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Derivatization method of carboxyl metabolites and non-targeted metabonomics efficient analysis method

An analysis method and a derivatization technology, applied in the field of metabolomics analysis, can solve the problems that fatty acids and phospholipids cannot be detected simultaneously, low ionization efficiency, low abundance of fatty acids, etc., to achieve increased detection sensitivity and metabolite coverage High, the effect of eliminating noise interference

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 CN111610246A discloses a carboxyl derivatization reagent composition, including I compound and amide condensing agent, the derivatization reagent composition and the sample are subjected to a derivatization reaction to obtain a derivatized sample liquid; The mass spectrometry analysis of the purified sample liquid is carried out in the positive ion mode, and the spectrum and peak data of the mass spectrum are obtained, so as to detect the fatty acids and phospholipids in the sample to be tested at the same time, so as to solve the problem of low abundance of fatty acids and low ionization efficiency, which are difficult to be detected. problems, and the problem that fatty acids and phospholipids cannot be detected synchronously caused by the difference between positive and negative ion modes

Method used

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  • Derivatization method of carboxyl metabolites and non-targeted metabonomics efficient analysis method
  • Derivatization method of carboxyl metabolites and non-targeted metabonomics efficient analysis method
  • Derivatization method of carboxyl metabolites and non-targeted metabonomics efficient analysis method

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Experimental program
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Effect test

Embodiment 1

[0045] 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 2

[0056] For urine samples, centrifuge for 15 minutes at 4°C under the condition of centrifugal force >15000g (12000rpm), and take 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.

[0057] 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.

[0058] For the derivatization method of dried samples and the high-efficiency analysis method of non-targeted metabolomics, see Example 1.

Embodiment 3

[0060] 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, 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 centrifugal con...

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Abstract

The invention relates to a derivatization method of carboxyl metabolites and a non-targeted metabonomics efficient analysis method. The derivatization method of the carboxyl metabolites comprises the steps of: step 1, enabling a sample to be detected to be subjected to carboxyl metabolite extraction through a first solvent, then performing drying, removing the first solvent, and obtaining a dried sample; step 2, mixing the dry sample with a second solvent to dissolve the dry sample, then adding triethanolamine and 2-bromo-1-(4-dimethylaminophenyl) ethanone, uniformly mixing, and then heating for incubation; after incubation is completed, adding triglycine, then performing heating, performing incubation, and obtaining a derivatization 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, so that the influence caused by instrument drift and matrix is reduced, and the quantification is more accurate.

Description

technical field [0001] The invention relates to metabolomics analysis technology, in particular to a method for derivatization of carboxyl 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 compa...

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 赵爽韩伟
Owner 厦门市迈理奥科技有限公司
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