A method and kit for detecting homocysteine and its metabolically related substances

Abstract

The present application relates to the technical field of metabolite detection, and particularly relates to a method and a kit for detecting homocysteine and metabolically related substances. The present application provides a method for detecting homocysteine and metabolically related substances, comprising the following steps: mixing the to-be-detected sample, S1-S6 standard, high-value quality control and low-value quality control with reagent D respectively, standing, then mixing with a reducing agent for reaction, and then mixing with a formic acid acetonitrile solution to obtain a mixture; mixing the mixture with water, centrifuging, taking the supernatant for liquid chromatography-tandem mass spectrometry detection. The method can detect three types of compounds including homocysteine, cysteine and methionine indicators at one time, and comprehensively reflects the reasons for the increase of homocysteine.

Description

Technical Field

[0001] This invention relates to the field of metabolite detection technology, and in particular to a method and kit for detecting homocysteine ​​and its metabolic-related substances. Background Technology

[0002] In recent years, with the establishment of the metabolic pathway of homocysteine ​​in the body and the continuous deepening of biomedical research, it has been found that homocysteine ​​plays a crucial role in the metabolism of cysteine ​​and methionine in body fluids, and its biological characteristics are closely related to those of methionine and cysteine. Studies have shown that homocysteine ​​participates in methylation reactions together with methionine, playing a key role in DNA methylation and demethylation; at the same time, the concentration and ratio of methionine and cysteine ​​can affect the production and concentration of homocysteine. Deficiency or excess of methionine and cysteine ​​can lead to abnormal accumulation or decrease in homocysteine. However, elevated blood homocysteine ​​levels are associated with a variety of diseases. Numerous studies have shown that the higher the homocysteine ​​level in the blood, the greater the risk of arteriosclerosis and thrombosis; mild to severe elevations in homocysteine ​​levels can increase the risk of death from cardiovascular disease by more than four times; homocysteine ​​levels can serve as an independent risk factor for macrovascular disease in patients with type 2 diabetes; and patients with chronic renal failure commonly have high homocysteine ​​levels, with an incidence rate several times higher than normal. These reports all demonstrate the significant role of serum homocysteine ​​levels in human health. Testing for homocysteine ​​and its related metabolites can clarify the causes of elevated homocysteine ​​levels, rule out neurological diseases, cardiovascular diseases, diabetic complications, respiratory diseases, and screen for and monitor pregnancy-related diseases. This, in turn, can guide precise clinical interventions, effectively improve homocysteine ​​levels, and ultimately prevent disease occurrence or delay disease progression.

[0003] Currently, commonly used clinical detection methods both domestically and internationally include enzymatic cycling (ECA), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay (CIA). Each of these methods has its own advantages and disadvantages. ECA is currently the most commonly used method for homocysteine ​​detection in clinical laboratories; however, it suffers from poor sensitivity, a narrow linear range, complex operation, long detection cycle, and high cost, making it unsuitable for large-scale testing. Furthermore, studies have shown that high levels of endogenous substances can significantly interfere with the results. With the introduction of mass spectrometry equipment into hospital testing centers at all levels, it has become an important tool for metabolomics research and clinical diagnostics. Although HPLC-MS is expensive and requires professional operation, it offers higher stability, characteristic features, sensitivity, and accuracy, faster analysis speed, and requires less sample volume. To provide doctors with accurate test results for accurate diagnoses, it is essential to establish accurate and efficient detection methods.

[0004] Traditional testing methods only detect homocysteine, often requiring empirical medication and thus lacking precision. Mass spectrometry, however, simultaneously detects methionine, homocysteine, and cysteine, clarifying the pathogenic mechanism and enabling more targeted treatment. Metabolic pathway analysis reveals that vitamins B6, B12, and folic acid play crucial roles in the homocysteine ​​metabolic pathway. Elevated levels of homocysteine, methionine, and cysteine ​​may be markers of vitamin deficiency; simultaneous detection of all three can guide precise clinical treatment. For example, patent CN113341012A detects seven substances: homocysteine, folic acid, 5-methyltetrahydrofolate, methionine, vitamin B6, vitamin B12, and cysteine. The gradient elution method is as follows: 0-1 min 1% mobile phase B, 1-2 min to 98% mobile phase B, 2-5 min maintain 98% mobile phase B, 5-5.5 min decrease to 1% mobile phase B, 5.5-7 min maintain 1% mobile phase B, and then stop. Although the detection scope covers a wide range of substances, the liquid chromatography gradient time is 7 minutes, resulting in a relatively long measurement time for a single sample. Patent CN106442836A detects folic acid, homocysteine, methionine, and cysteine ​​using a derived pretreatment method, increasing the difficulty of pretreatment, and the liquid chromatography time is 8 minutes, also leading to a long measurement time for a single sample. Patents CN112964808A, CN111983057A, and CN1979155A all detect only one substance, homocysteine. CN1979155A, in particular, requires the addition of a stabilizer to the sample tubes, and uses dialyzed serum as calibrators and quality control samples, making the processing of clinical samples cumbersome.

[0005] Therefore, there is an urgent need for a method that can simultaneously detect homocysteine, methionine, and cysteine ​​in a highly efficient, accurate, and simple manner. Summary of the Invention

[0006] The purpose of this invention is to provide a simple, efficient, accurate method for simultaneously detecting homocysteine, methionine, and cysteine.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a kit for detecting homocysteine ​​and its metabolic-related substances, comprising calibrators, high-value control samples, low-value control samples, reagent D, PBS buffer, reducing agent, water, and formic acid acetonitrile solution; The metabolism-related substances include methionine and cysteine; The reducing agent is prepared by mixing 0.03~0.05g of dithiothreitol and 0.5~1.5mL of water; The reagent D comprises the following components at final concentrations: 2-4 μmol / L homocysteine-D4, 5-7 μmol / L methionine-D3, and 7-9 μmol / L cysteine-D3. The calibrator comprises the following components at final concentrations: 500 μmol / L homocysteine, 1000 μmol / L methionine, and 2500 μmol / L cysteine. The high-value quality control materials include 400 μmol / L homocysteine, 800 μmol / L methionine, and 2000 μmol / L cysteine. The low-value quality control materials include 50 μmol / L homocysteine, 100 μmol / L methionine, and 250 μmol / L cysteine.

[0008] The present invention also provides the application of the kit in the detection of homocysteine ​​and its metabolically related substances, including methionine and cysteine.

[0009] The present invention also provides a method for detecting homocysteine ​​and its metabolic-related substances, comprising the following steps: (1) Mix the test sample, S1~S6 standard, high value quality control sample, and low value quality control sample with reagent D and let stand for 4~6 min. Then mix with reducing agent and let stand to react to obtain reduction product. (2) The reduction product and formic acid acetonitrile solution are mixed to obtain a mixture; (3) Mix the mixture with water, centrifuge, and take the supernatant for detection by liquid chromatography-tandem mass spectrometry; The S1 to S6 standards are obtained by gradient dilution of calibrators and PBS buffer.

[0010] Preferably, the amount of the sample to be tested is 40-60 μL; the amount of each gradient standard among the S1-S6 standards is 40-60 μL; the amount of the high-value quality control is 40-60 μL; the amount of the low-value quality control is 40-60 μL; the amount of reagent D is 40-60 μL; and the amount of the reducing agent is 40-60 μL.

[0011] Preferably, the temperature of the static reaction is 30~40℃; and the time of the static reaction is 15~25min.

[0012] Preferably, the amount of formic acid acetonitrile solution added is 350~450 μL.

[0013] Preferably, the amount of water added is 200~300μL; the centrifugation speed is 8000~12000rpm; and the centrifugation time is 4~6min.

[0014] Preferably, the liquid chromatography-tandem mass spectrometry detection process includes the following conditions: Mobile phase A is 0.02% formic acid in water, and mobile phase B is 0.02% formic acid in methanol; The elution conditions were: 0–1.5 min: 5%–40% B, 1.6–1.8 min: 80%–80% B, 1.81–2.5 min: 5%–5% B; The injection volume is 1~10μL, the mobile phase flow rate is 0.3~0.4mL / min, the injection temperature is 3~5℃, and the column temperature is 35~45℃.

[0015] Preferably, the mass spectrometry conditions during the liquid chromatography-tandem mass spectrometry detection process include: The ion source was an ESI+ source, the ion source voltage was 5500V, the ion source temperature was 500℃, and the scanning mode was MRM. Air curtain gas 40psi, impact gas 8psi, atomizing gas 50psi, auxiliary heater gas 60psi.

[0016] Beneficial effects: This invention provides a method for detecting homocysteine ​​and its metabolic related substances. The method employs high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to detect homocysteine, methionine, and cysteine ​​in serum. Deuterated isotopes corresponding to these three substances are used as internal standards. Through a reduction reaction, bound homocysteine ​​in the blood sample is reduced to free homocysteine. The target analytes are then purified using protein precipitation. The processed sample is analyzed using HPLC-MS / MS, and the peak areas of homocysteine, methionine, cysteine, and their corresponding internal standards are recorded. Using a calibration curve method, the concentrations of homocysteine, methionine, and cysteine ​​in the sample are calculated based on the ratio of the peak area of ​​the substance in the sample to the peak area measured by its internal standard. HPLC-MS / MS is a highly sensitive mass spectrometry analysis method that combines a mass spectrometer with specific reagents. Its core principle is to determine the composition of the sample by analyzing the molecular weight of the sample using mass spectrometry. This method can simultaneously detect three classes of compounds, including homocysteine, cysteine, and methionine, comprehensively reflecting the causes of elevated homocysteine ​​levels. This provides strong support for differentiating different types of hyperhomocysteinemia and lays the foundation for subsequent precision treatment.

[0017] This invention also provides a kit for detecting homocysteine ​​and its metabolic related substances, which can be applied to mainstream brands of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and has strong versatility. Furthermore, the pretreatment is simple and requires minimal expertise from testing personnel. It organically combines simple and efficient pretreatment techniques with mass spectrometers of different sensitivities and models, establishing a new method for the same test using different instruments. This product requires no nitrogen blowing or derivatization for pretreatment, and the liquid chromatography gradient elution time is only 2.5 minutes, resulting in faster elution, reduced sample detection time, and decreased contamination of equipment (expensive maintenance and upkeep) and wear and tear on the chromatographic column (a high-value consumable). From a clinical application perspective, this product selects homocysteine, methionine, and cysteine ​​as initial screening indicators, completing diagnosis and subtyping in one step. This avoids repeated drug trials, reduces patient treatment costs, and shortens treatment time. Compared to other manufacturers who focus on large packages of transient B vitamins (mainly folic acid) that are greatly affected by dietary fluctuations and pay more attention to type 2 hypermethionine, we focus more on type 1 hypermethionine and the subtyping diagnosis of primary hypermethioninemia, which also indirectly confirms the diagnosis of type 2 hypermethionine. Furthermore, regarding the nationally leading indicator test (cysteine), it focuses on both the disease itself and the protection of health (natural antioxidants, relief of liver damage, and respiratory mucosal protection); secondly, it can guide precise clinical intervention measures and provide clinicians with reference for disease diagnosis and human health monitoring. It can be applied to the diagnosis of hyperhomocysteinemia and the detection of its abnormal causes, the diagnosis of methionineemia, the auxiliary screening and monitoring of thioether β synthase deficiency, and the auxiliary diagnosis of folic acid and VB12 deficiency. It is suitable for patients with cardiovascular and cerebrovascular diseases and high-risk groups: patients with hypertension, coronary heart disease, ischemic heart disease, deep vein thrombosis, stroke, chronic kidney disease, diabetic complications, suspected homocysteine ​​metabolic pathway disorders, and special high-nutrition groups such as the elderly, pregnant women, and children; thirdly, it fills a market gap by providing a test kit for the in vitro quantitative detection of homocysteine, methionine, and cysteine ​​concentrations in human serum. Clinically, it is mainly used for the auxiliary diagnosis of hyperhomocysteinemia and the evaluation of cardiovascular disease risk. Attached Figure Description

[0018] Figure 1 Chromatograms of serum samples containing solvent blank, low-concentration standard solution, and isotope internal standard; Figure 2 Calibration curves for three amino acids; Figure 3 The results are for the stability test of the standard solution stored at -20℃. Detailed Implementation

[0019] This invention provides a kit for detecting homocysteine ​​and its metabolic-related substances, comprising calibrators, high-value control samples, low-value control samples, reagent D, PBS buffer, reducing agent, water, and formic acid acetonitrile solution; The metabolism-related substances include methionine and cysteine; The reducing agent is prepared by mixing 0.03~0.05g of dithiothreitol and 0.5~1.5mL of water; The preferred amount of dithiothreitol added is 0.04 g; the preferred amount of water added is 1 mL; The reagent D comprises the following components at final concentrations: 2-4 μmol / L homocysteine-D4, 5-7 μmol / L methionine-D3, and 7-9 μmol / L cysteine-D3. The concentration of homocysteine-D4 is preferably 3 μmol / L, the concentration of methionine-D3 is preferably 6 μmol / L, and the concentration of homocysteine-D3 is preferably 8 μmol / L. The calibrator comprises the following components at final concentrations: 500 μmol / L homocysteine, 1000 μmol / L methionine, and 2500 μmol / L cysteine. The high-value quality control materials include 400 μmol / L homocysteine, 800 μmol / L methionine, and 2000 μmol / L cysteine. The low-value quality control materials include 50 μmol / L homocysteine, 100 μmol / L methionine, and 250 μmol / L cysteine.

[0020] The present invention also provides the application of the kit in the detection of homocysteine ​​and its metabolically related substances, including methionine and cysteine.

[0021] The present invention also provides a method for detecting homocysteine ​​and its metabolic-related substances, comprising the following steps: (1) Reduction reaction: Mix the test sample, S1~S6 standard, high value quality control sample, and low value quality control sample with reagent D and let stand for 4~6 min. Then mix with reducing agent and let stand to react to obtain reduction product; (2) Protein precipitation: The reduction product is mixed with formic acid acetonitrile solution to obtain a mixture; (3) Centrifugation: Mix the mixture with water, centrifuge, and take the supernatant for detection by liquid chromatography-tandem mass spectrometry; The S1 to S6 standards are obtained by gradient dilution of calibrators and PBS buffer.

[0022] In this invention, the method for mixing the test sample, calibrator, high-value quality control sample, and low-value quality control sample with reagent D is vortex mixing; the mixing time is 25-35 seconds, preferably 28-32 seconds, and more preferably 30 seconds. The preferred settling time is 5 minutes; The method of mixing with the reducing agent is capped vortex mixing, and the mixing time is 1-3 minutes, preferably 2 minutes; The sample to be tested was serum.

[0023] In this invention, the amount of the sample to be tested is 40~60μL, preferably 45~55μL, and more preferably 50μL; The amount of each grade of standard S1 to S6 used is 40 to 60 μL, preferably 45 to 55 μL, and more preferably 50 μL. The amount of the high-value quality control material used is 40~60μL, preferably 45~55μL, and more preferably 50μL; The amount of the low-value quality control sample used is 40~60μL; preferably 45~55μL, and more preferably 50μL. The amount of reagent D used is 40-60 μL, preferably 45-55 μL, and more preferably 50 μL. The amount of reducing agent used is 40~60μL, preferably 45~55μL, and more preferably 50μL.

[0024] In this invention, the temperature of the static reaction is 30~40℃, preferably 33~37℃, and more preferably 35℃; the time of the static reaction is 15~25min, preferably 18~22min, and more preferably 20min.

[0025] In this invention, the amount of formic acid acetonitrile solution added is 350~450μL, preferably 380~420μL, and more preferably 400μL.

[0026] In this invention, the method for mixing the reduction product and the formic acid acetonitrile solution is vortex mixing for 4-6 minutes, preferably 5 minutes.

[0027] In this invention, the amount of water added is 200~300μL, preferably 230~270μL, and more preferably 250μL; the centrifugation speed is 8000~12000rpm, preferably 9000~11000rpm, and more preferably 10000rpm; the centrifugation time is 4~6min, preferably 5min.

[0028] In this invention, the liquid chromatography-tandem mass spectrometry detection process includes the following conditions: Mobile phase A is 0.02% formic acid in water, and mobile phase B is 0.02% formic acid in methanol; The elution conditions were: 0–1.5 min: 5%–40% B, 1.6–1.8 min: 80%–80% B, 1.81–2.5 min: 5%–5% B; The injection volume is 1~10μL, preferably 4~7μL, more preferably 5μL; the mobile phase flow rate is 0.3~0.4mL / min, preferably 0.35mL / min; the injection temperature is 3~5℃, preferably 4℃; and the column temperature is 35~45℃, preferably 38~42℃, more preferably 40℃.

[0029] Preferably, the mass spectrometry conditions during the liquid chromatography-tandem mass spectrometry detection process include: The ion source was an ESI+ source, the ion source voltage was 5500V, the ion source temperature was 500℃, and the scanning mode was MRM. Curtain gas 40 psi, impact gas 8 psi, atomizing gas 50 psi, auxiliary heater gas 60 psi In this invention, the preparation method of the S1~S6 standards is as follows: S6: Take 40 μL of calibrator into a 1.5 mL centrifuge tube, add 160 μL of PBS buffer, and vortex to mix. S5: Take 50 μL of S6 into a 1.5 mL centrifuge tube, add 50 μL of PBS buffer, and vortex to mix. S4: Take 20 μL of S6 into a 1.5 mL centrifuge tube, add 80 μL of PBS buffer, and vortex to mix. S3: Take 5 μL of S6 into a 1.5 mL centrifuge tube, add 95 μL of PBS buffer, and vortex to mix. S2: Take 2 μL of S6 into a 1.5 mL centrifuge tube, add 98 μL of PBS buffer, and vortex to mix. S1: Take 1 μL of S6 into a 1.5 mL centrifuge tube, add 99 μL of PBS buffer, and vortex to mix. The calibrator comprises the following components at final concentrations: 500 μmol / L homocysteine, 1000 μmol / L methionine, and 2500 μmol / L cysteine. In this invention, the following steps are included before the detection: Dilution of quality control sample: Take 10 μL of high-value quality control sample and add it to 40 μL of PBS buffer, then vortex to mix. Add 10 μL of low-value quality control sample to 40 μL of PBS buffer and vortex to mix. The high-value quality control materials include 400 μmol / L homocysteine, 800 μmol / L methionine, and 2000 μmol / L cysteine. The low-value quality control materials include 50 μmol / L homocysteine, 100 μmol / L methionine, and 250 μmol / L cysteine.

[0030] In this invention, the method for mixing the mixture and water is vortex mixing for 1 to 3 minutes, preferably 2 minutes.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1: A kit for detecting homocysteine ​​and its metabolic-related substances.

[0033] The components of the kit are shown in Table 1; Table 1. Components of the kit

[0034] Example 2: A method for detecting homocysteine ​​and its metabolic related substances

[0035] The detection was performed using the kit described in Example 1, and the specific steps are as follows: S6: Take 40 μL of calibrator into a 1.5 mL centrifuge tube, add 160 μL of PBS buffer, and vortex to mix. S5: Take 50 μL of S6 into a 1.5 mL centrifuge tube, add 50 μL of PBS buffer, and vortex to mix. S4: Take 20 μL of S6 into a 1.5 mL centrifuge tube, add 80 μL of PBS buffer, and vortex to mix. S3: Take 5 μL of S6 into a 1.5 mL centrifuge tube, add 95 μL of PBS buffer, and vortex to mix. S2: Take 2 μL of S6 into a 1.5 mL centrifuge tube, add 98 μL of PBS buffer, and vortex to mix. S1: Take 1 μL of S6 into a 1.5 mL centrifuge tube, add 99 μL of PBS buffer, and vortex to mix. The calibrator comprises the following components at final concentrations: 500 μmol / L homocysteine, 1000 μmol / L methionine, and 2500 μmol / L cysteine. Dilution of quality control sample: Take 10 μL of high-value quality control sample and add it to 40 μL of PBS buffer, then vortex to mix. Add 10 μL of low-value quality control sample to 40 μL of PBS buffer and vortex to mix. Reducing agent: Add 0.04g DTT to 1mL of ultrapure water; (1) Take 50 μL of the sample to be tested, S1~S6 standard, high value quality control and low value quality control and place them in a 96-well plate respectively. Add 50 μL of reagent D to each well, vortex mix evenly, and let stand for 5 min. Then add 50 μL of reducing agent to each well, cover and vortex mix for 2 min, and let stand at 35℃ for 20 min to obtain the reduction product. (2) Add 400 μL of formic acid acetonitrile solution to each well (reduction product), vortex mix for 5 min to obtain a mixture; (3) Add 250 μL of ultrapure water to the mixture and vortex mix for 2 min; centrifuge at 10000 prm for 5 min, take 200 μL of the supernatant into a new 96-well plate, and then perform liquid chromatography-tandem mass spectrometry detection; the liquid chromatography conditions are shown in Table 2, the mass spectrometry conditions are shown in Table 3, and the compound parameters are shown in Table 4. Table 2 Liquid Chromatography Conditions

[0036] Table 3 Mass Spectrometry Conditions

[0037] Table 4 Compound Parameters

[0038] Note: a represents quantitative ions, and b represents qualitative ions.

[0039] Example 3 Method Validation

[0040] 1. Exclusivity: To evaluate the specificity of this method, blank solvent (PBS), low-concentration standard solutions (homocysteine ​​10 μmol / L, methionine 20 μmol / L, cysteine ​​50 μmol / L), serum samples, low-concentration standard solutions with added internal standards, and serum samples with added internal standards (containing homocysteine-D4, methionine-D3, and cysteine-D3) were analyzed. Potential interferences from sulfur-containing amino acids (such as cystathionine and glutathione), metabolites (such as methylmalonic acid and 2-methylcitric acid), and matrix components (such as proteins and lipids) were specifically evaluated. To eliminate such interferences, chromatographic parameters (including column type, elution gradient, and mobile phase composition) were optimized to achieve efficient separation of homocysteine, cysteine, and methionine. Furthermore, multiple reaction monitoring (MRM) technology in tandem mass spectrometry was employed to improve selectivity by selecting specific precursor / product ions. According to the method described in Example 2, solvent blanks, low-concentration standard solutions, low-concentration standard solutions containing isotopic internal standards, serum samples, and serum samples containing isotopic internal standards were processed and then tested. The results are as follows: Figure 1 As shown; The results showed that the target analyte and potential interfering compounds were well separated, confirming that the method has high specificity and is reliable for the quantitative analysis of homocysteine ​​(Hcy), cysteine ​​(Cys), and methionine (Met) in complex matrices.

[0041] 2. Evaluation of matrix effect: Matrix effect (ME) refers to the interference caused by components in the sample other than the target analyte. It generally includes two types: matrix inhibition and matrix enhancement. The calculation formula is ME = (AC) / B × 100%, where A is the response value of the sample-matched standard solution; B is the response value of the pure solvent standard solution; and C is the sample background response value. If the matrix effect is within the range of 80%–120%, it can be ignored. If it exceeds this range, it indicates either matrix inhibition or matrix enhancement. In this study, phosphate-buffered saline (PBS) was used as an alternative matrix. Spiked matrix samples containing low, medium, and high concentration levels were prepared by adding mixed standard solutions to the prepared blank matrix (homocysteine ​​concentrations were 10, 40, and 80 μmol / L; methionine concentrations were 20, 80, and 160 μmol / L; and cysteine ​​concentrations were 50, 200, and 400 μmol / L). Three samples were prepared in parallel for each concentration level, and each sample was measured three times. ME was quantified by the ratio of the analytical response signals of the matrix-spiked sample to the solvent standard, and the results are shown in Table 5. Table 5. Matrix effect after matrix-matched isotope internal standard correction

[0042] The results showed that the matrix effect of the three amino acids in serum ranged from 45.8% to 62.4%, indicating a strong matrix inhibition effect. The matrix effects of the three amino acids after matrix-matched isotope internal standard correction were as follows: homocysteine ​​95.2%–103.3%, methionine 96.3%–101.2%, and cysteine ​​92.6%–98.8%, all within the range of 85%–115%, indicating that using PBS as an alternative to matrix-matched isotope internal standard correction can eliminate the influence of the matrix effect. No interfering peaks were observed at the retention times of the three compounds and their isotope internal standards, and other reagent components used had no effect on their retention times, indicating that this method has good specificity.

[0043] 3. Linearity, limit of detection, and limit of quantitation: Cysteine ​​was used at concentrations of 5, 10, 25, 100, 250, and 500 μmol / L; methionine at concentrations of 2, 4, 10, 40, 100, and 200 μmol / L; and homocysteine ​​at concentrations of 1, 2, 5, 20, 50, and 100 μmol / L. Matrix-matched calibration curves were prepared using fixed concentrations of internal standards (cysteine-D3, 8 μmol / L; methionine-D3, 6 μmol / L; homocysteine-D4, 3 μmol / L) to evaluate linearity. Calibration curves were determined by plotting the peak area ratios of cysteine, methionine, and homocysteine ​​with their corresponding internal standards. The correlation coefficient (r) was used to assess linearity. 2 A good linear relationship was obtained when the signal-to-noise ratio (S / N > 0.995); the signal-to-noise ratio (S / N > 3) was determined as the limit of detection (LOD); the signal-to-noise ratio (S / N > 10) was determined as the limit of quantitation (LOQ). The standard curve was processed, and a calibration curve was created using the internal standard method with concentration as the x-axis and peak area ratio as the y-axis. The fitted calibration curve is shown below. Figure 2 As shown in the figure; the linear range, linear equation, detection limit and quantitation limit of the three amino acids are shown in Table 6. Table 6. Linear range, linear equation, limit of detection, and limit of quantitation for the three amino acids.

[0044] The results showed that the linear ranges for homocysteine, cysteine, and methionine were between 1 and 500 μmol / L, with correlation coefficients r0. 2 The detection limit is between 0.9991 and 0.9997 μmol / L, the quantitation limit is between 0.006 and 0.016 μmol / L, and the linear relationship is between 0.020 and 0.055 μmol / L, showing good linearity and meeting the requirements for clinical analysis and detection.

[0045] 4. Recovery rate and precision: Recovery and precision were analyzed using matrix addition at three concentration levels: low, medium, and high. The concentrations were: cysteine ​​50, 100, and 500 μmol / L; methionine 10, 40, and 160 μmol / L; and homocysteine ​​10, 40, and 80 μmol / L. Analysis was performed continuously for 6 days, with each level repeated 6 times. Recovery was calculated by comparing the added sample concentration with the background concentration. Precision was expressed as relative standard deviation (RSD). Recovery was required to be between 85% and 115%, and precision was required to be ≤15%. The results are shown in Table 7. The formula for calculating the recovery rate is: , Table 7. Average recovery and precision of the three amino acids

[0046] The results showed that the average recoveries of homocysteine, methionine, and cysteine ​​were 94.6%–98.8%, 91.6%–96.9%, and 91.1%–98.8%, respectively. The intra-day precision of the three substances at all spiking levels was 3.1%–7.2%, and the inter-day precision was 4.9%–7.9%, indicating that the method has good accuracy and precision.

[0047] 5. Stability: (1) Stability of injection cycle at 4℃ Three matrix-matched standard solutions (low, medium, and high levels: cysteine ​​50, 100, and 500 μmol / L; methionine 10, 40, and 160 μmol / L; homocysteine ​​10, 40, and 80 μmol / L) and two human serum samples (from a hospital in Shijiazhuang) were used. The solutions were processed according to the method described in Example 2, and then placed in an autosampler at 4°C. Detection was performed at 0, 2, 6, 12, 24, and 48 hours. The response values ​​of the compounds at each time point were recorded, and the deviations between the response values ​​at each time point and the response value at 0 hours were calculated, as shown in Table 8. If the deviation between the response value at each time point and the initial value is within ±15%, it indicates that the analyte is stable during the injection and detection process. The formula for calculating the deviation is: (response value at each time point - response value at 0h) / response value at 0h × 100%; Table 8. Stability of injection cycle (4℃)

[0048] The results showed that the peak response values ​​of homocysteine, methionine, and cysteine ​​at six time points deviated from the peak response value at 0h by -12.2% to 5.1%, which met the stability requirements, indicating that the three substances were stable in an autosampler at 4℃ with a 48h injection cycle.

[0049] (2) Stability of standard solution stored at -20℃

[0050] A mixed standard stock solution of methionine (1000 μmol / L), cysteine ​​(2500 μmol / L), and homocysteine ​​(500 μmol / L) was frozen at -20°C, thawed at room temperature, and then analyzed in the sample prepared according to the method described in Example 2. The freeze-thaw cycle stability (n=3) was investigated at 0, 1, 2, 4, 8, 16, and 32 weeks. The results are as follows: Figure 3 As shown; if the response of the standard in each time period deviates from the initial response by ±15%, it indicates that the compound is stable under the corresponding storage conditions and time period. The results showed that the response values ​​of the mixed standard stock solution of methionine, cysteine ​​and homocysteine ​​at each time point within 32 weeks deviated from the initial response values ​​by ±15%, indicating that the mixed standard stock solution of the three compounds is stable at -20℃.

[0051] 6. Carryover effect

[0052] The carryover effect is a discontinuity in the response of one test sample carried over to the response of another test sample by the measurement system, thereby incorrectly affecting the performance of the other test sample. Carryover contamination is one of the clear sources of error in clinical testing, and the resulting erroneous results may have an adverse impact on patient medical outcomes. Ten sets of low-concentration standard solutions (cysteine, 8 μmol / L; methionine, 2.5 μmol / L; homocysteine, 1.4 μmol / L) were continuously injected and their peak areas were recorded (Series A). These ten sets of low-concentration standard solutions were then analyzed along with serum samples supplemented at high concentrations (cysteine ​​400 μmol / L; methionine 160 μmol / L; homocysteine ​​80 μmol / L). The peak areas of the low-concentration standard solutions were recorded in a sequence of high, high, high, low, high, high, low, high, high, low, high, high, high, low... (Series B). A t-test was performed on the peak areas of the low-concentration solutions in Series A and Series B to determine if there was a significant difference. If there was no significant difference, it indicated that there was no carryover effect during sample testing. The results are shown in Table 9. Table 9. Carrying effect of the three compounds

[0053] Note: A: Analysis of ten low-concentration samples; B: Analysis of high-concentration and low-concentration samples.

[0054] The results showed that the independent samples t-tests for the three compounds in the two groups yielded p-values ​​of 0.26, 0.06, and 0.82, respectively, all of which were greater than the significance level α=0.05. This indicates that there was no significant difference in the peak areas of cysteine, methionine, and homocysteine ​​between series A and series B, suggesting that the three compounds did not exhibit a carryover effect during the sample injection analysis.

[0055] 7. Validation of total homocysteine ​​standard material and clinical samples in lyophilized human serum: This method was used to pre-treat and detect total homocysteine ​​in lyophilized human serum at four different concentration levels. Each level was treated three times. Since this national standard reference material has been verified by nine laboratories (including five JCTLM-certified laboratories) using four measurement methods and nine measurement systems, including enzymatic method, enzyme cycling, Roche enzyme colorimetric method, and Roche enzyme method, this national standard reference material was selected to verify the results of homocysteine ​​detection. Sample detection (1) Detection of total homocysteine ​​standard substance in freeze-dried human serum The method described in Example 2 was used to pretreat and detect total homocysteine ​​standards (8.0 μmol / L, 14.4 μmol / L, 17.9 μmol / L, and 28.8 μmol / L) in lyophilized human serum at four different concentration levels. Each level was treated three times, and the results are shown in Table 10. Table 10 Results of the detection of total homocysteine ​​standard substances in freeze-dried human serum

[0056] The deviations of the homocysteine ​​test values ​​at the four levels from the standard values ​​ranged from -6.3% to 4.5%, all within ±10%. Since this national standard reference material has been verified by nine laboratories (including five JCTLM-certified laboratories) using four measurement methods—enzymatic method, enzyme cycling, Roche enzyme colorimetric method, and Roche enzyme method—and nine measurement systems, the results of this method for homocysteine ​​detection are accurate and reliable.

[0057] (2) Clinical sample testing

[0058] Blood samples were collected from patients in a hospital in Shijiazhuang, including males and females aged 11 to 85 years. The samples of different genders and age groups were tested using the method described in Example 2 to verify the reliability of the product in clinical application. The results are shown in Table 11. Table 11 Concentrations of homocysteine, methionine, and cysteine ​​in serum samples from 26 patients

[0059] The results showed that the concentrations of homocysteine ​​in serum ranged from 5.9 to 27.1 μmol / L, methionine from 10.1 to 36.2 μmol / L, and homocysteine ​​from 166.6 to 463.8 μmol / L. This indicates that the detection method can be widely used in clinical sample testing.

[0060] As can be seen from the above embodiments, the present invention provides a method and kit for detecting homocysteine ​​and its metabolic related substances. This method can simultaneously detect three types of compounds, including homocysteine, cysteine ​​and methionine, to comprehensively reflect the reasons for elevated homocysteine ​​levels.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting homocysteine ​​and its metabolic-related substances, characterized in that, Includes calibrators, high-value quality control products, low-value quality control products, reagent D, PBS buffer, reducing agent, water, and formic acid acetonitrile solution; The reducing agent is prepared by mixing 0.03~0.05g of dithiothreitol and 0.5~1.5mL of water; The reagent D comprises the following components at final concentrations: 2-4 μmol / L homocysteine-D4, 5-7 μmol / L methionine-D3, and 7-9 μmol / L cysteine-D3. The calibrator comprises the following components at final concentrations: 500 μmol / L homocysteine, 1000 μmol / L methionine, and 2500 μmol / L cysteine. The high-value quality control materials include 400 μmol / L homocysteine, 800 μmol / L methionine, and 2000 μmol / L cysteine; The low-value quality control materials include 50 μmol / L homocysteine, 100 μmol / L methionine, and 250 μmol / L cysteine.

2. The use of the kit according to claim 1 in the detection of homocysteine ​​and its metabolic-related substances, wherein the metabolic-related substances include methionine and cysteine.

3. A method for detecting homocysteine ​​and its metabolic-related substances, characterized in that, Includes the following steps: (1) Mix the test sample, S1~S6 standard, high value quality control sample, and low value quality control sample with reagent D and let stand for 4~6 min. Then mix with reducing agent and let stand to react to obtain reduction product. (2) The reduction product and formic acid acetonitrile solution are mixed to obtain a mixture; (3) Mix the mixture with water, centrifuge, and take the supernatant for detection by liquid chromatography-tandem mass spectrometry; The S1 to S6 standards are obtained by gradient dilution of calibrators and PBS buffer.

4. The method according to claim 3, characterized in that, The amount of the sample to be tested is 40-60 μL; the amount of each gradient standard in the S1-S6 standards is 40-60 μL; the amount of the high-value quality control is 40-60 μL; the amount of the low-value quality control is 40-60 μL; the amount of reagent D is 40-60 μL; and the amount of the reducing agent is 40-60 μL.

5. The method according to claim 3, characterized in that, The temperature for the static reaction is 30~40℃; the time for the static reaction is 15~25min.

6. The method according to claim 3, characterized in that, The amount of formic acid acetonitrile solution added is 350~450μL.

7. The method according to claim 3, characterized in that, The amount of water added is 200~300μL; the centrifugation speed is 8000~12000rpm; and the centrifugation time is 4~6min.

8. The method according to claim 3, characterized in that, During the liquid chromatography-tandem mass spectrometry detection process, the liquid chromatography conditions include: Mobile phase A is 0.02% formic acid in water, and mobile phase B is 0.02% formic acid in methanol; The elution conditions were: 0–1.5 min: 5%–40% B, 1.6–1.8 min: 80%–80% B, 1.81–2.5 min: 5%–5% B; The injection volume is 1~10μL, the mobile phase flow rate is 0.3~0.4mL / min, the injection temperature is 3~5℃, and the column temperature is 35~45℃.

9. The method according to claim 3, characterized in that, During the liquid chromatography-tandem mass spectrometry detection process, the mass spectrometry conditions include: The ion source is an ESI+ source, the ion source voltage is 5500V, the ion source temperature is 500℃, and the scanning mode is MRM. Air curtain gas 40psi, impact gas 8psi, atomizing gas 50psi, auxiliary heater gas 60psi.

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