A bilirubin linear high-value freeze-dried solution, freeze-dried powder, and their preparation methods and applications

Abstract

The present invention discloses a bilirubin linear high-value freeze-dried solution, freeze-dried powder, and their preparation methods and applications. The bilirubin linear high-value freeze-dried solution comprises the following components in parts by mass: 60-70 parts of serum, 30-40 parts of PB buffer solution with a pH of 6.5-8.0, 0.5-1.5 parts of sodium chloride, 0.015-0.05 parts of direct bilirubin, 0.01-0.05 parts of indirect bilirubin, 1-5 parts of excipient, 1-5 parts of stabilizer, and 0.01-0.5 parts of preservative. The preparation method of the freeze-dried powder prepared from this freeze-dried solution is: sequentially mix the corresponding amounts of PB buffer solution, human serum, direct bilirubin, indirect bilirubin, excipient, stabilizer, and preservative. After all components are completely dissolved, subject the obtained freeze-dried solution to freeze-drying to obtain the bilirubin linear high-value freeze-dried powder. The bilirubin linear high-value freeze-dried solution and freeze-dried powder provided by the present invention are simple to operate, have good stability, are easy to store, can also replace the linear high value prepared from concentrated mixed calibration solution, and are simultaneously applied in the determination of the linear range of bilirubin detection reagents.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and particularly to a bilirubin linear high-value freeze-dried solution, freeze-dried powder, and their preparation methods and applications. Background Art

[0002] The determination of bilirubin is the most commonly used detection index in current liver function tests, especially important for the detection of jaundice samples. Bilirubin has four components: namely, unconjugated bilirubin, monoglucuronide-conjugated bilirubin, diglucuronide-conjugated bilirubin, and bilirubin covalently bound to albumin, which is called δ-bilirubin. Generally, the total of these four components is called total bilirubin (TB), and the latter three are called direct bilirubin (DB). The first two of the direct bilirubin are also called conjugated bilirubin.

[0003] Currently, there are also various bilirubin detection reagents or kits on the market, and the linear range of the kit is an important performance index for evaluating the quality of these reagents. The experiment for testing the linear range of these reagents requires a series of bilirubin samples with concentrations from low to high. The lowest-value sample is the linear low value, and the highest-value sample is the linear high value. It is difficult to obtain a stable bilirubin linear high-value sample.

[0004] Currently, there is a lack of bilirubin linear high-value products on the market. Users usually obtain them through the following two ways: One way is to use a concentrated mixed calibration solution to replace the linear high value. However, the concentrated calibration solution is generally relatively viscous, the solution is not very homogeneous, and the concentrated calibration solution is unstable. The measured value and color will change over time. Another way is to prepare and use the bilirubin raw material on-site, which not only has low efficiency but also a very short storage time.

[0005] Therefore, the prior art needs to be further improved. Summary of the Invention

[0006] In view of the above problems, the present invention provides a bilirubin linear high-value freeze-dried solution, freeze-dried powder, and their preparation methods and applications. The bilirubin linear high-value freeze-dried powder is simple to operate, has good stability, is easy to store, and can also replace the linear high value prepared by the concentrated mixed calibration solution, saving costs.

[0007] To solve the above problems, the present application provides the following technical solutions:

[0008] In the first aspect, the present application provides a bilirubin linear high-value freeze-dried solution, and the freeze-dried solution comprises the following components:

[0009] 60 to 70 parts of serum, 30 to 40 parts of PB buffer solution with a pH of 6.5 - 8.0, 0.5 to 1.5 parts of sodium chloride, 0.015 to 0.05 parts of direct bilirubin, 0.01 to 0.05 parts of indirect bilirubin, 1 to 5 parts of excipient, 1 to 5 parts of stabilizer, 0.01 to 0.5 parts of preservative.

[0010] At present in the market, most bilirubin raw materials are poorly soluble and must be dissolved with the help of organic solvents such as dimethyl sulfoxide, and they are mostly mixtures or isomers of various bilirubins, which are not suitable for the effective determination of direct bilirubin. In this application, direct bilirubin of the Frontier Scientific brand is preferably used, and the content of direct bilirubin can be accurately determined.

[0011] Preferably, in the bilirubin linear high-value freeze-dried solution, the excipient is one or both of sucrose and trehalose. Further preferably, the excipient is sucrose.

[0012] Preferably, in the bilirubin linear high-value freeze-dried solution, the stabilizer is one or a combination of more than one of BSA, Triton 405 or mannitol.

[0013] In a further preferred scheme, the bilirubin linear high-value freeze-dried solution and freeze-dried powder prepared by the compound combination of sucrose and BSA have better stability.

[0014] Preferably, in the bilirubin linear high-value freeze-dried solution, the preservative is one or a combination of more than one of fungal preservatives, sodium azide or Proclin 300.

[0015] Preferably, the bilirubin linear high-value freeze-dried solution comprises the following components:

[0016] 60 parts of human serum, 30 parts of 0.2M PB buffer solution, 0.02 parts of direct bilirubin, 0.02 parts of indirect bilirubin, 2.5 parts of sucrose, 1.5 parts of BSA, 0.1 part of sodium azide, 1.2 parts of sodium chloride.

[0017] In the second aspect, this application provides a bilirubin linear high-value freeze-dried powder, which is prepared from the aforementioned bilirubin linear high-value freeze-dried solution.

[0018] In the third aspect, this application provides a preparation method of the aforementioned bilirubin linear high-value freeze-dried powder, specifically: mixing the corresponding amounts of PB buffer solution, human serum, direct bilirubin, indirect bilirubin, excipient, stabilizer and preservative in sequence. After all components are completely dissolved, the obtained freeze-dried solution is freeze-dried to obtain the bilirubin linear high-value freeze-dried powder.

[0019] The experimental results prove that the addition sequence of the above components, especially bilirubin, will directly affect its solubility, and thus indirectly affect the size of the linear high value of bilirubin obtained by preparation and the stability of the lyophilized powder.

[0020] Fourthly, the present application also provides an application of the above bilirubin linear high-value lyophilized solution or bilirubin linear high-value lyophilized powder in the determination of the linear range of bilirubin detection reagents.

[0021] In this application, the mixed detection of direct bilirubin and total bilirubin can be realized. Bilirubin detection generally includes two detections of direct bilirubin and total bilirubin. The bilirubin linear high-value lyophilized solution and lyophilized powder provided by the present invention can be simultaneously applied to these two tests, further reducing the detection cost.

[0022] The present invention has the following beneficial effects:

[0023] 1. The bilirubin linear high-value lyophilized solution and lyophilized powder provided by the present invention are simple to operate, have good stability, are easy to store, and can also replace the linear high value prepared by the concentrated mixed calibration solution, saving costs.

[0024] 2. The shelf life of the bilirubin linear high-value lyophilized solution and lyophilized powder prepared by the formula of the present application is greatly extended, and its stability is greatly improved; it can also save the detection cost and can realize the mixed detection of two indicators of direct bilirubin and total bilirubin. In addition, the preparation method of the linear high-value lyophilized solution and lyophilized powder is simple to operate, does not involve related pretreatment processes such as serum centrifugation, retains the original state of the serum, and reduces the matrix effect. Detailed implementation manners

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the protection scope of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0026] Example 1

[0027] 1. This embodiment provides a bilirubin linear high-value lyophilized powder, which is prepared from a lyophilized solution. The lyophilized solution includes the following components:

[0028]

[0029]

[0030] 2. This embodiment also provides a method for preparing the above bilirubin linear high-value lyophilized powder, and the specific steps are as follows:

[0031] Using a buffer solution as a solvent, add and mix all the above other raw materials in sequence. After all components are completely dissolved, dispense the obtained lyophilized solution into brown bottles and perform freeze-drying to obtain the bilirubin linear high-value lyophilized powder.

[0032] Example 2

[0033] 1. This embodiment provides a bilirubin linear high-value lyophilized powder, which is prepared from a lyophilized solution. The lyophilized solution includes the following components:

[0034]

[0035] 2. This embodiment also provides a method for preparing the above bilirubin linear high-value lyophilized powder, and the specific steps are as follows:

[0036] Using a buffer solution as a solvent, add and mix all the above other raw materials in sequence. After all components are completely dissolved, dispense the obtained lyophilized solution into brown bottles and perform freeze-drying to obtain the bilirubin linear high-value lyophilized powder.

[0037] Example 3

[0038] 1. This embodiment provides a bilirubin linear high-value lyophilized powder, which is prepared from a lyophilized solution. The lyophilized solution includes the following components:

[0039]

[0040] 2. This embodiment also provides a method for preparing the above bilirubin linear high-value lyophilized powder, and the specific steps are as follows:

[0041] Using a buffer solution as a solvent, add and mix all the above other raw materials in sequence. After all components are completely dissolved, dispense the obtained lyophilized solution into brown bottles and perform freeze-drying to obtain the bilirubin linear high-value lyophilized powder.

[0042] Example 4

[0043] 1. This embodiment provides a bilirubin linear high-value lyophilized powder, which is prepared from a lyophilized solution. The lyophilized solution includes the following components:

[0044]

[0045]

[0046] 2. This embodiment also provides a method for preparing the above bilirubin linear high-value lyophilized powder, and the specific steps are as follows:

[0047] Using a buffer solution as the solvent, add and mix all the other raw materials in the above order. After all components are completely dissolved, dispense the obtained freeze-drying solution into brown bottles and perform freeze-drying to obtain the high-value bilirubin linear freeze-dried powder.

[0048] Example 5

[0049] 1. This example provides a high-value bilirubin linear freeze-dried powder, which is prepared from a freeze-drying solution. The freeze-drying solution includes the following components:

[0050]

[0051] 2. This example also provides a preparation method for the above high-value bilirubin linear freeze-dried powder. The specific steps are as follows:

[0052] Using a buffer solution as the solvent, add and mix all the other raw materials in order. After all components are completely dissolved, dispense the obtained freeze-drying solution into brown bottles and perform freeze-drying to obtain the high-value bilirubin linear freeze-dried powder.

[0053] Example 6

[0054] 1. This example provides a high-value bilirubin linear freeze-dried powder, which is prepared from a freeze-drying solution. The freeze-drying solution includes the following components:

[0055]

[0056] 2. This example also provides a preparation method for the above high-value bilirubin linear freeze-dried powder. The specific steps are as follows:

[0057] Using a buffer solution as the solvent, add and mix all the other raw materials in order. After all components are completely dissolved, dispense the obtained freeze-drying solution into brown bottles and perform freeze-drying to obtain the high-value bilirubin linear freeze-dried powder.

[0058] Stability Test of High-Value Bilirubin Linear Freeze-Dried Powder in Example 7

[0059] 1. Test Object:

[0060] The high-value bilirubin linear freeze-dried powder prepared in Examples 1-6.

[0061] 2. Experimental Method

[0062] Place each test object in an environment of 2-8°C. Take out the samples at the 0th, 3rd, 6th, 9th, and 12th months respectively. Each time, take 2 bottles of high-value linear samples, reconstitute them, let them stand at room temperature for 15 minutes, mix well, and then perform a stability test on a Hitachi 7600 biochemical analyzer using a certified kit for direct bilirubin / total bilirubin. Test each bottle 3 times and calculate the average value of the measurements.

[0063] Requirement for meeting the standard of relative deviation: The relative deviation between the measured mean value and the labeled value is within ±10%.

[0064] Taking the collected linear high and low value samples (the remaining samples from the detection of biochemical liver function diseases in a certain domestic hospital) as control group 1, and the concentrated calibration solution as control group 2 for comparison.

[0065] Among them, the preparation method of the concentrated calibration solution is as follows: Select the composite calibration serum (lyophilized powder) of the commercially available Randox Laboratories Limited in the UK. Its theoretical reconstitution volume is 5 mL. In this experiment, for the preparation of the concentrated calibration solution, the reconstitution volume is required to be smaller. Therefore, according to the labeled value, the total bilirubin is concentrated to about 1 mL with distilled water, and the direct bilirubin is concentrated to about 2 mL with distilled water to prepare the concentrated calibration solution.

[0066] 2.1 Stability test method for TB / DB

[0067] (1) Real-time stability test method:

[0068] Set the item parameters of total bilirubin / direct bilirubin on the Hitachi 7600 biochemical analyzer according to the instructions of the certified kit. Then, calibrate the detection system with the calibration product supporting the certified kit, and use the calibrated detection system to test the linear high value. Test 2 bottles, and each bottle is tested 3 times to obtain the mean value (M) of the test results. Calculate the relative deviation (B) according to the following formula, and the obtained relative deviation is required to be within ±10%.

[0069]

[0070] In the formula:

[0071] M - Mean value of test results; T - Labeled value.

[0072] (2) Reconstitution stability test method

[0073] Reconstitute the linear high value lyophilized powder at 0, 1, 3, 5, and 7 days. Test 2 bottles at each time point, and each bottle is repeatedly measured 3 times; calculate the relative deviation, and its calculation method is the same as that of the real-time stability.

[0074] 3. Experimental results and analysis

[0075] (1) Real-time stability of TB / DB

[0076] The experimental results are shown in Table 1 and Table 2. From the results, it can be seen that:

[0077] ① Compared with control group 1 and 2, the real-time stability of the bilirubin linear high value lyophilized powder of this application has been greatly improved; from the comparison of real-time stability, it can be seen that the stability of Example 6 is the best.

[0078] ②The bilirubin linear high-value lyophilized powder prepared in Example 6 has the best stability, with a deviation within 10%; the stability still meets the standard when stored until December;

[0079] ③From the comparison between Example 1 and Example 2, it can be seen that the stability of Scheme 2 with sucrose as the excipient is better;

[0080] ④From the comparison between Example 2 - 4, it can be seen that for the compound combinations of sucrose - mannitol, sucrose - Triton 405, and sucrose - BSA, among them, the compound combination of sucrose - BSA has better stability for the bilirubin linear high-value lyophilized powder prepared;

[0081] ⑤The designs of Example 4, Example 5, and Example 6 are to study the effects of direct bilirubin raw materials, concentrations, and addition sequences on the measured values of the bilirubin linear high-value lyophilized powder. From the comparison of the results of Example 4, Example 5, and Example 6, it can be seen that the operation of adding bilirubin in the later addition sequence will affect its solubility, making it unable to reach the linear high value; at the same time, a relatively large amount of bilirubin added will not improve the stability. Instead, reducing the addition amount can also reduce costs. On this basis, increasing the amount of sucrose used can greatly improve the stability.

[0082] Table 1 TB real-time stability test results

[0083]

[0084] Table 2 DB real-time stability test results

[0085]

[0086]

[0087] (2) Reconstitution stability of TB / DB

[0088] From the results in Table 3 and Table 4, it can be seen that the measured values of the bilirubin linear high-value lyophilized powder in Example 6 after reconstitution are relatively stable within 5 days, with the smallest relative deviation; while the measured values of the linear high-value serum sample and the concentrated calibrator change greatly within 5 days, with a relatively high deviation and poor stability.

[0089] Table 3 Comparison of TB stability between reconstituted linear high-value lyophilized powder and linear high-value sample

[0090]

[0091] Table 4 Comparison results of DB stability between reconstituted linear high-value lyophilized powder and linear high-value sample

[0092]

[0093]

[0094] Example 8 Linear Test of Bilirubin

[0095] 1. Test Object:

[0096] The reconstituted high-value freeze-dried powder solution of bilirubin prepared in Examples 1-6 and distilled water.

[0097] 2. Experimental Method

[0098] Use a high-concentration reconstituted freeze-dried powder solution close to the upper limit of the linear range and distilled water close to the lower limit of the linear range to mix into at least 5 dilution concentrations. The 5 concentrations are high value, high value: low value = 5:1, high value: low value = 1:1, high value: low value = 1:5, and low value. Among them, the linear high-value range of total bilirubin (TB) is 616-684 μmol / L, and the linear low value of total bilirubin is 0 μmol / L; the linear high value of direct bilirubin (DB) is 308-342 μmol / L, and the linear low value of direct bilirubin is 0 μmol / L. Then calibrate the detection system with a certified kit calibration product on the Hitachi 7600 biochemical analyzer, and use the calibrated detection system to test the 5 dilution concentrations. Each dilution concentration is tested 3 times, and the mean value of the measurement results is calculated respectively. Taking the dilution concentration as the independent variable and the mean value of the measurement results as the dependent variable, a linear regression equation is obtained, and the correlation coefficient r≥0.9900. Among them, when the total bilirubin is lower than 30 μmol / L, the linear absolute deviation should not exceed ±3 μmol / L; within the range of 30-684 μmol / L, the linear relative deviation should not exceed ±10%; when the direct bilirubin is lower than 80 μmol / L, the linear absolute deviation should not exceed ±8 μmol / L; within the range of 80-342 μmol / L, the linear relative deviation should not exceed ±10%.

[0099] Experimental Group Settings: The experimental group is the reconstituted linear high value and distilled water, the control group 1 is the collected linear high value samples and distilled water, and the control group 2 is the concentrated calibration solution and distilled water. The three groups are diluted into 5 different concentrations according to the ratio respectively. Operate according to the above method.

[0100] 3. Experimental Results and Analysis

[0101] (1) TB Linear Test Results and Analysis

[0102] The results are shown in Table 5 below. According to the linear indicators, the linear tests of both the control group 1 and Example 6 meet the linear requirements, but Example 6 is better with the smallest deviation.

[0103] The absolute deviation of other groups does not meet the requirements within the low-value range. Among them, the overall deviation of the control group 2 is large, which is related to the high viscosity of its solution itself, and it is easy to appear uneven phenomena, thus affecting the measured values of this group.

[0104] Table 5 TB Linear Test Results

[0105]

[0106] (2) DB Linear Test Results and Analysis

[0107] The results are shown in Table 6. According to the linear index, Examples 5, 6, Control Group 1 and Control Group 2 all meet the linear requirements, but Example 6 is better with the smallest deviation.

[0108] Table 6 DB Linear Test Results

[0109]

[0110]

[0111] It can be understood that for those of ordinary skill in the art, equivalent substitutions or changes can be made according to the technical solution of the present invention and the inventive concept of the present invention, and all such changes or substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A bilirubin linear high-value freeze-dried solution, characterized in that, the freeze-dried solution comprises the following components in parts by mass: 60-70 parts of serum, 30-40 parts of PB buffer solution with a pH of 6.5-8.0, 0.5-1.5 parts of sodium chloride, 0.015-0.05 parts of direct bilirubin, 0.01-0.05 parts of indirect bilirubin, 1-5 parts of sucrose, 1-5 parts of BSA, 0.01-0.5 parts of preservative; the preservative is a fungal preservative, sodium azide or Proclin 300.

2. The bilirubin linear high-value freeze-dried solution according to claim 1, characterized in that, it comprises the following components: 60 parts of human serum, 30 parts of 0.2M PB buffer solution, 0.02 parts of direct bilirubin, 0.02 parts of indirect bilirubin, 2.5 parts of sucrose, 1.5 parts of BSA, 0.1 part of sodium azide, 1.2 parts of sodium chloride.

3. A bilirubin linear high-value freeze-dried powder, characterized in that, it is prepared from the bilirubin linear high-value freeze-dried solution according to any one of claims 1 to 2.

4. The preparation method of the bilirubin linear high-value freeze-dried powder according to claim 3, characterized in that, specifically: mix the corresponding amounts of PB buffer solution, human serum, direct bilirubin, indirect bilirubin, excipient, stabilizer and preservative in sequence. After the added amounts of direct bilirubin and indirect bilirubin are completely dissolved, then add the excipient, stabilizer and preservative to prevent the solution from being saturated and the linear high value from not being achieved. Then, subject the obtained freeze-dried solution to freeze-drying to obtain the bilirubin linear high-value freeze-dried powder.

5. The application of the bilirubin linear high-value freeze-dried solution according to any one of claims 1 to 2 or the bilirubin linear high-value freeze-dried powder according to claim 3 in the determination of the linear range of bilirubin detection reagents.

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