CN112924566A - 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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- 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 85
- 238000000034 method Methods 0.000 title claims abstract description 53
- 238000006911 enzymatic reaction Methods 0.000 title claims abstract description 31
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- 238000001514 detection method Methods 0.000 claims abstract description 61
- 238000010521 absorption reaction Methods 0.000 claims abstract description 25
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 19
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- 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
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- YPWSLBHSMIKTPR-UHFFFAOYSA-N Cystathionine Natural products OC(=O)C(N)CCSSCC(N)C(O)=O YPWSLBHSMIKTPR-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UWTATZPHSA-N D-Serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-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
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 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
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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
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
- G01N30/62—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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Abstract
The invention relates to the field of biological analysis, in particular to a method for simultaneously detecting glycine and serine in an enzymatic reaction solution. 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 method realizes the simultaneous detection of the glycine content and the serine content in the enzymatic reaction solution, can provide reliable data support for the real-time monitoring of the serine production, 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 glycine and serine in an enzymatic reaction solution.
Background
Serine (Ser), also known as beta-hydroxy alanine, has the chemical formula C3H7NO3(ii) a Glycine (Gly), also known as aminoacetic acid, has the chemical formula C2H5NO2. They are non-essential amino acids, are widely applied to the fields of cosmetics, medicines, foods and the like, and play a key role. For example, serine plays an important role in the metabolism of fats and fatty acids and in muscle growth. It also aids in the production of immunohemoglobin and antibodies and maintains the immune system healthy. Serine is also a nutritional supplement, a serine derivative material, an eye drop pH regulator, and a natural humectant. Glycine is an important organic chemical intermediate, and is mainly used for synthesizing glyphosate. The glycine has the functions of regulating food flavor, inhibiting escherichia coli propagation, inhibiting dairy product oxidation and the like. With the wide application of the special functions of serine and glycine, the market demand is also increasing year by year.
At present, amino acid is mainly produced by methods such as chemical synthesis, extraction, enzyme method, fermentation and the like. The enzyme method can obtain a product with a single structure and high purity, and is widely applied. The method for producing serine needs to be established for separating serine and glycine and quantitatively detecting serine and glycine, and the method needs to have the characteristics of accuracy, rapidness, simplicity and the like.
In the related art, the literature "determination of the contents of L-serine and glycine in a fermentation broth by a paper chromatography-spectrophotometry" (Lufa, Changwei, food and fermentation industry, 3 rd 2005) mainly includes the steps of centrifugation, spotting, blow-drying, color development, elution and the like, and the detection time is long. The method needs to be configured with the color developing agent, optimizes a plurality of experimental factors, and 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 a method for high performance liquid chromatography of serine, which employs derivatization reagent to derivatize a sample to be detected, and then uses high performance liquid chromatography to indirectly measure the content of serine. The method has complicated derivation steps and inconvenient operation. In addition, derivatization produces many by-products, making chromatographic separation difficult, which in turn affects the accuracy of serine quantitation. And the derivative reagents have various types, are difficult to screen, are expensive, have high detection cost and are not suitable for the detection of multiple samples. Patent ZL201310591734.7, entitled method for quantitatively detecting serine, adopts cystathionine lyase (CBL) to carry out enzymolysis on serine to generate pyruvic acid, and indirectly measures the content of serine in a sample by measuring the content of the pyruvic acid. The method also has the problems of troublesome sample treatment, fussy experimental operation, inaccurate experimental result and the like. In the literature, "content of glycine in blood products is measured by using a post-column derivatization HPLC method and content of glycine in dairy products is measured by using a pre-column derivatization high performance liquid chromatography" and in patent application CN201710050705.8 (the name of the invention is a method for detecting glycine and impurities thereof by using high performance liquid chromatography), the content of glycine is indirectly measured by using a derivatization method. The detector adopted in patent application CN201811635412.7 (entitled new glycine content detection method) is an ELSD detector, and the method has high requirements on chromatographic detection instruments.
The method can only be established for detecting the conventional samples at the laboratory level, and is not suitable for enterprises with more detected samples and large workload. Moreover, the content of only one amino acid can be detected by a high-precision method among the above methods. Therefore, it is urgently required to establish an accurate and convenient method for simultaneously determining the glycine content and the serine content in the enzymatic reaction solution.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting glycine and serine in an enzymatic reaction solution.
In order to realize the purpose of the invention, the technical scheme is as follows:
the invention provides a method for simultaneously detecting glycine and serine 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 210nm, 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 a glycine reference substance in the mixed reference substance solution is 0.1-5 g/L, and the concentration of a serine reference substance is 0.1-5 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 (2) respectively taking the mixed reference substance solution and the test sample solution, directly injecting into a high performance liquid chromatograph, separating by adopting a C18 column, detecting a separated glycine absorption peak and a separated serine absorption peak by utilizing an ultraviolet absorption detector, and calculating by using an external standard method to obtain the contents of glycine and serine in the enzymatic reaction solution to be detected.
Optionally, in step (1), a glycine reference substance storage solution and a serine reference substance storage solution are prepared respectively, and before detection, the glycine reference substance storage solution and the serine reference substance storage solution are respectively taken and mixed, and diluted with distilled water to obtain a mixed reference substance solution;
the preparation method of the glycine reference substance storage solution is preferably as follows: weighing a glycine reference substance, and diluting with distilled water to obtain a glycine reference substance storage solution with the concentration of 1-10 g/L;
the preparation method of the serine reference substance storage solution is preferably as follows: weighing a serine reference substance, and diluting with distilled water to obtain a serine reference substance storage solution with the concentration of 1-10 g/L.
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 glycine content and the serine content in the enzymatic reaction solution, can provide reliable data support for the real-time monitoring of the serine production, and has the following technical effects:
(1) the invention can simultaneously detect the content of glycine and serine 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 serine by an 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 glycine in example 3 and a linear curve of glycine;
FIG. 4 is a graph showing the peak area of serine concentration and the linearity of serine in example 4;
FIG. 5 is a detection spectrum of the mobile phase D1 detection mixed control solution in example 6;
FIG. 6 is a detection spectrum of the mobile phase D2 detection mixed control solution 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 glycine and serine in an enzymatic reaction solution. Specifically, the method adopts high performance liquid chromatography, the enzymatic reaction solution containing glycine and serine is injected into a high performance liquid chromatograph, C18 column is adopted for separation, and an ultraviolet absorption detector is utilized to detect the separated glycine absorption peak and serine absorption peak. Because glycine and serine have poor absorption in the ultraviolet spectrum, glycine or serine can be converted into other substances having absorption in the ultraviolet spectrum only by a derivation method and an enzymatic hydrolysis method or other detectors in the prior art for detection. The embodiment of the invention realizes the invention by keenly researching and not requiring other processing steps, so that the glycine and the serine have obvious and separated absorption peaks on an 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 glycine and serine contents in the 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 provided by the embodiment of the invention not only enables glycine and serine to have good absorption peaks on an ultraviolet absorption detector, but also enables the glycine absorption peaks and the serine absorption peaks to have good separation degrees, thereby realizing simultaneous detection of glycine and serine.
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: saimei fly Syncronis C18 column, Elite Hypersil ODS 2C 18 column, Saimei 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 210 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 glycine absorption peak and the serine 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 a glycine reference substance in the mixed reference substance solution is 0.1-5 g/L, and the concentration of a serine reference substance is 0.1-5 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 (2) respectively taking the mixed reference substance solution and the test sample solution, directly adding the mixed reference substance solution and the test sample solution into a high performance liquid chromatograph, separating by adopting a C18 column, detecting a separated glycine absorption peak and a separated serine absorption peak by using an ultraviolet absorption detector, and calculating by using an external standard method to obtain the contents of glycine and serine in the enzymatic reaction liquid to be detected.
According to the research of the embodiment of the invention, when the concentration ranges of the reference substance and the test solution are 0.1-5 g/L, the peak areas and the concentrations of the glycine and the serine at all concentrations have good linear relation. The contents of serine and glycine 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 the step (1), a glycine reference stock solution and a serine reference stock solution are prepared respectively, and before detection, the glycine reference stock solution and the serine reference stock solution are mixed respectively and diluted with distilled water to obtain a mixed reference stock solution.
Specifically, the preparation method of the glycine reference stock solution comprises the following steps: weighing a glycine reference substance, and diluting with distilled water to obtain a glycine reference substance storage solution with the concentration of 1-10 g/L;
the preparation method of the serine reference substance storage solution comprises the following steps: weighing a serine reference substance, and diluting with distilled water to obtain a serine reference substance storage solution with the concentration of 1-10 g/L.
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 or an enzymatic hydrolysis method, and detection can be performed 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: 210 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 the standard glycine and serine are as follows:
1. preparing a mixed reference substance solution:
glycine control stock solution: accurately weighing 1g of glycine reference substance, and dissolving and diluting the glycine reference substance by using distilled water to obtain a glycine reference stock solution with the 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 a serine reference stock solution with the concentration of 10 g/L;
2. mixing the reference solution: before use, appropriate amount of glycine reference stock solution and serine reference stock solution are respectively taken, and after the glycine reference stock solution and the serine reference stock solution are mixed, the two are diluted by distilled water to obtain mixed reference stock solutions with the concentrations of glycine and serine being 0.5g/L respectively.
3. 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: 210 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 glycine and serine in the enzymatic reaction solution are as follows:
1. 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.
2. The solution was filtered through a 0.45 μm aqueous membrane, and the filtrate was used as a sample test solution.
3. 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: 210 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 glycine linearity test are as follows:
1. taking appropriate amount of glycine reference stock solution, and diluting with distilled water to obtain test solution with concentration of 0.1g/L, 0.5g/L, 1g/L, 2.5g/L, and 5 g/L.
2. Taking 10 μ L of test solution, injecting into liquid chromatograph for determination, recording chromatogram, performing linear regression with glycine peak area as ordinate Y and glycine concentration as abscissa X to obtain correlation coefficient R of linear equation 21. The peak area and glycine linearity for each concentration are shown in FIG. 3. As can be seen from the glycine line diagram of FIG. 3: glycine is linear in the concentration range of 0.1-5 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: 210 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 serine linearity test are as follows:
1. taking a proper amount of serine reference stock solution, and diluting with distilled water to obtain test solution with concentration of 0.1g/L, 0.5g/L, 1g/L, 2.5g/L, and 5 g/L.
2. Taking 10 μ L of test solution, injecting into liquid chromatograph for determination, recording chromatogram, performing linear regression with serine peak area as ordinate Y and glycine concentration as abscissa X to obtain correlation coefficient R of linear equation 21. The peak area and serine linearity at each concentration are shown in FIG. 4. From the serine linear graph 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 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, 210nm, 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 0.01mol/L dipotassium hydrogen phosphate and 0.001mol/L sodium decane sulfonate, the pH value is 2.0, and the mobile phase B is absolute ethyl alcohol;
g, the mobile phase A is a mixed solution of 0.1mol/L dipotassium hydrogen phosphate and 0.01mol/L sodium decane sulfonate, the pH value is 2.5, 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).
A mixed control with glycine and serine concentrations of 2.5g/L respectively was prepared and tested according to the procedure of example 1. A test sample with the theoretical content of glycine of 1.0g/L and the theoretical concentration of serine of 1.2g/L is prepared and detected according to the operation steps of the example 2. The results of the glycine and serine content measurements 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 average serine content of the samples was 1.13g/L, which is very close to the theoretical serine content (1.2g/L) and the RSD value of serine was < 2%. The average value of the glycine content of the samples is 0.95g/L, the detection result is very close to the theoretical content (1g/L) of the glycine, and the RSD value of the glycine is less than 2%. In conclusion, the method provided by the embodiment of the invention has good precision and accuracy.
Example 6
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: saimeifei Syncronis C18(5 μm, 4.6X 250 mm);
column temperature: 30 ℃;
wavelength: 210 nm;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
the mobile phase employs two conventional mobile phase conditions:
d1: 10% aqueous methanol;
d2: 0.05mol/L potassium dihydrogen phosphate buffer solution;
d3: 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;
d4: 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;
the specific operation steps of the detection are as in example 5, and the detection is carried out on a test sample solution with the theoretical content of glycine of 1.0g/L and the theoretical concentration of serine of 1.2 g/L.
The high performance liquid chromatograms obtained by detection with mobile phases D1 and D2 are respectively shown in fig. 5 and fig. 6, and it can be seen from fig. 5 and fig. 6 that glycine and serine cannot be separated under the above two conventional chromatographic conditions, and only one liquid chromatogram peak is present on the chromatogram.
Detection with mobile phases D3 and D4 was also not possible. When sodium decanesulfonate is not added into the mobile phase, the separation time of the sample in the chromatographic column is short, and the retention time of the detected sample is influenced, so that an effective experimental result cannot be qualitatively and quantitatively detected. When the addition amount of the sodium decane sulfonate is too large, the separation effect of the chromatographic column is seriously influenced, and a sample cannot be detected.
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 simultaneously detecting glycine and serine in an enzymatic reaction solution 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 210nm, 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 a glycine reference substance in the mixed reference substance solution is 0.1-5 g/L, and the concentration of a serine reference substance is 0.1-5 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 substance solution and the test solution, injecting the mixed reference substance solution and the test solution into a high performance liquid chromatograph, separating by adopting a C18 column, detecting a separated glycine absorption peak and a separated serine absorption peak by using an ultraviolet detector, and calculating by using an external standard method to obtain the contents of glycine and serine in the enzymatic reaction solution to be detected.
9. The method according to claim 8, wherein in step (1), a glycine reference stock solution and a serine reference stock solution are prepared separately, and the glycine reference stock solution and the serine reference stock solution are mixed separately before detection and diluted with distilled water to obtain the mixed reference stock solution;
the preparation method of the glycine reference substance storage solution is preferably as follows: weighing a glycine reference substance, and diluting with distilled water to obtain a glycine reference substance storage solution with the concentration of 1-10 g/L;
the preparation method of the serine reference substance storage solution is preferably as follows: weighing a serine reference substance, and diluting with distilled water to obtain a serine reference substance storage solution with the concentration of 1-10 g/L.
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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