CN106596749B - Method that is a kind of while measuring DL-ATC in enzymatic reaction solution, L-cysteine and l-cysteine content - Google Patents
Method that is a kind of while measuring DL-ATC in enzymatic reaction solution, L-cysteine and l-cysteine content Download PDFInfo
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- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 title claims abstract description 154
- 235000013878 L-cysteine Nutrition 0.000 title claims abstract description 73
- 239000004201 L-cysteine Substances 0.000 title claims abstract description 73
- 238000006911 enzymatic reaction Methods 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 49
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 135
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims description 53
- 235000019393 L-cystine Nutrition 0.000 claims description 53
- 239000004158 L-cystine Substances 0.000 claims description 53
- 229960003067 cystine Drugs 0.000 claims description 53
- 238000001514 detection method Methods 0.000 claims description 33
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 31
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 31
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 31
- 239000011550 stock solution Substances 0.000 claims description 24
- 239000012071 phase Substances 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 11
- 150000002009 diols Chemical class 0.000 claims description 11
- 239000000741 silica gel Substances 0.000 claims description 11
- 229910002027 silica gel Inorganic materials 0.000 claims description 11
- 239000000523 sample Substances 0.000 claims description 10
- 239000012488 sample solution Substances 0.000 claims description 10
- 239000012085 test solution Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 239000008346 aqueous phase Substances 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 230000001737 promoting effect Effects 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 238000010812 external standard method Methods 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 239000012074 organic phase Substances 0.000 claims description 4
- 239000013558 reference substance Substances 0.000 claims description 4
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
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- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000012088 reference solution Substances 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- VHPXSBIFWDAFMB-UHFFFAOYSA-N 2-amino-Delta(2)-thiazoline-4-carboxylic acid Chemical compound NC1=[NH+]C(C([O-])=O)CS1 VHPXSBIFWDAFMB-UHFFFAOYSA-N 0.000 description 48
- 239000012295 chemical reaction liquid Substances 0.000 description 9
- 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 8
- 239000000126 substance Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
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- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
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- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000012430 stability testing Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 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
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The present invention provides a kind of methods for measuring DL-ATC in enzymatic reaction solution, L-cysteine and l-cysteine content simultaneously, this method includes first adjusting the pH value of the enzymatic reaction solution, then separated DL-ATC, L-cysteine and l-cysteine in enzymatic reaction solution and at the same time being measured with HPLC method.In method of the invention, by the pH value range for adjusting enzymatic reaction solution, the concentration for realizing DL-ATC in enzymatic reaction solution, L-cysteine and l-cysteine keeps stable within a certain period of time, again by high performance liquid chromatography, it is able to achieve the quick of DL-ATC in enzymatic reaction solution, L-cysteine and l-cysteine content while measures.
Description
Technical Field
The invention relates to a high performance liquid chromatography for simultaneously determining the contents of DL-ATC, L-cysteine and L-cystine in an enzymatic reaction solution by an HPLC method, belonging to the field of biological analysis.
Background
L-cysteine is an important sulfur-containing amino acid, and is α -amino acid which only contains sulfhydryl in more than 20 amino acids composing protein.
The market demand of L-cysteine is large, the main production mode of the L-cysteine is a hair hydrolysis method at present, although the process is relatively simple, the energy consumption is large, and the environmental pollution is serious. In recent years, the enzymatic conversion method has been increasingly emphasized due to its advantages of good stereoselectivity, availability of single configuration products, high product purity, mild reaction conditions, environmental friendliness and the like, and has gradually become one of the research hotspots.
At present, three main process routes are available for producing L-cysteine by an enzymatic conversion method, wherein one of the three process routes is to produce L-cysteine by using DL-2-amino-thiazoline-4-carboxylic acid (DL-ATC) as a starting material and performing an enzymatic reaction. This method was first invented in 1977 by Sano et al, who screened some Pseudomonas in soil and succeeded in synthesizing L-cysteine by adding glycerol and DL-ATC as substrates. The research on the optimum culture conditions and enzymatic reaction conditions for the enzymatic conversion of DL-ATC into L-cysteine was also completed by Ryu et al, a Korean scholarizer in 1990. In these studies, the conditions for the enzymatic reaction were mostly alkaline, while L-cysteine was relatively stable under acidic conditions and was highly susceptible to air oxidation to L-cystine in neutral and alkaline solutions. In the reaction liquid for producing the L-cysteine by the enzymatic reaction, DL-ATC, L-cysteine, L-cystine and the like exist at the same time, the DL-ATC is converted into the L-cysteine under the catalytic action of enzyme, and the L-cysteine is oxidized by air to generate the L-cystine under the alkaline condition, so that the dynamic change brings great difficulty for the accurate quantification of the contents of the DL-ATC, the L-cysteine and the L-cystine.
At present, conventional L-cysteine measuring methods include spectrophotometry, paper chromatography, electrochemical measurement and the like, but because the components of an enzymatic reaction solution are complex and impurity interference is difficult to remove, the method is difficult to be applied to the detection of L-cysteine in the enzymatic reaction solution. The contents of DL-ATC, L-cysteine and L-cystine in alkaline enzymatic reaction liquid are extremely unstable, and the common method is difficult to meet the requirement of simultaneously accurately and quantitatively detecting the DL-ATC, the L-cysteine and the L-cystine in the alkaline enzymatic reaction liquid, so that the development of a simple and convenient method for stabilizing the contents of the DL-ATC, the L-cysteine and the L-cystine in the enzymatic reaction liquid and simultaneously quickly and accurately realizing the simultaneous determination of the contents of the DL-ATC, the L-cysteine and the L-cystine has very important practical significance.
Through literature search, no report is found in the existing method for simultaneously determining the contents of DL-ATC, L-cysteine and L-cystine in an enzymatic reaction solution by an HPLC method.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring the contents of DL-ATC, L-cysteine and L-cystine in an enzymatic reaction solution.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for simultaneously determining the contents of DL-ATC, L-cysteine and L-cystine in an enzymatic reaction solution is characterized by comprising the steps of firstly adjusting the pH value of the enzymatic reaction solution, and then separating and simultaneously determining the DL-ATC, the L-cysteine and the L-cystine in the enzymatic reaction solution by using an HPLC method.
In the present invention, the column used for the detection by the HPLC method is a normal phase silica gel bonded diol column.
In the invention, when the HPLC method is used for detection, the detection conditions are as follows: the pH value of the aqueous phase solution is 1.0-5.0, the volume ratio of the aqueous phase to the organic phase is 15: 85-30: 70, the detection wavelength is 190-300nm, the column temperature is 20-40 ℃, and the sample injection amount is 5-20 mu L; preferably, the detection wavelength is 200 nm.
In the present invention, the acid used in adjusting the pH of the enzymatic reaction solution is one or more of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, or formic acid.
The enzymatic reaction solution is alkaline enzymatic reaction solution; preferably, the pH value of the alkaline enzymatic reaction solution is in the range of 7.5 to 9.5; more preferably, the pH value of the alkaline enzymatic reaction solution is in the range of 7.5 to 9.5, and the alkaline enzymatic reaction solution is a reaction solution in which DL-ATC is enzymatically converted into L-cysteine.
In the invention, the pH value of the enzymatic reaction solution is adjusted to be 0.5-5.0; preferably, the pH of the enzymatic reaction solution is adjusted to 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0.
The method specifically comprises the following steps:
(1) preparation of mixed control solution:
l-cysteine control stock solution: weighing an L-cysteine reference substance, adding an ammonium dihydrogen phosphate solution, and dissolving to obtain an L-cysteine reference stock solution;
DL-ATC mixed with L-cystine control stock solution: weighing appropriate amounts of DL-ATC and L-cystine reference substances, mixing, adding ammonium dihydrogen phosphate solution, and dissolving to obtain mixed reference storage solution of DL-ATC and L-cystine;
mixing the control solution: respectively taking an L-cysteine reference stock solution, a DL-ATC and L-cystine mixed reference stock solution before use, mixing, adding an ammonium dihydrogen phosphate solution for dissolving, and diluting to obtain a mixed reference solution;
(2) preparing a test solution:
taking an L-cysteine enzyme reaction promoting solution produced by an enzyme method, immediately adding hydrochloric acid to adjust the pH value of the solution, taking a proper amount of the solution, adding an ammonium dihydrogen phosphate solution to dissolve and dilute the solution to the concentration of the mixed control solution, filtering, and taking a filtrate as a test solution;
(3) and (3) detection:
respectively measuring a mixed control solution and a test solution, injecting the mixed control solution and the test solution into a high performance liquid chromatograph, and respectively detecting under the condition of HPLC detection; the detection conditions are as follows: the pH value of the aqueous phase solution is 1.0-5.0, the volume of the aqueous phase-organic phase is 15: 85-30: 70, the detection wavelength is 200nm, and the column temperature is 20-40 ℃.
In the step (2) of the present invention, a 0.45 μm aqueous filtration membrane is used for filtration, and the filtrate is continuously taken as a sample solution.
In the step (1), when the mixed control solution is prepared, the ammonium dihydrogen phosphate solution is dissolved and diluted into the mixed control solution containing DL-ATC, L-cysteine and L-cystine with the concentrations of 0.2-0.5, 0.1-0.5 and 0.1-0.5 mg/mL respectively.
The method of the invention also comprises the step of calculating the contents of DL-ATC, L-cysteine and L-cystine by an external standard method in a linear range.
In the method, the concentration of DL-ATC, L-cysteine and L-cystine in the enzymatic reaction liquid is kept stable for a certain time by adjusting the pH value range of the enzymatic reaction liquid, and then the content of DL-ATC, L-cysteine and L-cystine in the enzymatic reaction liquid can be rapidly and simultaneously determined by the high performance liquid chromatography.
The enzymatic reaction solution studied in the present invention is an alkaline enzymatic reaction solution. DL-ATC, L-cysteine and L-cystine all exist in alkaline enzymatic reaction liquid at the same time, DL-ATC can be converted into L-cysteine under the catalytic action of enzyme under the condition of enzymatic reaction with pH of 8.0, and L-cysteine is easily oxidized into L-cystine under alkaline condition. Therefore, to accurately measure the contents of DL-ATC, L-cysteine and L-cystine, the concentrations of the three substances in the solution must be kept stable for a certain period of time. Through several experimental studies, we found that the speed of enzymatic reaction can be effectively reduced by adjusting the pH value of the enzymatic reaction solution. The stability of DL-ATC, L-cysteine and L-cystine in the enzymatic reaction solution is studied under the condition that the pH value is 0.5-5.0, and experimental data show that the stability of DL-ATC, L-cysteine and L-cystine is good in a certain period of time. The present inventors have unexpectedly found through a large number of experiments that under the condition of pH 0.5 to 5.0, the pH includes pH 0.5 to 5.0, 0.5 to 4.0, 0.5 to 3.0, 0.5 to 2.0, 0.5 to 1.0, 0.5 to 0.8, 0.8 to 5.0, 0.8 to 4.0, 0.8 to 3.0, 0.8 to 2.0, 0.8 to 1.2, 0.8 to 1.0, 1.0 to 5.0, 1.0 to 4.0, 1.0 to 3.0, 1.0 to 2.0, 1.0 to 1.5, 1.0 to 1.2, 1.5 to 5.0, 1.5 to 4.0, 1.5 to 3.0, 1.5 to 2.0, 1.0 to 1.5, 2.5 to 5, 3.0 to 3.5, 3.5 to 2.0, 3.5 to 5, 3.0 to 5, 3.5 to 2.0, 3.5 to 5, 3.0, 3.5 to 5 to 2.0, 3.0, 3.5 to 5, 3.0, 3.5 to 5, 3.0 to 5, 3.0 to 5, 0 to 5, 0 to 5, 0, 4.5-5.0, the stability of DL-ATC, L-cysteine and L-cystine is good. Preferably, DL-ATC, L-cysteine and L-cystine are particularly well-stabilized at pH 0.5, 1.0, 2.0, 3.0, 4.0, 5.0. In addition, since it is difficult for the conventional column in the art to satisfy the simultaneous detection requirements of three substances, we found that the normal phase silica gel bonded diol column can effectively separate and measure three substances, DL-ATC, L-cysteine and L-cystine, through a large number of experiments.
The method is simple and rapid, has high accuracy and good reproducibility, can be applied to simultaneous detection of the contents of DL-ATC, L-cysteine and L-cystine in an enzymatic reaction solution, and provides a basis for quality control in the intermediate process of producing L-cysteine by an enzymatic method.
Drawings
FIG. 1DL-ATC, L-cysteine and L-cystine mixed control HPLC profile
FIG. 2 HPLC chromatogram of simultaneous determination of DL-ATC, L-cysteine and L-cystine in enzymatic reaction solution
FIG. 3L-cysteine Linearity plot
FIG. 4 DL-ATC Linear graph
FIG. 5 Linear graph of L-cystine
Detailed Description
Example 1
This example relates to the detection of a Mixed control solution
Firstly, instrument equipment and detection conditions:
the instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 deg.C
Sample introduction amount: 20 μ L
II, specific steps:
l-cysteine control stock solution: accurately weighing an L-cysteine control substance in a 50 mg-10 mL measuring flask, adding 0.01M ammonium dihydrogen phosphate solution (pH is 3.0) to dissolve, fixing the volume, and shaking up to obtain an L-cysteine control stock solution of 5.0 mg/mL.
DL-ATC and L-cystine mixed control stock solution is prepared by precisely weighing 50mg each of DL-ATC and L-cystine control, placing into a 100mL measuring flask, adding 0.01M ammonium dihydrogen phosphate solution (pH 3.0), dissolving, and metering to obtain 0.25mg/mL mixed control stock solution of DL-ATC and L-cystine.
Mixing the control solution: before use, 1mL of 5.0mg/mL L-cysteine control stock solution and 2mL of 0.25mg/mL DL-ATC and L-cystine mixed control stock solution are respectively taken into a 10mL measuring flask, 0.01M ammonium dihydrogen phosphate solution (pH is 3.0) is added for dissolution and dilution to a scale mark, and the volume is determined to be used as mixed control solution.
And (5) taking 20 mu L of the mixed control solution, immediately injecting the mixed control solution into a liquid chromatograph, and recording a chromatogram.
The results are shown in figure 1, wherein peak No. 1 is L-cysteine peak, peak No. 2 is DL-ATC peak, and peak No. 3 is L-cystine peak.
Example 2
The present example relates to the detection of a test solution
Firstly, instrument equipment and detection conditions:
the instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 deg.C
Sample introduction amount: 20 μ L
II, specific steps:
taking an enzyme-produced L-cysteine enzymatic reaction promoting solution, immediately adding hydrochloric acid to adjust the pH of the solution to 2.0, taking a measuring flask from 1mL to 10mL of the solution, adding a 0.01M ammonium dihydrogen phosphate solution (pH is 3.0) to dissolve and fix the volume, taking the solution, filtering the solution by using a 0.45 mu M water-based filter membrane, and continuously taking a filtrate as a sample solution. And (4) taking 20 mu L of sample solution, immediately injecting into a liquid chromatograph, and recording the chromatogram.
The results are shown in figure 2, in which peak No. 1 is L-cysteine peak, peak No. 2 is DL-ATC peak, and peak No. 3 is L-cystine peak.
Example 3
This example relates to the stability testing of enzymatic reaction solutions at different pH values
Detecting equipment and condition
The instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 deg.C
Sample introduction amount: 20 μ L
Taking 50mL of L-cysteine enzymatic reaction promoting solution produced by an enzyme method, immediately adding hydrochloric acid to adjust the pH of the solution, respectively adjusting the pH of the solution to 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0, standing at room temperature, respectively taking 1mL to 10mL of the solution in measuring bottles of 0 h, 4 h and 8h, adding 0.01M ammonium dihydrogen phosphate solution (pH is 3.0) to dissolve and fix the volume, taking the solution, filtering the solution by using a 0.45 mu M water-based filter membrane, and continuously taking filtrate as a test solution. And (3) taking 20 mu L of sample solution, immediately injecting into a liquid chromatograph, recording a chromatogram, and calculating the content of DL-ATC, L-cysteine and L-cystine at different times according to an external standard method.
The results of the different pH stability experiments are shown in Table 1. As can be seen from Table 1, the enzymatic reaction solutions were particularly stable at pH values of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0.
Table 1 different pH stability experimental data
Example 4
Detecting equipment and condition
The instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 deg.C
Sample introduction amount: 20 μ l
II, specific steps: accurately weighing an L-cysteine control substance in a 50 mg-10 mL measuring flask, adding 0.01M ammonium dihydrogen phosphate solution (pH is 3.0) to dissolve, fixing the volume, and shaking up to obtain an L-cysteine control stock solution of 5.0 mg/mL. The stock solutions were each diluted to 0.0005, 0.001, 0.002, 0.005, 0.01, 0.002, 0.05, 0.10, 0.20, 0.50, 1.0, 2.0, and 5.0mg/mL by adding 0.01M ammonium dihydrogen phosphate solution (pH 3.0), 20 μ L was measured precisely, and the solution was injected into a high performance liquid chromatograph, and the peak area was recorded and plotted as peak area versus concentration.
The results are shown in figure 3, the concentration of L-cysteine is in the range of 0.0005-5.0 mg/mL, and the peak area and the concentration form a good linear relationship.
Example 5
Detecting equipment and condition
The instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 deg.C
Sample introduction amount: 20 μ L
II, specific steps:
DL-ATC control 50mg to 100mL measuring flask was weighed accurately, 0.01M ammonium dihydrogen phosphate solution (pH 3.0) was added to dissolve, constant volume was set, and shaking was performed to obtain 0.5mg/mL control stock solution. A solution prepared by diluting the stock solutions to 0.001, 0.002, 0.004, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.04, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50mg/mL with 0.01M ammonium dihydrogen phosphate solution (pH 3.0) was added to each stock solution in an appropriate amount, 20 μ l was measured precisely, the solution was injected into a high performance liquid chromatograph, and the peak area was recorded and plotted as the peak area against the concentration.
The results are shown in figure 4, the DL-ATC concentration is in the range of 0.001-0.5 mg/mL, and the peak area and the concentration form a good linear relationship.
Example 6
First, experimental equipment and detection conditions
The instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 ℃; sample introduction amount: 20 μ L
Accurately weighing L-cystine control substance 50mg to 100mL, adding 0.01M ammonium dihydrogen phosphate solution pH 3.0), dissolving, diluting to desired volume, and shaking to obtain 0.5mg/mL control stock solution. A solution prepared by diluting the stock solutions to 0.001, 0.002, 0.004, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.04, 0.05, 0.10, 0.20, 0.30 and 0.40mg/mL with 0.01M ammonium dihydrogen phosphate solution (pH 3.0) was added to an appropriate amount of each stock solution, 20. mu.L of the solution was measured precisely, and the solution was injected into a high performance liquid chromatograph, and the peak area was recorded and plotted as the peak area against the concentration.
The results are shown in figure 5, the concentration of L-cystine is in the range of 0.001-0.4 mg/mL, and the peak area and the concentration have good linear relationship.
Comparative example 1
This comparative example relates to the stability test of the enzymatic reaction solution at a pH below 0.5
Detecting equipment and condition
The instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 ℃; sample introduction amount: 20 μ L
Taking 50mL of L-cysteine enzymatic reaction promoting solution produced by an enzyme method, immediately adding hydrochloric acid to adjust the pH of the solution, respectively adjusting the pH of the solution to 0.1 and 0.3, standing at room temperature, respectively taking 1mL to 10mL of the solution in measuring bottles at 0, 4 and 8h, adding 0.01M ammonium dihydrogen phosphate solution (pH is 3.0) to dissolve and fix the volume, taking the solution, filtering the solution by using a 0.45 mu M water-based filter membrane, and continuously taking filtrate as a sample solution. And (3) taking 20 mu L of sample solution, immediately injecting into a liquid chromatograph, recording a chromatogram, and calculating the contents of DL-ATC, L-cysteine and L-cystine at different times according to an external standard method.
The results of the stability test at pH below 0.5 of the enzymatic reaction solution are shown in Table 2. As can be seen from Table 2, the enzymatic reaction solution was not stable well at pH 0.1 and 0.3.
TABLE 2 Experimental data for different pH stabilities at pH lower than 0.5
Comparative example 2
This comparative example relates to the stability test of the enzymatic reaction solution at pH values above 5.0
Detecting equipment and condition
The instrument comprises the following steps: dionex Ultimate 3000 high performance liquid chromatograph
A chromatographic column: normal phase silica gel bonded diol chromatographic column
Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH 3.0) (80:20)
Detection wavelength: 200nm
Flow rate: 1.5mL/min
Column temperature: 35 deg.C
Sample introduction amount: 20 μ L
Taking 50mL of L-cysteine enzymatic reaction promoting solution produced by an enzyme method, immediately adding hydrochloric acid to adjust the pH of the solution, respectively adjusting the pH of the solution to 5.5, 6.0 and 6.5, standing at room temperature, respectively taking 1mL to 10mL of the solution in measuring bottles at 0, 4 and 8 hours, adding 0.01M ammonium dihydrogen phosphate solution (pH is 3.0) to dissolve and fix the volume, taking the solution, filtering the solution by using a 0.45 mu M water-based filter membrane, and continuously taking the filtrate as a sample solution. And (3) taking 20 mu L of sample solution, immediately injecting into a liquid chromatograph, recording a chromatogram, and calculating the content of DL-ATC, L-cysteine and L-cystine at different times according to an external standard method.
The results of the stability test at pH above 5.0 of the enzymatic reaction solution are shown in Table 3. As can be seen from Table 3, the enzymatic reaction solution was not stable well at pH 5.5, 6.0 and 6.5.
TABLE 3 Experimental data for different pH stabilities at pH above 5.0
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (12)
1. A method for simultaneously determining the contents of DL-ATC, L-cysteine and L-cystine in an enzymatic reaction solution is characterized by comprising the steps of firstly adjusting the pH value of the enzymatic reaction solution to be 0.5-5.0, and then separating and simultaneously determining the DL-ATC, the L-cysteine and the L-cystine in the enzymatic reaction solution by using an HPLC method; wherein,
the chromatographic column used for HPLC detection is a normal phase silica gel bonded diol chromatographic column.
2. The method according to claim 1, wherein the detection conditions in the HPLC method are as follows: the pH of the aqueous phase solution is 1.0-5.0, the volume ratio of the aqueous phase to the organic phase is 15: 85-30: 70, the detection wavelength is 190-300nm, the column temperature is 20-40 ℃, and the sample injection amount is 5-20 mu L.
3. The method of claim 2, wherein the detection wavelength is 200 nm.
4. The method according to any one of claims 1 to 3, wherein the acid used in adjusting the pH of the enzymatic reaction solution is one or more of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, or formic acid.
5. The method according to any one of claims 1 to 4, wherein the enzymatic reaction solution is an alkaline enzymatic reaction solution.
6. The method according to claim 5, wherein the pH of the alkaline enzymatic reaction solution is in the range of 7.5 to 9.5.
7. The method according to claim 5, wherein the pH of the alkaline enzymatic reaction solution is in the range of 7.5 to 9.5, and the alkaline enzymatic reaction solution is a reaction solution in which DL-ATC is enzymatically converted into L-cysteine.
8. The method according to any one of claims 1 to 7, wherein the pH of the enzymatic reaction solution is adjusted to 0.5, 1.0, 2.0, 3.0, 4.0 or 5.0.
9. The method according to any one of claims 1 to 8, characterized in that it comprises in particular the steps of:
(1) preparation of mixed control solution:
l-cysteine control stock solution: weighing an L-cysteine reference substance, adding an ammonium dihydrogen phosphate solution, and dissolving to obtain an L-cysteine reference stock solution;
DL-ATC mixed with L-cystine control stock solution: weighing appropriate amounts of DL-ATC and L-cystine reference substances, mixing, adding ammonium dihydrogen phosphate solution, and dissolving to obtain mixed reference storage solution of DL-ATC and L-cystine;
mixing the control solution: respectively taking an L-cysteine reference stock solution, a DL-ATC and L-cystine mixed reference stock solution before use, mixing, adding an ammonium dihydrogen phosphate solution for dissolving, and diluting to obtain a mixed reference solution;
(2) preparing a test solution:
taking an L-cysteine enzyme reaction promoting solution produced by an enzyme method, immediately adding hydrochloric acid to adjust the pH value of the solution, taking a proper amount of the solution, adding an ammonium dihydrogen phosphate solution to dissolve and dilute the solution to the concentration of the mixed control solution, filtering, and taking a filtrate as a test solution;
(3) detection of
Respectively measuring a mixed control solution and a test solution, injecting the mixed control solution and the test solution into a high performance liquid chromatograph, and respectively detecting under the condition of HPLC detection; the detection conditions are as follows: the pH value of the aqueous phase solution is 1.0-5.0, the volume ratio of the aqueous phase to the organic phase is 15: 85-30: 70, the detection wavelength is 200nm, and the column temperature is 20-40 ℃.
10. The method according to claim 9, wherein in the step (2), the sample solution is obtained by filtering the sample solution with a 0.45 μm aqueous filter membrane.
11. The method according to claim 9 or 10, wherein in the step (1), the ammonium dihydrogen phosphate solution is dissolved and diluted to prepare the mixed control solution containing DL-ATC, L-cysteine, and L-cystine at concentrations of 0.2-0.5, 0.1-0.5, and 0.1-0.5 mg/ml, respectively.
12. The method of claim 9, further comprising calculating the DL-ATC, L-cysteine, L-cystine content using external standard methods in the linear range.
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