A blood lipid control solution

Abstract

This application discloses a lipid control solution, comprising a buffer solution, cholesterol or cholesterol and glycerol, cholic acid or deoxycholic acid and its salts, and cyclodextrin. The lipid control solution provided in this application uses cholic acid or deoxycholic acid and its salts and cyclodextrin as solubilizers to form a complex, allowing cholesterol to dissolve in a water-soluble matrix. It is prepared by direct stirring at room temperature, without the involvement of any organic flammable or harmful reagents. The process is simple, safe, reliable, and exhibits good stability with a shelf life of up to 12 months.

Description

Technical Field

[0001] This application relates to the field of diagnostic reagent technology, and in particular to a blood lipid control solution. Background Technology

[0002] Blood lipids are a collective term for neutral fats (triglycerides) and lipids (phospholipids, glycolipids, sterols, and steroids) in blood plasma, and 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. Triglycerides participate in energy metabolism in the human body, while cholesterol is mainly used to synthesize cell membranes, steroid hormones, and bile acids.

[0003] Hyperlipidemia, also known as high blood lipids or dyslipidemia, typically refers to elevated levels of triglycerides and / or total cholesterol in the blood plasma. It also includes elevated low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol. High blood lipid levels can lead to thickened blood, which can deposit on blood vessels to form atherosclerotic plaques. In severe cases, this can block blood vessels, causing stroke, coronary heart disease, and other complications. Furthermore, high blood lipids can also lead to hypertension, gallstones, and other diseases. Monitoring blood lipids and making targeted adjustments to diet or taking medication can help prevent these diseases.

[0004] Currently, blood lipid testing is typically performed using automated instruments that require a control solution to simulate blood for testing, ensuring accurate results. This control solution contains cholesterol, an alcohol that requires an organic solvent to dissolve. Isopropanol is a commonly used organic solvent, and its preparation requires heating. Long-term inhalation of isopropanol can negatively impact health, potentially causing respiratory adverse reactions such as throat discomfort, coughing, and shortness of breath, and may also lead to allergies. Furthermore, isopropanol is highly flammable and poses a fire hazard. Therefore, the use of isopropanol requires good ventilation, placing high demands on the production workshop and processes for preparing the control solution, and imposing stricter protective measures on operators. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the purpose of this invention is to provide a blood lipid control solution. The blood lipid control solution provided in this application uses cholic acid or deoxycholic acid and its salts and cyclodextrin as solubilizers to form a complex, which dissolves cholesterol in a water-soluble matrix. It is directly stirred at room temperature, without the participation of organic flammable and harmful reagents. The process is simple, safe and reliable, with good stability and a shelf life of up to 12 months.

[0006] The technical solution provided by this invention is as follows:

[0007] A lipid control solution, a buffer solution, cholesterol or cholesterol and glycerol, bile acids or deoxycholic acid and their salts, and cyclodextrin.

[0008] Preferably, the cholic acid or deoxycholic acid and its salts include any one or more of cholic acid, sodium cholate, deoxycholic acid, and sodium deoxycholate.

[0009] Preferably, the cyclodextrin is any one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0010] Preferably, the buffer solution is one or more of phosphate buffer and Tris buffer.

[0011] Preferably, it also includes any one or more of thickeners, preservatives, and stabilizers.

[0012] Preferably, the thickener is one or more of polyethylene oxide, chitosan, gelatin, and methylcellulose.

[0013] Preferably, the preservative is one or more of the Proclin series preservatives and sodium azide.

[0014] Preferably, the stabilizer is one or more of polysaccharide stabilizers, protein stabilizers, and amino acid stabilizers.

[0015] Preferably, the polysaccharide stabilizer is one or more of trehalose, sucrose, lactose, and dextran.

[0016] Preferably, it comprises the following components:

[0017] Buffer solution 20-100 mmol / L;

[0018] Thickener 1-6g / L;

[0019] Preservative 0.5-4g / L;

[0020] stabilizer 1-8g / L

[0021] Cholesterol 0.1-1.5%, or cholesterol 0.1-1.5% and glycerol 0.02-0.25%.

[0022] Cholic acid or deoxycholic acid and its salts 5-16 g / L;

[0023] Cyclodextrin 8-15 g / L;

[0024] Water balance.

[0025] This application provides a lipid control solution that uses cholic acid or deoxycholic acid and its salts, along with cyclodextrin, as a solubilizer to form a complex that dissolves cholesterol in a water-soluble matrix. The process involves direct stirring at room temperature, without the use of any flammable or harmful organic reagents, making it simple, safe, and reliable. Experiments conducted by the applicant have verified that the above lipid control solution exhibits good stability and a shelf life of up to 12 months. Furthermore, the use of cholic acid or deoxycholic acid and its salts, along with cyclodextrin, as a solubilizer significantly increases the solubility of cholesterol.

[0026] As is known in the art, triglyceride detection also involves converting triglycerides into glycerol before measurement. Therefore, glycerol can be added to the quality control solution as a substitute to verify the accuracy of triglyceride detection. Furthermore, compared to triglycerides, glycerol has good solubility, which facilitates the preparation and storage of the quality control solution. The lipid control solution provided in this application allows for the addition of only cholesterol without glycerol when verifying the accuracy of cholesterol detection. Adding both cholesterol and glycerol simultaneously allows for the simultaneous verification of the accuracy of both cholesterol and triglyceride detection results.

[0027] Preferably, the buffer solution used in this application has a pH of 5-9. More preferably, the pH is 7-8, such as pH 7.5, 7.6 or 7.7.

[0028] The preferred lipid control solution provided in this application comprises the following components:

[0029] The buffer solution is 20-100 mmol / L, more preferably 50-60 mmol / L;

[0030] Thickener 1-6 g / L, more preferably 3-4 g / L;

[0031] Preservative: 0.5-4 g / L; more preferably 2-3 g / L;

[0032] Stabilizer 1-8 g / L; more preferably 3-4 g / L;

[0033] 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%;

[0034] 5-16% cholic acid or deoxycholic acid and its salts, more preferably 14-15%;

[0035] Cyclodextrin 8-15%, more preferably 11-12%;

[0036] Water balance. Detailed Implementation

[0037] 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 a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0038] Example 1-1

[0039] The lipid control solution contains the following components: cholesterol 0.78g, glycerol 0.15g, sodium cholate 10g, β-cyclodextrin 8g, purified water, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0040] Preparation method: Glycerol, sodium cholate and β-cyclodextrin were added to 50g of purified water and stirred to dissolve. Then cholesterol was added and stirred to dissolve at room temperature (25℃). Sodium phosphate buffer, polyethylene oxide, Proclin 300 and trehalose were diluted to the above concentration with purified water to prepare a 200ml mixed solution. The two were mixed and stirred for 2 hours to obtain a homogeneous and stable blood lipid control solution.

[0041] Examples 1-2

[0042] The total cholesterol control solution contains the following components: cholesterol 0.78g, sodium cholate 10g, β-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0043] Prepared according to the method of Example 1-1.

[0044] Examples 1-3

[0045] The total cholesterol control solution contains the following components: cholesterol 0.78g, sodium deoxycholate 10g, β-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0046] Prepared according to the method of Example 1-1.

[0047] Examples 1-4

[0048] The total cholesterol control solution comprises the following components: cholesterol 0.78g, bile acid 10g, β-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin 300 2g / L, and trehalose 3g / L.

[0049] Prepared according to the method of Example 1-1.

[0050] Examples 1-5

[0051] The total cholesterol control solution contains the following components: cholesterol 0.78g, sodium cholate 10g, γ-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0052] Prepared according to the method of Example 1-1.

[0053] Examples 1-6

[0054] The total cholesterol control solution contains the following components: cholesterol 0.78g, sodium deoxycholate 10g, γ-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0055] Prepared according to the method of Example 1-1.

[0056] Examples 1-7

[0057] The total cholesterol control solution contains the following components: cholesterol 0.78g, bile acid 10g, γ-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0058] Prepared according to the method of Example 1-1.

[0059] Examples 1-8

[0060] The total cholesterol control solution comprises the following components: cholesterol 0.78g, sodium cholate 10g, α-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin 300 2g / L, and trehalose 3g / L.

[0061] Prepared according to the method of Example 1-1.

[0062] Examples 1-9

[0063] The total cholesterol control solution contains the following components: cholesterol 0.78g, sodium deoxycholate 10g, α-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0064] Prepared according to the method of Example 1-1.

[0065] Examples 1-10

[0066] The total cholesterol control solution contains the following components: cholesterol 0.78g, bile acid 10g, α-cyclodextrin 8g, purified water 50g, sodium phosphate buffer 50mmol / L, pH 7.6; polyethylene oxide 3g / L, Proclin 300 2g / L, trehalose 3g / L.

[0067] Prepared according to the method of Example 1-1.

[0068] Comparative Example 1-1

[0069] The lipid control solution comprises the following components: 0.78g cholesterol, 0.15g glycerol, 10g sodium cholate, and 58g purified water. Preparation method: Glycerol and sodium cholate were dissolved in purified water by stirring. Then, cholesterol was added and stirred at room temperature (25℃) to dissolve. The results showed that the cholesterol could not be completely dissolved, and a clear, transparent liquid was not obtained.

[0070] Comparative Examples 1-2

[0071] The lipid control solution comprises the following components: 0.78g cholesterol, 0.15g glycerol, 8g β-cyclodextrin, and 60g purified water. Preparation method: Glycerol and β-cyclodextrin were added to purified water and stirred to dissolve. Then, cholesterol was added and stirred at room temperature (25℃) to dissolve. The results showed that cholesterol could not be completely dissolved, and a clear, transparent liquid could not be obtained.

[0072] The comparison between Examples 1-1 to 1-10 and Comparative Examples 1-1 and 1-2 shows that, for the same weight of cholesterol, neither sodium cholate nor cyclodextrin alone can completely dissolve it. However, the combination of cholic acid or deoxycholic acid and its salts with cyclodextrin can completely dissolve cholesterol, indicating that the combination of cholic acid or deoxycholic acid and its salts with cyclodextrin has a synergistic effect in promoting the dissolution of cholesterol at room temperature.

[0073] Comparative Examples 1-3

[0074] The lipid control solution contains the following components: cholesterol 0.78g, glycerol 0.15g, isopropanol 51g, Triton X-100 13g, phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin 300 2g / L, and trehalose 3g / L.

[0075] Preparation method: Cholesterol and glycerol are added to isopropanol, and then Triton X-100 is added. The mixture is stirred at room temperature (25℃) for 45 minutes to dissolve. Separately, phosphate buffer, polyethylene oxide, Proclin 300 and trehalose are mixed to prepare a 200ml mixed solution and each substance is brought to the above concentration. The two are mixed and the solution becomes turbid. After stirring and dissolving at 50℃ for 60 minutes, the solution becomes clear, and the blood lipid control solution is obtained.

[0076] Comparison of preparation and operation: The lipid control solutions in Comparative Examples 1-3 used the organic solvent isopropanol, which is a highly flammable liquid and vapor, and also causes severe eye irritation, drowsiness, and dizziness. It also requires heating to dissolve, placing high demands on the production environment and process. The lipid control solution in Example 1 uses sodium chitosan and β-cyclodextrin as solubilizers to form a complex, allowing cholesterol to dissolve in a water-soluble matrix. Direct stirring at room temperature is used, without the involvement of any flammable or harmful organic reagents, making the process simple, safe, and reliable.

[0077] Example 2-1

[0078] The lipid control solution contains 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, Proclin 300 2g / L, and trehalose 3g / L.

[0079] Glycerin, cholesterol, and β-cyclodextrin were added to 100g of purified water and stirred to dissolve. Then cholesterol was added and stirred to dissolve at room temperature (25℃) to obtain the mother liquor.

[0080] Separately, use purified water to dilute sodium phosphate buffer, polyethylene oxide, Proclin 300 and trehalose to the above concentrations as diluents;

[0081] Different amounts of stock solution were added to three 200ml diluents (Level 1: 23g stock solution added; Level 2: 46g stock solution added; Level 3: 69g stock solution added) to obtain three different concentration levels of blood lipid control solutions for experimental groups.

[0082] Example 2-2

[0083] The total cholesterol control solution contains 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, Proclin 300 2g / L, and trehalose 3g / L.

[0084] Glycerin, cholesterol, and β-cyclodextrin were added to 100g of purified water and stirred to dissolve. Then cholesterol was added and stirred to dissolve at room temperature (25℃) to obtain the mother liquor.

[0085] Separately, use purified water to dilute sodium phosphate buffer, polyethylene oxide, Proclin 300 and trehalose to the above concentrations as diluents;

[0086] Different amounts of stock solution were added to three 200ml dilution solutions (Level 1: 23 g stock solution; Level 2: 46 g stock solution; Level 3: 69 g stock solution) to obtain three different concentration levels of total cholesterol control solutions for the experimental groups.

[0087] Comparative Example 2

[0088] The lipid control solution contains the following components: cholesterol 1.56g, glycerol 0.31g, isopropanol 102g, Triton X-100 26g, phosphate buffer 50mmol / L, pH 7.6, polyethylene oxide 3g / L, Proclin 300 2g / L, and trehalose 3g / L.

[0089] Preparation method: Add cholesterol and glycerol to isopropanol, then add Triton X-100, stir at room temperature (25℃) for 45 minutes to dissolve, and use as the mother liquor;

[0090] Separately, use purified water to dilute sodium phosphate buffer, polyethylene oxide, Proclin 300 and trehalose to the above concentrations as diluents;

[0091] Different amounts of stock solution were added to three 200ml diluents (Level 1: 21.6g stock solution added; Level 2: 43.2g stock solution added; Level 3: 64.8g stock solution added) to obtain three control groups with different concentration levels of blood lipid control solution.

[0092] I. Accuracy and Repeatability Comparison

[0093] The lipid control solution of Comparative Example 2 served as the control group, while the lipid control solutions of Example 2-1 and Example 2-2 served as the experimental groups. The lipid control solutions at the three concentration levels were dispensed into 4 mL vials, sealed, and stored at 25°C. Ten vials of lipid control solutions at each concentration level were randomly selected. All samples were tested using a Roche Cobas c501 biochemical analyzer, using a Roche total cholesterol assay kit (enzyme colorimetric method). After testing, the accuracy (relative deviation of the mean value of 10 biochemical tests from the theoretical value) and repeatability CV of each control solution were calculated. The test results are shown in Tables 1 and 2.

[0094] Table 1. Repeatability test results of blood lipid control solution-total cholesterol accuracy in the control and experimental groups.

[0095]

[0096] Table 2. Results of repeatability test of blood lipid control solution-triglyceride ratio in control and experimental groups.

[0097]

[0098] As shown in Tables 1 and 2, the biochemical test results of total cholesterol and triglycerides in the lipid control solutions of both the experimental and control groups showed high agreement with the theoretical values, with very small deviations. However, the repeatability of the 10 tests in the experimental group was better than that in the control group, with the CV of the experimental group all within 4%. This indicates that the lipid control solution provided in this application can achieve test results with the same accuracy as existing conventional quality control solutions, and its repeatability is superior to existing lipid control solutions.

[0099] II. Stability Comparison

[0100] The lipid control solution of Comparative Example 2 served as the control group, while the lipid control solutions of Example 2-1 and Example 2-2 served as the experimental groups. The lipid control solutions at the three concentration levels were dispensed into 4 mL vials, sealed, and placed in incubators at 25°C and 60°C. Every week, one vial of lipid control solution at each concentration level was taken from both the incubators at 25°C and 60°C, and tested using a Roche Cobas c501 biochemical analyzer. The test kit was a Roche total cholesterol assay kit (enzyme colorimetric method). After testing, the deviation of the biochemical analyzer readings for the lipid control solutions placed at 60°C and those placed at room temperature was calculated for each week, for a total of 7 weeks. The test results are shown in Tables 3 and 4 below:

[0101] Table 3. Results of blood lipid control solution-total cholesterol test in control group and experimental group

[0102]

[0103] Table 4. Results of blood lipid control solution-triglyceride test in control group and experimental group

[0104]

[0105] The results in Tables 3 and 4 show that the lipid control solution in the experimental group under accelerated aging at 60℃ has good storage stability. After 7 weeks of storage, the deviations in total cholesterol and triglycerides were both within 5% compared with the control solution under normal temperature conditions.

[0106] In contrast, the control group's lipid control solution, after being stored at 60℃ for 7 weeks, showed the largest deviation in total cholesterol (-16.10%) and triglycerides (-21.43%) compared to the control group's solution at room temperature. This demonstrates that the experimental group's lipid control solution exhibits superior stability and a longer shelf life compared to the control group.

[0107] Based on the fundamental principles and methods of determining shelf life using accelerated aging experiments, specifically the simplified accelerated aging experimental protocol established using the Arrhenius model, a shelf life of one year is equivalent to 52 weeks. Acceleration is performed at 60°C, requiring 6.5 weeks of acceleration. Therefore, the experimental results of this application sufficiently demonstrate the good stability of the lipid control solution provided, maintaining stability within 12 months.

[0108] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A blood lipid control solution, characterized in that, Includes the following components: Phosphate buffer 20-100 mmol / L; Thickener 1-6 g / L; the thickener is polyethylene oxide; Preservative 0.5-4 g / L; the preservative is Proclin 300; Stabilizer 1-8 g / L; the stabilizer is trehalose; Cholesterol 0.1-1.5%; or cholesterol 0.1-1.5% and glycerol 0.02-0.25%; Sodium cholate 5-16%; β-Cyclodextrin 8-15%; Water balance.

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