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Detection method for sugar in intracellular and extracellular fluids through gas chromatography-mass spectrometry

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

Inactive Publication Date: 2014-04-09
LIUZHOU LIANHAI TECH
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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 sugars 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 broth.

[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 protei...

Embodiment 2

[0030] Get the lactic acid bacteria fermentation broth.

[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 acetonitri...

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 to...

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Abstract

The invention relates to a detection method for sugar 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 [0001] The invention belongs to the technical field of biochemical testing and determination, in particular to a method for detecting sugar in intracellular and extracellular liquid by gas chromatography-mass spectrometry. Background technique [0002] Regarding the detection of sugar metabolites by gas chromatography-mass spectrometry, there are related technical reports, such as patent 201210046953.2, which discloses a method for the detection of glucose in urine by gas chromatography-mass spectrometry, including the following steps: (1) Complete the urine sample to be tested Urine enzyme treatment; (2) Add internal standard, use frozen ethanol to precipitate protein, and dry the sample; (3) Perform methyl silylation derivatization on the above sample; (4) Use the above steps 1-3 Process standard sugars to obtain standard sugar samples; (5) Use gas chromatography-mass spectrometry to detect standard sugar samples and urine samples to be tested; (6) Use the ...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/06
Inventor 陈东梁远雄
Owner LIUZHOU LIANHAI TECH
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