Kit for quantitatively detecting homocysteine and application thereof

Abstract

The invention provides a kit for quantitatively detecting homocysteine and application of the kit. The kit comprises a derivative reagent, an internal standard solution and a standard substance working solution, according to the present invention, the homocysteine-monobrominated diamine derivative is generated through the derivatization reaction of the monobrominated diamine and the homocysteine, such that the problems of easy oxidation and poor stability of the homocysteine are solved, and the reliability of the determination result is improved; the polarity of the homocysteine-monobrominated diamine derivative is smaller than that of homocysteine, so that the problem that the homocysteine is not reserved in a conventional C18 chromatographic column is solved; homocysteine is accurately quantified by detecting derivative products, and tedious reduction reaction operation is omitted.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical analysis, and particularly relates to a kit for quantitatively detecting homocysteine and its application. Background Art

[0002] Homocysteine is a sulfur-containing amino acid and an important intermediate product generated during the metabolism of methionine and cysteine. Under normal circumstances, homocysteine can be catabolized in the body, and its concentration is maintained at a relatively low level. However, in daily life, for various reasons, the metabolism of blood homocysteine will be affected, resulting in an increase in the concentration of homocysteine, which will greatly increase the incidence risks of coronary heart disease, peripheral vascular diseases and cerebrovascular diseases. Hyperhomocysteinemia is a common metabolic disease in China. Through the accurate detection of homocysteine, the precise diagnosis and treatment of cardiovascular diseases can be achieved.

[0003] Currently, the commonly used detection methods for homocysteine and its related metabolites mainly include enzyme-linked immunosorbent assay, chemiluminescence assay, high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. The enzyme-linked immunosorbent assay and chemiluminescence assay are simple to operate and have a short detection time. However, due to being based on the antibody-antigen reaction, the detection specificity is poor and the simultaneous detection of multiple metabolites cannot be achieved. High-performance liquid chromatography can separate multiple metabolites by liquid phase, but the sensitivity is relatively low. When performing multi-index joint detection, it cannot meet the detection requirements of indicators at different concentration levels. Liquid chromatography-tandem mass spectrometry combines the advantages of chromatographic separation and mass spectrometry detection, has high sensitivity and accuracy, and can detect multiple metabolites at one time, providing more abundant information for clinical diagnosis, and has received more and more attention and recognition in the detection of homocysteine and its related metabolites.

[0004] CN115436536A discloses a detection method for detecting homocysteine and its related metabolites by liquid chromatography-tandem mass spectrometry. However, the homocysteine sample can only remain stable at room temperature for 8 hours.

[0005] Homocysteine contains a mercapto group in its structure. The mercapto group has relatively high biological activity and is easily oxidized during sample acquisition, preservation, and pretreatment. Currently, there is no method or kit that can effectively maintain the stability of homocysteine for a long time and accurately quantify it. In addition, due to the instability of homocysteine, standard samples, internal standards, etc. required for detection need to be prepared and used immediately, making it difficult to store and transport, and adding a lot of cumbersome pre-solution preparation processes to the detection process. Therefore, there is an urgent need for a kit that can accurately detect homocysteine and has the advantages of long-term preservation, suitability for transportation, simple operation, high processing efficiency, high recovery rate, and low cost, so as to make the method for detecting homocysteine in serum by high-performance liquid chromatography-tandem mass spectrometry have higher sensitivity, the detection process is more simple and efficient, and the accuracy and stability of the detection results can be guaranteed. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides a kit for detecting homocysteine by high-performance liquid chromatography-tandem mass spectrometry and its detection method. By making homocysteine undergo a derivatization reaction with monobromodiamine (MBBR) to generate a homocysteine-monobromodiamine derivative, homocysteine is transformed from an unstable thiol form into a stable thioether form, and at the same time, vitamin C is added to keep homocysteine and its internal standard in the internal solution and standard solution stable; homocysteine is accurately quantified through the derivative reaction product, avoiding cumbersome reduction reaction operations, improving the accuracy and precision of detection, and meeting the detection requirements. The derivatization reaction is shown in the following formula.

[0007]

[0008] The technical solution of the present invention is as follows: On the one hand, the present invention provides a kit for detecting homocysteine in serum by high-performance liquid chromatography-tandem mass spectrometry. The kit includes an internal standard solution, a standard working solution, and a derivatization reagent. The internal standard solution and the standard working solution contain a stabilizer, the stabilizer is vitamin C, and the derivatization reagent is an acetonitrile solution of monobromodiamine. Monobromodiamine is used to make homocysteine undergo a derivatization reaction to generate a homocysteine-MBBR derivative.

[0009] The present invention has established a kit for detecting homocysteine in human serum, which is used for the diagnosis of hyperhomocysteinemia, assisting in the investigation of etiologies and disease typing. In order to simplify the operation steps, for the standard sample used to prepare the standard curve in the kit, in order to ensure the stability of the standard sample, we added the antioxidant vitamin C.

[0010] Meanwhile, the internal standard solution in the kit contains homocysteine-d4 and also has the problems of instability and difficulty in storage. Therefore, in order to antioxidize, vitamin C is also added so that the prepared standard samples and internal standard solution can be stored at 4°C for more than two years, which is convenient for use.

[0011] Furthermore, the mass concentration of the vitamin C is 5-20 mg / ml, preferably 10 mg / ml, and it is dissolved in water.

[0012] The homocysteine standard stock solution is prepared with deionized water and has a concentration of 2 mg / mL.

[0013] Furthermore, it also includes a precipitant liquid chromatography mobile phase; the precipitant is a 10% trichloroacetic acid methanol-water (1:1) solution; the liquid chromatography mobile phase includes mobile phase A and mobile phase B. Mobile phase A is 0.2% formic acid water, and mobile phase B is methanol. The derivatization reagent is a monobromo diamine acetonitrile solution. Monobromo diamine is used to cause a derivatization reaction of homocysteine to generate homocysteine-MBBR derivatives. The concentration of the monobromo diamine acetonitrile solution is 0.5-5 mg / mL.

[0014] On the other hand, the present invention provides a method for detecting homocysteine in serum by high performance liquid chromatography-tandem mass spectrometry. The method uses the kit as described above for detection; it includes the following steps: taking 100 μL of the sample, adding 5-20 μL of the internal standard solution and mixing evenly, adding 10-40 μL of the derivatization reagent, reacting for 20 minutes under light-shielded conditions, then adding the precipitant and vortexing to mix evenly, centrifuging, taking the supernatant for filtration, and detecting with high performance liquid chromatography-tandem mass spectrometry.

[0015] Furthermore, when preparing the standard curve, the sample is a standard curve sample, and the standard curve sample is obtained by diluting the standard working solution with a PBS solution containing BSA.

[0016] Homocysteine has strong polarity and is basically not retained on the C18 chromatographic column. It is eluted simultaneously with the polar impurities in the serum matrix, and a serious matrix suppression effect will occur during mass spectrometry detection, thus affecting the detection sensitivity, the symmetry of the chromatographic peak, and the accuracy of the quantitative result. We choose the HSS T3 chromatographic column for analysis, which greatly enhances the retention of homocysteine. Moreover, due to the smaller polarity of the homocysteine-MBBR derivative, the retention is further enhanced, greatly improving the detection sensitivity and the accuracy of the quantitative result.

[0017] Meanwhile, the present invention also simulates the matrix effect of clinical samples by adding BSA to the standard samples.

[0018] The present invention has the following beneficial effects: (1) For the first time, the detection of homocysteine was realized by using homocysteine derivatization reaction, which improved the stability and reliability of the detection method and avoided cumbersome operations such as reduction reaction. The accurate monitoring of homocysteine was achieved, which can be used for the etiological analysis and typing diagnosis of hyperhomocysteinemia; (2) By adding stabilizers to the standard samples and internal standard solutions, the oxidation of homocysteine was avoided, and it could be stored at 4 °C for more than 2 years; (3) The pretreatment method is simple, fast, and has good stability, and can realize the detection of analytes at different concentration levels; detected by LC-MS, the analysis time is only 5 min, with high throughput and low cost. Description of the Drawings

[0019] Figure 1 It is the detection spectrum of the clinical serum sample in Example 1; Figure 2 It is the standard curve drawn in Example 1. Among them, R 2 represents the coefficient of determination, which is used to measure the linear fitting degree of the standard curve. When R² is close to 1 (such as ≥0.99), it indicates that the linear relationship between concentration and signal value is good and can be used for accurate quantification. Detailed Embodiments

[0020] The present invention will be further described below with reference to the drawings. The following examples are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention.

[0021] Example 1 Sample Preparation, Pretreatment and Detection I. Sample Preparation 1. Preparation of derivatization reagent: Dissolve 150 mg of monobromo diamine in 100 ml of acetonitrile.

[0022] 2. Preparation of PBS solution: Take 8 g of NaCl, 0.2 g of KCL, 1.44 g of disodium hydrogen phosphate and 0.24 g of potassium dihydrogen phosphate, add water to make up to 1 liter to obtain 1×PBS solution.

[0023] 3. Preparation of standard curve: Prepare a homocysteine stock solution with deionized water at a concentration of 4 mg / mL. For the standard curve samples, an appropriate amount of the standard stock solution needs to be taken, made into a standard working solution with a 10 mg / mL VC solution, and then diluted with a standard diluent (1×PBS solution containing 0.5% BSA) to prepare standard solutions with the following concentrations: 781.25 ng / mL, 1562.5 ng / mL, 3125 ng / mL, 6250 ng / mL, 12500 ng / mL, 25000 ng / mL, 50000 ng / mL.

[0024] 4. Preparation of internal standard solution: Prepare a stock solution of homocysteine-D4 internal standard at 2 mg / mL with deionized water. Take 0.1 mL of the stock solution of the internal standard and dilute it with a 10 mg / mL VC solution to obtain an internal standard solution at 15 μg / mL.

[0025] 5. Preparation of the precipitant: Dissolve trichloroacetic acid in a 50% methanol aqueous solution to prepare a concentration of 10%.

[0026] II. Sample pretreatment Take 100 μL of the serum sample / standard solution, add 20 μL of the internal standard solution and vortex for 1 min, then add 70 μL of PBS solution and 30 μL of monobromodiamine derivatization reagent, vortex for 1 min to mix well, let stand for 20 min in a light-proof environment, then add 280 μL of the precipitant and vortex for 5 min, centrifuge at 13000 r / min for 10 min, take the supernatant and filter it through a 0.2 μm filter membrane into an injection vial for injection preparation.

[0027] III. Sample detection LC-MS / MS detection Liquid chromatography conditions: Mobile phase A is 0.2% formic acid water; mobile phase B is methanol. The chromatographic column is ACQUITY UPLC HSS T3, the column temperature of the chromatographic column: 40 °C, the sample chamber temperature: 10 °C, the flow rate: 0.4 mL / min, the injection volume is 5 μL, and the elution gradient is shown in Table 1.

[0028] Table 1 Elution gradient

[0029] Mass spectrometry conditions: Detection is carried out in the ESI positive ion mode, capillary voltage: 0.5 kV, ion source temperature: 150 °C, desolvation gas flow rate: 1000 L / hr, desolvation gas temperature: 500 °C. The multiple reaction monitoring mode is adopted, and the ion pair parameters are shown in Table 2.

[0030] Table 2 Ion pair parameters

[0031] IV. Calculation of the measurement results The detection spectrum of the clinical low-concentration serum sample is as Figure 1 shown. As Figure 1 can be seen, according to the method provided in this embodiment, homocysteine can be accurately detected. According to the retention time and ion pair, read the mass spectrometry response signal of the analyte in the calibration curve and the sample and the corresponding internal standard peak area value. Take the ratio of the peak area of the calibration curve to the internal standard peak area as the Y value and the calibration curve concentration as the X value, and draw a fitting curve as Figure 2As shown, the linear correlation coefficients of homocysteine are all greater than 0.99. Substituting the peak area ratio of the analyte and the internal standard substance in the sample into the corresponding curve equation, the concentration of the analyte in the corresponding serum sample can be calculated.

[0032] Example 2 Investigation of Sample Stability The pretreated samples were placed at room temperature, 4°C, 10°C, and -20°C respectively, and the deviations of the test results at 8, 16, 24, 48, and 72 hours from the test results at 0 hour were investigated respectively. The results are shown in Table 3, indicating that the stability of homocysteine is greatly increased after derivatization, and the sample can remain stable for at least 72 hours at room temperature, improving the stability and reliability of the homocysteine test results.

[0033] Table 3

[0034] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and deformations can be made, and these improvements and deformations should also be regarded as the protection scope of the present invention.

Claims

1. A kit for quantitatively detecting homocysteine, characterized in that: The kit comprises an internal standard solution, a standard working solution and a derivatization reagent, wherein the derivatization reagent is an acetonitrile solution of monobromodiamine, and the monobromodiamine is used to cause homocysteine ​​to undergo a derivatization reaction to generate a homocysteine-monobromodiamine derivative.

2. The kit according to claim 1, characterized in that The internal standard solution and the standard working solution contain a stabilizer, and the stabilizer is vitamin C, and the mass concentration of vitamin C is 5-20 mg / ml.

3. The kit according to claim 1, characterized in that The internal standard solution comprises a stabilizer and an internal standard working solution; the internal standard working solution is composed of an isotope internal standard solution containing a metabolite to be measured, and the isotope internal standard solution is a homocysteine-d4 solution.

4. The kit according to claim 1, characterized in that The concentration of the monobromodiamine acetonitrile solution is 0.5-5 mg / mL.

5. The kit according to claim 1, characterized in that The homocysteine ​​standard stock solution was prepared with deionized water at a concentration of 2 mg / mL.

6. The kit according to claim 1, characterized in that It also includes a precipitant and a liquid chromatography mobile phase; the precipitant is a methanol-water solution of 10% trichloroacetic acid, and the volume ratio of methanol-water is 1:1; the liquid chromatography mobile phase includes mobile phase A and mobile phase B, mobile phase A is 0.2% formic acid water, and mobile phase B is methanol.

7. A method for quantitatively detecting homocysteine, characterized in that: The detection is performed using the kit described in any one of claims 1 to 6, comprising the following steps: taking 100 μL of sample, adding 5 to 20 μL of internal standard solution and mixing, adding 10 to 40 μL of derivatization reagent, reacting for 20 minutes under light-proof conditions, then adding a precipitant and vortex mixing, centrifuging, taking the supernatant and filtering, and detecting by high performance liquid chromatography tandem mass spectrometry.

8. The method according to claim 7, characterized in that When preparing a standard curve, the sample is a standard curve sample, and the standard curve sample is obtained by diluting the standard working solution with a PBS solution containing BSA.

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