Method for determining one-carbon metabolic pathway substance in plasma sample based on LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention provides a method for determining a carbon metabolic pathway substance in a plasma sample based on LC-MS / MS. The method comprises the following steps: S1, establishing a standard curve by adopting an internal standard method; s2, taking a plasma sample, adding an isotope internal standard and an extracting solution to precipitate protein, centrifuging, taking supernatant, and adding an activating reagent and a derivatization reagent to carry out a derivatization reaction, so as to obtain a loading solution; s3, centrifuging the on-machine solution, taking supernate for on-machine detection, and substituting a detection result into the standard curve to obtain the content of the to-be-detected carbon metabolic pathway substance in the plasma. The method provided by the invention can be used for detecting 22 one-carbon metabolic pathway substances in plasma, is comprehensive in variety, rapid in extraction, low in cost and high in efficiency, can be used for easily realizing automatic detection of substances, and can be used for well assisting research and judgment of various metabolic diseases due to the fact that an Amide chromatographic column is used and a derivatization reagent does not retain in the chromatographic column to protect a mass spectrum from being polluted.

Description

Technical Field

[0001] The present invention belongs to the technical field of metabolite detection, and particularly relates to a method for determining one-carbon metabolic pathway substances in plasma samples based on LC-MS / MS. Background Art

[0002] One-carbon metabolism is a metabolic process in cells involving one-carbon units, which include methyl (-CH 3 ), methylene (-CH 2 -), methenyl (-CH=), formyl (-CHO) and formimidoyl (-CH=NH), and they play important roles in the biosynthesis process. The one-carbon metabolic pathway mainly includes the folate cycle, the methionine cycle, as well as choline metabolism and the transsulfuration pathway. The main sources of one-carbon metabolism include the metabolism of amino acids such as serine, glycine, tryptophan and methionine. One-carbon units are important precursors for nucleic acid synthesis metabolism, participate in methylation reactions of DNA, RNA, proteins, lipids, neurotransmitters, etc. and the synthesis of glutathione, have important effects on cell epigenetics and redox status, and are closely related to the metabolic process in organisms. Therefore, simultaneous detection of metabolites related to the one-carbon metabolic pathway has important clinical significance in the study of related diseases. At present, the detection methods clinically are mostly the development of kit methods around the biomarkers related to homocysteine biochemical metabolism, and the coverage of detected substances is relatively small.

[0003] Currently, the detection of one-carbon metabolism-related metabolites mainly faces two problems: First, a variety of independent analytical methods are used to detect one-carbon related metabolites. Amino acids such as glycine and serine are detected using the hydrophilic interaction liquid chromatography (HILIC) mode; S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), folic acid, vitamin B12, etc. are generally detected in a reversed-phase C18 system. Due to the different detection modes, a large amount of manpower and material resources are required for sample pretreatment extraction and instrument detection. The method reported in the literature (A sensitive UPLC-MS / MS method for simultaneous quantification of one-carbon metabolites & co-factors in human plasma) for simultaneously detecting these substances is as follows: using an ion-pair reagent to derivatize substances such as glycine and then detecting them in a reversed-phase system. However, after adding the ion-pair reagent, the mass spectrometer will cause persistent contamination and interfere with the detection of the negative mode of other products, making the mass spectrometer no longer suitable for the detection of other products. Second, the method disclosed in the existing patent CN 117589916 A is as follows: using reducing agents (dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and β-mercaptoethanol) to hydrolyze homocystine in plasma into homocysteine and measure the total homocysteine, and cysteine is also measured for the total cysteine content. However, oxidized homocystine, cystine, and oxidized glutathione are also important substances in the one-carbon metabolic pathway, and these substances are not involved in the existing clinical detection methods. They are also highly related to cellular immunity, cell protection functions, etc., and have important significance in scientific research.

[0004] In addition, patent CN 113341012 B discloses a method, kit, and application for simultaneously detecting multiple metabolites in the homocysteine metabolic pathway, by Shandong Yingsheng Biotechnology Co., Ltd.: only 7 substances are detected. Basic substances such as SAM, SAH, and the one-carbon source substance glycine are not detected. Moreover, using a reducing agent for extraction can only measure homocysteine and cysteine, and cannot detect homocystine and cystine. Patent CN 115436536 B discloses a method for detecting markers related to the biochemical metabolism of homocysteine and a detection kit based on this method, by Beijing Hausi Biotechnology Co., Ltd.: the patent detects 18 substances, and SAM and SAH are not detected. Patent CN 117589916 A discloses a method and application for simultaneously detecting multiple metabolites in the one-carbon metabolic pathway: the patent detects 11 substances, without oxidized homocystine and cystine, and without substances such as glycine, serine, and choline.

[0005] In addition, the types of metabolites that can be simultaneously detected in the above methods are still not comprehensive enough and the quantity is small. Based on this, it is necessary to develop a method that can simultaneously detect more metabolites. Summary of the Invention

[0006] In view of this, the present invention provides a method for determining one-carbon metabolic pathway substances in plasma samples based on LC-MS / MS.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for determining one-carbon metabolic pathway substances in plasma samples based on LC-MS / MS, comprising the following steps: S1. Prepare a series of concentrations of a mixed standard of one-carbon metabolic pathway substances, add the corresponding isotope internal standard, and then add an activation reagent and a derivatization reagent to carry out a derivatization reaction to obtain a standard working solution; subject the standard working solution to on-machine detection to establish a standard curve; S2. Take a plasma sample, add the isotope internal standard corresponding to the one-carbon metabolic pathway substance to be detected, then add an extraction solution to precipitate proteins, filter, and add an activation reagent and a derivatization reagent to the supernatant to carry out a derivatization reaction to obtain an on-machine solution; S3. Centrifuge the on-machine solution, take the supernatant for on-machine detection, and substitute the detection result into the standard curve to obtain the content of the one-carbon metabolic pathway substance to be detected in the plasma; Wherein, the one-carbon metabolic pathway substances include folic acid, 5-methyltetrahydrofolic acid, dihydrofolic acid, folinic acid, S-adenosylmethionine, S-adenosylhomocysteine, cysteine, cystine, cystathionine, homocysteine, homocystine, glycine, glutamic acid, methionine, serine, tryptophan, glutathione, vitamin B12, choline, vitamin B2, dimethylglycine, acetylcholine; The isotope internal standards include 5-methyltetrahydrofolic acid-D3, N,N-dimethylglycine-D6, S-adenosylmethionine-D3, S-adenosylhomocysteine-D4, cysteine-D3,15N, glycine-D5, glutathione-D5, cystine-D6, cystathionine-D4, methionine-D3, choline chloride-D4, tryptophan-D5, serine-D7, homocysteine-D4, homocystine-D8, vitamin B2-D7, folinic acid-D4, acetylcholine chloride-D4; The extraction solution is a methanol solution containing 1% vitamin C; The activation reagent is ammonia water; The derivatization reagent includes any one of iodoacetamide, chloroacetamide, iodoacetic acid, and N-ethylmaleimide.

[0008] Further, the concentration of the isotope internal standard in the standard working solution is 100 μg / L, the concentration of the activation reagent is 0.02% - 0.2% (v / v), and the concentration of the derivatization reagent is 0.1 - 0.5 mg / mL; The concentration of the isotope internal standard in the upper machine solution is 100 μg / L, the concentration of the extraction solution is 70 - 100% (v / v), the concentration of the activation reagent is 0.02% - 0.2% (v / v), and the concentration of the derivatization reagent is 0.1 - 0.5 mg / mL.

[0009] Further, the derivatization reactions in steps S1 and S2 are specifically as follows: After adding the activation reagent and the derivatization reagent, vortex at 1500 - 2500 rpm for 2 - 5 min, and then place it in the dark at room temperature for reaction for 8 - 12 min.

[0010] Further, the centrifugation conditions of the upper machine solution in step S3 are: temperature 4°C, rotation speed 10000 - 14000 r / min, time 8 - 12 min.

[0011] In some specific embodiments, preferably, the chromatographic conditions are as follows: Chromatographic column: amide column, 2.6 µm, 100 mm × 2.1 mm; Mobile phase A: ultrapure water containing 5 - 20 mM ammonium acetate and 0.01% - 0.2% formic acid; Mobile phase B: 90% acetonitrile aqueous solution; Needle washing solution: 50% acetonitrile aqueous solution treated by ultrasonic degassing for 10 min; Column temperature: 40°C; flow rate: 0.6 mL / min; injection volume: 2 μL; Elution program: 0 - 1 min, 95% B; 5 min, 50% B; 6 min, 50% B; 6.1 - 9 min, 95% B; The mass spectrometry conditions are as follows: Ion mode: Positive; curtain gas: 35 psi; collision gas: Medium; spray voltage: 5500 V; atomization temperature: 500°C; atomizing gas: 50 psi; auxiliary gas: 60 psi; scan mode: Scheduled MRM; scan time: 0.8 s.

[0012] In some specific embodiments, preferably, the series of concentrations in step S1 are specifically 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, 1000 μg / L, 2000 μg / L, 5000 μg / L.

[0013] Further, the standard curve in step S1 is established with the ratio of the concentration of the one-carbon metabolic pathway substance to the corresponding isotope internal standard concentration as the x-axis and the ratio of the concentration of the one-carbon metabolic pathway substance to the peak area of the corresponding isotope internal standard as the y-axis.

[0014] Further, the specific conditions for precipitating proteins in step S2 are as follows: rotation speed of 1500 - 2500 rpm, temperature of 20 - 30 °C, and time of 3 - 6 min.

[0015] The application of the above method in detecting one-carbon metabolic pathway substances in plasma samples.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The detection method of the present invention can simultaneously detect 22 metabolites, with complete categories, rapid extraction, low cost, high efficiency, and extremely easy to achieve automated detection of substances, which can well assist the research and judgment of various metabolic diseases.

[0017] (2) The present invention uses a hilic mode chromatographic column. On the basis of ensuring accurate detection of substances such as folic acid and SAH, it has strong retention for homocysteine and amino acid substances, ensuring a high coverage rate of various substances on the one-carbon pathway; in addition, using an Amide chromatographic column, the derivatization reagent has no retention on this chromatographic column and can be directly cut to waste liquid without entering the mass spectrometer, protecting the mass spectrometer from contamination. Description of the Drawings

[0018] Figure 1 It is the extracted ion chromatogram for detecting 22 one-carbon metabolic pathway substances.

[0019] Figure 2 It is the detection chromatogram of 5-methyltetrahydrofolic acid (5-MTHF).

[0020] Figure 3 It is the detection chromatogram of folic acid (FA).

[0021] Figure 4 It is the detection chromatogram of cystine (Cys-Cys).

[0022] Figure 5 It is the detection chromatogram of S-adenosylmethionine (SAM) under different mobile phases; among them, the formic acid content in mobile phase A in the upper figure is 0.1%, and the formic acid content in mobile phase A in the lower figure is 0.02%. Detailed Embodiments

[0023] The following further detailed description of the present invention is provided in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0024] Example 1 This example provides a method for determining one-carbon metabolic pathway substances in plasma samples based on LC-MS / MS, which specifically includes the following steps: S1. Preparation of the standard working solution: Mix 22 standards of one-carbon metabolism pathway substances (folic acid, 5-methyltetrahydrofolic acid, dihydrofolic acid, folinic acid, S-adenosylmethionine, S-adenosylhomocysteine, cysteine, cystine, cystathionine, homocysteine, homocystine, glycine, glutamate, methionine, serine, tryptophan, glutathione, vitamin B12, choline, vitamin B2, dimethylglycine, acetylcholine), and successively prepare a series of concentrations of 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, 1000 μg / L, 2000 μg / L, 5000 μg / L. Then add isotope internal standards with a concentration of 100 μg / L (5-methyltetrahydrofolic acid-D3, N,N-dimethylglycine-D6, S-adenosylmethionine-D3, S-adenosylhomocysteine-D4, cysteine-D3,15N, glycine-D5, glutathione-D5, cystine-D6, cystathionine-D4, methionine-D3, choline chloride-D4, tryptophan-D5, serine-D7, homocysteine-D4, homocystine-D8, vitamin B2-D7, folinic acid-D4, acetylcholine chloride-D4); then add ammonia water with a concentration of 0.2% (v / v) and iodoacetamide with a concentration of 0.2 mg / mL, vortex at 2000 rpm for 3 min, and then place it in the dark at room temperature for reaction for 10 min to obtain the standard working solution. Detect the standard working solution on the machine, and establish a standard curve with the ratio of the concentration of one-carbon metabolism pathway substances to the concentration of the corresponding isotope internal standard as the x-axis and the ratio of the peak area of one-carbon metabolism pathway substances to the concentration of the corresponding isotope internal standard as the y-axis. The specific chromatographic conditions, mass spectrometry conditions, qualitative and quantitative ion information, and standard curve equation are shown in Tables 1-4.

[0025] Table 1 Chromatographic conditions

[0026] Table 2 Mass spectrometry conditions

[0027] Table 3 Qualitative and quantitative ion pair information

[0028] Table 4 Standard curve equation

[0029] S2. Preparation of the test sample: Take a plasma sample, add isotope internal standards (5-methyltetrahydrofolic acid-D3, N,N-dimethylglycine-D6, S-adenosylmethionine-D3, S-adenosylhomocysteine-D4, cysteine-D3,15N, glycine-D5, glutathione-D5, cystine-D6, cystathionine-D4, methionine-D3, choline chloride-D4, tryptophan-D5, serine-D7, homocysteine-D4, homocystine-D8, vitamin B2-D7, folinic acid-D4, acetylcholine chloride-D4) with a concentration of 100 μg / L, then add a methanol solution containing 1% vitamin C with a concentration of 90% (v / v). Vortex at a rotation speed of 2000 rpm and a temperature of 20 °C for 3 min to precipitate proteins, filter, and obtain the supernatant; add an activation reagent and a derivatization reagent to the supernatant for a derivatization reaction to obtain the solution for injection; add ammonia water with a concentration of 0.2% (v / v) and iodoacetamide with a concentration of 0.2 mg / mL to the supernatant, vortex at 2000 rpm for 3 min, and then place it in the dark at room temperature for a reaction for 10 min to obtain the solution for injection.

[0030] S3. Sample detection: Take the solution for injection in S2, centrifuge at 12000 r / min at 4 °C for 10 min, and take the supernatant after centrifugation for injection detection (the detection conditions are the same as those in step S1); integrate the peak areas of each substance in the obtained chromatogram, calculate the ratio of the peak area of the corresponding internal standard, substitute the obtained peak area ratio into the standard curve to calculate the concentration, and then calculate the content of the substance to be detected in each biological sample according to the mass of the biological sample used. Among them, the spiked recovery rate and precision of the plasma sample are shown in Table 5 specifically, and the specific relevant detection results are shown in Figures 1-4 .

[0031] Table 5 Spiked recovery rate and precision of plasma sample

[0032] Furthermore, the present invention also explored adjusting the contents of formic acid and ammonium acetate in mobile phase A to improve the tailing phenomenon of the substances to be detected on the chromatographic column: adjust the formic acid in mobile phase A to 0.1% and the ammonium acetate to 5 mM (v / v) (the other conditions are the same as those in Example 1 above), and the specific results are shown in Figure 5 .

[0033] It can be Figure 5 seen that by adjusting the contents of formic acid and ammonium acetate in mobile phase A, the tailing phenomenon of SAM and SAH on the Amide chromatographic column can be significantly improved.

[0034] The above experimental results show that: the method provided by this application can well detect the concentrations of substances in the one-carbon metabolic pathway in plasma, and the detected types are as many as 22, which can better serve the research on related metabolic diseases.

[0035] In the present invention, the specific raw materials not described are all existing substances and can be directly purchased from the market.

[0036] The above are only the preferred implementation schemes of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for determining one-carbon metabolic pathway substances in plasma samples based on LC-MS / MS, characterized in that: The following steps are involved: S1. Prepare a series of concentrations of a mixed standard of substances in the one-carbon metabolic pathway, add the corresponding isotope internal standard, and then add an activation reagent and a derivatization reagent to perform a derivatization reaction to obtain a standard working solution; test the standard working solution on a machine and establish a standard curve; S2, taking a plasma sample, adding an isotope internal standard corresponding to the substance in the one-carbon metabolic pathway to be tested, then adding an extract to precipitate the protein, filtering, adding an activation reagent and a derivatization reagent to the supernatant to perform a derivatization reaction, and obtaining an upper solution; S3, centrifuging the solution on the machine, taking the supernatant for machine testing, and inserting the test results into the standard curve to obtain the content of the substance in the one-carbon metabolic pathway to be tested in the plasma; The one-carbon metabolic pathway substances include folic acid, 5-methyltetrahydrofolate, dihydrofolate, folinic acid, S-adenosylmethionine, S-adenosylhomocysteine, cysteine, cystine, cystathionine, homocysteine, homocystine, glycine, glutamic acid, methionine, serine, tryptophan, glutathione, vitamin B12, choline, vitamin B2, dimethylglycine, and acetylcholine; The isotope internal standard includes 5-methyltetrahydrofolate-D3, N,N-dimethylglycine-D6, S-adenosylmethionine-D3, S-adenosylhomocysteine-D4, cysteine-D3,15N, glycine-D5, glutathione-D5, cystine-D6, cystathionine-D4, methionine-D3, choline chloride-D4, tryptophan-D5, serine-D7, homocysteine-D4, homocystine-D8, vitamin B2-D7, folinic acid-D4, and acetylcholine chloride-D4; The extract is a methanol solution containing 1% vitamin C; The activation reagent is ammonia water; The derivatization reagent includes any one of iodoacetamide, chloroacetamide, iodoacetic acid and N-ethylmaleimide.

2. The method according to claim 1, characterized in that The isotope internal standard concentration in the standard working solution is 100 μg / L, the activation reagent concentration is 0.02%~0.2% (v / v), and the derivatization reagent concentration is 0.1~0.5 mg / mL; The isotope internal standard concentration in the upper solution is 100 μg / L, the extract concentration is 70-100% (v / v), the activation reagent concentration is 0.02%-0.2% (v / v), and the derivatization reagent concentration is 0.1-0.5 mg / mL.

3. The method according to claim 1, characterized in that The derivatization reaction in steps S1 and S2 is specifically as follows: after adding the activation reagent and the derivatization reagent, vortex at 1500-2500 rpm for 2-5 minutes, and then place in a dark place for reaction at room temperature for 8-12 minutes.

4. The method according to claim 1, characterized in that: The centrifugal conditions of the solution in step S3 are as follows: temperature 4° C., rotation speed 10000-14000 r / min, and time 8-12 min.

5. The method according to claim 1, characterized in that The chromatographic conditions were as follows: Chromatographic column: amide column, 2.6µm, 100mm×2.1mm; Mobile phase A: ultrapure water containing 5-20 mM ammonium acetate and 0.01%-0.2% formic acid; Mobile phase B: 90% acetonitrile in water; Needle washing solution: 50% acetonitrile aqueous solution after ultrasonic degassing for 10 minutes; Column temperature: 40°C; flow rate: 0.6 mL / min; injection volume: 2 μL; Elution program: 0-1min, 95%B; 5min, 50%B; 6min, 50%B; 6.1-9min, 95%B; The mass spectrometry conditions were as follows: Ion mode: Positive; Air curtain gas: 35psi; Collision gas: Medium; Spray voltage: 5500V; Atomization temperature: 500℃; Nebulizer gas: 50psi; auxiliary gas: 60psi; scanning mode: Scheduled MRM; scanning time: 0.8s.

6. The method according to claim 1, characterized in that The series of concentrations in step S1 are specifically 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, 1000 μg / L, 2000 μg / L, and 5000 μg / L.

7. The method according to claim 1, characterized in that The standard curve in step S1 is established with the ratio of the concentration of the one-carbon metabolic pathway substance to the corresponding isotope internal standard concentration as the x-axis and the ratio of the peak area of ​​the one-carbon metabolic pathway substance to the corresponding isotope internal standard concentration as the y-axis.

8. The method according to claim 1, characterized in that The specific conditions for protein precipitation in step S2 are: rotation speed 1500-2500 rpm, temperature 20-30° C., and time 3-6 min.

9. Use of the method according to any one of claims 1 to 8 in detecting substances in the one-carbon metabolic pathway in plasma samples.

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