions were the same as in example 1).
Volume ratio of mobile phase a and mobile phase B: 1000: 50. 1000: 70. 1000: 90, respectively; (when the volume ratio of the mobile phase A and the mobile phase B was changed, other chromatographic conditions were the same as in example 1).
1. A mixed control solution was prepared according to the method of example 1:
2. preparing a test solution with the content of alpha-ketoglutaric acid of 0.2g/L, L and the concentration of glutamic acid of 1.2 g/L;
3. and respectively taking 10 mu L of the mixed reference substance solution and the test sample solution, injecting the mixed reference substance solution and the test sample solution into a liquid chromatograph for measurement, and recording the chromatogram.
The results of the content measurements of alpha-ketoglutaric acid and L-glutamic acid under different chromatographic conditions are reported in Table 1.
TABLE 1
As can be seen from Table 1, the chromatographic conditions were within the range of the examples of the present invention, and the accuracy of the measurement was maintained. The method of the invention has good durability. The average value of the content of the L-glutamic acid in the sample is 1.178g/L, the detection result is very close to the theoretical content of the L-glutamic acid (1.2g/L), and the RSD value of the L-glutamic acid is 0.65 percent. The average value of the content of the alpha-ketoglutaric acid of the sample is 0.193g/L, the detection result is very close to the theoretical content (0.2g/L) of the alpha-ketoglutaric acid, and the RSD value of the alpha-ketoglutaric acid is 0.5 percent. In conclusion, the experimental method has good precision and accuracy.
Example 6
In this embodiment, the influence of the mobile phase is examined, and the following instruments and detection conditions are adopted:
the instrument comprises the following steps: a Dionex Ultimate 3000 hplc; a VWD ultraviolet detector;
a chromatographic column: saimeifei Syncronis C18(5 μm, 4.6X 250 mm);
column temperature: 30 ℃;
wavelength: 215 nm;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
the mobile phase was used as follows:
d1: the mobile phase A is 0.05mol/L dipotassium hydrogen phosphate, the pH value is 2.3, the mobile phase B is absolute ethyl alcohol, and the volume ratio of the mobile phase A to the mobile phase B is 1000: 70;
d2: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.02mol/L sodium decane sulfonate, the pH value is 2.3, the mobile phase B is absolute ethyl alcohol, and the volume ratio of the mobile phases is 1000: 70;
d3: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, the mobile phase B is 10% methanol, and the volume ratio of the mobile phase is 1000: 70;
d4: the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate, the pH value is 2.3, the mobile phase B is acetonitrile, and the volume ratio of the mobile phases is 1000: 70.
the specific procedure of the detection was as in example 5, and the test solution with an α -ketoglutaric acid content of 0.2g/L, L and a glutamic acid concentration of 1.2g/L was examined. The results of the experiment are shown in Table 2.
Table 2: mobile phase investigation test results
As can be seen from the experimental data in table 2, when the mobile phase D1 is adopted and sodium decane sulfonate is not added to the mobile phase a, the retention time of the detection sample is affected, and thus an effective experimental result cannot be detected. In mobile phase D3, no valid experimental results could be detected using 10% methanol as mobile phase B, and the high performance liquid chromatogram is shown in fig. 5.
When the mobile phase D2 is adopted, the addition amount of sodium decane sulfonate is too large, the separation effect of a chromatographic column on a sample is seriously influenced, and the detection result is inaccurate. In the mobile phase D4, acetonitrile is selected as the mobile phase B, the instrument balance consumes long time, and the base line is not stable, so that the accuracy of the detection result is influenced. The difference between the detection result of the content of the sample L-glutamic acid and the theoretical content of the L-glutamic acid (1.2g/L) is large, and the difference between the detection result of the content of the sample alpha-ketoglutaric acid and the theoretical content of the alpha-ketoglutaric acid (0.2g/L) is large. The baseline results for acetonitrile as mobile phase B are shown in figure 6. Therefore, the embodiment of the invention selects the ethanol with the lowest toxicity and stable baseline, so the ethanol is selected as the mobile phase B.
Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.
Claims (10)
1. A method for detecting alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction solution simultaneously is characterized in that the method adopts high performance liquid chromatography, the enzymatic reaction solution is injected into a high performance liquid chromatograph, a C18 column is adopted for separation, and an ultraviolet absorption detector is adopted for detection;
the mobile phase A of the high performance liquid chromatography is a mixed solution of 0.01-0.1 mol/L dipotassium hydrogen phosphate and 0.001-0.01 mol/L sodium decane sulfonate, and the pH value is 1-5; and the mobile phase B of the high performance liquid chromatography is absolute ethyl alcohol.
2. The method according to claim 1, wherein the mobile phase A is a mixed solution of 0.05mol/L dipotassium hydrogen phosphate and 0.005mol/L sodium decane sulfonate.
3. The method according to claim 1, characterized in that the mobile phase a has a pH value of 2.3.
4. The method of claim 1, wherein the volume ratio of the mobile phase a to the mobile phase B is 1000: 50-1000: 90, preferably 1000: 70.
5. the method according to claim 1, characterized in that the mobile phase A further contains a pH value regulator, and the pH value regulator is preferably at least one of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid and acetic acid.
6. 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 muL.
7. The method of claim 1, wherein 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 215nm, the sample flow rate is 0.8-1 mL/min, preferably 1mL/min, and the sample injection amount is 8-15 muL, preferably 10 muL.
8. A method according to any of claims 1 to 7, characterized in that the method comprises at least the following steps:
(1) preparing a mixed reference solution: the concentration of the alpha-ketoglutaric acid reference substance in the mixed reference substance solution is 0.2-1.83 g/L, L-the concentration of the glutamic acid reference substance is 1.2-10.79 g/L;
(2) preparing a test solution: diluting an enzymatic reaction solution to be detected to the concentration of 0.1-5 g/L to serve as a test solution;
(3) and (3) detection: and (3) respectively taking the mixed reference solution and the test solution, injecting the mixed reference solution and the test solution into a high performance liquid chromatograph, separating by adopting a C18 column, detecting a separated alpha-ketoglutaric acid absorption peak and an L-glutamic acid absorption peak by using an ultraviolet detector, and calculating by using an external standard method to obtain the contents of alpha-ketoglutaric acid and L-glutamic acid in the enzymatic reaction solution to be detected.
9. The method according to claim 8, wherein in step (1), a mixed stock solution containing both a-ketoglutarate control and an L-glutamic acid control is prepared, and the mixed stock solution is diluted with distilled water before the detection to obtain the mixed control solution;
the preparation method of the mixed stock solution is preferably as follows: weighing alpha-ketoglutaric acid reference substance and L-glutamic acid reference substance in the same volumetric flask respectively, adding acid for dissolving, and diluting with distilled water to obtain mixed stock solution of alpha-ketoglutaric acid with concentration of 2g/L and L-glutamic acid with concentration of 12g/L respectively.
10. The method according to claim 8, wherein in the step (2), the enzymatic reaction solution to be tested is diluted and then filtered, and the filtration is performed by using a 0.45 μm aqueous membrane.
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