Method for detecting bacterial endotoxin capable of degrading polyester

Abstract

The invention discloses a method for detecting bacterial endotoxin of degradable polyester, and relates to the technical field of insoluble substance impurity detection.The method comprises the steps that the degradable polyester is dissolved with dichloromethane, and an organic phase solution is obtained; bET water is added, and water-phase extract liquor is extracted through oscillation; and carrying out bacterial endotoxin detection on the test solution by using the tachypleus amebocyte lysate. According to the method, the degradable polyester is dissolved by adopting dichloromethane, so that the problem that the material is difficult to dissolve is solved, the interference effect on bacterial endotoxin detection is eliminated, the detection result is accurate and reliable, the reproducibility is good, and the method has important significance in guaranteeing the safety of the polymer medical material; more powerful technical support can be provided for quality control of related medical implant materials, and the safety and reliability in medical application are further improved.

Description

Technical Field

[0001] This invention provides a method for detecting bacterial endotoxins in biodegradable polyester, relating to the field of detection technology for poorly soluble impurities. Background Technology

[0002] Biodegradable polyesters, as a class of high-performance medical biodegradable materials, play an irreplaceable role in many medical fields such as surgical sutures, skin regeneration and repair, medical aesthetic fillers, bone and soft tissue defect repair, and bioresorbable scaffolds. The safety and reliability of these medical materials that are directly implanted into the human body are related to the patient's postoperative recovery and even life and health. Therefore, it is crucial to conduct strict testing on bacterial endotoxins in them. This is not only a mandatory requirement of the Good Manufacturing Practice for Medical Devices for implantable medical devices, but also a key link in controlling medical risks from the source. However, in actual testing, the unique physicochemical properties of biodegradable polyesters present significant challenges: traditional water extraction methods cannot effectively dissolve these materials—the dense ester bonds and high crystallinity in their molecular structure make it difficult for water molecules to penetrate, resulting in a large number of bacterial endotoxins encapsulated inside the material not being released into the extract, causing serious false negatives and failing to accurately reflect the endotoxin contamination level of the material; although there have been attempts to use organic solvents (such as dichloromethane, acetone, etc.) to assist in dissolution in existing technologies, these organic solvents are prone to residues during the dissolution process, and the residual solvents can interfere with the enzymatic reactions in the Limulus Amebocyte Lysate (LAL) reagent detection system, impair the sensitivity of the reagent, and thus lead to false negative or false positive results, seriously affecting the accuracy and reliability of the detection. In their research published in *Chinese Journal of Drug Surveillance*, Volume 21, Issue 3, 2024, Zhang Chenxue, Pei Yusheng, Cai Tong, and other scholars clearly pointed out that the dissolution method for poorly soluble raw materials, excipients, and preparations needs to be determined comprehensively based on their physicochemical properties and interference test results. Common dissolution pathways include organic solvent dissolution and surfactant-assisted dissolution. However, while this research provided direction for solving the dissolution problem of poorly soluble materials, it failed to address the synergistic challenge between dissolution efficiency and detection interference specific to the molecular structure of biodegradable polyesters. It could neither achieve complete dissolution of the material to release all encapsulated endotoxins nor avoid the interference of substances introduced during dissolution on bacterial endotoxin detection. With the continuous expansion of the application of biodegradable polyesters in high-end medical fields such as minimally invasive surgery and tissue engineering, the requirements for controlling their bacterial endotoxin limits are becoming increasingly stringent. Developing a method that can efficiently dissolve biodegradable polyesters, ensure complete release of encapsulated endotoxins, and guarantee no interference with the bacterial endotoxin detection system, thereby ensuring reliable test results, has become a key issue urgently needing breakthroughs in the field of medical material quality control. Summary of the Invention

[0003] To address the above problems, this invention provides a method for detecting biodegradable polyester bacterial endotoxins, comprising the following steps:

[0004] (a) The biodegradable polyester was dissolved in dichloromethane to obtain an organic phase solution;

[0005] (b) Add BET water to the organic phase solution, shake to extract, centrifuge, and collect the aqueous phase extract;

[0006] (c) Dilute the aqueous extract with BET water to a biodegradable polyester concentration of 4-10 mg / mL to obtain the test solution;

[0007] (d) The bacterial endotoxins in the test solution were detected by photometry using horseshoe crab reagent at a wavelength of 405 nm.

[0008] Preferably, in step (b), the amount of BET water used is in a volume ratio of (1-3):1 to the organic phase solution; the shaking time is 10-15 min, and the centrifugation conditions are 2000-2500 r / min for 5-10 min; the concentration of degradable polyester in the resulting aqueous extract is 12.5-50 mg / mL.

[0009] Preferably, the concentration of the diluted biodegradable polyester in step (c) is 5 mg / mL.

[0010] Preferably, the detection method has a sensitivity of 0.02 EU / mL and a reaction time of 60 min.

[0011] Preferably, the above method further includes an interference verification step: dichloromethane interference test: no interference with bacterial endotoxins in the sample at the standard curve concentration.

[0012] Preferably, the dilution factor in step (c) is no greater than 10 times.

[0013] Preferably, all glassware must be sterilized by dry heat at 250°C for at least 0.5 hours.

[0014] Preferably, the biodegradable polyester includes polylactic acid, polycaprolactone, or copolymers thereof.

[0015] Furthermore, the detection range of the Limulus amebocyte lysate (LAL) reagent is 0.01-10 EU / mL.

[0016] Preferably, the minimum sensitivity of the Limulus amebocyte lysate (LAL) reagent is 0.02 EU / mL.

[0017] The beneficial effects of this invention are:

[0018] This method, employing a system combining dichloromethane dissolution and BET water extraction, successfully overcomes the dissolution bottleneck of biodegradable polyesters, enabling the complete release of bacterial endotoxins encapsulated within the material. Simultaneously, a dilution strategy of a certain factor effectively eliminates potential interference from organic solvents and the material matrix on the detection of Limulus Amebocyte Lysate (LAL) reagents. The measured recovery rate of this method is within the ideal range, with good reproducibility and a high level of sensitivity, significantly outperforming traditional methods which have a weak detection capability for encapsulated endotoxins. This method provides a reliable means for the quality control of implantable medical devices. The biodegradable polyester bacterial endotoxin detection method provided in this application has significant effects. Using dichloromethane to dissolve biodegradable polyester not only solves the problem of poor solubility but also eliminates interference with bacterial endotoxin detection, ensuring accurate, reliable, and reproducible results. This is of great significance for ensuring the safety of polymeric medical materials and can provide stronger technical support for the quality control of related medical implant materials, further improving the safety and reliability in medical applications. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0022] Experimental materials and instruments

[0023] Experimental materials: Limulus amebocyte lysate (LAL) reagent 1 (Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2407030, specification: 0.35 mL / vial, detection range: 10–0.01 EU / mL); LAL reagent 2 (Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number: 24030318, specification: 0.35 mL / vial, detection range: 10–0.01 EU / mL); Bacterial endotoxin working standard 1 (Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2301041, specification: 10 EU / vial); Bacterial endotoxin working standard 2 (Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number: 240202, specification: 10 EU / vial); BET water (Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2404170, specification: 50 mL); Bacterial endotoxin test reaction plate (Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2024022701, specification: 96 wells). Dichloromethane (Chengdu Kelong Chemical Co., Ltd., analytical grade, batch number 2023062501).

[0024] Instruments: Microplate reader (MuLtiskan FC / ET, Thermo Fisher Scientific); Electronic analytical balance (FA2204, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); Automatic vortex mixer (Vortex-M, Shanghai Huxi Industrial Co., Ltd.); Electric centrifuge (80-2, Changzhou Jintan District Baitaxinbao Instrument Factory).

[0025] Example 1: Reliability Verification of Standard Curve

[0026] Dissolve one vial of bacterial endotoxin working standard in 1.0 mL of BET water, seal the vial, and vortex for 10 minutes using an automatic vortex mixer to prepare a 10 EU / mL working standard solution. Then, serially dilute with BET water to prepare bacterial endotoxin standard solutions of 2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL, respectively. Prepare three parallel tubes for each concentration. Use BET water as a negative control, in two parallel tubes. The assay was performed using an ELISA reader. The reaction times were 60 min for Zhanjiang Andus horseshoe crab reagent and 90 min for Fuzhou Xinbei horseshoe crab reagent. The detection sensitivity was 0.02 EU / mL, the detection wavelength was 405 nm, and the preset OD values ​​were 0.1 for Zhanjiang Andus horseshoe crab reagent and 0.02 for Fuzhou Xinbei horseshoe crab reagent. A standard curve was plotted with the logarithm of bacterial endotoxin concentration (LgC) on the x-axis and the logarithm of average reaction time (LgT) on the y-axis. The results are shown in Table 1 below. Horseshoe crab reagent 1: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2407030; Horseshoe crab reagent 2: Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number: 24030318; Bacterial endotoxin working standard 1: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2301041; Bacterial endotoxin working standard 2: Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number: 240202.

[0027] Table 1

[0028]

[0029]

[0030] The results in Table 1 above indicate that the reaction time of the negative control was longer than that of the lowest concentration in the standard curve, with a coefficient of variation (CV) ≤ 20%. Linear regression analysis was performed on all data, yielding a regression equation with a correlation coefficient |γ| > 0.980 (see Table 1). The standard curve was valid, and the experiment was effective.

[0031] Example 2: Dichloromethane Interference Test

[0032] Dissolve one vial of bacterial endotoxin working standard in 1.0 mL of BET water, seal, and vortex for 10 min on an automatic vortex mixer to prepare a 10 EU / mL working standard solution A. Add 0.5 mL of solution A to 0.5 mL of dichloromethane, mix well, and extract by shaking. The endotoxin content in the aqueous solution is equivalent to 5 EU / mL. After vortexing, centrifuge at 2500 rpm for 5 min. Dilute the aqueous extract to prepare bacterial endotoxin standard solutions with concentrations of 2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL, respectively, for bacterial endotoxin recovery determination. Detection was performed using an ELISA reader with a reaction time of 60 min, a detection sensitivity of 0.02 EU / mL, a detection wavelength of 405 nm, and a preset OD value of 0.1. A standard curve was plotted with the logarithm of bacterial endotoxin concentration (LgC) on the x-axis and the logarithm of average reaction time (LgT) on the y-axis. The reaction time for each concentration was measured, and the bacterial endotoxin content and recovery rate were automatically calculated. The results are shown in Table 2 below. Limulus amebocyte lysate (LAL) reagent: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2407030; Bacterial endotoxin working standard: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2301041.

[0033] Table 2

[0034]

[0035] The results in Table 2 above demonstrate that when using dichloromethane to extract the bacterial endotoxin working standard solution via shaking, no interference was observed during the bacterial endotoxin recovery test. This indicates that dichloromethane dissolved in water does not interfere with the Limulus Amebocyte Lysate (LAL) reagent, and the method of dissolving the sample in dichloromethane and then extracting with water can be used for subsequent experiments. This embodiment, through a dichloromethane interference test, eliminated background interference from dichloromethane, confirming that the standard curve prepared by this method can be used for regression calculations of sample detection data.

[0036] Example 3: Preliminary Test of Bacterial Endotoxin Interference with Degradable Polyester

[0037] Preparation of experimental solutions: a) Test sample group: Weigh 100 mg of biodegradable polyester, dissolve it in 1 mL of dichloromethane, extract with an equal volume of BET water, centrifuge at 2500 r / min for 5 min, and take the aqueous extract to obtain a 50 mg / mL test sample solution. Dilute the aqueous extract with BET water by 5, 10, and 12.5 times respectively to prepare a concentration of 10 mg / mL (S 10 Test solutions of 5 mg / mL (S5) and 4 mg / mL (S4).

[0038] b) Positive control group of test sample: Take S 20 S 10The S8 test solution was mixed with an equal volume of 0.4 EU / mL bacterial endotoxin standard solution to prepare a solution containing S. 10 (E 0.2 ), S5(E 0.2 ), S4(E 0.2 Positive solution of bacterial endotoxin standard.

[0039] c) Positive control group: Take one vial of bacterial endotoxin standard (10 EU / vial), add 1 mL of BET water to reconstitute, prepare a bacterial endotoxin standard solution with a concentration of 10 EU / mL, and dilute with BET water to E... 2.0 E 0.2 E 0.02 Bacterial endotoxin standard solution.

[0040] d) Negative control group: BET water.

[0041] Detection: Take 25 μL of each concentration solution from the test sample group, positive control group, positive control group, and negative control group, and place them in an ELISA plate. Take two aliquots of each solution, and add 25 μL of reconstituted Limulus amebocyte lysate (LAL) reagent (10–0.01 EU) to each. Immediately after addition, place the plate in an ELISA reader for detection. The reaction time is 60 min, the detection sensitivity is 0.02 EU / mL, the detection wavelength is 405 nm, and the preset OD value is 0.1. Plot a standard curve with the logarithm of bacterial endotoxin concentration (LgC) on the x-axis and the logarithm of average reaction time (LgT) on the y-axis. Measure the reaction time for each concentration, and automatically calculate the bacterial endotoxin content and recovery rate of the test sample. Record the results (see Table 3). Limulus amebocyte lysate reagent: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2407030; Bacterial endotoxin working standard: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2301041.

[0042] Table 3

[0043]

[0044] The results in Table 3 above indicate that, using dichloromethane to dissolve the sample and extracting with water, diluting the aqueous extract by more than 5 times, the recovery rate of the positive control group for the test sample was in the range of 50%-200%, and the test sample had no interfering effect at this concentration. Therefore, a 10-fold dilution of the aqueous extract was chosen for the formal interference test.

[0045] Example 4: Degradable Polyester Bacterial Endotoxin Interference Test

[0046] Preparation of experimental solutions: a) Test sample group: Weigh 100 mg of biodegradable polyester, dissolve it in 1 mL of dichloromethane, extract with an equal volume of BET water, centrifuge at 2500 r / min for 5 min, and take the aqueous extract to obtain a 50 mg / mL test sample solution. Dilute the aqueous extract with BET water by 5 and 10 times respectively to prepare a concentration of 10 mg / mL (S 10 ), 5 mg / mL (S5) of the test solution.

[0047] b) Positive control group of test sample: Take S 10 The test solution was mixed with an equal volume of 0.4 EU / mL bacterial endotoxin standard solution to prepare S 5(E0.2) Positive solution of bacterial endotoxin standard.

[0048] c) Positive control group: Take one vial of bacterial endotoxin standard (10 EU / vial), add 1 mL of BET water to reconstitute, prepare a bacterial endotoxin standard solution with a concentration of 10 EU / mL, and dilute with BET water to E... 2.0 E 0.2 E 0.02 Bacterial endotoxin standard solution.

[0049] d) Negative control group: BET water.

[0050] Detection: (1) Take 25 μL of each concentration solution of the test sample group, the positive control group, the positive control group, and the negative control group, and place them in an ELISA plate. Take two portions of each solution and add 25 μL of reconstituted Limulus amebocyte lysate (LAL) reagent (10-0.01 EU) to each. After adding, immediately place the plate in an ELISA reader for detection. The reaction time is 60 min, the detection sensitivity is 0.02 EU / mL, the detection wavelength is 405 nm, and the preset OD value is 0.1. Plot a standard curve with the logarithm of bacterial endotoxin concentration (LgC) as the abscissa and the logarithm of average reaction time (LgT) as the ordinate. Measure the reaction time of each concentration and automatically calculate the bacterial endotoxin content and recovery rate of the test sample. Record the results, see Table 4. Limulus amebocyte lysate reagent: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2407030; Bacterial endotoxin working standard: Zhanjiang Andus Biotechnology Co., Ltd., batch number: 2301041;

[0051] (2) Take 25 μL of each concentration solution from the test sample group, positive control group, positive control group, and negative control group, and place them in an ELISA plate. Take two portions of each solution, and add 25 μL of reconstituted Limulus amebocyte lysate (LAL) reagent (10–0.01 EU) to each. Immediately after addition, place the plate in an ELISA reader for detection. The reaction time is 90 min, the detection sensitivity is 0.02 EU / mL, the detection wavelength is 405 nm, and the preset OD value is 0.02. Plot a standard curve with the logarithm of bacterial endotoxin concentration (LgC) on the x-axis and the logarithm of average reaction time (LgT) on the y-axis. Measure the reaction time for each concentration, and automatically calculate the bacterial endotoxin content and recovery rate of the test sample. Record the results, as shown in Table 4. Limulus amebocyte lysate reagent: Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number: 24030318; Bacterial endotoxin working standard: Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number: 240202.

[0052] Table 4

[0053]

[0054]

[0055] The results in Table 4 above show that: by dissolving the sample in dichloromethane and extracting with water, and diluting the aqueous extract 10 times, the recovery rate of the positive control group of the test sample was in the range of 50%-200%. After the test sample was treated with this method, the interference effect could be effectively eliminated. Therefore, this method can be used to test for bacterial endotoxins in biodegradable polyesters.

[0056] Example 4 uses the complete scheme obtained from the above examples for sample testing.

[0057] 1. Establishing the standard curve:

[0058] Bacterial endotoxin standards were diluted with BET water to 2.0, 0.2, and 0.02 EU / mL.

[0059] Take 25 μL of each concentration solution + 25 μL of Limulus amebocyte lysate (Zhanjiang Andus, batch number 2407030);

[0060] Monitor with an ELISA reader (405nm) for 60 minutes and record the time to reach the limit.

[0061] A curve was fitted with LgC as the x-axis and LgT as the y-axis.

[0062] result:

[0063]

[0064] 2. Testing of biodegradable polyester

[0065] Dissolve 100 mg of polylactic acid in 1 mL of dichloromethane; add 1 mL of BET water, vortex for 5 min, and centrifuge at 2500 r / min for 5 min; take the aqueous phase and dilute it 10 times with BET water (final concentration 5 mg / mL); take 25 μL of the solution and add 25 μL of 0.2 EU / mL endotoxin standard → test sample positive solution;

[0066] The detection method is the same as in Example 1.

[0067] result:

[0068] Test sample Spiked concentration (EU / mL) Measured value (EU / mL) Recovery rate (%) Polylactic acid 0.2 0.195 97.5

[0069] 3. Verification of dichloromethane interference:

[0070] Take 0.5 mL of 10 EU / mL endotoxin standard solution + 0.5 mL of dichloromethane; vortex for 5 min, centrifuge at 2500 r / min for 5 min; take the aqueous phase and dilute to 0.2 EU / mL for detection.

[0071] result:

[0072] Spiked concentration (EU / mL) Measured value (EU / mL) Recovery rate (%) 0.2 0.203 101.5

[0073] Comparative Example 1 (Replacement Solvent):

[0074] Ethanol was used instead of dichloromethane to dissolve polylactic acid, and the rest was the same as in Example 4.

[0075] Results: Recovery rate was 32.1% (ethanol residue inhibited the reaction of Limulus amebocyte lysate (LAL) reagent).

[0076] Comparative Example 2 (without dilution):

[0077] The aqueous extract was directly detected (concentration 50 mg / mL), and the rest was the same as in Example 4.

[0078] Results: Recovery rate 215% (high concentration matrix interference).

[0079] Comparative Example 3 (Changing Centrifugal Force):

[0080] The centrifugation conditions were changed to 1000 r / min for 5 min, and the rest were the same as in Example 4.

[0081] Results: Recovery rate 68.3% (residual dichloromethane in aqueous phase).

[0082] Comparative Example 4 (Dilution beyond the range):

[0083] Dilute 20 times (concentration 2.5 mg / mL), otherwise the same as in Example 4.

[0084] Results: Recovery rate was 41.7% (endotoxin concentration was below the detection limit of 0.02 EU / mL).

[0085] The statistical results of the proportions obtained in Example 2 are shown in the table below:

[0086] plan Dissolved solvent Dilution factor Centrifugation (r / min) Recovery rate (%) Example 2 dichloromethane 10 2000 97.5 Comparative Example 1 ethanol 10 2000 32.1 Comparative Example 2 dichloromethane Undiluted 2000 215.0 Comparative Example 3 dichloromethane 10 1000 68.3 Comparative Example 4 dichloromethane 20 2000 41.7

[0087] The core of this invention lies in the synergistic effect of four steps: dichloromethane dissolution, BET water extraction, 10-fold dilution, and centrifugation at 2500 r / min.

[0088] Solvent selection: Dichloromethane has a significantly better solubility efficiency (>99%) for polyester than ethanol (<60%), and its low polarity ensures complete release of endotoxin (98.5% release rate in encapsulated form vs. 42.3% for ethanol).

[0089] Dilution factor: A 10-fold dilution reduced the polyester concentration to 5 mg / mL, which eliminated matrix interference (recovery rate 97.5%) and avoided excessive dilution that would cause the endotoxin concentration to fall below the detection limit (comparative example 4, recovery rate 41.7%).

[0090] Centrifugation parameters: Centrifugation at 2500 r / min completely separated dichloromethane residue (residual amount <0.01%), while at 1000 r / min the residual solvent inhibited the activity of the Limulus amebocyte lysate reagent (comparative example 3 recovery rate 68.3%).

[0091] Synergistic effect:

[0092] The residual solvent during ethanol dissolution directly inhibited the coagulation cascade reaction of the horseshoe crab reagent (comparative example 1, recovery rate 32.1%).

[0093] High concentrations of polyester in undiluted samples adsorbed endotoxins, leading to false positives (Comparative Example 2, recovery rate 215.0%).

[0094] This method achieves a recovery rate of 97.5% (RSD = 3.2%) and a sensitivity of 0.02 EU / mL through the triple synergy of solvent-dilution-centrifugation, overcoming the detection blind spot (false negative rate > 50%) of traditional methods for encapsulated endotoxins. By precisely synergizing various technical features, it solves the coupling problem of "dissolution-release-interference removal" in the detection of endotoxins in biodegradable polyesters.

[0095] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0096] The present invention and its embodiments have been described above. This description is not restrictive, but merely one embodiment of the present invention, and the actual application is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for detecting bacterial endotoxins in biodegradable polyester, characterized in that, Includes the following steps: (a) The biodegradable polyester was dissolved in dichloromethane to obtain an organic phase solution; (b) Add BET water to the organic phase solution, shake to extract, centrifuge, and collect the aqueous phase extract; (c) Dilute the aqueous extract with BET water to a biodegradable polyester concentration of 4-10 mg / mL to obtain the test solution; (d) The bacterial endotoxins in the test solution were detected by photometry using horseshoe crab reagent at a wavelength of 405 nm.

2. The method according to claim 1, characterized in that, In step (b), the amount of BET water used is in a volume ratio of (1-3):1 to the organic phase solution; the shaking time is 5-15 min, and the centrifugation conditions are 2000-2500 r / min for 5-10 min; the concentration of degradable polyester in the resulting aqueous extract is 12.5-50 mg / mL.

3. The method according to claim 1, characterized in that, The concentration of the diluted biodegradable polyester in step (c) is 5 mg / mL.

4. The method according to claim 1, characterized in that, The detection method has a sensitivity of 0.02 EU / mL and a reaction time of 60 min.

5. The method according to claim 1, characterized in that, It also includes interference verification steps: Dichloromethane interference test: No interference with bacterial endotoxins in the sample at the standard curve concentration.

6. The method according to claim 1, characterized in that, The dilution factor in step (c) is no greater than 10 times.

7. The method according to claim 1, characterized in that, All glassware must be sterilized by dry heat at 250°C for at least 0.5 hours.

8. The method according to claim 1, characterized in that, The biodegradable polyester includes polylactic acid, polycaprolactone, or copolymers thereof.

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