CN113376267A - Method and kit capable of rapidly detecting TMAO biomarker - Google Patents
Method and kit capable of rapidly detecting TMAO biomarker Download PDFInfo
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- CN113376267A CN113376267A CN202110508077.XA CN202110508077A CN113376267A CN 113376267 A CN113376267 A CN 113376267A CN 202110508077 A CN202110508077 A CN 202110508077A CN 113376267 A CN113376267 A CN 113376267A
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- UYPYRKYUKCHHIB-UHFFFAOYSA-N trimethylamine N-oxide Chemical compound C[N+](C)(C)[O-] UYPYRKYUKCHHIB-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000000090 biomarker Substances 0.000 title claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims abstract description 12
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- 239000000243 solution Substances 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000010790 dilution Methods 0.000 claims description 14
- 239000012895 dilution Substances 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 150000002500 ions Chemical class 0.000 claims description 12
- 238000004949 mass spectrometry Methods 0.000 claims description 12
- 230000010355 oscillation Effects 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 238000002203 pretreatment Methods 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 230000006920 protein precipitation Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 54
- 210000002381 plasma Anatomy 0.000 abstract description 33
- 238000001819 mass spectrum Methods 0.000 abstract description 3
- 239000012472 biological sample Substances 0.000 abstract description 2
- 239000012086 standard solution Substances 0.000 description 4
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 4
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 230000002526 effect on cardiovascular system Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000002249 Diabetes Complications Diseases 0.000 description 1
- 206010012655 Diabetic complications Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 108010092282 Trimethylamine dehydrogenase Proteins 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
Classifications
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- 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
- G01N30/62—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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the technical field of biological sample detection, and particularly relates to a method and a kit capable of quickly detecting a TMAO biomarker. The method is based on liquid chromatography-mass spectrometry, and relates to (1) setting liquid chromatography conditions; (2) setting mass spectrum conditions; (3) preparing a standard sample; (4) establishing a standard curve; (5) pretreating blood plasma to be detected; (6) pre-treating plasma sample, detecting and the like. The invention further provides a kit and a using method thereof, which are convenient to operate. According to the method for rapidly detecting the TMAO biomarker, provided by the invention, the sample is reasonably pretreated, and reasonable conditions of liquid chromatography and mass spectrum are optimized, so that a detection result with high accuracy and good stability is obtained; the kit provided by the invention provides the internal standard and the standard sample with accurate concentration, so that the actual operation steps are reduced, and the detection efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of biological sample detection, and particularly relates to a method and a kit capable of quickly detecting a TMAO biomarker.
Background
Trimethylamine oxide (TMAO) is involved in many important biological functions in the human body, and after eating choline-containing foods such as beans and eggs, common people convert to trimethylamine in the large intestine, and the trimethylamine is further converted to trimethylamine oxide by trimethylamine oxidase in the liver. In recent years, much attention has been paid to the research on TMAO, and researchers have associated TMAO concentration with various chronic cardiovascular and cerebrovascular diseases and used TMAO as one of early warning indicators for chronic cardiovascular and cerebrovascular diseases. In addition, the team is working on researching and constructing the action relationship between TMAO and common diseases such as diabetic complications, bladder cancer, kidney cancer, prostate cancer, breast cancer and the like. Therefore, how to rapidly and accurately detect the concentration of TMAO in a sample is the basis for a series of studies in practice.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method and a kit capable of rapidly detecting a TMAO biomarker.
The method for rapidly detecting the TMAO biomarker comprises the following steps:
(S1) establishing a standard curve: taking a blank plasma sample, adding methanol, performing vortex oscillation, then loading into a centrifuge for centrifugal treatment, and taking supernatant and adding water for dilution; then adding TMAO and an internal standard to prepare a series of standard samples containing the internal standard with the same concentration and different TMAO concentrations; testing a series of standard samples by liquid chromatography-mass spectrometry, calculating to obtain a ratio (Y) of a TMAO peak area corresponding to each standard sample to an internal standard peak area and a ratio (X) of a TMAO concentration corresponding to each standard sample to an internal standard concentration, and making a change curve of Y along with X to obtain a standard curve;
(S2) pretreatment sample detection: detecting the pretreated plasma sample by liquid chromatography-mass spectrometry, calculating to obtain the ratio of the TMAO peak area to the internal standard peak area, substituting into a standard curve to obtain the ratio of the TMAO concentration in the pretreated sample to the internal standard concentration; calculating the concentration of TMAO in the original plasma sample according to the dilution times in the pretreatment process;
the plasma sample pretreatment method comprises the following steps: and adding a methanol solution containing an internal standard into a plasma sample, performing vortex oscillation, then loading into a centrifuge for centrifugal treatment, and adding water into a certain amount of supernatant for dilution to obtain a pretreated plasma sample.
Further, the plasma sample pretreatment method comprises the following steps: adding the plasma sample into methanol solution containing internal standard for protein precipitation, centrifuging to separate the sample, and diluting with water.
Furthermore, the plasma sample pretreatment method comprises the following steps: taking 35 mu L of plasma sample, adding 100 mu L of methanol solution containing the internal standard, performing vortex oscillation for 30-45s, then placing the plasma sample into a centrifuge for centrifugal treatment, wherein the separation factor RCF of the centrifuge is 3600-8000, and taking 50 mu L of supernatant, adding 200 mu L of deionized water for dilution to obtain the pretreated plasma sample.
In the pretreatment process of the plasma sample, the methanol solution of the internal standard is a methanol solution with d9-TMAO concentration of 30ng/mL (d 9-TMAO refers to a deuterated trimethylamine oxide isotope label).
Further, in the step (S1), 350 μ L of blank plasma sample is taken, 1000 μ L of methanol is added, vortex oscillation is carried out for 30-45S, and then the blank plasma sample is placed into a centrifuge for centrifugal treatment, wherein the separation factor RCF of the centrifuge is 3600-8000; adding 2000 mu L of deionized water into 500 mu L of supernatant for dilution, then adding TMAO, gradually diluting with water to obtain 10 solutions containing different concentrations of TMAO, wherein the TMAO concentrations are as follows: 10. 25, 55, 80, 100, 200, 400, 500, 800, 1000 ng/mL; mu.L of each solution was taken and 100. mu.L of a methanol solution containing 30ng/mL of d9-TMAO was added to each solution to obtain a series of standard samples.
Further, the liquid chromatography conditions were as follows:
a chromatographic column: an Agilent ZORBAX SB-C18 liquid chromatography column;
mobile phase: acetonitrile and 0.1% formic acid aqueous solution with the volume ratio of 88: 12;
flow rate: 0.3-0.5 mL.min-1；
Column temperature: 35-40 ℃;
sample introduction amount: 5 mu L of the solution;
and (3) an elution mode: isocratic elution.
Further, the mass spectrometry conditions were as follows:
mass spectrometry: a triple quadrupole mass spectrometer;
scanning mode: electrospray positive ion mode;
an acquisition mode: mass spectrometry Multiple Reaction Monitoring (MRM);
ion source voltage: 4000V;
ion source temperature: 400 ℃;
air curtain air: 25 psi;
atomizing gas (gas 1): 50 psi;
auxiliary gas (gas 2): 40 psi.
The invention not only provides the method capable of rapidly detecting the TMAO biomarker, but also provides a kit which can be matched with the method for use, wherein the kit comprises the following reagents:
reagent A: d9-TMAO methanol solution with concentration of 30 ng/mL;
a series of reagents B: the standard sample used in claim 5.
The application of the kit is as follows: the kit is used for detecting the TMAO content in blood by liquid chromatography-mass spectrometry, the reagent A is used as an internal standard substance, and the series reagent B is used as a standard sample to obtain a standard curve under the determined chromatographic condition.
Has the advantages that: the method for rapidly detecting the TMAO biomarker is a high-efficiency detection method which is based on liquid chromatography-mass spectrometry and can carry out batch rapid detection, and a detection result with high accuracy and good stability is obtained by reasonably preprocessing a sample and matching and optimizing reasonable conditions of liquid chromatogram and mass spectrum; in addition, the invention also provides a kit which can be used by matching with the method, and provides an internal standard and a standard sample with accurate concentration so as to reduce actual operation steps and improve detection efficiency.
Detailed Description
The invention is further illustrated by the following specific examples, which are illustrative and intended to illustrate the problem and explain the invention, but not limiting.
Example 1
(1) Setting the conditions of liquid chromatography
A chromatographic column: an Agilent ZORBAX SB-C18 liquid chromatography column;
mobile phase: acetonitrile and 0.1% formic acid aqueous solution with the volume ratio of 88: 12;
flow rate: 0.4 mL.min-1；
Column temperature: 35 ℃;
sample introduction amount: 5 mu L of the solution;
and (3) an elution mode: isocratic elution.
(2) Setting Mass Spectrometry conditions
Mass spectrometry: a triple quadrupole mass spectrometer;
scanning mode: electrospray positive ion mode;
an acquisition mode: mass spectrometry Multiple Reaction Monitoring (MRM);
ion source voltage: 4000V;
ion source temperature: 400 ℃;
air curtain air: 25 psi;
atomizing gas (gas 1): 50 psi;
auxiliary gas (gas 2): 40 psi.
(3) Preparing a standard sample
Taking 350 mu L of blank plasma sample, adding 1000 mu L of methanol, performing vortex oscillation for 45s, and then loading into a centrifuge for centrifugal treatment, wherein the separation factor RCF of the centrifuge is 8000; adding 2000 mu L of deionized water into 500 mu L of supernatant for dilution, and then adding TMAO to make the concentration of the TMAO 1000 ng/mL; gradually diluting with water to obtain 10 solutions containing TMAO with different concentrations, wherein the TMAO concentrations are respectively as follows: 10. 25, 55, 80, 100, 200, 400, 500, 800, 1000 ng/mL; a series of standards were prepared by adding 100. mu.L of each internal standard solution to 300. mu.L of each solution.
Internal standard solution: d9-TMAO was used as an internal standard to prepare a 30ng/mL methanol solution.
(4) Establishing a standard curve
And (3) testing a series of standard samples by liquid chromatography-mass spectrometry, calculating to obtain the ratio (Y) of the TMAO peak area corresponding to each standard sample to the internal standard peak area and the ratio (X) of the TMAO concentration corresponding to each standard sample to the internal standard concentration, and drawing a change curve of Y along with X to obtain the standard curve.
Linear regression equation: y = 1.256X-0.01192;
coefficient of correlation r2：0.9995；
Linear range of X: 1-100.
(5) Pretreatment of blood plasma to be tested
Taking 35 mu L of plasma sample, adding 100 mu L of methanol solution containing internal standard, carrying out vortex oscillation for 45s, then loading into a centrifuge for centrifugal treatment, wherein the separation factor RCF of the centrifuge is 8000, and taking 50 mu L of supernatant, adding 200 mu L of deionized water, and diluting to obtain the pretreated plasma sample.
(6) Pretreated plasma sample detection
Detecting the pretreated plasma sample by liquid chromatography-mass spectrometry, calculating to obtain the ratio of the TMAO peak area to the internal standard peak area, substituting into a standard curve to obtain the ratio of the TMAO concentration in the pretreated sample to the internal standard concentration; the concentration of TMAO in the original plasma sample was calculated from the dilution factor during the pretreatment.
Example 2
(1) Setting the conditions of liquid chromatography
A chromatographic column: an Agilent ZORBAX SB-C18 liquid chromatography column;
mobile phase: acetonitrile and 0.1% formic acid aqueous solution with the volume ratio of 88: 12;
flow rate: 0.3 mL.min-1；
Column temperature: 40 ℃;
sample introduction amount: 5 mu L of the solution;
and (3) an elution mode: isocratic elution.
(2) Setting Mass Spectrometry conditions
Mass spectrometry: a triple quadrupole mass spectrometer;
scanning mode: electrospray positive ion mode;
an acquisition mode: mass spectrometry Multiple Reaction Monitoring (MRM);
ion source voltage: 4000V;
ion source temperature: 400 ℃;
air curtain air: 25 psi;
atomizing gas (gas 1): 50 psi;
auxiliary gas (gas 2): 40 psi.
(3) Preparing a standard sample
Taking 350 mu L of blank plasma sample, adding 1000 mu L of methanol, performing vortex oscillation for 30s, and then putting the blank plasma sample into a centrifuge for centrifugal treatment, wherein the separation factor RCF of the centrifuge is 3600; adding 2000 mu L of deionized water into 500 mu L of supernatant for dilution, and then adding TMAO to make the concentration of the TMAO 1000 ng/mL; gradually diluting with water to obtain 10 solutions containing TMAO with different concentrations, wherein the TMAO concentrations are respectively as follows: 10. 25, 55, 80, 100, 200, 400, 500, 800, 1000 ng/mL; a series of standards were prepared by adding 100. mu.L of each internal standard solution to 300. mu.L of each solution.
Internal standard solution: d9-TMAO was used as an internal standard to prepare a 30ng/mL methanol solution.
(4) Establishing a standard curve
And (3) testing a series of standard samples by liquid chromatography-mass spectrometry, calculating to obtain the ratio (Y) of the TMAO peak area corresponding to each standard sample to the internal standard peak area and the ratio (X) of the TMAO concentration corresponding to each standard sample to the internal standard concentration, and drawing a change curve of Y along with X to obtain the standard curve.
Linear regression equation: y = 1.249X-0.01204;
coefficient of correlation r2：0.9992；
Linear range of X: 1-100.
(5) Pretreatment of blood plasma to be tested
Taking 35 mu L of plasma sample, adding 100 mu L of methanol solution containing the internal standard, performing vortex oscillation for 30s, then putting the plasma sample into a centrifuge for centrifugation, wherein the separation factor RCF of the centrifuge is 3600, and taking 50 mu L of supernatant, adding 200 mu L of deionized water and diluting to obtain the pretreated plasma sample.
(6) Pretreated plasma sample detection
Detecting the pretreated plasma sample by liquid chromatography-mass spectrometry, calculating to obtain the ratio of the TMAO peak area to the internal standard peak area, substituting into a standard curve to obtain the ratio of the TMAO concentration in the pretreated sample to the internal standard concentration; the concentration of TMAO in the original plasma sample was calculated from the dilution factor during the pretreatment.
As can be seen from the standard curves established in examples 1 and 2, the method provided by the invention can establish a very linear standard curve, and the correlation coefficient r2The TMAO concentration range exceeds 0.999, is optimally 0.9995, and the linear range spans two complete orders of magnitude, so that the detection result with high reliability and accuracy can be obtained in a larger TMAO concentration range. In contrast, example 1 provides the optimum operating parameters.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.
Claims (9)
1. A method capable of rapidly detecting a TMAO biomarker, comprising the steps of:
(S1) establishing a standard curve: taking a blank plasma sample, adding methanol, performing vortex oscillation, then loading into a centrifuge for centrifugal treatment, and taking supernatant and adding water for dilution; then adding TMAO and an internal standard to prepare a series of standard samples containing the internal standard with the same concentration and different TMAO concentrations; testing a series of standard samples by liquid chromatography-mass spectrometry, calculating to obtain a ratio (Y) of a TMAO peak area corresponding to each standard sample to an internal standard peak area and a ratio (X) of a TMAO concentration corresponding to each standard sample to an internal standard concentration, and making a change curve of Y along with X to obtain a standard curve;
(S2) pretreatment sample detection: detecting the pretreated plasma sample by liquid chromatography-mass spectrometry, calculating to obtain the ratio of the TMAO peak area to the internal standard peak area, substituting into a standard curve to obtain the ratio of the TMAO concentration in the pretreated sample to the internal standard concentration; calculating the concentration of TMAO in the original plasma sample according to the dilution times in the pretreatment process;
the plasma sample pretreatment method comprises the following steps: and adding a methanol solution containing an internal standard into a plasma sample, performing vortex oscillation, then loading into a centrifuge for centrifugal treatment, and adding water into a certain amount of supernatant for dilution to obtain a pretreated plasma sample.
2. The method of claim 1, wherein the plasma sample pretreatment method comprises: adding the plasma sample into methanol solution containing internal standard for protein precipitation, centrifuging to separate the sample, and diluting with water.
3. The method of claim 1, wherein the plasma sample pretreatment method comprises: taking 35 mu L of plasma sample, adding 100 mu L of methanol solution containing the internal standard, performing vortex oscillation for 30-45s, then placing the plasma sample into a centrifuge for centrifugal treatment, wherein the separation factor RCF of the centrifuge is 3600-8000, and taking 50 mu L of supernatant, adding 200 mu L of deionized water for dilution to obtain the pretreated plasma sample.
4. The method of claim 3, wherein the internal standard methanol solution is a methanol solution with d9-TMAO concentration of 30ng/mL during the pretreatment of the plasma sample.
5. The method as claimed in claim 1, wherein in the step (S1), 350 μ L of blank plasma sample is taken, 1000 μ L of methanol is added, vortex oscillation is carried out for 30-45S, and then the blank plasma sample is put into a centrifuge for centrifugal processing, wherein the separation factor RCF of the centrifuge is 3600-8000; adding 2000 mu L of deionized water into 500 mu L of supernatant for dilution, then adding TMAO, gradually diluting with water to obtain 10 solutions containing different concentrations of TMAO, wherein the TMAO concentrations are as follows: 10. 25, 55, 80, 100, 200, 400, 500, 800, 1000 ng/mL; mu.L of each solution was taken and 100. mu.L of a methanol solution containing 30ng/mL of d9-TMAO was added to each solution to obtain a series of standard samples.
6. The method of claim 1, wherein the liquid chromatography conditions are as follows:
a chromatographic column: an Agilent ZORBAX SB-C18 liquid chromatography column;
mobile phase: acetonitrile and 0.1% formic acid aqueous solution with the volume ratio of 88: 12;
flow rate: 0.3-0.5 mL.min-1；
Column temperature: 35-40 ℃;
sample introduction amount: 5 mu L of the solution;
and (3) an elution mode: isocratic elution.
7. The method of claim 1, wherein the mass spectrometry conditions are as follows:
mass spectrometry: a triple quadrupole mass spectrometer;
scanning mode: electrospray positive ion mode;
an acquisition mode: mass spectrometry Multiple Reaction Monitoring (MRM);
ion source voltage: 4000V;
ion source temperature: 400 ℃;
air curtain air: 25 psi;
atomizing gas (gas 1): 50 psi;
auxiliary gas (gas 2): 40 psi.
8. A kit comprising the following reagents:
reagent A: d9-TMAO methanol solution with concentration of 30 ng/mL;
a series of reagents B: the standard sample used in claim 5.
9. Use of the kit according to claim 8, wherein the kit is used in LC-MS for detecting TMAO content in blood, reagent A is used as an internal standard substance, and reagent B is used as a standard sample to obtain a standard curve under defined chromatographic conditions.
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