CN103698450A - Method for detecting intracellular organic acid through gas chromatography-mass spectrometry - Google Patents
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Abstract
The invention relates to a method for detecting intracellular organic acid through gas chromatography-mass spectrometry, which belongs to the technical field of biochemical test and measurement. The detecting method provided by the invention comprises the following steps: (1) high speed centrifuging fermentation liquor, and respectively collecting cells and extracellular fluid; (2) preparing an intracellular metabolin sample, cleaning the collected thallus through normal saline, dissolving the thallus in cold methanol, ultrasonic crushing, low temperature extracting, centrifuging to collect extract supernatant, adding internal standard substance, and vacuum drying at a low temperature; (3) deriving the sample, and adding a sugar derivating agent and an amino acid derivating agent; and (4) carrying out gas chromatography-mass spectrometry analysis and collecting data. The detecting method provided by the invention has the advantages of simple sample treatment, simple and convenient operation, short organic acid detection time, high sensitivity, high repeatability of test results, capability of preparing a plurality of 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 middle organic acid method in cell.
Background technology
Utilizing gas chromatography-mass spectrum to detect aspect organic acid metabolite, 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, 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 intracellular metabolite analysis detection method limitation, be used for specific certain class material at present, can not realize multiclass metabolin and detect simultaneously.Because each component content complexity in cell is various, disposal route for sample is very crucial, also do not have at present in relevant cell, the system of extracellular metabolin processes 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 method in 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 method in cell, and 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) in sample I analyte derivative: in step 2) obtaining, add respectively 50 ~ 150ul sugar derivating agent, constant temperature is placed 3 ~ 6h, add respectively more amino acid derived dose of 50 ~ 150ul, constant temperature is placed and is spent the night, derivative complete, sample I after centrifugal deriving, collects supernatant and obtains sample I in born of the same parents to be measured;
(4) utilize sample I in the born of the same parents to be measured that gas chromatography-mass spectrum obtains step 3), analyze and data acquisition.
As further restriction of 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 the intracellular organic acid 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.
Substantive distinguishing features of the present invention and marked improvement are: (1) the method can detect the detection of middle organic acid substance in cell within a cell, 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 born of the same parents' intracellular metabolite flow.(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 the organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid be realized and being detected.
Embodiment
To the inventive method, further describe by embodiment below, 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 the organic acids such as succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid are detected.
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 3h, 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
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 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 6.0h, 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
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 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 4.5h, 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 5.0h, 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 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 0.5g/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 6.0h, 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 an organic acid method in 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) in sample I analyte derivative: in step 2) obtaining, add respectively 50 ~ 150ul sugar derivating agent, constant temperature is placed 3 ~ 6h, add respectively more amino acid derived dose of 50 ~ 150ul, constant temperature is placed and is spent the night, derivative complete, sample I after centrifugal deriving, collects supernatant and obtains sample I in born of the same parents to be measured;
(4) utilize sample I in the born of the same parents to be measured that gas chromatography-mass spectrum obtains step 3), analyze and data acquisition.
2. a kind of gas chromatography-mass spectrum according to claim 1 detects organic acid method in 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 method in 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 method in cell, 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 method in cell according to claim 4, it is characterized in that, the method is applied to detect the intracellular organic acid 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 method in cell, it is characterized in that, described organic acid is succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid.
7. a kind of gas chromatography-mass spectrum according to claim 3 detects organic acid method in cell, it is characterized in that, described organic acid is succinic acid, malic acid, citric acid, pyruvic acid, fumaric acid.
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Application publication date: 20140402 |