CN115047087A - Method for simultaneously detecting trimethylamine oxide substances in serum sample - Google Patents
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Abstract
The invention provides a method for extracting, separating and identifying trimethylamine oxide, which can realize the rapid extraction, accurate separation and mass spectrometric identification of the trimethylamine oxide in a serum sample.
Description
Technical Field
The application relates to detection of metabolites in serum, and belongs to the field of biological detection.
Background
TMAO is generated by metabolism of choline-rich or Trimethylamine (TMA) -structure-rich substances by intestinal microorganisms, and plays an important role in occurrence and development of cardiovascular diseases such as coronary heart disease, atherosclerosis, hypertension and the like.
Cardiovascular diseases are the leading cause of death of urban and rural residents in China at present, and are higher than tumors and other diseases. The newly released data of 'Chinese cardiovascular disease report 2018' show that: the morbidity and mortality of cardiovascular diseases in China are still in a continuous rising stage, 2.9 million patients with cardiovascular diseases are calculated, wherein 1300 million stroke, 1100 million coronary heart disease, 500 million pulmonary heart disease, 45 million heart failure, 250 million rheumatic heart disease and 2.7 million hypertension exist in the whole country, the cardiovascular diseases are main killers of human health, the cardiovascular disease prevention and treatment work in China has primary effect but still faces serious challenges, the burden of the cardiovascular diseases is gradually increased, the cardiovascular disease prevention and treatment is a major public health problem, and the prevention and treatment of the cardiovascular diseases are not slow enough. How to early prevent and treat cardiovascular diseases, find effective targets of drug action, open up new therapeutic approaches, and become a hot spot of attention.
Metabolomics, which reflects the direct relationship of genes, proteins and metabolic activities themselves by measuring the change in the concentration of cellular, tissue and body metabolites, is another new branch of omics research that has emerged following genomics, transcriptomics, proteomics. Since metabonomics changes are the final reflection of the body on the effects of genes, diseases, environments, drugs and the like, and endogenous metabolites of the metabonomics are the key or end-point reactions of a series of life events of the body, metabonomics can help people to better understand various complex interactions and the essence thereof in the body and can be used for diagnosing human diseases. The high performance liquid chromatography-mass spectrometry (LC-MS/MS) is one of the most widely applied technical platforms in metabonomics research, and has the characteristics of high sensitivity, high flux, wide linear range and the like.
The human intestinal flora is a complex community, and the intestinal microbiota plays an important role in immunity and defense, digestion and metabolism, inflammation, and cell proliferation. The main nutrients choline, betaine and carnitine from red meat, eggs, dairy products and saltwater fish are involved in biological activities such as energy metabolism in human bodies. After ingestion, fermentation of these nutrients by gut microbes results in the release of Trimethylamine (TMA), which is converted by host liver enzymes, flavin-containing monooxygenase 3(FMO3) into trimethylamine oxide (TMAO). There is increasing evidence that: TMAO, one of small molecules in intestinal microorganism metabolism, participates in cholesterol metabolism, promotes platelet high aggregation, increases thrombus formation, and promotes vascular inflammatory reaction to cause arterial plaque formation. However, there are no metabolic markers currently available that are relevant for the diagnosis of cardiovascular diseases, in particular heart failure.
Liquid chromatography tandem mass spectrometry (LC-MS/MS) is increasingly widely applied to various fields of medicines, foods, environments, forensic science, clinics and the like. In the liquid chromatogram tandem mass spectrum, the liquid chromatogram is responsible for separating an object to be detected and an interfering substance, and the mass spectrum is responsible for detection. After sample introduction, the sample firstly enters a chromatographic column under the carrying of a mobile phase, and enters a mass spectrum for detection after being separated by the chromatographic column. The mass spectrum is detected according to the mass-to-charge ratio (m/z) of a detected object, the detected object is converted into gas phase ions in an ion source to enter the mass spectrum, first-stage mass spectrum in a triple quadrupole scans ions in a specific range or allows the specific ions to enter a collision chamber, molecular ions are collided and cracked in the collision chamber to form daughter ions to enter a second-stage mass spectrum, and the second-stage mass spectrum scans the ions in the specific range or allows the specific ions to enter a detector. The LC-MS/MS has the characteristics of high sensitivity, strong selectivity, good accuracy and the like, and the application range of the LC-MS/MS in clinical detection far exceeds the range of radioactive immunoassay and chemical detection, and is incomparable with other methods. At present, no quantitative and qualitative detection method for trimethylamine oxide in a serum sample exists in the prior art, and the purpose of the present application is to perform technical exploration for overcoming the above defects.
Disclosure of Invention
The invention provides a method for detecting trimethylamine oxide in a sample; the method is to detect the trimethylamine oxide in the serum sample by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. In a specific embodiment, the method comprises:
1) extracting trimethylamine oxide from a serum sample;
2) setting the parameter conditions of LC-MS/MS;
3) data processing: and correcting the mass spectrum peak detected in the sample by each substance according to the retention time of the trimethylamine oxide and the ion pair information, performing quality control analysis on the sample and obtaining a standard curve.
In a specific embodiment, the sample extraction method comprises: taking a serum sample, and according to the sample: adding 70% methanol into a solvent with the volume ratio of 1:5, mixing, sucking 1/2 of the mixed solution into a centrifuge tube, standing at-20 ℃, centrifuging at low temperature, and taking the supernatant to pass through a 96-well protein precipitation plate for later use.
In another embodiment, the data acquisition instrument system for LC-MS/MS detection comprises ultra high performance liquid chromatography (Waters HClass) and tandem mass spectrometry MS/MS (Applied Biosystems6500Quadrupole Trap); wherein
A chromatographic column: ACQUITY UPLC BEH Amide column
Mobile phase A: ultrapure water (10mM ammonium acetate, 0.3% ammonia);
mobile phase B: 90% acetonitrile/water (V/V);
needle washing liquid: 50% acetonitrile (ultrasonic degassing for 10 min);
column temperature: 30 ℃; flow rate: 0.4 mL/min; sample introduction amount: 2 mu L of the solution;
in another specific example, the elution gradient of LC-MS/MS: 0-1.2min A/B is 5:95(V/V), 8min A/B is 30:70(V/V), 9.0-11min A/B is 50:50(V/V), 11.1-15min A/B is 5:95 (V/V).
In another specific embodiment, the mass spectrometry conditions are:
| Ion Mode | ESI+ | Curtain |
20 |
| Nebulizer Cμrrent | 3 | Temperatμre | 350 |
| Ion Source |
20 | Ion Source Gas2 | 0 |
| Collision Gas | Medium | Scan type | MRM |
| Entrance Potential | 10 | Collision Cell Exit Potential | 13 |
| MRM detection window | 120 | Target Scan Time | 0.3s |
。
in another specific embodiment, the ion pair information is specifically:
| Compound | parent ion | Daughter ion | Retention time |
| Trimethylamine | 60.3 | 44.0 | 6.6 |
| Trimethylamine oxide | 76.0 | 58.1 | 4.83 |
| Choline | 104.1 | 60.1 | 6.6 |
| Betaine | 118.1 | 58.0 | 2.97 |
| Creatinine | 114.1 | 86.1 | 1.49 |
| L-Carnitine | 162.1 | 85.1 | 5.2 |
。
In another specific embodiment, the standard curve is:
| substance(s) | Linear equation of equations | Correlation coefficient |
| Trimethylamine | y＝4.20756e4 x+1.11621e5 | r＝0.99999 |
| Trimethylamine oxide | y＝3.39928e6 x+2.50231e5 | r＝0.99542 |
| Choline | y＝2.83918e6 x+1.03597e7 | r＝0.99970 |
| Betaine | y＝1.21803e6 x+9.57365e7 | r＝0.99278 |
| Creatinine | y＝8.21413e4 x+5.15596e6 | r＝0.99651 |
| L-Carnitine | y＝1.43101e6 x+6.98585e6 | r＝0.99545 |
。
In a second aspect, the invention provides the use of the method of the first aspect for detecting the amount of trimethylamine oxide in serum, wherein said use is a non-diagnostic use.
The beneficial effects of the invention are as follows:
1. the sample is simple to process, and the trimethylamine oxide in the serum sample can be quantified quickly and efficiently.
2. The Amide chromatographic column is adopted, so that the condition that trimethylamine oxide is weakly retained on a conventional reversed-phase chromatographic column due to high polarity is improved.
3. The mass spectrum detection has strong specificity and high sensitivity.
Drawings
FIG. 1 shows an overlay of nine glycosides XIC.
Detailed Description
1. The extraction method of the trimethylamine oxide in the serum sample comprises the following steps:
taking 50 mu L of serum sample, adding 250 mu L of 70% methanol, vortexing for 5min, centrifuging for 15min at 12000r/min at 4 ℃, transferring 150 mu L of supernatant into another numbered centrifuge tube, standing for 30min at-20 ℃ in a refrigerator, centrifuging for 5min at 4 ℃, further centrifuging for 5min at 12000r/min, transferring 150 mu L of supernatant after centrifugation, passing through a 96-well protein precipitation plate, and then using the supernatant for on-machine analysis.
2. The instrument parameter conditions are as follows:
the data acquisition instrument system used for LC-MS/MS detection mainly comprises ultra high performance liquid chromatography (Waters HClass) and tandem mass spectrometry MS/MS (Applied Biosystems6500Quadrupole Trap).
Wherein, the ultra-high performance liquid chromatography conditions mainly comprise:
a chromatographic column: ACQUITY UPLC BEH Amide column
Mobile phase A: ultrapure water (10mM ammonium acetate, 0.3% ammonia);
mobile phase B: 90% acetonitrile/water (V/V);
needle washing liquid: 50% acetonitrile (ultrasonic degassing for 10 min);
column temperature: 30 ℃; flow rate: 0.4 mL/min; sample introduction amount: 2 mu L of the solution;
elution gradient: 0-1.2min A/B is 5:95(V/V), 8min A/B is 30:70(V/V), 9.0-11min A/B is 50:50(V/V), 11.1-15min A/B is 5:95 (V/V);
the mass spectrometry conditions were as follows:
3. the data processing method comprises the following steps:
the mass spectral data was processed using software Analyst 1.6.3. The mass spectrum peak detected in the sample for each substance was corrected based on the trimethylamine oxide retention time and ion pair information to ensure accuracy of qualitative and quantitative determination. And (3) carrying out qualitative and quantitative analysis on the trimethylamine oxide in the sample, wherein the Peak Area (Peak Area) of each chromatographic Peak represents the relative content of the corresponding substance, and finally obtaining the qualitative and quantitative analysis result of the trimethylamine oxide in the sample.
Ion pair information is shown in the following table
(1) Sample quality control analysis: the repeatability of extraction and detection of the trimethylamine oxide is judged by performing overlapping display analysis on a total ion flow chart (TIC chart) of mass spectrometric detection analysis of different quality control samples, and the result is shown in figure 1.
(2) Drawing a standard curve: preparing trimethylamine oxide standard substance solutions with different concentrations, and obtaining mass spectrum peak intensity data of corresponding quantitative signals of the standard substances with different concentrations; standard curves of different substances are drawn by taking the concentration (mug/mL) of the standard substance as an abscissa and the Peak Area (Peak Area) of a mass spectrum Peak as an ordinate, and the obtained standards are selected from the following tables:
| substance(s) | Linear equation of equations | Correlation coefficient |
| Trimethylamine | y＝4.20756e4 x+1.11621e5 | r＝0.99999 |
| Trimethylamine oxide | y＝3.39928e6 x+2.50231e5 | r＝0.99542 |
| Choline | y＝2.83918e6 x+1.03597e7 | r＝0.99970 |
| Betaine | y＝1.21803e6 x+9.57365e7 | r＝0.99278 |
| Creatinine | y＝8.21413e4 x+5.15596e6 | r＝0.99651 |
| L-Carnitine | y＝1.43101e6 x+6.98585e6 | r＝0.99545 |
(3) Absolute quantification: and respectively substituting the detected integrated peak areas of the trimethylamine oxide of all the samples into a standard curve linear equation to calculate, and further substituting the calculation result into a content calculation formula to calculate to obtain the content of each substance in the actual sample.
Although the present invention has been described with reference to the preferred embodiments, it is not intended to limit the present invention, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the present invention.
Claims (8)
1. A method for detecting trimethylamine oxide in a sample; the method is to detect trimethylamine oxide in a serum sample by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method; in a specific embodiment, the method comprises:
1) extracting trimethylamine oxide from a serum sample;
2) setting the parameter conditions of LC-MS/MS;
3) data processing: and correcting the mass spectrum peak detected in the sample by each substance according to the retention time of the trimethylamine oxide and the ion pair information, performing quality control analysis on the sample and obtaining a standard curve.
2. The method of claim 1, wherein the sample extraction method comprises: taking a serum sample, and according to the sample: adding 70% methanol into a solvent with the volume ratio of 1:5, mixing, sucking 1/2 of the mixed solution into a centrifuge tube, standing at-20 ℃, centrifuging at low temperature, and taking the supernatant to pass through a 96-well protein precipitation plate for later use.
3. The method of claim 1 or 2, wherein the data collection instrument system for the LC-MS/MS detection comprises ultra high performance liquid chromatography (Waters HClass) and tandem mass spectrometry MS/MS (Applied Biosystems6500Quadrupole Trap); wherein
A chromatographic column: an ACQUITY UPLC BEH Amide column;
mobile phase A: ultrapure water (10mM ammonium acetate, 0.3% ammonia);
mobile phase B: 90% acetonitrile/water (V/V);
needle washing liquid: 50% acetonitrile (ultrasonic degassing for 10 min);
column temperature: 30 ℃; flow rate: 0.4 mL/min; sample introduction amount: 2 μ L.
4. The method of claim 1, wherein the elution gradient of LC-MS/MS is: 0-1.2min A/B is 5:95(V/V), 8min A/B is 30:70(V/V), 9.0-11min A/B is 50:50(V/V), 11.1-15min A/B is 5:95 (V/V).
5. The method of claim 1, wherein the mass spectrometry conditions are:
。
6. the method of claim 1, wherein the ion pair information is specifically:
。
7. In another specific embodiment, the method of claim 1, wherein the standard curve is:
8. use of the method according to any one of claims 1 to 7 for the detection of the amount of trimethylamine oxide in serum, said use being a non-diagnostic use.
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| CN111505132A (en) * | 2020-01-10 | 2020-08-07 | 中国医学科学院阜外医院 | Method for detecting novel cardiovascular disease risk marker by liquid chromatography-tandem mass spectrometry |
| CN111650285A (en) * | 2019-09-24 | 2020-09-11 | 上海透景诊断科技有限公司 | Detection method and detection kit for content of trimethylamine oxide in blood plasma |
| CN112505228A (en) * | 2021-01-04 | 2021-03-16 | 深圳市绘云生物科技有限公司 | Metabolic marker combination for assessing cardiovascular disease risk of subject and application thereof |
| CN112730706A (en) * | 2021-02-04 | 2021-04-30 | 麦特绘谱生物科技（上海）有限公司 | Method for detecting biological small molecule marker by liquid chromatography-tandem mass spectrometry |
| US20220050090A1 (en) * | 2018-09-11 | 2022-02-17 | Metabolon, Inc. | Mass spectrometry assay method for detection and quantitation of microbiota related metabolites |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220050090A1 (en) * | 2018-09-11 | 2022-02-17 | Metabolon, Inc. | Mass spectrometry assay method for detection and quantitation of microbiota related metabolites |
| CN111650285A (en) * | 2019-09-24 | 2020-09-11 | 上海透景诊断科技有限公司 | Detection method and detection kit for content of trimethylamine oxide in blood plasma |
| CN111505132A (en) * | 2020-01-10 | 2020-08-07 | 中国医学科学院阜外医院 | Method for detecting novel cardiovascular disease risk marker by liquid chromatography-tandem mass spectrometry |
| CN112505228A (en) * | 2021-01-04 | 2021-03-16 | 深圳市绘云生物科技有限公司 | Metabolic marker combination for assessing cardiovascular disease risk of subject and application thereof |
| CN112730706A (en) * | 2021-02-04 | 2021-04-30 | 麦特绘谱生物科技（上海）有限公司 | Method for detecting biological small molecule marker by liquid chromatography-tandem mass spectrometry |
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