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Unsaturated fatty acid quantitative method based on chemical derivatization and HPLC-MS

A technology for unsaturated fatty acids and quantitative detection methods, applied in the direction of measuring devices, scientific instruments, instruments, etc., can solve problems affecting the application of liquid chromatography-mass spectrometry detection technology and complex products, so as to improve the detection effect, ensure singleness, and result Improved effect

Inactive Publication Date: 2019-06-14
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the traditional fatty acid epoxidation method fails to fully epoxidize fatty acids, and the products after epoxidation become more complex, which affects the application of liquid chromatography-mass spectrometry detection technology.

Method used

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  • Unsaturated fatty acid quantitative method based on chemical derivatization and HPLC-MS
  • Unsaturated fatty acid quantitative method based on chemical derivatization and HPLC-MS
  • Unsaturated fatty acid quantitative method based on chemical derivatization and HPLC-MS

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Embodiment 1

[0067] Embodiment 1 The present invention extracts the method for fatty acid sample in blood plasma

[0068] Thaw the frozen human plasma solution at 4°C, take 500 μL plasma sample, add 1500 μL methanol, 750 μL PBS buffer solution (pH=7.4), add 69 μL 1M formic acid to acidify, mix well after shaking, and let stand for 15 minutes. Add 2.5ml of ethyl acetate, oscillate evenly, centrifuge at 4°C, 3000g for 5min, and take the supernatant; add 2.5ml of ethyl acetate to the centrifuged precipitate, shake well and repeat the centrifugation. The centrifuged supernatant was blown dry with ethyl acetate using a nitrogen blower, and reconstituted by adding 500 μL of acetonitrile for use.

Embodiment 2

[0069] Embodiment 2 The method for fatty acid epoxidation of the present invention

[0070] Among the present invention, the oxidation of fatty acid carbon-carbon double bond relies on potassium monopersulfate compound salt (Oxone) as oxidant, tetrahydrothiopyran-4 ketone 1,1-dioxide as catalyzer, and this method has reaction speed fast, and reaction product Advantages such as high rate. The reaction principle is as figure 1 shown.

[0071] Specific operation steps: the epoxidation reaction was operated in a 1.5mL plastic vial. Take 200 μL of fatty acid acetonitrile solution, then add 75 μL of 500 mM potassium persulfate to the solution to provide oxygen for the epoxidation reaction, and then add 50 μL of 100 mM tetrahydrothiopyran-4-one 1,1-dioxide to the solution as a catalyst to promote the epoxidation reaction. At the same time, 75 μL of 400 μM EDTA disodium salt solution and 100 μL of 1 M sodium bicarbonate solution were added to the reaction system. The whole reacti...

Embodiment 3

[0072] Embodiment 3 The method for the quantitative detection of fatty acids by liquid chromatography-mass spectrometry of the present invention

[0073] For the absolute quantification of fatty acids, 100 μM heptadecenic acid (FA 17:1 10Z) was used as the internal standard (IS) in the present invention. Standard curves for fatty acids and internal standards need to be established before processing samples, as well as cone voltage and collision energy voltage optimized for each FA standard. Taking oleic acid as an example, direct mass spectrometry analysis was first performed to optimize the cone voltage and collision energy voltage of the fatty acid, and then 100 μM heptadecanoic acid was added as a reactant to 50, 100, 200, 500 and 1000 μM oleic acid solutions, respectively. After the epoxidation reaction, compare the intensities of the epoxidized product of oleic acid (m / z 297) and the product of IS (m / z 283) to calculate the intensity ratio (I 297 / I 283 ). In multiple ...

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Abstract

The invention discloses an unsaturated fatty acid quantitative method based on chemical derivatization and HPLC-MS. The method comprises the following steps: 1) determining a standard optimization cone voltage and collision energy voltage of fatty acids by direct mass spectrometry; 2) using an unsaturated fatty acid A as an internal standard, performing carbon-carbon double-bond epoxidation on theinternal standard and a standard product fatty acid, performing detection through an HPLC-ESI-MS method, and establishing a standard curve of the standard product fatty acid and the internal standard; 3) performing carbon-carbon double-bond epoxidation on the fatty acid to be tested; and 4) detecting the epoxidized fatty acid to be tested in an internal standard method through the HPLC-ESI-MS method, detecting the content of the fatty acid to be tested, and performing regression on the standard curve obtained in the step 1) to obtain the content of the fatty acid to be tested. According to the unsaturated fatty acid quantitative method disclosed by the invention, accurate quantitative detection is performed on isomers of the fatty acids, the quantitative process is simplified, and the accuracy of the quantitative detection is improved.

Description

technical field [0001] The invention relates to the technical field of liquid mass spectrometry, in particular to a liquid mass spectrometry method for qualitative and quantitative detection using fatty acid epoxidation derivatives. Background technique [0002] Fatty acids are the main components of neutral fats, phospholipids and glycolipids. In the presence of sufficient oxygen supply, fatty acids can be oxidized and decomposed into CO 2 and H 2 O, and release a lot of energy, so fatty acid is one of the main energy sources of the body 1 . Fatty acids are usually present in the human body in esterified form as components of lipids such as neutral lipids, phospholipids and glycolipids. Esterified fatty acids are hydrolyzed under the action of phospholipase to obtain free fatty acids (FFA), which are non-esterified fatty acids and are important components of lipid metabolites. On the one hand, exogenous fat is transported through plasma, enters fat cells in the form of...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 蒋宇扬高丹宋超李上富
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV