Mass spectrum qualitative and quantitative analysis method for free fatty acids based on double-derivatization technology

Abstract

The invention relates to a mass spectrum qualitative and quantitative analysis method for free fatty acids based on a double-derivatization technology. The mass spectrum qualitative and quantitative analysis method comprises the following steps: 1) a photochemical derivatization reaction of double bonds of unsaturated fatty acids; 2) an N,N-diethylethylenediamine derivatization reaction of carboxyl terminals of the fatty acids; 3) a qualitative method for precise structures of the fatty acids; and 4) a quantitative method for the fatty acids. The method disclosed the invention is used for precise qualitative and quantitative analysis of the free fatty acids in actual samples, so that the problems that double-bond positions of the unsaturated fatty acids, particularly polyunsaturated fattyacids, are difficult to authenticate and the fatty acids are low in ionization efficiency and poor in detection sensitivity under a negative ion mode are solved, and high-sensitivity and accurate qualitative and quantitative analysis for the free fatty acids in the actual samples is realized.

Description

technical field

[0001] The invention relates to a qualitative and quantitative analysis method of free fatty acid mass spectrometry based on double derivatization technology, and its application in the qualitative and quantitative analysis of free fatty acids in different biological samples. technical background

[0002] Fatty acid (FA) refers to a series of organic substances with long aliphatic hydrocarbon chains containing a carboxyl group at one end. The length of the carbon chain varies (C4-C36). It is divided into saturated and unsaturated fatty acids according to whether they contain double bonds. FA is the simplest class of lipid compounds and is an important component of other lipids. The diversity of fatty acids in mammals plays a variety of key roles in cellular functions through: for example, the composition of various functional lipid molecules in biological membranes; the regulation of membrane fluidity and the interaction between signaling molecules; The prod...

Examples

Embodiment 1

[0046] Qualitative and Quantitative Analysis of Free Fatty Acids in Example 1 Human Gastric Cancer Cells

[0047] A method for qualitative and quantitative analysis of free fatty acids in human gastric cancer cells based on double derivatization technology, the specific steps are as follows:

[0048] 1) Take one million cells in a 16mm×125mm glass tube, add 150μL of 10μmol / L C17:1(n-7) and 1ml PBS, centrifuge for 2 minutes at 3000 rpm, and discard the upper layer; Then add 1mL DPBS, then add 2mL methanol, acidify with hydrochloric acid, and the final concentration of hydrochloric acid reaches 25mM; then add 1ml isooctane, centrifuge at 3000 rpm for 1 minute, separate layers, and move the upper layer to a 10mm×75mm glass tube medium, blow dry with nitrogen; after the fatty acid extract was reconstituted with 1ml of acetone:water (6:4), take 50 μL of diluted liquid nitrogen and blow dry, and finally reconstitute with 950 μL of acetone:water (6:4), add 50 μL of ethanol, and use ...

Embodiment 2

[0051] Qualitative and quantitative analysis of free fatty acids in embodiment 2 human plasma

[0052] Qualitative and quantitative analysis of free fatty acids in human plasma: Take 10 μL into a 16mm×125mm glass tube, add 150 μL of 10 μmol / L C17:1(n-7), add 1ml of PBS, centrifuge for 2 minutes, 3000 rpm, and then Discard the supernatant. Add 1 mL of DPBS to the cells, followed by 2 mL of methanol, and acidify with hydrochloric acid to a final concentration of 25 mM. After adding 1ml of isooctane, the sample was centrifuged at 3000 rpm for 1 minute, separated into layers, and the upper layer was transferred to a 10mm×75mm glass tube, blown dry with nitrogen, and used for derivatization reaction. After the fatty acid extract was reconstituted with 1ml of acetone:water (6:4), take 50 μL of dilute liquid nitrogen to blow dry, reconstitute with 950 μL of acetone:water (6:4), add 50 μL of ethanol for the subsequent photochemical derivatization reaction and N, N-diethylethylenedia...

Embodiment 3

[0055] Qualitative and quantitative analysis of free fatty acids in the mouse liver in embodiment 3

[0056] Qualitative and quantitative analysis of free fatty acids in mouse liver: Take 1 mg into a 16mm×125mm glass tube, add 100 μL of 40 μmol / L C17:1(n-7), add 1ml of PBS, centrifuge for 2 minutes, 3000 rpm, Then discard the upper aqueous layer. Add 1 mL of DPBS to the cells, followed by 2 mL of methanol, and acidify with hydrochloric acid to a final concentration of 25 mM. After adding 1ml of isooctane, the sample was centrifuged at 3000 rpm for 1 minute, separated into layers, and the upper layer was transferred to a 10mm×75mm glass tube, blown dry with nitrogen, and used for derivatization reaction. After the fatty acid extract was reconstituted with 1ml of acetone:water (6:4), take 50 μL of dilute liquid nitrogen to blow dry, reconstitute with 950 μL of acetone:water (6:4), add 50 μL of ethanol for the subsequent photochemical derivatization reaction and N, N-diethyleth...

technical field

[0001] The invention relates to a qualitative and quantitative analysis method of free fatty acid mass spectrometry based on double derivatization technology, and its application in the qualitative and quantitative analysis of free fatty acids in different biological samples. technical background

[0002] Fatty acid (FA) refers to a series of organic substances with long aliphatic hydrocarbon chains containing a carboxyl group at one end. The length of the carbon chain varies (C4-C36). It is divided into saturated and unsaturated fatty acids according to whether they contain double bonds. FA is the simplest class of lipid compounds and is an important component of other lipids. The diversity of fatty acids in mammals plays a variety of key roles in cellular functions through: for example, the composition of various functional lipid molecules in biological membranes; the regulation of membrane fluidity and the interaction between signaling molecules; The prod...

Examples

Embodiment 1

[0046] Qualitative and Quantitative Analysis of Free Fatty Acids in Example 1 Human Gastric Cancer Cells

[0047] A method for qualitative and quantitative analysis of free fatty acids in human gastric cancer cells based on double derivatization technology, the specific steps are as follows:

[0048] 1) Take one million cells in a 16mm×125mm glass tube, add 150μL of 10μmol / L C17:1(n-7) and 1ml PBS, centrifuge for 2 minutes at 3000 rpm, and discard the upper layer; Then add 1mL DPBS, then add 2mL methanol, acidify with hydrochloric acid, and the final concentration of hydrochloric acid reaches 25mM; then add 1ml isooctane, centrifuge at 3000 rpm for 1 minute, separate layers, and move the upper layer to a 10mm×75mm glass tube medium, blow dry with nitrogen; after the fatty acid extract was reconstituted with 1ml of acetone:water (6:4), take 50 μL of diluted liquid nitrogen and blow dry, and finally reconstitute with 950 μL of acetone:water (6:4), add 50 μL of ethanol, and use ...

Embodiment 2

[0051] Qualitative and quantitative analysis of free fatty acids in embodiment 2 human plasma

[0052] Qualitative and quantitative analysis of free fatty acids in human plasma: Take 10 μL into a 16mm×125mm glass tube, add 150 μL of 10 μmol / L C17:1(n-7), add 1ml of PBS, centrifuge for 2 minutes, 3000 rpm, and then Discard the supernatant. Add 1 mL of DPBS to the cells, followed by 2 mL of methanol, and acidify with hydrochloric acid to a final concentration of 25 mM. After adding 1ml of isooctane, the sample was centrifuged at 3000 rpm for 1 minute, separated into layers, and the upper layer was transferred to a 10mm×75mm glass tube, blown dry with nitrogen, and used for derivatization reaction. After the fatty acid extract was reconstituted with 1ml of acetone:water (6:4), take 50 μL of dilute liquid nitrogen to blow dry, reconstitute with 950 μL of acetone:water (6:4), add 50 μL of ethanol for the subsequent photochemical derivatization reaction and N, N-diethylethylenedia...

Embodiment 3

[0055] Qualitative and quantitative analysis of free fatty acids in the mouse liver in embodiment 3

[0056] Qualitative and quantitative analysis of free fatty acids in mouse liver: Take 1 mg into a 16mm×125mm glass tube, add 100 μL of 40 μmol / L C17:1(n-7), add 1ml of PBS, centrifuge for 2 minutes, 3000 rpm, Then discard the upper aqueous layer. Add 1 mL of DPBS to the cells, followed by 2 mL of methanol, and acidify with hydrochloric acid to a final concentration of 25 mM. After adding 1ml of isooctane, the sample was centrifuged at 3000 rpm for 1 minute, separated into layers, and the upper layer was transferred to a 10mm×75mm glass tube, blown dry with nitrogen, and used for derivatization reaction. After the fatty acid extract was reconstituted with 1ml of acetone:water (6:4), take 50 μL of dilute liquid nitrogen to blow dry, reconstitute with 950 μL of acetone:water (6:4), add 50 μL of ethanol for the subsequent photochemical derivatization reaction and N, N-diethyleth...

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