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Method for simultaneously detecting vitamins K1, MK4 and MK7

A technology of vitamins and isotopes, applied in the field of analysis, can solve the problems of decreased coagulation factor concentration, bleeding, and simultaneous detection of multiple sample types, etc., and achieves the effects of short analysis time, strong specificity, and good chromatographic resolution

Active Publication Date: 2022-01-14
GUANGZHOU KINGMED DIAGNOSTICS CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various studies have shown that vitamin K deficiency can lead to lower concentrations of active clotting factors in the blood, which can lead to bleeding
At present, there is no report on the simultaneous detection of VK1, MK4 and MK7 in multiple sample types

Method used

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  • Method for simultaneously detecting vitamins K1, MK4 and MK7
  • Method for simultaneously detecting vitamins K1, MK4 and MK7
  • Method for simultaneously detecting vitamins K1, MK4 and MK7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 A method for simultaneously detecting vitamin K1, MK4 and MK7

[0093] Preparation of mixed internal standard solution of VK1, MK4, MK7 three substances to be detected: using ethanol as solvent, prepare a mixed internal standard solution containing 1.4ng / mL VK1-d7, 1.2ng / mL MK4-d7 and 2.0ng / mL MK7-d7 standard solution.

[0094] Sample pretreatment: Add 400 μL of VK1, MK4, MK7 mixed internal standard solution to 200 μL mixed patient serum test sample to precipitate the protein in the test sample, shake for 5 minutes, let stand for 15 minutes, add n-hexane Extract with alkane, centrifuge at 10,000 rpm for 10 min to obtain the supernatant, blow dry with nitrogen, add 80 μL of complex solution in methanol to dissolve, and obtain the sample to be tested.

[0095] Liquid chromatography tandem mass spectrometry is used to detect the sample to be tested, and the chromatographic conditions are:

[0096] Use a BEH C18 column with a particle size of 1.7 μm, an inner di...

Embodiment 2

[0131] Because MK7 contains a relatively long isoprenoid chain, its physical and chemical properties are quite different from those of VK1 and MK4, and there are many limitations in its performance in mass spectrometry and chromatography. The common problems are that the mass spectrometry parameters of MK7 are difficult to obtain and the degree of chromatographic separation is not good. Good, low response, high baseline, severe matrix interference, etc. The inventors of the present invention unexpectedly found in research that when using a C18 column to detect MK7, the particle size and length of the liquid chromatography column have a great influence on the peak shape of the MK7 chromatographic peak. This example specifically analyzes and compares the effects of C18 columns with different particle sizes (1.7μm, 2.5μm, 2.7μm) and C18 columns with different lengths (50mm, 100mm) on the results of MK7 mass spectrometry and chromatographic separation (test samples are mixed serum ...

Embodiment 3

[0135] The initial concentration of the liquid phase elution organic phase and the elution gradient program in the detection method of the present invention affect the chromatographic behavior of VK1, MK4, and MK7. In this example, the mixed standard solution of VK1, MK4 and MK7 prepared in Example 1 was used for testing. When the initial organic phase concentration of the liquid phase gradient was lower than 88%, it was found that the MK4 peaking time was delayed, it was difficult to effectively separate from interfering substances, and the response intensity decreased. Such as Figure 5 As shown, the analysis and comparison of the influence of the initial organic phase concentration of 85% and 90% liquid phase gradient on the MK4 peaking, under the condition of 85% initial organic phase, the peaking time of MK4 is delayed by about 0.45min, and the response intensity is relatively reduced by about 45%. It is difficult to effectively separate the target substance from the int...

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Abstract

The invention relates to a method for simultaneously detecting vitamins K1, MK4 and MK7. The method comprises the following steps of taking a biological sample, adding an isotope internal standard mixed solution of three substances to be detected, adding an extraction solvent for extraction, centrifuging, taking supernate, drying, dissolving with a reconstitution fluid to obtain a sample to be detected, and detecting by adopting a liquid chromatography-tandem mass spectrometry method, a chromatographic column adopted by the liquid chromatography tandem mass spectrometry being a C18 chromatographic column, the particle size of the C18 chromatographic column being 1.6 to 2.2 mu m, and the length of the C18 chromatographic column being 40 to 60mm. The detection method provided by the invention not only can detect the vitamins K1, MK4 and MK7 at the same time, but also has strong specificity, obtains good chromatographic resolution (retention time difference is greater than 0.5 min) for the vitamins K1, MK4 and MK7, and has advantages of low baseline, less matrix interference, low detection limit, high sensitivity and strong stability.

Description

technical field [0001] The invention relates to the field of analysis, in particular to a method for simultaneously detecting vitamin K1, MK4 and MK7. Background technique [0002] Vitamin K is the general term for 2-methyl-1,4-naphthoquinone and its derivatives. There are two types of natural fat-soluble vitamin K that have been discovered so far: one is the oily substance proposed in alfalfa—vitamin K1 (VK1) , also known as phylloquinone; the other is to obtain crystalline vitamin K2 in spoiled fish. Vitamin K2 includes several different forms, the most common forms of vitamin K2 in the human diet are menadione-4 (MK4) and menadione-7 (MK7). MK4 is mainly found in animal products such as egg yolks, meat, liver and butter. MK7 is synthesized by bacteria and mainly found in fermented foods. [0003] Vitamin K, also known as coagulation vitamin, is a necessary cofactor for the synthesis of various coagulation factors and plays an indispensable role in blood homeostasis. V...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/34G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/34G01N30/72G01N30/8634G01N2030/045
Inventor 赵蓓蓓佘旭辉林清娜董衡李卓阳
Owner GUANGZHOU KINGMED DIAGNOSTICS CENT
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