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Method for detecting amino acids, saccharides and organic acids in cells through gas chromatography-mass spectrometry

A gas chromatography and mass spectrometry detection technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of narrow detection range, high test cost, and low sensitivity, and achieve the effects of saving time, excellent extraction effect, and simplifying operation steps

Active Publication Date: 2014-03-26
神州医疗科技股份有限公司 +1
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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 detecting intracellular amino acids, sugars and organic acids by gas chromatography-mass spectrometry, to make up for the gap in the current detection method for metabolites in microbial fermentation, and to overcome the many interference factors and cumbersome operation of the existing detection technology , high test cost, low sensitivity, narrow detection range and other shortcomings

Method used

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  • Method for detecting amino acids, saccharides and organic acids in cells through gas chromatography-mass spectrometry

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Get the lactic acid bacteria fermentation broth.

[0024] (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.

[0025] (2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 60ul of supernatant II, add acetonitrile to superna...

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] Get the yeast fermentation liquid.

[0037] (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.

[0038] (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...

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Abstract

The present invention relates to a method for detecting amino acids, saccharides and organic acids in cells through gas chromatography-mass spectrometry, and belongs to the technical field of biochemical analysis determination. The detection method comprises: (1) taking a fermentation broth, carrying out high-speed centrifugation, and respectively collecting cells and extracellular fluid; (2) preparing an intra-cellular metabolite sample, wherein the collected bacteria are washed by using physiological saline, dissolving is performed with cold methanol, ultrasound crushing and low temperature extraction are sequentially performed, centrifugation is performed to collect the supernatant, an internal standard substance is added, and vacuum drying is performed at a low temperature; (3) carrying out sample derivatization, wherein a saccharide derivatization agent and an amino acid derivatization agent are added; and (4) carrying out gas chromatography-mass spectrometry analysis and data acquisition. The method has advantages of simple sample treatment, easy operation, short amino acid, saccharide and organic acid detection time, high sensitivity, high reproducibility of the detection result, multiple sample preparation achievement and the like.

Description

technical field [0001] The invention belongs to the technical field of biochemical detection and measurement, and in particular relates to a method for detecting amino acids, sugars and organic acids in cells 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; (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 陈东
Owner 神州医疗科技股份有限公司
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