Determination of 13 core metabolic regulatory molecules under the same chromatographic system and construction of their detection and discrimination model

Abstract

The invention discloses a method for determining 13 metabolic regulation core molecules under a same chromatographic system and the construction of a detection discrimination model thereof; 13 metabolic regulation core molecules are methionine, homocysteine, cysteine, vitamin B2, 5-methyltetrahydrofolate, folic acid, pyridoxal phosphate, 4-pyridoxic acid, methylmalonic acid, betaine, choline, S-adenosylmethionine, S-adenosylhomocysteine. Low concentration acetic acid is used as a mobile phase additive in the detection system of the present invention, so that choline with very high polarity can be well retained on the chromatographic column, the response is also greatly increased, and the S-adenosylmethionine matrix effect is greatly reduced by WCX enrichment, and trace S-adenosylmethionine in serum can be accurately determined, further combined with specific chromatographic column selection, etc., it is achieved that 13 compounds are accurately determined under the same chromatographic system, and a discrimination model is established simultaneously, and a rapid interpretation of the clinical determination results of the 13 compounds is achieved.

Description

Technical Field

[0001] The invention belongs to the field of detection technology and relates to a method for determining 13 metabolic regulation core molecules under the same chromatographic system and the construction of a detection discrimination model thereof. Background Art

[0002] Hyperhomocysteinemia (HHcy) is a disease caused by elevated homocysteine ​​(Hcy) levels. It is closely related to cardiovascular and cerebrovascular diseases, hypertension, diabetes, kidney diseases, and pregnancy-related diseases, and affects almost every organ in the body. Hcy affects cell function and protein synthesis and regulation through multiple mechanisms such as oxidative damage, DNA methylation, and abnormal metabolism of sulfur-containing compounds, leading to pathological changes in tissues and organs. Hcy is metabolized in the body mainly through two pathways: remethylation and transsulfurization. The remethylation pathway is completed through two pathways: folic acid and betaine. The two complement each other but cannot replace each other; the transsulfurization pathway is carried out with the participation of vitamin B6 as a coenzyme. Hyperhomocysteinemia may cause diseases through multiple mechanisms such as inhibiting the generation of S-adenosylmethionine, affecting the synthesis and repair of DNA and RNA, causing oxidative damage, and promoting the proliferation of vascular smooth muscle. Risk factors for HHcy include genetic factors (such as related gene mutations), nutritional deficiencies (such as folic acid, vitamin B6 and B12 deficiency), unhealthy lifestyle habits (such as smoking and drinking), age and gender differences, and the effects of certain diseases and drugs. The management and treatment of HHcy include healthy lifestyle intervention, nutritional therapy and personalized precision supplementation programs. Specific measures include supplementation of nutrients such as folic acid, vitamin B12, vitamin B6, betaine and choline, and the development of personalized intervention programs based on genetic polymorphisms and individual nutritional status. In short, by detecting the levels of Hcy and its metabolic related compounds, HHcy can be better evaluated and managed, and the occurrence of related diseases can be prevented or their progression can be delayed. The whole process is of great significance for health and disease management, but if health and disease status monitoring is to be achieved, the following biochemical indicators must be accurately detected.

[0003] Thirteen compounds that need to be tested simultaneously are: Methionine, Homocysteine, Cysteine, Riboflavin, 5-Methyltetrahydrofolate, Folate, Pyridoxal Phosphate (PLP), 4-Pyridoxic Acid, Methylmalonic Acid (MMA), Betaine, Choline, S-Adenosylmethionine (SAM), and S-Adenosylhomocysteine ​​(SAH).

[0004] The significance and importance of the detection of the above 13 compounds are as follows:

[0005] 1) Methionine: Methionine is the precursor of homocysteine ​​and is converted to homocysteine ​​through demethylation. Therefore, measuring the level of methionine helps to understand the source and metabolism of homocysteine.

[0006] 2) Homocysteine: Elevated homocysteine ​​levels are considered to be an independent risk factor for a variety of diseases, such as cardiovascular and cerebrovascular diseases, diabetes, hypertension, etc. Measuring its level is of great significance for assessing the risk of these diseases.

[0007] 3) Cysteine: Cysteine ​​is a product of the homocysteine ​​transsulfurization pathway. Measuring its level can help understand the metabolism of homocysteine ​​and the function of the transsulfurization pathway.

[0008] 4) Vitamin B2 (Riboflavin): Vitamin B2 is an important coenzyme in the metabolism of homocysteine, especially in the folic acid pathway. Vitamin B2 deficiency may lead to elevated homocysteine ​​levels, so measuring its level is important for evaluating the overall status of homocysteine ​​metabolism.

[0009] 5) 5-Methyltetrahydrofolate: 5-Methyltetrahydrofolate is the active form of folate metabolism and participates in the remethylation of homocysteine ​​to methionine. Measuring its level helps to understand the efficiency of the folate metabolic pathway and the remethylation capacity of homocysteine.

[0010] 6) Folic acid: Folic acid plays a key role in the remethylation of homocysteine. Folic acid deficiency can lead to elevated homocysteine ​​levels. Measuring folate levels helps assess an important aspect of homocysteine ​​metabolism.

[0011] 7) Pyridoxal Phosphate: Pyridoxal phosphate is the active form of vitamin B6 and is involved in the transsulfurization pathway of homocysteine. Measuring its level helps to understand the function of the transsulfurization pathway.

[0012] 8) 4-Pyridoxic Acid: 4-Pyridoxic Acid is the end product of vitamin B6 metabolism. Measuring its level can reflect the metabolism of vitamin B6.

[0013] 9) Methylmalonic Acid: Methylmalonic Acid is a marker of vitamin B12 metabolism. Measuring its level helps to assess the status of vitamin B12 and the function of the homocysteine ​​remethylation pathway.

[0014] 10) Betaine: Betaine is an important methyl donor for homocysteine ​​remethylation. Measuring its level helps to understand the capacity of the methylation pathway.

[0015] 11) Choline: Choline can be converted into betaine in the liver and kidneys and participate in the remethylation of homocysteine. Measuring its level can reflect the supply of methyl donors.

[0016] 12) S-Adenosylmethionine: S-Adenosylmethionine is a methyl donor and participates in the methylation reaction of DNA, RNA and protein. Measuring its level can help understand the cell methylation capacity and the balance of homocysteine ​​metabolism.

[0017] 13) S-Adenosylhomocysteine: S-Adenosylhomocysteine ​​is the product of methyl transfer reaction. Measuring its level helps to understand the efficiency of intracellular methylation reaction and the state of homocysteine ​​metabolism.

[0018] The structural formulas of the 13 compounds are as follows:

[0019]

[0020]

[0021] Properties and analytical difficulties of the 13 compounds:

[0022] There are many difficulties to overcome when analyzing these 13 compounds (methionine, homocysteine, cysteine, vitamin B2, 5-methyltetrahydrofolate, folic acid, pyridoxal phosphate, 4-pyridoxic acid, methylmalonic acid, betaine, choline, S-adenosylmethionine, S-adenosylhomocysteine). These compounds have significant differences in chemical properties, polarity, structural active groups, charge, in vivo content, chromatographic retention and mass spectrometry ionization response, which poses a huge challenge to liquid chromatography-mass spectrometry (HPLC-MS / MS).

[0023] The Chinese and English names and abbreviations of the 13 compounds are listed below:

[0024]

[0025]

[0026] First, the chemical properties and polarity of these compounds vary greatly. For example, methionine, homocysteine, and cysteine ​​have medium polarity, while vitamin B2, folic acid, and its metabolites (such as 5-methyltetrahydrofolate) have high polarity. In addition, betaine and choline have very high polarity, and this difference in polarity will affect their retention behavior on the chromatographic column, which may lead to overlapping chromatographic peaks and difficulty in separation.

[0027] In addition, there are significant differences in the content of these compounds in the body. For example, the normal content of homocysteine ​​in the body is 5-15μmol / L, while the content of vitamin B2 and folic acid may be at the ng / mL level. This difference in content requires different sensitivity optimization in sample pretreatment and detection methods. In addition, some compounds (such as 5-methyltetrahydrofolate and pyridoxal phosphate) are unstable during sample processing and storage, and require special storage conditions and treatment methods to prevent degradation and loss. In particular, S-adenosylmethionine is not only a low-content compound, but also easily affected by the matrix. The accurate and sensitive determination of S-adenosylmethionine in biological samples has always been a great challenge. For S-adenosylmethionine, in actual analysis, matrix effects and sample complexity will also greatly affect the accuracy of the analysis results. Furthermore, because the content of S-adenosylmethionine is low, it is greatly affected by the matrix. One solution to the problem is to use a more sensitive liquid mass spectrometer, such as SCIEX (AB / API) 5000 or 5500 or 6500, Waters TQS or TQ-XS and other high-end liquid mass spectrometers. Although the use of high-end liquid mass spectrometers as much as possible can make S-adenosylmethionine detection relatively easy, these high-end liquid mass spectrometers are not affordable for every analytical unit, and expensive equipment can make the detection of S-adenosylmethionine daunting. The present invention, through technical innovation, enables S-adenosylmethionine to be accurately detected on low-end liquid mass spectrometers.

[0028] Furthermore, there are great challenges in chromatographic retention and separation. For example, betaine and choline have very high polarity, so their retention time is short and the separation effect is poor in conventional reversed-phase chromatography. Special chromatographic columns and mobile phase conditions are required to achieve good retention. However, when 13 compounds are measured simultaneously, it is impossible to design a special chromatographic column only for betaine and choline. A special chromatographic column means that during the analysis of a sample, the chromatographic column needs to be replaced to achieve analysis, which is basically impossible in actual inspection scenarios.

[0029] The simultaneous analysis of these 13 compounds requires a lot of work and innovation in multiple links such as sample pretreatment, chromatographic separation, and mass spectrometry detection to overcome challenges in chemical properties, polarity, charge, in vivo content, and matrix effects to achieve accurate and sensitive detection.

[0030] After searching the existing patent documents, it was found that CN 117589916 A discloses a method for extracting 11 one-carbon metabolic pathway-related metabolites (homocysteine, methionine, cysteine, S-adenosylhomocysteine, S-adenosylmethionine disulfate, riboflavin, pyridoxal phosphate, pyridoxic acid, 5-methylmethylhydrofolate, folic acid, methylmalonic acid) in plasma using a high concentration of reducing agent and an acidic precipitant. However, this method cannot detect betaine and choline. Betaine is a methyl donor for Hcy to be remethylated to generate methionine, and choline is a precursor of betaine. Changes in betaine and choline levels may affect the methylation process of homocysteine, thereby affecting the methylation reaction and metabolic health. Without the betaine and choline content, important evaluation indicators of homocysteine ​​metabolism cannot be obtained. Furthermore, CN 117589916A does not solve the problem of the influence of the matrix effect of S-adenosylmethionine. The method described in CN 117589916 A can only detect the standard curve of S-adenosylmethionine (Table 2 of CN 117589916 A) and the content of the standard curve added to the serum (Table 3 of CN 117589916 A), but cannot detect the serum of healthy people or patients without the addition of the standard curve, because the standard solution added to the serum from the outside does not bind to the serum matrix and has a high content, which is easy to detect, while S-adenosylmethionine in the serum samples of healthy people and patients will be inhibited by a large amount of matrix. For this reason, S-adenosylmethionine in the serum is difficult to detect. The peak area of ​​S-adenosylmethionine in the serum sample after direct protein precipitation is very small due to matrix inhibition, which makes it difficult to accurately determine S-adenosylmethionine in the serum. However, S-adenosylmethionine is one of the most critical indicators in the entire metabolism and plays an important role in the later evaluation of the disease. Deviations in its content will directly lead to diagnostic errors.

[0031] CN 115436536 A discloses a method for detecting biochemical metabolism-related markers of homocysteine. The method comprises the following steps: detecting a sample to be tested and a standard solution of biochemical metabolism-related markers of homocysteine ​​by HPLC-MS / MS, respectively, to obtain a chromatogram and a mass spectrum; then processing the obtained chromatogram and mass spectrum, and performing qualitative and quantitative analysis on 18 biochemical metabolism-related markers of homocysteine ​​(homocysteine, methionine, cystathionine, cysteine, serine, glycine, arginine, choline, betaine, dimethylglycine, creatinine, asymmetric dimethylarginine, symmetric dimethylarginine, methylmalonic acid, pyridoxic acid, riboflavin, 5-methyltetrahydrofolate and folic acid), respectively, to obtain the contents of the 18 biochemical metabolism-related markers of homocysteine ​​in the sample to be tested. The detection method can simultaneously detect 18 markers related to hyperhomocysteinemia, and can exclude the influence of the interfering substance succinic acid in serum samples on the detection results of methylmalonic acid, and has the technical advantages of accurate results, low price, simple operation, and easy promotion. However, the invention only targets compounds with high polarity in the biochemical metabolism of homocysteine. The method uses a HILIC column, so the compounds covered by the method are all highly polar compounds that can be analyzed using a HILIC column. Due to the limitation of this column, compounds with weak polarity that cannot be analyzed using a HILIC column are discarded, such as vitamin B2, pyridoxal phosphate, S-adenosylmethionine and S-adenosylhomocysteine, which cannot be analyzed in this method, and are far from covering the key indicators.

[0032] Furthermore, based on the accurate determination of 13 compounds, the clinical interpretation of the determination results of these compounds is a huge problem, because there are complex upstream and downstream and conversion relationships between these compounds. In clinical practice, the clinical interpretation of multiple indicators is itself a big problem, not to mention that all these compounds are related to each other. In the metabolic pathways of the human body, there are complex upstream and downstream conversion relationships between the following 13 key compounds. These compounds are converted into each other through a series of enzymatic reactions to form a complex metabolic network. The complex relationship between these compounds and their clinical significance are as follows 1) Methionine and S-adenosylmethionine (SAM, S-adenosylmethionine): Methionine is converted to SAM by methionine adenosyltransferase (MAT). SAM is a methyl donor and participates in a variety of biological methylation reactions. If the methionine content increases, it may lead to an increase in SAM, further affecting the methylation reaction. High SAM levels may indicate abnormal protein metabolism and methylation processes. 2) S-adenosyl homocysteine ​​(SAH): SAM is demethylated to produce SAH, which is then hydrolyzed to produce homocysteine ​​(Hcy) and adenosine. Increased SAH levels may hinder the methylation reaction because SAH is a competitive inhibitor of the methylation reaction. 3) Homocysteine ​​(Hcy) and cysteine: Hcy can be converted to cysteine, which reacts with CBS (cystathionine-β-synthase) to produce cystathionine, and then to cysteine. High Hcy levels may lead to an increased risk of atherosclerosis, while high cysteine ​​levels may indicate changes in the antioxidant defense system. 4) Folic acid and 5-methyltetrahydrofolate (5-MTHF): Folic acid is converted to 5-MTHF by MTHFR (5,10-methylenetetrahydrofolate reductase). 5-MTHF acts as a methyl donor during the remethylation of Hcy to methionine. Abnormal levels of 5-MTHF may lead to elevated Hcy levels, which are associated with an increased risk of cardiovascular disease. 5) Vitamin B2 and Pyridoxal Phosphate (PLP): Vitamin B2 is a coenzyme for multiple enzymes, including MTHFR. PLP is the active form of vitamin B6 and is involved in the conversion of Hcy to cysteine. Insufficient vitamin B2 can affect folate metabolism, while insufficient PLP can lead to abnormal accumulation of Hcy and increase the risk of cardiovascular disease.6) Methylmalonic Acid (MMA): MMA is a marker of vitamin B12 deficiency, and its accumulation indicates a disorder in vitamin B12-dependent metabolic processes. High MMA levels may indicate vitamin B12 deficiency, leading to disorders in methylation reactions and Hcy metabolism. 7) Betaine and Choline: Betaine is a methyl donor for Hcy remethylation to generate methionine. Choline is a precursor of betaine. Changes in betaine and choline levels may affect the methylation process of Hcy, thereby affecting methylation reactions and metabolic health.

[0033] Based on the above background, these 13 compounds have complex transformation relationships. When interpreting them clinically, they are likely to face the following challenges and complexities:

[0034] 1) Mutual influence of compound levels: When the level of a certain compound increases, the level of its downstream metabolites is likely to increase as well. For example, an increase in homocysteine ​​(Hcy) levels may lead to an increase in methionine and cysteine ​​levels. Such a concentration change between upstream and downstream often makes the abnormal level of a single compound indicate a broader metabolic disorder. 2) The effect of enzyme or protein dysfunction on metabolic pathways: If the key enzyme or protein function responsible for the mutual conversion of these compounds is impaired, it may lead to the accumulation of upstream compounds and the reduction of downstream products. For example, vitamin B2 deficiency may affect the activity of 5,10-methylenetetrahydrofolate reductase (MTHFR), thereby causing abnormal accumulation of 5-methyltetrahydrofolate (5-MTHF). This enzyme dysfunction can lead to the cascade effect of multiple metabolic pathways, making clinical interpretation complicated. 3) Systemic changes caused by metabolic pathway problems: If a metabolic pathway has a problem, it may not only affect the content of compounds on the pathway, but also affect the levels of compounds in other related metabolic pathways. For example, disorders in S-adenosylmethionine (SAM) metabolism may affect multiple methylation reactions, thereby changing the levels of a wide range of biological processes and compounds related to methylation. 4) The clinical significance of high and low changes in multiple compounds: When we can accurately detect the levels of these 13 compounds in the human body, their high and low changes will reveal a lot of information about physical abnormalities. The changes in each compound not only provide clues to its own metabolic state, but also reflect the health of the entire metabolic network. Therefore, interpreting the changes in these compounds is crucial for diagnosis and treatment, but due to the complex interactions between them, the interpretation has become extremely complicated.

[0035] The complex relationship between these compounds poses a huge challenge to clinicians when interpreting test results. Doctors need to comprehensively consider the dynamic balance and potential interactions between the various compounds, rather than just changes in a single indicator. For example: 1) Elevated homocysteine ​​may not only be a sign of folic acid or vitamin B12 deficiency, but may also be caused by abnormal enzyme activity or other metabolic pathway disorders. This complexity requires doctors to have an in-depth understanding of metabolic networks. 2) Changes in S-adenosylmethionine: may affect multiple key methylation reactions, leading to widespread changes in gene expression and cell function. This wide range of effects makes it more difficult to interpret changes at the level of a single compound.

[0036] Due to the interrelationships and complex metabolic relationships between these compounds, abnormal changes in one compound may cause abnormalities in multiple other compounds, thus forming a "butterfly effect". This effect requires doctors to comprehensively consider the health of the entire metabolic network when interpreting test results, rather than just focusing on changes in a single compound. However, such a complex relationship makes it too difficult for each clinician to analyze. In order to help clinicians quickly and accurately obtain valuable clinical information from the content of these compounds, it is urgent to develop simpler and more effective discrimination methods. These methods should be able to simplify complex metabolic networks, provide intuitive and easy-to-understand clinical guidance, and help doctors make rapid and reliable diagnostic and treatment decisions in complex metabolic environments. In this way, these biomarkers can be better utilized to provide more reliable support and guidance for patient health management.

[0037] There is no public report on how to evaluate the clinical test results of these 13 compounds. Summary of the invention

[0038] The object of the present invention is to provide a method for determining 13 metabolic regulation core molecules under the same chromatographic system and the construction of a detection and discrimination model thereof. The 13 metabolic regulation core molecules are methionine, homocysteine, cysteine, vitamin B2, 5-methyltetrahydrofolate, folic acid, pyridoxal phosphate, 4-pyridoxic acid, methylmalonic acid, betaine, choline, S-adenosylmethionine, and S-adenosylhomocysteine.

[0039] The objective of the present invention is achieved through the following technical solutions:

[0040] The present invention provides a method for determining 13 metabolic regulation core molecules under the same chromatographic system, wherein the 13 metabolic regulation core molecules are methionine, homocysteine, cysteine, vitamin B2, 5-methyltetrahydrofolate, folic acid, pyridoxal phosphate, 4-pyridoxic acid, methylmalonic acid, betaine, choline, S-adenosylmethionine, and S-adenosylhomocysteine; the method comprises the following steps:

[0041] S1. Take plasma or serum samples and divide them into three samples: A, B, and C;

[0042] S2. Add a reducing agent and an internal standard solution to sample A, precipitate with methanol, and centrifuge to obtain supernatant C; the internal standards are cysteine, methionine, betaine, choline, homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, S-adenosylhomocysteine, vitamin B2, 4-pyridoxalic acid, and folic acid;

[0043] S3, add internal standard solution to sample B, precipitate with 5-20% TCA, centrifuge and take supernatant B; the internal standard is pyridoxal phosphate;

[0044] Internal standard solution was added to the S4 and C samples, the pH value was adjusted with ammonium acetate, and the supernatant was obtained by centrifugation, and supernatant C was obtained by enrichment and purification with WCX; the internal standard was S-adenosylmethionine;

[0045] S5, supernatant A, supernatant B, and supernatant C were respectively injected into a liquid chromatography-mass spectrometer for analysis, and low-concentration acetic acid was added to the mobile phase used in the chromatographic detection; the chromatographic detection was performed without changing the chromatographic system, and a C18 chromatographic column with a specification of 150 mm*4.6 mm was used;

[0046] S6. Calculate the contents of 13 core metabolic regulatory molecules in the sample based on the isotope dilution method.

[0047] As an embodiment, the low concentration acetic acid is a 0.02%-0.05% acetic acid aqueous solution.

[0048] As an embodiment, in the mobile phase used in the chromatographic detection, the aqueous phase is a 0.02%-0.05% (volume ratio) acetic acid aqueous solution, and the organic phase is a methanol solution containing 0.05%-0.1% formic acid.

[0049] As an embodiment, in step S2, the reducing agent is DTT or TCEP.

[0050] As an embodiment, in step S4, an unconventional single elution method is used during WCX solid phase extraction. As an embodiment, the single elution solution is a 20 mM ammonium acetate aqueous solution.

[0051] As an embodiment, in step S4, the plasma or serum sample is added with internal standard and pH adjusting solution and the supernatant obtained by centrifugation is enriched by WCX solid phase purification and processed by a special single elution method (conventional WCX purification is two elutions) before sample injection and detection.

[0052] As an embodiment, in step S4, during WCX solid phase extraction, an additive that suppresses electron-withdrawing groups is added to the loading solution.

[0053] As an embodiment, the additive for suppressing electron-withdrawing groups is an aqueous solution of ammonium acetate. In some implementation examples, the additive is a 40 mM aqueous solution of ammonium acetate (only this concentration can successfully improve the sensitivity of SAM).

[0054] As an embodiment, in step S2, the chromatographic column used for chromatographic detection is an XSelect HSS T3 chromatographic column.

[0055] As an embodiment, the XSelect HSS T3 chromatographic column has a length of 150 mm and an inner diameter of 4.6 mm.

[0056] As an embodiment, in step S5, the supernatant A is purified by a C18 filler purification column before entering the chromatographic column.

[0057] As an embodiment, in step S2, the internal standard uses an isotope internal standard, including Hcy-d4, SAH-d4, methionine-d3, 5-methyltetrahydrofolate (13C5), VB2 (13C4, 15N2), 4-pyridoxalic acid-D3, betaine-d9, methylmalonic acid-d3, cysteine-d3, choline-d4, and folic acid (13C5, 15N).

[0058] As an embodiment, in step S3, the internal standard adopts an isotope internal standard, including PLP-d3.

[0059] As an embodiment, in step S4, the internal standard adopts an isotope internal standard, including S-adenosylmethionine-d3.

[0060] In some implementation examples, the elution gradient is as follows:

[0061] Time(min) Flow(ml / min) A Conc / % B Conc / % 1 / 0.6 95 5 2 1.0 0.6 95 5 3 4.0 0.6 5 95 4 6.0 0.6 5 95 5 6.1 0.6 95 5 6 9.0 0.6 95 5

[0062] In some implementation examples, the mass spectrometry conditions are as follows:

[0063]

[0064]

[0065] As an implementation scheme, the linear and regression equations of the 13 metabolic regulation core molecules are as follows (where y is the ratio of the peak area of ​​the target compound and its corresponding isotope internal standard, and x is the concentration of the target compound):

[0066]

[0067]

[0068] The present invention also provides a method for constructing a detection and discrimination model based on the concentrations of 13 metabolic regulation core molecules detected by the aforementioned method, the method comprising:

[0069] A1. Standardize each metabolic regulation core molecule

[0070] The formula for standardization is: Zi = (Xi-μ) / σ, where Xij is the concentration of the jth metabolic regulation core molecule in the i-th sample, μ is the average concentration of the metabolic regulation core molecule in all samples, σ is the standard deviation of the metabolic regulation core molecule in all samples, and Zij is the standardized value of the i-th metabolic regulation core molecule in the j-th sample;

[0071] A2. Constructing a discriminant model

[0072] The discriminant model is Among them, HIj is the comprehensive health index of the jth sample, N is the number of metabolic regulatory core molecules, and wi is the weight of the i-th metabolic regulatory core molecule;

[0073] HI j A value below -0.938 or above 0.938 indicates metabolic abnormalities.

[0074] As an embodiment, the Wi value is as follows:

[0075]

[0076]

[0077] Compared with the prior art, the present invention has the following beneficial effects:

[0078] (1) The detection system of the present invention uses low-concentration acetic acid as an additive, which first achieves a) improving the retention behavior of choline, a highly polar compound, so that choline with high polarity can be well retained on the chromatographic column. Choline is a typical highly polar compound containing amino groups. Choline, a compound with weakly basic groups, is better retained by selecting acetic acid with a higher pH value instead of conventional mobile phase additives such as formic acid. At the same time, the response is greatly increased. The retention of choline on the chromatographic column is significantly enhanced, reaching 2.12 minutes. Such retention allows choline to be accurately analyzed, avoiding the dead time of the chromatographic system and avoiding a large amount of matrix interference that peaks at the dead time of the chromatographic system; b) The use of low-concentration acetic acid as a mobile phase additive significantly improves the sensitivity of S-adenosylmethionine, a compound with a very low content among the 13 compounds, because they are easier to remain ionized at a higher pH value, thereby enhancing the chromatographic separation effect and obtaining a good peak shape and separation; the response of the test compound is greatly improved, which is very suitable for the compounds in the present invention, and maintains a more stable form in an environment closer to neutral.

[0079] (2) The use of WCX solid phase extraction plates to enrich and purify the compound S-adenosylmethionine in blood samples has not been reported in any public patents or literature. The present invention uses WCX as a solid phase extraction material to carry out targeted enrichment of the compound S-adenosylmethionine. The structure of the compound S-adenosylmethionine is relatively complex. The structure of the compound belongs to a highly polar compound. The structure of the compound contains both cationic groups and anionic groups, and has both electron-withdrawing centers and electron-donating centers. The present invention has tried a variety of different enrichment materials, but none of them achieved satisfactory results. The final experiment found that using WCX as an enrichment material and further adding an additive that inhibits electron-withdrawing groups (such as ammonium acetate aqueous solution) to the loading solution to inhibit the electron-withdrawing groups on the structure of S-adenosylmethionine can allow S-adenosylmethionine to be adsorbed on the solid phase extraction plate of WCX, achieving a good purification and enrichment effect.

[0080] (3) Betaine and choline have very high polarity. It is difficult to obtain appropriate chromatographic behavior and retention time for betaine and choline in conventional chromatographic separation, and peaks will appear during dead time. In order to solve the chromatographic retention problem of betaine and choline, the present invention tried various chromatographic columns. The results show that the selection of XSelect HSS T3 can obtain appropriate chromatographic behavior for betaine and choline. However, when using the XSelect HSS T3 chromatographic column, a chromatographic column with a column length of 150 mm and a column inner diameter of 4.6 mm must be used to obtain the best chromatographic separation system for betaine and choline. Chromatographic columns with a length of less than 150 mm or a column inner diameter of less than 4.6 mm cannot successfully carry the high concentrations of betaine and choline in the blood, while obtaining the best chromatographic behavior for these two compounds.

[0081] (4) Interference is very likely to occur in the mass spectrometry ion channel of betaine. At a retention time of about 1.7 minutes, obvious inverted peak interference will occur. Such interference will directly affect the accurate determination of betaine, because the chromatographic peak of betaine begins to emerge just at the position where the inverted peak of the interference ends. This situation causes the accurate detection of betaine to be significantly interfered. Finally, a large number of experiments have proved that it comes from the mobile phase. In order to solve this problem, the present invention specially connects a purification column of C18 filler in the mobile phase system pipeline for simultaneous analysis of 13 compounds. The mobile phase will first be purified by the column and then enter the chromatographic column to separate the target compound. The access to the purification column directly solves the problem of interference with betaine during liquid chromatography-mass spectrometry analysis. The stability and sensitivity of betaine analysis are greatly improved.

[0082] (5) Due to the interrelationships and complex metabolic relationships among these compounds, abnormal changes in one compound may cause abnormalities in multiple other compounds, thus forming a "butterfly effect". This effect requires doctors to comprehensively consider the health of the entire metabolic network when interpreting the test results, rather than just focusing on the changes in a single compound. Such a complex relationship makes it too difficult for each clinician to analyze. The present invention establishes a detection and discrimination model for these 13 compounds:

[0083] (6) The present invention realizes the unified and accurate detection of 13 compounds under the same chromatographic system, and the rapid identification of the detection results: methionine, homocysteine, cysteine, vitamin B2 (riboflavin), 5-methyltetrahydrofolate, folic acid (Folate), pyridoxal phosphate (Pyridoxal Phosphate, PLP), 4-pyridoxic acid (4-pyridoxic Acid), methylmalonic acid (Methylmalonic Acid, MMA), betaine (Betaine), choline (Choline), S-adenosylmethionine (S-adenosylmethionine, SAM), S-adenosylhomocysteine ​​(S-adenosylhomocysteine, SAH). BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0085] Figure 1 The choline chromatogram corresponding to the conventional mobile phase additive formic acid in Experimental Example 1;

[0086] Figure 2 This is the choline chromatogram corresponding to Experimental Example 1, using 0.05% acetic acid as the mobile phase additive;

[0087] Figure 3 The chromatogram of S-adenosylmethionine corresponding to the conventional mobile phase additive formic acid in Experimental Example 1;

[0088] Figure 4 This is the S-adenosylmethionine chromatogram corresponding to Experimental Example 1, using 0.05% acetic acid as the mobile phase additive;

[0089] Figure 5 This is the S-adenosylmethionine chromatogram corresponding to the protein precipitation pretreatment in Experimental Example 2;

[0090] Figure 6 This is the S-adenosylmethionine chromatogram corresponding to the WCX pretreatment in Experimental Example 2;

[0091] Figure 7 This is the corresponding betaine chromatogram in Experimental Example 3, where the mobile phase enters the chromatographic column directly without being purified;

[0092] Figure 8In Experiment 3, the mobile phase will first be purified by a C18 filler column before entering the chromatographic column, and the corresponding betaine chromatogram;

[0093] Fig. 9 Chromatograms for simultaneous detection of methionine, homocysteine, cysteine, vitamin B2, 5-methyltetrahydrofolate, folic acid, 4-pyridoxic acid, methylmalonic acid, betaine, choline, S-adenosylmethionine, and S-adenosylhomocysteine;

[0094] Fig.10 This is the S-adenosylmethionine detection chromatogram;

[0095] Fig.11 This is the chromatogram for the detection of pyridoxal phosphate. DETAILED DESCRIPTION

[0096] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0097] In the examples, if the specific conditions are not specified, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0098] The 13 metabolic regulation core molecules involved in the present invention are methionine, homocysteine, cysteine, vitamin B2, 5-methyltetrahydrofolate, folic acid, pyridoxal phosphate, 4-pyridoxic acid, methylmalonic acid, betaine, choline, S-adenosylmethionine, and S-adenosylhomocysteine.

[0099] The present invention is based on the same chromatography system, and the method for uniformly measuring 13 metabolic regulation core molecules comprises the following steps:

[0100] S1. Take plasma or serum samples and divide them into three samples: A, B, and C;

[0101] S2, A sample was added with reducing agent and internal standard solution, and after methanol precipitation, centrifugation was performed to obtain supernatant C; the internal standards were cysteine, methionine, betaine, choline, homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, S-adenosylhomocysteine, vitamin B2, 4-pyridoxalic acid, folic acid ( Fig. 9 );

[0102] Internal standard solution was added to samples S3 and B, and after 10% TCA precipitation, supernatant B was obtained by centrifugation; the internal standard was pyridoxal phosphate ( Fig.11 );

[0103] Internal standard solution was added to the S4 and C samples, the pH value was adjusted by ammonium acetate, and the supernatant was obtained by centrifugation, and then enriched and purified by WCX to obtain supernatant C; the internal standard was S-adenosylmethionine ( Fig.10 );

[0104] S5, supernatant A, supernatant B, and supernatant C were respectively injected into a liquid chromatography-mass spectrometer for analysis, and low-concentration acetic acid was added to the mobile phase used in the chromatographic detection; the chromatographic detection was performed without changing the chromatographic system, and a chromatographic column with a specification of 150 mm*4.6 mm was used;

[0105] S6. Calculate the contents of 13 core metabolic regulatory molecules in the sample based on the isotope dilution method.

[0106] Test Example 1

[0107] Configure different mobile phase ratios to examine the effects of different mobile phase additives;

[0108] 1) When low concentration acetic acid is used as a mobile phase additive, the chromatographic retention of choline is significantly enhanced, see Figure 1 and Figure 2 ;

[0109] 2) When low concentration of acetic acid is used as a mobile phase additive, a significant effect is produced, which significantly enhances the response of S-adenosylmethionine. The test results are shown in the table below.

[0110]

[0111] In summary, the use of conventional formic acid as a mobile phase additive cannot meet the requirements of the analytical task of the present invention. The test example of the present invention uses acetic acid as an additive, instead of conventional formic acid, as a mobile phase additive, and determines that a low concentration of acetic acid, that is, an acetic acid concentration of 0.05% is the most suitable concentration. Acetic acid itself is a weak acid. Acetic acid at a low concentration makes the pH of the mobile phase higher, about 4-5. Using low concentrations of acetic acid as an additive first achieves a) improving the retention behavior of highly polar compounds such as choline, so that choline with high polarity can be well retained on the chromatographic column. Choline is a typical highly polar compound containing amino groups. Choline, a compound with a higher pH value, is selected to replace conventional formic acid, so that choline, a compound with a weakly basic group, is better retained, and the response is greatly increased, and the attachment Figure 1 and 2It was shown that after replacing formic acid with low-concentration acetic acid, the retention of choline on the chromatographic column was significantly enhanced to 2.12 minutes. Such retention allows choline to be accurately analyzed, avoiding the dead time of the chromatographic system and the large amount of matrix interference that peaks at the dead time of the chromatographic system. In addition, although citric acid is sometimes used as a mobile phase additive in chromatography, when the mass spectrometer is used as a detector, citric acid is relatively less volatile. During mass spectrometry analysis, it is difficult to evaporate completely and may accumulate in the ion source or mass spectrometer, resulting in contamination and signal interference. In addition, as a tricarboxylic acid, citric acid has a strong buffering capacity and can significantly change the pH value of the mobile phase, which may affect the selectivity of chromatographic separation and the stability of mass spectrometry signals. In addition, high buffering capacity may lead to undesirable buffering effects in mass spectrometry analysis and affect the ionization process. Citric acid has a multi-carboxyl structure and is prone to form multiple charged ions in mass spectrometry analysis, which may lead to complex ion suppression effects and reduce the detection sensitivity and signal intensity of mass spectrometry. b) The use of low-concentration acetic acid as a mobile phase additive significantly improved the sensitivity of S-adenosylmethionine, a compound with a very low content among the 13 compounds ( Figure 3 , Figure 4 ), because they are easier to remain ionized at higher pH values ​​(according to experiments, even if formic acid adjusts the pH value to low concentration of acetic acid, it cannot achieve such obvious changes as low concentration of acetic acid. The lower buffer capacity of acetic acid will not significantly change the pH of the mobile phase at low concentrations, thereby reducing the impact on the chromatographic column and maintaining good separation performance. As a weaker acid, acetic acid has a weaker interaction with highly polar compounds such as choline and SAM, allowing these compounds to maintain a longer retention time in the mobile phase. Lower concentrations of acetic acid provide a more ideal polar environment in the mobile phase, promoting the separation and detection of these highly polar compounds. In addition, acetic acid can sometimes increase the signal intensity of certain compounds in mass spectrometry analysis because it provides a milder proton donor and promotes the protonation process), thereby enhancing the chromatographic separation effect and obtaining good peak shape and separation; the response of the compound to be tested is greatly improved, which is very suitable for the compounds in the present invention, maintaining a more stable form in an environment closer to neutral.

[0112] Figure 1 For the use of formic acid, a traditional mobile phase additive, the retention time was only 1.72 minutes; Figure 2 Using 0.05% acetic acid as additive - choline retention was significantly enhanced to 2.12 minutes. Figure 3 When using traditional additive formic acid, S-adenosylmethionine, Figure 4 Using 0.05% acetic acid as an additive, the response of S-adenosylmethionine was significantly enhanced.

[0113] Test Example 2

[0114] Mix a certain amount of serum, and purify and extract the same amount of serum using protein precipitation, HLB (C18) solid phase extraction, and WCX (additives added to the loading solvent and single elution). Then adjust the injection volume according to the different dilution multiples of each method, so that the amount of serum injected is equivalent to the same amount, ensuring that when comparing, it is the same amount of serum. The results of the three purification and enrichment are shown in the table below.

[0115]

[0116] Note: The single eluent is 20 mM ammonium acetate aqueous solution, and the additive is 40 mM ammonium acetate aqueous solution.

[0117] The content of S-adenosylmethionine in blood samples is very low, and special purification and enrichment treatment are required to achieve the requirements of accurate detection. Test Example 2 of the present invention uses WCX as a solid phase extraction material to carry out targeted enrichment of S-adenosylmethionine. The structure of S-adenosylmethionine is relatively complex, and the structure belongs to a highly polar compound. There are both cationic groups and anionic groups in the structure, and both electron-withdrawing centers and electron-donating centers. The present invention has tried a variety of different enrichment materials, but none of them has achieved satisfactory results. The final experiment found that using WCX as an enrichment material, and further adding additives that inhibit electron-withdrawing groups to the sample solution, inhibiting the electron-withdrawing groups on the S-adenosylmethionine structure, can allow S-adenosylmethionine to be adsorbed on the solid phase extraction plate of WCX, achieving good purification and enrichment. The use of WCX solid phase extraction plates to enrich and purify S-adenosylmethionine in blood samples has not been reported in published patents or literature. Figure 5 The S-adenosylmethionine peak was obtained by protein precipitation pretreatment; Figure 6 S-adenosylmethionine peak obtained by WCX pretreatment.

[0118] Test Example 3

[0119] Betaine and choline have very high polarity, and it is difficult to obtain appropriate chromatographic behavior and retention time for betaine and choline in conventional chromatographic separation, and peaks will appear during dead time. In order to solve the chromatographic retention problem of betaine and choline, various chromatographic columns were tried in Experimental Example 3 of the present invention. The results show that the selection of XSelect HSS T3 can obtain appropriate chromatographic behavior for betaine and choline. However, when using the chromatographic column XSelect HSS T3, a chromatographic column with a column length of 150 mm and a column inner diameter of 4.6 mm must be used to obtain the best chromatographic separation system for betaine and choline. Chromatographic columns with a length of less than 150 mm or a column inner diameter of less than 4.6 mm cannot successfully carry the high concentrations of betaine and choline in the blood, while obtaining the best chromatographic behavior for these two compounds.

[0120] Test Example 4

[0121] Interference is very likely to occur in the mass spectrometry ion channel of betaine, and obvious inverted peak interference will be produced at a retention time of about 1.7 minutes. Such interference will directly affect the accurate determination of betaine, because the chromatographic peak of betaine begins to emerge just at the position where the inverted peak of interference ends. This situation causes the accurate detection of betaine to be significantly interfered. It was finally proved by a large number of experiments that it comes from the mobile phase. In order to solve this problem, the present invention specially accesses a purification column of C18 filler in the mobile phase system pipeline for simultaneous analysis of 13 compounds. The mobile phase will first be purified by the column, and then enter the chromatographic column to separate the target compound. The access of the purification column directly solves the problem of interference of betaine during liquid chromatography-mass spectrometry analysis. The stability and sensitivity of betaine analysis are greatly improved. Figure 7 It is the inverted peak of betaine. Figure 8 This is the chromatogram of betaine after adding the trapping column.

[0122] Example 1: Linear and regression equations for detecting 13 compounds

[0123] A series of standard solutions of 13 compounds were prepared, and the selected concentrations were designed based on the concentrations of the 13 compounds in serum, which were required to cover and be larger than the range required for actual sample determination.

[0124] Step 1:

[0125] Accurately pipette 50 μL of calibration solution (the compounds and concentration ranges in the standard solution are shown in Table 1) and the supernatant of the serum sample into a 2 mL centrifuge tube, add 10 μL of isotope internal standards (Hcy-d4, SAH-d4, methionine-d3, 5-methyltetrahydrofolate (13C5), VB2 (13C4, 15N2), 4-pyridoxalic acid-D3, betaine-d9, methylmalonic acid-d3, cysteine-d3, choline-d4, folic acid (13C5, 15N)), 10 μL of 0.5 M DTT, add 150 μL of methanol, vortex mix at 1400 rpm for 3 min, centrifuge at 14000 rpm for 3 min at 4 °C, pipette 50 μL of supernatant into the sample injection plate, add 50 μL of 0.05% acetic acid solution, and mix well;

[0126] serial number name Linear upper limit μg / mL Linear lower limit μg / mL 1 Methionine 20 0.1 2 Homocysteine 10 0.05 3 Cysteine 100 0.5 4 S-adenosylhomocysteine 0.4 0.002 5 5-Methyltetrahydrofolate 0.4 0.002 6 4-Pyridoxic acid 0.2 0.001 7 Betaine 50 0.25 8 choline 10 0.05 9 Folic acid 0.1 0.0005 10 Methylmalonic acid 1 0.005 11 Vitamin B2 0.1 0.0005 12 Pyridoxal phosphate 0.4 0.002 13 S-adenosylmethionine 1 0.005

[0127] Step 2:

[0128] Accurately pipette 50 μL of the calibrator solution (for information, see Table 1) and the supernatant of the serum sample into a 2 mL centrifuge tube, add 20 μL of the isotope internal standard (PLP-d3) and 200 μL of 10% trichloroacetic acid aqueous solution, shake for 1 hour, and centrifuge at 14000 rpm for 3 min at 4 °C;

[0129] Step 3:

[0130] Accurately pipette 200 μL of the calibration solution (for information, see Table 1) and the supernatant of the serum sample into a 2 mL centrifuge tube, add 10 μL of the isotope internal standard (S-adenosylmethionine-d3) and 200 μL of 40 mM ammonium acetate aqueous solution, vortex mix at 1400 rpm for 3 min, centrifuge at 14000 rpm for 3 min at 4 °C, and set aside;

[0131] Take Purphy WCX 96-Well Plate, and the solid phase extraction operation process is as follows (the pressure of the positive pressure instrument is less than 0.02MPa during the whole operation, and the flow rate is controlled at 5-10 drops / minute):

[0132] ① Add 200 μL of methanol to each well, vertically in the middle.

[0133] ② Add 200 μL of ultrapure water to each well, vertically in the middle.

[0134] ③ Add 350 μL of the supernatant sample after centrifugation and slowly add it to the wall.

[0135] ④ Add 200 μL of 20 mM ammonium acetate aqueous solution vertically to the center.

[0136] ⑤ Place the solid phase extraction plate on a 1mL 96-well receiving plate, add 100μL of 5% formic acid acetonitrile solution vertically in the middle, and wash under pressure;

[0137] Step 4: Take the final solutions of steps 1, 2 and 3 and inject them into liquid chromatography-mass spectrometry for analysis.

[0138] The chromatographic mass spectrometry conditions used for detection are as follows:

[0139] Chromatographic conditions

[0140] Chromatographic column: Waters XSelect HSS T3 (150 * 4.6mm)

[0141] Column temperature: 40°C

[0142] Injection volume: 10 μL

[0143] Mobile Phase:

[0144] Phase A: 0.05% acetic acid in water

[0145] Phase B: 0.1% formic acid in methanol

[0146] Elution gradient:

[0147] HPLC gradient table:

[0148] Time(min) Flow(ml / min) A Conc / % B Conc / % 1 / 0.6 95 5 2 1.0 0.6 95 5 3 4.0 0.6 5 95 4 6.0 0.6 5 95 5 6.1 0.6 95 5 6 9.0 0.6 95 5

[0149] Mass spectrometry conditions:

[0150] name Precursor ion Product ions Methionine 150.2 132.9 Homocysteine 136 117.9 Cysteine 122 75.9 S-adenosylmethionine 399.2 249.9 S-adenosylhomocysteine 385.2 133.8 Vitamin B2 377.2 243.2 5-Methyltetrahydrofolate 460.2 313.2 4-Pyridoxic acid 184.1 165.9 Betaine 118 58 choline 104 60 Folic acid 442 295 Pyridoxal phosphate 248.1 150.1 Methylmalonic acid 117 72.8

[0151] The linear and regression equations of the 13 compounds are as follows:

[0152]

[0153]

[0154] Example 2 Precision and accuracy of testing 13 compounds

[0155] The method of the present invention was used to add high and low concentrations of the following compounds to serum to simulate real samples, and the accuracy and precision of the method at different concentrations were investigated:

[0156] Methionine: concentrations are 0.4 μg / mL and 4 μg / mL.

[0157] Homocysteine: concentration is 0.2μg / mL, 2μg / mL.

[0158] Cysteine: concentration is 2μg / mL, 20μg / mL.

[0159] S-Adenosylhomocysteine ​​(SSAH): concentration is 0.008μg / mL, 0.08μg / mL.

[0160] 5-Methyltetrahydrofolate: concentrations are 0.008μg / mL and 0.08μg / mL.

[0161] 4-Pyridoxic Acid: concentrations are 0.004μg / mL, 0.04μg / mL.

[0162] Betaine: concentration is 1μg / mL, 10μg / mL.

[0163] Choline: concentration is 0.2μg / mL and 2μg / mL.

[0164] Folic acid (Folate): concentration is 0.002μg / mL, 0.02μg / mL.

[0165] Methylmalonic Acid (MMA): concentrations are 0.02 μg / mL and 0.2 μg / mL.

[0166] Vitamin B2 (Riboflavin): concentration is 0.002μg / mL, 0.02μg / mL.

[0167] Pyridoxal 5-Phosphate (PLP): concentration is 0.008μg / mL, 0.08μg / mL.

[0168] S-Adenosylmethionine (SAM): concentration is 0.02μg / mL, 0.2μg / mL.

[0169] The specific detection steps are the same as those in Example 1, except that the serum sample in Example 1 is replaced by the above-mentioned simulated sample.

[0170] The test was divided into 3 batches, with 6 repetitions for each concentration, and the precision and accuracy of the method were systematically investigated. The precision was investigated by calculating the coefficient of variation %CV at each concentration level, and the accuracy was investigated by calculating the average value of each concentration level and the deviation %Bias of the theoretical concentration. The results of the precision and accuracy tests are as follows:

[0171] Accuracy and Precision Results

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Note: The unit of concentration, average concentration and standard deviation of methionine, homocysteine, cysteine, betaine and choline is μg / mL; the unit of concentration, average concentration and standard deviation of S-adenosylhomocysteine, S-adenosylmethionine, 5-methyltetrahydrofolate, folic acid, 4-pyridoxalic acid, pyridoxal phosphate, vitamin B2 and methylmalonic acid is ng / mL.

[0180] Example 3: Sample detection

[0181] 20 real samples were selected to detect 13 compounds; the detection steps were the same as in Example 1.

[0182] The test results are as follows.

[0183] Sample test results-1

[0184]

[0185]

[0186] Sample test results-2

[0187]

[0188]

[0189] Example 4 Establishment of a detection and discrimination model for 13 compounds

[0190] Data on 13 compounds from 111 healthy people were collected to form a 111*13 data matrix, and a clinical rapid detection and discrimination model was established to help clinicians quickly interpret the test results of the 13 compounds.

[0191] 1) Data Standardization

[0192] The concentration of each compound was first normalized.

[0193] The standardized formula is as follows:

[0194] Zi=(Xi-μ) / σ

[0195] in:

[0196] Xi is the concentration of the compound in the ith sample.

[0197] μ is the average concentration of the compound in all samples.

[0198] σ is the standard deviation of the compound in all samples.

[0199] Zi is the normalized value.

[0200] The following table shows the mean values ​​and standard deviations of the 13 compounds in all samples (111 healthy subjects).

[0201]

[0202] Note: The unit of concentration, average concentration and standard deviation of methionine, homocysteine, cysteine, betaine and choline is μg / mL; the unit of concentration, average concentration and standard deviation of S-adenosylhomocysteine, S-adenosylmethionine, 5-methyltetrahydrofolate, folic acid, 4-pyridoxalic acid, pyridoxal phosphate, vitamin B2 and methylmalonic acid is ng / mL.

[0203] 2) Calculate the evaluation score according to the following formula

[0204] After obtaining the standardized values ​​of all compounds, the present invention obtains the evaluation score of each sample by calculating the average of these standardized values.

[0205] The calculation formula of the comprehensive health index is as follows:

[0206] in:

[0207] HIj is the evaluation score of the jth sample

[0208] N is the number of compounds (in this example, N=13).

[0209] Zij is the normalized value of the ith compound in the jth sample.

[0210] wi is the weight of the ith compound.

[0211] The weight is a key constant in the evaluation of these 13 compounds, which depends on the clinical importance and clinical significance of these compounds and their mutual relationship. The present invention first gives the calculated weights as the weight coefficients that can be used at present. In the future, it can be updated according to clinical experience and cognition.

[0212]

[0213] 3) Determine the evaluation score

[0214] Distribution of health interval data based on normal distribution assumption

[0215] Mean (μ): 6.101×10 -17 (very close to 0, consistent with the characteristics of standardized data)

[0216] Standard deviation (σ): 0.479

[0217] Using a 95% confidence interval, we calculate:

[0218] Lower limit: -0.938

[0219] Upper limit: 0.938

[0220] An evaluation score within this range indicates that the individual's metabolic status is roughly within the average level.

[0221] Any index below -0.938 or above 0.938 may indicate metabolic abnormalities.

[0222] 4) Use real samples to verify the discriminant scores (models and diagnostic cut points)

[0223] A. The following are the measured data and clinical interpretation data of 13 compounds from a randomly selected subject (healthy person), where the original values ​​are the measured values ​​of the 13 compounds;

[0224]

[0225]

[0226] Note: The unit of concentration, average concentration and standard deviation of methionine, homocysteine, cysteine, betaine and choline is μg / mL; the unit of concentration, average concentration and standard deviation of S-adenosylhomocysteine, S-adenosylmethionine, 5-methyltetrahydrofolate, folic acid, 4-pyridoxalic acid, pyridoxal phosphate, vitamin B2 and methylmalonic acid is ng / mL.

[0227] The discrimination result is -0.08552, which is between -0.938 and 0.938, indicating that the subject's discrimination score is at the average level and is in good health.

[0228] B. The following are the measured data and clinical interpretation data of 13 compounds from a randomly selected subject (patient), where the original values ​​are the measured values ​​of the 13 compounds;

[0229]

[0230]

[0231] =Note: The unit of concentration, average concentration and standard deviation of methionine, homocysteine, cysteine, betaine and choline is μg / mL; the unit of concentration, average concentration and standard deviation of S-adenosylhomocysteine, S-adenosylmethionine, 5-methyltetrahydrofolate, folic acid, 4-pyridoxalic acid, pyridoxal phosphate, vitamin B2 and methylmalonic acid is ng / mL.

[0232] The discrimination result was 1.92065, which was greater than the diagnostic cut-off point of 0.938, indicating a potential disease.

[0233] According to the indicators of 13 compounds of this subject, the following problems may be indicated: 1) Cardiovascular disease risk: High levels of homocysteine ​​and methionine are important risk factors for cardiovascular disease. 2) Methylation disorder: High levels of SAM, SAH, choline and betaine indicate abnormal methylation reactions, which may affect gene expression and DNA repair. 3) Vitamin metabolism disorder: High levels of 4-pyridoxalic acid, PLP and MMA indicate problems with vitamin B6 and B12 metabolism, which may be related to vitamin supplementation or metabolic disorders. 4) Abnormal liver function: Abnormal levels of multiple metabolites may also reflect disorders of liver metabolic function.

[0234] In summary, the detection system of the present invention uses low-concentration acetic acid as a mobile phase additive, so that choline with high polarity can be well retained on the chromatographic column, the response is greatly increased, and the enrichment of WCX greatly reduces the influence of the S-adenosylmethionine matrix, and the trace S-adenosylmethionine in serum can be accurately determined. In addition, combined with the selection of a specific chromatographic column, etc., the 13 compounds can be accurately determined under the same chromatographic system. On the premise of accurate detection of the 13 compounds, a digital matrix of 111 healthy people multiplied by 13 is used to establish a discrimination model for healthy people, and the rapid interpretation of the determination results of the 13 compounds in clinical practice is achieved.

[0235] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for determining 13 core molecules of metabolic regulation under the same chromatographic system, characterized in that: The 13 core metabolic regulation molecules are methionine, homocysteine, cysteine, vitamin B2, 5-methyltetrahydrofolate, folic acid, pyridoxal phosphate, 4-pyridoxic acid, methylmalonic acid, betaine, choline, S-adenosylmethionine, and S-adenosylhomocysteine; the method comprises the following steps: S1. Take plasma or serum samples and divide them into three samples: A, B, and C; S2, A sample is added with a reducing agent and an internal standard solution, and after methanol precipitation, the supernatant A is obtained by centrifugation; the internal standards are cysteine, methionine, betaine, choline, homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, S-adenosylhomocysteine, vitamin B2, 4-pyridoxalic acid, and folic acid; S3. Add internal standard solution to sample B, precipitate with 5-20% TCA, centrifuge and take supernatant B; the internal standard is pyridoxal phosphate; Internal standard solution was added to the S4 and C samples, the pH value was adjusted with ammonium acetate, and the supernatant was obtained by centrifugation, and then enriched, purified and eluted with WCX to obtain supernatant C; the internal standard was S-adenosylmethionine; S5, supernatant A, supernatant B, and supernatant C were respectively injected into a liquid chromatography-mass spectrometer for analysis, and low-concentration acetic acid was added to the mobile phase used in the chromatographic detection; the chromatographic detection was performed without changing the chromatographic system, and a C18 chromatographic column with a specification of 150mm*4.6mm was used; in the mobile phase used in the chromatographic detection, the aqueous phase was a 0.02%-0.05% acetic acid aqueous solution, and the organic phase was a methanol solution containing 0.05%-0.1% formic acid; S6. Calculate the contents of 13 core metabolic regulatory molecules in the sample based on the isotope dilution method.

2. The method according to claim 1, characterized in that In step S4, during the enrichment and purification of WCX, an additive for suppressing electron-withdrawing groups is added to the loading solution; the elution is a single elution.

3. The method according to claim 1, characterized in that The chromatographic column used for chromatographic detection was XSelect HSS T3 column.

4. The method according to claim 1, characterized in that: In step S5, the supernatant A is purified by a C18 filler purification column before entering the chromatographic column.

5. The method according to claim 1, characterized in that In step S2, the internal standard adopts an isotope internal standard, including Hcy-d4, SAH-d4, methionine-d3, 5-methyltetrahydrofolate (13C5), VB2 (13C4, 15N2), 4-pyridoxalic acid-D3, betaine-d9, methylmalonic acid-d3, cysteine-d3, choline-d4, and folic acid (13C5, 15N); in step S3, the internal standard adopts an isotope internal standard, including PLP-d3; in step S4, the internal standard adopts an isotope internal standard, including S-adenosylmethionine-d3.

6. The method according to claim 1, characterized in that The linear and regression equations of the 13 core metabolic regulatory molecules are as follows:

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