Preparation method and detection method of TMAO biological negative sample and biological standard substance

A biological sample and detection method technology, applied in the detection field, can solve the problems of stimulation, severe reaction process, negative samples cannot pass, etc., achieve the effect of mild and controllable reaction process, accurate quantitative detection process, and batch preparation

Active Publication Date: 2020-09-22
CHANGSHA DUXACT BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some studies have shown that samples with low TMAO values ​​can be found. However, due to the limited number of such samples and the complicated search process, the practicability is not strong
Chinese invention patent application document CN109387410A discloses a TMAO-negative sample and its preparation method and application. It specifically discloses that TMAO contained in the sample can be removed by adding Reynolds salt to the biological sample. Although this method can eliminate the TMAO in the blank matrix For the purpose of TMAO background interference, there are obvious defects in the reaction of chemical complexing agents, such as: 1. The reaction process is relatively violent, and a large amount of precipitation will be produced after the reaction, and these

Method used

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  • Preparation method and detection method of TMAO biological negative sample and biological standard substance
  • Preparation method and detection method of TMAO biological negative sample and biological standard substance
  • Preparation method and detection method of TMAO biological negative sample and biological standard substance

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Experimental program
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Example Embodiment

[0033] The first embodiment of the present invention is: a method for preparing a biologically negative sample for TMAO detection, including the following steps:

[0034] 1.1 Biological sample collection: clinically collect whole blood samples of 22 Chinese healthy people (tested for related pathogenic microorganisms, no related infections). Take 5mL of blood from each sample. After separating the plasma, get 1~2mL of plasma. The plasma of the sample is mixed to obtain 40 mL of the biological sample mixture.

[0035] 1.2 Standard curve drawing: the methanol aqueous solution is equipped with different gradient verification standard solutions with TMAO concentrations of 2, 5, 10, 20, 50, 100, 200 and 500 ng / mL, and the precipitation agent acetonitrile and internal standard TMAO-D are added 9 Afterwards, HPLC-MS detects the content and draws a TMAO verification standard curve. The volume percentage of methanol in the methanol aqueous solution is 40% (30-60% is acceptable).

[0036] 1.3...

Example Embodiment

[0070] The second embodiment of the present invention is an application of TMAO bio-negative samples:

[0071] 2.1 Drawing a quantitative standard curve: Take an appropriate amount of the biologically negative sample prepared in Example 1, and add TMAO to prepare a quantitative standard solution containing TMAO of different concentrations (2, 5, 10, 20, 50, 100, 500 ng / mL). In addition to the different content of TMAO in each standard solution, the content of other components is the same), TMAO and internal standard substances in each standard solution detected by HPLC-MS. Take the sample of the 1.4 part of Group1 group enzyme reaction in Example 1 after 12 minutes and add the internal standard substance for HPLC-MS test. The test results of TMAO and internal standard substance are as follows figure 2 with 3 As shown, figure 2 Is the spectrum of TMAO in the sample, the result of the internal standard is as image 3 Shown. Take the ratio of the chromatographic peak area of ​​TMA...

Example Embodiment

[0075] The quantitative standard curve equation of the negative sample in Example 1 is Y=0.417x+0.0002, r 2 Is 0.9999. It shows that when the biologically negative samples prepared by the method of the present invention are used for the quantitative detection of TMAO at a concentration of 2 to 500 ng / mL, the linear correlation is good, which indicates that the negative samples prepared by the method of the present invention can be used for quantitative detection. Wide linear range and lower sensitivity.

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Abstract

The invention discloses a preparation method and a detection method of a TMAO biological negative sample and a biological standard substance. The method comprises the following steps: taking a biological sample, adding a biological enzyme to act on TMAO so as to reduce or eliminate the content of TMAO in the biological sample, and terminating the reaction between the biological enzyme and TMAO soas to obtain the biological negative sample. The method comprises the following steps: preparing a TMAO biological negative sample by using a biological enzyme, so that the endogenous TMAO backgroundinterference can be effectively eliminated, the reaction specificity is strong, the efficiency is high, the operation is convenient, and the economic benefit is high; by means of the scheme, endogenous TMAO background interference can be effectively eliminated, an enzyme reaction is ingeniously utilized, oxidative TMAO is reduced, metabolized and decomposed or groups are added to change structuralmodification, and therefore background interference caused by TMAO in a blank matrix is eliminated; and excessive bio-enzyme can be removed by subsequently inactivating or filtering the bio-enzyme, so that the bio-enzyme can be further applied to preparation of TMAO standard substances.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a preparation method and a detection method of a TMAO biological negative sample and a biological standard. Background technique [0002] Trimethylamine oxide (Trimetlyl amine oxide, TMAO) is a product produced by the metabolism of alkaloid components such as choline or carnitine through intestinal flora. Related studies have shown that TMAO is closely related to the occurrence and development of various cardiovascular diseases, such as atherosclerosis, heart failure and hypertension. Therefore, it is of great significance to detect TMAO. When establishing a TMAO detection method, it is necessary to use human plasma, urine, etc. as a blank matrix. However, almost all human plasma contains TMAO. Therefore, it is difficult to obtain a blank biological matrix during method validation. Blank biological matrices such as water, bovine serum albumin solution, phosphate buffer saline...

Claims

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Application Information

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IPC IPC(8): G01N33/49G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N1/28G01N1/38
CPCG01N1/28G01N1/38G01N30/02G01N30/06G01N30/30G01N30/32G01N30/34G01N30/72G01N33/49G01N2030/324
Inventor 陈露露欧阳冬生谢秀芬李超鹏侯利平李晓晖
Owner CHANGSHA DUXACT BIOTECH CO LTD
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