Liquid stable system and application thereof

Abstract

The invention discloses a liquid-state stable system and application thereof, a solute in the liquid-state stable system comprises one or more of homocysteine metabolic pathway compounds, a stabilizer contains DTT and ascorbic acid and / or erythorbic acid, and a solvent is a methanol aqueous solution. The stabilizer disclosed by the invention is simple in component, can play a role in stabilizing homocysteine metabolic pathway compounds at the same time, particularly can play a very good role in stabilizing homocysteine metabolic pathway compounds with different stabilizer requirements at the same time, and effectively and greatly reduces the degradation rate of each compound. When the liquid stable system is applied to preparation of a homocysteine metabolic pathway compound detection kit, the liquid stable system can be used as a calibration product, a quality control product, an internal standard working solution and other components, has good stability, is beneficial to prolonging the shelf life of a kit product and reducing the storage cost, and is suitable for industrial production. When the kit is used for quantitative detection of homocysteine metabolic pathway compounds, the detection result is more reliable, and more valuable data support is provided for research and application in related fields.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a liquid stable system and its application. Background Technology

[0002] Homocysteine ​​(Hcy), an intermediate product of methionine metabolism, is crucial for maintaining normal metabolism in the body. When enzymes involved in Hcy metabolism malfunction or are deficient, it leads to abnormally high Hcy levels, inducing hyperhomocysteinemia (HHcy). HHcy not only weakens the body's antioxidant and methylation capabilities but also has direct or indirect adverse effects on human health. Clinical studies have shown that HHcy is closely related to the development of various diseases, including but not limited to cardiovascular disease, chronic kidney disease, fatty liver, diabetes, skeletal muscle injury, and gastrointestinal diseases. Epidemiological surveys show that the prevalence of HHcy in my country is as high as 37.2%. For every 5 mmol / L increase in plasma Hcy levels, the risk of all-cause mortality increases by 33.6%. Given the serious harm that HHcy causes to human health, in-depth exploration of its pathogenesis is of great significance for the effective diagnosis and treatment of this disease.

[0003] The causes of hyperhomocysteinemia are mainly divided into congenital and acquired types. Congenital types are primarily caused by deficiencies in cystathionine β-synthetase (CBS), impaired cobalamin (vitamin B12) synthesis, and methylenetetrahydrofolate reductase (MTHFR). These enzyme deficiencies affect the metabolic pathway of homocysteine, leading to its abnormal accumulation in the body. Acquired types can be caused by lifestyle factors, dietary habits, underlying diseases (such as diabetes, stroke, chronic kidney disease, and tumors), and medications. In addition, malnutrition, nutrient deficiencies (such as folic acid, vitamin B6, cobalamin, and betaine deficiencies), aging, chronic gastrointestinal diseases, liver disease, kidney disease, malignant tumors, medications (such as isoniazid and methotrexate), and unhealthy lifestyle habits (such as long-term smoking and excessive alcohol consumption) can also lead to hyperhomocysteinemia.

[0004] The necessity of detecting substances involved in the homocysteine ​​cycle in the body stems from the fact that homocysteine ​​is an intermediate product in methionine metabolism, and its metabolism is influenced by various enzymes and cofactors. Abnormally elevated homocysteine ​​levels in the blood are associated with an increased risk of various diseases, such as cardiovascular disease, neural tube defects, and certain cancers. By detecting compounds in the homocysteine ​​metabolic pathway, it is possible to identify whether secondary hyperhomocysteinemia is caused by nutritional deficiencies, thus providing important evidence for clinical diagnosis and treatment. Furthermore, for hereditary hyperhomocysteinemia, differential diagnosis through genetic analysis and other examinations helps to clarify the cause and enable individualized, precise treatment.

[0005] Generally, compounds associated with hyperhomocysteinemia (homocysteine ​​metabolic pathway compounds) mainly include amino acids and their derivatives, as well as water-soluble vitamins and their derivatives. Amino acids and their derivatives include thiol-containing amino acids and their derivatives, taurine, glutathione, glycine, dimethylglycine (DMG), and trimethylglycine (betaine). Water-soluble vitamins and their derivatives include vitamin B2 (riboflavin), vitamin B6, folic acid, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, and methylmalonic acid. Additionally, thiol-containing amino acids and their derivatives include methionine, homocysteine, cysteine, glutathione, S-adenosylmethionine (SAM), and S-adenosylhomocysteine ​​(SAH). However, different compounds have different stability requirements. For example, amino acids (e.g.) contain amino and carboxyl groups, and their amino (-NH2) groups and α-carbon positions are prone to oxidation. Furthermore, because it contains both -NH2 and -COOH groups, under strong acid conditions, -NH2 may be protonated to form -NH3. + Under strong acid conditions, -COOH may deprotonate to form (-COO). -This can lead to further decarboxylation or condensation reactions, making them unstable. Therefore, amino acid stabilizers need to possess both antioxidant properties and a suitable pH environment. Simultaneously, for thiol-containing amino acids and their derivatives, mass spectrometry detection requires the disulfide bonds to be opened to form free thiol groups, which then need to be protected. Therefore, these compounds require the presence of compounds in the environment that can open disulfide bonds to form free thiol groups and protect the thiol groups. Water-soluble vitamins and their derivatives, due to their inherently oxidizable groups or active sites (such as unsaturated bonds, reactive -OH groups that are easily oxidized and dehydrated), are prone to degradation under conditions such as light, high temperature, and unsuitable pH. Furthermore, the pH requirements for these compounds vary. Therefore, stabilizers for water-soluble vitamins and their derivatives also require suitable antioxidants and a suitable pH environment. In summary, stabilizers that simultaneously satisfy the stability requirements of compounds in the homocysteine ​​metabolic pathway have become one of the technical problems that need to be solved in this field. For example, Chinese patents (CN111175406A) and (CN115326957A) use methanol, acetonitrile, and ascorbic acid (VC) as stabilizers to prepare mixed standard solutions of water-soluble vitamins at various concentrations. However, this preparation method is not conducive to the stability of the target compounds methionine and homocysteine ​​in this invention. Chinese patent (CN114935619A) uses three stabilizers (ascorbic acid, sodium metabisulfite, and tea polyphenols) to stabilize the target water-soluble vitamin compounds. The stabilizer composition is relatively complex and is not conducive to the stability of the target compound methionine. Chinese patent (CN 116381084A) detects eight substances simultaneously, but it uses a variety of stabilizers (including ammonium acetate, ascorbic acid, and glacial acetic acid) prepared in a certain proportion of methanol. It also requires dilution before use, which increases the complexity of reagent preparation in practical applications and is not conducive to the promotion and maintenance of efficient and stable production and detection technologies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a liquid stable system and its application, wherein the stabilizer has a simple composition and good stability for homocysteine ​​metabolic pathway compounds, especially with the fewest types of stabilizers, while simultaneously satisfying the stabilizing effect on homocysteine ​​metabolic pathway compounds with different stabilizer requirements; when applied to reagent kits, it helps to improve the shelf life of the kits and reduce storage costs.

[0007] To address the aforementioned technical problems, a first aspect of the present invention is to provide a liquid-stabilized system, comprising:

[0008] Solute: One or more compounds from the homocysteine ​​metabolic pathway;

[0009] Stabilizer: Contains DTT (dithiothreitol) and at least one of two compounds, namely ascorbic acid and isoascorbic acid;

[0010] Solvent: Aqueous methanol solution.

[0011] In this invention, DTT and ascorbic acid / isoascorbic acid are used as stabilizers. They can play a good stabilizing role among one or more homocysteine ​​metabolic pathway compounds with different pH stability requirements. In particular, they can simultaneously meet the stabilizing effect of homocysteine ​​metabolic pathway compounds with different stabilizer requirements with the fewest types of stabilizers. The compounds have low degradation rate and good stability under accelerated aging conditions of 3 days at 37°C and 75% and storage conditions of 4°C for one year.

[0012] In one specific embodiment, the homocysteine ​​metabolic pathway compounds include amino acids and their derivatives, as well as water-soluble vitamins and their derivatives. Preferably, the amino acids and their derivatives include thiol-containing amino acids and their derivatives, taurine, glycine, dimethylglycine, and trimethylglycine, wherein the thiol-containing amino acids and their derivatives include methionine, homocysteine, cysteine, glutathione, S-adenosylmethionine, and S-adenosylhomocysteine; preferably, the water-soluble vitamins and their derivatives include vitamin B2, vitamin B6, folic acid, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, and methylmalonic acid.

[0013] In this invention, DTT primarily functions to open the disulfide bonds of thiol-containing amino acids and their derivatives, forming free thiol groups, and also protects these thiol groups, thereby stabilizing the stability of thiol-containing amino acids and their derivatives. The hydroxyl group in the molecular structure of ascorbic acid can provide hydrogen atoms, interrupting the oxidation chain reaction. On the one hand, it directly eliminates free radicals in the system through its own antioxidant properties; on the other hand, it assists in the regeneration of DTT, enhancing its stabilizing effect on thiol-containing amino acids and their derivatives. The synergistic effect of DTT and ascorbic acid, through the regulation of the redox potential of the homocysteine ​​metabolic pathway compound liquid mixture system, provides stable chemical reduction for at least one of them, inhibiting degradation reactions and achieving stable maintenance.

[0014] In one specific embodiment, the final mass concentration of DTT is 0.1%-10%, preferably 0.5%-5%, more preferably 1%-2%, and the final mass concentration of ascorbic acid is 0.01%-5%, preferably 0.1%-1%.

[0015] In one specific embodiment, the volume concentration of the methanol-water solution is 20-80%, preferably 50%.

[0016] To address the aforementioned technical problems, a second aspect of the present invention is to provide an application of the aforementioned liquid stable system in the preparation of a reagent kit, particularly, the aforementioned liquid stable system is used as an internal standard solution and a standard working solution in the preparation of the reagent kit.

[0017] In this invention, the liquid stable system has excellent stability. Therefore, when used as a component of internal standard working solution, calibrator and quality control, it helps to improve the shelf life of the reagent kit and reduce storage costs. Moreover, when using the reagent kit for quantitative detection of homocysteine ​​metabolic pathway compounds, the detection results are more reliable, providing more valuable data support for research and application in related fields. Detailed Implementation

[0018] The technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] Example 1: Preparation of a Liquid Stable System

[0020] 1. Preparation of a liquid-stable system containing VB2, VB6, 5-MTHF, FA, MMA, Cth, Hcy, and Met.

[0021] S1. Preparation of stock solutions for each solute: Prepare stock solutions for VB2, VB6, 5-MTHF, FA, MMA, Cth, Hcy, and Met according to Table 1:

[0022] Table 1

[0023]

[0024] S2. Dissolve the stock solution from step S1, VC (ascorbic acid, abbreviated as VC in subsequent examples), and DTT in a 50% (v / v) methanol aqueous solution as required, forming the liquid stabilizers shown in Table 2. The concentrations of each solute are shown as C1-C7, as well as LQC and HQC. VC and DTT are stabilizers, with a final concentration of 0.1% for VC and 1% for DTT. Among the nine liquid stabilizers with different concentrations, C1-C7 can be used as standards in the kit, LQC can be used as a low-value control solution, and HQC can be used as a high-value control solution.

[0025] Table 2

[0026]

[0027]

[0028] 2. Preparation of a product containing VB2- 13 C4 15 N2, VB6-d3, 5-MTHF- 13 C5, FA- 13 Liquid stable system of C5, MMA-d3, Cth-d4, Hcy-d4, Met-d3

[0029] The preparation steps are the same as those for the aforementioned liquid stable system. For example, based on Table 1, first prepare stock solutions of each solute, then dissolve the stock solutions, VC, and DTT in a 50% (v / v) methanol solution to form a liquid stable system. Among these, VB2- 13 C4 15 N2, VB6-d3, 5-MTHF- 13 C5, FA- 13 The final concentrations of C5, MMA-d3, Cth-d4, Hcy-d4, and Met-d3 were 240 ng / mL, 120 ng / mL, 144 ng / mL, 1440 ng / mL, 240 ng / mL, 14 μM, 64 μM, and 120 μM, respectively. The final concentrations of VC and DTT were the same as those in the aforementioned liquid stable system. This liquid stable system was used as the internal standard solution in the kit.

[0030] Example 2: Stability Test of Liquid Stable System

[0031] 1. Accelerated aging stability test

[0032] S1. Preparation of liquid stable samples: The preparation method is the same as in Example 1, except that the type and content of stabilizer are different, and the final concentration of solute is the same as that of the low-value quality control solution and the high-value quality control solution in Example 1.

[0033] S2. The sample obtained in step S1 was aged for 3 days in an incubator at 37°C and 75% humidity. 200 μL of the aged sample was taken, and 10 μL of the internal standard solution prepared in Example 1 and 10 μL of reducing agent (120 mM dithiothreitol aqueous solution) were added. The mixture was vortexed for 1 min, allowed to stand at room temperature for 15 min, and then 600 μL of 50% methanol solution was added. The mixture was vortexed for 1 min and centrifuged at 4°C and 14000 rpm for 10 min. 500 μL of the supernatant was taken, dried under nitrogen at 40°C, and 130 μL of the reconstitution solution (0.1% formic acid aqueous solution) was added. The mixture was shaken for 5 min and then centrifuged at 4000 r / min for 5 min in a low-speed centrifuge. The sample concentration was detected by LC-MS / MS. The stability deviation of the solute was calculated based on the measured concentration using the following method: Deviation = (Detected value - Theoretical value) / Theoretical value * 100%.

[0034] In this embodiment, the stability deviations of the compounds in different samples are as follows:

[0035] 1. The results of samples with different VC concentrations and only VC as stabilizer are shown in Table 3. The final concentration of solute is the same as that of the high-value quality control solution in Example 1.

[0036] Table 3

[0037]

[0038] It is evident that when the stabilizer is only VC, the stability deviation is large and the sample stability is poor (VB2 and VB6 showed no significant degradation after accelerated aging, and are not shown here).

[0039] 2. The results of samples with DTT as the only stabilizer and different DTT concentrations are shown in Table 4. The final concentration of the solute is the same as that of the high-value quality control solution in Example 1.

[0040] Table 4

[0041]

[0042] It is evident that when DTT is used as a stabilizer, the stability of Met, Cth, MMA, 5-MTHF and FA is significantly improved and the stability is good, except for Hcy which has poor stability. (VB2 and VB6 showed no significant degradation after accelerated aging and are not shown here).

[0043] 3. When the stabilizers are DTT and VC, the final concentration of DTT is fixed at 1%, and the results of different final concentrations of VC are shown in Table 5. The final concentration of the solute is the same as that of the low-value quality control solution and the high-value quality control solution in Example 1.

[0044] Table 5

[0045]

[0046] It is evident that using DTT and VC together as stabilizers significantly improves the stabilization effect on all eight solutes, especially when the concentration of VC is in the range of 0.1%-5%, the stabilization effect is even better.

[0047] In this embodiment, the liquid chromatography conditions are as follows:

[0048] Mobile phase A: 0.1% formic acid aqueous solution;

[0049] Mobile phase B: A 0.1% (v / v) aqueous methanol solution;

[0050] Chromatographic column: C18 (3μm);

[0051] Column temperature: 40℃;

[0052] Sample manager: 10℃;

[0053] Flow rate: 0.5 mL / min;

[0054] Injection volume: 10 μL;

[0055] The elution gradient is shown in Table 6:

[0056] Table 6

[0057]

[0058] In this embodiment, the mass spectrometry conditions are as follows:

[0059] Ion source: electrospray ion source; Ion mode: negative ion mode (MMA), the other 7 compounds use positive ion mode;

[0060] Detection mode: Multiple reaction monitoring;

[0061] The ion source temperature is 550℃;

[0062] The spray voltage for positive ions is 5500V;

[0063] The air curtain gas pressure is 35 psi;

[0064] The pressure of atomizing gas Gas1 and auxiliary heating gas Gas2 is 55 psi.

[0065] The ion pair parameters are shown in Table 7:

[0066] Table 7

[0067] Compound Q1 (m / z) Q3 (m / z) Met 150.3 103.9 Met-d3 154 107.9 L-Cth 223 134 L-Cth-d4 227.2 138.1 Hcy 136.4 90.1 Hcy-d4 140 94.1 VB6 184.1 166.1 VB6-d3 187.1 169.1 5M-HTF 460.31 313.3 5M-HTF-d3 463.2 316 MMA 117.2 73.1 MMA-D3 120.4 75.8 FA 442.2 295.1 [[ID=十六]]FA-13C5 447.2 295 VB2-d6 383.1 249 VB2 It should be noted that there seems to be a misspelling in "[[ID=十六]]", it should probably be "". If this is a real error, please correct it according to the actual situation. 377 243

[0068] 2. Stability test at 4℃

[0069] Stability test samples stored at 4℃: The stabilizers were 0.1% final mass concentration VC and 1.0% final mass concentration DTT. The final concentrations of the solutes were the same as those of the low-value and high-value quality control solutions in Example 1. The samples were stored at 4℃ for one year. After storage, the sample concentrations were detected by LC-MS / MS. The stability deviation of the solutes was calculated based on the measured concentrations. The results are shown in Table 8.

[0070] Table 8

[0071]

[0072] It is evident that when DTT and VC are used together as stabilizers, all eight solutes exhibit excellent stability at 4°C, regardless of whether the quality control samples are low-value or high-value.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A liquid stable system, characterized in that, Comprise: Solute: one or more of homocysteine metabolic pathway compounds; Stabilizer: DTT, and at least one of ascorbic acid and erythorbic acid; Solvent: methanol aqueous solution.

2. The liquid stable system of claim 1, wherein, The homocysteine metabolic pathway compounds include amino acids and their derivatives, and water-soluble vitamins and their derivatives.

3. The liquid stable system of claim 2, wherein, The amino acids and their derivatives include sulfhydryl-containing amino acids and their derivatives, taurine, glycine, dimethylglycine, and trimethylglycine.

4. The liquid stable system of claim 3, wherein, The sulfhydryl-containing amino acids and their derivatives include methionine, homocysteine, cysteine, glutathione, S-adenosylmethionine, and S-adenosylhomocysteine.

5. The liquid stable system of claim 2, wherein, The water-soluble vitamins and their derivatives include vitamin B2, vitamin B6, folic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, and methylmalonic acid.

6. The liquid stable system of claim 1, wherein, In the liquid stabilizing system, the final concentration of DTT is 0.1%-10% by mass, and the final concentration of ascorbic acid and / or erythorbic acid is 0.01%-5% by mass.

7. The liquid stable system of claim 1, wherein, The volume concentration of the methanol aqueous solution is 20-80%.

8. Use of the liquid stabilizing system according to any one of claims 1-7 in the preparation of a kit.

9. Use according to claim 8, wherein the compound is ###0002### The liquid stabilizing system is used as an internal standard solution and a standard and quality control working solution in the preparation of a kit.

10. Use according to claim 9, wherein The kit further comprises a reducing agent, which is an aqueous solution of DTT; The reconstitution solution is an aqueous formic acid solution.

Images