Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for detecting intracellular organic acid through gas chromatography-mass spectrometry

A gas chromatography and mass spectrometry detection technology, applied in the field of gas chromatography-mass spectrometry detection of organic acids in cells, can solve the problems of many interference factors, cumbersome operation, low sensitivity and the like of the detection technology, achieve excellent extraction effect, simplify operation steps, and analyze good effect

Inactive Publication Date: 2014-04-02
LIUZHOU LIANHAI TECH
View PDF3 Cites 2 Cited by
  • 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 organic acids by gas chromatography-mass spectrometry, which can make up for the blank of the current detection method for metabolites in microbial fermentation, and overcome the many interference factors, cumbersome operation and high test cost of the existing detection technology , low sensitivity, narrow detection range and other shortcomings

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Get the yeast fermentation broth.

[0023] (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.

[0024] (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

[0028] Get the yeast fermentation broth.

[0029] (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.

[0030] (2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 350ul of supernatant II, add acetonitrile to supernatant I...

Embodiment 3

[0034] Get the lactic acid bacteria fermentation broth.

[0035] (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.

[0036](2) Preparation of extracellular fluid samples: take the fermentation broth, centrifuge to obtain supernatant II, take 60ul of supernatant II, add acetonitrile to supernat...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 [0001] The invention belongs to the technical field of biochemical detection and determination, in particular to a method for detecting organic acids in cells 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 gas chromatography-mass spectrometry detection of urinary organic acids, including the following steps: (1) the urine to be tested The sample has been treated with urine enzyme; (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 1-3 (5) Use gas chromatography-mass spectrometry to detect standard organic acid samples and urine samples to be tested; (6) Use the test results of standard organic acid samples as With reference to th...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N30/88
Inventor 陈东梁远雄
Owner LIUZHOU LIANHAI TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com