阅读说明：本技术 一种同时检测同型半胱氨酸及其代谢相关物质的方法 (Method for simultaneously detecting homocysteine and metabolic related substances thereof ) 是由 刘霞 王猛 米文珍 张继钢 孙海燕 孙翠霞 靳海娇 时瑞海 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种同时检测同型半胱氨酸及其代谢相关物质的方法。所述方法步骤包括：将血液样本经过前处理,在采用液相色谱-串联质谱法进行分析检测,最后采用内标法进行定量。该方法可以同时检测人血清中同型半胱氨酸及其代谢相关物质(甲硫氨酸、吡哆醇、叶酸和5-甲基四氢叶酸)的含量,并进行精确的定性和定量分析,是一种样本处理简单、通量高、结果可靠的检测方法。(The invention relates to a method for simultaneously detecting homocysteine and relevant metabolic substances thereof. The method comprises the following steps: the blood sample is pretreated, and is analyzed and detected by adopting a liquid chromatography-tandem mass spectrometry method, and finally, an internal standard method is adopted for quantification. The method can simultaneously detect the content of homocysteine and relevant metabolic substances (methionine, pyridoxine, folic acid and 5-methyltetrahydrofolic acid) in human serum, and performs accurate qualitative and quantitative analysis, and is a detection method with simple sample processing, high flux and reliable result.)

1. A method for simultaneously detecting homocysteine and metabolic related substances thereof is characterized in that: the method comprises the following steps: the blood sample is pretreated, and is analyzed and detected by adopting a liquid chromatography-tandem mass spectrometry method, and finally, an internal standard method is adopted for quantification.

2. The method of claim 1, wherein the blood sample is plasma or serum.

3. The method of claim 1, wherein the homocysteine and its metabolic-related substances are: homocysteine Hcy, methionine Met, methylmalonic acid MMA, pyridoxine PN, folic acid FA, and 5-methyltetrahydrofolic acid 5-MTHF.

4. The method of claim 1, wherein the isotopic internal standards for each analyte to be quantified using the internal standard method are: homocysteine-D4, methionine-¹³C5, methylmalonic acid-D3, pyridoxine-¹³C4, folic acid-¹³C5 and 5-methyltetrahydrofolate-¹³C5。

5. The method of claim 1, wherein the sample pre-processing operation is to: adding 150 mu L of a calibrator and a serum sample into a 2mL centrifuge tube, adding 20 mu L of an internal standard solution, adding 20 mu L of a reducing agent solution, performing vortex incubation for 30min, adding 460 mu L of methanol to precipitate protein, performing vortex oscillation for 3min, sucking 500 mu L of a sample mixed solution, filtering by a protein phospholipid filter plate, drying filtrate by nitrogen, and then re-dissolving by ultrapure water.

6. The method of claim 1, wherein the step of performing analytical detection by liquid chromatography-tandem mass spectrometry comprises the following steps:

(1) preparing a standard working solution: preparing standard analyte stock solutions with water, preparing mixed standard solutions with different concentrations with BSA-PBS solution, subpackaging, and storing at-20 deg.C for use;

(2) preparing an internal standard solution: preparing isotope internal standard storage liquid of each analyte by using water, diluting the isotope internal standard storage liquid by using ultrapure water, mixing to obtain mixed isotope internal standard product solution, subpackaging, and storing at-20 ℃ for later use;

(3) liquid chromatography tandem mass spectrometry detection: after a sample to be detected enters a chromatographic column for separation through a gradient elution mode, a positive ion electric spray ionization multi-ion reaction monitoring mode is adopted to carry out qualitative and quantitative detection on a substance to be detected; chromatographic conditions are as follows: the mobile phase A is water containing formic acid, the mobile phase B is acetonitrile containing formic acid, and gradient elution is carried out; mass spectrum conditions: multiple ion reaction monitoring of positive ion electrospray ionization.

7. The method according to claim 6, wherein the chromatographic conditions are in particular: a chromatographic column: column T3C 18, column temperature: 40 ℃; mobile phase A: 0.1% aqueous formic acid, mobile phase B: 0.1% formic acid acetonitrile solution; gradient elution conditions: 0-1 min, 99% A, 1-1.5 min, 99-30% A, 1.5-2.5 min, 30-5% A, 2.5-3.5 min, 5% A, 3.5-5.0 min, 99% A, flow rate of 0.4mL/min, sample injection amount of 10 muL.

8. The method according to claim 6, wherein the mass spectrometry conditions are in particular: multi-ion reaction monitoring mode using positive ion electrospray ionization, capillary voltage: 3.90kV, taper hole voltage: 45V, ion source temperature: 150 ℃, desolventizing gas flow: 650L/Hr, desolvation gas temperature: at 200 ℃.

9. The method of claim 5, wherein the reducing agent is dithiothreitol.

10. The method of claim 6, wherein the BSA-PBS solution is 1.0% bovine serum albumin phosphate buffered saline.

Technical Field

The invention belongs to the technical field of joint detection of amino acid and vitamin, and particularly relates to a method for simultaneously detecting homocysteine and metabolic related substances thereof.

Background

Homocysteine is a sulfur-containing amino acid, mainly derived from methionine metabolism in dietary intake, is an important intermediate product in the methionine metabolism process, and does not participate in protein synthesis per se. Methionine is one of essential amino acids constituting the human body and is involved in protein synthesis. If methionine is deficient, protein synthesis in vivo is hindered, causing body damage. There are two major metabolic pathways for homocysteine metabolism within the cell: one is the re-methylation of methionine, i.e. the re-synthesis of methionine by homocysteine through the cycle of folic acid; another route is the transthio route.

The normal content of homocysteine in human blood is generally 5-15 mu mol/L, and when the concentration of total homocysteine exceeds 15 mu mol/L, hyperhomocysteinemia can be caused. The hyperhomocysteinemia is caused by various reasons, mainly due to poor high methionine dietary habits, folic acid and vitamin B12 deficiency, renal insufficiency and the like. Many studies show that hyperhomocysteinemia is an independent risk factor of atherosclerosis and coronary heart disease, and is proved to have close relationship with respiratory system diseases, pregnancy-related diseases, diabetes, senile dementia, liver diseases, tumors, senile osteoporosis and the like.

At present, the commonly used detection methods of amino acids or vitamins mainly comprise an enzyme-linked immunoassay method, a chemiluminescence method, a high performance liquid chromatography, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the like. The antibody used in the immunoassay can also identify other vitamin derivatives, and the antibody antigen reaction is usually less than 100%, so the result difference is large, the specificity is poor, the reliability of the immunoassay is not high, and especially for detecting vitamins with low content. The liquid chromatography is mainly used for measuring samples with high amino acid and vitamin contents due to limited detection limit, and generally can only measure single components or a plurality of components with high contents at the same time. The LC-MS/MS has the advantages of high detection sensitivity, good accuracy, easy high flux, capability of detecting various metabolism related substances at one time and the like, so the LC-MS/MS is gradually popularized in the aspect of detecting amino acids and water-soluble vitamins. The current LC-MS/MS detection method can only carry out quantitative analysis on one amino acid, three sulfur-containing amino acids or a plurality of single water-soluble vitamins of homocysteine. Due to the complex components of blood plasma/serum and low content of water-soluble vitamins, impurities seriously affect the accuracy of a detection result when quantitative analysis is carried out. Therefore, in order to reduce the matrix effect during detection, the detector performs detection analysis on different amino acids and vitamins one by one. This has the following disadvantages: the detection cost is high, the operation is complex, the consumed time is long, and the blood volume required by detection is large. At present, an effective method capable of simultaneously detecting homocysteine, methionine and water-soluble vitamins in serum/plasma does not exist.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, the present application provides a method for simultaneously detecting homocysteine and its metabolic related substances. The method can simultaneously detect the content of homocysteine and relevant metabolic substances (methionine, pyridoxine, folic acid and 5-methyltetrahydrofolic acid) in human serum, and performs accurate qualitative and quantitative analysis, and is a detection method with simple sample processing, high flux and reliable result.

A method for simultaneously detecting homocysteine and metabolic related substances thereof, said method comprising the steps of: the blood sample is pretreated, and is analyzed and detected by adopting a liquid chromatography-tandem mass spectrometry method, and finally, an internal standard method is adopted for quantification.

Further, the blood sample is plasma or serum.

Further, the homocysteine and relevant metabolic substances thereof are: homocysteine Hcy, methionine Met, methylmalonic acid MMA, pyridoxine PN, folic acid FA, and 5-methyltetrahydrofolic acid 5-MTHF.

Further, the isotope internal standard of each analyte for quantification by the internal standard method is as follows: homocysteine-D4, methionine-¹³C5, methylpropanediacid-D3, pyridoxine-¹³C4, folic acid-¹³C5 and 5-methyltetrahydrofolate-¹³C5。

Further, the sample preprocessing operation is: adding 150 mu L of a calibrator and a serum sample into a 2mL centrifuge tube, adding 20 mu L of an internal standard solution, adding 20 mu L of a reducing agent solution, performing vortex incubation for 30min, adding 460 mu L of methanol to precipitate protein, performing vortex oscillation for 3min, sucking 500 mu L of a sample mixed solution, filtering by a protein phospholipid filter plate, drying filtrate by nitrogen, and then re-dissolving by ultrapure water.

Further, the specific operation steps of analyzing and detecting by adopting the liquid chromatography-tandem mass spectrometry method comprise:

(1) preparing a standard working solution: preparing standard analyte stock solutions with water, preparing mixed standard solutions with different concentrations with BSA-PBS solution, subpackaging, and storing at-20 deg.C for use;

(2) preparing an internal standard solution: preparing isotope internal standard storage liquid of each analyte by using water, diluting the isotope internal standard storage liquid by using ultrapure water, mixing to obtain mixed isotope internal standard product solution, subpackaging, and storing at-20 ℃ for later use;

(3) liquid chromatography tandem mass spectrometry detection: after a sample to be detected enters a chromatographic column for separation through a gradient elution mode, a positive ion electric spray ionization multi-ion reaction monitoring mode is adopted to carry out qualitative and quantitative detection on a substance to be detected; chromatographic conditions are as follows: the mobile phase A is water containing formic acid, the mobile phase B is acetonitrile containing formic acid, and gradient elution is carried out; mass spectrum conditions: multiple ion reaction monitoring of positive ion electrospray ionization.

Further, the chromatographic conditions are specifically as follows: a chromatographic column: column T3C 18, column temperature: 40 ℃; mobile phase A: 0.1% aqueous formic acid, mobile phase B: 0.1% formic acid acetonitrile solution; gradient elution conditions: 0-1 min, 99% A, 1-1.5 min, 99-30% A, 1.5-2.5 min, 30-5% A, 2.5-3.5 min, 5% A, 3.5-5.0 min, 99% A, flow rate of 0.4mL/min, sample injection amount of 10 muL.

Further, the mass spectrometry conditions are specifically as follows: multi-ion reaction monitoring mode using positive ion electrospray ionization, capillary voltage: 3.90kV, taper hole voltage: 45V, ion source temperature: 150 ℃, desolventizing gas flow: 650L/Hr, desolvation gas temperature: at 200 ℃.

Further, the reducing agent is dithiothreitol.

Further, the BSA-PBS solution is 1.0% bovine serum albumin phosphate buffer solution.

Has the advantages that:

the invention establishes a method for simultaneously detecting homocysteine and relevant metabolic substances thereof by optimizing a sample pretreatment method and conditions of liquid chromatography-tandem mass spectrometry. The method can simultaneously detect the content of homocysteine and relevant metabolic substances (methionine, pyridoxine, folic acid and 5-methyltetrahydrofolic acid) in human serum, and performs accurate qualitative and quantitative analysis, thereby being a detection method with simple sample processing, high flux and reliable result.

The extraction method used in the invention can remove more impurities, reduce the matrix effect during detection, has simple and rapid operation, and can sensitively detect the content of each analyte, especially folic acid and pyridoxine with lower content in human body.

The invention can simultaneously detect the content of 6 substances related to homocysteine and metabolism thereof, reduces the detection cost, saves time, reduces the blood volume of a detected person, can assist a clinician in judging the Hcy level, assists in analyzing possible reasons causing the abnormal Hcy level, and provides a basis for supplementing vitamin B6 and folic acid.

The invention uses the liquid chromatogram-tandem mass spectrometry to detect homocysteine and relevant metabolic substances thereof in serum, has strong specificity, high sensitivity, high flux and objective result, is easy to analyze, and is particularly suitable for clinical popularization and application.

The method of the invention selects specific parent ions and specific daughter ions respectively for each analyte, takes the relative retention time of the analytes as a qualitative basis, and prepares a standard curve for quantification by using a standard substance, thereby ensuring the accuracy of the detection result.

The invention realizes the purpose of simultaneously detecting Hcy and 5 metabolism related substances in a serum sample by applying the LC-MS technology for the first time, and the specific parent ions and daughter ions of the analyte are selected to ensure the specificity of the detected substance and reduce the influence of interferents. The method is simple, convenient and quick to operate, the analysis time is only 6min, the flux is high, the cost is low, the homocysteine and related metabolite levels in a human body can be effectively monitored, the possible reasons of the Hcy level abnormality can be subjected to auxiliary analysis, the method has guiding significance for reasonable and safe supplement of vitamin B6 and folic acid, and the method is easy to popularize and popularize in clinic.

Drawings

FIG. 1 is a total ion flow diagram of an embodiment of the present invention;

FIG. 2 is a chromatogram of homocysteine and its internal standard according to an embodiment of the present invention;

FIG. 3 is a chromatogram of methionine and its internal standard of an embodiment of the present invention;

FIG. 4 is a chromatogram of pyridoxine and its internal standard according to an embodiment of the present invention;

FIG. 5 is a chromatogram of methylmalonic acid and its internal standard according to an example of the present invention;

FIG. 6 is a chromatogram of folic acid and its internal standard according to an embodiment of the present invention;

FIG. 7 is a chromatogram of 5-methyltetrahydrofolic acid and its internal standard according to an example of the present invention.

Detailed Description

Examples

A method for simultaneously detecting homocysteine and metabolic related substances thereof comprises the following steps:

(1) sample treatment: taking 120 mu L of serum sample, adding the serum sample into a 2mL centrifuge tube, adding 20 mu L of internal standard solution, adding 20 mu L of dithiothreitol solution, performing vortex incubation for 30min, adding 460 mu L of methanol to precipitate protein, performing vortex oscillation for 3min, sucking 500 mu L of sample mixed solution, filtering the sample mixed solution by a 96-pore protein phospholipid filter plate, collecting filtrate, drying the filtrate by nitrogen, and re-dissolving the dried filtrate by ultrapure water. Through the treatment, the precipitated protein is effectively separated, impurities are removed, ion interference is reduced, matrix effect is reduced, and meanwhile, the analyte can be retained to the maximum extent, so that the analyte is prevented from being decomposed or oxidized, and the most real content of the analyte to be detected is detected.

(2) Preparing a standard working solution: hcy, Met and MMA standards were dissolved with ultrapure water, PN standard was dissolved with 0.1mol/L hydrochloric acid solution, and FA and 5-MTHF standards were dissolved with 0.1mol/L sodium hydroxide solution to obtain stock solutions of the respective analyte standards. Then diluting with an aqueous solution containing an antioxidant to prepare a mixed standard solution of 6 gradients (Hcy: 2, 4, 8, 16, 32, 64 mu mol/L; Met: 2, 4, 8, 16, 32, 64 mu mol/L; MMA: 2, 4, 8, 16, 32, 64 mu mol/L; PN: 8, 16, 32, 64, 128, 256 nmol/L; FA: 8, 16, 32, 64, 128, 256 nmol/L; 5-MTHF: 8, 16, 32, 64, 128, 256nmol/L) and subpackaging the mixed standard solution into a 2mL brown bottle and preserving at-70 ℃ and below for later use;

(3) LC-MS detection: after a sample to be detected enters a chromatographic column for separation through a gradient elution mode (the gradient elution condition is shown in table 1), a positive ion electrospray ionization multi-ion reaction monitoring mode (MRM mass spectrum parameters are shown in table 2) is adopted to carry out quantitative detection on the substance to be detected.

TABLE 1 gradient elution conditions

TABLE 2 MRM Mass Spectrometry parameters

As shown in fig. 1 to 7, a standard is used to prepare a standard curve, a ratio (y) of each analyte in each standard to the peak area of the corresponding internal standard is calculated, a standard curve is drawn for the ratio (y) of the identification concentration (x) of each analyte in the calibrator to the corresponding peak area, a standard curve equation is fitted, and the ratio of the peak area of each analyte in the sample to be measured to the peak area of the corresponding internal standard is substituted into the fitted standard curve equation, so that the concentration of each analyte in the sample can be calculated.

Through specific experiments, a general mobile phase and a flow velocity gradient are optimized, so that homocysteine and 5 metabolic related substances can be detected simultaneously. In addition, each analyte selects specific parent ions, specific daughter ions, specific cone hole voltage and specific collision energy, so that the signal intensity of each analyte is improved, the influence of matrix effect is reduced, and the method is favorable for detecting the content of each analyte, especially for detecting the water-soluble vitamins with low content.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments of the invention are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.