High performance liquid chromatography tandem mass spectrometry detection method of 25-hydroxy vitamin D in serum

A high-performance liquid chromatography and hydroxyvitamin technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., to reduce costs, improve automation, and improve the throughput of analysis

CN108645942AInactive Publication Date: 2018-10-12杭州凯莱谱精准医疗检测技术有限公司
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2018-10-12
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a high performance liquid chromatography tandem mass spectrometry detection method of 25-hydroxy vitamin D in serum. The method comprises the following steps that (1) a sampleis pre-treated, specifically, an internal standard working solution and a sodium hydroxide solution are sequentially added into a serum sample and mixed to be uniform; (2) solid-liquid extraction treatment is conducted, specifically, the sample treated in the step (1) is loaded to an SLE 96 pore plate, and eluted with normal hexane, and then the eluant is blown to be dried by using nitrogen; (3) aderivatization reaction is conducted, a derivatization reagent 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD) solution is added to redissolve, a thermostatic reaction is conducted, then ethyl alcoholis added to terminate the reaction, and the product is blown to be dried by using nitrogen; and (4) redissolution is conducted, and analytical detection is conducted by adopting the high performance liquid chromatography tandem mass spectrometry based on a multiplexing system (MPX). The high performance liquid chromatography tandem mass spectrometry detection method is high in detection sensitivity, high in specificity and low in cost, meanwhile, the pre-treatment time and analysis time of the sample are effectively shortened, and the detection efficiency of the sample is improved.
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Description

technical field

[0001] The invention relates to the technical field of analysis and detection, in particular to a high-throughput, high-sensitivity, and low-cost detection method for 25-hydroxyvitamin D in serum by high performance liquid chromatography tandem mass spectrometry. Background technique

[0002] Vitamin D is a steroid hormone that can regulate the body's calcium and phosphorus homeostasis, and its level in the body has an important impact on the normal physiological functions of the body. Vitamin D is converted into the metabolite 25-hydroxyvitamin D in the liver. 25-hydroxyvitamin D can reflect the level of vitamin D in the human body and is an important indicator for evaluating vitamin D. At present, the methods for determining 25-hydroxyvitamin D in serum mainly include enzyme immunoassay, competitive protein binding method, high performance liquid chromatography, etc.; these methods have the following problems and shortcomings: enzyme immunology methods cann...

Examples

Embodiment

[0030] Example: A high performance liquid chromatography tandem mass spectrometry detection method for 25-hydroxyvitamin D in serum

[0031] 1. Solution configuration

[0032] Standard working solution configuration: Accurately weigh 25-OHD respectively 2and 25-OHD 3 Standard, add ethanol to dissolve to obtain a primary stock solution with a concentration of 1mg / mL, and then dilute the primary stock solution with ethanol to 25-OHD 2 At a concentration of 10 μg / mL and 25-OHD 3 Secondary stock solution with a concentration of 50 μg / mL; precisely draw a certain volume of ethanol and place it in a 2mL EP tube, and then precisely draw a certain volume of 25-OHD in turn 2 Secondary stock solution and 25-OHD 3 The secondary stock solution was placed in the EP tube, vortexed for 10s to mix well, and the concentration of 25-OHD was 800ng / mL 2 and 2000ng / mL of 25-OHD 3 Mix the standard working solution and name it as the standard working solution SD6. Dilute the standard working ...