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High-throughput detection pretreatment method for fat-soluble vitamins in blood plasma

A fat-soluble vitamin, high-throughput technology, which is applied in the preparation of test samples, measuring devices, instruments, etc., can solve the problems of increased operation time, a lot of labor, and is not suitable for large-scale high-throughput fat-soluble vitamin extraction work. , to achieve the effect of easy automation, shortening extraction time, and achieving high-throughput detection pretreatment

Pending Publication Date: 2020-12-11
SUZHOU ENRICHING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The liquid-liquid extraction technology is simple to operate, but the disadvantage is that it needs to be operated in EP tubes, and a large number of high-speed vortex oscillations and centrifugation work are required, resulting in increased operating time and a lot of labor. It is not suitable for large-scale high-throughput fat-soluble vitamins. extract work

Method used

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  • High-throughput detection pretreatment method for fat-soluble vitamins in blood plasma
  • High-throughput detection pretreatment method for fat-soluble vitamins in blood plasma
  • High-throughput detection pretreatment method for fat-soluble vitamins in blood plasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Preparation of experimental reagents

[0087] 1) Plasma sample to be processed: centrifuge the anticoagulated whole blood at 3000 rpm for 10 min, collect the upper layer of plasma, and obtain the plasma sample to be processed.

[0088] 2) Internal standard solution configuration: vitamin A internal standard, 25-hydroxyvitamin D2 internal standard, 25-hydroxyvitamin D3 internal standard, vitamin E internal standard and vitamin K1 internal standard were all purchased from China National Institutes for Food and Drug Control. The five vitamins were mixed according to a certain ratio and dissolved in methanol, so that the final internal standard concentration of vitamin A was 200ng / mL, the final internal standard concentration of 25-hydroxyvitamin D2 was 40ng / mL, and the final internal standard concentration of 25-hydroxyvitamin D3 was 40ng / mL , the final internal standard concentration of vitamin E was 200 ng / mL, and the final internal standard concentration of vitamin K1 w...

Embodiment 2

[0091] Pretreatment for high-throughput detection of fat-soluble vitamins in plasma, including the following steps:

[0092] S1. Take 200 μL of the untreated plasma sample obtained in Example 1 in a 96-well plate, add 200 μL of the internal standard solution obtained in Example 1, use a magnetic bar cover to mix the sample evenly by shaking up and down, and incubate with ultrasonic vibration for 1 minute, and the balance is sufficient;

[0093] S2. Add extraction reagent to the sample treated in step S1, oscillate up and down with a magnetic bar cover, mix evenly, ultrasonicate for 5 minutes, ultrasonic power 300W, frequency 35KHz, fully balanced, precipitate protein and macromolecular hydrocarbons;

[0094] Wherein, the extraction reagent comprises acetonitrile of 65 parts by volume, n-hexane of 15 parts by volume, ethyl acetate of 20 parts by volume;

[0095] S3. Add the first magnetic bead suspension to the sample treated in step S2, oscillate up and down with a magnetic rod ...

Embodiment 10

[0114] Determination of fat-soluble vitamins in embodiment 2 to embodiment 9, comparative example 1

[0115] HPLC-MS / MS analysis, specific detection conditions are as follows:

[0116] Chromatographic column: C18 liquid chromatography column (Waters ACQUITY HPLC CSH C18);

[0117] Organic phase: methanol (containing 0.1% formic acid), aqueous phase: water (containing 0.1% formic acid);

[0118] Column temperature: 40°C; total collection time: 3min; injection volume: 8μL.

[0119] The mass spectrum adopts APCI ion source and is collected in positive ion mode. The target compound fat-soluble vitamin and its isotope internal standard are simultaneously scanned by multiple reaction monitoring to obtain the integrated peak area.

[0120] The extraction efficiencies (peak area ratios of target substance / internal standard) and repeatability CV% of the five fat-soluble vitamins in the plasma samples to be treated in Example 2 to Example 9 and Comparative Example 1 are shown in Table...

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Abstract

The invention provides a high-throughput detection pretreatment method for fat-soluble vitamins in blood plasma. The method comprises the following steps: S1, uniformly mixing a blood plasma sample tobe treated with an internal standard solution, and performing ultrasonic oscillation incubation; S2, adding an extraction reagent, precipitated protein and macromolecular hydrocarbon; S3, adding a first magnetic bead suspension, performing ultrasonic oscillation, combining the first magnetic beads with protein precipitation, macromolecular hydrocarbon precipitation and phospholipid, performing magnetic separation, and reserving liquid; and S4, performing nitrogen blowing concentration, and adding an organic solvent for redissolution for detection and analysis. The invention also provides a kit. According to the detection pretreatment method, protein, macromolecular hydrocarbon and phospholipid in blood plasma are removed through adoption of a paramagnetic particle method, centrifugation or filtration is not needed, and high-throughput operation can be achieved. The kit disclosed by the invention is simple and convenient to operate, the magnetic beads have magnetic separability and canbe quickly separated from a solution under the action of an external magnetic field, high-flux operation can be realized by matching with an automatic instrument, and the extraction working efficiency is improved.

Description

technical field [0001] The invention relates to a technology for extracting fat-soluble vitamins in blood plasma, in particular to a pretreatment method and a kit for high-throughput detection of fat-soluble vitamins in blood plasma. Background technique [0002] Vitamin (vitamin) is a class of compounds that cannot be synthesized or synthesized in insufficient amounts by humans and animals and must be ingested directly or indirectly from food. and play an important role in development. Vitamins are a class of organic compounds necessary to maintain good health. These substances are neither raw materials for body tissue nor sources of energy in the body, but a class of regulatory substances that play an important role in substance metabolism. If there is a long-term lack of certain vitamins, it will cause physiological dysfunction and certain diseases, which are collectively referred to as vitamin deficiency. At present, according to the difference in solubility of vitamin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N30/08G01N30/88
CPCG01N1/28G01N30/08G01N30/88
Inventor 刘栋然尚春庆
Owner SUZHOU ENRICHING BIOTECH CO LTD
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