A method for detecting bacterial endotoxin in a lactide compound

Abstract

The application discloses a kind of lactide compounds bacterial endotoxin detection method, it is related to the technical field of difficult-soluble hydrolysis detection.The method of the present application comprises: using anhydrous ethanol to dissolve lactide compounds, obtain primary dissolution solution;0.1 mol / L sodium carbonate solution is added to the primary dissolution solution, after mixing evenly, dilute with BET water, obtain test sample stock solution;Test sample stock solution is mixed with calcium ion buffer, and then diluted to a set concentration with BET water, to obtain test sample solution;Using photometric method, limulus reagent is used to detect bacterial endotoxin of test sample solution.The application overcomes the technical difficulties of strong hydrophobicity, easy hydrolysis and inhibition interference of limulus reagent reaction of lactide compounds by four-step synergistic scheme of anhydrous ethanol dissolution-sodium carbonate cooperative treatment-calcium ion buffer adaptation-80-fold dilution, and the detection result is accurate and reliable, reproducible, the recovery rate is stable in the required range of pharmacopoeia, suitable for bacterial endotoxin detection of L-lactide, glycolide, D,L-lactide and other lactide compounds, and provides reliable technical support for medical polymer raw material safety detection.

Description

Technical Field

[0001] This invention provides a method for detecting bacterial endotoxins derived from lactide compounds, relating to the field of detection technology for poorly soluble substances that are easily hydrolyzed. Background Technology

[0002] Lactose compounds, including L-lactide, D-lactide, DL-lactide, glycolide, and their derivatives, are core monomers for synthesizing biodegradable medical polymers such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-co-glycolic acid copolymer (PLGA). Their quality and safety directly determine the clinical safety of the final medical implant materials, drug-release carriers, and absorbable surgical instruments. Bacterial endotoxins are a key quality control indicator for medical raw materials. Materials with excessive endotoxin levels can easily cause serious adverse reactions such as fever, inflammatory reactions, and sepsis after implantation. Therefore, it is essential to establish strict, accurate, and stable methods for detecting bacterial endotoxins, meeting the relevant quality control requirements of Section 1143, "Bacterial Endotoxin Test Method," in the 2025 edition of the Chinese Pharmacopoeia. However, lactones possess unique physicochemical properties: they are readily hydrolyzed, highly hydrophobic, and poorly water-soluble. Conventional bacterial endotoxin detection uses pure water to directly dissolve and dilute samples, which easily leads to premature hydrolysis and degradation of lactones. The hydrolysis products are acidic, not only damaging the sample's chemical structure and causing detection distortion, but also inhibiting the enzymatic activity of the horseshoe crab reagent. Simultaneously, high-concentration lactone solutions significantly inhibit and interfere with the aggregation reaction of the horseshoe crab reagent. The hydrolysis products of lactones react with divalent cations such as Ca in the reaction system. 2+ Chelation occurs, leading to the release of free Ca. 2+Insufficient concentration inhibits the activity of the horseshoe crab reagent. Furthermore, the strong hydrophobicity of lactide compounds leads to incomplete dissolution or the formation of turbid and uneven suspensions in conventional aqueous solutions, severely affecting the accuracy and reproducibility of the detection results. Existing technologies for bacterial endotoxin detection are mostly designed for water-soluble drugs, injections, and other conventional samples. Literature searches indicate that while methods for detecting endotoxins in poorly soluble samples exist, such as using DMSO, ethanol, methanol, and Tween 80 as solvents, these general methods only address the solubility issue and do not specifically optimize for the easy hydrolysis of lactide compounds and their chelation interference with the horseshoe crab reagent. Existing review literature mentions general approaches such as "adjusting pH and supplementing divalent cations to eliminate interference," but these are merely theoretical guidelines and do not provide specific feasible technical solutions for lactide compounds, nor do they address the unpredictable impact of different cation types on detection results. In addition, the poor compatibility of horseshoe crab reagents from different manufacturers and the large differences in detection results further limit the universality and reliability of lactide compound endotoxin detection. Therefore, there is currently a lack of bacterial endotoxin detection methods that are specifically optimized for the physicochemical properties of lactide compounds, have simple pretreatment, are interference-free, provide accurate results, and comply with pharmacopoeia standards. This results in technical loopholes in the endotoxin quality control of lactide raw materials, making it difficult to meet the stringent quality control requirements for medical implant-grade lactide. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a method for detecting bacterial endotoxins from lactide compounds, comprising the following steps: (a) dissolving the lactide compound in anhydrous ethanol to obtain a primary solution; (b) adding sodium carbonate solution to the primary solution, mixing thoroughly, and then diluting with BET water to obtain a test sample stock solution; (c) mixing the test sample stock solution with calcium ion buffer, and then diluting with BET water to a set concentration to obtain a test sample solution; and (d) using a spectrophotometric method with Limulus amebocyte lysate (LAL) reagent to detect bacterial endotoxins in the test sample solution.

[0004] Preferably, the concentration of the sodium carbonate solution in step (b) is 0.1 mol / L, the volume ratio of the primary solution to the sodium carbonate solution is 1:3, and the concentration of the lactone compound in the test sample stock solution is 8 mg / mL.

[0005] Preferably, the dilution with BET water is as follows: take the mixture of the primary solution and the sodium carbonate solution, add BET water to dilute, so that the concentration of the lactone compound in the test sample stock solution is 8 mg / mL; the concentration of the test sample stock solution is 8 mg / mL, which is prepared by the following method: take 400 μL of the mixture of the primary solution and the sodium carbonate solution, add 850 μL of BET water, and mix evenly.

[0006] Preferably, the concentration set in step (c) is 1.25 mg / mL, the volume ratio of the test sample stock solution to the calcium ion buffer is 1:1, and the calcium ion buffer is an endotoxin-free calcium ion buffer with a bacterial endotoxin content of less than 0.015 EU / mL.

[0007] Preferably, the concentration of the test sample solution is 1.25 mg / mL, which is prepared by the following method: take 200 μL of the test sample stock solution, add 200 μL of calcium ion buffer, mix well, and then add 880 μL of BET water and mix well.

[0008] Preferably, the photometric method in step (d) is a dynamic colorimetric method with a detection wavelength of 405 nm and a detection sensitivity of 0.02 EU / mL.

[0009] Preferably, the reaction time of the horseshoe crab reagent in the method is 90 min, and the detection range of the horseshoe crab reagent in step (d) is 0.01-10 EU / mL.

[0010] Preferably, all glassware used in the method must be sterilized by dry heat at 250°C for more than 0.5 hours.

[0011] Preferably, in step (d), the preset OD value of the microplate reader is 0.02 or 0.1.

[0012] Preferably, the amount of anhydrous ethanol used in step (a) is 1.0 mL of anhydrous ethanol per 100 mg of lactide compound.

[0013] Preferably, the dissolution method in step (a) is as follows: add the lactone compound and anhydrous ethanol into a centrifuge tube, tighten the centrifuge tube cap, place it on a vortex mixer and continuously vortex at maximum speed until the solid is completely dissolved and the solution is homogeneous, clear and transparent; the vortex oscillation time is 3 min.

[0014] Preferably, the mixing method in step (b) is as follows: after mixing the primary solution with the sodium carbonate solution, the container is tightly covered and placed on a vortex mixer for continuous vortex oscillation until the mixture is uniform; the vortex oscillation time is 3 min.

[0015] Preferably, the mixing method in step (c) is as follows: after mixing the test sample stock solution with the calcium ion buffer, mix them on a vortex mixer for at least 10 s.

[0016] Preferably, in step (c), the mixing method after dilution with BET water is: mixing on a vortex mixer for at least 30 seconds.

[0017] Preferably, the lactide compound is selected from at least one of L-lactide, D-lactide, DL-lactide, glycolide, or derivatives thereof.

[0018] Preferably, the horseshoe crab reagent in step (d) is a horseshoe crab reagent that has been reconstituted with BET water.

[0019] Preferably, the detection method in step (d) includes: taking 25 μL each of the test sample solution, the test sample positive control solution, the positive control solution and the negative control solution and placing them in an ELISA plate, then adding 25 μL of reconstituted Limulus amebocyte lysate (LAL) reagent, and immediately placing it in an ELISA reader for detection.

[0020] Preferably, the method further includes the preparation of a positive control solution: take a bacterial endotoxin working standard, add BET water to dissolve and vortex mix to prepare a stock solution with a concentration of 10 EU / mL, and then dilute it stepwise with BET water to prepare bacterial endotoxin standard solutions with concentrations of 2.0 EU / mL, 0.2 EU / mL and 0.02 EU / mL.

[0021] Preferably, the method further includes the preparation of a positive control solution for the test sample: the test sample stock solution is mixed with calcium ion buffer at a volume ratio of 1:1, and then diluted with a mixture containing endotoxin standard solution and BET water to make the final concentration of the test sample 1.25 mg / mL and the added endotoxin concentration 0.2 EU / mL.

[0022] Preferably, the negative control in the method is BET water.

[0023] Preferably, in the method, the standard curve is plotted with the logarithm of bacterial endotoxin concentration as the abscissa and the logarithm of average reaction time as the ordinate, and the standard curve is valid when the correlation coefficient |γ|>0.980.

[0024] Preferably, step (b) further includes adding disodium ethylenediaminetetraacetate to the mixture of the primary solution and the sodium carbonate solution, wherein the final concentration of disodium ethylenediaminetetraacetate in the mixture is 0.5-5.0 mmol / L.

[0025] Preferably, the final concentration of the disodium ethylenediaminetetraacetate in the mixture is 1.0 mmol / L, 2.0 mmol / L, or 3.0 mmol / L.

[0026] Preferably, in step (a), polysorbate-20 is further added to the anhydrous ethanol, wherein the volume fraction of polysorbate-20 in the primary solution is 0.01%-0.1%.

[0027] Preferably, the volume fraction of polysorbate-20 in the primary solution is 0.05%.

[0028] Preferably, the calcium ion buffer in step (c) is further supplemented with a lactate-sodium lactate buffer system, wherein the pH value of the lactate-sodium lactate buffer system is 4.5-6.0, and the molar ratio of lactate to sodium lactate is 1:2 to 2:1.

[0029] Preferably, the pH value of the lactic acid-sodium lactate buffer system is 5.2, and the molar ratio of lactic acid to sodium lactate is 1:1.

[0030] Preferably, step (b) further includes adding disodium ethylenediaminetetraacetate to the mixture of the primary dissolving solution and the sodium carbonate solution to a final concentration of 0.5-5.0 mmol / L; adding polysorbate-20 to the anhydrous ethanol in step (a) to a volume fraction of 0.01%-0.1%; and supplementing the calcium ion buffer in step (c) with a lactate-sodium lactate buffer system, wherein the pH of the lactate-sodium lactate buffer system is 4.5-6.0, and the molar ratio of lactate to sodium lactate is 1:2 to 2:1.

[0031] Preferably, the final concentration of the disodium ethylenediaminetetraacetate is 1.0 mmol / L, 2.0 mmol / L, or 3.0 mmol / L, the volume fraction of the polysorbate-20 is 0.05%, the pH value of the lactic acid-sodium lactate buffer system is 5.2, and the molar ratio of lactic acid to sodium lactate is 1:1.

[0032] The beneficial effects of this invention are: First, addressing the complex technical challenges posed by the strong hydrophobicity, easy hydrolysis, and chelation inhibition of horseshoe crab reagents by lactide compounds, this invention establishes a novel method specifically for the detection of bacterial endotoxins from lactide compounds. This method employs a four-step synergistic approach: dissolving in anhydrous ethanol, adjusting with a 1:3 volume ratio of 0.1 mol / L sodium carbonate, adapting with calcium ion buffer, and diluting 80-fold to 1.25 mg / mL. Comparative data from Examples 1-6 show that direct dissolution with BET water or omission of sodium carbonate adjustment resulted in zero recovery; omitting calcium ion buffer or replacing it with magnesium ion buffer reduced the recovery to 22%-34%; excessive dilution to 0.4 mg / mL directly reduced the recovery to 0%; and using sodium bicarbonate or sodium hydroxide instead of sodium carbonate also resulted in zero recovery. In comparison, Examples 3-5 showed recoveries of 81.01%-88.81% for L-lactide, 93.64% for glycolide, and 84.72% for D,L-lactide, with coefficients of variation less than 5.5%, fully meeting the requirements of the 2025 edition of the Chinese Pharmacopoeia. Furthermore, they demonstrated good compatibility with Limulus amebocyte lysate (LAL) reagents from two different manufacturers. These comparisons fully demonstrate that the four-step synergistic approach of Examples 3-5 successfully overcomes the three major technical bottlenecks in the detection of lactide compounds: difficulty in dissolution, easy hydrolysis, and strong interference. Secondly, this invention further introduces a triple additive combination of disodium ethylenediaminetetraacetate (EDTA), polysorbate-20, and lactate-sodium lactate buffer. The recovery rate of the triple-component solution increased from 83.81% to 98.0%-100.5%, closer to the theoretical optimum of 100%; the coefficient of variation decreased from 5.3% to 2.8%-3.5%; and the average time to reach the limit was shortened from 1464 seconds to 1155-1205 seconds, representing an increase in reaction rate of approximately 18%-21%. Data from Comparative Examples 7-12 showed that when any one of the triple components was used alone, the recovery rate was only 69%-86%, with the coefficient of variation increasing to 5.0%-6.5%; when any two components were combined, the recovery rate was only 65%-83%, comparable to or worse than in Example 3; only when all three components were present simultaneously did all indicators simultaneously reach optimal levels. This indicates that disodium EDTA, polysorbate-20, and lactate-sodium lactate buffer constitute a synergistic functional chain of "dissociation-dispersion-stabilization," and none of them can be omitted. In summary, this invention has achieved a breakthrough in the detection of bacterial endotoxins from lactide compounds at the basic scheme level, and has achieved a leap from existing to superior detection accuracy, precision and efficiency at the triple additive scheme level, providing a reliable and efficient technical means for the quality control of medical polymer raw materials. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Experimental materials: Limulus amebocyte lysate (LAL) reagent 1, produced by Zhanjiang Andus Biotechnology Co., Ltd., batch number 2407030, specification 0.35 mL / vial, detection range 10~0.01 EU / mL; Limulus amebocyte lysate (LAL) reagent 2, produced by 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, produced by Zhanjiang Andus Biotechnology Co., Ltd., batch number 2502061, specification 10 EU / vial; Bacterial endotoxin working standard 2, produced by Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number 240202, specification 10 EU / vial; BET water, produced by Zhanjiang Andus Biotechnology Co., Ltd., batch numbers 2404170 and 2502140, specification 50... mL; Bacterial endotoxin test reaction plate, manufactured by Zhanjiang Andus Biotechnology Co., Ltd., batch number 2025061303, specification 96 wells; Anhydrous ethanol, manufactured by Sinopharm Chemical Reagent Co., Ltd., analytical grade, batch number 20251011; Sodium carbonate, manufactured by Sinopharm Chemical Reagent Co., Ltd., analytical grade, batch number 20250627; Calcium ion buffer, manufactured by Zhanjiang Andus Biotechnology Co., Ltd., batch number 2502250, specification 2.1mL / vial; Calcium ion buffer or calcium-magnesium ion regulator with bacterial endotoxin content less than 0.015 EU / mL; Calcium-magnesium ion regulator, manufactured by Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number 250401, specification 2 mL / vial; Calcium ion buffer or calcium-magnesium ion regulator should be used according to the manufacturer's instructions.

[0037] Instruments: Microplate reader, Thermo Fisher Scientific Multiskan FC / ET; Electronic analytical balance, Shanghai Lichen Bangxi Instrument Technology Co., Ltd. FA2204; Automatic vortex mixer, Shanghai Huxi Industrial Co., Ltd. Vortex-M; Volumetric flasks and beakers used in the experiment were baked at 250℃ for more than 30 minutes to remove pyrogens; Sterile enzyme-free centrifuge tubes; Pyrogen-free glass test tubes; Pipette tips were commercially available pyrogen-removed tips.

[0038] Preparation of standard solutions Bacterial endotoxin working standard stock solution: Take one vial of bacterial endotoxin working standard (10 EU / vial), add 1.0 mL of BET water to dissolve it, seal it, place it on an automatic vortex mixer and vortex at maximum speed for 10 minutes to ensure complete and uniform dissolution, remove it, and you will get a working standard stock solution with a concentration of 10 EU / mL.

[0039] Preparation of bacterial endotoxin standard curve solutions: The 10 EU / mL working standard stock solution was serially diluted with BET water to prepare bacterial endotoxin standard solutions with concentrations of 2.0 EU / mL, 0.2 EU / mL and 0.02 EU / mL respectively.

[0040] Preparation of positive control solution (for positive control of test sample): Take 500 μL of 10 EU / mL stock solution, add 2000 μL BET water, mix for 30 seconds to obtain a 2.0 EU / mL solution; take 400 μL of the 2.0 EU / mL solution, add 1600 μL BET water, mix for 30 seconds to obtain a 0.4 EU / mL endotoxin standard solution; take 200 μL of the 2.0 EU / mL solution, add 1800 μL BET water, mix for 30 seconds to obtain a 0.2 EU / mL endotoxin standard solution; take 200 μL of the 0.2 EU / mL solution, add 1800 μL BET water, mix for 30 seconds to obtain a 0.02 EU / mL endotoxin standard solution.

[0041] Standard curve reliability verification method (testing method in Example 1) Dissolve one vial of bacterial endotoxin working standard in 1.0 mL of BET water, seal, and vortex for 10 minutes on 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, with two parallel tubes. Measure using a microplate reader. The reaction time should be set according to the horseshoe crab reagent manufacturer's specifications: 90 minutes for Zhanjiang Andus horseshoe crab reagent and 90 minutes for Fuzhou Xinbei horseshoe crab reagent. The detection sensitivity is 0.02 EU / mL, the detection wavelength is 405 nm, and the preset OD values ​​are: 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 mean reaction time (LgT) on the y-axis. The correlation coefficient γ was calculated using linear regression with the least squares method. The reaction time of the negative control should be greater than the reaction time of the lowest concentration on the standard curve. The standard curve is valid when the coefficient of variation (CV) is ≤20% and |γ|>0.980.

[0042] Statistical testing methods for analyzing data In each embodiment and comparative example, parallel samples were set for each test, and the data are expressed as average values. The standard curve was established by performing linear regression using the least squares method after double logarithmic transformation, and the correlation coefficient γ was calculated, requiring |γ|>0.980 and the coefficient of variation CV≤20%. The bacterial endotoxin content in the test sample was calculated by substituting the measured reaction time (time to limit) into the standard curve regression equation. The recovery rate was calculated using the formula: Recovery rate (%) = (Measured endotoxin concentration in the positive control group of the test sample - Measured endotoxin concentration in the test sample group) / Added endotoxin concentration in the positive control group of the test sample × 100%. According to the provisions of General Chapter 1143 of Part IV of the 2025 edition of the Chinese Pharmacopoeia, when the recovery rate of the positive control group of the test sample is in the range of 50%~200%, it is determined that the test sample solution has no interfering effect on the reaction of the horseshoe crab reagent, and this method can be used for bacterial endotoxin testing.

[0043] Example 1: Reliability Verification of Standard Curve One vial of bacterial endotoxin working standard (batch number 2502061) produced by Zhanjiang Andus Biotechnology Co., Ltd. was dissolved in 1.0 mL of BET water. After sealing, the solution was placed on an automatic vortex mixer and vortexed at maximum speed for 10 minutes to prepare a 10 EU / mL working standard solution. The working standard solution was serially diluted with BET water to prepare bacterial endotoxin standard solutions of three concentrations: 2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL. Three parallel tubes were prepared for each concentration. BET water was used as a negative control, with two parallel tubes prepared.

[0044] Add 25 μL of each of the above-mentioned standard solutions to a 96-well bacterial endotoxin test plate. Take one vial of Limulus Amebocyte Lysate (LAL) reagent (batch number 2407030) from Zhanjiang Andus Biotechnology Co., Ltd., and redissolve it in 0.35 mL of BET water. Gently shake until the solution is clear. Add 25 μL of the redissolved LAL reagent to each well and immediately place the plate in a microplate reader for detection. Microplate reader settings: detection mode: dynamic colorimetric method; detection wavelength: 405 nm; reaction time: 90 minutes; detection sensitivity: 0.02 EU / mL; preset OD value: 0.1. The instrument automatically records the time required for the absorbance of each well to reach the preset OD value during the reaction (i.e., time to reach limit, in seconds).

[0045] Two vials of bacterial endotoxin working standards (batch number 240202) produced by Fuzhou Xinbei Biochemical Industry Co., Ltd. were dissolved in 1.0 mL of BET water, sealed, and vortexed at maximum speed for 10 minutes on an automatic vortex mixer to prepare a 10 EU / mL working standard solution. This solution was serially diluted with BET water to prepare bacterial endotoxin standard solutions of 2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL, with three parallel tubes for each concentration. BET water was used as a negative control, with two parallel tubes. One vial of horseshoe crab reagent (batch number 24030318) from Fuzhou Xinbei Biochemical Industry Co., Ltd. was reconstituted in 0.35 mL of BET water, gently shaken until the solution was clear, and 25 μL of the reconstituted horseshoe crab reagent was added to each well. The wells were immediately placed in a microplate reader for detection. Microplate reader settings: detection wavelength 405 nm, reaction time 90 minutes, detection sensitivity 0.02 EU / mL, preset OD value 0.02.

[0046] After the test was completed, the average time to reach the limit for each concentration was exported from the ELISA reader. The correlation coefficient γ was calculated by performing linear regression using the least squares method with the logarithm of bacterial endotoxin concentration (LgC) as the x-axis and the logarithm of average reaction time (LgT) as the y-axis.

[0047] The results in Table 1 show that the time to reach the limit of free radicals (LPR) for the negative control was longer than that for the lowest concentration of the standard curve (0.02 EU / mL), with a coefficient of variation (CV) ≤ 20%. After double logarithmic transformation of all data, linear regression analysis was performed using the least squares method. The regression equation for the standard curve of the Zhanjiang Andus horseshoe crab reagent was LgT = -0.2357LgC + 3.2512, with a correlation coefficient |γ| = 0.9989; the regression equation for the standard curve of the Fuzhou Xinbei horseshoe crab reagent was LgT = -0.2412LgC + 3.1836, with a correlation coefficient |γ| = 0.9976. Both |γ| values ​​are greater than 0.980. The results indicate that the standard curves for both manufacturers' horseshoe crab reagents are valid, demonstrating the effectiveness of the method and its universality for horseshoe crab reagents from different sources.

[0048] Example 2: Preliminary Experiment on L-lactide Bacterial Endotoxin Interference Dissolve sodium carbonate in BET water in a pyrogen-free beaker, and then dilute it in a pyrogen-free volumetric flask to obtain a 0.1 mol / L sodium carbonate solution.

[0049] Accurately weigh 100.0 mg of L-lactide sample using an electronic analytical balance and place it in a 10 mL sterile, enzyme-free centrifuge tube. Accurately add 1.0 mL of anhydrous ethanol using a micropipette. Immediately cap the centrifuge tube and place it on a vortex mixer. Vortex continuously at maximum speed for 3 minutes until all solid sample at the bottom and sides of the centrifuge tube is completely dissolved, and the solution is homogeneous, clear, and transparent.

[0050] Using a micropipette, accurately add 3.0 mL of 0.1 mol / L sodium carbonate solution (i.e., the volume ratio of primary solution to sodium carbonate solution is 1:3) to the above primary solution. Tightly cap the tube and vortex continuously for 3 minutes until homogeneous. Then, using a micropipette, accurately measure 400 μL of the above mixture into another centrifuge tube, add 850 μL of BET water, and vortex until homogeneous. This solution is labeled as test stock solution S8, in which the theoretically calculated concentration of L-lactide is approximately 8 mg / mL.

[0051] Preparation of test sample solution: S2 solution (2 mg / mL): Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution. Mix on a vortex mixer for 10 seconds. Then add 400 μL of BET water to the mixture and mix again on a vortex mixer for 30 seconds to obtain the final test sample solution of 2 mg / mL.

[0052] S1.25 solution (1.25 mg / mL): Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution. Vortex mix for 10 seconds. Then add 880 μL of BET water to the mixture and vortex mix again for 30 seconds to obtain the final test sample solution of 1.25 mg / mL.

[0053] S1 solution (1 mg / mL): Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution. Mix on a vortex mixer for 10 seconds. Then add 1200 μL of BET water to the mixture and mix again on a vortex mixer for 30 seconds to obtain the final test sample solution of 1 mg / mL.

[0054] S0.8 solution (0.8 mg / mL): Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution. Vortex mix for 10 seconds. Then add 1600 μL of BET water to the mixture and vortex mix again for 30 seconds to obtain a test sample solution with a concentration of 0.8 mg / mL.

[0055] Preparation of positive control solution: Take one vial of bacterial endotoxin working standard (10 EU / vial), add 1.0 mL of BET water to dissolve, and vortex at maximum speed for 10 minutes to prepare a stock solution with a concentration of 10 EU / mL. Then, perform serial dilutions with BET water: Take 500 μL of the 10 EU / mL stock solution, add 2000 μL of BET water, mix for 30 seconds to obtain a 2.0 EU / mL solution; take 400 μL of the 2.0 EU / mL solution, add 1600 μL of BET water, mix for 30 seconds to obtain a 0.4 EU / mL endotoxin standard solution; take 200 μL of the 2.0 EU / mL solution, add 1800 μL of BET water, mix for 30 seconds to obtain a 0.2 EU / mL endotoxin standard solution; take 200 μL of the 0.2 EU / mL solution, add 1800 μL of BET water, mix for 30 seconds to obtain a 0.02 EU / mL endotoxin standard solution.

[0056] Preparation of positive control solutions for the test samples: Positive controls for each concentration of the test sample solution (S2, S1.25, S1, S0.8) were prepared. For each concentration, one glass test tube was used, and 200 μL of the test sample stock solution S8 and 200 μL of calcium ion buffer solution were precisely added. The mixture was then vortexed for 10 seconds. Then, the corresponding volumes of endotoxin standard solution and BET water were added to the mixture, and the mixture was vortexed again for 30 seconds. Specific addition protocol: 400 μL of 0.4 EU / mL endotoxin standard solution was added to the S2 positive control; 640 μL of 0.4 EU / mL endotoxin standard solution and 240 μL of BET water were added to the S1.25 positive control; 800 μL of 0.4 EU / mL endotoxin standard solution and 400 μL of BET water were added to the S1 positive control; and 200 μL of 2.0 EU / mL endotoxin standard solution and 1400 μL of BET water were added to the S0.8 positive control. The final positive solutions of the test sample were obtained with final concentrations of 2 mg / mL, 1.25 mg / mL, 1 mg / mL and 0.8 mg / mL, respectively, and the standard endotoxin concentration in each solution was 0.2 EU / mL.

[0057] Negative control group: BET water.

[0058] Take 25 μL each of the following solutions: test sample group, positive control group, positive control group (0.02 EU / mL, 0.2 EU / mL, 2.0 EU / mL standard solutions), and negative control group. Place each solution in a microplate, with two aliquots. Take one vial of Limulus Amebocyte Lysate (LAL) reagent (batch number 2407030) from Zhanjiang Andus Biotechnology Co., Ltd., and redissolve it in 0.35 mL of BET water. Gently shake until the solution is clear. Add 25 μL of the redissolved LAL reagent to each well. Immediately after addition, place the plate in the microplate reader for detection. Microplate reader settings: detection wavelength 405 nm, reaction time 90 minutes, detection sensitivity 0.02 EU / mL, preset OD value 0.1. The instrument automatically records the limit of attainment time for each well and automatically calculates the bacterial endotoxin content and recovery rate of the test sample.

[0059] The results in Table 2 above show that when the concentration of the L-lactide test solution was diluted to 2 mg / mL or below, the recovery rate of the positive control group was between 58% and 74%, all within the range of 50% to 200% specified in the pharmacopoeia. This indicates that L-lactide has no significant interference effect on the Limulus Amebocyte Lysate (LAL) reagent reaction within this concentration range. The average time to limit of reaction (TLOR) at a concentration of 1.25 mg / mL was 1404.5 seconds, with a recovery rate of 63.87%. The recovery rate was relatively stable across all concentrations. Considering that a lower test concentration would further reduce the matrix effect, 1.25 mg / mL was ultimately selected as the non-interfering dilution concentration for the formal interference test.

[0060] Example 3: L-lactide bacterial endotoxin interference test Use the horseshoe crab reagent from Zhanjiang Andus Biotechnology Co., Ltd. (follow the calcium ion buffer instructions): Accurately weigh 100.0 mg of L-lactide and place it in a 10 mL centrifuge tube. Add 1.0 mL of anhydrous ethanol, tighten the cap, and vortex continuously at maximum speed for 3 minutes until the solid is completely dissolved, obtaining the primary solution. Add 3.0 mL of 0.1 mol / L sodium carbonate solution to the primary solution (primary solution to sodium carbonate solution volume ratio 1:3), tighten the cap, and vortex continuously for 3 minutes until well mixed. Take 400 μL of the above mixture and add 850 μL of BET water, mix well, and obtain a test sample stock solution with a concentration of 8 mg / mL.

[0061] Test sample group: Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution, and mix on a vortex mixer for 10 seconds. Then add 880 μL of BET water to the mixture and mix again on a vortex mixer for 30 seconds to obtain a test sample solution of 1.25 mg / mL (S1.25). Prepare 3 different test samples for a total of 3 samples (Test Sample 1, Test Sample 2, and Test Sample 3).

[0062] Positive control group for test sample: Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution, and mix on a vortex mixer for 10 seconds. Then add 640 μL of 0.4 EU / mL endotoxin standard solution and 240 μL of BET water to the mixture, and mix again on a vortex mixer for 30 seconds to obtain a positive control solution of 1.25 mg / mL (S1.25, E0.2), in which the added endotoxin concentration is 0.2 EU / mL. Prepare 3 different test samples for a total of 3 samples.

[0063] Positive control group: Take one tube of bacterial endotoxin working standard (10 EU / tube), add 1.0 mL of BET water to reconstitute it, and prepare a bacterial endotoxin standard solution with a concentration of 10 EU / mL. Dilute it with BET water to prepare bacterial endotoxin standard solutions of 2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL. Prepare two parallel tubes for each concentration.

[0064] Negative control group: BET water, 2 parallel tubes.

[0065] Take the horseshoe crab reagent (Zhanjiang Andus Biotechnology Co., Ltd., batch number 2407030), open it, and add 0.35 mL of bacterial endotoxin test water to reconstitute it. Gently shake until the solution is clear. Add 25 μL of the sample to each well of a 96-well bacterial endotoxin test plate, with two replicates per sample. Add 25 μL of the reconstituted horseshoe crab reagent to each well and immediately place the plate into a microplate reader for detection. Microplate reader settings: detection wavelength 405 nm, reaction time 90 minutes, detection sensitivity 0.02 EU / mL, preset OD value 0.1. The instrument automatically records the limit time for each well and automatically calculates the bacterial endotoxin content and recovery rate.

[0066] Using horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. (operated according to the calcium and magnesium ion regulator instructions): The preparation methods for the test sample group, the positive control group, the positive control group, and the negative control group are exactly the same as those for the Zhanjiang Andus section above.

[0067] Take the horseshoe crab reagent (Fuzhou Xinbei Biochemical Industry Co., Ltd., batch number 24030318), open the package, and add 0.35 mL of calcium and magnesium ion regulator for bacterial endotoxin testing to reconstitute it. Gently shake until the solution is clear. Add 25 μL of the sample to be tested to each well of a 96-well bacterial endotoxin test plate, with two replicates per sample. Add 25 μL of the reconstituted horseshoe crab reagent to each well and immediately place the plate into a microplate reader for detection. Microplate reader settings: detection wavelength 405 nm, reaction time 90 minutes, detection sensitivity 0.02 EU / mL, preset OD value 0.02;

[0068] The results in Table 3 above show that, after treatment using the method of the present invention, the recoveries of the three test batches in the Zhanjiang Andus horseshoe crab reagent system were 81.62%, 81.01%, and 88.81%, respectively, all within the 50%–200% recovery range specified in the 2025 edition of the Chinese Pharmacopoeia, with a CV of 5.3% ≤ 20%. The negative control reaction time was > 5400.0 seconds, which is greater than the reaction time at the lowest concentration of the standard curve, indicating the validity of the experiment. In the Fuzhou Xinbei horseshoe crab reagent system, the measured recoveries of the three test batches were 164.43%, 131.90%, and 162.18%, respectively, also within the 50%–200% recovery range specified in the pharmacopoeia, indicating the validity of the experiment. The detection results of two different manufacturers' horseshoe crab reagents both showed that the test samples at a concentration of 1.25 mg / mL had no interfering effect on the bacterial endotoxin test, proving that the method of the present invention has good adaptability and universality to horseshoe crab reagents from different manufacturers.

[0069] Example 4 Detection of bacterial endotoxins in glycolide Accurately weigh 100.0 mg of glycolide and place it in a 10 mL centrifuge tube. Add 1.0 mL of anhydrous ethanol, tighten the cap, and vortex continuously at maximum speed for 3 minutes until the solid is completely dissolved, obtaining the primary solution. Add 3.0 mL of 0.1 mol / L sodium carbonate solution to the primary solution (primary solution to sodium carbonate solution volume ratio 1:3), tighten the cap, and vortex continuously for 3 minutes until well mixed. Take 400 μL of the above mixture and add 850 μL of BET water, mix well, and obtain a test sample stock solution with a concentration of 8 mg / mL.

[0070] Test sample group: Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution. Mix on a vortex mixer for 10 seconds. Then add 880 μL of BET water to the mixture and mix again on a vortex mixer for 30 seconds to obtain a test sample solution of 1.25 mg / mL.

[0071] Positive control group for the test sample: Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution, and mix on a vortex mixer for 10 seconds. Then add 640 μL of 0.4 EU / mL endotoxin standard solution and 240 μL of BET water to the mixture, and mix again on a vortex mixer for 30 seconds to obtain a positive control solution of 1.25 mg / mL, in which the added endotoxin concentration is 0.2 EU / mL.

[0072] Positive control group: Take one vial of bacterial endotoxin working standard (10 EU / vial), add 1.0 mL of BET water to reconstitute it, and prepare a bacterial endotoxin standard solution with a concentration of 10 EU / mL. Dilute with BET water to prepare bacterial endotoxin standard solutions with concentrations of 2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL.

[0073] Negative control group: BET water.

[0074] One vial of Limulus Amebocyte Lysate (LAL) reagent (batch number 2407030) from Zhanjiang Andus Biotechnology Co., Ltd. was opened and reconstituted with 0.35 mL of bacterial endotoxin test water. The solution was gently shaken until clear. In a 96-well bacterial endotoxin test plate, 25 μL of the sample to be tested was added to each well, with two replicates per sample. 25 μL of the reconstituted LAL reagent was added to each well, and the plate was immediately placed in a microplate reader for detection. Microplate reader settings: detection wavelength 405 nm, reaction time 90 minutes, detection sensitivity 0.02 EU / mL, preset OD value 0.1. The instrument automatically recorded the time to reach the limit for each well and automatically calculated the bacterial endotoxin content and recovery rate.

[0075] The results in Table 4 above show that, at a concentration of 1.25 mg / mL, the recovery rate of the positive control group of the glycolide test sample was 93.64%, which is within the 50%–200% range specified in the pharmacopoeia. The reaction time of the negative control was >5400.0 seconds, which is greater than the reaction time at the lowest concentration of the standard curve, indicating that the test is effective. This demonstrates that the method of the present invention does not interfere with the detection of bacterial endotoxins in glycolide, and this method is also applicable to the detection of endotoxins in glycolide.

[0076] Example 5: Detection of bacterial endotoxins in D,L-lactide Accurately weigh 100.0 mg of D,L-lactide and place it in a 10 mL centrifuge tube. Add 1.0 mL of anhydrous ethanol, tighten the cap, and vortex continuously at maximum speed for 3 minutes until the solid is completely dissolved, obtaining the primary solution. Add 3.0 mL of 0.1 mol / L sodium carbonate solution to the primary solution (primary solution to sodium carbonate solution volume ratio 1:3), tighten the cap, and vortex continuously for 3 minutes until well mixed. Take 400 μL of the above mixture and add 850 μL of BET water, mix well, and obtain a test sample stock solution with a concentration of 8 mg / mL.

[0077] Test sample group: Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution. Mix on a vortex mixer for 10 seconds. Then add 880 μL of BET water to the mixture and mix again on a vortex mixer for 30 seconds to obtain a test sample solution of 1.25 mg / mL.

[0078] Positive control group for the test sample: Take a glass test tube and accurately add 200 μL of test sample stock solution S8 and 200 μL of calcium ion buffer solution, and mix on a vortex mixer for 10 seconds. Then add 640 μL of 0.4 EU / mL endotoxin standard solution and 240 μL of BET water to the mixture, and mix again on a vortex mixer for 30 seconds to obtain a positive control solution of 1.25 mg / mL, in which the added endotoxin concentration is 0.2 EU / mL.

[0079] Positive control group: Take one vial of bacterial endotoxin working standard (10 EU / vial), add 1.0 mL of BET water to reconstitute it, and prepare a bacterial endotoxin standard solution with a concentration of 10 EU / mL. Dilute with BET water to prepare bacterial endotoxin standard solutions with concentrations of 2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL.

[0080] Negative control group: BET water.

[0081] One vial of Limulus Amebocyte Lysate (LAL) reagent (batch number 2407030) from Zhanjiang Andus Biotechnology Co., Ltd. was opened and reconstituted with 0.35 mL of bacterial endotoxin test water. The solution was gently shaken until clear. In a 96-well bacterial endotoxin test plate, 25 μL of the sample to be tested was added to each well, with two replicates per sample. 25 μL of the reconstituted LAL reagent was added to each well, and the plate was immediately placed in a microplate reader for detection. Microplate reader settings: detection wavelength 405 nm, reaction time 90 minutes, detection sensitivity 0.02 EU / mL, preset OD value 0.1. The instrument automatically recorded the time to reach the limit for each well and automatically calculated the bacterial endotoxin content and recovery rate.

[0082] The results in Table 5 above show that, at a concentration of 1.25 mg / mL, the recovery rate of the positive control group of the D,L-lactide test sample was 84.72%, which is within the 50%–200% range specified in the pharmacopoeia. The reaction time of the negative control was >5400.0 seconds, which is greater than the reaction time at the lowest concentration of the standard curve, indicating that the test is effective. This demonstrates that the method of the present invention does not interfere with the bacterial endotoxin test of D,L-lactide and is applicable to the endotoxin detection of various lactide compounds.

[0083] Example 6: Triple Synergy Accurately weigh 100.0 mg of L-lactide and place it in a 10 mL centrifuge tube. Add 1.0 mL of anhydrous ethanol (containing 0.05% polysorbate-20, i.e., 0.5% stock solution diluted 10 times with anhydrous ethanol before use). Tightly cap the tube and place it on a vortex mixer at maximum speed for 3 minutes until the solid is completely dissolved, obtaining the primary solution. Add 2.6 mL of 0.1 mol / L sodium carbonate solution to the primary solution (the volume ratio of primary solution to sodium carbonate solution is 1:3). Tightly cap the tube and place it on a vortex mixer for 3 minutes until well mixed.

[0084] Preparation of disodium EDTA stock solution: Weigh 372.2 mg of disodium EDTA, dissolve it in BET water, and bring the volume to 100 mL to obtain a 10 mmol / L stock solution. Filter the solution through a 0.22 μm filter for sterilization. Add EDTA stock solution to the above mixture to achieve a final EDTA concentration of 1.0 mmol / L (total volume approximately 4.0 mL, requiring 400 μL of 10 mmol / L EDTA stock solution). Vortex for 10 seconds. Take 400 μL of the above EDTA-containing solution and add 850 μL of BET water. Mix thoroughly to obtain a test sample stock solution S8-Triple with a concentration of 8 mg / mL.

[0085] Preparation of lactate-sodium lactate buffer: Weigh 90.08 mg lactate (molecular weight 90.08) and 112.06 mg sodium lactate (molecular weight 112.06), dissolve in BET water and bring the volume to 100 mL. Adjust the pH to 5.2 with 0.1 mol / L NaOH or HCl. Filter through a 0.22 μm filter membrane for sterilization to obtain a buffer solution with pH 5.2 and a lactate / sodium lactate molar ratio of 1:1.

[0086] Test sample group: Take a glass test tube and accurately add 200 μL of test sample stock solution S8-Triple, 200 μL of calcium ion buffer solution, and 100 μL of the above lactate-sodium lactate buffer solution. Vortex mix for 10 seconds. Then add 780 μL of BET water to the mixture and vortex mix again for 30 seconds to obtain a final test sample solution of 1.25 mg / mL.

[0087] Positive control group for the test sample: Take a glass test tube and accurately add 200 μL of test sample stock solution S8-Triple, 200 μL of calcium ion buffer solution, and 100 μL of lactate-sodium lactate buffer solution. Vortex mix for 10 seconds. Then add 640 μL of 0.4 EU / mL endotoxin standard solution and 140 μL of BET water to the mixture and vortex mix again for 30 seconds to obtain a positive control solution of 1.25 mg / mL, with an added endotoxin concentration of 0.2 EU / mL.

[0088] The preparation and detection parameters of the positive and negative control groups were the same as in Example 3. Limulus amebocyte lysate (LAL) reagent from Zhanjiang Andus Biotechnology Co., Ltd. was used, and the detection was performed according to the aforementioned method. Three parallel tubes were prepared for each batch, and the average value was taken.

[0089] Example 7: Triple Synergy The procedure is essentially the same as in Example 6, except that the final concentration of EDTA is adjusted to 2.0 mmol / L. Specifically, 800 μL of 10 mmol / L EDTA stock solution (total volume approximately 4.0 mL) is added to the mixture of the primary dissolving solution and the sodium carbonate solution (2.2 mL). All other steps are exactly the same.

[0090] Example 8: Triple Synergy The procedure is essentially the same as in Example 6, except that the final concentration of EDTA is adjusted to 3.0 mmol / L. Specifically, 1200 μL of 10 mmol / L EDTA stock solution (total volume approximately 4.0 mL) is added to the mixture of the primary dissolving solution and sodium carbonate solution (1.8 mL), and the remaining steps are exactly the same.

[0091] Comparative Example 1: The sample was dissolved using BET water instead of anhydrous ethanol. Accurately weigh 100.0 mg of lactide and place it in a 10 mL centrifuge tube. Add 1.0 mL of BET water instead of anhydrous ethanol, tighten the cap, and place the tube on a vortex mixer at maximum speed for 3 minutes. Observation revealed that the sample could not be completely dissolved, forming a turbid and opaque suspension.

[0092] Add 3.0 mL of 0.1 mol / L sodium carbonate solution directly to the above suspension, tighten the cap, and shake vigorously to form an unstable particulate suspension. Take 400 μL of the above mixed solution, add 850 μL of BET water, mix well, and obtain the test sample stock solution.

[0093] Take a glass test tube and accurately add 200 μL of the test sample stock solution and 200 μL of calcium ion buffer solution. Vortex mix for 10 seconds. Then add 880 μL of BET water to the mixture and vortex mix again for 30 seconds to obtain a test sample solution with a concentration of 1.25 mg / mL.

[0094] Preparation of the positive control group for the test sample: Take a glass test tube and accurately add 200 μL of the test sample stock solution and 200 μL of calcium ion buffer solution, and mix on a vortex mixer for 10 seconds. Then add 640 μL of 0.4 EU / mL endotoxin standard solution and 240 μL of BET water to the mixture, and mix again on a vortex mixer for 30 seconds to obtain a positive control solution of 1.25 mg / mL, in which the added endotoxin concentration is 0.2 EU / mL.

[0095] The preparation and detection parameters of the positive and negative control groups were the same as in Example 3. Limulus amebocyte lysate (LAL) reagent from Zhanjiang Andus Biotechnology Co., Ltd. was used for detection according to the aforementioned method.

[0096] Results: The measured endotoxin concentration in the positive control group of the test sample was below the detection limit (<0.00283 EU / mL), with the measured value approaching 0, and the recovery rate was 0.00%. Analysis of the reasons: BET water cannot effectively dissolve lactide, resulting in insufficient sample dispersion; simultaneously, lactide undergoes hydrolysis in BET water to generate lactic acid, lowering the system pH to the acidic range (pH≈3-4). Acidic conditions severely inhibit the enzymatic activity of the Limulus Amebocyte Lysate (LAL) reagent. Furthermore, undissolved sample particles may also cause physical interference, leading to complete inactivation of the LAL reagent and making endotoxin undetectable. This comparative example demonstrates that anhydrous ethanol as a dissolving solvent is an indispensable technical feature of this invention.

[0097] Comparative Example 2: The sodium carbonate solution was not used to adjust the pH. Accurately weigh 100.0 mg of lactide and place it in a 10 mL centrifuge tube. Add 1.0 mL of anhydrous ethanol, tighten the cap, and vortex continuously at maximum speed for 3 minutes until the solid is completely dissolved to obtain the primary solution. Dilute the primary solution directly with 3.0 mL of BET water, without adding sodium carbonate solution. Take 400 μL of the above mixture, add 850 μL of BET water, and mix well to obtain the test sample stock solution.

[0098] Take a glass test tube and accurately add 200 μL of the test sample stock solution and 200 μL of calcium ion buffer solution. Vortex mix for 10 seconds. Then add 880 μL of BET water to the mixture and vortex mix again for 30 seconds to obtain a test sample solution with a concentration of 1.25 mg / mL.

[0099] Preparation of the positive control group for the test sample: Take a glass test tube and accurately add 200 μL of the test sample stock solution and 200 μL of calcium ion buffer solution, and mix on a vortex mixer for 10 seconds. Then add 640 μL of 0.4 EU / mL endotoxin standard solution and 240 μL of BET water to the mixture, and mix again on a vortex mixer for 30 seconds to obtain a 1.25 mg / mL positive control solution for the test sample, in which the added endotoxin concentration is 0.2 EU / mL.

[0100] The preparation and detection parameters of the positive and negative control groups were the same as in Example 3.

[0101] Results: The measured endotoxin concentration in the positive control group of the test sample was below the detection limit (<0.00283 EU / mL), with the measured value approaching 0, and the recovery rate was 0.00%. Analysis: Although anhydrous ethanol can effectively dissolve lactide, lactide still undergoes hydrolysis after subsequent mixing with BET water. The lactic acid generated by hydrolysis lowers the pH of the system to the acidic range (pH≈4-5). The acidic conditions strongly inhibit the enzymatic activity of the Limulus Amebocyte Lysate (LAL) reagent, preventing it from responding to the endotoxin in the system. This comparative example demonstrates that the addition of sodium carbonate solution is an indispensable technical feature of this invention for maintaining a neutral pH, preventing sample hydrolysis, and inhibiting the activity of the LAL reagent.

[0102] Comparative Example 3: Calcium ion buffer solution removed Accurately weigh 100.0 mg of lactide and place it in a 10 mL centrifuge tube. Add 1.0 mL of BET water, tighten the cap, and vortex continuously at maximum speed for 3 minutes (the sample will not completely dissolve, forming a turbid suspension). Add 3.0 mL of 0.1 mol / L sodium carbonate solution directly to the above suspension, tighten the cap, and shake vigorously to form a suspension. Take 400 μL of the above mixture, add 850 μL of BET water, and mix well to obtain the test sample stock solution.

[0103] Take a glass test tube and accurately add 200 μL of the test sample stock solution and 1080 μL of BET water (without calcium ion buffer). Mix on a vortex mixer for 30 seconds to obtain a test sample solution of 1.25 mg / mL.

[0104] Preparation of the positive control group for the test sample: Take a glass test tube and accurately add 200 μL of test sample stock solution, 640 μL of 0.4 EU / mL endotoxin standard solution and 440 μL of BET water. Mix again on a vortex mixer for 30 seconds to obtain a test sample positive solution of 1.25 mg / mL, in which the added endotoxin concentration is 0.2 EU / mL.

[0105] The preparation and detection parameters of the positive and negative control groups were the same as in Example 3.

[0106] Results: The measured endotoxin concentration in the positive control group of the test sample was 0.04705 EU / mL, with a recovery rate of 22.11% (theoretically, the recovery rate should reach 100% when an additional 0.2 EU / mL is added). Analysis of the cause: After removing the calcium ion buffer, the divalent cation Ca required for the reaction of lactide hydrolysis products (lactic acid and its oligomers) with the Limulus amebocyte lysate (LAL) reagent was lost. 2+ Chelation occurs, leading to the release of free Ca2+ in the reaction system. 2+ Insufficient concentration, Limulus amebocyte lysate (LAL) reagent depends on Ca 2+ The coagulation cascade reaction was inhibited, and the activity of the Limulus amebocyte lysate (LAL) reagent was significantly reduced, thus failing to respond properly to endotoxins, resulting in a significantly lower endotoxin detection level. This comparative example demonstrates that calcium ion buffer is crucial for compensating for the chelation of Ca by lactone compounds. 2+ Ensuring the proper functioning of the horseshoe crab reagent is an indispensable technical feature of this invention.

[0107] Comparative Example 4: Over-dilution Accurately weigh 100.0 mg of lactide and place it in a 10 mL centrifuge tube. Add 1.0 mL of anhydrous ethanol, tighten the cap, and vortex continuously at maximum speed for 3 minutes until the solid is completely dissolved, obtaining the primary solution. Add 3.0 mL of 0.1 mol / L sodium carbonate solution to the primary solution, tighten the cap, and vortex continuously for 3 minutes until well mixed. Take 400 μL of the above mixture and add 850 μL of BET water, mix well, and obtain a test stock solution with a concentration of 8 mg / mL.

[0108] Take a glass test tube and accurately add 200 μL of the test sample stock solution and 200 μL of calcium ion buffer solution. Mix well on a vortex mixer for 10 seconds. Then add a measured amount of BET water to the mixture to make the final test sample concentration 0.4 mg / mL (i.e., over-diluted to about 1 / 3 of the concentration of 1.25 mg / mL): for example, to prepare 0.4 mg / mL, the volume of BET water to be added is (200 × 8 / 0.4 - 200 - 200) = 3600 μL, resulting in a 0.4 mg / mL test sample solution.

[0109] Preparation of the positive control group for the test sample: Take a glass test tube and accurately add 200 μL of the test sample stock solution and 200 μL of calcium ion buffer solution. Vortex mix for 10 seconds. Then add 200 μL of 2.0 EU / mL endotoxin standard solution and the corresponding volume of BET water to the mixture (to make the final concentration 0.4 mg / mL). Vortex mix again for 30 seconds to obtain a 0.4 mg / mL positive control solution for the test sample. The concentration of the added endotoxin, calculated according to the dilution factor, is approximately 0.02 EU / mL (below the detection limit of 0.02 EU / mL).

[0110] The preparation and detection parameters of the positive and negative control groups were the same as in Example 3.

[0111] Result: Recovery rate 0.00%. Analysis of the cause: Excessive dilution makes the sample acidic, directly inhibiting the reaction of the Limulus amebocyte lysate (LAL) reagent.

[0112] Comparative Example 5: pH adjustment using different alkaline solutions instead of sodium carbonate solution. This comparative example included four experimental groups, each using a different alkaline solution instead of sodium carbonate solution for pH adjustment: Group 1 (Comparative Group of the present invention, i.e., the method of Example 3): Accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol to dissolve it, add 3.0 mL of 0.1 mol / L sodium carbonate solution (volume ratio 1:3), and the remaining steps are the same as in Example 3.

[0113] Group 2 (Sodium bicarbonate group): Accurately weigh 100.0 mg of L-lactide, dissolve it in 1.0 mL of anhydrous ethanol, add 3.0 mL of 0.1 mol / L sodium bicarbonate solution (volume ratio 1:3), and follow the same steps as in Example 3.

[0114] Group 3 (Sodium hydroxide group): Accurately weigh 100.0 mg of L-lactide, dissolve it in 1.0 mL of anhydrous ethanol, add 3.0 mL of 0.1 mol / L sodium hydroxide solution (volume ratio 1:3), and follow the same steps as in Example 3.

[0115] Group 4 (low concentration sodium bicarbonate group): Accurately weigh 100.0 mg of L-lactide, dissolve it in 1.0 mL of anhydrous ethanol, add 3.0 mL of 0.05 mol / L sodium bicarbonate solution (volume ratio 1:3), and follow the same steps as in Example 3;

[0116] The results in Table 6 above show that Group 1, using only 0.1 mol / L sodium carbonate solution as a pH adjuster, achieved good detection results with a recovery rate of 88.81%. In contrast, Group 2, using 0.1 mol / L sodium bicarbonate, had a recovery rate of 0.00%; Group 3, using 0.1 mol / L sodium hydroxide, also had a recovery rate of 0.00%; and Group 4, using 0.05 mol / L sodium bicarbonate, had a recovery rate of 0.00%. This indicates that not all alkaline solutions can achieve good detection results. Sodium bicarbonate is less alkaline than sodium carbonate, and the chelating ability of bicarbonate ions for calcium ions differs from that of carbonate ions. Although sodium hydroxide is more alkaline, its strong alkalinity may cause violent hydrolysis of lactide compounds, and high concentrations of OH- may interfere with the enzymatic reaction of the horseshoe crab reagent. Therefore, the effect of sodium carbonate is not predictable due to its common function of pH adjustment; 0.1 mol / L sodium carbonate is a key technical feature of this invention.

[0117] Comparative Example 6: Magnesium ion buffer was used instead of calcium ion buffer. This comparative example was set up with two experimental groups: Group 1 (Comparative Group of the present invention, i.e., the method of Example 3): Using calcium ion buffer, accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol to dissolve it, add 3.0 mL of 0.1 mol / L sodium carbonate solution (volume ratio 1:3) to obtain the test sample stock solution, take 200 μL of the test sample stock solution and mix it with 200 μL of calcium ion buffer, and then add 880 μL of LBET water to dilute to 1.25 mg / mL. The preparation and detection steps of the positive control of the test sample are the same as in Example 3.

[0118] Group 2 (Magnesium Ion Buffer Group): Accurately weigh 100.0 mg of L-lactide, dissolve it in 1.0 mL of anhydrous ethanol, and add 3.0 mL of 0.1 mol / L sodium carbonate solution (volume ratio 1:3) to obtain the test sample stock solution. Take a glass test tube, accurately add 200 μL of the test sample stock solution and 200 μL of magnesium ion buffer, and mix on a vortex mixer for 10 seconds. Then add 880 μL of BET water to the mixture, and mix again on a vortex mixer for 30 seconds to obtain a final test sample solution of 1.25 mg / mL. Preparation of the positive control group: Take a glass test tube, accurately add 200 μL of the test sample stock solution and 200 μL of magnesium ion buffer, and mix on a vortex mixer for 10 seconds. Then, 640 μL of 0.4 EU / mL endotoxin standard solution and 240 μL of BET water were added to the mixture, and the mixture was vortexed again for 30 seconds to obtain a positive solution of 1.25 mg / mL test sample, in which the added endotoxin concentration was 0.2 EU / mL.

[0119] The results in Table 7 show that the recovery rate of group 1 using calcium ion buffer reached 88.81%, while the recovery rate of group 2 using magnesium ion buffer was only 34.42%, significantly lower than the 50% lower limit specified in the pharmacopoeia. The role of magnesium ions in the coagulation cascade reaction of the horseshoe crab reagent differs from that of calcium ions. Calcium ions are essential cofactors for several key enzymes in the coagulation cascade (such as the activation of factor C and the conversion of coagulase to coagulase, all of which depend on calcium ions). 2+ Magnesium ions cannot replace Ca. 2+ The function in these key steps; simultaneously, the hydrolysis products of lactones on Ca 2+ The chelation effect is specific, and magnesium ion compensation cannot effectively restore the activity of the Limulus amebocyte lysate (LAL) reagent. This comparative example demonstrates that lactones have a specific effect on Ca2+. 2+ The chelation effect is specific; calcium ion buffer is effective in replenishing chelated Ca. 2+ Ensuring the normal response of the horseshoe crab reagent to endotoxin plays an irreplaceable role.

[0120] Comparative Example 7: EDTA only (1.0 mmol / L), no polysorbate-20, no lactate-sodium lactate buffer. Accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol (without polysorbate-20), dissolve, then add 2.6 mL of 0.1 mol / L sodium carbonate solution, mix well, and add EDTA stock solution to a final concentration of 1.0 mmol / L (total volume: 4.0 mL), vortex to mix. Take 400 μL and add 850 μL of BET water to obtain an 8 mg / mL stock solution. Test sample group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 880 μL of BET water, mix well. Test sample positive control group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 640 μL of 0.4 EU / mL endotoxin standard solution + 240 μL of BET water, mix well. Do not add polysorbate-20 and lactate-sodium lactate buffer. The rest is the same as in Example 3.

[0121] Comparative Example 8: Polysorbate-20 (0.05%) added only, no EDTA, no lactate-sodium lactate buffer. Accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol (containing 0.05% polysorbate-20), dissolve, then add 3.0 mL of 0.1 mol / L sodium carbonate solution and mix well (without EDTA). Take 400 μL and add it to 850 μL of BET water to obtain an 8 mg / mL stock solution. Test sample group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 880 μL of BET water and mix well. Positive control group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 640 μL of 0.4 EU / mL endotoxin standard solution + 240 μL of BET water and mix well. Do not add EDTA or lactate-sodium lactate buffer. The rest is the same as in Example 3.

[0122] Comparative Example 9: Lactate-sodium lactate buffer (pH 5.2, 1:1) was added only; no EDTA and no polysorbate-20 were added. Accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol (without polysorbate-20), dissolve, then add 3.0 mL of 0.1 mol / L sodium carbonate solution and mix well (without EDTA). Take 400 μL and add 850 μL of BET water to obtain an 8 mg / mL stock solution. Test sample group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 100 μL of lactate-sodium lactate buffer + 780 μL of BET water and mix well. Positive control group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 100 μL of lactate-sodium lactate buffer + 640 μL of 0.4 EU / mL endotoxin standard solution + 140 μL of BET water and mix well. Do not add EDTA or polysorbate-20. The rest is the same as in Example 3.

[0123] Comparative Example 10: Added EDTA and polysorbate-20, lactate-sodium lactate-free buffer. Accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol (containing 0.05% polysorbate-20), dissolve, then add 2.6 mL of 0.1 mol / L sodium carbonate solution, mix well, and then add EDTA stock solution to a final concentration of 1.0 mmol / L (total volume: 4.0 mL), and vortex to mix. Take 400 μL and add 850 μL of BET water to obtain an 8 mg / mL stock solution. Test sample group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 880 μL of BET water, and mix well. Test sample positive control group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 640 μL of 0.4 EU / mL endotoxin standard solution + 240 μL of BET water, and mix well. Do not add lactate-sodium lactate buffer. The rest is the same as in Example 3.

[0124] Comparative Example 11: Added EDTA, lactate-sodium lactate buffer, and polysorbate-20-free. Accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol (without polysorbate-20), dissolve, then add 2.6 mL of 0.1 mol / L sodium carbonate solution, mix well, and add EDTA stock solution to a final concentration of 1.0 mmol / L (total volume: 4.0 mL), vortex to mix. Take 400 μL and add 850 μL of BET water to obtain an 8 mg / mL stock solution. Test sample group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 100 μL of lactate-sodium lactate buffer + 780 μL of BET water, mix well. Positive control group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 100 μL of lactate-sodium lactate buffer + 640 μL of 0.4 EU / mL endotoxin standard solution + 140 μL of BET water, mix well. Do not add polysorbate-20. The rest is the same as in Example 3.

[0125] Comparative Example 12: Polysorbate-20 and lactate-sodium lactate buffer were added; EDTA-free. Accurately weigh 100.0 mg of L-lactide, add 1.0 mL of anhydrous ethanol (containing 0.05% polysorbate-20), dissolve, then add 3.0 mL of 0.1 mol / L sodium carbonate solution and mix well (without EDTA). Take 400 μL and add it to 850 μL of BET water to obtain an 8 mg / mL stock solution. Test sample group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 100 μL of lactate-sodium lactate buffer + 780 μL of BET water and mix well. Positive control group: Take 200 μL of stock solution + 200 μL of calcium ion buffer + 100 μL of lactate-sodium lactate buffer + 640 μL of 0.4 EU / mL endotoxin standard solution + 140 μL of BET water and mix well. No EDTA is added. The rest is the same as in Example 3.

[0126] In all examples and comparative examples, the test solution, positive control solution, positive control solution, and negative control solution were detected using a uniform dynamic colorimetric method. A 96-well bacterial endotoxin test plate was used, with 25 μL of the test sample added to each well, and two replicates per sample. Limulus amebocyte lysate (LAL) reagent (batch number 2407030, detection range 10-0.01 EU / mL) from Zhanjiang Andus Biotechnology Co., Ltd. was reconstituted in 0.35 mL of BET water, and then 25 μL was added to each well. The plate was immediately placed in a Multiskan FC / ET microplate reader for detection. Detection parameters: wavelength 405 nm, reaction time 90 minutes, preset OD value 0.1, detection sensitivity 0.02 EU / mL. The instrument automatically recorded the time required for the absorbance of each well to reach the preset OD value (time to limit, in seconds) and automatically calculated the endotoxin concentration of each well based on a pre-established standard curve. The standard curve was plotted using the time to reach the limit of bacterial endotoxin (TIC) for three concentrations (2.0 EU / mL, 0.2 EU / mL, and 0.02 EU / mL, two parallel tubes for each concentration) and a negative control (BET water, two tubes). A linear regression was performed with the logarithm of bacterial endotoxin concentration (LgC) on the x-axis and the logarithm of the average time to reach the limit (LgT) on the y-axis. A valid standard curve was defined as follows: |γ| > 0.980, the TIC time for the negative control was greater than the TIC time for the 0.02 EU / mL concentration, and the coefficient of variation (CV) ≤ 20%. The recovery rate was calculated as follows: Recovery rate (%) = (Measured endotoxin concentration in the positive control group - Measured endotoxin concentration in the test group) / Added endotoxin concentration (0.2 EU / mL) × 100%. The mean and coefficient of variation (CV) were calculated for parallel samples (e.g., three batches of test samples) under each condition. All examples and comparative examples obtained their data using the same method described above to ensure data comparability.

[0127] The reliability verification results of the standard curve in Example 1 showed that the correlation coefficients of the two different manufacturers' Limulus Amebocyte Lysate (LAL) reagents were both greater than 0.980, the time to reach the limit of concentration for the negative control was greater than the time to reach the limit of concentration for the lowest concentration point of the standard curve, and the coefficient of variation was less than 20%, proving the effectiveness of the experimental system. The pre-interference test in Example 2 determined that L-lactide at a concentration of 1.25 mg / mL had no significant interference with the LAL reagent. The average recovery rate of L-lactide for the three parallel samples in Example 3 was 83.81%, with a coefficient of variation of 5.3%; the recovery rate of glycolide in Example 4 was 93.64%; and the recovery rate of D,L-lactide in Example 5 was 84.72%, all within the 50%-200% range specified in the pharmacopoeia, and the negative control met the requirements. The above results demonstrate that the four-step synergistic scheme established in Examples 3-5—anhydrous ethanol dissolution, sodium carbonate 1:3 volume ratio adjustment, calcium ion buffer adaptation, and 80-fold dilution to 1.25 mg / mL—effectively overcomes the strong hydrophobicity, easy hydrolysis, and chelation interference of lactoferrin reagent in lactide compounds. The detection results are accurate, reliable, and applicable to a variety of lactide compounds. Comparative Examples 1 to 6 verified the necessity of each step in the above four-step synergistic scheme from different perspectives: direct dissolution with BET water or omission of sodium carbonate adjustment resulted in zero recovery; omission of calcium ion buffer or replacement with magnesium ion buffer resulted in a significant decrease in recovery to 22%-34%; over-dilution to 0.4 mg / mL directly reduced the recovery to 0%; and using sodium bicarbonate or sodium hydroxide instead of sodium carbonate also resulted in zero recovery. The recovery rates of Comparative Examples 1-6 were all significantly lower than the 50% pharmacopoeia lower limit, strongly refuting the technical necessity of the four-step synergistic scheme in Examples 3-5.

[0128] Building upon the scheme in Example 3, Examples 6-8 further introduced disodium ethylenediaminetetraacetate (final concentrations of 1.0, 2.0, and 3.0 mmol / L, respectively), polysorbate-20 (0.05% by volume), and lactate-sodium lactate buffer (pH 5.2, lactate / sodium lactate molar ratio 1:1). Compared to Example 3, the recoveries in Examples 6-8 increased from 83.81% to 98.0%-100.5%, closer to the theoretical optimum of 100%; the coefficient of variation decreased from 5.3% to 2.8%-3.5%, significantly improving precision; and the time to reach the limit was shortened, indicating an increased reaction rate. This demonstrates that the triple additive not only eliminated matrix interference, bringing the recoveries closer to the ideal value, but also significantly improved the precision and reaction kinetics of the detection. The synergistic mechanism may be as follows: Disodium EDTA chelates divalent metal ions, promoting the dissociation of endotoxin lipopolysaccharide from its polymeric state to its monomeric state; Polysorbate-20 inserts into the intermolecular space of lipopolysaccharide molecules to form mixed micelles, preventing the dissociated monomers from re-aggregating, while improving the contact efficiency between hydrophobic substances and the Limulus amebocyte lysate (LAL) reagent; The lactate-sodium lactate buffer system maintains a stable weakly acidic microenvironment (pH 5.2), avoiding pH fluctuations caused by EDTA and PS-20, while lactate ions help maintain the EDTA dissociation equilibrium. These three components constitute a complete functional chain of "dissociation-dispersion-stabilization": If the lactate-sodium lactate buffer (Comparative Example 10) is missing, pH fluctuations and decreased EDTA dissociation efficiency cause the overall effect to fall back to baseline; if polysorbate-20 is missing (Comparative Example 11), endotoxin monomers are prone to re-aggregate and become ineffective; if disodium EDTA (Comparative Example 12) is missing, there are insufficient endotoxin monomers in the system for detection, and the situation may even worsen due to interference from PS-20. Based on the known properties of each component (PS-20 interferes with endotoxin detection, EDTA chelates calcium ions and inhibits the reaction of the horseshoe crab reagent, and changes in the pH of the buffer solution may affect the reaction system), those skilled in the art could not have foreseen that the combination of the three components could simultaneously achieve a synergistic effect of nearly 100% recovery, reduced coefficient of variation, and accelerated reaction rate, and that no combination of any two components could achieve the level of the triple combination.

[0129] 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.

[0130] 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 derived from lactide compounds, characterized in that, Includes the following steps: (a) Dissolve the lactone compound in anhydrous ethanol to obtain a primary solution; (b) Add sodium carbonate solution to the primary solution, mix well, and dilute with BET water to obtain a test stock solution; (c) Mix the test stock solution with calcium ion buffer, and then dilute with BET water to the set concentration to obtain a test solution; (d) Use a spectrophotometric method with Limulus amebocyte lysate (LAL) reagent to detect bacterial endotoxins in the test solution.

2. The method according to claim 1, characterized in that, The concentration of the sodium carbonate solution in step (b) is 0.1 mol / L, the volume ratio of the primary solution to the sodium carbonate solution is 1:3, and the concentration of the lactone compound in the test sample stock solution is 8 mg / mL.

3. The method according to claim 1, characterized in that, The concentration set in step (c) is 1.25 mg / mL, the volume ratio of the test sample stock solution to the calcium ion buffer is 1:1, and the calcium ion buffer is an endotoxin-free calcium ion buffer with a bacterial endotoxin content of less than 0.015 EU / mL.

4. The method according to claim 1, characterized in that, The photometric method described in step (d) is a dynamic colorimetric method with a detection wavelength of 405 nm and a detection sensitivity of 0.02 EU / mL.

5. The method according to claim 1, characterized in that, The reaction time of the Limulus amebocyte lysate (LAL) reagent in the method is 90 min, and the detection range of the LAL reagent in step (d) is 0.01-10 EU / mL.

6. The method according to claim 1, characterized in that, The amount of anhydrous ethanol used in step (a) is 1.0 mL of anhydrous ethanol per 100 mg of lactide compound.

7. The method according to claim 1, characterized in that, The lactide compound is selected from at least one of L-lactide, D-lactide, DL-lactide, glycolide, or their derivatives.

8. The method according to claim 1, characterized in that, The method also includes the preparation of a positive control solution: take a bacterial endotoxin working standard, add BET water to dissolve and vortex mix to prepare a stock solution with a concentration of 10 EU / mL, and then dilute it stepwise with BET water to prepare bacterial endotoxin standard solutions with concentrations of 2.0 EU / mL, 0.2 EU / mL and 0.02 EU / mL.

9. The method according to claim 1, characterized in that, The method further includes the preparation of a positive control solution for the test sample: the test sample stock solution is mixed with calcium ion buffer at a volume ratio of 1:1, and then diluted with a mixture containing endotoxin standard solution and BET water to make the final concentration of the test sample 1.25 mg / mL and the added endotoxin concentration 0.2 EU / mL.

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