Method for detecting bacterial endotoxin of natural high-molecular polysaccharide freeze-dried product
By adding 0.9% sodium chloride solution during the testing process and mixing it in a specific order, the false positive problem in the detection of bacterial endotoxins in freeze-dried natural high-molecular-weight polysaccharides was solved, achieving accuracy and reliability of the test results and ensuring the safety of polysaccharide injections.
Patent Information
- Application Number
- CN202511223114.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-11-28
AI Technical Summary
The existing technology for detecting bacterial endotoxins in freeze-dried natural polymer polysaccharides has a false positive problem, which leads to a deviation between the test results and the true values, affecting product quality control.
When preparing the test solution, add 0.9% sodium chloride solution as an auxiliary reagent and mix it with the polysaccharide freeze-dried product in a specific order. Then add endotoxin standard for detection and use dynamic colorimetric method for detection.
It effectively eliminates false positive interference, improves the accuracy and reliability of detection, and ensures the safety of polysaccharide injections.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial detection technology, specifically relating to a method for detecting bacterial endotoxins in freeze-dried natural high-molecular-weight polysaccharide products. Background Technology
[0002] In the development of injectable products, bacterial endotoxin control is a crucial step in ensuring drug safety. Bacterial endotoxins, as a major component of the cell wall of Gram-negative bacteria (lipopolysaccharide), are the core substances that trigger pyrogenic reactions. When large amounts of endotoxins enter the bloodstream, they can induce serious clinical reactions such as fever, abnormal white blood cell counts, endotoxin shock, and even disseminated intravascular coagulation. Therefore, strict control over the endotoxin content in raw materials is essential.
[0003] Currently, the spectrophotometric method for bacterial endotoxins has become the mainstream detection technology due to its precise quantitative advantages. This method achieves quantitative analysis by monitoring changes in the optical signal (turbidity or chromogenic groups) generated by the reaction of Limulus amebocyte lysate (LAL) reagent with endotoxin using a spectrophotometer. It mainly includes two types of detection systems: turbidity methods and chromogenic matrix methods. Chromogenic matrix methods utilize the amount of chromogenic groups released from a specific substrate by coagulating enzymes produced during the reaction of LAL reagent with endotoxins to determine the endotoxin content. Based on the detection principle, it is further divided into endpoint colorimetric methods and dynamic colorimetric methods. The endpoint colorimetric method determines the endotoxin content based on the quantitative relationship between the endotoxin concentration in the reaction mixture and the amount of chromogenic groups released at the end of incubation. The dynamic colorimetric method detects the reaction time required for the absorbance or transmittance of the reaction mixture to reach a predetermined detection value, or the rate of increase in the detection value.
[0004] Compared to traditional gel electrophoresis, spectrophotometry offers advantages such as precise quantification, high sensitivity, strong anti-interference capabilities, and good accuracy. For spectrophotometric bacterial endotoxin testing, at least three concentration gradient solutions of standard endotoxin must first be prepared, with the lowest concentration ≥ the detection limit indicated by the Limulus Amebocyte Lysate (LAL) reagent. A negative control (endotoxin-free solution) is set up, and linear regression analysis is performed, requiring a correlation coefficient |r| ≥ 0.980; otherwise, the test must be repeated. Then, four solutions are prepared according to the characteristics of the analyte: A: test solution, B: test solution + standard endotoxin (selecting the midpoint concentration of the standard curve), C: a series of standard endotoxin concentrations (for standard curve reliability), and D: negative control. The recovery rate is calculated as (B endotoxin concentration - A endotoxin concentration) / added endotoxin concentration × 100%, and should be within the range of 50%–200%. However, in practical applications, false positive results often occur in the endotoxin detection of lyophilized natural high-molecular-weight polysaccharide preparations, with deviations between the detection results and the true values, which is detrimental to the accurate detection of bacterial endotoxins. Existing research indicates that changing the manufacturer of the horseshoe crab reagent or adding conventional auxiliary reagents cannot solve this problem, which poses a challenge to the quality control of freeze-dried products.
[0005] Therefore, designing a method that can accurately and reliably detect bacterial endotoxins in freeze-dried products containing natural high-molecular-weight polysaccharides is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention
[0006] The purpose of this invention is to provide a method for detecting bacterial endotoxins in freeze-dried natural high-molecular-weight polysaccharide products, which solves the problem of false positives in the prior art and effectively improves the accuracy of detecting bacterial endotoxins in freeze-dried natural high-molecular-weight polysaccharide products.
[0007] To achieve the above technical objectives, this application adopts the following technical solution:
[0008] A method for detecting bacterial endotoxins in a freeze-dried natural high-molecular-weight polysaccharide product, wherein when preparing the test solution, an auxiliary reagent is first added and mixed with the freeze-dried polysaccharide product, and then an endotoxin standard is added for detection.
[0009] Specifically, the preparation process of the above-mentioned test solution is as follows: Weigh the lyophilized polysaccharide product, add water for bacterial endotoxin testing to dilute and bring to volume, and mix to obtain Sogen; take a certain amount of Sogen and dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S1; take a certain amount of S1 and dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S2; take S2 and dilute it with water for bacterial endotoxin testing, and mix to obtain S3; take S3 and dilute it with water for bacterial endotoxin testing, and mix to obtain the test solution.
[0010] More specifically, in the above implementation scheme, the mixing method is vortex mixing;
[0011] Furthermore, the vortex mixing time is 30-60.
[0012] In some preferred embodiments, the auxiliary reagent is selected from 0.9% sodium chloride solution.
[0013] In some preferred embodiments, when preparing S1 and S2 as described above, the volume ratio of water for bacterial endotoxin testing to 0.9% sodium chloride solution is 1:2.
[0014] In some more specific implementations, the detection method includes the following steps:
[0015] a. Dissolve the bacterial endotoxin standard in water for bacterial endotoxin testing, and then dilute it serially to obtain a series of standard solutions.
[0016] b. Weigh the lyophilized polysaccharide product, dilute it with water for bacterial endotoxin testing, and mix to obtain Sogen; take a certain amount of Sogen, dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S1; take a certain amount of S1, dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S2; take S2, dilute it with water for bacterial endotoxin testing, and mix to obtain S3; take S3, dilute it with water for bacterial endotoxin testing, and mix to obtain the test solution.
[0017] c. Add bacterial endotoxin standard solution, then dilute with bacterial endotoxin test water, then add a certain volume of S3, mix, and obtain the positive control solution of the test sample.
[0018] d. Water used for bacterial endotoxin testing is a negative control solution;
[0019] e. Take a certain amount of the solution prepared in steps a, b, c, and d above and add it into the ELISA plate. Add an equal volume of Limulus amebocyte lysate (LAL) reagent solution. After preparation, place the plate into an ELISA reader for reaction and use the dynamic colorimetric method to detect the content of bacterial endotoxins.
[0020] More specifically, in the above implementation scheme, the mixing method is vortex mixing.
[0021] Furthermore, the vortex mixing time is 30-60 seconds.
[0022] In some preferred embodiments of this application, in step b above, the polysaccharide freeze-dried product is weighed, diluted to volume with water for bacterial endotoxin testing, and vortexed for 30 seconds to obtain Sorigin; 0.3 mL of Sorigin is diluted with 0.9 mL of water for bacterial endotoxin testing and 1.8 mL of 0.9% sodium chloride solution, and vortexed for 30 seconds to obtain S1; 0.3 mL of S1 is diluted with 0.9 mL of water for bacterial endotoxin testing and 1.8 mL of 0.9% sodium chloride solution, and vortexed for 30 seconds to obtain S2; 0.2 mL of S2 is diluted with 1.4 mL of water for bacterial endotoxin testing, and vortexed for 30 seconds to obtain S3; 0.5 mL of S3 is diluted with 0.5 mL of water for bacterial endotoxin testing, and vortexed for 30 seconds to obtain the test solution.
[0023] In some preferred embodiments of this application, in step c above, the bacterial endotoxin working standard is diluted to 0.2 EU / ml with BET water; 0.2 ml of S3 is added to 0.2 ml (0.2 EU / ml) and vortexed for 30 seconds to obtain solution B, which is the positive control solution for the test sample.
[0024] In some preferred embodiments of this application, in step e above, the reaction conditions for the dynamic colorimetric method are: 36-38°C and the detection wavelength is 405nm.
[0025] More specifically, the polysaccharide freeze-dried product is selected from sodium carboxymethyl cellulose or carboxymethyl chitosan.
[0026] In some preferred embodiments of this application, in step a above, the bacterial endotoxin standard is dissolved in water for bacterial endotoxin testing and serially diluted to prepare a series of standard solutions with concentrations of 1 EU / mL, 0.1 EU / mL, and 0.01 EU / mL.
[0027] Compared with the prior art, the technical solution of the present invention has the following advantages:
[0028] Unexpectedly, it was discovered that the addition of 0.9% sodium chloride solution, and the order of its addition, plays a crucial role in suppressing false positives. This eliminates the interference of natural high-molecular-weight polysaccharide freeze-dried products in the detection of bacterial endotoxin limits, avoids the problem of false positive interference in existing technologies, and provides accurate and reliable test results. The operation is simple and reproducible, which is of great significance for ensuring the safety of polysaccharide injections. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the following specific embodiments will describe the present invention in detail.
[0030] 1. Experimental materials and instruments
[0031] Experimental materials:
[0032] Limulus amebocyte lysate (LAL) reagent 1: Zhanjiang Andus Biotechnology Co., Ltd., specification: 0.35mL / vial, batch number: 2404090; Limulus amebocyte lysate (LAL) reagent 2: Xiamen Limulus amebocyte lysate Biotechnology Co., Ltd., specification: 0.5ml / vial, batch number: 24010116; Bacterial endotoxin working standard 1: Zhanjiang Andus Biotechnology Co., Ltd., specification: 1ml / vial, potency: 10EU / vial, batch number: 2402200; Bacterial endotoxin working standard 2: Xiamen Limulus amebocyte lysate Biotechnology Co., Ltd., specification: 1ml / vial, potency: 100 EU / vial, batch number: 23086005; National standard for bacterial endotoxins: China National Institutes for Food and Drug Control, potency: 9000 EU / vial, batch number: 150800-201601; Water for bacterial endotoxin testing (BET water) 1: Zhanjiang Andus Biotechnology Co., Ltd., specification: 100 mL / bottle, batch number: 2407250; Water for bacterial endotoxin testing (BET water) 2: Xiamen Limulus Amebocyte Lysate Reagent Biotechnology Co., Ltd., specification: 10 mL / vial, batch number: 23086005.
[0033] Instruments: forced-air drying oven, fully automated bacterial endotoxin detection system, medium-sized glass tubes, etc.
[0034] Example 1: Screening Test for Auxiliary Reagents
[0035] Because auxiliary reagents can affect the detection, a screening test for auxiliary reagents was conducted.
[0036] Solution D (negative control solution): BET water.
[0037] Dilution of bacterial endotoxin working standard: Take one vial of bacterial endotoxin working standard, reconstitute it with BET water, vortex mix for 15 minutes, and then dilute it to 100 EU / ml.
[0038] Solution C (standard curve): Dilute the 100 EU / ml bacterial endotoxin working standard with BET water to 1 EU / ml, 0.1 EU / ml, and 0.01 EU / ml.
[0039] Test solution A:
[0040] Test solution A1:
[0041] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of factor G inhibitor, vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing and 600 μl of factor G inhibitor, vortex for 30 seconds to obtain S. 100 Take S 100 Add 100 μl of 900 μl of Tris buffer and vortex for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0042] Solution B1 (positive control solution for the test sample):
[0043] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0044] Test solution A2:
[0045] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of factor G inhibitor, vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing and 600 μl of factor G inhibitor, vortex for 30 seconds to obtain S. 100 Take S 100 Add 100 μl of 500 μl of Tris buffer and 400 μl of water for bacterial endotoxin testing, vortex for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds.2000 .
[0046] Solution B2 (positive control solution for the test sample):
[0047] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0048] Test solution A3:
[0049] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of factor G inhibitor, vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing and 600 μl of factor G inhibitor to S, vortex mix for 30 seconds. 100 Take S 100 Add 100 μl of calcium-magnesium ion buffer to 900 μl of water, vortex for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0050] Solution B3 (positive control solution for the test sample):
[0051] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0052] Test solution A4:
[0053] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of factor G inhibitor, vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing and 600 μl of factor G inhibitor to S, vortex mix for 30 seconds. 100 Take S 100 Add 100 μl of 500 μl of calcium-magnesium ion buffer and 400 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0054] Solution B4 (positive control solution for the test sample):
[0055] Take S 1000It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0056] Test solution A5:
[0057] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of factor G inhibitor, vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing and 600 μl of factor G inhibitor to S, vortex mix for 30 seconds. 100 Take S 100 Add 100 μl of 0.9% sodium chloride injection to 900 μl of the solution, vortex for 30 seconds to obtain solution S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0058] Solution B5 (positive control solution for the test sample):
[0059] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0060] Test solution A6:
[0061] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of factor G inhibitor, vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing and 600 μl of factor G inhibitor to S, vortex mix for 30 seconds. 100 Take S 100 Add 100 μl of 0.9% sodium chloride injection and 400 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0062] Solution B6 (positive control solution for the test sample):
[0063] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0064] Test solution A7:
[0065] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source and add 900 μl of Tris buffer, vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl to 900 μl of Tris buffer and vortex for 30 seconds to obtain S. 100 Take S 100 Add 100 μl to 900 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0066] Solution B7 (positive control solution for the test sample):
[0067] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0068] Test solution A8:
[0069] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of Tris buffer, and vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing to 300 μl of Tris buffer, vortex for 30 seconds to obtain the final product (S). 100 Take S 100 Add 100 μl to 900 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0070] Solution B8 (positive control solution for the test sample):
[0071] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0072] Test solution A9:
[0073] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source and add 900 μl of calcium magnesium ion buffer. Vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl to 900 μl of calcium-magnesium ion buffer and vortex for 30 seconds to obtain S. 100 Take S 100Add 100 μl to 900 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0074] Solution B9 (positive control solution for the test sample):
[0075] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0076] Test solution A10:
[0077] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of calcium magnesium ion buffer, and vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing to 300 μl of calcium magnesium ion buffer, and vortex for 30 seconds to obtain S. 100 Take S 100 Add 100 μl to 900 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0078] Solution B10 (positive control solution for the test sample):
[0079] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0080] Test solution A11:
[0081] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the sulfur source. Take 100 μl of the sulfur source and add 900 μl of 0.9% sodium chloride injection, vortex mix for 30 seconds to obtain the sulfur source. 10 Take S 10 Add 100 μl of 0.9% sodium chloride injection to 900 μl of solution and vortex for 30 seconds to obtain solution S. 100 Take S 100 Add 100 μl to 900 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0082] Solution B11 (positive control solution for the test sample):
[0083] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0084] Test solution A12:
[0085] Take an appropriate amount of the test sample, dissolve it completely, and mix it to obtain the S source. Take 100 μl of the S source, add 300 μl of bacterial endotoxin test water and 600 μl of 0.9% sodium chloride injection, and vortex mix for 30 seconds to obtain the S source. 10 Take S 10 Add 100 μl of water for bacterial endotoxin testing to 300 μl of 0.9% sodium chloride injection, vortex for 30 seconds to obtain solution S. 100 Take S 100 Add 100 μl to 900 μl of water for bacterial endotoxin testing, vortex mix for 30 seconds to obtain S. 1000 Take S 1000 Add 300 μl of water for bacterial endotoxin testing to S, vortex mix for 30 seconds. 2000 .
[0086] Solution B12 (positive control solution for the test sample):
[0087] Take S 1000 It is obtained by mixing with a 0.2 EU / ml endotoxin standard solution at a 1:1 ratio.
[0088] Add 25 μl each of the above solutions A1-A12, B1-B12, C, and D to the microplate, then add an equal volume of Limulus amebocyte lysate (LAL) reagent solution. Prepare two parallel tubes for each concentration. Place the prepared tubes into the microplate reader for reaction. The reaction temperature is (37±1)℃, and the working wavelength is [not specified].
[0089] The absorbance was set at 405 nm and 0.1, and the data were recorded. Table 1 shows the screening test for auxiliary reagents.
[0090]
[0091] In summary, based on the results in Table 1, the screening results indicate that without adding a G factor inhibitor, using 0.9% sodium chloride injection as the auxiliary reagent yields the best experimental results. Therefore, this condition was selected for the interference experiment.
[0092] Example 2: Optimization Experiment of Auxiliary Reagent Addition Order
[0093] Because auxiliary reagents were added, in order to ensure that the auxiliary reagents could effectively eliminate interference and would not cause the endotoxin to lose its activity, it was necessary to investigate the effect of 0.9% sodium chloride solution on the endotoxin. However, in the actual experiment, it was found that the method and dilution factor were not suitable for the dilution of samples from Zhanjiang Andus Biotechnology Co., Ltd., and the order affected the detection. Therefore, relevant experiments were conducted on the dissolution and dilution order of the samples.
[0094] Solution D (negative control solution): BET water.
[0095] Dilution of national standard for bacterial endotoxin: Take one vial of national standard for bacterial endotoxin, reconstitute it with BET water, vortex mix for 15 minutes, and then dilute it to 100 EU / ml.
[0096] Solution C (standard curve): Dilute the 100 EU / ml bacterial endotoxin national standard with BET water to 1 EU / ml, 0.1 EU / ml, and 0.01 EU / ml (each dilution step should be mixed for 30 seconds on a vortex mixer).
[0097] Solution A1 (test solution):
[0098] Take an appropriate amount of the test sample and dissolve it in water for bacterial endotoxin testing. After dissolution, mix the samples to obtain S1. Take 0.3 ml of S1, add 0.9 ml of water for bacterial endotoxin testing and 1.8 ml of 0.9% sodium chloride solution, and vortex mix for 30 seconds to obtain S2. Take 0.3 ml of S1, add 0.9 ml of water for bacterial endotoxin testing and 1.8 ml of 0.9% sodium chloride solution, and vortex mix for 30 seconds to obtain S2. Take 0.2 ml of S2, add 1.4 ml of water for bacterial endotoxin testing, and vortex mix for 30 seconds to obtain S3. Take 0.5 ml of S3, add 0.5 ml of water for bacterial endotoxin testing, and vortex mix for 30 seconds to obtain the final product.
[0099] Solution B1 (positive control solution for test sample): Dilute the bacterial endotoxin working standard to 0.2 EU / ml with BET water; add 0.2 ml of S3 to 0.2 ml (0.2 EU / ml) and vortex mix for 30 seconds to obtain solution B (positive control solution for test sample).
[0100] Solution A2 (test solution):
[0101] Take an appropriate amount of the test sample and dissolve it in water for bacterial endotoxin testing. After dissolution, mix the samples to obtain S1. Take 0.3 ml of S1 and add 2.7 ml of water for bacterial endotoxin testing, vortex and mix for 30 seconds to obtain S2. Take 0.3 ml of S1, add 0.9 ml of water for bacterial endotoxin testing and 1.8 ml of 0.9% sodium chloride solution, vortex and mix for 30 seconds to obtain S2. Take 0.2 ml of S2 and add 1.4 ml of water for bacterial endotoxin testing, vortex and mix for 30 seconds to obtain S3. Take 0.5 ml of S3 and add 0.5 ml of water for bacterial endotoxin testing, vortex and mix for 30 seconds to obtain the final product.
[0102] Solution B2 (positive control solution for test sample): Dilute the bacterial endotoxin working standard to 0.2 EU / ml with BET water; add 0.2 ml of S3 to 0.2 ml (0.2 EU / ml) and vortex for 30 seconds to obtain solution B (positive control solution for test sample).
[0103] Add 25 μl each of solutions A1, A2, B1, B2, C, and D to an ELISA plate, followed by an equal volume of Limulus Amebocyte Lysate (LAL) reagent solution. Prepare two parallel tubes for each concentration. Place the prepared tubes into an ELISA reader for reaction. The reaction temperature is (37±1)℃, the working wavelength is 405 nm, and the preset absorbance is 0.1. Record the data.
[0104] Table 2 Results of the experiment on optimization of dilution factor and order of addition of auxiliary reagents
[0105]
[0106] In summary, based on the results in Table 2, the dilution order of the samples affects the detection. The auxiliary reagent used is 0.9% sodium chloride injection. After adding the sample, water, and sodium chloride, they should be mixed first to allow the sodium chloride to react with the sample preferentially. After mixing, the endotoxin standard is added. No false positives were observed in the test results. Therefore, this condition was selected for the interference test.
[0107] Example 3: Effect of 0.9% Sodium Chloride Solution on Endotoxins
[0108] When using suitable reagent solutions for dissolution, an interference test must be performed to verify the recovery of endotoxins. In this verification process, 0.9% sodium chloride solution was used to dissolve the lyophilized carboxymethyl chitosan product. Therefore, it was necessary to investigate the effect of 0.9% sodium chloride solution on endotoxins. Thus, an interference test was conducted on one batch of lyophilized carboxymethyl chitosan product using Limulus amebocyte lysate (LAL) reagents from two different manufacturers.
[0109] The methods were based on the provisions of the Chinese Pharmacopoeia 2020 edition, Part IV, 1143, and solutions A, B, C, and D were prepared according to the table below.
[0110] Solution D (negative control solution): BET water.
[0111] Dilution of national standard for bacterial endotoxin: Take one vial of national standard for bacterial endotoxin, reconstitute it with BET water, vortex mix for 15 minutes, and then dilute it to 100 EU / ml.
[0112] Solution C (standard curve): Dilute the 100 EU / ml bacterial endotoxin national standard with BET water to 1 EU / ml, 0.1 EU / ml, and 0.01 EU / ml.
[0113] Solution A (test solution):
[0114] Take an appropriate amount of the test sample and dissolve it in water for bacterial endotoxin testing. After dissolution, mix the solutions to obtain S source. Take 0.3 ml of S source, add 0.9 ml of water for bacterial endotoxin testing and 1.8 ml of 0.9% sodium chloride solution, and vortex mix for 30 seconds to obtain S1. Take 0.3 ml of S1, add 0.9 ml of water for bacterial endotoxin testing and 1.8 ml of 0.9% sodium chloride solution, and vortex mix for 30 seconds to obtain S2. Take 0.2 ml of S2, add 1.4 ml of water for bacterial endotoxin testing, and vortex mix for 30 seconds to obtain S3. Take 0.5 ml of S3, add 0.5 ml of water for bacterial endotoxin testing, and vortex mix for 30 seconds to obtain test sample solution A.
[0115] Solution B (positive control solution for the test sample):
[0116] Take 0.3 ml of S, add 0.42 ml of BET water and 1.8 ml of 0.9% sodium chloride solution, vortex mix for 30 seconds, then add E. 100 0.48 ml of solution is vortexed for 30 seconds to obtain S1; 0.3 ml of S1 is added to 0.9 ml of BET water and 1.8 ml of 0.9% sodium chloride solution, and vortexed for 30 seconds to obtain S2; 0.2 ml of S2 is added to 1.4 ml of BET water, and vortexed for 30 seconds to obtain S3; 0.2 ml of S3 is added to 0.2 ml of BET water, and vortexed for 30 seconds to obtain solution B.
[0117] Add 25 μl each of solutions A, B, C, and D to the microplate, followed by an equal volume of Limulus Amebocyte Lysate (LAL) reagent solution. Prepare two parallel tubes for each concentration. Place the tubes into the microplate reader for reaction. The reaction temperature is (37±1)℃, the working wavelength is 405 nm, and the preset absorbance is 0.1. Record the data.
[0118] Table 3. Effects of 0.9% sodium chloride solution on endotoxins (experimental study)
[0119]
[0120] If the endotoxin recovery rate is between 50% and 200%, it is considered that the freeze-dried natural polymer polysaccharide solution does not have an interfering effect under these test conditions. If the endotoxin recovery rate is outside the specified range, the interfering factors must be removed according to the method in the "Gel Interference Test", and the interference test must be repeated to verify the effectiveness of the treatment.
[0121] Table 4. Results of the experiment on the effect of 0.9% sodium chloride solution on endotoxins.
[0122]
[0123] In summary, the interference tests of this invention show that if the endotoxin recovery rate is within the range of 50% to 200%, then under these test conditions, the freeze-dried natural polymer polysaccharide product solution does not have an interfering effect. When the endotoxin recovery rate is outside the specified range, the interfering factors must be removed according to the method in the "Gel Method Interference Test," and the interference test must be repeated to verify the effectiveness of the treatment.
[0124] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. The understanding of the present invention specification should be based on those skilled in the art. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still make modifications or equivalent substitutions to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.
Claims
1. A method for detecting bacterial endotoxins in freeze-dried natural high-molecular-weight polysaccharide products, characterized in that, In the preparation of the test solution, the detection method first adds auxiliary reagents and mixes them with the lyophilized polysaccharide product, and then adds endotoxin standards for detection.
2. The detection method according to claim 1, characterized in that: The preparation process of the test solution is as follows: Weigh the lyophilized polysaccharide product, add water for bacterial endotoxin testing to dilute and bring to volume, and mix to obtain Sogen; take a certain amount of Sogen and dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S1; take a certain amount of S1 and dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S2; take S2 and dilute it with water for bacterial endotoxin testing, and mix to obtain S3; take S3 and dilute it with water for bacterial endotoxin testing, and mix to obtain the test solution.
3. The detection method according to claim 1 or 2, characterized in that: The auxiliary reagent is selected from 0.9% sodium chloride solution.
4. The detection method according to claim 3, characterized in that: In the preparation of S1 and S2, the volume ratio of water used for bacterial endotoxin testing to 0.9% sodium chloride solution was 1:
2.
5. The detection method according to any one of claims 1-4, characterized in that: It also includes the following steps: a. Dissolve the bacterial endotoxin standard in water for bacterial endotoxin testing, and then dilute it serially to obtain a series of standard solutions. b. Weigh the lyophilized polysaccharide product, dilute it with water for bacterial endotoxin testing, and mix to obtain Sogen; take a certain amount of Sogen, dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S1; take a certain amount of S1, dilute it with water for bacterial endotoxin testing and auxiliary reagents, and mix to obtain S2; take S2, dilute it with water for bacterial endotoxin testing, and mix to obtain S3; take S3, dilute it with water for bacterial endotoxin testing, and mix to obtain the test solution. c. Add bacterial endotoxin standard solution, then dilute with bacterial endotoxin test water, then add a certain volume of S3, mix, and obtain the positive control solution of the test sample. d. Water used for bacterial endotoxin testing is a negative control solution; e. Take a certain amount of the solution prepared in steps a, b, c, and d above and add it into the ELISA plate. Add an equal volume of Limulus amebocyte lysate (LAL) reagent solution. After preparation, place the plate into an ELISA reader for reaction and use the dynamic colorimetric method to detect the content of bacterial endotoxins.
6. The detection method according to any one of claims 1-5, characterized in that, The polysaccharide freeze-dried product is selected from sodium carboxymethyl cellulose or carboxymethyl chitosan.
7. The detection method according to claim 5, characterized in that, In step a, the bacterial endotoxin standard is dissolved in the water for bacterial endotoxin testing and serially diluted to prepare a series of standard solutions with concentrations of 1 EU / mL, 0.1 EU / mL, and 0.01 EU / mL.
8. The detection method according to claim 2 or 5, characterized in that, The mixing method is vortex mixing, and the vortex mixing time is 30-60s.
9. The detection method according to claim 5, characterized in that, In step b, weigh the polysaccharide freeze-dried product, add water for bacterial endotoxin testing to dilute and bring to volume, vortex for 30 seconds to obtain Sogen; take 0.3 ml of Sogen and dilute with 0.9 ml of water for bacterial endotoxin testing and 1.8 ml of 0.9% sodium chloride solution, vortex for 30 seconds to obtain S1. Take 0.3 mL of S1 and dilute it with 0.9 mL of water for bacterial endotoxin testing and 1.8 mL of 0.9% sodium chloride solution. Vortex for 30 s to obtain S2. Take 0.2 mL of S2 and dilute it with 1.4 mL of water for bacterial endotoxin testing. Vortex for 30 s to obtain S3. Take 0.5 mL of S3 and dilute it with 0.5 mL of water for bacterial endotoxin testing. Vortex for 30 s to obtain the test solution. In step c, the bacterial endotoxin working standard is diluted to 0.2 EU / ml with BET water; 0.2 ml of S3 is added to 0.2 ml (0.2 EU / ml) and vortexed for 30 seconds to obtain solution B, which is the positive control solution for the test sample.
10. The detection method according to claim 5, characterized in that, In step e, the reaction conditions for the dynamic colorimetric method are: 36–38℃, and the detection wavelength is 405 nm.