Method for detecting vitamin D metabolite

A detection method and metabolite technology, applied in the field of analysis and detection, can solve the problems of large sample size, difficulty in obtaining such substrates, inconvenient transportation, etc., to eliminate detection interference, improve derivatization efficiency, and improve derivatization speed effect

Pending Publication Date: 2020-02-21
上海医药临床研究中心 +1
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

The invention relates to a liquid chromatography tandem mass spectrometry detection method for 25-hydroxyvitamin D in dried blood samples. The pretreatment process mainly includes sample pretreatment, derivation, and redissolution processes. At the same time, multiple reaction monitoring (MRM) scanning is adopted. The method realizes the high-throughput liquid chromatography-tandem mass spectrometry detection of 25-hydroxyvitamin D in dried blood slices, and solves the problems of large sample size, inconvenient sampling and transportation of traditional liquid chromatography-tandem mass spectrometry methods, but the high cost remains unsolved The problem
[0005] In addition, another problem in the detection of vitamin D by LC-MS / MS (liquid chromatography-tandem mass spectrometry) is the blank matrix
The ideal matrix for preparing standard curves and quality control samples for quantification is analyte-free human serum, but such matrices are difficult to obtain and need to find alternatives

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  • Method for detecting vitamin D metabolite
  • Method for detecting vitamin D metabolite
  • Method for detecting vitamin D metabolite

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Experimental program
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Effect test

Embodiment 1

[0030] The addition order of embodiment 1 derivative and the selection optimization of substituting matrix

[0031] 1.1. The solution of innovative chemical derivatization to the sensitivity, speed and cost of the method:

[0032] See the principle of innovative chemical derivatization reaction image 3 .Derivatization reagents DR1 and DR2 react to produce product A, and product A and vitamin D metabolites undergo the next step of derivatization reaction of Diels-Alder addition reaction (Diels-Alder reaction).

[0033] According to attached Figure 4A , it can be found that without derivatization, 1α,25-dihydroxyvitamin D3 in serum cannot be detected even under the instrument conditions of the high-end instrument ABQTRAP 5500 in current clinical research. After derivatization, the target components of the same samples were accurately detected ( Figure 4B ). The reason is that although the derivatization reagent used in the present invention is still PTAD in essence, it is g...

Embodiment 2

[0045] Example 2 Detection method of 1α, 25-dihydroxyvitamin D3

[0046] Taking the development process of the method of α,25-dihydroxyvitamin D3 in Example 1 as an example, the reagents and instrument information used in the method development are described in detail as follows. Other metabolites are applicable in principle, and the specific parameters should be determined by those skilled in the art according to the target of metabolite detection, through experiments to determine the mass spectrometry detection conditions and liquid phase separation conditions, and the derivatization conditions basically do not need to be adjusted.

[0047] 2.1. Reagent and instrument information:

[0048] 2.1.1 Reference compounds:

[0049] 1α, 25-dihydroxyvitamin D3 and 1α, 25-dihydroxyvitamin D3-d3 were purchased from Isosciences, and 1 mL of ethanol solution was placed in an ampoule.

[0050] 2.1.2 Reagents:

[0051] Methyl tert-butyl ether (HPLC grade), methanol (HPLC grade), ammoniu...

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Abstract

The invention provides a method for detecting a vitamin D metabolite. The method comprises the following steps that: an analyte, namely the vitamin D metabolite, in a serum sample, is extracted by using an organic solvent; and a derivative reagent 4-phenylurazole and iodobenzene diacetate are added into a blow-dried extraction residue, so that Diels-Alder reaction can be performed. According to the method, the vitamin D metabolite is detected by using a liquid chromatography-tandem mass spectrometry method. The method has the advantages of low cost, short reaction time, high sensitivity and high interference resistance.

Description

technical field [0001] The invention relates to the field of analysis and detection, in particular to a method for detecting metabolites. Background technique [0002] Vitamin D (including D2 and D3) is a type of fat-soluble vitamin necessary for the human body, which is derived from the synthesis and food supplementation in the human body under ultraviolet radiation. After being metabolized by the liver and kidney, a series of metabolites will be produced, whose names and chemical structures are as follows: figure 1 and 2 are shown. Among them, 25-hydroxyvitamin D3 and D2 have been extensively clinically studied, and have been proven to be closely related to many diseases such as children's rickets, coronary heart disease, diabetes, children's headaches, and cancer. 1α,25 dihydroxyvitamin D3 is one of the active metabolites. Recent studies have shown that 1α,25-dihydroxyvitamin D3 can promote the absorption of calcium and phosphorus in the small intestine, maintain the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/7266G01N30/06G01N2030/027G01N2030/067G01N2030/8822
Inventor 李金蓉杨文初许萍阮亮亮甘荣兴
Owner 上海医药临床研究中心
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