Detection method and kit for detecting 25-hydroxyvitamin D (25OHD) in human serum

A technology of hydroxyvitamin and detection method, applied in the field of biological detection, can solve the problems of matrix interference, can not effectively remove matrix interference, etc., achieves the effect of removing matrix interference, meeting clinical detection requirements, and improving sensitivity

Inactive Publication Date: 2019-05-21
上海墨煦科技发展合伙企业(有限合伙)
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, since 25-hydroxyvitamin D mainly binds to the serum binding protein DBP in the human body, and there are a large number of high-affinity binding proteins in the human body, there is serious matrix interference in the detection, while the traditional radioimmunoassay and competing protein binding In contrast, LC-MS / MS has the characteristics of strong specificity, high precision and accuracy, and is considered to be the gold standard method for evaluating the nutritional status of vitamin D in the body.

Method used

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  • Detection method and kit for detecting 25-hydroxyvitamin D (25OHD) in human serum
  • Detection method and kit for detecting 25-hydroxyvitamin D (25OHD) in human serum
  • Detection method and kit for detecting 25-hydroxyvitamin D (25OHD) in human serum

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Experimental program
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Embodiment 1

[0041] The kit for detecting 25-hydroxyvitamin D in human serum is characterized in that it includes: calibrator, quality control product, isotope internal standard solution, the concentration is 1mol / L sodium hydroxide aqueous solution, methyl tert-butyl ether, 0.2% Aqueous formic acid, 0.2% formic acid in methanol, 80% methanol in water.

[0042] The calibrator is 5 kinds of concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, all of which are bovine serum albumin aqueous solutions of 5, 10, 25, 50, 100ng / mL in turn, and the bovine serum albumin aqueous solution Concentration is 5% (mass fraction);

[0043] The quality control product is 2 kinds of concentration of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 respectively successively the bovine serum albumin aqueous solution of 7.5,15 and 15,30ng / mL, the concentration of described bovine serum albumin aqueous solution 5% (mass fraction).

Embodiment 2

[0045] The method for detecting 25-hydroxyvitamin D in human serum, the steps are as follows: add 10 μL sodium hydroxide aqueous solution to 300 μL human serum sample, vortex for 1 minute, then transfer to an ultrafiltration device and centrifuge for 30 minutes, then add 100 μL Add 10 μL 25-hydroxyvitamin D isotope internal standard solution to the ultrafiltrate, vortex for 30 seconds, add 1000 μL methyl tert-butyl ether, vortex for 4 minutes; after centrifugation, pipette 800 μL supernatant and blow dry with nitrogen , add 100 μL complex solution, and vortex for 4 minutes to obtain the sample to be tested; then transfer it to a 200 μL V-bottom liner tube, put it into a matching 1.5mL injection bottle, and transfer it to a liquid chromatography autosampler middle. The LC autosampler automatically loads 30 μL of sample onto the chromatographic column, which is then passed through the mobile phase to the mass spectrometer for detection.

[0046] The concentration of the sodium ...

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Abstract

The invention provides a detection method and a kit for detecting 25-hydroxyvitamin D (25OHD) in human serum. The method comprises the following steps: mixing a serum sample with a sodium hydroxide aqueous solution uniformly; performing ultrafiltration centrifugation; adding an isotope internal standard solution of 25-hydroxyvitamin D in the ultrafiltrate; after full and uniform mixing, adding a methyl tertiary butyl ether solvent and carrying out vortex shock extraction; after centrifugation, taking a supernatant and carrying out nitrogen drying, and carrying out redissolving to obtain a to-be-measured sample; and then carrying out detection. The detection method has advantages of simple pre treatment, high specificity and resistance to matrix interference, high detection precision and accuracy, and high detection sensitivity.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a high-performance liquid chromatography tandem mass spectrometry method for detecting 25-hydroxyvitamin D in human serum. technical background [0002] The amount of vitamin D detection is increasing exponentially every year. The reason for this demand is mainly due to the high rate of vitamin D deficiency in different populations around the world and research findings that vitamin D plays an important role in many physiological functions of the human body. If it can promote the absorption of calcium and phosphate in the diet and enhance bone mineral density, it can promote the formation of children's bones and maintain the strength of adult bones. In addition, vitamin D is also closely related to diseases such as cardiovascular disease, autoimmune disease, diabetes and malignant tumor. Vitamin D deficiency can lead to impaired bone development and an increased risk of the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 钱学庆秦炜吴小虎贺笑非
Owner 上海墨煦科技发展合伙企业(有限合伙)
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