CN111693686A - Preparation method and detection method of TMAO biological negative sample and biological standard substance - Google Patents

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

Info

Publication number
CN111693686A
CN111693686A CN202010465570.3A CN202010465570A CN111693686A CN 111693686 A CN111693686 A CN 111693686A CN 202010465570 A CN202010465570 A CN 202010465570A CN 111693686 A CN111693686 A CN 111693686A
Authority
CN
China
Prior art keywords
tmao
biological
sample
negative sample
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010465570.3A
Other languages
Chinese (zh)
Other versions
CN111693686B (en
Inventor
陈露露
欧阳冬生
谢秀芬
李超鹏
侯利平
李晓晖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Changsha Duxact Biotech Co ltd
Original Assignee
Changsha Duxact Biotech Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Changsha Duxact Biotech Co ltd filed Critical Changsha Duxact Biotech Co ltd
Priority to CN202010465570.3A priority Critical patent/CN111693686B/en
Publication of CN111693686A publication Critical patent/CN111693686A/en
Application granted granted Critical
Publication of CN111693686B publication Critical patent/CN111693686B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Ecology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a preparation method and a detection method of a TMAO biological negative sample and a biological standard substance, which comprises the following steps: and (3) taking a biological sample, adding a biological enzyme to act on the TMAO, so as to reduce or eliminate the content of the TMAO in the biological sample, and obtaining the biological negative sample after stopping the reaction between the biological enzyme and the TMAO. According to the method, biological enzyme is used for preparing the TMAO biological negative sample, so that endogenous TMAO background interference can be effectively eliminated, the reaction specificity is strong, the efficiency is high, the operation is convenient, and the economic benefit is high; and subsequently, the removal of the excessive biological enzyme can be realized through inactivation or filtration of the biological enzyme, so that the method can be further applied to the preparation of the TMAO standard product.

Description

Preparation method and detection method of TMAO biological negative sample and biological standard substance
Technical Field
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 substance.
Background
Trimethylamine oxide (TMAO) is a product produced by metabolizing alkaloid components such as choline or carnitine by intestinal flora. Relevant research shows that TMAO is closely related to the occurrence and development of various cardiovascular diseases, such as atherosclerosis, heart failure, hypertension and the like. Therefore, detection of TMAO is of great significance. When the TMAO detection method is established, human plasma, urine and the like are required to be used as blank matrixes, however, almost all human plasma contains TMAO, and therefore, the acquisition of white biological matrixes becomes a difficulty in methodological verification. In the traditional method, blank biological matrixes such as water, bovine serum albumin liquid, phosphate buffer, human serum albumin and the like are generally adopted. The blank biological matrix and the matrix of the sample to be detected have far component difference, so that the methodological verification persuasion is not high, and particularly, the explanation requirements on the matrix effect in the 'verification guiding principle of the quantitative analysis method of the biological sample' in pharmacopoeia regulations cannot be met, so that the reliability of the obtained detection result is poor.
Because the methylamine oxide is oxidizing and can be reduced into trimethylamine, the methylamine oxide can be reduced into the trimethylamine or other substances by adding a specific reducing agent by utilizing the redox property of the methylamine oxide, thereby eliminating TMAO background interference in a blank matrix. Some studies show that low TMAO value samples can be searched, however, the samples are limited in number and complicated in searching process, and therefore, the practicability is not strong. Chinese patent application CN109387410A discloses a TMAO negative sample and its preparation method and application, specifically discloses that adding raney salt into a biological sample to remove TMAO contained in the sample, although this method can achieve the purpose of eliminating TMAO background interference in blank matrix, there are obvious defects in using chemical complexing agent reaction, such as: 1. the reaction process is violent, a large amount of precipitates are generated after the reaction, and the precipitates can influence other components in the sample; 2. because the TMAO content in the biological sample is relatively low, the added reducing agent is easy to be excessive, and the excessive reducing agent is difficult to be completely removed or just completely reacted, the obtained negative sample cannot be added with TMAO in the later period to prepare the TMAO biological standard substance; 3. the Lee's salt is also highly toxic and is very easy to be inserted into eyes or adhered to skin to generate strong stimulation; the sample after reaction is extremely harmful to the water, even small-volume products can not contact underground water, water course or sewage system, therefore all there is very big potential safety hazard to experiment operating personnel and environment.
Therefore, the development of a preparation method of the biological negative sample which is efficient and safe, can eliminate the background interference of endogenous TMAO and is convenient for post-treatment is of great significance.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a preparation method of TMAO biological negative sample, and the biological negative sample prepared by the method can efficiently and safely eliminate endogenous TMAO background interference and is convenient for post-treatment.
The invention also provides a preparation method of the TMAO biological standard substance.
The invention also provides a TMAO detection method.
A method according to an embodiment of the first aspect of the invention, comprising the steps of:
and (3) taking a biological sample, adding a biological enzyme to act on the TMAO, so as to reduce or eliminate the content of the TMAO in the biological sample, and obtaining the biological negative sample after stopping the reaction between the biological enzyme and the TMAO.
According to some embodiments of the invention, the biological enzyme includes, but is not limited to, at least one of TMAO reductase (trimethyamine-N-oxide reductase), TMAO demethylase (trimethyamine oxide dehydrogenase), TMAO aldolase (trimethyamine oxide aldolase), TMAO formaldehyde lyase (trimethyamine oxide formaldehyde lyase).
According to some embodiments of the present invention, the reaction between the biological enzyme and TMAO is terminated by adding an enzyme inhibitor, adding an enzyme inactivator, changing the temperature of the reaction system (to a temperature above the effective temperature of the enzymatic reaction) or pH (e.g., adding a strong acid or strong base reagent to break the pH of the enzymatic reaction, about 1/10 of the reaction system, HCl, NaOH, or perchloric acid, etc.).
According to some embodiments of the invention, if included, the enzyme inhibitor is selected from at least one of a TMAO reductase inhibitor, a TMAO demethylase inhibitor, a TMAO aldolase inhibitor, a TMAO formaldehyde lyase inhibitor; if the enzyme inactivator is contained, the enzyme inactivator is a non-specific reagent for terminating the enzymatic reaction, and can be selected from at least one of methanol, acetonitrile or trichloroacetic acid (TCA) (the volume ratio of the addition amount to the solution of the reaction system is 1: 5-1: 1).
According to some embodiments of the invention, if included, the enzyme inactivator inhibitor is not less than a molar amount of the biological enzyme.
According to some embodiments of the invention, the amount of the biological enzyme added per milliliter of the biological sample is 1-10 ng; preferably 2-8 ng; more preferably 4 to 6 ng.
The preparation method according to the embodiment of the invention has at least the following beneficial effects: according to the method, the biological negative TMAO sample is prepared by using the biological enzyme, so that the endogenous TMAO background interference can be effectively eliminated, the reaction specificity is strong, the efficiency is high, the operation is convenient, the economic benefit is high, and the endogenous TMAO background interference can be effectively eliminated by using the scheme provided by the invention. The enzymatic reaction is skillfully utilized to reduce, metabolize and decompose TMAO with oxidability or add groups to change the structure modification, thereby eliminating background interference caused by TMAO in a blank matrix; and subsequently, the removal of the excessive biological enzyme can be realized through inactivation or filtration of the biological enzyme, so that the method can be further applied to the preparation of the TMAO standard product. By the scheme, the preparation of samples in large batch can be realized, and the defects of high difficulty in searching low-concentration TMAO and extremely small sample amount in the prior art are overcome; the technical scheme of the invention has ingenious design and strong practicability, the whole process does not need expensive instruments or complex operation processes, the rapid and simple TMAO negative sample preparation can be realized, the concentrated addition of biological enzymes after the mixing of a plurality of biological samples can be realized for unified treatment, the TMAO is eliminated, and compared with the method for searching samples with low TMAO values one by one, the method is more convenient and economic and can realize batch preparation.
The preparation method according to the second aspect embodiment of the present invention comprises the steps of:
s01, preparing a biological negative sample by adopting the method;
s02, TMAO is quantitatively added to the biological negative sample prepared in the step S01.
According to some embodiments of the present invention, the final concentration of the TMAO added in step S02 is between 1 ng/mL and 1000 ng/mL; preferably, the final concentration of the TMAO is between 2 and 500 ng/mL; more preferably, the final concentration of TMAO is selected from at least one of 2, 5, 10, 20, 50, 100, 200 and 500 ng/mL.
According to some embodiments of the present invention, the TMAO may be added in solid form or in solution form, and if in solution form, the solution may be prepared with a biological negative sample as a solvent or diluent.
The preparation method according to the embodiment of the invention has at least the following beneficial effects: the biological enzyme is utilized to prepare the negative sample, so that not only is the reaction process milder and controllable, but also the prepared negative sample can be further made into a biological standard product by inactivating the biological enzyme, so that the subsequent quantitative detection process is more accurate; the negative sample obtained after TMAO is directly removed by the biological sample, a series of trimethylamine oxide standard solutions are added, and when the sample to be detected is homologous with the sample to be detected, the matrix effect caused by the standard solution prepared by adopting buffer solution or other solvents in the prior art can be greatly reduced, and meanwhile, the defect that the detection result is interfered by the TMAO carried by other samples when other samples are adopted as negative quality control products or standard solution solvents can be avoided.
The detection method according to the third aspect embodiment of the present invention includes the steps of:
I. preparing a biological negative sample by adopting the method;
II. And (4) detecting the signal of the TMAO in the sample to be detected by taking the signal of the biological negative sample prepared in the step I as a background signal.
According to some embodiments of the present invention, the detection method is a quantitative detection method, a biological negative sample is added with a quantitative TMAO to prepare a biological standard in the detection process, the signal intensity of the TMAO in the biological standard is characterized, the TMAO signal intensity (which may be a TMAO detection method known in the art such as optical intensity, mass spectrum signal intensity or chromatographic signal intensity) at different concentrations is measured, and the TMAO in the sample to be detected is quantitatively analyzed.
The detection method provided by the embodiment of the invention has at least the following beneficial effects: the detection method of the invention does not aim at disease diagnosis and treatment, and the detection process directly prepares a biological negative sample from a homologous sample, thereby avoiding unreliable detection results caused by matrix effect when other simulation samples are used as negative quality control products or standard liquid solvents in the traditional technology; the detection method can realize quantitative detection and qualitative detection; the biological sample can be reduced, decomposed or modified by adding enzyme, so that TMAO in the biological sample is removed, and the biological sample without TMAO or the biological sample with residual TMAO which is negligible when used as negative sample contrast is obtained, thereby meeting the requirement of the invention. The enzyme added in the scheme of the invention has mild reaction conditions with the biological sample, high reaction efficiency and small damage degree to the biological sample, so that the matrix effect background of the obtained biological negative sample can be subtracted to the maximum extent. The enzyme has high specificity, does not affect other substances in the biological sample, does not generate other impurities, and simultaneously, a product after the reaction with the TMAO can be effectively separated from the TMAO through the optimization of detection conditions, thereby not causing interference to the quantitative detection of the TMAO. In addition, the action mechanism of the reaction of the enzyme and the TMAO is not limited to reduction or decomposition of the TMAO, and a new substance can be generated by modifying the TMAO, so that the TMAO can be separated from the TMAO in the detection process, the effect that the TMAO cannot be detected in a negative sample is achieved, and when the mechanism of treating the TMAO by the enzyme is different, a plurality of enzymes can be added simultaneously to perform synergistic reaction, so that the reaction effect is improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a graph showing the change of TMAO concentration after different reaction times in example 1 of the present invention;
FIG. 2 is a chromatogram of TMAO in a sample after a bio-enzyme reaction detected in example 2 of the present invention;
FIG. 3 is a chromatogram of an internal standard detected in example 2 of the present invention;
FIG. 4 is a standard curve measured in example 2 of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
The first embodiment of the invention is as follows: a method for preparing a biological negative sample for TMAO detection comprises the following steps:
1.1 biological sample collection: clinically collecting whole blood samples (subjected to relevant pathogenic microorganism detection and free of relevant infection) of 22 Chinese healthy people, taking 5mL of blood from each sample, separating plasma to obtain 1-2 mL of plasma, and uniformly mixing the plasma of each sample to obtain 40mL of a biological sample mixture.
1.2 standard curve drawing: preparing different gradient verification standard solutions with TMAO concentrations of 2, 5, 10, 20, 50, 100, 200 and 500ng/mL with methanol water solution, adding precipitant acetonitrile and internal standard TMAO-D9And detecting the content by HPLC-MS, and drawing a TMAO (TMAO validation standard) curve. Wherein the volume percentage of methanol in the methanol water solution is 40 percent (30-60 percent).
1.3 biological sample TMAO assay: the amount of TMAO in the biological sample mixture obtained in 1.1 was quantitatively determined, and the average value was obtained by repeating 10 times (the amount of each sample was 200. mu.L).
1.4 different in vitro incubation systems were prepared in groups as follows:
first Group (Group 1): preparing TMAO reductase enzymatic reaction buffer solution, accurately weighing 12.80g of sodium chloride, 1.54g of sodium acetate (dried), 0.127g of ascorbic acid, 0.036g of cysteine, 0.0038g of ferric chloride and 0.005g of methylene blue, dissolving the mixture to 290mL by using 1.46% (w/v) Triton X-100, and adjusting the pH value to 4.5 by using acetic acid. Adding 10mL of TMAO reductase extract (dissolved in a solution containing 0.1mol/L NaCl and 20mmol/L Tris-acetic acid) with a concentration of 0.05mg/mL, and mixing well.
② first Group control Group (Group 1-Con): preparing blank TMAO reductase enzymatic reaction buffer solution, accurately weighing 12.80g of sodium chloride, 1.54g of sodium acetate (dried), 0.127g of ascorbic acid, 0.036g of cysteine, 0.0038g of ferric chloride and 0.005g of methylene blue, dissolving the mixture to 290mL by using 1.46% (w/v) Triton X-100, and adjusting the pH value to 4.5 by using acetic acid. Adding 10mL solution containing 0.1mol/L NaCl and 20mmol/L Tris-acetic acid, and mixing well.
Third Group (Group 2): preparing TMAO demethylase enzymatic reaction buffer solution, precisely weighing 4.36g of Tris base, 0.087g of cysteine, 0.127g of ascorbic acid and 0.0143g of ferric chloride, adding double distilled water to dissolve the mixture to 290mL, and adjusting the pH value to 7.0 by acetic acid. 10mL of TMAO demethylase extract (dissolved in a solution containing 0.1mol/L NaCl, 20mmol/L of LTris-acetic acid) was added at a concentration of 0.05 mg/mL. Mixing well.
Fourth Group control Group (Group 2-Con): preparing blank TMAO demethylase enzymatic reaction buffer solution, precisely weighing 4.36g of Tris base, 0.087g of cysteine, 0.127g of VC and 0.0143g of iron dichloride, adding double distilled water to dissolve to 290mL, and adjusting the pH value to 7.0 by acetic acid. Add 10mL of a solution containing 0.1mol/L NaCl and 20mmol/L Tris-acetic acid and mix well.
Fifth Group (Group 3): preparing TMAO aldolase enzymatic reaction buffer solution, precisely weighing 4.36g of Tris alkali, 2.46g of sodium acetate (dried), 0.127g of ascorbic acid, 0.073g of cysteine and 0.0076g of ferric chloride, adding double distilled water to dissolve the mixture to 290mL, and adjusting the pH value to 7.0 by acetic acid. TMAO aldolase extract (dissolved in a solution containing 0.1mol/L NaCl, 20mmol/L Tris-acetic acid) was added at a concentration of 0.05mg/mL for 10 mL. Mixing well.
Sixthly, a third Group control Group (Group 3-Con): precisely weighing 4.36g of Tris base, 2.46g of sodium acetate (dried), 0.127g of ascorbic acid, 0.073g of cysteine and 0.0076g of iron dichloride, adding double distilled water to dissolve the mixture to 290mL, and adjusting the pH value to 7.0 by acetic acid. 10mL of a solution containing 0.1mol/L NaCl and 20mmol/L Tris-acetic acid was added thereto, and the mixture was thoroughly mixed.
1.5 reaction:
carrying out in-vitro enzyme incubation reaction: taking 6 parts of 5mL of 1.3 mixed sample, respectively adding 500 mu L of Group1, Group 1-Con, Group 2-Con, Group 3 and Group 3-Con enzymatic reaction solution, placing the mixture in a water bath at 50 ℃ for reaction in a warm bath, shaking the reaction solution up and down in 1min intervals, and sampling 200 mu L of the reaction solution at 2min intervals for TMAO content determination.
② fourth Group Leeb salt control (Group 4): taking 5mL of 1.3 mixed sample, adding 0.5mL of Rayleigh salt solution (the concentration is determined according to the analysis result of 1.3, the molar ratio of Rayleigh salt to TMAO is not lower than 1: 1), adjusting the pH value to 1 by using concentrated hydrochloric acid, and reacting at 4 ℃. The reaction solution was shaken up and down once every 1min, and 200. mu.L of the reaction solution was sampled every 2min for TMAO content determination.
1.6 termination reaction: group1, Group 1-Con, Group 2-Con, Group 3-Con, and 500. mu.L of 10% trichloroacetic acid (TCA) was added to the sample to terminate the reaction.
1.7 detection of TMAO content in plasma samples: 200 μ L of reacted plasma sample was takenAdding internal standard TMAO-D9Centrifuging at 4 deg.C and 4000rpm for 20min, collecting supernatant, directly performing HPLC-MS detection, and calculating TMAO content in the sample according to the standard curve.
If the TMAO content in the plasma sample is higher than 1.6ng/mL, the TMAO in the sample is not completely reacted, and at the moment, the sample reaction time needs to be prolonged and then the sampling detection is continued until the TMAO content is equal to or lower than 1.6 ng/mL.
1.8 negative sample preparation: the reaction sample which meets the requirement of 1.7 is the negative sample.
1.9TMAO quantitative standard solution preparation: and 1.7, adding TMAO with different concentrations to prepare TMAO quantitative standard solution with the concentration range of 2-500ng/mL (or diluting the negative sample prepared by 1.7 into TMAO quantitative standard solution with the concentration range of 2-500ng/mL after adding high-concentration TMAO to prepare high-concentration TMAO solution).
The equipment and parameter information adopted in the embodiment is as follows:
1) apparatus and device
Waters TQ-D mass spectrometry system; waters UPLC I-Class liquid phase system; waters UNIFI software; thermo Fisher Thermo ST16R model high speed refrigerated centrifuge (semer fly, usa); XW-80A vortex mixer (Ningbo Xinzhi Biotech Co., Ltd., China); pipettes (Gilson, usa). An electric heating constant temperature water tank (Shanghai sperm macro experimental facilities Co., Ltd., China).
2) Reagent and material
Trimethylamine oxide: tokyo chemical industry co., ltd, purity 99.5%, lot number: 3 EVJG-MC; deuterated trimethylamine oxide (TMAO-D)9): toronto Research Chemicals, purity 98%, batch number: 3-LXM-155-1; methanol: HPLC grade, Merck, lot number: 1.06007.4008, respectively; ammonium acetate: analytically pure, national medicine, batch number: 20160310, respectively; acetonitrile: gradient for liquid chromatography, Merck; formic acid: LC grade, ACS.
3) Conditions for liquid chromatography and mass spectrometry
Chromatographic conditions
A chromatographic column: ACQUITYBEH C18(2.1×50mm, 1.7 μm); mobile phase: 10mM ammonium acetate aqueous solution: 0.1% formic acid in acetonitrile by volume 80:20 (v/v); the flow rate is 0.2 mL/min; column temperature: 30 ℃; sample chamber temperature: 10 ℃; sample introduction amount: 3 mu L of the solution; needle washing liquid: 80% methanol water.
Conditions of Mass Spectrometry
Detecting in positive ion MRM mode by using electrospray ion source (ESI); trimethylamine oxide: monitor ion m/z 75.99 → m/z 58.6; taper hole voltage: 18V, collision energy: 12 eV; Oxetamine-D9: monitor ion m/z 85.1 → m/z 68, cone voltage: 18V, collision energy: 13 eV; capillary voltage: 0.5 kV; taper hole airflow: 60L/h; desolventizing the gas stream: 1000L/h; ion source temperature: 150 ℃; the temperature of the desolvation: at 500 ℃.
The results of this example were analyzed as follows:
the average TMAO concentration of the mixture measured in the step 1.3 of this example is 135.20ng/mL, and after the above steps, the TMAO content finally measured in each group 1.6 is shown in Table 1 and FIG. 1:
TABLE 1
Figure BDA0002512527050000081
According to the experimental results, the consumption rates of TMAO in the groups 1-3 are obviously higher than those in the Group 4, which indicates that the TMAO consumption efficiency in the enzymatic reaction is higher than that in the Rayleigh salt precipitation reaction. Wherein, the groups 2 and 3 are lower than the lower detection limit when the reaction is carried out for 10 minutes; the groups 1 and 4 reached the lower limit of detection by the time the reaction proceeded to the 12 th minute. Reaction was terminated by the time minute 12, when the concentration of TMAO in all groups was below the lower limit of detection. TMAO in the Group 1-3-Con Group has no significant change, so that the enzyme in the in vitro enzyme reaction system plays a main role in consuming TMAO.
After the experiment is finished, obtaining 3.5mL of TMAO negative samples from the groups 1-3 respectively; in group 5, a large amount of precipitate is generated due to the direct reaction of TMAO and Rayleigh salt, and only 2.8mL of TMAO negative sample is obtained.
In addition, the ledeburite also has great toxicity compared with biological enzymes, and the ledeburite is easy to enter eyes or adhere to skin to generate strong stimulation; the sample after the reaction of the raney salt is extremely harmful to the water body, and even a small amount of products cannot contact underground water, a water channel or a sewage system. The preparation of TMAO invisible samples by using biological enzymes is also superior to the Lee's salt precipitation method from the viewpoint of protecting operators and environment.
In conclusion, the scheme of the embodiment of the invention can quickly prepare a large amount of TMAO negative samples, and the method is obviously superior to the Reye salt precipitation method in terms of reaction speed, preparation efficiency and environmental protection.
The second embodiment of the invention is an application of TMAO biological negative samples:
2.1 drawing of quantitative standard curve: taking a proper amount of the biological negative sample prepared in example 1, adding TMAO to prepare quantitative standard solutions (except different TMAO content, the content of other components is consistent) containing TMAO with different concentrations (2, 5, 10, 20, 50, 100, 500ng/mL), and detecting TMAO and internal standard substance in each standard solution by HPLC-MS. And (3) adding an internal standard substance into a sample obtained after the 1.4 part of Group1 Group enzyme in example 1 is subjected to 12min of enzyme reaction, and performing HPLC-MS test, wherein the detection results of the TMAO and the internal standard substance are shown in figures 2 and 3, the figure 2 is a map of the TMAO in the sample, and the result of the internal standard substance is shown in figure 3. And (3) taking the ratio of the chromatographic peak area of the TMAO in each standard solution prepared from the negative sample after the Group1 Group enzyme reaction to the chromatographic peak area of the internal standard substance as a vertical coordinate, and taking the ratio of the concentration of the TMAO in the corresponding standard solution to the concentration of the internal standard substance as a horizontal coordinate to draw a standard curve, as shown in FIG. 4.
2.2 method accuracy verification: biological negative samples were prepared by repeating the procedure of example 1, and TMAO series samples were prepared at concentrations of 2, 5, 10, 20, 50, 100, 200 and 500ng/mL (sequentially labeled as cure 01 to cure 08), and low, medium and high quality control samples (sequentially labeled as Q1, Q2, Q3) at concentrations of 6, 75 and 400 ng/mL. Quantitative analysis of TMAO content of the system sample was performed according to quantitative standard curves obtained by plotting samples of example 1 in section 2.1, respectively, to verify the accuracy of the method.
2.3 analysis of results:
quantitative standard curve linear relationship:
example 1 the quantitative standard curve equation for negative samples is Y ═ 0.417x +0.0002, r2Is 0.9999. The biological negative sample prepared by the scheme of the invention has good linear correlation when used for quantitative detection of TMAO concentration at 2-500ng/mL, thereby showing that the negative sample prepared by the scheme of the invention can obtain wider linear range and lower sensitivity when used for quantitative detection.
And (3) testing accuracy:
TMAO with known concentration is added into the negative sample prepared by the 2.2, and quantitative analysis is carried out by combining with the quantitative standard curve of the 2.1 sample, so as to verify the stability and reliability of the scheme of the invention, and the data is shown in the following table 2:
table 2 accuracy testing experiment data table
Figure BDA0002512527050000091
Figure BDA0002512527050000101
As can be seen from Table 2, the accuracy is between 95% and 105%, and the error falls within a reasonable error range, so that the result of TMAO quantitative analysis of the biological negative sample prepared by the method for detecting TMAO is accurate and reliable.
The working curves obtained by other sets of measurements have similar effects, and are not described in detail herein to avoid redundancy.
In conclusion, the embodiment of the invention utilizes the specificity and high efficiency of the biological enzyme metabolism, and can selectively act on the TMAO to decompose the TMAO in a metabolic mode, thereby eliminating the TMAO background in the blank matrix, realizing accurate detection of the TMAO in the biological sample, and having simple and convenient preparation process. The enzyme reaction condition is mild and easy to inactivate, so that the influence on the biological sample can be reduced to the maximum extent. The enzyme can be added in one or more of the forms of dry powder, precipitation, solution and the like under the synergistic action, so that the operation modes are flexible and various. The scheme of the invention has the advantages of ingenious design, specificity, high efficiency, simple and convenient operation and strong practicability. In addition, the scheme of the invention utilizes the specific action of the biological enzyme on the TMAO reaction to decompose or metabolize the TMAO into other substances, thereby reducing or eliminating the content of the TMAO in the biological sample, and the TMAO negative sample can be prepared by adding an enzyme inhibitor or an enzyme inactivator to stop the enzymatic reaction. Expensive instrument equipment and complex operation processes are not needed, and the rapid, simple and convenient TMAO negative sample preparation can be realized; the method can directly mix a plurality of biological samples and then intensively add the biological enzyme for uniform treatment to eliminate the TMAO, compared with the method of searching samples with low TMAO value one by one, the method of the invention has the advantages of rapidness, convenience and economy, and can be prepared in large batch.
In the above embodiments, blood is used as a sample source, and other body fluids may be used as a sample source, which also has similar effects to the above samples.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims (9)

1. A preparation method of TMAO biological negative sample is characterized in that: the method comprises the following steps:
and (3) taking a biological sample, adding a biological enzyme to act on the TMAO, so as to reduce or eliminate the content of the TMAO in the biological sample, and obtaining the biological negative sample after stopping the reaction between the biological enzyme and the TMAO.
2. The method for preparing TMAO biological negative sample according to claim 1, wherein: the biological enzyme includes but is not limited to at least one of TMAO reductase, TMAO demethylase, TMAO aldolase, and TMAO formaldehyde lyase.
3. The method for preparing TMAO biological negative sample according to claim 1, wherein: the reaction between the biological enzyme and the TMAO is stopped by adding an enzyme inhibitor, adding an enzyme inactivator and changing the temperature or the pH value of a reaction system.
4. The method for preparing TMAO biological negative sample according to claim 3, wherein: if contained, the enzyme inhibitor is at least one selected from TMAO reductase inhibitor, TMAO demethylase inhibitor, TMAO aldolase inhibitor and TMAO formaldehyde lyase inhibitor.
5. The method for preparing TMAO biological negative sample according to claim 1, wherein: the addition amount of the biological enzyme in each milliliter of biological sample is 1-10 ng; preferably 2-8 ng; more preferably 4 to 6 ng.
6. A preparation method of TMAO biological standard substance is characterized in that: the method comprises the following steps:
s01, preparing a biological negative sample by the method of any one of claims 1 to 5;
s02, TMAO is quantitatively added to the biological negative sample prepared in the step S01.
7. The method for preparing TMAO biological standard according to claim 6, wherein: the final concentration of the TMAO added in the step S02 is 1-1000 ng/mL; preferably, the final concentration of TMAO is between 2 ng/mL and 500 ng/mL.
8. A TMAO detection method is characterized in that: the method comprises the following steps:
I. preparing a biological negative sample using the method of any one of claims 1 to 5;
II. And (4) detecting the signal of the TMAO in the sample to be detected by taking the signal of the biological negative sample prepared in the step I as a background signal.
9. The detection method according to claim 8, characterized in that: the detection method is a quantitative detection method, a biological standard substance is prepared by adding quantitative TMAO into a biological negative sample in the detection process, the signal intensity of the TMAO in the biological standard substance is characterized, the TMAO signal intensity under different concentrations is measured, and the TMAO in the sample to be detected is quantitatively analyzed.
CN202010465570.3A 2020-05-28 2020-05-28 Preparation method and detection method of TMAO biological negative sample and biological standard substance Active CN111693686B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010465570.3A CN111693686B (en) 2020-05-28 2020-05-28 Preparation method and detection method of TMAO biological negative sample and biological standard substance

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010465570.3A CN111693686B (en) 2020-05-28 2020-05-28 Preparation method and detection method of TMAO biological negative sample and biological standard substance

Publications (2)

Publication Number Publication Date
CN111693686A true CN111693686A (en) 2020-09-22
CN111693686B CN111693686B (en) 2021-09-14

Family

ID=72478710

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010465570.3A Active CN111693686B (en) 2020-05-28 2020-05-28 Preparation method and detection method of TMAO biological negative sample and biological standard substance

Country Status (1)

Country Link
CN (1) CN111693686B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112485438A (en) * 2020-11-10 2021-03-12 深圳市科曼医疗设备有限公司 Specific protein reaction detection method and device
CN114371239A (en) * 2022-03-22 2022-04-19 长沙都正医学检验有限责任公司 Kit for determining trimethylamine oxide and preparation method and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107099518A (en) * 2017-03-21 2017-08-29 苏州斯奈普生物科技有限公司 A kind of method of removal TAQ archaeal dna polymerase center acid pollutions
CN107340341A (en) * 2017-06-27 2017-11-10 长沙都正生物科技有限责任公司 A kind of TMAO immue quantitative detection reagent box and method
CN108507984A (en) * 2018-02-28 2018-09-07 山东大学第二医院 A kind of method and its application of enzyme process detection trimethylamine oxide TMAO
CN109387410A (en) * 2018-09-27 2019-02-26 长沙都正医学检验有限责任公司 A kind of TMAO negative sample and the preparation method and application thereof
CN110577936A (en) * 2019-08-19 2019-12-17 江南大学 Method for efficiently degrading tetracyclic and sulfonamide antibiotics by laccase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107099518A (en) * 2017-03-21 2017-08-29 苏州斯奈普生物科技有限公司 A kind of method of removal TAQ archaeal dna polymerase center acid pollutions
CN107340341A (en) * 2017-06-27 2017-11-10 长沙都正生物科技有限责任公司 A kind of TMAO immue quantitative detection reagent box and method
CN108507984A (en) * 2018-02-28 2018-09-07 山东大学第二医院 A kind of method and its application of enzyme process detection trimethylamine oxide TMAO
CN109387410A (en) * 2018-09-27 2019-02-26 长沙都正医学检验有限责任公司 A kind of TMAO negative sample and the preparation method and application thereof
CN110577936A (en) * 2019-08-19 2019-12-17 江南大学 Method for efficiently degrading tetracyclic and sulfonamide antibiotics by laccase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
PARKIN KIRK L.等: "Characterization of trimethylamine-N-Oxide(TMAO) demethylase activity from fish muscle microsomes", 《J BIOCHEM》 *
吴士筠 等主编: "《酶工程技术》", 31 December 2009, 华中师范大学出版社 *
宋丹阳 等: "氧化三甲胺酶研究进展", 《食品科学》 *
郭修娟: "氧化三甲胺代谢相关菌的研究", 《中国优秀硕士学位论文全文数据库》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112485438A (en) * 2020-11-10 2021-03-12 深圳市科曼医疗设备有限公司 Specific protein reaction detection method and device
CN114371239A (en) * 2022-03-22 2022-04-19 长沙都正医学检验有限责任公司 Kit for determining trimethylamine oxide and preparation method and application thereof
CN114371239B (en) * 2022-03-22 2022-07-15 长沙都正医学检验有限责任公司 Kit for determining trimethylamine oxide and preparation method and application thereof

Also Published As

Publication number Publication date
CN111693686B (en) 2021-09-14

Similar Documents

Publication Publication Date Title
US6355416B1 (en) Assay for the measurement of DNA synthesis rates
CN111693686B (en) Preparation method and detection method of TMAO biological negative sample and biological standard substance
Hu et al. Urinary analysis of 8-oxo-7, 8-dihydroguanine and 8-oxo-7, 8-dihydro-2′-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction: Study of 8-oxo-7, 8-dihydroguanine stability
Ruiz-Calero et al. Pressure-assisted capillary electrophoresis–electrospray ion trap mass spectrometry for the analysis of heparin depolymerised disaccharides
Yen et al. Quantitative analysis of the DNA adduct N2, 3‐ethenoguanine using liquid chromatography/electrospray ionization mass spectrometry
CN113933419A (en) Method for determining concentration of 5 immunosuppressive agents in human whole blood
CN111398449A (en) Kit for detecting 9 water-soluble vitamins in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology
Antia et al. Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring
CN111458417A (en) Method and kit for combined detection of multiple antibiotics in sample to be detected
Bond The determination of 4-hydroxy-3-methoxyphenylethylene glycol in urine and CSF using gas chromatography
CN116087373B (en) Detection method and pretreatment method for folic acid and 5-methyltetrahydrofolate in erythrocytes
CN111693687B (en) Preparation method of biological negative sample and biological standard substance and TMAO detection method
CN114264741B (en) Method for identifying whether pig-derived heparin is doped with ruminant heparin
Gao et al. Formation of cyclic acylphosphoramidates in mass spectra of N‐monoalkyloxyphosphoryl amino acids using electrospray ionization tandem mass spectrometry
Willems et al. Analysis of benzo [a] pyrene diol epoxide‐DNA adducts by capillary zone electrophoresis‐electrospray ionization‐mass spectrometry in conjunction with sample stacking
CN115586281A (en) System and method for extracting 25-hydroxy vitamin D from serum and combined LC-MSMS (liquid chromatography-metal Mass Spectrometry) detection method
CN111239291B (en) GC-MS-based method for detecting short-chain fatty acids in mouse intestinal-cerebral axis related tissue sample
Aigner-Held et al. Polyamine-pyridoxal Schiff bases in urine.
CN110261519B (en) Liquid chromatography-mass spectrometry analysis method for EGCG
Shrivastaw et al. A new method for spectrophotometric determination of thiomersal in biologicals
CN111198235B (en) Method for detecting content of isosinensetin in plasma
Schmid et al. Radiotracer study of the preatomization behavior of lead in the graphite furnace
CN103698425B (en) A kind of chemical degradation method obtains and detects the method for biogenetic derivation glycosaminoglycan disaccharides
CN112858518A (en) Method for extracting acadesine from urine and detection method
CN111812224A (en) Method for detecting concentration of anti-dementia drug in serum

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB02 Change of applicant information
CB02 Change of applicant information

Address after: Room 601, building c8-c10, jinruilugu science and Technology Park, No.28 Lutian Road, high tech Development Zone, Changsha City, Hunan Province, 410000

Applicant after: Changsha Duzheng Biotechnology Co., Ltd

Address before: 410000 Building C8-C10, Jinrui Lugu Science and Technology Park, 28 Lutian Road, Changsha High-tech Development Zone, Hunan Province

Applicant before: CHANGSHA DUXACT BIOTECH Co.,Ltd.

GR01 Patent grant
GR01 Patent grant