Method for detecting homocysteinemia related metabolites in serum by HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) combination

Abstract

The invention provides a method for detecting homocysteinemia related metabolites in serum through HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) combination. The method can be used for simultaneously detecting homocysteine, cysteine, methionine, cystathionine, glycine, serine, vitamin B2, vitamin B6, vitamin B9, 5-methyltetrahydrofolic acid and methylmalonic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biological detection. Specifically, the present invention relates to a method for detecting homocysteineemia-related metabolites in serum by HPLC-MS / MS coupling. Background Art

[0002] Homocysteine is a sulfur-containing amino acid, also known as homocysteine, abbreviated as Hcy. It is an essential amino acid for human nutrition (required in the body but cannot be synthesized by itself) and is an important intermediate product produced during the metabolism of methionine and cysteine. Homocysteine can be catabolized in the body, and its concentration is maintained at a relatively low level. The catabolism of homocysteine in cells mainly occurs through two pathways: the remethylation pathway and the transsulfuration pathway. The remethylation pathway refers to that about half of the homocysteine in cells, under the action of methionine synthase, with vitamin B12 as a coenzyme factor and N-5-methyltetrahydrofolate as a methyl donor, undergoes remethylation to synthesize methionine and tetrahydrofolate. The transsulfuration pathway refers to that the other about half of the homocysteine is condensed into cystathionine and water under the catalysis of cystathionine synthase, with vitamin B6 as a coenzyme. This reaction is irreversible under physiological conditions and is beneficial to the transport of Hcy. When the above two metabolic pathways are blocked, homocysteine accumulates in cells and enters the blood circulation, thereby causing chronic pathological damage. Homocysteine can damage blood vessels through multiple pathways. It can damage vascular endothelial cells and at the same time stimulate the growth of vascular smooth muscle cells. The damage caused by both leads to the obstruction of the blood flow pathway; homocysteine can also disrupt the normal coagulation mechanism, increase the formation of thrombi, and cause heart attacks and strokes. Therefore, the level of homocysteineemia-related metabolites in serum is closely related to cardiovascular diseases. Figure 1 It is a methionine metabolism diagram and also a homocysteine metabolism pathway diagram. The metabolism of homocysteine is a relatively complex process. Therefore, the detection of homocysteine and its metabolism-related substances is more meaningful for the diagnosis and treatment of hyperhomocysteinemia.

[0003] Currently, the main clinical detection methods for serum homocysteineemia are high-performance liquid chromatography (HPLC), fluorescence polarization immunoassay (FPLA), enzyme-linked immunosorbent assay (ELISA), chemiluminescence, and cyclic enzyme method, etc. However, these methods for detecting homocysteineemia have the disadvantages of being easily interfered by the matrix, unable to distinguish isomers, large result differences, insufficient sensitivity, small throughput, and generally only being able to detect one substance, homocysteine. After detecting that the homocysteine is on the high side, the cause cannot be determined, etc., thus limiting the accurate determination of homocysteineemia and the accurate health assessment of the homocysteine metabolism status in the human body.

[0004] Therefore, there is a need in the art to develop an accurate, rapid, and high-precision method for determining metabolites related to hyperhomocysteinemia, which can detect related products in the metabolic pathway while detecting homocysteine, so as to more clearly identify the causes of elevated homocysteine and facilitate the timely formulation of treatment plans. Summary of the Invention

[0005] The object of the present invention is to provide an accurate, rapid, and high-precision method for determining metabolites related to hyperhomocysteinemia, so as to alleviate the technical problems in the prior art that cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6 (pyridoxic acid), vitamin B2, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid cannot be detected or cannot be detected simultaneously.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] According to one aspect of the present invention, the present invention provides a method for detecting metabolites related to hyperhomocysteinemia in a sample by high performance liquid chromatography-mass spectrometry (HPLC-MS), characterized in that the metabolites related to hyperhomocysteinemia include cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6 (pyridoxic acid), vitamin B2, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid; the method includes:

[0008] Pretreatment: Mix the sample with a reducing agent, then add a protein precipitation solution and mix, centrifuge and take the supernatant.

[0009] Analysis: Use high performance liquid chromatography-mass spectrometry to detect the metabolites related to hyperhomocysteinemia in the supernatant, and quantify by a standard curve drawn from a known amount of standard products within a certain range to obtain the content of metabolites related to hyperhomocysteinemia.

[0010] In a further aspect, the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride / methanol solution.

[0011] In a further aspect, the concentration of the tris(2-carboxyethyl)phosphine hydrochloride / methanol solution is 5-15 mg / mL; preferably 10 mg / mL.

[0012] In a further aspect, the protein precipitation solution is 100% methanol solution.

[0013] In a further aspect, in the pretreatment, the volume ratio of the sample, the reducing agent, and the protein precipitation solution is 2:(0.5-2):(2-5); preferably 2:1:4.

[0014] In a further aspect, the protein precipitation solution further contains an internal standard. Preferably, the internal standard is an isotope internal standard labeled with an isotope.

[0015] In a further aspect, the high performance liquid chromatography detection conditions include:

[0016] Chromatographic column: reverse phase chromatographic column; preferably, octadecylsilane (C18) bonded silica gel;

[0017] Mobile phase: The mobile phase comprises mobile phase A and mobile phase B; mobile phase A is an aqueous formic acid solution with a concentration of 0.02 - 0.5%, preferably 0.1%; mobile phase B is a formic acid methanol solution with a concentration of 0.02 - 0.5%, preferably 0.1%.

[0018] In a further aspect, the mass spectrometry detection conditions include:

[0019] Ion source parameters: In the electrospray ionization (ESI) mode, multiple reaction monitoring (MRM) is used for simultaneous positive and negative ion mode scanning; the ionization voltage is 5500 (ESI+) / - 4500 (ESI-); the ion source temperature is 550 °C; the curtain gas is 30 psi, the collision gas is 6 psi, the spray gas is 50 psi, and the auxiliary heating gas is 50 psi.

[0020] In a further aspect, the sample is a blood sample of a human or non - human mammal (such as pig, dog, cat, sheep or cow), preferably whole blood, serum, plasma or dried blood spot; preferably, the sample is serum.

[0021] In a further aspect, in the method, first, high performance liquid chromatography is used to separate the metabolites related to homocystinemia from the interfering components in the sample, and then mass spectrometry is used to detect the mass - to - charge ratio of the metabolites related to homocystinemia and their corresponding internal standards. According to the internal standard method for quantitative analysis, the content of the metabolites related to homocystinemia is obtained.

[0022] The method of the present invention can accurately, rapidly and with high precision determine the metabolites related to homocystinemia, alleviating the technical problem in the prior art that homocysteine, cysteine, methionine, cystathionine, glycine, serine, vitamin B2, vitamin B6, vitamin B9, 5 - methyltetrahydrofolate, and methylmalonic acid cannot be detected simultaneously, and can accurately analyze the cause of homocystinemia.

[0023] Specifically, the present invention provides a method for detecting homocystinemia-related metabolites in a sample by high performance liquid chromatography coupled with mass spectrometry. The homocystinemia-related metabolites include cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6 (pyridoxic acid), vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid. The method includes using high performance liquid chromatography coupled with mass spectrometry to detect the homocystinemia-related metabolites in the pretreated sample to be tested, and quantifying by a standard curve to obtain the content of the homocystinemia-related metabolites.

[0024] Preferably, the pretreatment method uses the protein precipitation method.

[0025] Preferably, after mixing the sample with a reducing agent, add a protein precipitation solution and shake at 1000 - 3000 rpm for 5 - 15 min; centrifuge at 10000 - 15000 rpm at 2 - 8 °C for 5 - 15 min, and then perform high performance liquid chromatography coupled with mass spectrometry detection.

[0026] Preferably, after mixing the sample with a reducing agent, add a protein precipitation solution and shake at 2000 rpm for 10 min; centrifuge at 12000 rpm at 4 °C for 10 min, and then perform high performance liquid chromatography coupled with mass spectrometry detection.

[0027] Preferably, the reducing agent is a mixed solution of tris(2-carboxyethyl)phosphine hydrochloride and a diluent; the protein precipitation solution is the diluent.

[0028] Preferably, the diluent is a pure methanol solution.

[0029] Preferably, in the mixed solution of tris(2-carboxyethyl)phosphine hydrochloride and the diluent, the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 5 - 15 mg / mL; preferably 10 mg / mL.

[0030] Preferably, the mixing volume ratio of the sample, the reducing agent, and the protein precipitation solution is 2:(0.5 - 2):(2 - 5); preferably 2:1:4.

[0031] Preferably, the protein precipitation solution also contains an internal standard.

[0032] Preferably, the chromatographic column is a reverse chromatographic column.

[0033] Preferably, the reverse chromatographic column is octadecylsilane (C18) bonded silica gel.

[0034] Preferably, the mobile phase of the high performance liquid chromatography includes mobile phase A and mobile phase B; mobile phase A is an aqueous phase, mobile phase B is an organic phase, and the mobile phase contains formic acid.

[0035] Preferably, the content of formic acid in mobile phase A and mobile phase B is independently preferably 0.02-0.5%;

[0036] Preferably, the content of formic acid in mobile phase A and mobile phase B is independently preferably 0.1%;

[0037] Preferably, mobile phase B is methanol containing formic acid;

[0038] Preferably, the mobile phase elution is gradient elution, and the conditions of the gradient elution are as follows:

[0039] Time (min) ％A ％B 0.0 85～95 15～5 1.0 20～30 80～70 3.0 15～5 85～95 3.5 15～5 85～95 3.6 85～95 15～5 5.0 85～95 15～5

[0040] Among them, %A refers to the volume percentage of mobile phase A in the mobile phase, and %B refers to the volume percentage of mobile phase B in the mobile phase; the elution gradient process is: within 0-1.0 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from (85-95):(15-5) to (20-30):(80-70); within 1.0-3.0 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from (20-30):(80-70) to (15-5):(85-95); within 3.0-3.5 minutes, the volume ratio of mobile phase A and mobile phase B is (15-5):(85-95); within 3.5-3.6 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from (15-5):(85-95) to (85-95):(15-5); within 3.6-5.0 minutes, the volume ratio of mobile phase A and mobile phase B is (85-95):(15-5);

[0041] Preferably, the conditions of the gradient elution are as follows:

[0042] Time (min) ％A ％B 0.0 90 10 1.0 25 75 3.0 10 90 3.5 10 90 3.6 90 10 5.0 90 10

[0043] Among them, %A refers to the volume percentage of mobile phase A in the mobile phase, and %B refers to the volume percentage of mobile phase B in the mobile phase; the elution gradient process is: within 0-1.0 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 90:10 to 25:75; within 1.0-3.0 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 25:75 to 10:90; within 3.0-3.5 minutes, the volume ratio of mobile phase A and mobile phase B is 10:90; within 3.5-3.6 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 10:90 to 90:10; within 3.6-5.0 minutes, the volume ratio of mobile phase A and mobile phase B is 90:10.

[0044] Preferably, the ion source parameters are as follows: in the electrospray ionization (ESI) mode, multiple reaction monitoring (MRM) is used for positive (negative) ion mode scanning; the ionization voltage is 5500 (ESI+) / -4500 (ESI-); the ion source temperature is 550 °C; the curtain gas is 30 psi, the collision gas is 6 psi, the spray gas is 50 psi, and the auxiliary heating gas is 50 psi.

[0045] Preferably, the mass spectrometry parameters are as follows:

[0046]

[0047]

[0048] Among them, * represents the quantitative ion.

[0049] Preferably, the mobile phase flow rate is 0.2 - 0.4 mL / min, preferably 0.3 mL / min;

[0050] Preferably, the column temperature of the chromatographic column is 35 - 45 °C, preferably 40 °C;

[0051] Preferably, the injection volume is 1 - 5 μL, preferably 2 μL;

[0052] Preferably, the running time is 3 - 6 min, preferably 5 min;

[0053] Preferably, the corresponding relationship between the content of the analyte in the standard curve and the standard product and the detected value of the standard product; the detected value of the standard product is the peak ratio of the standard product to the internal standard, and the peak includes peak height or peak area;

[0054] Preferably, the standard product consists of a series of concentrations of several standard products, and the concentrations of each substance in the several standard products with a series of concentrations are as follows:

[0055] The concentrations of cysteine are 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, and 500 μmol / L in sequence;

[0056] The concentrations of homocysteine are 1.5 μmol / L, 3.0 μmol / L, 7.5 μmol / L, 15 μmol / L, 30 μmol / L, and 75 μmol / L in sequence;

[0057] The concentrations of methionine are 2 μmol / L, 4 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, and 100 μmol / L in sequence;

[0058] The concentrations of cystathionine are 0.2 μmol / L, 0.4 μmol / L, 1.0 μmol / L, 2 μmol / L, 4 μmol / L, and 10 μmol / L in sequence;

[0059] The concentrations of glycine are 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, and 500 μmol / L in sequence;

[0060] The concentrations of serine are 5 μmol / L, 10 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L, and 250 μmol / L in sequence;

[0061] The concentrations of vitamin B6 are 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL in sequence;

[0062] The concentrations of vitamin B2 are 1.5 ng / mL, 3 ng / mL, 7.5 ng / mL, 15 ng / mL, 30 ng / mL, and 75 ng / mL in sequence;

[0063] The concentrations of vitamin B9 are 2 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 100 ng / mL in sequence;

[0064] The concentrations of 5-methyltetrahydrofolate are 3 ng / mL, 6 ng / mL, 15 ng / mL, 30 ng / mL, 60 ng / mL, and 150 ng / mL in sequence;

[0065] The concentrations of methylmalonic acid are 100 nmol / L, 200 nmol / L, 500 nmol / L, 1000 nmol / L, 2000 nmol / L, and 5000 nmol / L in sequence;

[0066] Preferably, the internal standard is an isotope internal standard labeled with an isotope;

[0067] Preferably, the detection method further includes detecting a quality control sample:

[0068] Preferably, the quality control sample includes at least two of a low-value quality control sample, a mid-value quality control sample, and a high-value quality control sample; the low-value quality control sample is 2 to 4 times the lowest point of the linear range, the mid-value quality control sample is 40 to 50% of the highest point of the linear range, and the high-value quality control sample is 60 to 80% of the highest point of the linear range;

[0069] Preferably, the sample is a blood sample of a human or non-human mammal (such as a pig, dog, cat, sheep, or cow), preferably whole blood, serum, plasma, or a dried blood spot;

[0070] Preferably, the sample is serum;

[0071] Preferably, the matrix of the standard and the quality control product includes PBS*10 solution and BSA;

[0072] Preferably, the matrix has a BSA content of 0.01-10%, more preferably 0.05-5%, and most preferably 0.1-1%.

[0073] This method uses high-performance liquid chromatography to separate the metabolites related to hyperhomocysteinemia from the interfering components in the sample, and then uses mass spectrometry to detect the mass-to-charge ratio of the metabolites related to hyperhomocysteinemia and their corresponding internal standards. According to the internal standard method for quantitative analysis, the content of the metabolites related to hyperhomocysteinemia is obtained, which is used in the preparation of products for diagnosing hyperhomocysteinemia.

[0074] Compared with the prior art, the present invention has the following beneficial effects:

[0075] This method uses high performance liquid chromatography tandem mass spectrometry for high-capacity detection of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6 (pyridoxic acid), vitamin B2, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid in human serum. It can detect eleven compounds simultaneously in one test. After the sample is pretreated by the protein precipitation method, it enters the high performance liquid chromatography for separation and then enters the tandem mass spectrometer. The eleven-compound test can be completed within the optimal five minutes, which is convenient and fast. The detection method provided by the present invention has the characteristics of high sensitivity, good repeatability, high accuracy, and good specificity. The linear ranges of this method are as follows: cysteine 10 μmol / L - 500 μmol / L; homocysteine 1.5 μmol / L - 75 μmol / L; methionine 2 μmol / L - 100 μmol / L; cystathionine 0.2 μmol / L - 10 μmol / L; glycine 10 μmol / L - 500 μmol / L; serine 5 μmol / L - 250 μmol / L; vitamin B6 1 ng / mL - 50 ng / mL; vitamin B2 1.5 ng / mL - 75 ng / mL; vitamin B9 2 ng / mL - 100 ng / mL; 5-methyltetrahydrofolate 3 ng / mL - 150 ng / mL; methylmalonic acid 100 nmol / L - 5000 nmol / L, and the linear correlation coefficient r ≥ 0.990; the coefficient of variation (CV) of the repeatability of the low-value quality control sample ≤ 20%, the coefficient of variation (CV) of the repeatability of the medium-value quality control sample ≤ 15%, and the coefficient of variation (CV) of the repeatability of the high-value quality control sample ≤ 15%; the relative deviation (B) of the accuracy ≤ ±15%, and the spike recovery rate is 85% - 115%. The detection method of metabolites related to hyperhomocysteinemia provided by the present invention can meet the performance requirements of relevant regulations for the linearity, repeatability, inter-batch difference, and accuracy of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6 (pyridoxic acid), vitamin B2, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid, and plays an important role in the research on the pathological mechanism and treatment of hyperhomocysteinemia.

[0076] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferably technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0077] Figure 1 It is a methionine metabolism diagram and also a homocysteine metabolic pathway diagram.

[0078] Figure 2Chromatogram of cysteine in human serum sample, where the retention time of 1.19 min is the cysteine peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0079] Figure 3 Chromatogram of homocysteine in human serum sample, where the retention time of 1.26 min is the homocysteine peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0080] Figure 4 Chromatogram of methionine in human serum sample, where the retention time of 1.49 min is the methionine peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0081] Figure 5 Chromatogram of vitamin B6 in human serum sample, where the retention time of 2.27 min is the vitamin B6 peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0082] Figure 6 Chromatogram of cystathionine in human serum sample, where the retention time of 1.13 min is the cystathionine peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0083] Figure 7 Chromatogram of vitamin B2 in human serum sample, where the retention time of 2.69 min is the vitamin B2 peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0084] Figure 8 Chromatogram of vitamin B9 in human serum sample, where the retention time of 2.60 min is the vitamin B9 peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0085] Figure 9 Chromatogram of 5-methyltetrahydrofolic acid in human serum sample, where the retention time of 2.21 min is the 5-methyltetrahydrofolic acid peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0086] Figure 10 Chromatogram of glycine in human serum sample, where the retention time of 1.12 min is the glycine peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0087] Figure 11 Chromatogram of serine in human serum sample, where the retention time of 1.13 min is the serine peak. Note: The left figure is the analyte, and the right figure is the corresponding isotope internal standard of the analyte.

[0088] Figure 12 Chromatogram of methylmalonic acid in human serum sample, where the retention time of 2.42 min is the methylmalonic acid peak. Note: The left figure is the analyte, and the right figure is the corresponding isotopic internal standard of the analyte. Detailed implementation mode

[0089] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0091] As used herein, the terms "comprising", "including" and "containing" are interchangeable and include not only the open definition, but also the semi-closed and closed definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0092] As used herein, the term "Cys" refers to cysteine.

[0093] As used herein, the term "Hcy" refers to homocysteine.

[0094] As used herein, the term "Met" refers to methionine.

[0095] As used herein, the term "Cth" refers to cystathionine.

[0096] As used herein, the term "VB2" refers to vitamin B2.

[0097] As used herein, the term "VB6" refers to vitamin B6.

[0098] As used herein, the term "VB9" refers to vitamin B9.

[0099] As used herein, the term "5-MTHF" refers to 5-methyltetrahydrofolic acid.

[0100] As used herein, the term "Gly" refers to glycine.

[0101] As used herein, the term "Ser" refers to serine.

[0102] As used herein, the term "MMA" refers to methylmalonic acid.

[0103] As used herein, the term "high performance liquid chromatography" is abbreviated as HPLC.

[0104] As used herein, the term "mass spectrometry" is abbreviated as MS.

[0105] According to one aspect of the present invention, the present invention provides a detection method for detecting metabolites related to hyperhomocysteinemia in serum by HPLC-MS / MS. The detection indexes include: homocysteine, cysteine, methionine, cystathionine, glycine, serine, vitamin B2, vitamin B6, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid.

[0106] The detection method provided by the present invention includes using high performance liquid chromatography tandem mass spectrometry to detect the pretreated sample to be detected, and substituting the detected value into the standard curve to calculate the content of each analyte in the sample.

[0107] The test principle of the determination method provided by the present invention is to take the sample to be detected, such as standard products, quality control products and actual samples, and after pretreatment, perform liquid chromatography tandem mass spectrometry (HPLC-MS / MS) analysis. When using the internal standard method for quantification, an internal standard is also mixed in the sample to be detected. After the analyte is separated by chromatography, it is introduced into the mass spectrometer and ionized in the ion source to form charged ions. Under the action of the electric field, it is focused into the triple quadrupole mass analyzer. In the mass spectrometer analyzer, the parent ion and daughter ion of the analyte are separated, and finally enter the detector to generate a signal, record the detected value of the sample to be detected, and then obtain the content of the target substance in the sample to be detected through further analysis of the detected value.

[0108] In some optional embodiments, the determination method includes first pretreating the sample to remove impurities in the sample and / or enrich the target analyte in the sample. The pretreatment can be carried out in any acceptable manner in the art, including but not limited to one or more of protein precipitation, liquid-liquid extraction, solid-phase extraction, etc. The present invention does not make any restrictions. It can be understood that those skilled in the art can select the corresponding pretreatment method according to the general knowledge in the art and the different types of samples. For serum samples, we have made a comparison of different pretreatment methods. The comparison mainly verified indexes such as operation convenience, peak shape, sensitivity, addition recovery, etc. The verification summary is as follows:

[0109]

[0110] Since all 11 of our analytes are water-soluble substances and amino acids have very weak retention in the chromatographic column, combined with the verification results, the protein precipitation pretreatment method is recommended for serum samples.

[0111] In some alternative embodiments, protein precipitation is used for the pretreatment. The protein precipitation method can be carried out in EP tubes and 96-well plates, and the 96-well plate can realize fully automatic pretreatment operations subsequently. The preferred operation steps for protein precipitation are as follows: After mixing the sample with the reducing agent working solution, add the protein precipitation solution and shake at 1000 - 3000 rpm for 5 - 15 min; centrifuge at 10000 - 15000 rpm at 2 - 8 °C for 5 - 15 min and then perform high performance liquid chromatography - mass spectrometry (HPLC - MS) detection; preferably, after mixing the sample with the reducing agent, add the protein precipitation solution and shake at 2000 rpm for 10 min; centrifuge at 12000 rpm at 4 °C for 10 min and then perform HPLC - MS detection. The reducing agent working solution is a mixed solution of tris(2 - carboxyethyl)phosphine hydrochloride and a diluent, and the concentration of tris(2 - carboxyethyl)phosphine hydrochloride is 10 mg / mL. We made comparisons with different salt concentrations, and the comparison results are as follows:

[0112]

[0113]

[0114]

[0115] As shown in the results in the table, when the concentration of tris(2 - carboxyethyl)phosphine hydrochloride is 5 - 15 mg / mL, the detection results of each analyte are stable, and the preferred concentration of tris(2 - carboxyethyl)phosphine hydrochloride is 10 mg / mL.

[0116] In some alternative embodiments, the protein precipitation solution is a diluent, and the selected diluent is pure methanol solution. Preferably, the mixing volume ratio of the sample, the reducing agent, and the protein precipitation solution is 2:1:4. For example, take 100 μL of the sample, add 50 μL of the reducing agent working solution, and add 200 μL of the protein precipitation solution. The protein precipitation solution also contains an internal standard to facilitate the introduction of the internal standard during the pretreatment process. We also tried using acetonitrile as the diluent. Because for the same volume of methanol and acetonitrile, the protein precipitation effect of acetonitrile is better than that of methanol, but when using acetonitrile as the diluent, the chromatographic peak shapes of each analyte are not as good as those of methanol, especially for methionine and vitamins.

[0117] In some alternative embodiments, internal standard method is used for quantification. The internal standard is the isotope internal standard of the analyte. Using the corresponding isotope internal standard for quantification can not only greatly eliminate matrix interference, but also the results are not affected by conditions such as the pretreatment process and instrument response fluctuations, and can achieve accurate quantification. The preferred internal standards include: vitamin B2 - 13C4.15N2, vitamin B6 - d3, vitamin B9 - 13C5, methylmalonic acid - d3, 5 - methyltetrahydrofolic acid - 13C5, cysteine - d3, homocysteine - d4, methionine - d3, cystathionine - d4, serine - d3, glycine - d2.

[0118] In some alternative embodiments, the chromatographic conditions in high performance liquid chromatography tandem mass spectrometry are also optimized. The optimized mobile phase is as follows: the mobile phase comprises mobile phase A and mobile phase B; mobile phase A is an aqueous phase, mobile phase B is an organic phase, and formic acid is contained in the mobile phase; the content of formic acid in mobile phase A and mobile phase B is independently preferably 0.01-1%; for example, it can be but not limited to 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, preferably 0.05%-0.2%, and more preferably 0.1%. The chromatographic column is preferably octadecylsilane (C18) bonded silica gel. Mobile phase B is methanol containing formic acid. We compared the use of methanol and acetonitrile for mobile phase B and found that when using acetonitrile, the retention time of the overall analyte was earlier than that when using methanol, and amino acids were basically not retained, resulting in the inability to separate the target peak and impurity peaks. When acetonitrile containing formic acid was selected as mobile phase B, the sensitivity of most analytes decreased significantly and the internal standard substance did not elute, so methanol containing formic acid was selected as mobile phase B.

[0119] In some alternative embodiments, the elution program for high performance liquid chromatography detection is as follows:

[0120]

[0121]

[0122] Wherein, %A refers to the volume percentage of mobile phase A in the mobile phase, and %B refers to the volume percentage of mobile phase B in the mobile phase; the elution gradient process is as follows: within 0-1.0 minute, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 90:10 to 25:75; within 1.0-3.0 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 25:75 to 10:90; within 3.0-3.5 minutes, the volume ratio of mobile phase A and mobile phase B is 10:90; within 3.5-3.6 minutes, the volume ratio of mobile phase A and mobile phase B gradually changes uniformly from 10:90 to 90:10; within 3.6-5.0 minutes, the volume ratio of mobile phase A and mobile phase B is 90:10.

[0123] In some alternative embodiments, the mass spectrometry ion source parameters are as follows: in the electrospray ionization (ESI) mode, multiple reaction monitoring (MRM) is used for positive (negative) ion mode scanning; the ionization voltage is 5500 (ESI+) / -4500 (ESI-); the ion source temperature is 550 °C; the curtain gas is 30 psi, the collision gas is 6 psi, the spray gas is 50 psi, and the auxiliary heating gas is 50 psi.

[0124] In some alternative embodiments, the mass spectrometry parameters are as follows:

[0125]

[0126]

[0127] where * represents the quantitative ion.

[0128] In some alternative embodiments, the mobile phase flow rate is 0.2 - 0.4 mL / min, preferably 0.3 mL / min; the column temperature of the chromatographic column is 35 - 45 °C, preferably 40 °C, the injection volume is 1 - 5 μL, preferably 2 μL; the running time is 3 - 6 min, preferably 5 min;

[0129] In some alternative embodiments, the corresponding relationship between the content of the analyte in the standard curve and the standard product and the detected value of the standard product; the detected value of the standard product is the peak ratio of the standard product to the internal standard, and the peak includes peak height or peak area; preferably, taking the standard product concentration as the independent variable xi and the peak area ratio of the corresponding concentration standard product to the internal standard as the dependent variable yi, calculate the linear regression equation y = ax + b and the correlation coefficient r.

[0130] The standard product preferably consists of a number of standard products with a series of concentrations. The concentrations of each substance in the number of standard products with a series of concentrations are as follows:

[0131] The concentrations of cysteine are 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L in sequence;

[0132] The concentrations of homocysteine are 1.5 μmol / L, 3.0 μmol / L, 7.5 μmol / L, 15 μmol / L, 30 μmol / L, 75 μmol / L in sequence;

[0133] The concentrations of methionine are 2 μmol / L, 4 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 100 μmol / L in sequence;

[0134] The concentrations of cystathionine are 0.2 μmol / L, 0.4 μmol / L, 1.0 μmol / L, 2 μmol / L, 4 μmol / L, 10 μmol / L in sequence;

[0135] The concentrations of glycine are 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L in sequence;

[0136] The concentrations of serine are 5 μmol / L, 10 μmol / L, 25 μmol / L, 50 μmol / L, 100 μmol / L, 250 μmol / L in sequence;

[0137] The concentrations of vitamin B6 are 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL in sequence;

[0138] The concentrations of vitamin B2 are 1.5 ng / mL, 3 ng / mL, 7.5 ng / mL, 15 ng / mL, 30 ng / mL, and 75 ng / mL in sequence;

[0139] The concentrations of vitamin B9 are 2 ng / mL, 4 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 100 ng / mL in sequence;

[0140] The concentrations of 5-methyltetrahydrofolate are 3 ng / mL, 6 ng / mL, 15 ng / mL, 30 ng / mL, 60 ng / mL, and 150 ng / mL in sequence;

[0141] The concentrations of methylmalonic acid are 100 nmol / L, 200 nmol / L, 500 nmol / L, 1000 nmol / L, 2000 nmol / L, and 5000 nmol / L in sequence;

[0142] In some alternative embodiments, the detection method further includes detecting quality control samples: the quality control samples preferably include at least two of low-value quality control samples, medium-value quality control samples, and high-value quality control samples; the low-value quality control sample is 2 to 4 times the lowest point of the linear range, the medium-value quality control sample is 40 to 50% of the highest point of the linear range, and the high-value quality control sample is 60 to 80% of the highest point of the linear range. The preferred concentrations of each substance in the quality control samples are as follows:

[0143] Cysteine: low-value quality control 30 μmol / L, medium-value quality control 250 μmol / L, high-value quality control 400 μmol / L;

[0144] Homocysteine: low-value quality control 4.5 μmol / L, medium-value quality control 37.5 μmol / L, high-value quality control 60 μmol / L;

[0145] Methionine: low-value quality control 6 μmol / L, medium-value quality control 50 μmol / L, high-value quality control 80 μmol / L;

[0146] Cystathionine: low-value quality control 0.6 μmol / L, medium-value quality control 5 μmol / L, high-value quality control 8 μmol / L;

[0147] Glycine: low-value quality control 30 μmol / L, medium-value quality control 250 μmol / L, high-value quality control 400 μmol / L;

[0148] Serine: low-value quality control 15 μmol / L, medium-value quality control 125 μmol / L, high-value quality control 200 μmol / L;

[0149] Vitamin B6: low value quality control 3ng / mL, medium value quality control 25ng / mL, high value quality control 40ng / mL;

[0150] Vitamin B2: low value quality control 4.5ng / mL, medium value quality control 37.5ng / mL, high value quality control 60ng / mL;

[0151] Vitamin B9: low value quality control 6ng / mL, medium value quality control 50ng / mL, high value quality control 80ng / mL;

[0152] 5-Methyltetrahydrofolate: low value quality control 9ng / mL, mid value quality control 75ng / mL, high value quality control 120ng / mL;

[0153] Methylmalonic acid: low value quality control 300nmol / L, mid value quality control 2500nmol / L, high value quality control 4000nmol / L;

[0154] The method for detecting metabolites related to hyperhomocysteinemia provided by the present invention, the source of the sample to be tested is, for example, but not limited to, blood samples of humans or non-human mammals (such as pigs, dogs, cats, sheep or cattle). It should be noted that the sample for detecting metabolites related to hyperhomocysteinemia provided by the present invention can be, but not limited to, serum, plasma, extracts and body fluids of tissues or cells, etc. In some optional examples, the sample is serum. When the sample is serum, since human-derived samples without background can be obtained, the matrix of the standard and quality control products preferably includes PBS*10 solution and BSA mixed solution, and the mixed solution preferably has a BSA content of 0..05-5%. Use 50% methanol water to prepare the standard curve, measure the background of each test object with different blanks instead of matrices (different proportions of BSA content), and the verification results are as follows:

[0155]

[0156]

[0157] Because the blank surrogate matrix background response cannot exceed 20% of the first point of the calibration curve, the blank surrogate matrix is ​​most preferably 0.1% BSA.

[0158] According to another aspect of the present invention, the present invention also provides an application of the above-mentioned method for detecting metabolites related to hyperhomocysteinemia in the preparation of products for detecting metabolites related to hyperhomocysteinemia or products for diagnosing hyperhomocysteinemia. The above-mentioned determination method can be used as an evaluation criterion to evaluate the detection effect of metabolites related to hyperhomocysteinemia, or the reagent used in the above-mentioned detection method can be used as a supporting reagent in the detection product after presetting the above-mentioned detection method in the detection product; or the above-mentioned detection method can be preset in the detection product as an operation module to be used in combination with the detection product for the diagnosis, auxiliary diagnosis and treatment of hyperhomocysteinemia.

[0159] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0160] Examples

[0161] Example 1

[0162] 1. Experimental Materials and Instruments

[0163] 1.1 Materials

[0164] Samples: Human serum samples.

[0165] (1) Instruments: AB SCIEX Triple QuadTM (4500MD); High-performance liquid chromatography system (equipped with an auto-sampler); Refrigerated tabletop centrifuge (5810R, Eppendorf); Multi-tube vortex mixer (MTV-100, Hangzhou Ausheng); Adjustable pipette (Eppendorf 0.5 - 10 μL, 10 - 100 μL, 100 - 1000 μL); Glassware, measuring cylinders, etc.

[0166] (2) Reagents and Consumables: HPLC-grade methanol (Fisher, USA); Distilled water (Watsons, China); Formic acid (Anpel, China); Tris(2-carboxyethyl)phosphine hydrochloride (Aladdin, China); Bovine serum albumin (Sigma, USA); 10×PBS buffer (Anbixin, China); Ascorbic acid (Aladdin, China); Hydrochloric acid (Anpel, China); Sodium hydroxide (Aladdin, China); Chromatographic column: C18 alkylsilyl-bonded silica gel column (Shim-pack VP-ODS chromatographic column, 2.0×150 mm, 5.0 μm) (Shimadzu).

[0167] (3) Standards: cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolate, methylmalonic acid and the corresponding isotope internal standards cysteine-d3, homocysteine-d4, methionine-d3, cystathionine-d4, glycine-d2, serine-d3, vitamin B6-d3, vitamin B2-13C4.15N2, vitamin B9-13C5, 5-methyltetrahydrofolate-13C5, methylmalonic acid-d3 were purchased from Beijing Manhag Biotechnology Co., Ltd., Shanghai Aladdin Biochemical Technology Co., Ltd., and Tanmo Quality Inspection Technology Co., Ltd.

[0168] (4) Quality control products: blank matrices containing cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid, namely QC (QC1) and QC (QC2), respectively.

[0169] 2. Liquid Mass Spectrometry

[0170] 2.1 High performance liquid chromatography method:

[0171] Mobile phase: mobile phase A + mobile phase B; mobile phase A is 0.1% formic acid in water, mobile phase B is 0.1% formic acid in methanol;

[0172] Gradient elution: Mobile phase gradient elution see Table 1;

[0173] Chromatographic column: C18 alkylsilane bonded silica gel column (2.0×150mm, 5.0μm)

[0174] Flow rate: 0.3 mL / min;

[0175] Column temperature: 40°C;

[0176] Injection volume: 2 μL;

[0177] Table 1: HPLC gradient elution conditions

[0178]

[0179] Note: %A refers to the volume percentage of mobile phase A in the mobile phase, and %B refers to the volume percentage of mobile phase B in the mobile phase; the elution gradient process is as follows: within 0 - 1.0 minute, the volume ratio of mobile phase A to mobile phase B changes uniformly from 90:10 to 25:75; within 1.0 - 3.0 minutes, the volume ratio of mobile phase A to mobile phase B changes uniformly from 25:75 to 10:90; within 3.0 - 3.5 minutes, the volume ratio of mobile phase A to mobile phase B is 10:90; within 3.5 - 3.6 minutes, the volume ratio of mobile phase A to mobile phase B changes uniformly from 10:90 to 90:10; within 3.6 - 5.0 minutes, the volume ratio of mobile phase A to mobile phase B is 90:10.

[0180] 2.2 Mass spectrometry method:

[0181] In the electrospray ionization (ESI) mode, positive (negative) ion mode scanning is performed using multiple reaction monitoring (MRM); the ionization voltage is 5500 (ESI+) / - 4500 (ESI-); the ion source temperature is 550 °C; the curtain gas is 30 psi, the collision gas is 6 psi, the spray gas is 50 psi, and the auxiliary heating gas is 50 psi; 11 target substances and their corresponding isotope internal standard compounds are monitored simultaneously, and the mass spectrometry acquisition parameters for each target analyte are shown in Table 2.

[0182] Table 2: Mass spectrometry parameters

[0183]

[0184]

[0185] Where * represents the quantitative ion.

[0186] Among them: Cys is cysteine, and Cys-IS is the isotope internal standard Cys-d3 of Cys;

[0187] Hcy is homocysteine, and Hcy-IS is the isotope internal standard Hcy-d4 of Hcy;

[0188] Met is methionine, and Met-IS is the isotope internal standard Met-d3 of Met;

[0189] Cth is cystathionine, and Cth-IS is the isotope internal standard Cth-d4 of Cth;

[0190] VB2 is vitamin B2, and VB2-IS is the isotope internal standard VB2-13C4.15N2 of VB2;

[0191] VB6 is vitamin B6, and VB6-IS is the isotope internal standard VB6-d3 of VB6;

[0192] VB9 is vitamin B9, and VB9-IS is the isotope internal standard of VB9, VB9-13C5;

[0193] 5-MTHF is 5-methyltetrahydrofolic acid, and 5-MTHF-IS is the isotope internal standard of 5-MTHF, 5-MTHF-13C5;

[0194] Gly is glycine, and Gly-IS is the isotope internal standard of Gly, Gly-d2;

[0195] Ser is serine, and Ser-IS is the isotope internal standard of Ser, Ser-d3;

[0196] MMA is methylmalonic acid, and MMA-IS is the isotope internal standard of MMA, MMA-d3.

[0197] 3. Implementation operation

[0198] Step 101: Preparation of standards: Weigh 1 - 50 mg of each standard of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid into a volumetric flask, and add pure methanol, purified water, 0.1 mol / L hydrochloric acid solution, and 0.2 mol / L sodium hydroxide aqueous solution respectively until completely dissolved to prepare standard stock solutions with concentrations of 200 μg / mL - 50000 μg / mL; then use 0.1% BSA as a surrogate matrix to prepare a mixed working solution from each standard stock solution (see Table 3 for details), and mix evenly for standby.

[0199] Table 3 Preparation of the mixed working solution

[0200]

[0201] Step 102: Using the mixed working solution as the intermediate solution, gradually dilute it with 0.1% BSA to WS01 (see Table 4 for details), and the concentrations of each calibration curve point are listed in Table 4.

[0202] Table 4: Preparation and concentration of the calibration curve

[0203]

[0204] Step 103: Dilute and prepare QC (QC1) and QC (QC2) from the mixed working solution with 0.1% BSA according to Table 5.

[0205] Table 5 Preparation and concentration of the QC solution

[0206]

[0207]

[0208] Step 104: The mixed internal standard solution is prepared as follows: Weigh each isotope internal standard substance, and dilute them with purified water respectively to prepare isotope internal standard mother solutions with concentrations of cysteine 0.5 mg / mL, homocysteine 2 mg / mL, methionine 1 mg / mL, cystathionine 0.5 mg / mL, glycine 2 mg / mL, serine 1 mg / mL, vitamin B6 1 mg / mL, vitamin B2 1 mg / mL, vitamin B9 1 mg / mL, 5-methyltetrahydrofolic acid 1 mg / mL, and methylmalonic acid 1 mg / mL respectively;

[0209] Step 105: Then, the above isotope internal standard mother solutions are further diluted with purified water to prepare an isotope internal standard IS solution containing cysteine 50 mg / mL, homocysteine 5 mg / mL, methionine 5 mg / mL, cystathionine 5 mg / mL, glycine 37.5 mg / mL, serine 12.5 mg / mL, vitamin B6 0.75 mg / mL, vitamin B2 0.75 mg / mL, vitamin B9 2 mg / mL, 5-methyltetrahydrofolic acid 0.75 mg / mL, and methylmalonic acid 2.5 mg / mL;

[0210] Step 106: Dilute the internal standard solution 1:24 with the diluent, and vortex it with a vortex mixer for 1 min to obtain the internal standard-containing methanol solution.

[0211] Step 107: The reducing agent is prepared as follows:

[0212] Weigh 5 g of tris(2-carboxyethyl)phosphine hydrochloride into a volumetric flask, add 10 mL of water to completely dissolve it, and dilute it to 50 mL with water to obtain the reducing agent tris(2-carboxyethyl)phosphine hydrochloride;

[0213] The above reducing agent tris(2-carboxyethyl)phosphine hydrochloride is further diluted 1:9 with the diluent to obtain the working solution of the reducing agent. Step 108: Sample treatment for the calibration curve:

[0214] Precisely pipette 100 μL of the calibration curve sample into a 2 mL centrifuge tube, add 50 μL of the working solution of tris(2-carboxyethyl)phosphine hydrochloride, add 200 μL of the internal standard working solution, centrifuge (at 4 °C, 14000 rpm / min) for 10 min, transfer 100 μL of the supernatant to a new 96-well sample plate for injection, and perform detection on the machine.

[0215] Step 109: Sample treatment for QC samples:

[0216] Precisely pipette 100 μL of the QC sample into a 2 mL centrifuge tube, add 50 μL of the working solution of tris(2-carboxyethyl)phosphine hydrochloride, add 200 μL of the internal standard working solution, centrifuge (at 4 °C, 14000 rpm / min) for 10 min, transfer 100 μL of the supernatant to a new 96-well sample plate for injection, and perform detection on the machine.

[0217] Step 110: Serum sample treatment:

[0218] Precisely pipette 100 μL of serum sample into a 2 mL centrifuge tube, add 50 μL of tris(2-carboxyethyl)phosphine hydrochloride working solution, add 200 μL of internal standard working solution, centrifuge (at 4 °C, 14000 rpm / min) for 10 min, transfer 100 μL of the supernatant to a new 96-well injection plate, and perform on-machine detection.

[0219] Step 111: Calculate results: Use Analyst for data processing, generate a standard curve by linear regression weighted 1 / x or 1 / x2, where x is the concentration of the analyte and y is the ratio of the analyte peak area to the internal standard peak signal.

[0220] 4. Method validation:

[0221] As can be seen from Figures 2 to 12 , in the detection method, the standard products of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid have symmetric peak shapes with the dry blood spot samples, and there is no interference from impurity peaks, indicating that good detection can be obtained under these conditions.

[0222] 4.1 Calibration curve

[0223] Adopt the isotope internal standard quantification method, use Analyst for data processing, generate a standard curve by linear regression weighted 1 / x or 1 / x2, where x is the concentration of the analyte and y is the ratio of the analyte peak area to the internal standard peak area, establish a calibration curve, and calculate the concentrations of 11 analytes in serum. The linear fitting equations of the 11 analytes within their respective concentration ranges have good linearity, and the correlation coefficients are above 0.990. See Table 6 for details.

[0224] Table 6 Linear ranges of each analyte

[0225]

[0226]

[0227] 4.2 Investigation of spike recovery rate: Randomly select one serum sample, leave one without adding the standard product, and add two concentrations of QC standard products to the other two. Repeat the treatment and determination 6 times in the same steps. Quantify the concentrations of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid by the isotope internal standard method, calculate the recovery rate results, and repeat for three batches. See Table 10. The results show that the spike recovery rate results of each substance are between 85% and 115%, all meeting the requirements.

[0228] Table 10 Results of spike recovery rate of homocysteine

[0229]

[0230]

[0231] 4.3 Accuracy and precision test: Take the quality control samples and repeat the treatment for 6 batches within one day and for 3 days. The concentrations of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid are quantitatively determined by isotope internal standard method. The within-batch and between-batch precisions and accuracies are statistically analyzed for three consecutive days, and the calculation results are shown in Table 11 and Table 12. The results show that the within-batch / between-batch accuracy results are between 85% - 115%, and the RSD values are less than 15%, meeting the requirements.

[0232] Table 11: Within-batch precision and accuracy (n = 6)

[0233]

[0234]

[0235]

[0236] Table 12: Between-batch precision and accuracy (n = 18)

[0237]

[0238] 5. Conclusion

[0239] In this example, HPLC-MS / MS method was used to determine cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid in human serum. The method has the characteristics of high sensitivity, good repeatability, high accuracy, and good specificity. Quantitative determination by isotope internal standard method can greatly eliminate matrix effects and interference from other components, is not affected by the sample treatment process, and can achieve accurate quantification. At the same time, the protein precipitation method can use less blood samples to quickly and efficiently detect samples, and the treatment is convenient.

[0240] The spiked recoveries of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid in human serum over three days were all between 85% and 115%, meeting the requirements. At the same time, the accuracy and precision were investigated. The reproducibility results of the method showed that the within-day precision, between-day precision, and RSD of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid in human serum were less than 15%, and the accuracy was between 85% and 115%. The reproducibility of the method was good.

[0241] Compared with other LC-MS / MS methods, the method of the present invention has higher sensitivity, requires less sample volume, and can complete the detection of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid within 5 minutes. It can be used for the detection of cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6, vitamin B2, vitamin B9, 5-methyltetrahydrofolic acid, and methylmalonic acid in serum clinically.

[0242] It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for detecting homocysteineemia-related metabolites in serum by HPLC-MS / MS, characterized in that: The homocystinemia-related metabolites include cysteine, homocysteine, methionine, cystathionine, glycine, serine, vitamin B6 (pyridoxic acid), vitamin B2, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid; the method includes: Pretreatment: Mix the sample with a reducing agent, then add a protein precipitation solution, mix, centrifuge and take the supernatant; Analysis: The supernatant is subjected to high performance liquid chromatography coupled to mass spectrometry to detect the homocysteineemia-related metabolites, and the content of the homocysteineemia-related metabolites is obtained by quantification using a standard curve drawn from a certain range of known amounts of standard substances.

2. The method according to claim 1, characterized in that The reducing agent is tris(2-carboxyethyl)phosphine hydrochloride / methanol solution.

3. The method according to claim 2, characterized in that The concentration of the tris(2-carboxyethyl)phosphine hydrochloride / methanol solution is 5-15 mg / mL, preferably 10 mg / mL.

4. The method according to claim 1 or 2, characterized in that: The protein precipitation solution is a 100% methanol solution.

5. The method according to claim 1 or 2, characterized in that: In the pretreatment, the volume ratio of the sample, the reducing agent and the protein precipitation solution is 2:(0.5-2):(2-5); preferably 2:1:

4.

6. The method according to claim 1 or 2, characterized in that: The protein precipitation solution also contains an internal standard. Preferably, the internal standard is an isotope internal standard labeled with an isotope.

7. The method according to claim 1 or 2, characterized in that: The HPLC detection conditions include: Chromatographic column: reverse phase chromatographic column; preferably, carbon octadecyl (C18) alkylsilane bonded silica gel; Mobile phase: The mobile phase comprises mobile phase A and mobile phase B; mobile phase A is a formic acid aqueous solution with a concentration of 0.02-0.5%, preferably 0.1%; mobile phase B is a formic acid methanol solution with a concentration of 0.02-0.5%, preferably 0.1%.

8. The method according to claim 1 or 2, characterized in that: The mass spectrometry detection conditions include: Ion source parameters: In electrospray ionization (ESI) mode, multiple reaction monitoring (MRM) was used for simultaneous scanning in positive and negative ion modes; ionization voltage was 5500 (ESI+) / -4500 (ESI-); ion source temperature was 550°C; curtain gas was 30 psi, collision gas was 6 psi, spray gas was 50 psi, and auxiliary heating gas was 50 psi; Preferably, the mass spectrometry parameters are as follows: Wherein, * represents the quantitative ion.

9. The method according to claim 1 or 2, characterized in that: The sample is a blood sample of a human or non-human mammal (such as a pig, dog, cat, sheep or cow), preferably whole blood, serum, plasma or dried blood spot; preferably, the sample is serum.

10. The method according to claim 1 or 2, characterized in that: First, high performance liquid chromatography is used to separate homocysteineemia-related metabolites from interfering components in the sample, and then mass spectrometry is used to detect the charge-to-mass ratio of homocysteineemia-related metabolites and their corresponding internal standards. According to the internal standard method, quantitative analysis is performed to obtain the content of homocysteineemia-related metabolites.

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