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Method for simultaneously detecting contents of leucine, isoleucine and valine in blood

A technology of isoleucine and leucine, applied in the field of clinical chemistry, can solve the problems of complex, cumbersome and time-consuming processing operations, and achieve the effect of simple pretreatment

Pending Publication Date: 2020-02-18
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] It can be seen that the sample pretreatment operation of the existing detection method is complex, cumbersome and time-consuming

Method used

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  • Method for simultaneously detecting contents of leucine, isoleucine and valine in blood
  • Method for simultaneously detecting contents of leucine, isoleucine and valine in blood
  • Method for simultaneously detecting contents of leucine, isoleucine and valine in blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The embodiment of the present invention is used to obtain the standard curve equation.

[0092] 1.1 Preparation of standard stock solution

[0093] Standard stock solution A: Accurately weigh 9.61mg of leucine standard substance and place it in a 5mL volumetric flask, dissolve it in water containing 0.1% formic acid, and set the volume to 5mL to obtain standard stock solution A, and store it at -80°C Save, valid for one year.

[0094] Standard stock solution B: Accurately weigh 16.50 mg of isoleucine standard substance and place it in a 5mL volumetric flask, dissolve it in water containing 0.1% formic acid, and set the volume to 5mL to obtain standard stock solution B, and store at -80°C Save under the following, valid for one year.

[0095] Standard stock solution C: Accurately weigh 10.52mg of valine standard substance and place it in a 5mL volumetric flask, dissolve it in water containing 0.1% formic acid, and set the volume to 5mL to obtain standard stock solution...

Embodiment 2

[0136] The embodiment of the present invention is used to detect the contents of leucine, isoleucine and valine in the blood to be detected.

[0137] 2.1 Obtaining blood samples

[0138] The blood sample is obtained by processing at least 0.2mL of blood to be tested. After the blood sample is obtained, it can be pre-treated to obtain a corresponding sample to be tested that can be directly loaded.

[0139] 2.2 Blood sample pretreatment

[0140] Use a pipette gun to pipette 10 μL of mixed internal standard working solution into a 1.5 mL centrifuge tube, then add 10 μL of blood sample, then add 80 μL of methanol, vortex and mix at 2000 rpm for 3 minutes, then centrifuge at 14000 rpm for 5 minutes at high speed , take 20 μL of supernatant, add 180 μL of water to dilute 10 times, vortex and mix at 2000 rpm for 2 minutes, then pipette 200 μL of supernatant into a clean injection vial, and the pipetted 200 μL of supernatant is the Test samples.

[0141] 2.3 Detection of samples ...

Embodiment 3

[0150] The embodiments of the present invention are used to determine the limit of quantification and limit of detection.

[0151] 1% bovine serum albumin was used to prepare simulated blood samples containing 4.352 μmol / L of leucine, 4.352 μmol / L of isoleucine and 7.68 μmol / L of valine, and then diluted to different degrees, Please refer to Table 3 for the dilution factor, so as to prepare a series of simulated blood samples with different concentrations, and measure these simulated blood samples according to the blood sample pretreatment method and measurement conditions in Example 2.

[0152] Among them, the dilution factor and actual concentration are shown in Table 3.

[0153] After testing, it was found that the detection limit and quantification limit of leucine, isoleucine and valine are as follows:

[0154] Leucine

[0155] (1) Limit of detection (LOD): 0.054 μmol / L, S / N=7.

[0156] (2) Limit of quantification (LOQ): 0.070 μmol / L, S / N=10.

[0157] Isoleucine

[0...

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Abstract

The invention provides a method for simultaneously detecting contents of leucine, isoleucine and valine in blood. The method comprises the following steps: respectively detecting at least three standard solutions by using a high performance liquid chromatography tandem mass spectrometer and a PFP chromatographic column to obtain a chromatogram of each standard solution containing a standard substance and an internal standard substance of each branched chain amino acid with the known concentration; carrying out fitting based on the chromatogram of each standard solution to obtain a standard curve equation of each branched chain amino acid; adding the mixed internal standard working solution into a blood sample obtained by treating at least 0.2 mL of to-be-detected blood, adding a protein precipitation reagent, carrying out vortex mixing, carrying out centrifuging to obtain a supernatant, diluting the supernatant, carrying out vortex mixing again, and then taking the supernatant to obtain a to-be-detected sample; detecting the to-be-detected sample to obtain a chromatogram; and then calculating the contents of leucine, isoleucine and valine in the blood sample according to the chromatogram of the to-be-detected sample and each standard curve equation. The sample pretreatment is simple; the time is saved; and the operation becomes rapid.

Description

technical field [0001] The invention relates to the technical field of clinical chemistry, in particular to a method for simultaneously detecting the contents of leucine, isoleucine and valine in blood. Background technique [0002] Leucine, isoleucine and valine are called Branched-chain Amino Acids (BCAA) because they all contain branched chains in their molecular composition. Branched-chain amino acids, as essential amino acids in the human body, are not only raw materials for the synthesis of body proteins, but also have special physiological and biological functions. [0003] As an important nutrient in the body, branched-chain amino acids not only affect protein synthesis and decomposition, but also have unique anti-fatigue, reduce myocardial and peripheral muscle tissue damage, enhance the body's immune protection, and improve exercise capacity. An important amino acid involved in energy supply during long-term continuous exercise. When the content of branched-chain...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/86G01N30/06G01N30/34G01N30/72
CPCG01N30/06G01N30/34G01N30/7266G01N30/8624G01N30/88G01N2030/8818G01N2030/8822
Inventor 杨缓缓贾永娟倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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