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Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry

A hydroxyvitamin and ultra-high performance liquid phase technology, which is applied in the field of ultra-high performance liquid chromatography tandem mass spectrometry to detect serum 25-hydroxyvitamin D, can solve the problems of complex processing, sensitivity and precision that need to be further improved, matrix interference, etc. Easy to handle, meet the requirements of clinical testing, and remove the effect of matrix interference

Inactive Publication Date: 2016-04-27
袁洪
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are mainly the following problems: First, since 25-hydroxyvitamin D is mainly bound to serum-binding proteins in the human body, there are a large number of high-affinity binding proteins in the human body, so there is serious matrix interference in the detection
However, the "gold standard" processing process is complex, and the sensitivity and precision need to be further improved

Method used

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  • Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry
  • Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry
  • Method for detecting 25-hydroxy-vitamin D through ultra-performance liquid chromatography-tandem mass spectrometry

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Experimental program
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Effect test

Embodiment 1

[0049] The method for detecting 25-hydroxyvitamin D by the ultra-high performance liquid chromatography tandem mass spectrometer actually comprises the following steps:

[0050] (1) Pre-processing:

[0051] Add the internal standard of 25-hydroxyvitamin D to the human serum sample, add zinc sulfate solution and methanol solution to carry out protein precipitation; add n-hexane solvent for extraction after thorough mixing; further mix well and centrifuge, then pipette the supernatant and Carry out nitrogen blow-drying, add complex solution, obtain the sample to be tested;

[0052] Wherein, the volume ratio of the human serum sample to the zinc sulfate solution, methanol solution, and n-hexane extraction solution is 1:1:2:5; the volume ratio of the supernatant to the corresponding original solution is 1:1 ; The volume ratio of the complex solution to the human serum sample is 1:2.

[0053] 25 hydroxyvitamin D internal standard can include d 6 -25 hydroxyvitamin D 2 and / or d ...

Embodiment 2

[0108] Example 2 In order to enable those skilled in the art to better understand the present invention, an example is now provided to illustrate the specific implementation process of the detection method for 25-hydroxyvitamin D of the present invention.

[0109] Pipette 150ul human serum into a clean 2ml centrifuge tube, add 10ul internal standard solution, mix for 10s, add 150ul of 0.2M zinc sulfate solution, mix for 10s, add 300ul methanol, mix for 10s, add 750ul n-hexane, mix Homogenize for 30s, centrifuge (13000rpm, 5min), take 600ul of the supernatant, blow dry with nitrogen at 50°C, and finally reconstitute with 75ul of 70% methanol aqueous solution, vortex and mix, transfer to a glass liner, and load it into the liquid chromatography automatic in the sampler.

[0110] The LC autosampler automatically loads the sample onto the chromatographic column, which then passes through the mobile phase to the mass spectrometer LC-MS / MS. LC-MS / MS uses an electrospray ionization ...

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Abstract

The invention provides a method for detecting 25-hydroxy-vitamin D through a high-sensitivity high-throughput ultra-performance liquid chromatography-tandem mass spectrometry device. The method comprises adding an internal standard substance of 25-hydroxy-vitamin D into a serum sample, adding a zinc sulfate solution and a methanol solution into the mixture so that protein deposition is realized, carrying out full mixing, adding a n-hexane solvent into the mixture, carrying out extraction, carrying out full mixing, carrying out centrifugation, taking the supernatant, carrying out nitrogen blowing drying, adding a compounded solution into the supernatant to obtain a sample to be detected, detecting the sample to be detected through an ultra-performance liquid chromatography-tandem quadrupole mass spectrometry device and carrying out quantitation through an internal standard curve method based on 25-hydroxy-vitamin D2 and / or 25-hydroxy-vitamin D3 quantitative and qualitative ion pair retention time as qualitative basis. The method has advantages of simple pre-treatment process, good specificity, good matrix interference resistance, short detection time, high throughput, high detection precision, high sensitivity and low cost.

Description

technical field [0001] The invention relates to the technical field of vitamin D detection, in particular to a method for detecting serum 25-hydroxyvitamin D by ultra-high performance liquid chromatography tandem mass spectrometry. Background technique [0002] Vitamin D is a fat-soluble non-essential vitamin and is a sterol derivative. 25-hydroxyvitamin D is formed by the conversion of vitamin D under the action of 25-hydroxylase in liver microsomes. Compared with other metabolites of vitamin D, it is The concentration in the blood is high and the circulation period is long, so it becomes a good indicator of the concentration of vitamin D in the body and an evaluation index of the nutritional status of vitamin D in the human body. [0003] The most important member of the vitamin D family is vitamin D 2 and vitamin D 3 . Vitamin D 2 It is mostly contained in plant foods, and it is synthesized by plant ergosterol through sunlight. Vitamin D 3 Accounting for 90%-95% of ...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/06
Inventor 袁洪郭成贤江涛刘湘军
Owner 袁洪
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