CN111505132A

CN111505132A - Method for detecting novel cardiovascular disease risk marker by liquid chromatography-tandem mass spectrometry

Info

Publication number: CN111505132A
Application number: CN202010024358.3A
Authority: CN
Inventors: 周洲; 王恺隽; 武红梅; 赵丽平; 高小晶; 栗琳
Original assignee: Beijing Haosi Biotechnology Co ltd; Fuwai Hospital of CAMS and PUMC
Current assignee: Beijing Haosi Biotechnology Co ltd; Fuwai Hospital of CAMS and PUMC
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2020-08-07

Abstract

The invention belongs to the technical field of clinical biological sample detection, and particularly relates to a method for detecting trimethylamine oxide, choline, betaine, creatinine and L-carnitine in a biological sample by a high-throughput liquid chromatography-tandem mass spectrometry method; the method can be used for simultaneously detecting 5 substances in the biological sample, and has the advantages of high accuracy, high sensitivity, high specificity, small required sample volume, simple sample pretreatment, good tolerance and the like.

Description

Method for detecting novel cardiovascular disease risk marker by liquid chromatography-tandem mass spectrometry

Technical Field

The invention relates to a method for detecting trimethylamine oxide, choline, betaine, L-carnitine and creatinine in a biological sample by high-throughput liquid chromatography-tandem mass spectrometry.

Background

The prevalence and mortality of cardiovascular disease (CVD) in china continues to rise, with about 2.9 million patients. The CVD death rate is the first cause of various diseases and accounts for more than 40 percent of the death components of resident diseases. Meanwhile, the cardiovascular disease burden in China is getting more and more serious, and the cardiovascular disease becomes a great public health problem. With the development of social economy, the national lifestyle has been profoundly changed. Under the background of accelerated social aging and urbanization processes and the prevalence of unhealthy life styles of residents, the prevalence of national CVD risk factors is exposed, the prevalence of hypertension, dyslipidemia and diabetes is continuously increased, the prevalence and the mortality of the Chinese CVD are continuously increased, and the CVD is in a rapidly increasing trend in low-age and low-income groups at present. The number of CVD patients in China will continue to increase in the next decade, and the prevention and control work is very severe.

An important step in reducing the risk of CVD is the identification and treatment of known risk factors (e.g., smoking, dyslipidemia, hypertension, poor diet). New risk factors, such as the metabolite trimethylamine oxide (TMAO), help to refine CVD risk assessment and may be a new target substance for CVD therapeutic intervention.

TMAO levels in the blood and ingested nutrients such as Choline (Choline), Betaine (Betaine) and l-Carnitine (L-Carnitine) intestinal bacteria convert these precursor nutrients into Trimethylamine (TMA), which is then oxidized in the liver to TMAO studies have shown that in rodent models, TMAO precursor-rich diets increase foam cells caused by atherosclerosis, leading to aortic lesions, inhibit cholesterol transfer from tissues to the liver, and alter platelet properties increasing the risk of thrombosis.

Meta-analysis of the large prospective cohort showed that high concentrations of TMAO were associated with increased risk of Major Adverse Coronary Events (MACE) and mortality, and were independent of other risk factors. Clinical data support more accurate prediction of MACE risk using TMAO in coronary angiography and acute coronary syndrome patients. Clinical data also support the use of TMAO to refine risk assessment of all-cause mortality in patients with peripheral arterial disease, heart failure, chronic kidney disease, and myocardial infarction. Clinicians and researchers can define tools to guide dietary adjustments and treatments based on the effects of TMAO.

Creatinine (Creatinine) is a product of muscle metabolism in the human body, and its level is related to renal function, and long-term hypertension or blood-borne cardiac insufficiency may cause renal injury, which in turn increases Creatinine level.

At present, the invention discloses a liquid phase tandem mass spectrometry method for measuring trimethylamine oxide, which is applied to China patent 'a trimethylamine oxide quantitative detection kit and a method' of 27.6.2017 by Changsha, all Limited Biotechnology corporation, and application number 201710502265.5, but only one substance of the trimethylamine oxide can be measured, and application value is not clarified. The invention discloses a method for measuring small molecule biomarkers, which is applied by Suzhou panomike biomedicine science and technology Limited at 2018, 9/13 and has the application number of 201811068542.7, and discloses a liquid phase tandem mass spectrum method for measuring 11 small molecules (choline, betaine, L-carnitine, trimethylamine oxide, creatinine, homocysteine, leucine/isoleucine, valine, phenylalanine, tyrosine and tryptophan): since a plurality of substances are detected at one time, the pretreatment of a sample is complicated, and a reducing agent needs to be used. The invention discloses a Chinese patent 'a kit' applied in 2019, 4 and 8 days in first people hospital in Changzhou city, application number 201910274765.7, which discloses a kit for measuring the content of blood plasma TMAO, and only one substance of TMAO can be measured.

In summary, the main drawbacks of the prior art regarding the process of methodological detection of novel cardiovascular disease risk markers are:

1. most methods can only detect one substance of TMAO and cannot meet clinical requirements;

2. the pretreatment method of the biological sample is too complex and cannot be automated, so that the method is difficult to popularize in clinical application;

3. the clinical significance of the detection markers is unclear and there is not enough literature and data support.

4. Too many irrelevant markers are detected, and the cost is too high.

Disclosure of Invention

The invention aims to provide a method for detecting trimethylamine oxide, choline, betaine, L-carnitine and creatinine in a biological sample by high-throughput liquid chromatography-tandem mass spectrometry.

The purpose of the invention is realized by the following technical scheme:

preparation of standard solution

(a) Preparation of the Standard Curve

An analytical balance accurately weighs appropriate amounts of trimethylamine oxide, choline, betaine, creatinine and L-carnitine standard respectively, and the standard is dissolved by 50-90% methanol aqueous solution to prepare a standard stock solution. And diluting the standard substance stock solution by using 50-90% methanol aqueous solution to prepare mixed standard substance working solution.

The standard working solution is diluted and mixed by 50-90% methanol water solution to prepare a standard curve, and the concentration ranges of the standard curve are 0.01-10 mu g/m L of TMAO, 0.1-100 mu g/m L of Choline, 100-10000.1-100 mu g/m L of beta-amine and 0.1-100 mu g/m L of Creatine, 0.1-100 mu g/m L of Carnitine.

(b) Preparation of standard internal standard solution

An appropriate amount of internal standard of trimethylamine oxide, Choline, Betaine, Creatinine and L-Carnitine is accurately weighed by an analytical balance, dissolved by 50-90% methanol aqueous solution to prepare internal standard stock solution, and the internal standard stock solution is diluted by a protein precipitator to prepare mixed internal standard working solution with the concentrations of TMAO-d 95-50 ng/m L, Choline-d 45-50 ng/m L, Betaine-d 350-500 ng/m L, Creatine-d 35-50 ng/m L-Carnitine-d 35-50 ng/m L.

(c) Preparation of quality control product

An analytical balance accurately weighs appropriate amounts of trimethylamine oxide, choline, betaine, creatinine and L-carnitine standard substances respectively, and the appropriate amounts are dissolved by 50-90% methanol aqueous solution to prepare quality control substance stock solution. The quality control product stock solution is diluted by 50-90% methanol aqueous solution, and low-concentration, medium-concentration, high-concentration and three-concentration mixed quality control product working solution is prepared respectively.

Mixing 10-20 human biological samples, adding a mixed quality control working solution which is not more than 10% of the total volume of the biological samples, and uniformly mixing to obtain a low-concentration quality control product (L QC), a medium-concentration quality control product (MQC) and a high-concentration quality control product (HQC), wherein each of the low-concentration quality control product (L QC) and the high-concentration quality control product (HQC) is 30 mu L, and each of the high-concentration quality control products (HQC) is packed in an EP tube.

(II) treatment of biological samples

Taking at least 1m L of blood to be detected, centrifuging for 10-30 min at a centrifugation speed of 1500-3000 rpm to obtain a supernatant, and placing at-80 ℃ for later use;

(III) treatment of biological sample/standard curve/quality control material to be detected

And (C) transferring the mixed internal standard working solution 500-1000 mu L obtained in the step (b) into a 1.5m L centrifugal tube by a pipettor, adding a biological sample/standard curve/quality control product of 10-50 mu L into the centrifugal tube, uniformly mixing, centrifuging for 10-30 min at the centrifugation speed of 13000-24000 rpm, removing protein precipitates, and taking the supernatant by the pipettor for L C-MS/MS detection.

(IV) detection of sample to be tested

And (3) detecting the biological sample/standard curve/quality control product treated in the step (III) by using a high performance liquid chromatography triple quadrupole tandem mass spectrometer. And fitting to obtain a standard curve equation through the concentration and the peak area of the standard curve, and calculating the concentrations of trimethylamine oxide, choline, betaine, creatinine and L-carnitine in the biological sample to be detected through the peak area of the biological sample to be detected.

Preferably, in the above method, the biological sample comprises serum and plasma.

Preferably, in the above method, the standard curve is eight different concentration gradients.

Preferably, in the above method, the internal standards are deuterated isotope internal standards, specifically, trimethylamine oxide-d 9(TMAO-d9), Choline-d 4(Choline-d4), Betaine-d 3(Betaine-d3), Creatinine-d 3(Creatinine-d3), and L-Carnitine-d 3 (L-Carnitine-d 3), respectively.

Preferably, in the above method, the protein precipitant is used as an internal standard working solution solvent, and the protein precipitant comprises methanol/acetonitrile (100-0% -0-100%).

Preferably, in the above method, in the step (c) of preparing the quality control material, the concentrations of the biological samples are as follows:

analyte	LQC μg/mL	MQC μg/mL	HQC μg/mL
				TMAO	0.05～0.5	0.5～1	1～3
Choline	0.5～2	2～5	5～15
				Betaine	0.5～5	5～15	20～50
Creatinine	1～10	10～20	30～60
				L-Carnitine	1～10	10～30	30～100

Preferably, in the method, the analytical chromatographic column used by the high performance liquid chromatography triple quadrupole tandem mass spectrometer is a HI L IC chromatographic column or an Amide chromatographic column, the column temperature is 30-40 ℃, and the sample injection amount is 2-10 mu L;

preferably, in the method, the mobile phase of the analytical chromatographic column is an ammonium formate aqueous solution containing 5 mmol/L% of 0.1-0.5% of formic acid and an acetonitrile solution containing 0.1-0.5% of formic acid, the flow rate is 0.2-0.6 m L/min, the analytical chromatographic column adopts a gradient elution mode, and the volume ratio of the mobile phase A to the mobile phase B is 100-0% -0-100%.

Preferably, in the above method, the mass spectrometry conditions are as follows: an ion source: an electrospray ion source; ion source temperature: 300-600 ℃; ion source voltage: 4500-5000V; collision gas: 9-10 units; air curtain air: 40-50 psi; GS1 at 50-60 psi; GS 2: 50-60 psi; the detection mode is as follows: and detecting positive ions.

Preferably, in the above method, the high performance liquid chromatography triple quadrupole tandem mass spectrometer detection adopts an electrospray ion source (ESI) and a Multiple Reflection Monitoring (MRM) scanning mode, and specific ion pairs are as follows:

analyte	Ion pair	Internal standard	Ion pair
				TMAO	76.1→58.0	TMAO-d9	85.1→66.1
Choline	104.1→60.1	Choline-d4	108.3→60.0
				Betaine	118.1→58.1	Betaine-d3	121.1→61.1
Creatinine	114.1→44.1	Creatinine-d3	117.1→47.1
				L-Carnitine	162.2→103.0	L-Carnitine-d3	165.2→105.2

Compared with the prior art, the invention has the following beneficial effects:

1. the invention can detect 5 substances of trimethylamine oxide, choline, betaine, creatinine and L-carnitine in serum/plasma at one time;

2. the biological sample serum/plasma sample is directly injected after protein precipitation, the treatment method is simple, the efficiency is greatly improved, the automation is more easily realized, and the clinical popularization is facilitated;

3. the prediction of MACE risk for cardiovascular disease patients is proposed for the first time in the clinic.

Drawings

FIG. 1 Total chromatogram.

FIGS. 2 to 6 are chromatograms of trimethylamine oxide, choline, betaine, creatinine, and L-carnitine in this order.

Detailed Description

First, the present invention will be described in detail with reference to specific embodiments in order to better explain technical features and effects of the present invention, but the present invention is not limited thereto.

Calibration of standard solution

(a) And preparing a standard working solution:

respectively and accurately weighing 10.00mg of trimethylamine oxide, choline, betaine, creatinine and L-carnitine standard substance by an analytical balance, placing the standard substance in a 10ml volumetric flask, metering the volume to a scale by 50% methanol to obtain a standard stock solution with the concentration of 1.00mg/m L, and diluting the standard stock solution by 50% methanol solution to obtain a standard intermediate solution with the concentration of 100 mu g/m L;

sucking the above standard intermediate solution respectively to 20 μ L, 200 μ L, 200 μ L0, 200 μ L and 200 μ L, adding 180 μ L50% methanol solution, mixing, diluting with water solution, preparing standard working solution with various concentrations in the concentration range of 0.01-10 μ g/m L trimethylamine oxide, 0.1-100 μ g/m L choline, 0.1-100 μ g/m L betaine, 0.1-100 μ g/m L L-carnitine and 0.1-100 μ g/m L creatinine, which is specifically as follows:

μg/ml	STD1	STD2	STD3	STD4	STD5	STD6	STD7	STD8
									TMAO	0.01	0.05	0.1	0.2	0.5	1	5	10
Choline	0.1	0.5	1	2	5	10	50	100
									Betaine	0.1	0.5	1	2	5	10	50	100
Creatinine	0.1	0.5	1	2	5	10	50	100
									L-Carnitine	0.1	0.5	1	2	5	10	50	100

(b) and preparing standard internal standard solution:

accurately weighing 10.00mg of an isotope internal standard substance of trimethylamine oxide, choline, betaine, creatinine and L-carnitine, placing the isotope internal standard substance in a 10ml volumetric flask, metering the volume to a scale by using 50% methanol to obtain a standard stock solution, wherein the isotope concentration is 1.00mg/m L, and diluting the standard stock solution by using 50% methanol solution to obtain a standard intermediate solution, wherein the isotope concentration is 100 mu g/m L;

respectively sucking 10 mu L, 10 mu L, 100 mu L0, 50 mu L and 10 mu L from the standard intermediate solution, adding 820 mu L50% methanol solution, uniformly mixing, and diluting with aqueous solution to obtain standard internal standard solution containing 1 mu g/m L trimethylamine oxide isotope, 1 mu g/m L choline isotope, 10 mu g/m L betaine isotope, 5 mu g/m L L-carnitine isotope and 1 mu g/m L creatinine isotope;

and preparing a standard internal standard working solution, and diluting the mixed standard internal standard solution by 100 times with methanol for use.

(c) And preparing quality control products:

Mixing 10-20 biological samples, adding a mixed quality control product working solution which is not more than 10% of the total volume of the biological samples, and uniformly mixing to obtain a low-concentration quality control product (L QC), a medium-concentration quality control product (MQC) and a high-concentration quality control product (HQC), wherein each sample is 30 mu L, and the samples are respectively packed into an EP tube and stored in a refrigerator at the temperature of-80 ℃ for later use, and the adding concentration of specific standard products is as follows:

analyte	QCL	QCM	QCH
				TMAO	0.2	0.6	1.8
Choline	1	3	9
				Betaine	3	9	27
Creatinine	5	15	45
				L-Carnitine	6	18	54

(d) And (3) calibrating by using a standard solution to obtain a standard curve equation:

using a liquid transfer gun to respectively transfer at least three standard working solutions with different concentrations, wherein the standard working solutions with different concentrations are mixed with 800 mu L standard internal standard working solution, and are placed in a 1.5m L centrifuge tube to prepare at least three standard solutions, the substitute matrix is water for L C-MS/MS detection, chromatograms of trimethylamine oxide, choline, betaine, creatinine and L-carnitine in the at least three standard solutions and chromatogram of trimethylamine oxide isotope, choline isotope, betaine isotope, creatinine isotope and L-carnitine isotope in the corresponding standard internal standard solutions are respectively obtained, peak area ratios of trimethylamine oxide, choline, betaine isotope, creatinine isotope, and L-carnitine isotope in the at least three standard solutions are used as y-1, y-2, y-3, y-4 and y-5 of the standard solutions, the peak area ratios of the carnitine isotope, creatinine isotope and L-carnitine isotope are used as y-coordinates of the standard solutions, y-2, y-3, y-3684, the standard working solutions contain the concentrations of the trimethylamine oxide, choline, betaine isotope, creatinine and L-carnitine isotope, the standard solutions, the concentration of the betaine, the betaine isotope is used as x-8624, x-2, the standard isotope curves are respectively obtained by fitting, x- λ -;

(II) centrifugation of test blood

Centrifuging at least 1m L of blood to be detected at 3000rpm for 15min to obtain supernatant plasma, and standing at-80 deg.C;

(III) treatment of the sample to be tested

(e) Transferring the standard internal standard working solution 800 mu L obtained in the step (b) into a centrifuge tube with the diameter of 1.5m L by using a liquid transfer gun, adding the plasma with the diameter of 20 mu L obtained in the step (II) into the centrifuge tube, uniformly mixing, centrifuging for 5min at the centrifugation speed of 13000rpm, and taking the supernatant for L C-MS/MS detection

(IV) detection of sample to be tested

Taking the sample to be detected in the step (d), detecting the sample to be detected by using a high performance liquid chromatography triple quadrupole tandem mass spectrometer to obtain a chromatogram of trimethylamine oxide, choline, betaine, creatinine and L-carnitine of the sample to be detected and a chromatogram of trimethylamine oxide isotope, choline isotope, betaine isotope, creatinine isotope and L-carnitine isotope in a corresponding standard internal standard solution, substituting peak areas of trimethylamine oxide, choline, betaine, creatinine and L-carnitine in the chromatogram and peak areas of trimethylamine oxide isotope, choline isotope, betaine isotope, creatinine isotope and L-carnitine isotope in the corresponding standard internal standard solution, y1, y2, y3, y4 and y5 into a standard curve equation y1 of the step (c) a x1+ b, y2 c x2+ d, y3 e x3+ f, and y4 x4+ h and y5 x5+ j, the ratio of the concentration of the trimethylamine oxide, the choline, the betaine, the creatinine and the L-carnitine in the sample to be detected to the concentration of the trimethylamine oxide isotope, the choline isotope, the betaine isotope, the creatinine isotope and the L-carnitine isotope in the corresponding standard internal standard solution, x1, x2, x3, x4, x5 and x6, the concentration of the trimethylamine oxide isotope, the choline isotope, the betaine isotope, the creatinine isotope and the L-carnitine isotope in the standard internal standard solution are known, and the concentration of the trimethylamine oxide, the choline, the betaine, the creatinine and the L-carnitine isotope in the blood to be detected is obtained through calculation.

The mass spectrometer parameters of the high performance liquid chromatography triple quadrupole tandem mass spectrometry are as follows:

the multiple reaction monitoring parameters for mass spectrometric detection are as follows:

compound (I)	Q1	Q3	DWELL(ms)	DP	EP	CE	CXP
								TMAO	76.1	58.0	20	60	10	25	9
TMAO-d9	85.1	66.1	20	60	10	25	7
								Choline	104.1	60.1	20	80	10	25	9
Choline-d4	108.3	60.0	20	50	10	25	5
								Betaine	118.1	58.1	20	40	10	40	4
Betaine-d3	121.1	61.1	20	40	10	40	4
								Creatinine	114.1	44.1	20	55	10	30	6
Creatinine-d3	117.1	47.1	20	70	10	30	5
								L-Carnitine	162.2	103	20	30	10	25	7
L-Carnitine-d3	165.2	105.2	20	25	10	27	7

The mobile phase gradient elution program parameters of the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are as follows:

time (min)	Flow rate (ml/min)	Mobile phase A (%)	Mobile phase B (%)
				0.00	0.4	15	85
0.5	0.4	15	85
				2.8	0.4	100	0
2.9	0.4	15	85
				5	0.4	15	85

The technical scheme in the embodiment is demonstrated as follows:

(one) the linear relationship and quantitative limit of the method:

the mixed standard working solution prepared above is subjected to protein precipitation and then sample introduction, according to the determination conditions of the embodiment, determination is carried out according to the concentration from low to high, a standard curve is obtained by plotting the peak area-concentration of quantitative chromatography, and the result shows that the linear ranges and quantitative limits of trimethylamine oxide, choline, betaine, creatinine and L-carnitine are as follows:

(II) recovery and precision of the process

Preparing high, medium and low 3 concentrations of standard working solution of trimethylamine oxide, choline, betaine, creatinine and L-carnitine into sample recovery rate experiment and precision experiment, measuring according to the method of the embodiment, and repeatedly analyzing and measuring 3 batches, wherein the precision in each batch and among batches is as follows:

preparing high, medium and low 3 concentrations of standard working solution of trimethylamine oxide, choline, betaine, creatinine and L-carnitine into sample-adding recovery rate experiment and precision experiment, measuring according to the method of the embodiment, and repeatedly analyzing and measuring for 3 batches, wherein the recovery rate is as follows:

by combining the verification experiments, the detection limit, the recovery rate, the precision and other technical indexes of the embodiment meet the requirements, the method for detecting the contents of trimethylamine oxide, choline, betaine, creatinine and L-carnitine in the plasma has good reproducibility and high standard recovery rate, and the accuracy of the detection result is improved.

Chromatogram charts of trimethylamine oxide, choline, betaine, creatinine and L-carnitine in the plasma sample are shown in the figure, retention time of the trimethylamine oxide is 1.18min, retention time of the choline is 1.27min, retention time of the betaine is 2.05min, retention time of the nucleotide is 1.42min, and retention time of the L-carnitine is 1.46 min.

Claims

1. An analysis method for detecting the content of Trimethylamine Oxide (TMAO), Choline (Choline), Betaine (Betaine), Creatinine (Creatine) and L-Carnitine (L-Carnitine) in a biological sample by using a high performance liquid chromatography triple quadrupole tandem mass spectrometer (L C-MS/MS), which comprises the following steps:

preparation of standard solution

(a) Preparation of the Standard Curve

An analytical balance accurately weighs appropriate amounts of trimethylamine oxide, choline, betaine, creatinine and L-carnitine standard respectively, and the standard is dissolved by 50-90% methanol aqueous solution to prepare a standard stock solution. Diluting the standard substance stock solution by using 50-90% methanol aqueous solution to prepare mixed standard substance working solution;

(b) Preparation of standard internal standard solution

An analysis balance accurately measures appropriate internal standards of trimethylamine oxide, Choline, Betaine, Creatinine and L-Carnitine respectively, and the internal standards are dissolved by 50-90% methanol aqueous solution to prepare internal standard stock solution, and protein precipitant is used to dilute the standard stock solution to prepare mixed internal standard working solution, so that the concentrations of the internal standard working solution are TMAO-d 95-50 ng/m L, Choline-d 45-50 ng/m L, Betaine-d 350-500 ng/m L, Creatine-d 35-50 ng/m L-Carnitine-d 35-50 ng/m L;

(c) preparation of quality control product

Accurately weighing appropriate amounts of trimethylamine oxide, choline, betaine, creatinine and L-carnitine standard substances by an analytical balance, dissolving the appropriate amounts of the trimethylamine oxide, the choline, the betaine, the creatinine and the L-carnitine standard substances by 50-90% methanol aqueous solution, and preparing quality control substance stock solution; diluting the quality control product stock solution by using 50-90% methanol aqueous solution, and respectively preparing low-concentration, medium-concentration, high-concentration and three-concentration mixed quality control product working solution;

mixing 10-20 biological samples, adding a mixed quality control product working solution which is not more than 10% of the total volume of the biological samples, and uniformly mixing to obtain a low-concentration quality control product (L QC), a medium-concentration quality control product (MQC) and a high-concentration quality control product (HQC), wherein each 30 mu L is respectively packaged in an EP (European patent specification) tube and stored in a refrigerator at the temperature of-80 ℃ for later use;

(II) treatment of biological samples

(IV) detection of sample to be tested

2. The analytical method of claim 1, wherein: biological samples include dried blood sheets, serum, and plasma.

3. The analytical method of claim 1, wherein: in step (a), 5-9 standard curves with different concentrations are used.

4. The analysis method according to claim 1, wherein the internal standards are deuterated isotope internal standards, such as trimethylamine oxide-d 9(TMAO-d9), Choline-d 4(Choline-d4), Betaine-d 3(Betaine-d3), Creatinine-d 3(Creatinine-d3), and L-Carnitine-d 3 (L-Carnitine-d 3).

5. The analytical method of claim 1, wherein: the method uses a protein precipitator as an internal standard working solution solvent, wherein the protein precipitator comprises methanol/acetonitrile (100-0% -0-100%).

6. The analytical method of claim 1, wherein: in the step (c) of preparing the quality control product, the concentrations of the biological samples added with the standard are shown in the following table.

Analyte LQCμg/mL MQCμg/mL HQCμg/mL TMAO 0.05～0.5 0.5～1 1～3 Choline 0.5～2 2～5 5～15 Betaine 0.5～5 5～15 20～50 Creatinine 1～10 10～20 30～60 L-Carnitine 1～10 10～30 30～100

7. The analytical method according to claim 1, wherein the analytical column used in the high performance liquid chromatography triple quadrupole tandem mass spectrometer is a HI L IC column or an Amide column, the column temperature is 30-40 ℃, and the sample size is 2-10 μ L.

8. The analytical method according to claim 1, wherein the mobile phase of the analytical chromatographic column comprises an aqueous ammonium formate solution containing 0.1-0.5% formic acid 5 mmol/L% and an acetonitrile solution containing 0.1-0.5% formic acid at a flow rate of 0.2-0.6 m L/min, the analytical chromatographic column adopts a gradient elution mode, and the volume ratio of the mobile phase A to the mobile phase B is 100-0% -0-100%.

9. The analytical method of claim 1, wherein: the mass spectrometry conditions were as follows: an ion source: an electrospray ion source; ion source temperature: 300-600 ℃; ion source voltage: 4500-5000V; collision gas: 9-10 units; air curtain air: 40-50 psi; GS1 at 50-60 psi; GS 2: 50-60 psi; the detection mode is as follows: and detecting positive ions.

10. The analytical method of claim 1, wherein: the detection of the high performance liquid chromatography triple quadrupole tandem mass spectrometer adopts an electrospray ionization source (ESI) and a multi-reflection monitoring (MRM) scanning mode, and specific ion pairs are shown in the table below.