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Extraction method and detection method of vitamin D metabolite-25(OH)D3 in serum

A technology for vitamins and metabolites, applied in the field of biochemical detection, can solve the problems of the difference in detection methods affecting the clinical diagnosis and treatment of vitamin D, the consumption of organic solvents, environmental pollution, etc., and achieve easy automation, improved detection sensitivity, high extraction efficiency and The effect of recovery

Pending Publication Date: 2020-05-26
苏州康吉诊断试剂有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] 25-Hydroxyvitamin D (25(OH)D3) is the best indicator to reflect the status of vitamin D in the human body, but the large difference in results between detection methods affects the clinical diagnosis and treatment of vitamin D deficiency
[0021] At present, most of the extraction methods of 25(OH)D3 in serum are liquid-liquid extraction method. The method is: 1. Mixing: the solution and the extract are poured into the separating funnel; Oscillation; 3. Stand still: put the separating funnel on the iron stand and stand still; 4. Liquid separation: After the liquid is layered, open the glass plug at the upper end of the funnel, release the lower liquid from the lower end, and pour out the upper liquid from the upper end (extraction Different types of reagents, 25(OH)D3 or in the upper layer or in the lower layer) However, the above-mentioned liquid-liquid extraction method has certain defects: 1. The operation is cumbersome and time-consuming
2. It needs to consume a lot of organic solvents, resulting in high cost and environmental pollution; 3. It is difficult to extract highly water-soluble substances from water

Method used

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  • Extraction method and detection method of vitamin D metabolite-25(OH)D3 in serum
  • Extraction method and detection method of vitamin D metabolite-25(OH)D3 in serum
  • Extraction method and detection method of vitamin D metabolite-25(OH)D3 in serum

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preparation example Construction

[0066] The preparation method of activated carbon comprises the following steps:

[0067] Carbonize organic raw materials consisting of at least fruit shells, cores, coal, and wood at a high temperature of 600-900°C under air-isolated conditions to obtain carbonized products to reduce non-carbon components, oxygen elements such as H2O, CO, CO2 and other gases Precipitation (this process is called carbonization); reacting the carbonization with air causes its surface to be eroded, resulting in a microporous structure. Activated carbon can be bought on the market, or it can be obtained by entrusting a manufacturer to make it specially.

[0068] Activated carbon can effectively adsorb or remove phospholipids in serum samples. In addition, activated carbon can also adsorb many other substances due to its developed microporous structure, light weight, high porosity, and strong ability to absorb liquids. In this application, it is emphasized that activated carbon can more effective...

Embodiment 1

[0071] Example 1 of the present invention discloses a method for extracting vitamin D metabolite-25(OH)D3 in serum, comprising the following steps:

[0072] Step 1) Sample extraction: Take 100 μL of serum sample and add it to a centrifuge tube (1.5 mL sterile centrifuge tube), then add 100 μL of water / isopropanol mixture with a volume ratio of 50:50 into the centrifuge tube as a precipitated protein destroyer Destroy the precipitated protein in the serum, so that 25(OH)D3 is released from the precipitated protein, vortex and mix the serum sample with the precipitated protein destroyer at 2500rpm, and spin at 7000rpm for 10s;

[0073] After the first instant centrifugation, at least 100 μL of 50:50 water / isopropanol mixture was added to the centrifuge tube, and vortexed at 2500 rpm for 1 min, then uniformly mixed and then centrifuged at 7000 rpm for 10 s, thereby Finally, a mixture sample containing 25(OH)D3, phospholipid and protein was obtained;

[0074] Step 2) Sample loadi...

Embodiment 2

[0079] Example 2 of the present invention discloses a method for extracting vitamin D metabolite-25(OH)D3 in serum, comprising the following steps:

[0080] Step 1) Sample extraction: Take 150 μL serum sample and add it to a centrifuge tube (1.5 mL sterile centrifuge tube), then add 200 μL water / isopropanol mixture with a volume ratio of 50:50 into the centrifuge tube as a precipitated protein destroyer Destroy the precipitated protein in the serum, so that 25(OH)D3 is released from the precipitated protein, vortex and mix the serum sample and the precipitated protein destroyer at 2800rpm, and spin at 10000rpm for 30s;

[0081] After the first instant centrifugation, at least 200 μL of 50:50 water / isopropanol mixture was added to the centrifuge tube, and vortexed at 2800 rpm for 2 min, then mixed evenly and then centrifuged at 10000 rpm for 30 s. Finally, a mixture sample containing 25(OH)D3, phospholipid and protein was obtained;

[0082] Step 2) Sample loading: Load the mix...

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Abstract

The invention discloses an extraction method and a detection method of vitamin D metabolite-25(OH)D3 in serum. The extraction method comprises the following steps of: 1) releasing 25(OH)D3 from precipitated protein, and instantaneously separating for at least one time, so as to obtain a mixture sample containing 25(OH)D3, phospholipid and protein, 2) utilizing an activated carbon extraction plateto at least adsorb phospholipid and protein in the mixture sample, 3) eluting the activated carbon extraction plate, and pressing the activated carbon extraction plate and the collection plate to collect eluent containing 25(OH)D3, and 4) blow-drying the collection plate, and re-dissolving. The detection method comprises the following steps of: taking a reconstitution fluid of 25(OH)D3 obtained byan extraction method of vitamin D metabolite-25(OH)D3 in serum as a to-be-detected substance, and loading the to-be-detected object to a liquid chromatography tandem mass spectrometer for detection.According to the extraction method provided by the invention, sample enrichment and purification can be completed at the same time, the extraction rate is higher, and the sensitivity of subsequent detection can be greatly improved.

Description

technical field [0001] The invention relates to a biochemical detection method, in particular to an extraction method and detection method of vitamin D metabolite-25(OH)D3 in serum. Background technique [0002] Vitamin D (vitaminD) is a fat-soluble sterol substance, referred to as VD. Currently, vitamin D is considered to be a steroid hormone. The most important members of the vitamin D family are VD2 (ergocalciferol) and VD3 (cholecalciferol). . VD2 and VD3 have different side chains in the chemical structure, and there is no clear evidence to prove that the physiological functions of the two are different. There are not many natural foods containing vitamin D in nature, and wild salmon and mushrooms irradiated by ultraviolet rays contain VD. VD can also be ingested through VD-enhanced food. The most common VD-enhanced food is milk, followed by some cereals, fruit juices, and margarine. [0003] In the process of vitamin D metabolism, vitamin D precursors exist in the s...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/30G01N30/34G01N30/72
CPCG01N30/02G01N30/06G01N30/30G01N30/34G01N30/72G01N2030/062
Inventor 傅强谢利德程亚美丁浩
Owner 苏州康吉诊断试剂有限公司
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