CN103713076A - Detection method for organic acids and amino acids in intracellular and extracellular fluids through gas chromatography-mass spectrometry - Google Patents
Detection method for organic acids and amino acids in intracellular and extracellular fluids through gas chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention relates to a detection method for organic acids and amino acids in intracellular and extracellular fluids through gas chromatography-mass spectrometry and belongs to the technical field of biochemistry examination and determination. The detection method comprises steps: first, a fermentation solution is taken and centrifuged at a high speed and cells and an extracellular fluid are collected respectively; second, intracellular metabolite samples are prepared, the collected thalli are washed in normal saline, dissolved in cold methanol, and subjected to ultrasonication, low temperature extraction, and centrifugation collection, the supernate is extracted, internal standard substances are added, and vacuum drying is carried out at a low temperature; third, extracellular fluid samples are prepared, proteins in the extracellular fluid are removed with acetonitrile, vortex oscillation is carried out, then centrifugation is carried out, the supernate is collected, internal standard substances are added, and vacuum drying is carried out at a low temperature; fourth, sample derivatization is performed, and sugar derivative agents and amino acid derivative agents are added; fifth, gas chromatography-mass spectrometry analysis and data collection are carried out. The method has advantages of simple sample processing, easy and simple operation, short detection time, high sensitivity, high repeatability of detection results, realization of preparation of multiple samples and the like.
Description
Technical field
The invention belongs to biochemical test determination techniques field, particularly relate to a kind of gas chromatography-mass spectrum and detect organic acid, amino acid whose method in fluid of inside and outside cell.
Background technology
Utilizing gas chromatography-mass spectrum to detect inside and outside cell aspect metabolin, at present existing correlation technique report, as patent 201210046953.2, announced a kind of method that gas chromatography-mass spectrum detects urinary organic acid, comprise the steps: that (1) completes urine enzyme to urine specimen to be checked and processes; (2) add interior mark product, adopt frozen ethanol to carry out the processing of protein precipitation, sample is dried up; (3) above-mentioned sample is carried out to methyl-monosilane derivation process; (4) adopt the step process standard organic acid of above-mentioned 1-3, obtain standard organic acid sample; (5) adopt gas chromatograph-mass spectrometer (GCMS) to detect standard organic acid sample and urine specimen to be checked; (6) using the testing result of standard organic acid sample as reference curve, with conventional algorithm, the organic acid of urine specimen to be checked is carried out quantitatively.This patented invention main application fields is urine examination, and this detection method is only confined to the detection of organic acid metabolite.
Detection research for metabolin in extracellular microbial in fermentation liquor, outside born of the same parents, can disclose microorganism metabolic rule under fermentation conditions, especially fermentation condition for the rule that affects of microorganism target product.The output of grasping the promotion target product that the metabolic rule of microorganism can be not only favourable promotes, and can determine that endobacillary metabolic flux distributes, and then utilizing genetic engineering means to realize the optimization and reconstruction of bacterium passway of metabolism, realize target product high-performance bio is synthetic.
For the metabolite analysis detection method limitation in fermentation liquor, be used for specific certain class material at present, can not realize multiclass metabolin and detect simultaneously.Because each component content complexity in fermentation liquor is various, disposal route for sample is very crucial, in current also relevant fermentation liquor, cell system interior, extracellular metabolin is processed detection method, make to develop a kind of more sensitive, wide spectrum, general fermentating metabolism object detecting method, identify the compound structure of various spectrums peak mapping, and with the integration of other dummy model, become the focus of microbial metabolism group research.
Summary of the invention
The object of this invention is to provide a kind of gas chromatography-mass spectrum and detect organic acid, amino acid whose method in fluid of inside and outside cell, make up the blank to metabolin system detecting method in microorganism fermentation at present, overcome the shortcomings such as existing detection technique disturbing factor is a lot, complex operation, testing cost is high, sensitivity is low, sensing range is narrow.
The solution of the present invention is by such realization: a kind of gas chromatography-mass spectrum detects organic acid, amino acid whose method in fluid of inside and outside cell, it is characterized in that, detection method step comprises:
(1) get fermentation liquor, high speed centrifugation, collects respectively thalline and supernatant I.
(2) endocellular metabolism matter sample preparation: get the thalline that step 1) obtains, with physiological saline, clean 2 ~ 3 times, after cold methanol dissolves, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cracking supernatant II after centrifugal collection 50 ~ 200ul extraction, add internal standard compound ribitol solution, cracking supernatant II volume and internal standard compound ribitol part by weight are 50 ~ 200ul:20 μ g, and room temperature vacuum drying obtains endocellular metabolism matter sample I.
(3) extracellular fluid sample preparation: get the supernatant I that 60 ~ 350ul step 1) obtains, in clear liquid I, add acetonitrile to remove deproteinized, supernatant I and acetonitrile volume ratio are 1:0.5 ~ 1.5, vortex vibration, centrifugal collection supernatant III, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 50 ~ 300ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(4) analyte derivative: in step 2) and in step 3) the sample I and sample II that obtain, add respectively 50 ~ 150ul sugar derivating agent, constant temperature is placed 1.5 ~ 4h, then adds respectively amino acid derived dose of 50 ~ 150ul, and constant temperature is placed and spent the night, derivative complete,
Distinguish sample I and sample II after centrifugal deriving, correspondence is collected supernatant separately and is obtained sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(5) utilize sample I and the outer sample II of born of the same parents to be measured in the born of the same parents to be measured that gas chromatography-mass spectrum obtains step 4), analyze respectively and data acquisition.
As a further improvement on the present invention, its sampling amount of described thalline reaches 0.2 ~ 1.0g/ml for dissolve artifact amount dry weight with cold methanol; Described cold methanol is for being chilled in advance-40 ℃ through cryostat groove; Described low-temperature extraction extracts 2 ~ 7h at-40 ℃ to-50 ℃.
As a further improvement on the present invention, described sugared derivating agent is that 0.1 ~ 0.3mg methoxamine hydrochloric acid is dissolved in 10ml pyridine solution preparation and obtains; Described amino acid derived dose is that 50 ~ 200ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml.
As a further improvement on the present invention, its instrumental analysis condition of described gas chromatography-mass spectrum is: gas chromatography: chromatographic column is HP-5MS or the DB-5MS capillary column of 30 m * 0.25 mm, 300 ℃ of injector temperatures, 250 ℃ of detector temperatures, column temperature rise program equilibration time 3 min → 80 ℃ maintain 1min → 2 ℃/min and are warming up to 100 ℃ → 15 ℃/min and are warming up to 220 ℃ → 30 ℃/min and are warming up to 300 ℃ → 300 ℃ and maintain 3 min, sampling volume 1 μ L; Mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70 eV, 280 ℃ of ion gun surface temperatures.
As a further improvement on the present invention, the method is applied to detect organic acid, the amino acid of fluid of inside and outside cell in the fermentation liquor in the biological fermentation process of saccharomycete, lactic acid bacteria, clostridium, mould.
As a further improvement on the present invention, it is characterized in that, described organic acid is succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Described amino acid is glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline.
Substantive distinguishing features of the present invention and marked improvement are: (1) the method can synchronously detect the detection of organic acid, amino acids material in cell in fermentation liquor and in extracellular, do not need fermentation liquor to carry out repeatedly complex process, save time and simplify the operation step, obtain in time microbial metabolism rule in sweat, analyze the inside and outside metabolic flux of born of the same parents.(2) the method adopts cold methanol extraction endocellular metabolism thing method, and its effect of extracting is good, obtains more metabolites kinds in cell, is conducive to the analysis of metabolic rule.(3) the chromatographic peak analytical effect that sample analysis obtains is good, can be to amino acid such as glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline, the organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid, realize and detecting.
Embodiment
Below the method that a kind of gas chromatography-mass spectrum of the present invention is detected to organic acid in fluid of inside and outside cell, amino acid, sugar by embodiment is further described, these descriptions are not that content of the present invention is further limited.
In following examples, centrifugal condition is: centrifugal 10 min of 10000 rpm at-4 ℃.
In following examples, its instrumental analysis condition of gas chromatography-mass spectrum is: gas chromatography: chromatographic column is HP-5MS or the DB-5MS capillary column of 30 m * 0.25 mm, 300 ℃ of injector temperatures, 250 ℃ of detector temperatures, column temperature rise program equilibration time 3 min → 80 ℃ maintain 1min → 2 ℃/min and are warming up to 100 ℃ → 15 ℃/min and are warming up to 220 ℃ → 30 ℃/min and are warming up to 300 ℃ → 300 ℃ and maintain 3 min, sampling volume 1 μ L; Mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70 eV, 280 ℃ of ion gun surface temperatures.
Following examples all can realize amino acid such as glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline, and the organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid detect.
Embodiment 1
Get saccharomycetes to make fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, with physiological saline, clean thalline 2 times,-40 ℃ of cold methanols dissolve artifact amount dry weight and reach 0.2g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: at-40 ℃ to-50 ℃, extract 4h, cracking supernatant I after centrifugal collection 150ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 300ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:1.2, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 100ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) in the sample I and sample II analyte derivative: in step 1) and step 2) obtaining, add respectively 100ul sugar derivating agent (sugared derivating agent be 0.1mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 1.5h, add respectively more amino acid derived dose of 100ul (prepare and obtain for 100ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, distinguish sample I and sample II after centrifugal deriving, correspondence is collected supernatant separately and is obtained sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4), according to its instrumental analysis condition of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Embodiment 2
Extracting lactic acid fermented liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, with physiological saline, clean thalline 3 times,-40 ℃ of cold methanols dissolve artifact amount dry weight and reach 1.0g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: at-40 ℃ to-50 ℃, extract 5h, cracking supernatant I after centrifugal collection 100ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 100ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 350ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:0.7, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 200ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) in the sample I and sample II analyte derivative: in step 1) and step 2) obtaining, add respectively 50ul sugar derivating agent (sugared derivating agent be 0.3mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 2.0h, add respectively more amino acid derived dose of 150ul (prepare and obtain for 150ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, distinguish sample I and sample II after centrifugal deriving, correspondence is collected supernatant separately and is obtained sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4), according to its instrumental analysis condition of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Embodiment 3
Get clostridium fermentation liquor.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, with physiological saline, clean thalline 3 times,-40 ℃ of cold methanols dissolve artifact amount dry weight and reach 0.6g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: at-40 ℃ to-50 ℃, extract 3h, cracking supernatant I after centrifugal collection 200ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 200ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 60ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:1.5, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 50ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) in the sample I and sample II analyte derivative: in step 1) and step 2) obtaining, add respectively 150ul sugar derivating agent (sugared derivating agent be 0.15mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 2.5h, add respectively more amino acid derived dose of 50ul (prepare and obtain for 200ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, distinguish sample I and sample II after centrifugal deriving, correspondence is collected supernatant separately and is obtained sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4), according to its instrumental analysis condition of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Embodiment 4
Get mold fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, with physiological saline, clean thalline 2 times,-40 ℃ of cold methanols dissolve artifact amount dry weight and reach 0.8g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: at-40 ℃ to-50 ℃, extract 2h, cracking supernatant I after centrifugal collection 50ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 50ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 200ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:1.0, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) in the sample I and sample II analyte derivative: in step 1) and step 2) obtaining, add respectively 120ul sugar derivating agent (sugared derivating agent be 0.2mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 3.0h, add respectively more amino acid derived dose of 80ul (prepare and obtain for 50ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, distinguish sample I and sample II after centrifugal deriving, correspondence is collected supernatant separately and is obtained sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4), according to its instrumental analysis condition of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Embodiment 5
Get saccharomycetes to make fermentation liquid.
(1) endocellular metabolism matter sample preparation: get 5ml fermentation liquor, the centrifugal thalline that obtains, with physiological saline, clean thalline 3 times,-40 ℃ of cold methanols dissolve artifact amount dry weight and reach 0.5g/ml, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cryogenic conditions is: at-40 ℃ to-50 ℃, extract 3h, cracking supernatant I after centrifugal collection 200ul extraction, adds internal standard compound ribitol solution, and cracking supernatant I volume and internal standard compound ribitol part by weight are 150ul:20 μ g, room temperature vacuum drying, obtains endocellular metabolism matter sample I.
(2) extracellular fluid sample preparation: get fermentation liquor, the centrifugal supernatant II that obtains, get 300ul supernatant II, in clear liquid II, add acetonitrile to remove deproteinized, supernatant II and acetonitrile volume ratio are 1:0.5, vortex vibration, the centrifugal supernatant III of collecting to obtain, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 300ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II.
(3) in the sample I and sample II analyte derivative: in step 1) and step 2) obtaining, add respectively 100ul sugar derivating agent (sugared derivating agent be 0.2mg methoxamine hydrochloric acid be dissolved in preparation in 10ml pyridine solution obtain), constant temperature is placed 4.0h, add respectively more amino acid derived dose of 120ul (prepare and obtain for 120ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml for amino acid derived dose), constant temperature is placed and is spent the night, derivative complete, distinguish sample I and sample II after centrifugal deriving, correspondence is collected supernatant separately and is obtained sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured.
(4), according to its instrumental analysis condition of gas chromatography-mass spectrum, sample I in born of the same parents to be measured and the outer sample II of born of the same parents to be measured are analyzed and data acquisition.
Claims (7)
1. gas chromatography-mass spectrum detects organic acid, an amino acid whose method in fluid of inside and outside cell, it is characterized in that, detection method step comprises:
(1) get fermentation liquor, high speed centrifugation, collects respectively thalline and supernatant I;
(2) endocellular metabolism matter sample preparation: get the thalline that step 1) obtains, with physiological saline, clean 2 ~ 3 times, after cold methanol dissolves, ultrasonication is to lysis, obtain lysate, low-temperature extraction lysate, cracking supernatant II after centrifugal collection 50 ~ 200ul extraction, add internal standard compound ribitol solution, cracking supernatant II volume and internal standard compound ribitol part by weight are 50 ~ 200ul:20 μ g, and room temperature vacuum drying obtains endocellular metabolism matter sample I;
(3) extracellular fluid sample preparation: get the supernatant I that 60 ~ 350ul step 1) obtains, in clear liquid I, add acetonitrile to remove deproteinized, supernatant I and acetonitrile volume ratio are 1:0.5 ~ 1.5, vortex vibration, centrifugal collection supernatant III, adds internal standard compound ribitol solution again, and supernatant III volume and internal standard compound ribitol part by weight are 50 ~ 300ul:20 μ g, room temperature vacuum drying, obtains extracellular fluid sample II;
(4) analyte derivative: in step 2) and in step 3) the sample I and sample II that obtain, add respectively 50 ~ 150ul sugar derivating agent, constant temperature is placed 1.5 ~ 4h, add respectively more amino acid derived dose of 50 ~ 150ul, constant temperature is placed and is spent the night, derivative complete, centrifugal sample I and sample II after derivative respectively, correspondence is collected supernatant separately and is obtained sample I and the outer sample II of born of the same parents to be measured in born of the same parents to be measured;
(5) utilize sample I and the outer sample II of born of the same parents to be measured in the born of the same parents to be measured that gas chromatography-mass spectrum obtains step 4), analyze respectively and data acquisition.
2. a kind of gas chromatography-mass spectrum according to claim 1 detects organic acid, amino acid whose method in fluid of inside and outside cell, it is characterized in that, its sampling amount of described thalline reaches 0.2 ~ 1.0g/ml for dissolve artifact amount dry weight with cold methanol; Described cold methanol is for being chilled in advance-40 ℃ through cryostat groove; Described low-temperature extraction extracts 2 ~ 7h at-40 ℃ to-50 ℃.
3. a kind of gas chromatography-mass spectrum according to claim 1 and 2 detects organic acid, amino acid whose method in fluid of inside and outside cell, it is characterized in that, described sugared derivating agent is that 0.1 ~ 0.3mg methoxamine hydrochloric acid is dissolved in preparation in 10ml pyridine solution and obtains; Described amino acid derived dose is that 50 ~ 200ul trimethyl chlorosilane is dissolved in the trifluoroacetamide of 10ml.
4. a kind of gas chromatography-mass spectrum according to claim 3 detects organic acid in fluid of inside and outside cell, amino acid whose method, it is characterized in that, its instrumental analysis condition of described gas chromatography-mass spectrum is: gas chromatography: chromatographic column is HP-5MS or the DB-5MS capillary column of 30 m * 0.25 mm, 300 ℃ of injector temperatures, 250 ℃ of detector temperatures, column temperature rise program equilibration time 3 min → 80 ℃ maintain 1min → 2 ℃/min and are warming up to 100 ℃ → 15 ℃/min and are warming up to 220 ℃ → 30 ℃/min and are warming up to 300 ℃ → 300 ℃ and maintain 3 min, sampling volume 1 μ L, mass spectrum: ion gun EI, detecting device is level Four bar, ionization energy 70 eV, 280 ℃ of ion gun surface temperatures.
5. a kind of gas chromatography-mass spectrum detects organic acid, amino acid whose method in fluid of inside and outside cell according to claim 4, it is characterized in that, the method is applied to detect organic acid, the amino acid in the fermentation liquor fluid of inside and outside cell in the biological fermentation process of saccharomycete, lactic acid bacteria, clostridium, mould.
6. according to a kind of gas chromatography-mass spectrum described in claim 1 ~ 2 or 4 ~ 5 any one, detect organic acid, amino acid whose method in fluid of inside and outside cell, it is characterized in that, described organic acid is succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Described amino acid is glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline.
7. a kind of gas chromatography-mass spectrum according to claim 3 detects organic acid, amino acid whose method in fluid of inside and outside cell, it is characterized in that, described organic acid is succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid; Described amino acid is glutamic acid, glycocoll, halfcystine, serine, threonine, leucine, isoleucine, phenylalanine, valine, lysine, alanine, histidine, methionine, arginine, glutamine, asparatate, asparagine, tyrosine, proline.
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Application publication date: 20140409 |