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Method for detecting concentration of DHA in blood in human body

A detection method and blood technology, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of flammability, affect accuracy, and difficult to control adsorption concentration, and achieve the effect of simple operation and satisfactory precision.

Pending Publication Date: 2021-04-09
苏州和合医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the literature, DHA is analyzed by thin-layer chromatography, but most of them use gas chromatography or gas chromatography-mass spectrometry. Due to the strong polarity, volatility and low stability of DHA, it needs to be derivatized after treatment. Gas chromatographic detection, the derivatization method is mostly methyl ester derivatization method, which can be divided into two categories: acid catalysis and base catalysis. Relevant studies have found that acid catalysis is more suitable for esterifying free fatty acids such as DHA, acid catalysis is mainly Using hydrogen chloride-methanol esterification method, sulfuric acid-methanol esterification method and boron trifluoride-ether-methanol esterification method, the adsorption concentration of hydrogen chloride-methanol in the hydrogen chloride-methanol esterification method is not easy to control, which will affect the accuracy of the measurement The esterification rate of DHA in the sulfuric acid-methanol esterification method is not high enough; the boron trifluoride-ether complex in the boron trifluoride-ether-methanol esterification method is flammable, toxic, easy to decompose, inconvenient to operate, and unsafe , in addition, under electron bombardment of unsaturated fatty acid methyl esters, it is difficult to locate the double bond position correctly. Therefore, we propose a method for detecting the concentration of DHA in human blood

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] S1. Blood sample pretreatment:

[0021] (1) Weigh 0.3 g of blood, put it into a 10 mL centrifuge tube, add 40 μL of C23:0 internal standard solution with a mass concentration of 40 μg / mL, 1.5 mL of distilled water, 400 μL of methanol solution containing 0.05% antioxidant (BHT), 2mL of chloroform, oscillate evenly, centrifuge at 2500r / min for 10min, take the chloroform layer, repeat the above extraction process, combine the chloroform layer into a 20mL headspace sampling bottle, blow dry with nitrogen for later use;

[0022] (2) Under the protection of nitrogen, add 200μLAMP to the above-mentioned dried headspace sample vial, fill it with nitrogen and seal it, react in a constant temperature oil bath at 180°C for 120min, then cool to room temperature, add methanol to dilute to 1mL, and the sample is passed through After filtering through a 0.22 μm filter membrane, it was analyzed and detected by LC-MS / MS.

[0023] S2. Preparation of standard stock solution and standard ...

Embodiment 2

[0030] S1. Blood sample pretreatment:

[0031] (1) Weigh 0.3 g of blood, put it into a 10 mL centrifuge tube, add 40 μL of C23:0 internal standard solution with a mass concentration of 40 μg / mL, 1.5 mL of distilled water, 400 μL of methanol solution containing 0.05% antioxidant (BHT), 2mL of chloroform, oscillate evenly, centrifuge at 2500r / min for 10min, take the chloroform layer, repeat the above extraction process, combine the chloroform layer into a 20mL headspace sampling bottle, blow dry with nitrogen for later use;

[0032] (2) Under nitrogen protection, add 200 μLAMP to the above dried headspace sample bottle, fill it with nitrogen and seal it, react in a constant temperature oil bath at 180 °C for 100 min, then cool to room temperature, add methanol to dilute to 1 mL, and the sample is passed through After filtering through a 0.22 μm filter membrane, it was analyzed and detected by LC-MS / MS.

[0033] S2. Preparation of standard stock solution and standard solution: ...

Embodiment 3

[0040] S1. Blood sample pretreatment:

[0041](1) Weigh 0.3 g of blood, put it into a 10 mL centrifuge tube, add 40 μL of C23:0 internal standard solution with a mass concentration of 40 μg / mL, 1.5 mL of distilled water, 400 μL of methanol solution containing 0.05% antioxidant (BHT), 2mL of chloroform, oscillate evenly, centrifuge at 2500r / min for 10min, take the chloroform layer, repeat the above extraction process, combine the chloroform layer into a 20mL headspace sampling bottle, blow dry with nitrogen for later use;

[0042] (2) Under nitrogen protection, add 200 μLAMP to the above dried headspace sampling vial, fill it with nitrogen and seal it, react in a constant temperature oil bath at 210 °C for 75 min, then cool to room temperature, add methanol to dilute to 1 mL, and the sample is passed through After filtering through a 0.22 μm filter membrane, it was analyzed and detected by LC-MS / MS.

[0043] S2. Preparation of standard stock solution and standard solution:

...

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PUM

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Abstract

The invention discloses a method for detecting the concentration of DHA in blood in a human body. The method comprises the following steps: S1, pretreating a blood sample; S2, preparing a standard stock solution and a standard solution; and S3, conducting LC-MS / MS analysis and detection. The specific operation method of the step S1 comprises the steps of accurately weighing 0.3 g of blood, putting the blood into a 10mL centrifuge tube, sequentially adding 40 [mu]L of a C23:0 internal standard solution with a mass concentration of 40 [mu]g / mL, 1.5 mL of distilled water, 400 [mu]L of a methanol solution containing 0.05% of an antioxidant (BHT) and 2mL of chloroform, conducting oscillating uniformly, conducting centrifuging at a rotating speed of 2500r / min for 10min, taking a chloroform layer, repeating the above extraction process, merging the chloroform layers in a 20 mL headspace injection bottle, and conducting nitrogen blow-drying for later use. According to the method, DHA is analyzed under heterocycle derivatization combination, the method is applied to detection of DHA in human blood, an internal standard method is adopted for quantification instead of a normalization method for determining the relative content of DHA, and the method is easy and convenient to operate, high in practicability and suitable for analysis and detection of DHA content in human blood, and the accuracy, sensitivity and precision all reach satisfactory effects.

Description

technical field [0001] The invention relates to the technical field of unsaturated fatty acid detection, in particular to a method for detecting DHA concentration in human blood. Background technique [0002] Docosahexaenoic acid (DHA), commonly known as brain gold, is an n-3 polyunsaturated fatty acid and is the main lipid component in the human brain nerve cell membrane. DHA can promote the development of the central nervous system of infants and young children, and can protect vision. DHA also has the functions of preventing cardiovascular and cerebrovascular diseases, inhibiting tumor growth, anti-inflammation and inhibiting allergic reactions. Epidemiological surveys show that newborns, infants, pregnant women and the elderly are most prone to DHA deficiency in the population. However, the more intake of DHA, the better. Large intake will cause bleeding tendency, coagulation dysfunction, Patients with severe hypertension and autoimmune diseases must use it with cautio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06G01N30/72
CPCG01N30/06G01N30/72G01N30/88G01N2030/045G01N2030/067G01N2030/8822
Inventor 蒙晋宁邹皓月王贯宇
Owner 苏州和合医学检验有限公司