Method for detecting organic acid in intracellular fluid and extracellular fluid through gas chromatography-mass spectrometry

A gas chromatography and mass spectrometry detection technology, which is applied in the field of gas chromatography-mass spectrometry detection of organic acids in intracellular and extracellular fluids, can solve the problems of many interference factors, cumbersome operations, low sensitivity, etc. The effect of simplifying the operation steps

Inactive Publication Date: 2014-04-02
陈东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for gas chromatography-mass spectrometry detection of organic acids in intracellular and extracellular liquids, to make up for the blank of the current detection method for metabolites in microbial fermentation, and to overcome the many interference factors, cumbersome operation, and difficult testing of existing detection technologies. Disadvantages such as high cost, low sensitivity, and narrow detection range

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Get the yeast fermentation liquid.

[0025] (1) Sample preparation of intracellular metabolites: Take 5ml of fermentation broth, centrifuge to obtain the bacteria, wash the bacteria twice with normal saline, dissolve in cold methanol at -40°C, and the dry weight of the biomass reaches 0.2g / ml, and ultrasonically break down to the cells Cleavage to obtain a lysate, extract the lysate at low temperature, the low temperature condition is: extract at -40°C to -50°C for 4 hours, centrifuge to collect 150ul of the extracted lysate supernatant I, add internal standard ribitol solution, and lyse the supernatant I The ratio of the volume to the weight of the internal standard ribitol is 150ul:20μg, and vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0026] (2) Preparation of extracellular fluid samples: take fermentation broth and centrifuge to obtain supernatant II, take 300ul supernatant II, add acetonitrile to supernatant II to remove prote...

Embodiment 2

[0030] Get the lactic acid bacteria fermentation liquid.

[0031] (1) Sample preparation of intracellular metabolites: Take 5ml of fermentation broth, centrifuge to obtain the bacteria, wash the bacteria three times with normal saline, dissolve in cold methanol at -40°C, and the dry weight of the biomass reaches 1.0g / ml, and ultrasonically break down to the cells Cleavage to obtain a lysate, extract the lysate at low temperature, the low temperature conditions are: extract at -40°C to -50°C for 5 hours, centrifuge to collect 100ul of the extracted lysate supernatant I, add internal standard ribitol solution, and lyse the supernatant I The ratio of the volume to the weight of the internal standard ribitol is 100ul:20μg, and it is vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0032] (2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 350ul of supernatant II, add acetonitr...

Embodiment 3

[0036] Take the Clostridium fermentation broth.

[0037] (1) Sample preparation of intracellular metabolites: Take 5ml of fermentation broth, centrifuge to obtain the bacteria, wash the bacteria three times with normal saline, dissolve in cold methanol at -40°C, and the dry weight of the biomass reaches 0.6g / ml, and ultrasonically break down to the cells Cleavage to obtain a lysate, extract the lysate at low temperature, the low temperature condition is: extract at -40°C to -50°C for 3 hours, centrifuge to collect 200ul of the extracted lysate supernatant I, add internal standard ribitol solution, and lyse the supernatant I The ratio of the volume to the weight of the internal standard ribitol is 200ul:20μg, and vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

[0038] (2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 60ul of supernatant II, add acetonitrile to supernatant II ...

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Abstract

The invention relates to a method for detecting organic acid in intracellular fluid and extracellular fluid 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 intracellular fluid 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) preparing an extracellular fluid sample, removing protein in the extracellular fluid through acetonitrile, vortex oscillating, centrifuging to collect supernatant, adding internal standard substance, and vacuum drying at a low temperature; (4) deriving the sample, and adding a sugar derivating agent and an amino acid derivating agent; and (5) 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 detection time, high sensitivity, high repeatability of test results, capability of preparing a plurality of samples, and the like.

Description

technical field [0001] The invention belongs to the technical field of biochemical testing and determination, in particular to a method for detecting organic acids in intracellular and extracellular fluids by gas chromatography-mass spectrometry. Background technique [0002] In terms of using gas chromatography-mass spectrometry to detect organic acid metabolites, there are related technical reports, such as patent 201210046953.2, which discloses a method for detecting urine sugar by gas chromatography-mass spectrometry, including the following steps: (1) The urine sample to be tested Complete the urine enzyme treatment; (2) add internal standard, use frozen ethanol to process the precipitated protein, and dry the sample; (3) perform methyl silylation derivatization on the above sample; (4) use the above 1-3 (5) Use gas chromatography-mass spectrometry to detect the standard sugar sample and the urine sample to be tested; (6) Use the test results of the standard sugar sampl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
Inventor 陈东
Owner 陈东
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