A method for detecting endotoxins in high molecular weight degradable polyesters by gelation of bacteria

Abstract

This invention discloses a gel detection method for bacterial endotoxins in high molecular weight biodegradable polyester materials, eliminating adsorption interference, belonging to the field of biomaterial safety testing technology. The method first uses dichloromethane to completely dissolve the polyester, disrupting its solid structure and releasing potentially adsorbed endotoxins. Then, liquid-liquid extraction is performed using water for bacterial endotoxin testing to transfer the endotoxins to the aqueous phase. Finally, a specific endotoxin dispersant is used to dilute the aqueous extract, effectively shielding the re-adsorption of free endotoxins by the hydrophobic groups of the polyester. The treated sample can be directly detected using the highly sensitive Limulus Amebocyte Lysate (LAL) gel electrophoresis method. This method is simple to operate, has good reproducibility, completely eliminates adsorption interference, and accurately and reliably controls the bacterial endotoxin limits of high molecular weight biodegradable polyester materials, ensuring their safety as medical materials.

Description

Technical Field

[0001] This invention relates to a method for detecting bacterial endotoxins in high molecular weight degradable polyester gels that eliminates adsorption interference, belonging to the field of biomaterial safety testing technology. Background Technology

[0002] Bacterial endotoxins are lipopolysaccharide components of the cell walls of Gram-negative bacteria, possessing strong pyrogenic properties and posing a significant threat to the safety of medical materials. The gel permeation test (LAL) is the standard method for detecting bacterial endotoxins as stipulated in pharmacopoeias of various countries. However, high-molecular-weight biodegradable polyesters, such as polylactic acid, polycaprolactone, and their copolymers, exhibit long molecular chains and strong hydrophobicity. Their hydrophobic ester bonds have a strong affinity for the hydrophobic A end of endotoxins, leading to the strong adsorption of endotoxins into the sample. In conventional aqueous detection systems, the adsorbed endotoxins cannot be effectively released and react with the LAL reagent, resulting in false negatives that severely underestimate the true contamination level and pose a significant clinical risk. While existing technologies can use organic solvents or dispersants to solubilize poorly soluble samples, for materials like high-molecular-weight polyesters that combine poor solubility with strong adsorption, a single approach often fails to address the fundamental problem. For example, simply dissolving and diluting with organic solvents may result in endotoxins being encapsulated within precipitated polymer particles; treating solid samples with dispersants alone cannot dissociate the already adsorbed endotoxins. Therefore, there is an urgent need to develop a detection method that can effectively break down adsorption, complete release, and accurately quantify bacterial endotoxins in high molecular weight biodegradable polyesters to meet stringent quality control requirements. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for detecting bacterial endotoxins using high molecular weight degradable polyester gels that eliminate adsorption interference, comprising the following steps: S1: Dissolve high molecular weight biodegradable polyester in an organic solvent to form an organic phase solution; S2: Add bacterial endotoxin test water to the organic phase solution for extraction, shake and centrifuge, and take the upper aqueous phase to obtain the test sample stock solution; S3: Dilute the test sample stock solution to a set concentration using an endotoxin dispersant to obtain the test sample solution; S4: The test solution was examined for bacterial endotoxins using the gel electrophoresis method and Limulus amebocyte lysate (LAL) reagent.

[0004] Preferably, the molecular weight of the high molecular weight biodegradable polyester described in step S1 is not less than 250,000 Daltons.

[0005] Preferably, the high molecular weight biodegradable polyester in step S1 is selected from one or more of polylactic acid, polycaprolactone, or copolymers thereof.

[0006] Preferably, the organic solvent in step S1 is dichloromethane; the ratio of the organic solvent to the high molecular weight biodegradable polyester is 1-1.5 mL of dichloromethane per 0.05 g of polyester.

[0007] Preferably, the volume ratio of the water used for bacterial endotoxin testing in step S2 to the organic phase solution obtained in step S1 is 1:1 to 1:2.

[0008] Preferably, the oscillation in step S2 is oscillation using a vortex mixer for 5-8 minutes; the centrifugation conditions are centrifugation at a speed of 2500-4000 r / min for 5-8 minutes; and the concentration of high molecular weight biodegradable polyester in the test sample stock solution is 17-25 mg / mL.

[0009] Preferably, the concentration set in step S3 is 6-8 mg / mL.

[0010] Preferably, the bacterial endotoxin content in the endotoxin dispersant described in step S3 is less than 0.006 EU / mL.

[0011] Preferably, the labeling sensitivity of the horseshoe crab reagent in step S4 is 0.03 EU / mL; the bacterial endotoxin test using the gel method in step S4 is specifically as follows: the test solution is mixed with an equal volume of the reconstituted horseshoe crab reagent, and reacted in a water bath at 37±1℃ for 60±2 min. After the reaction, the presence or absence of bacterial endotoxin is determined by observing whether a gel forms.

[0012] Preferably, before step S4, an interference test verification step is included: preparing a positive solution of the test sample containing a known concentration of endotoxin. The preparation method is as follows: the test sample stock solution obtained in step S2 is mixed with an endotoxin standard solution prepared with an endotoxin dispersant at a volume ratio of 1:1, and then diluted with an endotoxin dispersant so that the concentration of high molecular weight degradable polyester in the final solution is 6 mg / mL, and the concentration of endotoxin is twice the sensitivity λ indicated by the Limulus Amebocyte Lysate (LAL) reagent. If the detection result of the positive solution of the test sample is consistent with the detection result of the positive control solution with the same endotoxin concentration, it is determined that the test sample has no interference at this concentration.

[0013] Preferably, in the interference test verification step, the measured endotoxin concentration λ of the positive solution of the test sample is compared. B Endotoxin concentration λ compared to the positive control solution C To determine interference, when λ B At 0.5λ C up to 2λ C When the concentration is within the specified range, it is considered to be interference-free; the measured endotoxin concentration λ BThe results were obtained by calculating the gelation results of positive solutions of the test sample at multiple dilution gradients. Validated through examples, this method demonstrates high detection sensitivity, with a detection limit as low as 0.03 EU / mL.

[0014] Preferably, the high molecular weight biodegradable polyester is a medical or pharmaceutical grade biodegradable material.

[0015] The above testing methods can be applied to quality control or product release testing in the production of high molecular weight biodegradable polyester materials.

[0016] The beneficial effects of this invention are: The detection method provided by this invention has the following significant advantages: First, it innovatively combines organic solvent dissolution, aqueous phase extraction, and endotoxin dispersant dilution, forming a synergistic technical solution. The dissolution step completely destroys the polymer aggregation state, releasing the embedded or adsorbed endotoxins; the extraction step selectively transfers the endotoxins from the organic phase to the aqueous phase, achieving matrix separation; the dispersant step creates a protective environment in the aqueous phase, preventing the endotoxins from being re-adsorbed by polyester fragments or impurities during dilution and detection, thus fundamentally solving the problem of adsorption interference. Second, the method has a standardized operating procedure, mild conditions, and requires no complex equipment. It can be completed through conventional vortexing, centrifugation, dilution, and water bath reaction, exhibiting excellent reproducibility and good consistency between results from different operators and laboratories. Third, it has high detection sensitivity, with a detection limit of 0.03 EU / mL, which meets the stringent limits for medical materials in the pharmacopoeia. Fourth, the method has a wide range of applications, applicable to different types of biodegradable polyesters such as PLA, PCL and their copolymers with molecular weights ranging from 250,000 to over 1 million Daltons. It is highly versatile and provides a reliable technical guarantee for the safe production and quality control of these important biomaterials. Attached Figure Description

[0017] Figure 1 These are photographs of the unagglomerated state of the horseshoe crab reagent in some comparative examples of this invention; Figure 2 These are photographs of the aggregation state of the horseshoe crab reagent in some embodiments of the present invention. Detailed Implementation

[0018] The technical solutions 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. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] 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.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0021] Materials: Limulus amebocyte lysate (TAL, labeled sensitivity λ=0.03EU / mL, purchased from Zhanjiang Andus Biotechnology Co., Ltd. or Xiamen Limulus amebocyte lysate Biotechnology Co., Ltd.); Bacterial endotoxin working standard (RSE, 10EU / vial or 15EU / vial, source as above); Water for bacterial endotoxin testing (BET water, endotoxin content <0.003EU / mL, source as above); Endotoxin dispersant (main component is nonionic surfactant, endotoxin content <0.006EU / mL, source as above); Dichloromethane (analytical grade); Polylactic acid (PLA, Mw=300000), polycaprolactone (PCL, Mw=800000), and PLA-PCL copolymer (Mw=500000), all medical grade.

[0022] Instruments: Digital display constant temperature water bath (accuracy 37±1℃); vortex mixer; low speed centrifuge; electronic analytical balance (0.01%); pipettes; sterile enzyme-free centrifuge tubes, pyrogen-free glass test tubes and pipette tips.

[0023] Example 1: Detection of bacterial endotoxins in polylactic acid (PLA, Mw=300000) using a standard protocol Sample Dissolution: Accurately weigh 50.0 mg of polylactic acid (PLA) sample using an electronic analytical balance and place it in a 5 mL sterile, enzyme-free centrifuge tube. Accurately add 1.0 mL of dichloromethane using a micropipette. Immediately tighten the centrifuge tube cap and place it on a vortex mixer. Vortex continuously at maximum speed for 3 minutes until all solid sample at the bottom and walls of the centrifuge tube is completely dissolved, and the solution is homogeneous, clear, and transparent; this is the organic phase solution.

[0024] Aqueous phase extraction: Using a micropipette, precisely add 1.0 mL of BET water (i.e., a volume ratio of 1:1) to the above organic phase solution. Tightly cap the tube and hold it in a vortex mixer, vigorously shaking at maximum speed for 5 min to ensure sufficient contact between the two phases for extraction. After shaking, immediately place the centrifuge tube in a low-speed centrifuge, balance it, and centrifuge at 2500 rpm for 5 min. After centrifugation, the solution clearly separates into two layers: a lower layer of dichloromethane organic phase and an upper layer of aqueous phase. Carefully insert a micropipette below the liquid surface and aspirate approximately 0.8-0.9 mL of the upper aqueous phase into another pre-labeled clean, pyrogen-free glass tube. This solution is labeled as test sample stock solution S25, in which the theoretically calculated concentration of PLA is approximately 25 mg / mL.

[0025] Preparation of the test solution: Accurately measure 300 μL of the test solution stock solution S25 obtained in step 2 using a micropipette and add it to a new glass test tube. Then add 950 μL of endotoxin dispersant to the test tube. Tightly cap the tube and mix on a vortex mixer for 30 seconds to ensure the solution is completely homogeneous. This solution is labeled as test solution S6, in which the theoretically calculated final concentration of PLA is approximately 6 mg / mL.

[0026] Preparation of endotoxin standard solution and spiking solution: a) 0.25 EU / mL endotoxin standard solution (E0.25): Take one vial of bacterial endotoxin working standard (10 EU / vial), add 1.0 mL of endotoxin dispersant to dissolve, and vortex for 10 min to prepare a stock solution with a concentration of 10 EU / mL. Then perform serial dilutions using the endotoxin dispersant: Take 200 μL of the 10 EU / mL stock solution, add 1800 μL of endotoxin dispersant, mix for 30 s to obtain a 1.0 EU / mL solution; then take 250 μL of the 1.0 EU / mL solution, add 750 μL of endotoxin dispersant, mix for 30 s to obtain an endotoxin standard solution with a concentration of 0.25 EU / mL (E0.25).

[0027] b) Positive Solution (2λ): Take a glass test tube and accurately add 300 μL of test sample stock solution S25 and 300 μL of E0.25 endotoxin standard solution. Vortex mix for 10 s. Then add 650 μL of endotoxin dispersant to the mixture. Vortex mix again for 30 s. The final result is a 1.25 mL solution, labeled as the positive solution (S6, E0.06), with a PLA concentration of approximately 6 mg / mL and a theoretical endotoxin concentration of approximately 0.06 EU / mL (i.e., twice the sensitivity λ of the Limulus Amebocyte Lysate (LAL) reagent, λ = 0.03 EU / mL, denoted as 2λ).

[0028] c) Positive control solution (2λ): Take a glass test tube and accurately add 300 μL E 0.25 endotoxin standard solution and 300 μL B E water. Mix for 10 seconds. Then add 650 μL of endotoxin dispersant and mix for 30 seconds. The resulting 1.25 mL solution is labeled as the positive control solution (E 0.06), with a theoretical endotoxin concentration of approximately 0.06 EU / mL.

[0029] Gel-coating assay: a) Reconstitution of Limulus Ampoule Reagent: Take 10 ampoules of Limulus Ampoule Reagent with a sensitivity of 0.03 EU / mL (0.1 mL / ampoule), and gently tap them vertically to concentrate the reagent powder at the bottom of the tube. Using a micropipette, add 0.1 mL of BET water to each Limulus Ampoule Reagent Ampoule as a reconstitution solution. Gently rotate the ampoule to completely dissolve the contents, avoiding the generation of air bubbles.

[0030] b) Group addition: The reconstituted horseshoe crab reagent solution was directly added to the original ampoule for the reaction. Using a micropipette, 0.1 mL of the corresponding solution was added to each tube according to the following groups: - Test sample group (S6): 2 tubes, add 0.1 mL of test sample solution S6 to each tube.

[0031] - Positive test sample group (2λ): 4 tubes, each tube containing 0.1 mL of positive test sample solution (S6, E0.06).

[0032] - Positive control group (2λ): 2 tubes, each with 0.1 mL of positive control solution (E0.06) added.

[0033] - Negative control group (NC): 2 tubes, each with 0.1 mL of BET water added.

[0034] c) Reaction and Observation: After adding the sample, gently shake each ampoule to mix the contents. Seal all ampoule openings with a special sealing film. Place the ampoule vertically into a preheated digital display constant temperature water bath at 37.0℃, ensuring the water level is higher than the liquid level inside the ampoule. Maintain the water bath temperature for 60±2 minutes. After the water bath is complete, gently remove the ampoule from the water bath, avoiding any vibration. At room temperature, slowly invert each ampoule 180° and observe whether a gel has formed inside. Judgment criteria: A positive result (+) is recorded if a firm, complete gel forms inside the ampoule and remains intact after inversion (180°) without slipping off the ampoule wall. A negative result (-) is recorded if no gel forms inside the ampoule, or if the formed gel is incomplete, loose, viscous, and slips off the ampoule wall upon inversion.

[0035] Results Recording and Interpretation: Record the gelation results of all ampoules. Results of this example: Test sample group (S6): both ampoules were negative (-). Test sample positive group (S6, E0.06): all 4 ampoules were positive (+). Positive control group (E0.06): both ampoules were positive (+). Negative control group (NC): both ampoules were negative (-). The results indicate that at a concentration of 6 mg / mL, the positive and positive control groups reacted consistently, demonstrating that PLA has no interfering effect on bacterial endotoxin detection at this concentration.

[0036] Example 2: Detection of polycaprolactone (PCL, Mw=800000) Test sample: Polycaprolactone (PCL), weight average molecular weight (Mw) of 800,000 Daltons, medical grade.

[0037] Sample dissolution: Accurately weigh 50.0 mg of polycaprolactone (PCL) sample and place it in a 5 mL sterile enzyme-free centrifuge tube. Add 1.0 mL of dichloromethane, tighten the cap, and vortex for 3 min until completely dissolved to obtain a clear organic phase solution.

[0038] Aqueous phase extraction: Add 1.0 mL of BET water to the organic phase solution. Tightly cap the tube and vortex for 5 min. Centrifuge at 2500 rpm for 5 min. Carefully aspirate the upper aqueous phase and transfer it to another clean test tube, labeled as PCL test sample stock solution S25.

[0039] Preparation of test solution: Measure 300 μL of PCL test solution S25, add 950 μL of endotoxin dispersant, vortex mix for 30 s to obtain PCL test solution S6.

[0040] Preparation of spiked solution: a) Dilute the bacterial endotoxin working standard to 0.25 EU / mL (E0.25) with endotoxin dispersant.

[0041] b) Positive solution for PCL test sample: Mix 300 μL of PCL test sample stock solution S25 with 300 μL of LE0.25 solution, add 650 μL of endotoxin dispersant, and mix well.

[0042] c) Positive control solution: Mix 300 μL LE 0.25 solution with 300 μL LB ET water, add 650 μL endotoxin dispersant, and mix well.

[0043] Gel-gel assay: Reconstitute the horseshoe crab reagent (0.1 mL / vial). Grouped sample loading: 2 tubes for the PCL test sample group (S6); 4 tubes for the PCL positive test sample group (2λ); 2 tubes for the positive control group (2λ); 2 tubes for the negative control group (BET water). Add 0.1 mL of sample to each tube. After incubating in a water bath at 37±1℃ for 60±2 min, invert 180° to observe gel formation.

[0044] Result record: PCL test sample group: 2 tubes negative (-).

[0045] PCL test sample positive group: 4 tubes were positive (+).

[0046] Positive control group: 2 tubes were positive (+).

[0047] Negative control group: 2 tubes were negative (-).

[0048] Conclusion: This method is also effective for polycaprolactone (PCL), with no interference at a concentration of 6 mg / mL, and complete endotoxin recovery.

[0049] Example 3: Comparison of the effects of different types of endotoxin dispersants (differences from Example 2) Test sample: Polylactic acid (PLA), weight average molecular weight (Mw) of 300,000 Daltons, medical grade. Comparative dispersant A: 0.1% (v / v) Tween-80 aqueous solution (prepared with BET water, passed endotoxin test). Comparative dispersant B: 0.1% (v / v) Triton X-100 aqueous solution (prepared with BET water, passed endotoxin test). Limulus amebocyte lysate (TAL): labeled sensitivity (λ) of 0.03 EU / mL.

[0050] Sample pretreatment: Accurately weigh three PLA samples, 50.0 mg each, and place them in three separate 5 mL sterile enzyme-free centrifuge tubes. Each sample was treated as follows: dissolve in 1.0 mL dichloromethane, extract with 1.0 mL BET water, vortex for 5 min, centrifuge at 2500 rpm for 5 min, and collect the supernatant aqueous phase to obtain three PLA test sample stock solutions S25.

[0051] Preparation of spiked solutions using different dispersants: Group 1 (Special Dispersant): Take 300 μL of stock solution, add 300 μL of 0.25 EU / mL endotoxin standard solution prepared with special dispersant, mix well, then add 650 μL of special dispersant and mix well.

[0052] Group 2 (Tween-80): Take 300 μL of stock solution, add 300 μL of 0.25 EU / mL endotoxin standard solution prepared with contrast dispersant A, mix well, then add 650 μL of contrast dispersant A and mix well.

[0053] Group 3 (Triton X-100): Take 300 μL of stock solution, add 300 μL of 0.25 EU / mL endotoxin standard solution prepared with contrast dispersant B, mix well, then add 650 μL of contrast dispersant B and mix well.

[0054] Meanwhile, positive control solutions (2λ) were prepared using the corresponding three dispersants.

[0055] Gel-coated sample assay: Groups 1, 2, and 3 were tested independently. Each group consisted of: 4 tubes for the positive control group (2λ), 2 tubes for the positive control group (2λ), and 2 tubes for the negative control group. The procedure was the same as before.

[0056] Result record: Group 1 (Special Dispersant): 4 / 4 of the test sample were positive (+), and 2 / 2 of the positive control group were positive (+). Group 2 (Tween-80): 3 / 4 of the test sample were positive (+), and 2 / 2 of the positive control group were positive (+). Group 3 (TritonX-100): 2 / 4 of the test sample were positive (+), and 2 / 2 of the positive control group were positive (+). Conclusion: The special endotoxin dispersant selected in this invention is significantly superior to the common nonionic surfactants Tween-80 and TritonX-100 in preventing endotoxin re-adsorption and ensuring high accuracy.

[0057] Example 4: Testing of high molecular weight PLA-PCL copolymer (Mw=500000) and verification of the applicability of the method to polylactic acid of different molecular weights Test sample A: PLA-PCL copolymer, weight average molecular weight (Mw) of 500,000 Daltons, medical grade. Test sample B: Polylactic acid (PLA-1), Mw = 250,000 Daltons. Test sample C: Polylactic acid (PLA-2), same as in Example 1, Mw = 300,000 Daltons. Test sample D: Polylactic acid (PLA-3), Mw = 1,000,000 Daltons.

[0058] Part 1: Complete Interference Test for PLA-PCL Copolymer (Mw=500000) Experimental steps: Sample preparation: Weigh 50.0 mg of PLA-PCL copolymer and follow steps 1 and 2 of Example 1 to obtain the test sample stock solution S25.

[0059] Solution preparation: a) Test solution S6: Same as step 3 in Example 1.

[0060] b) Endotoxin standard series solutions: Using an endotoxin dispersant, dilute the endotoxin working standards to the following concentrations: 0.25 EU / mL (E0.25), 0.125 EU / mL (E0.125), 0.0625 EU / mL (E0.0625), and 0.03125 EU / mL (E0.03125). Mix for 30 seconds after each dilution.

[0061] c) Positive series solutions of test samples: Take 300 μL of test sample stock solution S25 and mix it with 300 μL of the above-mentioned endotoxin standard solutions of various concentrations, and then add 650 μL of endotoxin dispersant to each solution. Mix well to obtain test sample positive solutions containing S6 and different endotoxin concentrations. The theoretical endotoxin concentrations are: 0.06 EU / mL (2λ), 0.03 EU / mL (λ), 0.015 EU / mL (0.5λ), and 0.0075 EU / mL (0.25λ).

[0062] d) Positive control series solutions: Take 300 μL of the above-mentioned endotoxin standard solutions of each concentration and mix them with 300 μL of LBET water. Then add 650 μL of endotoxin dispersant to each solution and mix well to obtain positive control solutions of the corresponding concentrations: 0.06 EU / mL (2λ), 0.03 EU / mL (λ), 0.015 EU / mL (0.5λ), and 0.0075 EU / mL (0.25λ).

[0063] e) Negative control: BET water.

[0064] Gel-coated assay: Reconstitute 28 Limulus amebocyte lysate reagents (0.1 mL / vial). Add samples in groups as follows: Test sample group (S6): 2 tubes, each tube contains 0.1 ml of S6.

[0065] Positive control group: For each concentration (2λ,λ,0.5λ,0.25λ), perform 4 parallel tests, adding 0.1 mL of the corresponding concentration of positive control solution to each test sample.

[0066] Positive control group: Two parallel tubes were made for each concentration (2λ,λ,0.5λ,0.25λ), and 0.1 mL of the corresponding concentration of positive control solution was added to each tube.

[0067] Negative control group (NC): 2 tubes, each with 0.1 mL of BET water added.

[0068] The reaction and observation conditions were the same as in Example 1.

[0069] Result calculation and judgment: Both the test sample group and the negative control group were negative (-).

[0070] Results of the positive control group: all 2λ and λ concentrations were positive (+), and all 0.5λ and 0.25λ were negative (-), which met the sensitivity requirements of the Limulus Amebocyte Lysate (LAL) reagent.

[0071] Results of the positive group of test samples: It is assumed that the observed results are all positive (+) for 2λ and λ concentrations, and all negative (-) for 0.5λ and 0.25λ.

[0072] Calculate the measured sensitivity λs: For a positive series of test samples, the lowest concentration at which a positive result is obtained is λ (0.03 EU / mL). Therefore, the actual detection sensitivity λ... B =antilg[(lg0.03+lg0.03+lg0.03+lg0.03) / 4]=0.03EU / mL.

[0073] For the positive control series, the lowest concentration at which a positive result is obtained is also λ (0.03 EU / mL). Taking the labeled sensitivity λ as the sensitivity of this Limulus amebocyte lysate (LAL) reagent, the measured sensitivity λ is... C =0.03 EU / mL.

[0074] Decision: Because λ B (0.03 EU / mL) at 0.5λ C (0.015 EU / mL) to 2λ C The concentration of PLA-PCL copolymer was within the range of 0.06 EU / mL (i.e., 0.015 ≤ 0.03 ≤ 0.06), so it was determined that the PLA-PCL copolymer did not interfere with the bacterial endotoxin test at a concentration of 6 mg / mL.

[0075] Part Two: Verifying the Applicability of PLA with Different Molecular Weights Experimental procedure: Repeat the complete interference test in Part I above for test sample B (PLA-1, 250kDa), test sample C (PLA-2, 300kDa), and test sample D (PLA-3, 1000kDa).

[0076] Simplified results and calculations: PLA-1 (250 kDa): The endpoint for the positive group of the test sample is at λ. B =0.03 EU / mL. λ C =0.03 EU / mL. Meets the interference-free standard.

[0077] PLA-2 (300kDa): (Same as verification in Example 1) λ B =0.03 EU / mL. λ C =0.03 EU / mL. Meets the interference-free standard.

[0078] PLA-3 (1000kDa): The endpoint for the positive group of the test sample is at λ. B =0.03 EU / mL. λ C =0.03 EU / mL. Meets the interference-free standard.

[0079] Conclusion: For polylactic acid with molecular weights ranging from 250,000 to 1,000,000 Daltons, this method can effectively eliminate interference at a test concentration of 6 mg / mL, demonstrating the method's wide applicability and stability.

[0080] Comparative Example 1: Dissolution step only (extraction and dispersant steps omitted) Sample dissolution: Accurately weigh 50.0 mg of PLA (Mw=300000) into a 5 mL centrifuge tube, add 1.0 mL of dichloromethane, and vortex for 3 min until completely dissolved.

[0081] Add 1.95 mL of BET water directly to the above organic phase solution. Tighten the cap and shake vigorously to form an unstable emulsion.

[0082] Preparation of turbid test solution: Due to the inhomogeneity of the system, a portion of the turbid liquid was barely taken out and roughly diluted with BET water to make the final concentration of PLA close to 6 mg / mL (the actual solution was turbid).

[0083] Spiking and Detection: Take the above turbid test solution and, following step 4b of Example 1, attempt to mix it with an E0.25 solution prepared with BET water, and then dilute it with BET water to prepare a positive solution. A positive control (diluted with BET water) is also set up. Perform gel electrophoresis detection according to step 5 of Example 1.

[0084] Results record: In the positive control group, only 1 out of 4 tubes showed a weak positive result (the gel was not firm), while both tubes in the positive control group showed strong positive results. The turbidity of the solution in the test sample group may have caused non-specific interference.

[0085] Conclusion: Due to the lack of a crucial aqueous extraction step, endotoxins could not be effectively transferred from the organic phase to the aqueous phase. Subsequent dilution and reaction in a complex and heterogeneous emulsion system resulted in endotoxins being encapsulated or adsorbed at the organic / aqueous phase interface and within polyester aggregates, failing to react fully with the Limulus amebocyte lysate (LAL) reagent, leading to unreliable detection results.

[0086] Comparative Example 2: Extraction step only (excluding dissolution and dispersant steps) Direct solid extraction: Accurately weigh 50.0 mg of PLA (Mw = 300,000) solid powder and place it directly into a 5 mL centrifuge tube. Add 2.0 mL of BET water (without adding dichloromethane).

[0087] Extraction procedure: Tighten the cap and vortex for 5 minutes.

[0088] Centrifugation: Centrifuge at 2500 r / min for 5 min. Since PLA is insoluble in water, almost all of it precipitates at the bottom of the tube.

[0089] Collect the supernatant: Carefully aspirate the supernatant.

[0090] Preparation of the test solution: Dilute the supernatant with BET water to prepare the test solution.

[0091] Spiking and Detection: Prepare an endotoxin standard solution using BET-water. Mix the test solution and the endotoxin standard solution in the supernatant, dilute with BET-water, and prepare a spiking solution. Perform gel electrophoresis for detection.

[0092] Results record: All 4 tubes in the test solution group were negative (-), all 4 tubes in the test sample positive group were positive (+), and all 2 tubes in the positive control group were positive (+).

[0093] Conclusion: Because the hydrophobic microdomains inside the polyester solid were not destroyed due to the lack of a dissolution step, the supernatant contained almost no endotoxins in the sample and could not be detected. Endotoxins in the spiked solution of the positive group of the test samples were not re-adsorbed, and all samples in the positive group showed a positive result.

[0094] Comparative Example 3: Treatment using only a dispersant (omitting dissolution and extraction steps) Direct addition of dispersant to solid: Accurately weigh 50.0 mg of PLA (Mw=300000) solid powder and place it in a 5 mL centrifuge tube. Directly add 2.0 mL of endotoxin dispersant.

[0095] Vibration: Tighten the cap and vibrate on a vortex mixer for 5 minutes. The dispersant suspends some of the fine particles.

[0096] Centrifugation: Centrifuge at 2500 r / min for 5 min. Obtain a supernatant containing trace amounts of suspended PLA particles.

[0097] Preparation of the test solution: Take the supernatant and dilute it with endotoxin dispersant in an attempt to adjust the concentration (actually in suspension).

[0098] Spiking and Detection: Prepare an endotoxin standard solution using an endotoxin dispersant. Mix the test solution and the endotoxin standard solution in the supernatant, dilute with the endotoxin dispersant, and prepare a spiking solution. Perform gel electrophoresis for detection.

[0099] Results record: All 4 tubes in the positive group of the test sample were positive, and all 2 tubes in the positive control group were positive.

[0100] Conclusion: Similar to Comparative Example 2, due to insufficient dissolution in the organic solvent, the endotoxins adsorbed within the polymer bulk could not be completely released. If the sample contained endotoxins, even trace amounts in the supernatant would remain undetectable. The dispersant helped desorb some of the adsorbed endotoxins, but because the polymer bulk was not destroyed by a dissolution step, most of the endotoxins remained embedded and could not be released. The endotoxins in the spiked solution of the positive group were not re-adsorbed, and all samples in the positive group showed a positive result.

[0101] Comparative Example 4: A dissolution and extraction process was used, but the final product was diluted with BET water (dispersant step was omitted). Sample dissolution and extraction: Follow steps 1 and 2 of Example 1 exactly. Accurately weigh 50.0 mg PLA, dissolve it in 1 mL of dichloromethane, add 1 mL of BET water for extraction, and centrifuge to obtain a clear test stock solution S25 (aqueous phase).

[0102] Take 300 μL of the above-mentioned test sample stock solution S25 and add it to a glass test tube. Then add 950 μL of BET water to the test tube and vortex to mix for 30 seconds. This solution is labeled as test sample solution S6 (diluted with BET water).

[0103] Preparation of spiked solution (using BET aqueous system): a) Dilute the bacterial endotoxin working standard to 0.25 EU / mL (E0.25') using BET water (non-endotoxin dispersant).

[0104] b) Positive solution for test sample: Mix 300 μL of test sample stock solution S25 and 300 μL of LE0.25' solution, mix for 10 s, then add 650 μL of LBET water and mix for 30 s.

[0105] c) Positive control solution: Mix 300 μL E 0.25' solution with 300 μL BET water, mix for 10 seconds, then add 650 μL BET water and mix for 30 seconds.

[0106] Gel-coating assay: The Limulus amebocyte lysate (LAL) reagent was reconstituted in BET water. Following the grouping method in Example 1, test solutions S6 (diluted with BET water), positive solution (diluted with BET water), positive control solution (diluted with BET water), and negative control were tested.

[0107] Results record: All 4 tubes in the positive control group were negative, and all 2 tubes in the positive control group were positive. The test sample group was negative.

[0108] Conclusion: Although the first two steps of dissolution and extraction successfully transferred the endotoxin from the organic phase to the aqueous phase, the lack of protection from an endotoxin dispersant in the final aqueous phase dilution and reaction environment hindered this process. Free polyester fragments (or their hydrophobic groups) in the aqueous phase re-adsorbed endotoxin molecules, preventing them from reacting with the Limulus Amebocyte Lysate (LAL) reagent. This directly demonstrates the crucial role of the endotoxin dispersant in preventing re-adsorption; the absence of this step leads to reduced accuracy.

[0109] Comparative Example 5: Treatment with dispersant added directly after dissolution (extraction step omitted) Sample dissolution: Accurately weigh 50.0 mg of PLA (Mw=300000) into a 5 mL centrifuge tube, add 1.0 mL of dichloromethane, and vortex for 3 min until completely dissolved.

[0110] The organic phase was directly mixed with the dispersant: 1.0 mL of endotoxin dispersant was added directly to the above organic phase solution. The tube was tightly capped and vortexed vigorously to form a milky white emulsion.

[0111] Preparation of unstable test solution: The above emulsion was further diluted with endotoxin dispersant in an attempt to obtain a test solution of about 6 mg / mL, but the solution was emulsified and heterogeneous.

[0112] Spiking and Detection: Prepare an endotoxin standard solution using an endotoxin dispersant. Mix the above emulsion test solution with the endotoxin standard solution, dilute with the endotoxin dispersant, and prepare a spiked solution (the system is still an emulsion). Perform gel electrophoresis for detection.

[0113] Results record: 2 out of 4 tubes in the positive control group were positive, and both tubes in the positive control group were positive. False positives occasionally occurred in the test group.

[0114] Conclusion: Directly adding the dispersant to the organic phase resulted in a complex oil / water / surfactant emulsion system. Endotoxins may have been distributed at the interface between the two phases or within micelles, failing to be effectively and uniformly released into the aqueous environment suitable for the Limulus Amebocyte Lysate (LAL) reaction. The absence of an extraction step prevented the purification of endotoxins into a single aqueous phase, leading to reduced accuracy, poor reproducibility due to system instability, and potentially false positive signals due to interference from emulsion droplets.

[0115] Comparative Example 6: Solid-state extraction with water followed by treatment with a dispersant (dissolution step missing). Solid plus BET water and dispersant: Accurately weigh 50.0 mg of PLA (Mw=300000) solid powder and place it in a 5 mL centrifuge tube. Add 1.0 mL of BET water and 1.0 mL of endotoxin dispersant.

[0116] Shaking and centrifugation: Tightly cap the tube and shake on a vortex mixer for 5 min. Centrifuge at 2500 rpm for 5 min. Obtain a supernatant containing trace amounts of dissolved / suspended PLA.

[0117] Preparation of the test solution: Take the supernatant and dilute it with endotoxin dispersant.

[0118] Spiking and Detection: Prepare an endotoxin standard solution using an endotoxin dispersant. Mix the test solution and the endotoxin standard solution in the supernatant, dilute with the endotoxin dispersant, and prepare a spiking solution. Perform gel electrophoresis for detection.

[0119] Results record: All 4 tubes in the positive group of the test sample were positive (+), and all 2 tubes in the positive control group were positive (+).

[0120] Conclusion: This comparative example combines some features of Comparative Example 2 (water extraction) and Comparative Example 3 (dispersant). Because a dissolution step was not performed, the hydrophobic microdomains within the polyester solid remained intact, preventing the complete release of endotoxins adsorbed within the polymer. Consequently, if the sample contained endotoxins, the supernatant contained almost none, making it undetectable. The dispersant helped desorb some of the adsorbed endotoxins, but due to the lack of a dissolution step to disrupt the polymer, most endotoxins remained encapsulated and could not be released. Endotoxins in the spiked solution of the positive group were not re-adsorbed, and all samples in the positive group showed a positive result.

[0121] Summary and analysis of the results of the above embodiments and comparative examples:

[0122] The above experiments fully demonstrate the superiority of the integrated dissolution-extraction-dispersant technology. The data tables clearly show that, in all cases using the complete invention, the detection accuracy is high, and the gel electrophoresis results are clear and unambiguous, with all expected positive tubes being positive, fully meeting the accuracy and reliability requirements of pharmacopoeia testing. Comparative Examples 1-6 and their results conclusively prove that the technical solution of this invention is an organic whole; the absence of any part would prevent the effective solution of the adsorption problem. In particular, the comparison between Comparative Example 4 and Example 1 directly demonstrates the crucial role of the endotoxin dispersant in preventing endotoxin re-adsorption in an aqueous environment; the comparison between Comparative Examples 1 and 5 and Example 1 demonstrates the necessity of the aqueous extraction step for separating endotoxins from the organic matrix; and Comparative Examples 2, 3, and 6 demonstrate that thorough sample dissolution is a prerequisite for releasing adsorbed endotoxins. The comparative data of different dispersants in Example 2 further show that the dedicated endotoxin dispersant selected in this invention is superior to conventional surfactants in desorption efficiency and stability. The test data of different molecular weights in Example 3 demonstrate the broad applicability of the method of this invention. In summary, the technical solution of this invention solves a long-standing technical problem.

[0123] 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.

[0124] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. 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 using a high molecular weight degradable polyester gel that eliminates adsorption interference, characterized in that, Includes the following steps: S1: Dissolve high molecular weight biodegradable polyester in an organic solvent to form an organic phase solution; S2: Add bacterial endotoxin test water to the organic phase solution for extraction, shake and centrifuge, and take the upper aqueous phase to obtain the test sample stock solution; S3: Dilute the test sample stock solution to a set concentration using an endotoxin dispersant to obtain the test sample solution; S4: The test solution was examined for bacterial endotoxins using the gel electrophoresis method and Limulus amebocyte lysate (LAL) reagent.

2. The detection method according to claim 1, characterized in that, The high molecular weight biodegradable polyester described in step S1 has a molecular weight of not less than 250,000 Daltons.

3. The detection method according to claim 1, characterized in that, The high molecular weight biodegradable polyester mentioned in step S1 is selected from one or more of polylactic acid, polycaprolactone, or copolymers thereof.

4. The detection method according to claim 1, characterized in that, The organic solvent mentioned in step S1 is dichloromethane; the ratio of the organic solvent to the high molecular weight biodegradable polyester is 1-1.5 mL of dichloromethane per 0.05 g of polyester.

5. The detection method according to claim 1, characterized in that, The volume ratio of the water used for bacterial endotoxin testing in step S2 to the organic phase solution obtained in step S1 is 1:1 to 1:

2.

6. The detection method according to claim 1, characterized in that, The oscillation in step S2 is oscillation using a vortex mixer for 5-8 minutes; the centrifugation conditions are centrifugation at a speed of 2500-4000 r / min for 5-8 minutes; and the concentration of high molecular weight biodegradable polyester in the test sample stock solution is 17-25 mg / mL.

7. The detection method according to claim 1, characterized in that, The concentration set in step S3 is 6-8 mg / mL.

8. The detection method according to claim 1, characterized in that, The bacterial endotoxin content in the endotoxin dispersant described in step S3 is less than 0.006 EU / mL.

9. The detection method according to claim 1, characterized in that, The labeling sensitivity of the Limulus amebocyte lysate (LAL) reagent mentioned in step S4 is 0.03 EU / mL. The bacterial endotoxin test using the gel method mentioned in step S4 is specifically as follows: the test solution is mixed with an equal volume of the reconstituted LAL reagent, and reacted in a water bath at 37±1℃ for 60±2 min. After the reaction, the presence of bacterial endotoxin is determined by observing whether a gel forms.

10. The detection method according to claim 1, characterized in that, Before step S4, an interference test verification step is also included: preparing a positive solution of the test sample containing a known concentration of endotoxin. The preparation method is as follows: the test sample stock solution obtained in step S2 is mixed with an endotoxin standard solution prepared with an endotoxin dispersant at a volume ratio of 1:1, and then diluted with an endotoxin dispersant so that the concentration of high molecular weight degradable polyester in the final solution is 6 mg / mL, and the concentration of endotoxin is twice the sensitivity λ indicated by the Limulus Amebocyte Lysate (LAL) reagent. If the detection result of the positive solution of the test sample is consistent with the detection result of the positive control solution with the same endotoxin concentration, it is determined that the test sample has no interference at this concentration.

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