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Analytical kit for detecting four free fatty acids in human blood spots through high performance liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatography and free fatty acid technology, applied in the field of analysis and detection, can solve the problems of low detection sensitivity and complicated operation

Inactive Publication Date: 2019-08-20
上海可力梅塔生物医药科技有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, although there are some detection methods for free fatty acids in the prior art, the operations are often complicated (such as extraction steps are required), the detection sensitivity is low, and it is difficult to quantitatively detect multiple free fatty acids at the same time
At present, there are no related reports on the methods and kits for simultaneous detection of these four substances.

Method used

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  • Analytical kit for detecting four free fatty acids in human blood spots through high performance liquid chromatography-tandem mass spectrometry
  • Analytical kit for detecting four free fatty acids in human blood spots through high performance liquid chromatography-tandem mass spectrometry
  • Analytical kit for detecting four free fatty acids in human blood spots through high performance liquid chromatography-tandem mass spectrometry

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] The analysis kits are listed in Table 5.

[0106] Table 5 Preparation of Analysis Kit Components

[0107]

[0108] The preparation method of the above-mentioned kit is as follows:

[0109] (1) Standard product:

[0110] The preparation of the standard substance with the concentration of J1: take 160 μL of 50 mM α-octadecatrienoic acid, 8 μL of 100 mM eicosapentaenoic acid, 20 μL of 100 mM ω3-docosapentaenoic acid, and 160 μL of 50 mM docosahexaenoic acid, Add 1652 μL of methanol respectively to prepare 4 μM α-octadecatrienoic acid, 0.4 μM eicosapentaenoic acid, 1 μM ω3-docosapentaenoic acid and 4 μM docosahexaenoic acid; Then take 150 μL each of 4 μM α-octadecatrienoic acid, 0.4 μM eicosapentaenoic acid, 1 μM ω3-docosapentaenoic acid and 4 μM docosahexaenoic acid, and use artificial rabbit The blood volume was adjusted to 3000 μL, and the obtained concentration was J1 standard;

[0111]Preparation of standard substance with concentration J2: Take 500 μL each of α...

Embodiment 2

[0129] 1. Pretreatment

[0130] Test sample processing: (1) Extraction: Take a dried blood spot sample with a diameter of 6mm in a 2mL centrifuge tube, add 200μL of the extraction solution containing the internal standard, and then vortex and shake at 2500rpm for 30min, take 150μL of the above mixed solution and use a nitrogen meter Blow dry; (2) Derivatization: add 200 μL derivatization reagent 1 (10vt% acetyl chloride in dichloromethane solution), derivatize with vortex shaking at 500rpm for 30min at room temperature, and dry; then add 200 μL derivatization reagent 2 (1vt% Acetonitrile solution of picolylamine), derivatized by vortexing at 500rpm for 5min at room temperature, and dried; (3) Reconstitution: add 400μL reconstitution solution (saturated monohydric alcohol), and vortex and mix at 2500rpm for 5min at room temperature .

[0131] Standard product: the processing method is the same as that of the test sample, and will not be repeated here;

[0132] Quality control...

Embodiment 3

[0162] Example 3: Performance Verification

[0163] figure 1 is the chromatogram of alpha-octadecatrienoic acid standard substance, figure 2 Octadecatrienoic acid- 13 C18 isotope internal standard chromatogram, image 3 is the chromatogram of eicosapentaenoic acid standard substance, Figure 4 is eicosapentaenoic acid-d5 isotope internal standard chromatogram, Figure 5 is the chromatogram of ω3-docosapentaenoic acid standard substance, Figure 6 It is the internal standard chromatogram of docosahexaenoic acid-d5 isotope (which is the internal standard of ω3-docosapentaenoic acid). Figure 7 is the docosahexaenoic acid standard chromatogram, Figure 8 Docosahexaenoic acid-d5 isotope internal standard chromatogram (internal standard for two compounds of docosahexaenoic acid). , the peak positions of each substance are 5.033min, 5.052min, 5.049min, 5.058min, 6.626min, 5.939min, 6.094min and 5.939min, although these 8 substances are not completely separated, but for LC-MS / ...

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Abstract

The invention provides an analytical kit for detecting four free fatty acids (alpha-octadecatrienoic acid, eicosapentaenoic acid, omega3-docosapentaenoic acid and docosahexaenoic acid) in human bloodspots through high performance liquid chromatography-tandem mass spectrometry. The analytical kit specifically comprises replacement matrix pretreatment, dried blood spot sample treatment and the like. The method has the advantages of high sensitivity, short time and improved detection efficiency.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection. Specifically, the invention relates to an analysis kit for detecting four kinds of free fatty acids in human dried blood spots using high performance liquid chromatography-tandem mass spectrometry technology and an application thereof. Background technique [0002] Free fatty acids (FFAs) play an important role in tissue energy supply, especially in the fasted state. Normal free fatty acid levels are crucial to human health, and different free fatty acids play different roles in physiological and pathological states, such as eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22 : 6), has a certain protective effect on inflammation, cancer, arteriosclerosis, insulin resistance and type 2 diabetes. [0003] Epidemiological studies have shown that fatty acids are related to insulin resistance, cardiovascular disease, type 2 diabetes, hypertension, obesity and other chronic dise...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/027
Inventor 朱监宝官培龙邱碧涵于嘉屏
Owner 上海可力梅塔生物医药科技有限公司
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