Blood fat quality control liquid

Abstract

The invention discloses a blood lipid quality control liquid, which comprises a buffer solution, cholesterol or cholesterol and glycerol, cholic acid or deoxycholic acid and salt thereof, and cyclodextrin. According to the blood fat quality control liquid, cholic acid or deoxycholic acid and salt thereof and cyclodextrin are used as solubilizers to form a compound, cholesterol is dissolved in a water-soluble matrix, direct normal-temperature stirring is performed, no organic flammable harmful reagent participates, the process is simple, safe and reliable, the stability is good, and the effective period can reach 12 months.

Description

Technical Field

[0001] The present application relates to the technical field of diagnostic reagents, and in particular to a blood lipid control solution. Background Art

[0002] Blood lipids are the general term for neutral fats (triglycerides) and lipids (phospholipids, glycolipids, sterols, steroids) in plasma, which are widely present in the human body. They are essential substances for the basic metabolism of living cells. Generally speaking, the main components of blood lipids are triglycerides and cholesterol, among which triglycerides participate in energy metabolism in the human body, while cholesterol is mainly used to synthesize cell plasma membranes, steroid hormones and bile acids.

[0003] Hyperlipidemia, also known as high blood lipids or dyslipidemia, usually refers to elevated triglycerides and / or total cholesterol in plasma, and also includes elevated low-density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol. If blood lipids are too high, blood thickening is likely to occur, and plaques will form on blood vessels, which can block blood vessels in severe cases, causing stroke, coronary heart disease, etc. In addition, high blood lipids may also lead to high blood pressure, gallstones and other diseases. Monitoring blood lipids and adjusting diet or taking medications in a targeted manner can help prevent the occurrence of diseases.

[0004] At present, blood lipid testing is usually carried out with automated instruments, and quality control fluid is required to simulate blood for testing to ensure that the instrument's test results are accurate. The quality control fluid contains cholesterol, which belongs to the alcohol class and requires an organic solvent to dissolve. The commonly used organic solvent is isopropyl alcohol, and heating is required during the preparation process. Long-term inhalation of isopropyl alcohol affects human health and may cause adverse respiratory reactions such as pharyngeal discomfort, coughing, shortness of breath, and may cause allergies. At the same time, isopropyl alcohol is highly flammable and has a fire hazard. Therefore, the use of isopropyl alcohol requires good ventilation, and there are greater requirements for the production workshop and process for the preparation of the quality control fluid, and the protection requirements for operators are also more stringent. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a blood lipid control solution; the blood lipid control solution provided by the present application uses bile acid or deoxycholic acid and its salts and cyclodextrin as solubilizers to form a complex, so that cholesterol is dissolved in a water-soluble matrix, and is directly stirred at room temperature without the participation of organic flammable and harmful reagents. The process is simple, safe and reliable, and the stability is good, and the shelf life can reach 12 months.

[0006] The technical solution provided by the present invention is as follows: A blood lipid control solution comprises a buffer, cholesterol or cholesterol and glycerol, bile acid or deoxycholic acid and salts thereof, and cyclodextrin.

[0007] Preferably, the bile acid or deoxycholic acid and salts thereof include any one or more of bile acid, sodium cholate, deoxycholic acid and sodium deoxycholate.

[0008] Preferably, the cyclodextrin is any one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0009] Preferably, the buffer is one or more of phosphate buffer and Tris buffer.

[0010] Preferably, any one or more of a thickener, a preservative and a stabilizer are also included.

[0011] Preferably, the thickener is one or more of polyethylene oxide, chitosan, gelatin and methyl cellulose.

[0012] Preferably, the preservative is one or more of Proclin series preservatives and sodium azide.

[0013] Preferably, the stabilizer is one or more of a polysaccharide stabilizer, a protein stabilizer, and an amino acid stabilizer.

[0014] Preferably, the polysaccharide stabilizer is one or more of trehalose, sucrose, lactose and dextran.

[0015] Preferably, the following components are included: Buffer 20-100mmol / L; Thickener 1-6g / L; Preservatives 0.5-4g / L; Stabilizer 1-8g / L Cholesterol 0.1-1.5%, or cholesterol 0.1-1.5% and glycerol 0.02-0.25% Cholic acid or deoxycholic acid and its salts 5-16 g / L; Cyclodextrin 8-15 g / L; Water balance.

[0016] The present application provides a blood lipid control liquid, which uses bile acid or deoxycholic acid and its salts and cyclodextrin as solubilizers to form a complex, so that cholesterol is dissolved in a water-soluble matrix, directly stirred at room temperature, without the involvement of organic flammable and harmful reagents, and the process is simple, safe and reliable. The applicant has verified through experiments that the above-mentioned blood lipid control liquid has good stability and a validity period of up to 12 months. Moreover, the present application uses bile acid or deoxycholic acid and its salts and cyclodextrin as solubilizers, which can greatly increase the solubility of cholesterol.

[0017] It is well known in the art that in the detection process of triglycerides, glycerol is generated from triglycerides and then measured. Therefore, glycerol can be added to the quality control solution as a substitute to verify the accuracy of triglyceride detection; at the same time, compared with triglycerides, glycerol also has good solubility, which is conducive to the preparation and preservation of quality control solution. When the blood lipid control solution provided by the present application needs to verify whether the cholesterol test is accurate, only cholesterol can be added without glycerol. By adding cholesterol and glycerol at the same time, it is possible to verify whether the test results of cholesterol and triglycerides are accurate at the same time.

[0018] Preferably, the buffer used in the present application has a pH of 5 to 9. More preferably, the pH is 7 to 8, such as pH 7.5, 7.6 or 7.7.

[0019] The blood lipid control solution provided in this application preferably comprises the following components: Buffer 20-100 mmol / L, more preferably 50-60 mmol / L; Thickener 1-6 g / L, more preferably 3-4 g / L; Preservative 0.5-4 g / L; more preferably 2-3 g / L; Stabilizer 1-8 g / L; more preferably 3-4 g / L; Cholesterol 0.1-1.5%, more preferably 1.1-1.2%; or cholesterol 0.1-1.5% and glycerol 0.02-0.25%, more preferably cholesterol 1.1-1.2% and glycerol 0.2-0.3%; Cholic acid or deoxycholic acid and salts thereof 5-16%, more preferably 14-15%; Cyclodextrin 8-15%, more preferably 11-12%; Water balance. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0021] Example 1-1 The blood lipid control solution includes the following components: 0.78g cholesterol, 0.15g glycerol, 10g sodium cholate, 8g β-cyclodextrin, purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0022] Preparation method: Add glycerol, sodium cholate and β-cyclodextrin to 50g purified water and stir to dissolve, then add cholesterol and stir to dissolve at room temperature (25°C); use purified water to dilute sodium phosphate buffer, polyethylene oxide, Proclin300 and trehalose to the above concentrations to prepare a 200mml mixed solution; mix the two and stir for 2h to obtain a uniform and stable blood lipid control solution.

[0023] Example 1-2 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g sodium cholate, 8g β-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0024] Prepared according to the method of Example 1-1.

[0025] Examples 1-3 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g sodium deoxycholate, 8g β-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0026] Prepared according to the method of Example 1-1.

[0027] Examples 1-4 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g bile acid, 8g β-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0028] Prepared according to the method of Example 1-1.

[0029] Examples 1-5 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g sodium cholate, 8g γ-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0030] Prepared according to the method of Example 1-1.

[0031] Examples 1-6 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g sodium deoxycholate, 8g γ-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0032] Prepared according to the method of Example 1-1.

[0033] Examples 1-7 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g bile acid, 8g γ-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0034] Prepared according to the method of Example 1-1.

[0035] Examples 1-8 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g sodium cholate, 8g α-cyclodextrin, 50g purified water. , 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, 3g / L trehalose.

[0036] Prepared according to the method of Example 1-1.

[0037] Examples 1-9 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g sodium deoxycholate, 8g α-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0038] Prepared according to the method of Example 1-1.

[0039] Examples 1-10 The total cholesterol quality control solution includes the following components: 0.78g cholesterol, 10g bile acid, 8g α-cyclodextrin, 50g purified water, 50mmol / L sodium phosphate buffer, pH 7.6; 3g / L polyethylene oxide, 2g / L Proclin300, and 3g / L trehalose.

[0040] Prepared according to the method of Example 1-1.

[0041] Comparative Example 1-1 The blood lipid control solution includes the following components: 0.78g cholesterol, 0.15g glycerol, 10g sodium cholate, and 58g purified water. Preparation method: Add glycerol and sodium cholate into purified water and stir to dissolve, then add cholesterol and stir to dissolve at room temperature (25°C). The results show that cholesterol cannot be completely dissolved and no clear liquid can be obtained.

[0042] Comparative Example 1-2 The blood lipid control solution includes the following components: 0.78g cholesterol, 0.15g glycerol, 8g β-cyclodextrin, and 60g purified water. Preparation method: Add glycerol and β-cyclodextrin to purified water and stir to dissolve, then add cholesterol and stir to dissolve at room temperature (25°C). The results show that cholesterol cannot be completely dissolved and no clear liquid can be obtained.

[0043] From the comparison between Examples 1-1 to 1-10 and Comparative Examples 1-1 and 1-2, it can be seen that the same gram weight of cholesterol cannot be completely dissolved by sodium cholate alone or cyclodextrin alone, but the combination of bile acid or deoxycholic acid and its salts with cyclodextrin can completely dissolve cholesterol, indicating that the combination of bile acid or deoxycholic acid and its salts with cyclodextrin has the effect of synergistically promoting the dissolution of cholesterol at room temperature.

[0044] Comparative Examples 1-3 The blood lipid control solution includes the following components: cholesterol 0.78g, glycerol 0.15g, isopropanol 51g, Triton X-10013g., phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin300 2g / L, trehalose 3g / L.

[0045] Preparation method: Add cholesterol and glycerol to isopropanol, then add Triton X-100, and stir at room temperature (25°C) for 45 minutes to dissolve; separately mix phosphate buffer, polyethylene oxide, Proclin300 and trehalose, configure into 200 ml mixed solution and make each substance reach the above concentration, mix the two, the solution becomes turbid, and after stirring and dissolving at 50°C for 60 minutes, it becomes clear to obtain blood lipid control solution.

[0046] Configuration and operation comparison: The blood lipid control liquid of comparative examples 1-3 uses an organic solvent, isopropanol, which is a highly flammable liquid and vapor, has severe eye irritation, can cause drowsiness and dizziness, and needs to be heated to dissolve, which has high requirements on the production environment and production process. The blood lipid control liquid of Example 1 uses sodium bile acid and β-cyclodextrin as solubilizers to form a complex, dissolves cholesterol in a water-soluble matrix, directly stirs at room temperature, does not involve organic flammable and harmful reagents, and has a simple, safe and reliable process.

[0047] Example 2-1 The blood lipid control solution includes the following components: cholesterol 1.56g, glycerol 0.31g, sodium cholate 20g, β-cyclodextrin 16g, purified water, phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin300 2g / L, trehalose 3g / L.

[0048] Add glycerol, cholesterol and β-cyclodextrin to 100 g purified water and stir to dissolve, then add cholesterol and stir to dissolve at room temperature (25°C) to prepare the mother solution; Separately, sodium phosphate buffer, polyethylene oxide, Proclin 300 and trehalose were diluted to the above concentrations using purified water as diluent; Different amounts of mother solution were added to three 200 ml dilutions (Level 1: 23 g mother solution; Level 2: 46 g mother solution; Level 3: 69 g mother solution) to obtain blood lipid control solutions for three experimental groups at different concentration levels.

[0049] Example 2-2 The total cholesterol quality control solution includes the following components: cholesterol 1.56g, sodium cholate 20g, β-cyclodextrin 16g, purified water, phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin300 2g / L, trehalose 3g / L.

[0050] Add glycerol, cholesterol and β-cyclodextrin to 100 g purified water and stir to dissolve, then add cholesterol and stir to dissolve at room temperature (25°C) to prepare the mother solution; Separately, sodium phosphate buffer, polyethylene oxide, Proclin 300 and trehalose were diluted to the above concentrations using purified water as diluent; Different amounts of mother solution were added to three 200 ml dilutions (Level 1: add 23 g mother solution; Level 2: add 46 g mother solution; Level 3: add 69 g mother solution) to obtain three total cholesterol quality control solutions for experimental groups with different concentration levels.

[0051] Comparative Example 2 The blood lipid control solution includes the following components: cholesterol 1.56g, glycerol 0.31g, isopropanol 102g, Triton X-10026g, phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin300 2g / L, and trehalose 3g / L.

[0052] Preparation method: Add cholesterol and glycerol to isopropanol, then add Triton X-100, stir at room temperature (25°C) for 45 minutes to dissolve, and use this as the mother solution; Separately, sodium phosphate buffer, polyethylene oxide, Proclin 300 and trehalose were diluted to the above concentrations using purified water as diluent; Different amounts of mother solution were added to three 200 ml dilutions (Level 1: 21.6 g mother solution; Level 2: 43.2 g mother solution; Level 3: 64.8 g mother solution) to obtain three different concentration levels of control group blood lipid control solutions.

[0053] 1. Comparison of accuracy and repeatability The blood lipid control solution of comparative example 2 was used as the control group, and the blood lipid control solution of Example 2-1 and Example 2-2 were used as the experimental group. The blood lipid control solutions of the above three concentration levels were divided into small bottles with a volume of 4 mL each, sealed and placed at 25°C. Randomly take 10 bottles of blood lipid control solutions of different concentration levels. They were all tested using the Roche Cobas c501 biochemical analyzer, and the test kit was the total cholesterol determination kit (enzyme colorimetric method) produced by Roche. After the test, the accuracy of the biochemical test values ​​of each quality control solution (the relative deviation between the mean value of 10 bottles of biochemical tests and the theoretical value) and the repeatability CV were calculated respectively. The test results are shown in Tables 1 and 2: Table 1 Results of accuracy and repeatability test of total cholesterol in the control group and experimental group

[0054] Table 2 Results of accuracy and repeatability test of blood lipid control liquid-triglyceride in control group and experimental group

[0055] As can be seen from Tables 1 and 2, the total cholesterol and triglyceride biochemical test results of the blood lipid control solution in the experimental group and the control group are highly consistent with the theoretical values, and the deviations are very small, but the repeatability of the 10 tests in the experimental group is better than that in the control group, and the CV of the experimental group is within 4%. This shows that the blood lipid control solution provided by this application can achieve the same accuracy test results as the existing conventional quality control solution, and the repeatability is better than the existing blood lipid control solution.

[0056] 2. Stability comparison The blood lipid control solution of comparative example 2 was used as the control group, and the blood lipid control solution of embodiment 2-1 and embodiment 2-2 were used as the experimental group. The blood lipid control solution of the above three concentration levels was divided into small bottles with a volume of 4 mL each, sealed and placed in a constant temperature box at 25°C and 60°C. Every other week, one bottle of blood lipid control solution of different concentration levels was taken from the constant temperature box at room temperature and 60°C, and they were tested using Roche Cobas c501 biochemical analyzer. The test kit was the total cholesterol determination kit (enzyme colorimetric method) produced by Roche. After the test, the deviation of the biochemical analyzer test value of the blood lipid control solution placed at 60°C and the blood lipid control solution placed at room temperature was calculated for each week, and the test was continued for 7 weeks. The test results are shown in Tables 3 and 4 below: Table 3 Results of blood lipid control-total cholesterol test in the control group and experimental group

[0057] Table 4 Blood lipid control-triglyceride test results of the control group and experimental group

[0058] From the results in Tables 3 and 4, it can be seen that the blood lipid control solution in the experimental group has good storage stability under accelerated aging at 60°C. After being placed for 7 weeks, the deviations of total cholesterol and triglycerides were within 5% compared with the quality control solution under room temperature conditions.

[0059] After the blood lipid control solution in the control group was placed at 60°C for 7 weeks, the maximum deviation of total cholesterol reached -16.10% compared with the blood lipid control solution at room temperature; the triglyceride deviation even reached -21.43%. It can be seen that the blood lipid control solution in the experimental group has excellent stability and a long storage period compared with the control group.

[0060] According to the basic principle and method of determining the shelf life through accelerated aging experiments, i.e., the simplified accelerated aging experimental scheme established by the Arrhenius model, the shelf life is one year, i.e., 52 weeks. If the temperature is accelerated at 60°C, the time required for acceleration is 6.5 weeks. Therefore, the above experimental results of the present application can fully prove that the blood lipid control liquid provided by the present application has good stability and can remain stable within 12 months.

[0061] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A blood lipid control solution, characterized in that: include: Buffer, cholesterol or cholesterol and glycerol, cholic acid or deoxycholic acid and their salts, and cyclodextrins.

2. The blood lipid control solution according to claim 1, characterized in that The bile acid or deoxycholic acid and salts thereof include any one or more of bile acid, sodium cholate, deoxycholic acid and sodium deoxycholate.

3. The blood lipid control solution according to claim 1, characterized in that: The cyclodextrin is any one or more of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin.

4. The blood lipid control solution according to claim 1, characterized in that The buffer is one or more of phosphate buffer and Tris buffer.

5. The blood lipid control solution according to any one of claims 1 to 4, characterized in that: It also includes any one or more of a thickener, a preservative, and a stabilizer.

6. The blood lipid control solution according to claim 5, characterized in that: The thickener is one or more of polyethylene oxide, chitosan, gelatin and methyl cellulose.

7. The blood lipid control solution according to claim 5, characterized in that: The preservative is one or more of Proclin series preservatives and sodium azide.

8. The blood lipid control solution according to claim 5, characterized in that: The stabilizer is one or more of a polysaccharide stabilizer, a protein stabilizer, and an amino acid stabilizer.

9. The blood lipid control solution according to claim 8, characterized in that: The polysaccharide stabilizer is one or more of trehalose, sucrose, lactose and dextran.

10. The blood lipid control solution according to claim 5, characterized in that: Includes the following components: Buffer 20 -100mmol / L; Thickener 1-6g / L; Preservatives 0.5-4g / L; Stabilizer 1-8g / L; Cholesterol 0.1-1.5%; or cholesterol 0.1-1.5% and glycerol 0.02-0.25%; Cholic acid or deoxycholic acid and its salts 5-16%; Cyclodextrin 8-15%; Water balance.

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