Hyaluronic acid assay lyophilized calibrators and methods of making same

Abstract

The embodiment of the application belongs to the technical field of detection, and relates to a hyaluronic acid determination freeze-dried calibrant and a preparation method thereof. The hyaluronic acid determination freeze-dried calibrant comprises hyaluronic acid, a phosphate buffer, potassium oxalate, bovine serum albumin, mannitol, sucrose, polyethylene glycol, disodium ethylenediaminetetraacetate and a preservative. The application provides the hyaluronic acid determination freeze-dried calibrant with good quality.

Description

Technical Field

[0001] This application relates to the field of detection technology, and in particular to lyophilized calibrators for the determination of hyaluronic acid and their preparation methods. Background Technology

[0002] Hyaluronic acid (HA), also known as hyaluronic acid or glucuronic acid, is an acidic mucopolysaccharide with a unique molecular structure and physicochemical properties. It is mainly composed of D-glucuronic acid and N-acetylglucosamine, with a molecular weight of 40-80 million, and possesses various physiological functions. In animals, HA can stabilize the physiological structure of the collagen network, regulate the motility of cells in the viscoelastic matrix, and thereby protect and lubricate cells. When hepatocytes are damaged, the expression level of HA in hepatic fibroblasts increases, while the HA clearance capacity of hepatic endothelial cells is inhibited, ultimately leading to high HA expression in human serum or plasma.

[0003] Currently, methods for determining hyaluronic acid include latex immunoturbidimetry and chemiluminescence immunoassay.

[0004] The former's reaction principle is that the hyaluronic acid in the sample combines with the hyaluronic acid antibody-sensitized latex in the reagent to form an insoluble immune complex. Turbidity is formed in a specific buffer environment. The turbidity is proportional to the content of the analyte within the detection range. By comparing it with the calibrator operated under the same conditions, the content of hyaluronic acid in the sample can be calculated.

[0005] The latter reaction principle involves adding the sample to be tested into a microplate coated with hyaluronic acid, then adding a certain amount of hyaluronic acid-binding protein (HABP). The hyaluronic acid coated on the microplate and the hyaluronic acid to be tested in the sample compete to bind with HABP, forming a solid-phase HA-HABP complex. Then, an enzyme-labeled anti-HABP antibody is added to react and form a solid-phase HA-HABP-anti-HABP antibody-enzyme complex. Finally, a substrate is added to measure the luminescence or absorbance value, and the sample concentration is determined by a calibration curve.

[0006] Whether using latex immunoturbidimetry or chemiluminescence immunoassay to detect hyaluronic acid content, a matching hyaluronic acid calibrator is required for multi-point calibration. A standard curve is obtained by fitting logit / log or spline to calculate the concentration of hyaluronic acid in the sample. Therefore, there is an urgent need to find lyophilized calibrators with good appearance, stability, and solubility, along with their manufacturing process. Summary of the Invention

[0007] The purpose of this application is to provide a lyophilized calibrator for hyaluronic acid determination and its preparation method. This application provides a lyophilized calibrator for hyaluronic acid determination of relatively high quality.

[0008] To address the aforementioned technical problems, this application provides a lyophilized calibrator for hyaluronic acid determination, employing the following technical solution:

[0009] A lyophilized calibrator for hyaluronic acid determination comprises: hyaluronic acid, phosphate buffer, potassium oxalate, bovine serum albumin, mannitol, sucrose, polyethylene glycol, disodium EDTA, and preservatives.

[0010] Furthermore, the potassium oxalate concentration is 2 mmol / L, bovine serum albumin is 10 g / L, mannitol is 15 g / L, sucrose is 50 g / L, polyethylene glycol is 0.48 g / L, disodium EDTA is 1 mmol / L, and the preservative concentration is 0.2 g / L.

[0011] Furthermore, the preservatives include sulfadiazine and dimethoprim, wherein the weight ratio of sulfadiazine to dimethoprim is 25:1.

[0012] Furthermore, the phosphate includes disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0013] To address the aforementioned technical problems, this application also provides a method for preparing a lyophilized calibrator for hyaluronic acid determination, employing the following technical solution:

[0014] A method for preparing a lyophilized calibrator for hyaluronic acid determination, characterized by comprising the following steps:

[0015] Take pure hyaluronic acid and dilute it with phosphate buffer to obtain a diluted solution;

[0016] Add potassium oxalate solution and bovine serum albumin to the diluent;

[0017] Add sucrose, polyethylene glycol, mannitol, disodium EDTA and preservatives, mix well and let stand to obtain calibrator material;

[0018] The calibrator material is dispensed into multiple lyophilized vials, and the lyophilized vials are placed in a vacuum freeze dryer for pre-freezing to obtain the initial lyophilized product;

[0019] The initial lyophilized product is subjected to vacuum freeze-drying to obtain an intermediate lyophilized product;

[0020] The intermediate freeze-dried product is subjected to a desorption and drying operation to obtain the target freeze-dried product;

[0021] Inert gas is introduced, the lyophilized sample is removed, and the lyophilized hyaluronic acid assay calibrator in lyophilized state is obtained.

[0022] Furthermore, the concentration of the phosphate buffer solution is 10 mM to 50 mM;

[0023] The concentration of hyaluronic acid in the diluent is 1000 ng / mL to 3000 ng / mL.

[0024] Furthermore, the step of pre-freezing the lyophilized bottle in a vacuum freeze dryer to obtain the initial lyophilized product includes:

[0025] The freeze-drying bottle was placed in a vacuum freeze dryer, and the temperature was set to -50°C and the atmospheric pressure to 30Pa.

[0026] Maintain the temperature for 4 hours, then gradually increase the temperature of the frozen environment by 10°C / hour until the temperature of the frozen environment reaches -30°C to obtain the initial freeze-dried product.

[0027] Furthermore, the step of vacuum freeze-drying the initial lyophilized product includes:

[0028] The initial freeze-dried product was placed in an environment with an atmospheric pressure of 30 Pa, and the freezing temperature was raised from -30°C to -15°C and maintained for 12 hours for sublimation drying.

[0029] Furthermore, the step of performing a desorption and drying operation on the intermediate lyophilized product to obtain the target lyophilized product includes:

[0030] The intermediate freeze-dried product was placed in an environment with an atmospheric pressure of 15 Pa, and the freezing temperature was raised from -15°C to 25°C. The product was kept at this temperature for 3 hours to desorb and dry, thereby obtaining the target freeze-dried product.

[0031] Furthermore, the inert gas is nitrogen.

[0032] Compared with the prior art, the embodiments of this application have the following main advantages:

[0033] This application provides a matching calibrator for the in vitro quantitative detection of hyaluronic acid (HA) content in human serum or plasma samples using a fully automated biochemical analyzer. The lyophilized calibrator manufacturing process ensures that the hyaluronic acid calibrator does not shrink after lyophilization and maintains its original shape; it does not stick to the bottle or stopper; it has good toughness and is not easily broken, resisting shock during transportation; its activity (i.e., concentration) remains essentially unchanged before and after lyophilization, within ±5%; the uniformity within and between bottles is within ±8%; the relative deviations in long-term stability, opening stability, and transportation stability are within ±15%; after reconstitution, it completely dissolves within 30 minutes of standing, resulting in a clear solution without lumps or foam; it extends shelf life; and as a high-value calibrator, its dilution process is more convenient. Attached Figure Description

[0034] To more clearly illustrate the solutions in this application, the accompanying drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a flowchart of an embodiment of the method for preparing lyophilized calibrators for hyaluronic acid determination according to this application;

[0036] Figure 2 A photograph of a lyophilized calibrator for hyaluronic acid determination prepared using an existing lyophilization formulation.

[0037] Figure 3 Another physical image of a lyophilized calibrator for hyaluronic acid determination prepared for an existing lyophilization formulation;

[0038] Figure 4 This is a photograph of the lyophilized calibrator for hyaluronic acid determination according to this application. Detailed Implementation

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application, are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0040] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0041] The following embodiments are provided to facilitate a better understanding of this application, but do not limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0042] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0043] I. Lyophilized Calibrators for Hyaluronic Acid Determination

[0044] The lyophilized calibrator for hyaluronic acid determination includes: hyaluronic acid, phosphate buffer, potassium oxalate, bovine serum albumin, mannitol, sucrose, polyethylene glycol, disodium EDTA, and preservatives.

[0045] The concentrations of potassium oxalate (2 mmol / L), bovine serum albumin (10 g / L), mannitol (15 g / L), sucrose (50 g / L), polyethylene glycol (0.48 g / L), disodium EDTA (1 mmol / L), and preservatives (0.2 g / L) are as follows:

[0046] As an option in this application, the preservative includes sulfadiazine and dimethoprim, wherein the weight ratio of sulfadiazine to dimethoprim is 25:1;

[0047] Optionally, the phosphate may include disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0048] The calibrators obtained in this application are in a lyophilized state.

[0049] In the lyophilized calibrators, apart from hyaluronic acid, the other substances are the matrix, and the specific matrix formulation is shown in Table 1.

[0050] Table 1. Matrix formulations of lyophilized calibrators

[0051]

[0052]

[0053] II. Preparation Method of Lyophilized Calibrator for Hyaluronic Acid Determination

[0054] Continue to refer to Figure 1 The flowchart illustrates an embodiment of a method for preparing a lyophilized calibrator for hyaluronic acid determination according to this application. The method for preparing the lyophilized calibrator for hyaluronic acid determination includes the following steps:

[0055] S1: Take pure hyaluronic acid and dilute it with phosphate buffer to obtain a diluted solution;

[0056] S2: Add potassium oxalate solution and bovine serum albumin to the diluent;

[0057] S3: Add sucrose, polyethylene glycol, mannitol, disodium EDTA and preservatives, mix well and let stand to obtain calibrator material.

[0058] S4: Dispense the calibrator material into multiple freeze-drying bottles, place the freeze-drying bottles in a vacuum freeze dryer for pre-freezing, and obtain the initial freeze-dried product;

[0059] S5: The initial freeze-dried product is subjected to vacuum freeze-drying to obtain an intermediate freeze-dried product;

[0060] S6: Perform a desorption and drying operation on the intermediate freeze-dried product to obtain the target freeze-dried product;

[0061] S7: Fill with inert gas, remove the lyophilized sample, and obtain the lyophilized hyaluronic acid determination calibrator in lyophilized state.

[0062] The concentration of the phosphate buffer solution is 10 mM to 50 mM; the concentration of hyaluronic acid in the diluent is 1000 ng / mL to 3000 ng / mL; and the inert gas is nitrogen.

[0063] In this embodiment, the concentration of the phosphate buffer is 10mM to 50mM, and can be selected from 10mM to 35mM or 35mM to 50mM; for example, 10mM, 20mM, 35mM, 50mM, etc.; the concentration of hyaluronic acid in the diluent is 1000ng / mL to 3000ng / mL, and can be selected from 1000ng / mL to 2000ng / mL or 2000ng / mL to 3000ng / mL; for example, 1000ng / mL, 2000ng / mL, 2500ng / mL, 3000ng / mL, etc.

[0064] Preferably, the concentration of hyaluronic acid in the diluent is 2000 ng / mL;

[0065] The hyaluronic acid prepared in this application has a concentration of 2000 ng / mL, is dispensed into 500 μL vials, lyophilized, and then reconstituted with 1 mL of pure water before further studies. Therefore, the target value for subsequent studies is 1000 ng / mL.

[0066] Furthermore, the step of pre-freezing the lyophilized bottle in a vacuum freeze dryer to obtain the initial lyophilized product includes:

[0067] The freeze-drying bottle was placed in a vacuum freeze dryer, and the temperature was set to -50°C and the atmospheric pressure to 30Pa.

[0068] Maintain the temperature for 4 hours, then gradually increase the temperature of the frozen environment by 10°C / hour until the temperature of the frozen environment reaches -30°C to obtain the initial freeze-dried product.

[0069] Furthermore, the step of vacuum freeze-drying the initial lyophilized product includes:

[0070] The initial freeze-dried product was placed in an environment with an atmospheric pressure of 30 Pa, and the freezing temperature was raised from -30°C to -15°C and maintained for 12 hours for sublimation drying.

[0071] In addition, the step of performing a desorption drying operation on the intermediate lyophilized product to obtain the target lyophilized product includes:

[0072] The intermediate freeze-dried product was placed in an environment with an atmospheric pressure of 15 Pa, and the freezing temperature was raised from -15°C to 25°C. The product was kept at this temperature for 3 hours to desorb and dry, thereby obtaining the target freeze-dried product.

[0073] The specific preparation process is as follows:

[0074] (1) Take pure hyaluronic acid, add phosphate buffer (the concentration of phosphate buffer is 10mM to 50mM) to dilute, shake to dissolve fully, and obtain a diluted solution. The concentration of hyaluronic acid calibrator in the diluted solution is not less than 2000ng / mL, specifically 2000ng / mL to 5000ng / mL.

[0075] (2) Add potassium oxalate solution and bovine serum albumin to the above diluent and mix thoroughly.

[0076] (3) Add sucrose, polyethylene glycol, mannitol, disodium EDTA and preservative, stir thoroughly and mix well, and store at 2-8℃ to obtain calibrator material (i.e. freeze-dried material);

[0077] (4) After stirring and mixing the above calibrator material (i.e., lyophilized material), it is evenly packaged into several brown lyophilized bottles, each with a net content of 500 μL; it is placed in a vacuum freeze dryer for pre-freezing, that is, the above lyophilized material is placed in a freezing environment at a temperature of -50℃ and an atmospheric pressure of 30 Pa for 4 hours, and then the freezing environment is gradually heated at a rate of 10℃ / hour until the temperature of the freezing environment is -30℃, thus obtaining the initial lyophilized product.

[0078] (5) The initial freeze-dried product was subjected to vacuum freeze-drying and placed in an environment with an atmospheric pressure of 30 Pa. The freezing environment temperature was raised from -30℃ to -15℃ and maintained for 12 hours for sublimation drying to obtain the intermediate freeze-dried product.

[0079] (6) The above intermediate freeze-dried product is then placed in an environment with an atmospheric pressure of 15 Pa, and the freezing environment temperature is raised from -15℃ to 25℃ and maintained for 3 hours for desorption and drying to obtain the target freeze-dried product.

[0080] (7) After the freeze-drying is complete, inert gas (nitrogen) is introduced, the freeze-dried sample is taken out, and the freeze-dried hyaluronic acid determination calibrator is obtained (i.e. the freeze-dried product of the hyaluronic acid determination calibrator).

[0081] The beneficial effects of this application are:

[0082] This application provides a matching calibrator for the in vitro quantitative detection of hyaluronic acid (HA) content in human serum or plasma samples using a fully automated biochemical analyzer. The lyophilized calibrator manufacturing process ensures that the hyaluronic acid calibrator does not shrink after lyophilization and maintains its original shape; it does not stick to the bottle or stopper; it has good toughness and is not easily broken, resisting shock during transportation; its activity (i.e., concentration) remains essentially unchanged before and after lyophilization, within ±5%; the uniformity within and between bottles is within ±8%; the relative deviations in long-term stability, opening stability, and transportation stability are within ±15%; after reconstitution, it completely dissolves within 30 minutes of standing, resulting in a clear solution without lumps or foam; it extends shelf life; and as a high-value calibrator, its dilution process is more convenient.

[0083] This application also provides lyophilization performance testing of the above-mentioned lyophilized calibrators for hyaluronic acid determination.

[0084] (1) Appearance and shape: After freeze-drying, it should be stored at 2-8℃ and should not shrink after freeze-drying and should be able to maintain the shape before freeze-drying; the freeze-dried powder should not stick to the bottle body and bottle cap; it should have good toughness and not be easily broken, etc.

[0085] (2) Resolubility: Add 1mL of pure water to resolubilize, let stand at room temperature for 30 minutes (no shaking or stirring required), the lyophilized product can be completely dissolved, the solution is clear, and there are no lumpy solid residues or foam.

[0086] (3) Activity and stability: The activity of HA calibrators remains essentially unchanged before and after lyophilization, with the difference in activity compared to before lyophilization within ±5%. The relative deviation of the test results after 12 months at 4℃, 7 days at 37℃, and 24 hours after opening / reconstitution at 4℃ should be within ±8%. Under repeatability conditions, the lyophilized calibrators should be tested using the same batch of kits, repeated 10 times, and the average of the 10 test results should be calculated using the formula. Standard deviation (SD) and coefficient of variation (CV). The calculation formulas are as follows:

[0087]

[0088] In the formula: SD represents the average of 10 measurements; i represents 1, 2, 3, ..., n; n represents the number of tests.

[0089] (4) Uniformity within and between bottles: Randomly select 15 calibrators and number them 1 to 15. Measure each calibrator 3 times in the following order: Measurement order: 1, 3, 5, 7, 9, 11, 13, 15, 2, 4, 6, 8, 10, 12, 14, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 12, 14, 1, 3, 5, 7, 9, 11, 13, 15. Record the measurement results and calculate F, Sbb, Sr, CV (within and between bottles) according to the following formulas. The results should meet the following requirements: CV (within) ≤ 8.0%, CV (between) ≤ 10.0%. The calculation formulas are as follows:

[0090]

[0091]

[0092] SS 瓶内 =SS 总和 -SS 瓶间 ,

[0093] v1 = a - 1,

[0094] v2 = Na,

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] In the formula: SS represents variance; x i This represents the i-th measurement or calculation result of the specified parameter; Indicates the overall average; x ij The result represents the j-th result of sample i; v represents the degrees of freedom; v1 represents the inter-bottle degrees of freedom; v2 represents the intra-bottle degrees of freedom; MS represents the mean square; F represents the F-test value; N represents the total number of tests; n i The number of measurements is represented by n; n0 represents the number of valid measurements; a represents the number of samples taken; S bb S represents the standard deviation between bottles; r This represents the standard deviation within the bottle (repeatability standard deviation).

[0103] F should be ≤10; when F≤1, the standard deviation within the bottle is used instead of the standard deviation between bottles, i.e., Sbb=Sr.

[0104] IV. Test Results

[0105] 1. Appearance and solubility of lyophilized calibrators, such as... Figures 2 to 4 As shown, where, Figure 2 A photograph of a lyophilized calibrator for hyaluronic acid determination prepared using an existing lyophilization formulation. Figure 3 Another physical image of a lyophilized calibrator for hyaluronic acid determination prepared for an existing lyophilized formulation. Figure 4 This is a photograph of the lyophilized calibrator for hyaluronic acid determination according to this application.

[0106] As can be seen from the figure, the lyophilized calibrator for hyaluronic acid determination in this application is stable and not easily broken during transportation.

[0107] The changes in activity of the calibrators before and after lyophilization are shown in Table 2.

[0108] Table 2. Changes in the activity of calibrators before and after freeze-drying.

[0109]

[0110]

[0111] 2. Stability of lyophilized calibrators

[0112] As shown in Tables 3, 4 and 5.

[0113] Table 3 Long-term stability of lyophilized calibrators

[0114]

[0115]

[0116] Table 4 Reconstitution Stability of Lyophilized Calibrators

[0117]

[0118] Table 5. Transport stability of freeze-dried calibrators

[0119]

[0120]

[0121] 3. Uniformity within and between calibration bottles

[0122] As shown in Table 6:

[0123] Table 6 Uniformity within and between calibration bottles

[0124]

[0125]

[0126] In summary, the lyophilization protective solution and lyophilization technology described in this application ensure that the hyaluronic acid calibrators do not shrink after lyophilization and can basically maintain their shape before lyophilization; they do not stick to the bottle body or stopper; they have good toughness and are not easily broken, resisting shock during transportation; the activity (i.e., concentration) before and after lyophilization is within ±5%; the uniformity within and between bottles is within ±8%; the relative deviations after 12 months of storage at 4℃, 7 days of storage at 37℃, and 24 hours after opening at 4℃ are all within ±15%; they have good solubility, and after reconstitution, they can completely dissolve within 30 minutes of standing, resulting in a clear solution without lumps or foam; they can extend the storage time; and as high-value calibrators, the dilution process is more convenient.

[0127] It should be understood that although the steps in the flowcharts of the accompanying figures are shown sequentially as indicated by the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the accompanying figures may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or in turn with other steps or at least some of the sub-steps or stages of other steps.

[0128] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A lyophilized calibrator for hyaluronic acid determination, characterized in that, It is composed of hyaluronic acid, phosphate buffer, potassium oxalate, bovine serum albumin, mannitol, sucrose, polyethylene glycol, disodium ethylenediaminetetraacetate, and preservatives; the lyophilized calibrator for hyaluronic acid determination is in a lyophilized state, and the other substances besides hyaluronic acid are the matrix; The concentrations of each component in the matrix are as follows: potassium oxalate 2 mmol / L, bovine serum albumin 10 g / L, mannitol 15 g / L, sucrose 50 g / L, polyethylene glycol 0.48 g / L, disodium EDTA 1 mmol / L, and preservative 0.2 g / L; the preservative is composed of sulfadiazine and dimethoprim, and the weight ratio of sulfadiazine to dimethoprim is 25:

1.

2. The lyophilized calibrator for hyaluronic acid determination according to claim 1, characterized in that, The phosphate buffer solution includes disodium hydrogen phosphate and sodium dihydrogen phosphate.

3. A method for preparing the lyophilized calibrator for hyaluronic acid determination according to claim 1, characterized in that, Includes the following steps: Take pure hyaluronic acid and dilute it with phosphate buffer to obtain a diluted solution; Add potassium oxalate solution and bovine serum albumin to the diluent; Add sucrose, polyethylene glycol, mannitol, disodium EDTA and preservatives, mix well and let stand to obtain calibrator material; The calibrator material is dispensed into multiple lyophilized vials, and the lyophilized vials are placed in a vacuum freeze dryer for pre-freezing to obtain the initial lyophilized product; The initial lyophilized product is subjected to vacuum freeze-drying to obtain an intermediate lyophilized product; The intermediate freeze-dried product is subjected to a desorption and drying operation to obtain the target freeze-dried product; Inert gas is introduced, the lyophilized sample is removed, and the lyophilized hyaluronic acid determination calibrator in lyophilized state is obtained.

4. The method for preparing the lyophilized calibrator for hyaluronic acid determination according to claim 3, characterized in that, The concentration of the phosphate buffer solution is 10 mM to 50 mM; The concentration of hyaluronic acid in the diluent is 1000 ng / mL to 3000 ng / mL.

5. The method for preparing the lyophilized calibrator for hyaluronic acid determination according to claim 3, characterized in that, The step of placing the freeze-drying bottle in a vacuum freeze dryer for pre-freezing to obtain the initial freeze-dried product includes: The freeze-drying bottle was placed in a vacuum freeze dryer, and the temperature was set to -50°C and the atmospheric pressure to 30Pa. Maintain the temperature for 4 hours, then gradually increase the temperature of the frozen environment by 10°C / hour until the temperature of the frozen environment reaches -30°C to obtain the initial freeze-dried product.

6. The method for preparing the lyophilized calibrator for hyaluronic acid determination according to claim 3, characterized in that, The step of vacuum freeze-drying the initial freeze-dried product includes: The initial freeze-dried product was placed in an environment with an atmospheric pressure of 30 Pa, and the freezing temperature was raised from -30°C to -15°C and maintained for 12 hours for sublimation drying.

7. The method for preparing the lyophilized calibrator for hyaluronic acid determination according to claim 3, characterized in that, The step of performing a desorption and drying operation on the intermediate lyophilized product to obtain the target lyophilized product includes: The intermediate freeze-dried product was placed in an environment with an atmospheric pressure of 15 Pa, and the freezing temperature was raised from -15°C to 25°C. The product was kept at this temperature for 3 hours to desorb and dry, thereby obtaining the target freeze-dried product.

8. The method for preparing the lyophilized calibrator for hyaluronic acid determination according to claim 3, characterized in that, The inert gas is nitrogen.

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