CN101738440A - Method for detecting sulfur-containing amino acid - Google Patents
Method for detecting sulfur-containing amino acid Download PDFInfo
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- CN101738440A CN101738440A CN 200910248427 CN200910248427A CN101738440A CN 101738440 A CN101738440 A CN 101738440A CN 200910248427 CN200910248427 CN 200910248427 CN 200910248427 A CN200910248427 A CN 200910248427A CN 101738440 A CN101738440 A CN 101738440A
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- 150000001413 amino acids Chemical class 0.000 title claims abstract description 22
- 229910052717 sulfur Inorganic materials 0.000 title claims abstract description 18
- 239000011593 sulfur Substances 0.000 title claims abstract description 18
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title abstract description 9
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 claims abstract description 28
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims abstract description 18
- 229930182817 methionine Natural products 0.000 claims abstract description 18
- 229960003067 cystine Drugs 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 11
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 6
- 239000007791 liquid phase Substances 0.000 claims abstract description 6
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000010521 absorption reaction Methods 0.000 claims abstract description 4
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 31
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000012071 phase Substances 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 18
- 238000007254 oxidation reaction Methods 0.000 claims description 13
- UCUNFLYVYCGDHP-BYPYZUCNSA-N L-methionine sulfone Chemical compound CS(=O)(=O)CC[C@H](N)C(O)=O UCUNFLYVYCGDHP-BYPYZUCNSA-N 0.000 claims description 12
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 claims description 11
- 229940001584 sodium metabisulfite Drugs 0.000 claims description 11
- 235000010262 sodium metabisulphite Nutrition 0.000 claims description 11
- LJGHYPLBDBRCRZ-UHFFFAOYSA-N 3-(3-aminophenyl)sulfonylaniline Chemical compound NC1=CC=CC(S(=O)(=O)C=2C=C(N)C=CC=2)=C1 LJGHYPLBDBRCRZ-UHFFFAOYSA-N 0.000 claims description 10
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- 230000003647 oxidation Effects 0.000 claims description 9
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
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- 239000012488 sample solution Substances 0.000 claims description 6
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- 235000019253 formic acid Nutrition 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- CBCINYXHBYPBBC-UHFFFAOYSA-N acetonitrile 1-fluoro-2,4-dinitrobenzene Chemical compound CC#N.[N+](=O)([O-])C1=C(C=CC(=C1)[N+](=O)[O-])F CBCINYXHBYPBBC-UHFFFAOYSA-N 0.000 claims description 3
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims description 3
- 229910021538 borax Inorganic materials 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 239000000470 constituent Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 235000019800 disodium phosphate Nutrition 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
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- 230000007062 hydrolysis Effects 0.000 claims description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 3
- 239000012982 microporous membrane Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000004328 sodium tetraborate Substances 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
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- 235000001014 amino acid Nutrition 0.000 description 16
- 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 description 12
- 238000004458 analytical method Methods 0.000 description 9
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
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- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
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- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
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- 235000004279 alanine Nutrition 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
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- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
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- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
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- 229910052742 iron Inorganic materials 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 229940066491 mucolytics Drugs 0.000 description 1
- 238000003822 preparative gas chromatography Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a method for detecting sulfur-containing amino acid. The method comprises the following steps of: oxidizing cystine and methionine and preparing cysteic acid and metson by acid hydrolysis; adding a derivatization reagent of 2,4-dinitrofluorobenzene to the cysteic acid and the metson for derivatization reaction to generate products having the function of ultraviolet absorption; and performing highly efficient liquid phase chromatographic analysis of the derivatives. The method has the advantages that: the amino acid analyzer with high price is unnecessary; the operation is simple; the detection cost is low; and the accuracy and the sensitivity are high.
Description
Technical field:
The invention belongs to the chemical analysis detection range, especially a kind of simple to operate, to detect cost low, has the detection method of high accuracy and highly sensitive sulfur-containing amino acid.
Background technology:
Sulfur-containing amino acid is often referred to methionine, cystine and halfcystine, has crucial effects in the biosome metabolic process.Methionine is the sulfur-bearing essential amino acid, closely related with the metabolism of various sulfocompounds in the biosome, when biosome lacks methionine, can cause anorexia, growth slow down or do not put on weight, phenomenons such as kidney enlargement and the accumulation of liver iron, cause hepatonecrosis or fiberization at last; Methionine also can utilize its with methyl, Toxic or medicine methylated and play the effect of detoxifcation.Therefore, methionine can be used for preventing and treating chronic or liver diseases such as oxyhepatitis, cirrhosis at present, also can be used for alleviating the toxic reaction of objectionable impuritiess such as arsenic, methenyl choloride, phenixin, benzene, pyridine and quinoline.Cystine and halfcystine are the nonessential amino acid of sulfur-bearing, can reduce the requirement of human body to methionine.Cystine is to form the indispensable material of skin, can quicken the rehabilitation of burn wound and the chemoproection of radioactive damage, stimulates red, leukocytic increase; Halfcystine with sulfydryl (SH) have many physiological actions, can alleviate the degree of intoxication of Toxic or toxic medicament (phenol, benzene, naphthalene, cryanide ion), radioactive ray are also had prevention effect.The derivant N-acetyl-L-cysteine of halfcystine is also because of the effect of sulfydryl, has the effect that reduces viscosity, can be used as mucolytic agent, is used to prevent and treat the discharge difficulty of expectoration such as bronchitis.Therefore, the mensuration to sulfur amino acid content has very important significance.
The detection method of using both at home and abroad has chemical analysis, electrochemical method, spectrophotometric method, high performance liquid chromatography, capillary electrophoresis and vapor-phase chromatography etc. at present.High performance liquid chromatography is to use the most general detection method at present, and ion exchange process in the high performance liquid chromatography and reversed phase chromatography are most widely used two kinds.For the national standard that sulfur-containing amino acid in the feed detects, employing be ion-exchange chromatography, use amino-acid analyzer.Sulfur-containing amino acid in a lot of bibliographical informations employing oxydrolysis-ion-exchange chromatography feeds is also arranged, promptly earlier sulfur-containing amino acid at first is oxidized to the stable product of acid, by acid hydrolysis oxidation product is discharged from albumen again, carry out analyzing and testing with ion-exchange chromatography, also need to use amino-acid analyzer.Because the amino-acid analyzer specificity is strong, cost is high, the cost that causes sulfur-containing amino acid to detect is higher, so the popularity rate that detects is lower.
Summary of the invention:
The present invention is in order to solve the above-mentioned technical matters of existing in prior technology, provide a kind of simple to operate, to detect cost low, has the detection method of high accuracy and highly sensitive sulfur-containing amino acid.
Technical solution of the present invention is: a kind of detection method of sulfur-containing amino acid, it is characterized in that by as
Following step is carried out:
A. with cystine and methionine oxidation,, produce cysteic acid and methionine sulfone again by acidolysis;
B. with derivative reagent 2,4-dinitrofluorobenzene and cysteic acid and methionine sulfone carry out derivative reaction, generate the product that uv absorption is arranged;
C. derivative products is carried out efficient liquid phase chromatographic analysis.
Described a step is to get the sample that contains albumen 7.5～25mg to put in the container, adds performic acid solutions 0.3mL, ice-water bath oxidation reaction 16h; Add 0.1mL Sodium Metabisulfite solution in oxidation liquid, directly add 6.8mol/L hydrochloric acid solution 2.5mL after fully shaking up again, container closure is also put 110 ± 3 ℃ of hydrolysis 24h of baking oven; Hydrolyzate is placed room temperature and opened container finish, hydrolyzate nitrogen is dried up, with the buffer solution washing container of deriving, cleansing solution is transferred to the 25mL volumetric flask, with the buffer solution constant volume of deriving, produces the sample solution that contains cysteic acid and methionine sulfone; Described performic acid solutions is that the hydrogen peroxide with concentration 30% mixed with the formic acid of concentration 88% in 1: 9 by volume, places 1h under room temperature, puts cooling 30min in the ice-water bath and makes; Described Sodium Metabisulfite solution is the 3.36g Sodium Metabisulfite to be added water be settled to 10mL; Described 6.8mol/L hydrochloric acid solution is to 50mL with pure hydrochloric acid 28mL thin up; The described buffer solution of deriving is to get boric acid 1.24g and borax 7.63g, puts to add the water constant volume in the 500mL volumetric flask and shake up and makes.
Described b step is a step gained sample solution to be filtered and gets the sample liquid 10mL that filtered in the dark-coloured volumetric flask of 50mL, adds the 5mL derivative reagent, puts into 60 ℃ of water-baths, is cooled to room temperature behind the reaction 60min, with equalizing and buffering solution constant volume; Described derivative reagent be concentration be 1% 2,4-dinitrofluorobenzene acetonitrile solution; Described equalizing and buffering solution is to get potassium dihydrogen phosphate 0.91g and sodium hydrogen phosphate 3.58g, puts to add the water constant volume in the 250mL volumetric flask and shake up.
The chromatogram flow phase of described c step: mobile phase A, Mobile phase B; Flow velocity: 1.2mL/min; Detect wavelength: 360nm; Described mobile phase A is that the acetonitrile with equal volume mixes ultrasonic degas 10～15min with water; Described Mobile phase B is to get moving phase anhydrous sodium acetate solid constituent 4.1g to add the about 950mL of water, transfers pH to 6.4～6.8 with glacial acetic acid, adds 10mL N, dinethylformamide, add water and be settled to 1000mL, shake up back with 0.45 μ m filtering with microporous membrane, ultrasonic degas 10～15min; Described eluent gradient condition such as table 1:
Table 1
The present invention is through the performic acid oxidation with the methionine in the sample, cystine, be converted into methionine sulfone and cysteic acid and from forage protein, discharge through acid hydrolysis again, with 2,4-dinitrofluorobenzene generation derivative reaction, to after deriving, carry out efficient liquid phase chromatographic analysis by sample, need not to use expensive amino-acid analyzer, simple to operate, to detect cost low, has high accuracy and high sensitivity.
Description of drawings:
Fig. 1 standard model analysis of spectra of the present invention.
Fig. 2 the present invention is routine in real time to feed sample analysis spectrogram.
Embodiment:
Below in conjunction with description of drawings the specific embodiment of the present invention:
1. instrument and equipment and reagent
UV1201 ultraviolet-visible detecting device, the P1201 high pressure constant flow pump, Elite AAK, 5 μ m, 4.6 * 250mm chromatographic column, ZW II type chromatographic column constant temperature oven, EC2006 chromatographic work station (more than be Dalian Yilite Analytical Instrument Co., Ltd's product), baking oven, spirit lamp, ampoule bottle, electric-heated thermostatic water bath, Nitrogen evaporator.
Methionine standard items (Beijing extensive and profound in meaning star biotechnology responsibility company limited), cystine standard items (Shanghai political affairs Xiang chemical reagent research institute), the feed sample, 30% hydrogen peroxide, 88% formic acid, hydrochloric acid (top grade is pure), Sodium Metabisulfite, Elite-AAK amino acid analysis system kit, deionized water etc. (except that special mark, it is pure that other is analysis).
2. detection method
2.1 method principle
Methionine is through the performic acid oxidation in the feed, and be converted into methionine sulfone and cysteic acid and from forage protein, discharge through acid hydrolysis again, with 2,4-dinitrofluorobenzene generation derivative reaction, sample carries out efficient liquid phase chromatographic analysis after deriving.
Oxidation reaction and derivatization reaction equation are as follows:
The oxidation equation formula:
The derivatization reaction equation:
Above-mentioned reaction equation shows: performic acid is oxidized to sulfonyl with the S in cystine and the methionine, but amino do not change, 2, the 4-dinitrofluorobenzene can with amino generation substitution reaction wherein, generate the product that uv absorption is arranged.
2.2 method step
Carry out as follows:
A. getting the sample that contains albumen 7.5～25mg places 5mL ampoule bottle (performic acid adds 2mL can guarantee that oxygenant is excessive) to add chilled performic acid solutions 0.3mL, ice-water bath oxidation reaction 16h; With the Sodium Metabisulfite is terminator, adds 0.1mL Sodium Metabisulfite solution in oxidation liquid, directly adds 6.8mol/L hydrochloric acid solution 2.5mL after fully shaking up again, and 110 ± 3 ℃ of hydrolysis 24h of baking oven are sealed and put in the wire drawing of spirit lamp high temperature; The ampoule bottle taking-up is placed to room temperature, opens the ampoule bottle bottleneck, hydrolyzate nitrogen dries up, buffer solution repeatedly washs ampoule bottle with deriving, cleansing solution is transferred to the 25mL volumetric flask, and with the buffer solution constant volume of deriving, it is standby to produce the sample solution that contains cysteic acid and methionine sulfone; Described performic acid solutions is that the hydrogen peroxide with concentration 30% mixed with the formic acid of concentration 88% in 1: 9 by volume, places 1h under room temperature, puts cooling 30min in the ice-water bath and makes; Described Sodium Metabisulfite solution is the 3.36g Sodium Metabisulfite to be added water be settled to 10mL; Described 6.8mol/L hydrochloric acid solution is to 50mL with pure hydrochloric acid 28mL thin up; The described buffer solution of deriving is to get boric acid 1.24g and borax 7.63g, puts to add the water constant volume in the 500mL volumetric flask and shake up and makes.
B. above-mentioned a step gained sample solution is filtered and gets the sample liquid 10mL that filtered in 50mL dead color (brown) volumetric flask, add the 5mL derivative reagent, put into 60 ℃ of water-baths, be cooled to room temperature behind the dark place reaction 60min, with equalizing and buffering solution constant volume; Described derivative reagent be concentration be 1% 2,4-dinitrofluorobenzene acetonitrile solution; Described equalizing and buffering solution is to get potassium dihydrogen phosphate 0.91g and sodium hydrogen phosphate 3.58g, puts to add the water constant volume in the 250mL volumetric flask and shake up.
C. sample carries out efficient liquid phase chromatographic analysis after deriving, chromatogram flow phase: mobile phase A, Mobile phase B; Flow velocity: 1.2mL/min; Detect wavelength: 360nm; Described mobile phase A is that the acetonitrile with equal volume mixes ultrasonic degas 10～15min with water; Described Mobile phase B is to get moving phase anhydrous sodium acetate solid constituent 4.1g to add the about 950mL of water, transfers pH to 6.4～6.8 with glacial acetic acid, adds 10mL N, dinethylformamide, add water and be settled to 1000mL, shake up back with 0.45 μ m filtering with microporous membrane, ultrasonic degas 10～15min; Described eluent gradient condition such as table 1.
3 testing results
3.1 cystine and methionine standard items analyzing and testing result
Take by weighing cystine standard items 3.7mg (concentration: 29.6mg/L), methionine standard items 2.0mg (concentration: 16.0mg/L), handle and analyze by 2.2 methods.
Cystine separates spectrogram such as Fig. 1 with the methionine standard items, among the figure 1, cysteic acid; 2, methionine sulfone; Sample sets submeter such as table 2:
Table 2
3.2 feed sample analysis result
Feed sample analysis spectrogram is seen Fig. 2, among the figure 1, cysteic acid; 2, aspartic acid; 3, proline; 4, methionine sulfone; 5, alanine; Sample size: 20 μ L.
Cysteic acid and methionine sulfone and chromatographic peak good separation on every side.
Same feed sample, one is divided into three, adopts 2.2 methods to handle 3 times continuously, and cystine and methionine content the results are shown in Table 3.
Table 3
In a feed sample, add cystine and methionine standard items,, calculate recovery of standard addition by 2.2 method Treatment Analysis samples.Recovery of standard addition the results are shown in Table 4.
Table 4
Claims (4)
1. the detection method of a sulfur-containing amino acid is characterized in that carrying out as follows:
A. with cystine and methionine oxidation,, produce acid stable cysteic acid and methionine sulfone again by acidolysis;
B. with derivative reagent 2,4-dinitrofluorobenzene and cysteic acid and methionine sulfone carry out derivative reaction, generate the product that uv absorption is arranged;
C. derivative products is carried out efficient liquid phase chromatographic analysis.
2. the detection method of sulfur-containing amino acid according to claim 1 is characterized in that described a step is to get the sample that contains albumen 7.5～25mg to put in the container, adds performic acid solutions 0.3mL, ice-water bath oxidation reaction 16h; Add 0.1mL Sodium Metabisulfite solution in oxidation liquid, directly add 6.8mol/L hydrochloric acid solution 2.5mL after fully shaking up again, container closure is also put 110 ± 3 ℃ of hydrolysis 24h of baking oven; Hydrolyzate is placed room temperature and opened container finish, hydrolyzate nitrogen is dried up, with the buffer solution washing container of deriving, cleansing solution is transferred to the 25mL volumetric flask, with the buffer solution constant volume of deriving, produces the sample solution that contains cysteic acid and methionine sulfone; Described performic acid solutions is that the hydrogen peroxide with concentration 30% mixed with the formic acid of concentration 88% in 1: 9 by volume, places 1h under room temperature, puts cooling 30min in the ice-water bath and makes; Described Sodium Metabisulfite solution is the 3.36g Sodium Metabisulfite to be added water be settled to 10mL; Described 6.8mol/L hydrochloric acid solution is to 50mL with pure hydrochloric acid 28mL thin up; The described buffer solution of deriving is to get boric acid 1.24g and borax 7.63g, puts to add the water constant volume in the 500mL volumetric flask and shake up and makes.
3. the detection method of sulfur-containing amino acid according to claim 2, it is characterized in that described b step is a step gained sample solution to be filtered and gets the sample liquid 10mL that filtered in the dark-coloured volumetric flask of 50mL, add the 5mL derivative reagent, put into 60 ℃ of water-baths, be cooled to room temperature behind the reaction 60min, with equalizing and buffering solution constant volume; Described derivative reagent be concentration be 1% 2,4-dinitrofluorobenzene acetonitrile solution; Described equalizing and buffering solution is to get potassium dihydrogen phosphate 0.91g and sodium hydrogen phosphate 3.58g, puts to add the water constant volume in the 250mL volumetric flask and shake up.
4. the detection method of sulfur-containing amino acid according to claim 3 is characterized in that the chromatogram flow phase of described c step: mobile phase A, Mobile phase B; Flow velocity: 1.2mL/min; Detect wavelength: 360nm; Described mobile phase A is that the acetonitrile with equal volume mixes ultrasonic degas 10～15min with water; Described Mobile phase B is to get moving phase anhydrous sodium acetate solid constituent 4.1g to add the about 950mL of water, transfers pH to 6.4～6.8 with glacial acetic acid, adds 10mLN, dinethylformamide, add water and be settled to 1000mL, shake up back with 0.45 μ m filtering with microporous membrane, ultrasonic degas 10～15min; Described eluent gradient condition such as following table:
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