Method for determining free vitamin D in sample through liquid chromatography tandem mass spectrometry

A liquid chromatography and tandem mass spectrometry technology, which is applied in the field of liquid chromatography tandem mass spectrometry for the determination of free vitamin D in samples, can solve the problems of no liquid chromatography tandem mass spectrometry detection of Free-25(OH)D, no supporting kits, etc. , to increase the detection specificity, ensure the steady state, and avoid the effect of interference

Active Publication Date: 2019-08-27
北京豪思生物科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no method for detecting Free-25(OH)D by liquid chromatography tandem mass spectrometry, and there is no matching kit

Method used

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  • Method for determining free vitamin D in sample through liquid chromatography tandem mass spectrometry
  • Method for determining free vitamin D in sample through liquid chromatography tandem mass spectrometry
  • Method for determining free vitamin D in sample through liquid chromatography tandem mass spectrometry

Examples

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Embodiment 1

[0039] Example 1 Method for Determination of Serum Free Vitamin D by Liquid Chromatography Tandem Mass Spectrometry

[0040] 1. Experimental reagents

[0041] The sample preparation solution includes: methanol solution, n-hexane, isotope internal standard, 1mol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution, 0.9% physiological saline, PTAD derivative reagent, and complex solution.

[0042] Isotopic internal standard: 25-hydroxyvitamin D3-d3 solution, the concentration is 50-2000pg / mL, and 500pg / mL is preferably used in this embodiment.

[0043] HEPES: Use HEPES to adjust the serum pH value, the sample volume is 50-200 μl, and finally use 100 μl HEPES to adjust the pH to 7.4±0.3.

[0044] PTAD derivatization reagent: PTAD solution containing 0.01-1 mg / mL, preferably 0.5 mg / mL PTAD solution in this embodiment.

[0045] Complex solution: a mixture of methanol solution containing 0.01%-1% v / v formic acid and water in a volume ratio of 1-10:1. In this embod...

Embodiment 2

[0058] The determination of free vitamin D in the actual serum sample of embodiment 2

[0059] Take 500 μl of sample serum / plasma and add 100 μl of HEPES buffer, adjust the pH to 7.4±0.3, vortex and mix well, bathe in water at 37°C for 30 minutes, take 500 μl of the mixture and add it to an ultrafiltration tube (the molecular weight cut-off of the ultrafiltration tube is 3KDa), centrifuge at 14000g Take the filtrate for 30 minutes, add 500 μl of normal saline to wash, centrifuge at 14,000 g for 30 minutes, and take the filtrate; wash the ultrafiltration membrane with 500 μl of 0.9% NaCl three times, and collect the filtrate; wash the membrane with 500 μl of anhydrous methanol, and combine the above filtrates; take 1ml of the filtrate, add 500μl isotope internal standard (500pg / mL 25-hydroxyvitamin D3-d3 solution), 200μl water and 2ml n-hexane; shake, centrifuge at 3000rpm for 10min; take supernatant; blow dry with nitrogen; add 50μl0.5mg / ml PTAD solution and place at room tempe...

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Abstract

The present invention provides a method for determining free vitamin D in a sample through liquid chromatography tandem mass spectrometry. The method comprises: preparing free vitamin D standard substance solutions of different concentrations; adding stable isotope internal standard substances to the standard substance solutions, uniformly mixing, then extracting with n-hexane, drying supernatantswith nitrogen, adding PTAD for derivatization and then drying with the nitrogen, and adding a complex solution for detection of liquid chromatography tandem mass spectrometry; drawing a standard curve according to the concentrations of the standard substance solutions and the detected signal intensity; replacing the standard substance solution in step B with a sample to be determined for conducting detection; and obtaining the concentration of the free vitamin D in the sample to be determined according to the detection result of the sample to be determined with reference to the standard curve, thereby realizing quantitative detection of the free vitamin D in the sample. The present invention successfully establishes the method for determining the free vitamin D in the sample through liquid chromatography tandem mass spectrometry. The method can quickly and specifically detect the content of the free vitamin D in the sample (especially serum).

Description

technical field [0001] The invention relates to liquid chromatography tandem mass spectrometry detection technology, in particular to a liquid chromatography tandem mass spectrometry method for determining free vitamin D in a sample. Background technique [0002] Vitamin D is a fat-soluble vitamin necessary to maintain human health. As a hormone, it regulates calcium and phosphorus metabolism and promotes healthy growth and reconstruction of bones. There are two main forms of vitamin D: ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Vitamin D cannot be synthesized by itself, and can only be synthesized by sunlight or external sources of vitamin D2 or D3. Vitamin D enters the liver through blood circulation and is transformed into 25(OH)D, which is then metabolized into dihydroxyvitamin D [1,25(OH)2D] by the renal 1α hydroxylase CYP27B1, which then acts on vitamin D receptors , play a biological effect. In addition to directly causing rickets and osteoporosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72G01N2030/027G01N2030/062
Inventor 王丹晨禹松林邱玲
Owner 北京豪思生物科技股份有限公司
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