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Carbonyl metabolite derivatization method and non-targeted metabonomics efficient analysis method

A derivatization and metabolite technology, applied in the field of metabolomics analysis, can solve the problem that the sensitivity needs to be improved, and achieve the effect of increasing detection sensitivity, eliminating noise interference and reducing instrument drift.

Pending Publication Date: 2021-07-23
厦门市迈理奥科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese invention patent application CN112125827A discloses a synthetic method for derivatizing reagents of carbonyl-containing steroids, first utilizing 1,3-dibromopropane and N-hydroxyphthalimide to react to synthesize 2-(3- Bromopropoxy) isoindoline-1,3-dione; then use 2-(3-bromopropoxy)isoindoline-1,3-dione to prepare 3-((1,3-dione Oxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide; finally to 3-((1,3-dioxo-2-isoindole hydrazinyl)-oxyl)-N,N,N-trimethylpropylammonium bromide for hydrazinolysis, the detection sensitivity of this method needs to be improved

Method used

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  • Carbonyl metabolite derivatization method and non-targeted metabonomics efficient analysis method
  • Carbonyl metabolite derivatization method and non-targeted metabonomics efficient analysis method
  • Carbonyl metabolite derivatization method and non-targeted metabonomics efficient analysis method

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Take a tissue sample, weigh the tissue, and place it in a centrifuge tube. If the sample is too large, cut the original sample so that the weight of the sample is around 150-200mg. Add pre-cooled methanol / water solution to the centrifuge tube, according to the ratio of 500 μL methanol / water solution: 100 mg tissue, the ratio of methanol / water solution is 4:1, v / v. Homogenize the tissue sample for 15 seconds using a homogenizer. The homogenization speed and time can be adjusted according to the sample type if necessary (eg: harder tissue requires higher speed and longer time). After homogenization, the samples were incubated at -20°C for 10 minutes. After incubation, centrifuge for 10 minutes at 4°C and centrifugal force >10000g. Take the supernatant into a centrifuge tube, and centrifuge each centrifuge tube at low speed, so that the sample droplets on the tube wall sink to the bottom of the centrifuge tube. Take the supernatant and dry it completely with a vacuum c...

Embodiment 2

[0056] Take the plasma sample and centrifuge for 15 minutes at 4°C under the condition of centrifugal force>15000g (12000rpm). After centrifugation, take the supernatant to a 0.5mL centrifuge tube; Add 90 μL of pre-cooled LC-MS or HPLC grade methanol to the centrifuge tube of 30 μL sample, fully vortex the centrifuge tube containing the sample and methanol to completely precipitate the protein, and then centrifuge the centrifuge tube at low speed to make the mixture in the tube sink to bottom of the centrifuge tube. Place the vortexed mixture in a -20°C refrigerator for at least 1 hour; after 1 hour, take the centrifuge tube out of the refrigerator, centrifuge at a centrifugal force >10,000g for 15 minutes, and pipette 90 μL of the supernatant into a new microcentrifuge tube. 90 μL supernatant was completely dried with a vacuum centrifugal concentrator, and the drying was stopped when there was no solvent at the bottom of the centrifuge tube, and the obtained dried sample was ...

Embodiment 3

[0059] Take the urine sample and centrifuge it for 15 minutes at 4°C under the conditions of centrifugal force >15000g (12000rpm), and transfer the supernatant to a 1.5mL centrifuge tube after centrifugation. Use a vacuum centrifugal concentrator to completely dry the obtained supernatant, stop drying when there is no solvent at the bottom of the centrifuge tube, and obtain a dry sample.

[0060] If the urine sample has not been filtered before, the supernatant needs to be filtered through a 0.2-0.3 μm filter. After filtration, take the filtrate into a centrifuge tube. Then use a vacuum centrifugal concentrator to dry, and stop drying when there is no solvent at the bottom of the centrifuge tube to obtain a dry sample.

[0061] For the derivatization method of dried samples and the high-efficiency analysis method of non-targeted metabolomics, see Example 1.

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Abstract

The invention relates to a carbonyl metabolite derivatization method and a non-targeted metabonomics efficient analysis method.The carbonyl metabolite derivatization method comprises the steps of: 1, performing carbonyl metabolite extraction on a sample to be detected through a first solvent, then performing drying to remove the first solvent, and obtaining a dried sample; and 2, dissolving the dried sample, adding a pH adjusting reagent to adjust the pH value, adding dansylhydrazide, uniformly mixing, heating, incubating, cooling to terminate the reaction, drying to obtain a solid, and redissolving the solid to obtain a derivatized sample. According to the non-targeted metabonomics efficient analysis method, 12C labeling and 13C labeling are synchronously carried out by adopting the derivatization method, so that the metabolite detection sensitivity is improved, more metabolite can be detected at the same time, and the metabolite coverage rate is higher; and the detected metabolite is a peak pair, the influence caused by instrument drift and matrix is reduced, noise interference is eliminated, and quantification is more accurate.

Description

technical field [0001] The invention relates to a metabolomics analysis technology, in particular to a method for derivatization of carbonyl metabolites and a non-targeted metabolomics high-efficiency analysis method. Background technique [0002] As an important part of systems biology, metabolomics has broad application prospects in the field of clinical medicine. Metabolomics analysis techniques include: NMR, GC-MS, CE-MS, and LC-MS. Although these techniques can detect metabolites to a certain extent, there are still many difficulties, such as low ionization efficiency of metabolites and mass spectrometry. Weak signal, lack of isotope internal standard for quantification, small number of detected metabolites, and many interferences. [0003] For example, LC-MS uses liquid chromatography-mass spectrometry for analysis, and compares the respective metabolites in different samples to determine all the metabolites therein. Essentially, metabolic fingerprinting involves com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/88
CPCG01N30/06G01N30/88G01N2030/067G01N2030/027G01N2030/8813
Inventor 赵爽
Owner 厦门市迈理奥科技有限公司
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