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Method for detecting amino acid in intracellular and extracellular fluids by gas chromatography-mass spectrum

A gas chromatography and mass spectrometry detection technology, which is applied in the field of gas chromatography-mass spectrometry detection of amino acids in intracellular and extracellular fluids, can solve the problems of many interference factors, cumbersome operation, and low sensitivity of the detection technology, and achieve excellent extraction effects, good analysis effects, and simplified The effect of the action steps

Inactive Publication Date: 2014-03-26
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 gas chromatography-mass spectrometry method for detecting amino acids in the intracellular and extracellular liquids, which can make up for the gap in the current detection method for metabolites in microbial fermentation, and overcome the many interference factors, cumbersome operation, and testing costs of the existing detection technology. High, low sensitivity, narrow detection range and other shortcomings

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 three times with normal saline, dissolve in cold methanol at -40°C, and the dry weight of the biomass reaches 0.5g / 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 150ul:20μg, and vacuum-dried at room temperature to obtain the intracellular metabolite sample I.

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

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 amino acid in intracellular and extracellular fluids by gas chromatography-mass spectrum, belonging to a biochemistrical examination and determination technology field. The detection method comprises the following steps of: (1) high-speed centrifuging fermentation broth, and respectively collecting cells and the extracellular liquid; (2) preparing metabolin sample of the cell, cleaning the collected thallus with normal saline, ultrasonically breaking thallus after the thallus is dissolved by a cold methanol, extracting under a low temperature, centrifugally collecting an extracted supernatant, adding an internal standard substance, and vacuum-drying under a low temperature; (3) preparing a extracellular liquid sample, removing protein in the extracellular liquid by acetonitrile, vortex-oscillating, then centrifugally collecting a supernatant, adding an internal standard substance, and vacuum-drying under a low temperature; (4) deriving the sample, adding a sugar derivating agent and an amino acid derivating agent; and (5) analyzing by gas chromatography-mass spectrum, and acquiring data. The method provided by the invention has advantages of simple sample treatment, simple operation, short detection time, high sensitivity, and high detection result repeatability, and can implement preparation of a plurality of samples.

Description

technical field [0001] The invention belongs to the technical field of biochemical detection and determination, and in particular relates to a method for detecting amino 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...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 陈东
Owner 陈东
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