CN113686998A - Method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology - Google Patents
Method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology Download PDFInfo
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- 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
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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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
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Abstract
The invention discloses a method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS technology, which has the technical scheme that: s1, collecting a sample; s2, sample processing; s3, LC-MS/MS measurement; the method for determining the trimethylamine oxide in the dried blood spots by the LC-MS/MS technology has the beneficial effects that: the method is free from derivatization, simple and quick, shortens the pretreatment time, and simultaneously avoids the influence of high temperature on trimethylamine oxide in the derivatization process, so that the detection is more accurate; acetonitrile has a good protein removing effect, trimethylamine oxide is dissolved in the acetonitrile, and detection of the trimethylamine oxide in the dry blood spots by the LC-MS/MS technology is a quick, accurate and sensitive method, can be applied to clinical detection and scientific research, and is particularly suitable for population research of large samples.
Description
Technical Field
The invention relates to the technical field of medical biological detection, in particular to a method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS (liquid chromatography-mass spectrometry) technology.
Background
Cardiovascular and cerebrovascular diseases are the general names of cardiovascular and cerebrovascular diseases, which generally refer to ischemic or hemorrhagic diseases of heart, brain and systemic tissues caused by hyperlipidemia, blood viscosity, atherosclerosis, hypertension and the like, and the prepared clinical detection means of the cardiovascular diseases is trimethylamine oxide detection, and the trimethylamine oxide (TMAO) is a polar small molecular compound produced by digesting red meat, eggs, dairy products and salted fish by intestinal bacteria and then processing the red meat, eggs, dairy products and salted fish by liver. In recent years, with the progress of research on intestinal flora, scientists have found that TMAO is closely related to various diseases such as cardiovascular diseases, stroke, diabetes, chronic kidney diseases, and cancer, and thus TMAO is receiving much attention. TMAO is a novel biomarker for predicting cardiovascular and cerebrovascular disease risk, and can well and independently predict the cardiovascular and cerebrovascular disease risk. The TMAO quantitative analysis can provide reliable basis for diagnosis, risk stratification, prediction and the like of cardiovascular and cerebrovascular diseases. The elevated TMAO concentration is significantly correlated with the risk of major adverse cardiovascular events, and the relative increase in risk does not vary with changes in body mass index, diabetes, cardiovascular disease history, renal dysfunction, etc., and can be used as an accurate screening tool for predicting the risk of future heart attack, stroke, and death in people who are not identified by traditional risk factors and blood tests. The detection of the metabolic small molecule trimethylamine oxide is helpful for guiding clinicians to provide individualized nutrition suggestions for preventing cardiovascular diseases like cholesterol, triglyceride or blood glucose levels, the traditional TMAO detection methods are spectrophotometry, capillary electrophoresis and chromatography, the sensitivity and accuracy are limited, the repeatability is poor, and a liquid chromatography tandem mass spectrometry (LC-MS/MS) has the advantages of short analysis time, high sensitivity, high specificity and the like, shows extremely high superiority and application prospect, and is the most commonly used method for TMAO detection at present.
The existing trimethylamine oxide detection method which is applied more is a gas chromatography-mass spectrometry combined method, although the gas chromatography-mass spectrometry combined method has high sensitivity, the operation is complicated and long in time consumption, and the trimethylamine oxide can be degraded in the sample derivatization process, so that the accuracy of the determination result is influenced.
Disclosure of Invention
Therefore, the invention provides a method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS technology, which aims to solve the problems of complicated operation, high time consumption and inaccurate detection result of the trimethylamine oxide detection.
In order to achieve the above purpose, the invention provides the following technical scheme: a method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS technology is characterized by comprising the following steps: the method comprises the following specific steps:
s1, collecting samples: pricking a finger with a needle, dropping a small amount of fingertip blood on filter paper, collecting fingertip blood or venous blood by using blood collection filter paper, wherein 3 blood spots are required for a qualified filter paper dried blood sheet, the diameter of each blood spot is more than 8 mm, the blood drops naturally permeate, the blood spots on the front side and the back side of the filter paper are consistent and pollution-free, the blood spots have no blood seepage ring, drying is carried out at room temperature, and after drying is finished, a trimethylamine oxide sample is placed into a sealing bag containing a drying agent for storage;
s2, sample processing: utilizing a puncher to punch a blood part on a trimethylamine oxide sample and mark the blood part as an A sample, taking the A sample with the diameter of 6mm, adding ultrapure water and protein removing liquid, performing ultrasonic treatment, adding 2mL of water, performing vortex for 30s, adding an ammonium formate solution, performing vortex mixing for often, performing reciprocating oscillation after vortex mixing, and performing solution centrifugation;
s3, LC-MS/MS measurement: and (3) taking the upper organic phase, drying the upper organic phase in another test tube under 40 ℃ air flow, sucking 90 mu L of the upper organic phase, transferring the upper organic phase to a sealed sample injection bottle, loading the sealed sample injection bottle on the machine for detection, and performing qualitative determination by using retention time and a mass spectrum and quantitative determination by using an internal standard method to detect the trimethylamine oxide.
Specifically, the LC-MS/MS technology is a technology in which a sample is subjected to chromatographic separation, ionized by an ion source to generate ions with a certain charge and mass number, the ions are separated according to mass-to-charge ratio (m/z) by using different mass analyzers to obtain mass spectrograms arranged in a mass sequence, and qualitative and quantitative analysis is performed by analyzing and processing the mass spectrograms, and the whole blood volume involved in the manufacture of dried blood spots is less.
Preferably, in the step S1, drying is carried out at room temperature for 120-150 min.
Specifically, the dried blood spot blood sample is coagulated by drying at room temperature.
Preferably, in the step S2, the amount of the ultrapure water added is 200-260. mu.L, and the amount of the protein-removing liquid is 10-14. mu.L.
Specifically, different amounts of ultrapure water are used for soaking dry blood spot samples, so that blood samples with different concentrations are obtained, and the protein in the blood can be precipitated by the protein removing liquid.
Preferably, in the step S2, the ultrasonic condition is controlled at 40kHz and 200W, and the ultrasonic treatment is carried out for 15-17 min.
Specifically, the protein is coagulated by ultrasound.
Preferably, in step S2, the ammonium formate solution is added in an amount of 3-5mL, and vortex mixing is performed for 1min, reciprocal shaking is performed for 10min (240 times. min-1), and centrifugation is performed for 5min (3500 r. min-1).
Specifically, the protein was precipitated by centrifugation.
Preferably, in step S3, the a sample solution is placed at 4 ℃ after centrifugation, and the measurement is performed at 0h, 24h and 72h after separation and the concentration change is measured.
Specifically, the sample detection after different times is carried out, and better trimethylamine oxide detection data are obtained.
Preferably, the deproteinizing solution is obtained by diluting acetonitrile solution with ultrapure water.
In particular, acetonitrile solutions have excellent solvent properties and can dissolve a variety of organic, inorganic, and gaseous materials.
Preferably, the preparation method of the ammonium formate solution comprises the following steps: 0.6306g of ammonium formate was weighed out, 10mmol/L ammonium formate solution was prepared with ultrapure water, the pH was adjusted to 3.0 with formic acid, filtration was carried out with 0.45m filter membrane, and a suitable ammonium formate solution was obtained by ultrasonic degassing treatment.
Specifically, ammonium formate is colorless and crystalline, deliquescent, soluble in water and ethanol, and its aqueous solution is acidic.
Preferably, the LC-MS/MS determination of chromatographic conditions: the chromatographic column is as follows: UltimateSiO, column (2.1mm x 100mm, 5Ixm, WelchMaterials, Inc), mobile phase: acetonitrile: 10mmol/L, ammonium formate (ph3.0) ═ 70%: 30% (v/v), isocratic elution, flow rate: 0.4mL/min, sample size: 5 μ L, column temperature: at 30 ℃.
Preferably, the LC-MS/MS determines mass spectrometry conditions: the ion source is an electrospray ionization source (ESI source), the spraying voltage is 4.2kV, the ion source temperature is 300 ℃, the pressure of ion source gas 1(N2) is 0.3MPa, the pressure of ion source gas 2(N2) is 0.3MPa, the pressure of gas curtain gas (N2) is 0.07MPa, the pressure of collision gas (N2) is 0.03MPa, the positive ion mode detection is carried out, the cluster removal voltage (DP) is 54V, the scanning mode is multi-reaction monitoring (MRM), and the Collision Energy (CE) is 22V.
The embodiment of the invention has the following advantages:
the method is free from derivatization, simple and quick, shortens the pretreatment time, and simultaneously avoids the influence of high temperature on trimethylamine oxide in the derivatization process, so that the detection is more accurate; acetonitrile has a good protein removing effect, trimethylamine oxide is dissolved in the acetonitrile, the detection of the trimethylamine oxide in dried blood spots by the high performance liquid chromatography tandem mass spectrometry is a quick, accurate and sensitive method, and can be applied to clinical detection and research, particularly to the research of large-sample crowds, the influence of the storage temperature and time on the concentration of the trimethylamine oxide in the dried blood spots is noticed when samples are stored, the pretreatment process of the samples determined by the LC-MS/MS technology is simpler, the consumed time is short, the cost is reduced, the problem that the large samples are limited during determination is avoided, and the whole blood volume involved in the manufacture of the dried blood spots is less.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the invention provides a method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS technology, which comprises the following steps:
s1, collecting samples: pricking a finger with a needle, dropping a small amount of fingertip blood on filter paper, collecting fingertip blood or venous blood by using blood collection filter paper, wherein 3 blood spots are required for a qualified filter paper dried blood sheet, the diameter of each blood spot is more than 8 mm, the blood drops naturally permeate, the blood spots on the front side and the back side of the filter paper are consistent and pollution-free, the blood spots have no blood seepage ring, drying is carried out at room temperature, and after drying is finished, a trimethylamine oxide sample is placed into a sealing bag containing a drying agent for storage;
s2, sample processing: using a puncher to punch a blood part on a trimethylamine oxide sample and mark the blood part as an A sample, taking the A sample with the diameter of 6mm, adding 200 mu L of ultrapure water, 10 mu L of protein removing liquid, performing ultrasound, controlling the ultrasonic conditions at 40kHz and 200W, performing ultrasound for 15min, adding 2mL of water, performing vortex mixing for 30s, adding 3mL of ammonium formate solution, performing vortex mixing for 1min, performing reciprocating oscillation for 10min (240 times. min < -1 >), and centrifuging for 5min (3500 r. min < -1 >);
s3, LC-MS/MS measurement: and (3) after centrifugation, placing the sample solution A in 4 ℃, measuring the separated sample solution for 0h, 24h and 72h, measuring the concentration change, separating an upper organic phase in another test tube, drying the separated upper organic phase under air flow at 40 ℃, sucking 90 mu L of the separated sample solution, transferring the separated upper organic phase to a sealed sample bottle, and then detecting the sample solution on a machine, wherein the retention time and mass spectrogram are used for qualitative determination, and an internal standard method is used for quantitative determination, so that the trimethylamine oxide is measured.
Further, the protein removing liquid is obtained by diluting an acetonitrile solution with ultrapure water.
Further, the preparation method of the ammonium formate solution comprises the following steps: 0.6306g of ammonium formate was weighed out, 10mmol/L ammonium formate solution was prepared with ultrapure water, the pH was adjusted to 3.0 with formic acid, filtration was carried out with 0.45m filter membrane, and a suitable ammonium formate solution was obtained by ultrasonic degassing treatment.
Further, the LC-MS/MS determines the chromatographic conditions: the chromatographic column is as follows: UltimateSiO, column (2.1mm x 100mm, 5Ixm, WelchMaterials, Inc), mobile phase: acetonitrile: 10mmol/L, ammonium formate (ph3.0) ═ 70%: 30% (v/v), isocratic elution, flow rate: 0.4mL/min, sample size: 5 μ L, column temperature: at 30 ℃.
Further, the LC-MS/MS determines mass spectrometry conditions: the ion source is an electrospray ionization source (ESI source), the spraying voltage is 4.2kV, the ion source temperature is 300 ℃, the pressure of ion source gas 1(N2) is 0.3MPa, the pressure of ion source gas 2(N2) is 0.3MPa, the pressure of gas curtain gas (N2) is 0.07MPa, the pressure of collision gas (N2) is 0.03MPa, the positive ion mode detection is carried out, the cluster removal voltage (DP) is 54V, the scanning mode is multi-reaction monitoring (MRM), and the Collision Energy (CE) is 22V.
Example 2:
the invention provides a method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS technology, which comprises the following steps:
s1, collecting samples: pricking a finger with a needle, dripping a small amount of fingertip blood on filter paper, drying at room temperature for 135min, and storing a trimethylamine oxide sample in a sealed bag containing a desiccant for later use;
s2, sample processing: using a puncher to punch a blood part on a trimethylamine oxide sample and mark the blood part as an A sample, taking the A sample with the diameter of 6mm, adding 230 mu L of ultrapure water, adding 12 mu L of protein removing liquid, performing ultrasound, controlling the ultrasonic conditions at 40kHz and 200W, performing ultrasound for 16min, adding 2mL of water, performing vortex mixing for 30s, adding 4mL of ammonium formate solution, performing vortex mixing for 1min, performing reciprocating oscillation for 10min (240 times. min < -1 >), and centrifuging for 5min (3500 r. min < -1 >);
s3, LC-MS/MS measurement: and (3) after centrifugation, placing the sample solution A in 4 ℃, measuring the separated sample solution for 0h, 24h and 72h, measuring the concentration change, separating an upper organic phase in another test tube, drying the separated upper organic phase under air flow at 40 ℃, sucking 90 mu L of the separated sample solution, transferring the separated upper organic phase to a sealed sample bottle, and then detecting the sample solution on a machine, wherein the retention time and mass spectrogram are used for qualitative determination, and an internal standard method is used for quantitative determination, so that the trimethylamine oxide is measured.
Further, the protein removing liquid is obtained by diluting an acetonitrile solution with ultrapure water.
Further, the preparation method of the ammonium formate solution comprises the following steps: 0.6306g of ammonium formate was weighed out, 10mmol/L ammonium formate solution was prepared with ultrapure water, the pH was adjusted to 3.0 with formic acid, filtration was carried out with 0.45m filter membrane, and a suitable ammonium formate solution was obtained by ultrasonic degassing treatment.
Further, the LC-MS/MS determines the chromatographic conditions: the chromatographic column is as follows: UltimateSiO, column (2.1mm x 100mm, 5Ixm, WelchMaterials, Inc), mobile phase: acetonitrile: 10mmol/L, ammonium formate (ph3.0) ═ 70%: 30% (v/v), isocratic elution, flow rate: 0.4mL/min, sample size: 5 μ L, column temperature: at 30 ℃.
Further, the LC-MS/MS determines mass spectrometry conditions: the ion source is an electrospray ionization source (ESI source), the spraying voltage is 4.2kV, the ion source temperature is 300 ℃, the pressure of ion source gas 1(N2) is 0.3MPa, the pressure of ion source gas 2(N2) is 0.3MPa, the pressure of gas curtain gas (N2) is 0.07MPa, the pressure of collision gas (N2) is 0.03MPa, the positive ion mode detection is carried out, the cluster removal voltage (DP) is 54V, the scanning mode is multi-reaction monitoring (MRM), and the Collision Energy (CE) is 22V.
Example 3:
the invention provides a method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS technology, which comprises the following steps:
s1, collecting samples: pricking a finger with a needle, dripping a small amount of fingertip blood on filter paper, drying at room temperature for 150min, and storing a trimethylamine oxide sample in a sealed bag containing a drying agent for later use;
s2, sample processing: using a puncher to punch a blood part on a trimethylamine oxide sample and mark the blood part as an A sample, taking the A sample with the diameter of 6mm, adding 260 mu L of ultrapure water, 14 mu L of protein removing liquid, performing ultrasound, controlling the ultrasonic conditions at 40kHz and 200W, performing ultrasound for 17min, adding 2mL of water, performing vortex mixing for 30s, adding 5mL of ammonium formate solution, performing vortex mixing for 1min, performing reciprocating oscillation for 10min (240 times. min < -1 >), and centrifuging for 5min (3500 r. min < -1 >);
s3, LC-MS/MS measurement: and (3) after centrifugation, placing the sample solution A in 4 ℃, measuring the separated sample solution for 0h, 24h and 72h, measuring the concentration change, separating an upper organic phase in another test tube, drying the separated upper organic phase under air flow at 40 ℃, sucking 90 mu L of the separated sample solution, transferring the separated upper organic phase to a sealed sample bottle, and then detecting the sample solution on a machine, wherein the retention time and mass spectrogram are used for qualitative determination, and an internal standard method is used for quantitative determination, so that the trimethylamine oxide is measured.
Further, the protein removing liquid is obtained by diluting an acetonitrile solution with ultrapure water.
Further, the preparation method of the ammonium formate solution comprises the following steps: 0.6306g of ammonium formate was weighed out, 10mmol/L ammonium formate solution was prepared with ultrapure water, the pH was adjusted to 3.0 with formic acid, filtration was carried out with 0.45m filter membrane, and a suitable ammonium formate solution was obtained by ultrasonic degassing treatment.
Further, the LC-MS/MS determines the chromatographic conditions: the chromatographic column is as follows: UltimateSiO, column (2.1mm x 100mm, 5Ixm, WelchMaterials, Inc), mobile phase: acetonitrile: 10mmol/L, ammonium formate (ph3.0) ═ 70%: 30% (v/v), isocratic elution, flow rate: 0.4mL/min, sample size: 5 μ L, column temperature: at 30 ℃.
Further, the LC-MS/MS determines mass spectrometry conditions: the ion source is an electrospray ionization source (ESI source), the spraying voltage is 4.2kV, the ion source temperature is 300 ℃, the pressure of ion source gas 1(N2) is 0.3MPa, the pressure of ion source gas 2(N2) is 0.3MPa, the pressure of gas curtain gas (N2) is 0.07MPa, the pressure of collision gas (N2) is 0.03MPa, the positive ion mode detection is carried out, the cluster removal voltage (DP) is 54V, the scanning mode is multi-reaction monitoring (MRM), and the Collision Energy (CE) is 22V.
EXAMPLES 1-3 determination of trimethylamine oxide concentration data in dried blood spots by LC-MS/MS technique based on different concentrations of blood sample solutions and different ratios of deproteinizing solution and ammonium formate solution added
Thus, it can be seen that the concentration of trimethylamine oxide measured by the method of measuring trimethylamine oxide in dried blood spots by LC-MS/MS technique in example 1 is the highest and most accurate.
The above table shows the effect of repeated tests on the determination of trimethylamine oxide in dried blood spots by LC-MS/MS technique of example 1 on the determination of the LC-MS/MS technique after different standing times at the same temperature:
from the above table, it can be seen that the blood sample trimethylamine oxide concentration change is not obvious after being stored for 24 hours at 4 ℃, and the concentration change is within 10%, but the concentration rise is obvious after being stored for 72 hours, and the concentration change is 15.82%, so the sample should be subjected to LC-MS/MS technical determination as soon as possible after centrifugation.
The above description is only a preferred embodiment of the present invention, and any person skilled in the art may modify the present invention or modify it into an equivalent technical solution by using the technical solution described above. Therefore, any simple modifications or equivalent substitutions made in accordance with the technical solution of the present invention are within the scope of the claims of the present invention.
Claims (10)
1. A method for determining trimethylamine oxide in dried blood spots by an LC-MS/MS technology is characterized by comprising the following steps: the method comprises the following specific steps:
s1, collecting samples: pricking a finger with a needle, dropping a small amount of fingertip blood on filter paper, collecting fingertip blood or venous blood by using blood collection filter paper, wherein 3 blood spots are required for a qualified filter paper dried blood sheet, the diameter of each blood spot is more than 8 mm, the blood drops naturally permeate, the blood spots on the front side and the back side of the filter paper are consistent and pollution-free, the blood spots have no blood seepage ring, drying is carried out at room temperature, and after drying is finished, a trimethylamine oxide sample is placed into a sealing bag containing a drying agent for storage;
s2, sample processing: utilizing a puncher to punch a blood part on a trimethylamine oxide sample and mark the blood part as an A sample, taking the A sample with the diameter of 6mm, adding ultrapure water and protein removing liquid, performing ultrasonic treatment, adding 2mL of water, performing vortex for 30s, adding an ammonium formate solution, performing vortex mixing for often, performing reciprocating oscillation after vortex mixing, and performing solution centrifugation;
s3, LC-MS/MS measurement: and (3) taking the upper organic phase, drying the upper organic phase in another test tube under 40 ℃ air flow, sucking 90 mu L of the upper organic phase, transferring the upper organic phase to a sealed sample injection bottle, loading the sealed sample injection bottle on the machine for detection, and performing qualitative determination by using retention time and a mass spectrum and quantitative determination by using an internal standard method to detect the trimethylamine oxide.
2. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: in the step S1, drying is carried out at room temperature for 120-150 min.
3. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: in the step S2, the amount of ultrapure water added is 200-260. mu.L, and the amount of protein-removing liquid is 10-14. mu.L.
4. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: in the step S2, the ultrasonic condition is controlled at 40kHz and 200W, and ultrasonic treatment is carried out for 15-17 min.
5. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: in the step S2, the ammonium formate solution is added in an amount of 3-5mL, vortex mixing is performed for 1min, reciprocating oscillation is performed for 10min (240 times. min-1), and centrifugation is performed for 5min (3500 r. min-1).
6. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: in the step S3, the A sample solution is placed at 4 ℃ after centrifugation, and the measurement is performed at 0h, 24h and 72h after separation and the concentration change is measured.
7. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: the protein removing liquid is obtained by diluting an acetonitrile solution with ultrapure water.
8. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: the preparation method of the ammonium formate solution comprises the following steps: 0.6306g of ammonium formate was weighed out, 10mmol/L ammonium formate solution was prepared with ultrapure water, the pH was adjusted to 3.0 with formic acid, filtration was carried out with 0.45m filter membrane, and a suitable ammonium formate solution was obtained by ultrasonic degassing treatment.
9. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: the chromatographic conditions are as follows: the chromatographic column is as follows: UltimateSiO, column (2.1mm x 100mm, 5Ixm, WelchMaterials, Inc), mobile phase: acetonitrile: 10mmol/L, ammonium formate (ph3.0) ═ 70%: 30% (v/v), isocratic elution, flow rate: 0.4mL/min, sample size: 5 μ L, column temperature: at 30 ℃.
10. The method for determining trimethylamine oxide in dried blood spots by LC-MS/MS technology according to claim 1, wherein: the mass spectrum conditions are as follows: the ion source is an electrospray ionization source (ESI source), the spraying voltage is 4.2kV, the ion source temperature is 300 ℃, the pressure of ion source gas 1(N2) is 0.3MPa, the pressure of ion source gas 2(N2) is 0.3MPa, the pressure of gas curtain gas (N2) is 0.07MPa, the pressure of collision gas (N2) is 0.03MPa, the positive ion mode detection is carried out, the cluster removal voltage (DP) is 54V, the scanning mode is multi-reaction monitoring (MRM), and the Collision Energy (CE) is 22V.
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