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Method for simultaneously detecting vitamin K1 and vitamin K2 (MK4) in blood

A vitamin and blood technology, applied in the field of vitamin detection

Pending Publication Date: 2019-09-06
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the determination methods of vitamin K1 and vitamin K2 (MK4) at home and abroad are mainly to detect vitamin K1 and vitamin K2 (MK4) separately. At present, there is no report on the simultaneous determination of vitamin K1 and vitamin K2 (MK4) in blood.

Method used

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  • Method for simultaneously detecting vitamin K1 and vitamin K2 (MK4) in blood
  • Method for simultaneously detecting vitamin K1 and vitamin K2 (MK4) in blood
  • Method for simultaneously detecting vitamin K1 and vitamin K2 (MK4) in blood

Examples

Experimental program
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Effect test

Embodiment 1

[0117] Embodiment 1: Prepare the mixed standard solution of series concentration

[0118] Preparation of vitamin K1 standard stock solution: Accurately weigh 10 mg of vitamin K1 standard product, place it in a 10 mL volumetric flask, dissolve it with methanol, and set the volume to 10 mL, store at -80 °C, and the validity period is 6 months;

[0119] Preparation of vitamin K2 (MK4) standard stock solution: Accurately weigh 10 mg of vitamin K2 (MK4) standard product, place it in a 10 mL volumetric flask, dissolve it with methanol, and set the volume to 10 mL, store it at -80°C, and the validity period is 6 days moon;

[0120] Prepare vitamin K1-d7 internal standard stock solution: take 1mg of vitamin K1-d7 standard product and place it in a 10mL volumetric flask, dissolve it with methanol, and set the volume to 10mL to obtain standard stock solution D, and store it at -80°C , valid for 1 year;

[0121] Vitamin K2 (MK4)-d7 internal standard stock solution: Take 0.25 mg of vita...

Embodiment 2

[0125] Embodiment 2: fitting standard curve equation

[0126] After the nine kinds of concentration mixed standard solutions obtained in the above-mentioned Example 1 were vortexed and mixed for 1-2min at a speed of 1500-2500rpm respectively, the fourth supernatant corresponding to each concentration mixed standard solution was pipetted, and the high-efficiency solution was used to Phase tandem mass spectrometer detects the fourth supernatant corresponding to each concentration mixed standard solution; obtain vitamin K1, vitamin K2 (MK4) and vitamin K1-d7 internal standard substance, vitamin K2 (MK4) of nine kinds of different concentration mixed standard solutions )-d7 internal standard chromatogram;

[0127] Obtain the chromatographic peak area of ​​vitamin K1, vitamin K2 (MK4), vitamin K1-d7 internal standard substance, vitamin K2 (MK4)-d7 internal standard substance respectively from the chromatogram of the mixed standard solution of above-mentioned each concentration, res...

Embodiment 3

[0135] Embodiment 3: Based on the standard curve equation given in Example 2, simultaneously detect vitamin K1 and vitamin K2 (MK4) in whole blood

[0136] Pipette 10 μL of the mixed internal standard solution prepared in Example 1 above, and sequentially add 50-300 μL of the first supernatant of the whole blood sample to be detected after centrifugation to 10 μL of the mixed internal standard solution (the whole blood sample to be detected Centrifuge to take at least 5mL of the blood to be tested, centrifuge at a centrifugation speed of 3500rpm for 10min, take the first supernatant, and store the first supernatant at -20°C for storage until analysis) and precipitate protein reagents. After vortex mixing at a speed of 1500-2500rpm for 2-5min, add the extractant, vortex mix at a speed of 1500-2500rpm for 3-10min, then centrifuge at a speed of 10000-15000rpm for 4-6min at a high speed to obtain the second For the supernatant, pipette a certain amount of the second supernatant af...

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Abstract

The invention provides a method for simultaneously detecting vitamin K1 and vitamin K2 (MK4) in blood. The method comprises the following steps: detecting mixed standard solutions in at least three concentrations, wherein mixed standard solution is the mixed standard solutions of the vitamin K1 and vitamin K2 (MK4) with internal standard subtances, and fitting a standard curve equation; adding a first supernatant and protein precipitation reagent after centrifuging a to-be-detected sample in the to-be-detected sample, and adding extraction agent, uniformly mixing, and high-speed centrifuging,and taking supernatant; drying the second supernatant through a nitrogen-blowing way, dissolving the dried leftover by using the re-dissolving solution, centrifuging at high speed, and taking the third supernatant; detecting the third supernatant by utilizing a high performance liquid chromatograph tandem mass spectrometer; and obtaining the concentrations of the vitamin K1 and vitamin K2 (MK4) inthe to-be-detected sample based on the detection result and the standard curve equation. Through the scheme provided by the invention, the vitamin K1 and vitamin K2 (MK4) can be detected at the sametime.

Description

technical field [0001] The invention relates to the technical field of vitamin detection, in particular to a method for simultaneously detecting vitamin K1 and vitamin K2 (MK4) in blood. Background technique [0002] Both vitamin K1 and vitamin K2 are fat-soluble vitamins, both of which are necessary to maintain the normal functioning of human body. Among them, vitamin K1 participates in the synthesis of intrahepatic coagulation factors II, VII, IX, X, etc., and is a factor that promotes the transformation of fibrinogen into fibrin during the coagulation process. Vitamin K2, as a coenzyme of glutamic acid-γ-carboxylase, participates in the normal blood coagulation process of the body. [0003] In particular, vitamin K1 deficiency can cause blood coagulation disorders in humans, resulting in hypothrombin, vitamin K1 deficiency, and neonatal spontaneous hemorrhage. And studies have shown that excessive vitamin K1 in the human body can lead to serious adverse reactions, inclu...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/72G01N30/04G01N30/06G01N30/86G01N30/88
CPCG01N30/02G01N30/04G01N30/06G01N30/72G01N30/8634G01N30/88G01N2030/045G01N2030/062G01N2030/8822
Inventor 许丽雒琴贾永娟倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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