Method for inspecting bacterial endotoxin in edaravone raw material

Abstract

The invention discloses an edaravone raw material bacterial endotoxin inspection method, and belongs to the technical field of medicine quality control. According to the method, a gel method tachypleus amebocyte lysate is taken as a core, and the interference of an edaravone molecular pyrazolone group and a residual intermediate on detection is effectively eliminated by optimizing the dilution concentration of a test sample, establishing a gradient dilution interference test system, treating a turbid sample by combining a 0.22 mu m pyrogen-free filter membrane, retesting double-batch reagent verification and other key technologies. Triple quality control is set to ensure reaction effectiveness, complex instrument analysis is not needed, whether bacterial endotoxin is qualified or not can be accurately judged, the blank of exclusive detection of edaravone raw material endotoxin is filled, and the method is suitable for industrial quality control.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical quality control technology, specifically relating to methods for detecting pyrogens in chemical raw materials, and is particularly applicable to the bacterial endotoxin limit test of edaravone raw materials. Background Technology

[0002] Edaravone, chemically known as 3-methyl-1-phenyl-2-pyrazolin-5-one, is widely used as a free radical scavenger in the treatment of acute ischemic stroke. According to the 2020 edition of the Chinese Pharmacopoeia, the content of bacterial endotoxins in injectable raw materials must be strictly controlled to avoid triggering pyrogen reactions.

[0003] Current methods for detecting bacterial endotoxins primarily utilize the Limulus Amebocyte Lysate (LAL) assay. However, the pyrazolone group in the edaravone molecule may react non-specifically with coagulant proteinogen in the LAL assay, leading to false positives. Furthermore, residual synthetic intermediates in the raw materials may inhibit coagulase activity, resulting in false negatives. Existing technologies have not optimized detection conditions for edaravone characteristics, resulting in incomplete interference elimination and poor result repeatability.

[0004] Therefore, it is of great significance to establish a method for testing bacterial endotoxins in edaravone raw materials. Summary of the Invention

[0005] Given the lack of existing detection methods, this application overcomes the interference of edaravone raw materials on the Limulus amebocyte lysate (LAL) reagent method and establishes a bacterial endotoxin detection method that meets pharmacopoeia standards, is controllable in operation, and provides reliable results.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for detecting bacterial endotoxins in edaravone raw materials, characterized by comprising the following steps:

[0008] Prepare gel-method Limulus amebocyte lysate (LAL) reagent with a sensitivity of 0.06 EU / mL-0.5 EU / mL, bacterial endotoxin working standards of 50 EU / vial-100 EU / vial, pyrogen-free water, edaravone raw material samples, and pyrogen-free pipettes, test tubes, and a constant temperature water bath with an accuracy of ±0.1℃, all of which have been sterilized by dry heat at 250℃ for 1 hour.

[0009] Take edaravone raw material samples, dilute with pyrogen-free water to 1.0 mg / mL-5.0 mg / mL, and when turbid, filter through a 0.22 μm pyrogen-free filter membrane to collect the filtrate;

[0010] Prepare a series of test sample dilutions at concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, and 1.0 mg / mL. Add bacterial endotoxin to each solution to achieve a concentration of 2λ (λ is the sensitivity of the Limulus Amebocyte Lysate (LAL) reagent), thus obtaining the spiked test sample solutions. Simultaneously, set up a positive control (2λ concentration standard solution) and a negative control (pyrogen-free water). Incubate the spiked test sample solutions with the positive and negative controls simultaneously. A recovery rate of 50%-200% indicates no interference.

[0011] Take a test solution with no interference concentration, and set up a positive control (including λ concentration standard solution and 2λ concentration standard solution) and a negative control (pyrogen-free water). React the test solution, positive control and negative control with the Limulus amebocyte lysate (LAL) reagent respectively, and incubate at 37℃±0.5℃ for 60min±2min. Judge the results according to the integrity of the gel. If the first judgment does not meet the requirements, a retest is required.

[0012] Furthermore, the preferred dilution concentration of the test sample diluent is 2.0 mg / mL.

[0013] Furthermore, the preferred concentration gradients of the test sample diluent are 0.2 mg / mL, 0.5 mg / mL, and 1.0 mg / mL.

[0014] Furthermore, the positive control in the interference test is a 2λ concentration standard solution, and in the endotoxin test, in addition to the λ and 2λ concentration standard solutions, an additional spiked solution of the test sample is required (the test sample solution without interference concentration is supplemented with bacterial endotoxin to make the concentration 2λ).

[0015] Furthermore, in addition to dry heat sterilization, pyrogen-free instruments can be sterilized with ethylene oxide and then rinsed three times with pyrogen-free water.

[0016] Furthermore, the bacterial endotoxin working standard was diluted in a Class 100 cleanroom pyrogen-free operating table and stored at 4°C for no more than 24 hours after dilution.

[0017] Furthermore, the incubation process uses pyrogen-free test tubes with sealed caps, with the test tube openings 3 cm below the water bath surface and not in contact with the water surface.

[0018] Furthermore, when observing the results, the gel is slowly inverted at a tilt angle of 180°. If the gel remains intact and does not flow, it is considered a positive result for coagulation.

[0019] Furthermore, the filter membrane needs to be soaked in pyrogen-free water for 30 minutes before use, and the initial filtrate of 5 mL should be discarded before collecting the subsequent filtrate.

[0020] Furthermore, the sample size for the retest is twice that of the first test, and the batches of horseshoe crab reagent and standard are changed. If both results are unqualified, it is determined that the test does not meet the requirements.

[0021] The Limulus amebocyte lysate (LAL) reagent used in the gel electrophoresis method was selected with a sensitivity of 0.25 EU / mL as the preferred value, matching the endotoxin limit of ≤0.5 EU / mg for edaravone raw materials. The working standard prepared by the China National Institutes for Food and Drug Control was used, with an optimal potency of 80 EU / vial. The matrix concentration of the test sample was reduced through gradient dilution (0.1 mg / mL-1.0 mg / mL), and the minimum non-interfering dilution factor (usually 0.5 mg / mL) was determined by combining spiked recovery verification (using 2λ concentration standards). Special treatment was applied to address potential sample turbidity during the synthesis process, using a 0.22 μm polyethersulfone filter membrane. This membrane has a pyrogen adsorption rate of <1% and does not affect the solubility of edaravone. A triple quality control system was established, including a negative control (pyrogen-free water), a positive control (2λ standard), and a spiked control of the test sample (test sample + 2λ standard) to ensure the effectiveness of the reaction system.

[0022] Beneficial technical effects

[0023] A method for detecting bacterial endotoxins in edaravone raw materials is disclosed. The core of this application lies in fundamentally addressing the inherent interference of edaravone molecular characteristics on the Limulus Amebocyte Lysate (LAL) reagent method. Through a gradient dilution design, the method leverages the characteristic that the interference of edaravone on the LAL coagulation reaction weakens with decreasing concentration within a specific concentration range. Combined with spike verification, this precisely identifies the interference-free concentration, fundamentally avoiding non-specific false positives caused by the pyrazolone group and false negatives due to the inhibition of coagulation enzyme activity by residual intermediates. For turbid samples, the filter membrane treatment utilizes the physical retention of a pyrogen-free filter with a specific pore size to remove suspended impurities while preventing the adsorption of bacterial endotoxins or the retention of edaravone, thus achieving separation of the target substance from interfering substances. The retesting process enhances representativeness by increasing the sample size and eliminates reagent batch fluctuations, constructing a double quality control barrier to avoid misjudgments due to operational errors or accidental contamination. Employing the core technology of gelation, this method eliminates the need for complex standard curves and instrumental analysis. Qualitative determination can be made solely through the gel solidification state, meeting routine quality control requirements. The overall design revolves around the physicochemical properties and detection reaction mechanism of edaravone, forming a complete technical system that ensures scientific rigor and accuracy. It does not rely on existing general method frameworks and is highly innovative and targeted. Attached Figure Description

[0024] Figure 1 Flowchart of a method for detecting bacterial endotoxins in edaravone raw materials Detailed Implementation

[0025] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application will be provided below.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of this application. However, this application may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0027] Example 1

[0028] like Figure 1 As shown in the figure, this embodiment provides a method for testing bacterial endotoxins in edaravone raw materials, including the following steps:

[0029] Limulus amebocyte lysate (LAL) gel electrophoresis reagent (λ=0.25 EU / mL, batch number 20240105), bacterial endotoxin standard (80 EU / vial, batch number 20240312, diluted to 2λ=0.5 EU / mL), pyrogen-free water (resistivity ≥18.2 MΩ•cm); constant temperature water bath (37℃±0.5℃), pyrogen-free pipettes / test tubes (sterilized at 250℃ for 1 h); test sample (batch number 20240501, purity 99.8%).

[0030] Take 100 mg of the test sample and dilute it with pyrogen-free water to a stock solution of 2.0 mg / mL. Then dilute it to 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL and 1.0 mg / mL, and make 3 parallel samples for each.

[0031] Each dilution was incubated with 2λ standard and simultaneously with the positive control (2λ standard) and negative control (pyrogen-free water) for 60 min ± 2 min. Results: The recovery rate of the 0.1-0.5 mg / mL group was 100% (within 50%-200%), and the recovery rate of the 1.0 mg / mL group was 66.7% (poor stability). The concentration with no interference was determined to be 0.5 mg / mL.

[0032] Take 0.5 mg / mL of the test solution and react it with λ (0.25 EU / mL), 2λ standard and negative control. Result: The test solution did not coagulate, the negative control did not coagulate, and the positive control coagulated, so it was deemed qualified.

[0033] Example 2

[0034] like Figure 1 As shown in the figure, this embodiment provides a method for testing bacterial endotoxins in edaravone raw materials, including the following steps:

[0035] Limulus amebocyte lysate (LAL) gel electrophoresis reagent (λ=0.25 EU / mL, batch number 20240106), bacterial endotoxin standard (80 EU / vial, batch number 20240313, diluted to 2λ=0.5 EU / mL), pyrogen-free water (resistivity ≥18.2 MΩ•cm), 0.22 μm pyrogen-free filter membrane (batch number 20240320); constant temperature water bath (37℃±0.5℃), pyrogen-free pipettes / test tubes (sterilized at 250℃ for 1 h), pyrogen-free filter; test sample (batch number 20240502, purity 99.5%, turbid after dissolution).

[0036] Take 100 mg of the test sample, dissolve it in 50 mL of pyrogen-free water (the solution will be turbid), filter it through a 0.22 μm pyrogen-free filter membrane (discard 5 mL of the initial filtrate) to obtain a 1.98 mg / mL treatment solution; then dilute the treatment solution to 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL and 1.0 mg / mL dilutions, and make 3 parallel samples for each.

[0037] Each dilution was incubated with 2λ standard and simultaneously with the positive control (2λ standard) and negative control (pyrogen-free water) for 60 min ± 2 min. Results: The recovery rate of the 0.1-0.5 mg / mL group was 100% (within 50%-200%), while the recovery rate of the 0.5 mg / mL group in the pre-filtration turbid solution was 66.7% (significant interference). The concentration with no interference was determined to be 0.5 mg / mL.

[0038] Take a 0.5 mg / mL test solution and react it with λ (0.25 EU / mL), 2λ standard and negative control. Result: The test solution did not coagulate, the negative control did not coagulate, and the positive control coagulated. The result was deemed acceptable, and the endotoxin level was <0.5 EU / mg.

[0039] Example 3

[0040] like Figure 1 As shown in the figure, this embodiment provides a method for testing bacterial endotoxins in edaravone raw materials, including the following steps:

[0041] Limulus amebocyte lysate (LAL) gel electrophoresis reagent (λ=0.25 EU / mL, batch number 20240107), bacterial endotoxin standard (80 EU / vial, batch number 20240314, diluted to 2λ=0.5 EU / mL), pyrogen-free water (resistivity ≥18.2 MΩ•cm); constant temperature water bath (37℃±0.5℃), pyrogen-free pipettes / test tubes (sterilized at 250℃ for 1 h, new batch); test sample (batch number 20240503, purity 99.7%, 2 samples coagulated for the first test).

[0042] Take 20 mg of the test sample (twice the amount of the first sample), dilute it with 10 mL of pyrogen-free water to a stock solution of 2.0 mg / mL, and then dilute it to a test solution of 0.5 mg / mL. Prepare 3 parallel samples for each.

[0043] Add 0.5 mg / mL of test solution to 2λ standard, and incubate simultaneously with the positive control (2λ standard) and negative control (pyrogen-free water) for 60 min ± 2 min. Results: Recovery rate 100% (within 50%-200%), no interference.

[0044] Take 0.5 mg / mL of the test solution and react it with λ (0.25 EU / mL), 2λ standard and negative control. Results: The test solution did not coagulate, the negative control did not coagulate, and the positive control coagulated. The result was considered acceptable. The first false positive was due to cross-contamination.

[0045] Comparative Example 1

[0046] This embodiment provides a method for testing bacterial endotoxins in edaravone raw materials, including the following steps:

[0047] Limulus amebocyte lysate (LAL) gel electrophoresis reagent (λ=0.25 EU / mL, batch number 20240108), bacterial endotoxin standard (80 EU / vial, batch number 20240315, diluted to 2λ=0.5 EU / mL), pyrogen-free water (resistivity ≥18.2 MΩ•cm); constant temperature water bath (37℃±0.5℃), pyrogen-free pipettes / test tubes (sterilized at 250℃ for 1 h); test sample (batch number 20240501, purity 99.8%).

[0048] Take 100 mg of the test sample and dilute it with pyrogen-free water to a stock solution of 2.0 mg / mL. Use this stock solution directly as the test solution. Prepare 3 parallel samples for each sample.

[0049] The 2.0 mg / mL test solution was reacted with the positive control (2λ standard) and the negative control (pyrogen-free water). Results: The test solution did not coagulate, and the initial judgment was qualified; subsequent interference tests were performed, and the recovery rate of the 2.0 mg / mL group was 30% (<50%, indicating significant inhibition). If the endotoxin content was 0.3 EU / mg, a false negative would occur, and the results would be unreliable.

[0050] Comparative Example 2

[0051] This embodiment provides a method for testing bacterial endotoxins in edaravone raw materials, including the following steps:

[0052] Limulus amebocyte lysate (LAL) reagent for dynamic turbidity method (λ=0.03 EU / mL, batch number 20240201), bacterial endotoxin standard (80 EU / vial, batch number 20240316, diluted to a series of concentrations of 0.03-0.5 EU / mL), pyrogen-free water (resistivity ≥18.2 MΩ•cm); bacterial endotoxin spectrophotometer, constant temperature water bath (37℃±0.5℃), pyrogen-free pipettes / test tubes (sterilized at 250℃ for 1 h); test sample (batch number 20240501, purity 99.8%).

[0053] Take 100 mg of the test sample, dilute it with pyrogen-free water to a stock solution of 2.0 mg / mL, and then dilute it to a diluted solution of 0.5 mg / mL. Prepare three parallel samples for each.

[0054] Plot a standard curve (0.03-0.5 EU / mL, R²=0.998), take 0.5 mg / mL of the test sample solution to measure the reaction time, substitute it into the curve to obtain endotoxin 0.08±0.02 EU / mL (recovery rate 80-120%); operation time 90 min.

[0055] Examples 1-3 focus on the bacterial endotoxin test of edaravone raw materials, all using the gel-gel method with Limulus amebocyte lysate (LAL) reagent as the core. Through a gradient dilution design, the interference of edaravone on the LAL reagent coagulation reaction decreases with decreasing concentration in the 0.1-0.5 mg / mL range. Combined with spiked verification, the interference-free concentration (preferably 0.5 mg / mL) is precisely locked, fundamentally avoiding non-specific false positives caused by the pyrazolone group and false negatives caused by the inhibition of coagulation enzyme activity by residual intermediates. For turbid samples (Example 2), 0. The physical retention effect of the 22μm pyrogen-free filter membrane removes suspended impurities while avoiding the adsorption of bacterial endotoxins or the retention of edaravone, thus achieving the separation of target substances from interfering substances. The retesting process (Example 3) improves sample representativeness by doubling the sample size and eliminates reagent fluctuations by changing reagent batches, thus constructing a dual quality control barrier to avoid misjudgments caused by operational errors or accidental contamination. In the end, accurate detection was achieved, verifying the adaptability and reliability of the method for edaravone raw materials in different states (normal, turbid, and abnormal initial detection).

[0056] Comparative Example 1 did not undergo interference testing and directly used 2.0 mg / mL of the test sample stock solution for detection. Although it was initially deemed qualified, subsequent verification showed that edaravone had a significant inhibitory effect on the detection at this concentration (recovery rate 30% < 50%), which could not avoid the risk of false negatives. This highlights the necessity of the interference testing step in this application. Only by locking in the non-interfering concentration through gradient dilution and spike verification can the inherent interference of edaravone molecular characteristics on the detection be eliminated in principle, thus ensuring the accuracy of the results.

[0057] Comparative Example 2 uses dynamic turbidimetry (photometry) instead of the gel method of this application. Although it can measure the endotoxin concentration, it requires the plotting of a standard curve, which is complicated and time-consuming (90 min). In contrast, the gel method of this application does not require complicated instrument analysis. The qualitative determination can be made by directly observing the gel solidification state, which meets the requirements of routine quality control for ease of operation and efficiency. At the same time, it avoids the absorbance interference that may be introduced by the slight color or turbidity of edaravone in the photometric method. This further demonstrates the advantages of the method of this application in terms of specificity and practicality.

[0058] It should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit it. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the spirit and scope of the technical solutions of this application, and all such modifications and substitutions should be covered within the scope of the claims of this application.

Claims

1. A method for the bacterial endotoxin test of edaravone raw material, characterized by, It comprises the following steps: Prepare the gel method limulus reagent with sensitivity of 0.06 EU / mL-0.5 EU / mL, bacterial endotoxin working standard with 50 EU / branch-100 EU / branch, pyrogen-free water, edaravone raw material sample, and pyrogen-free pipette, test tube and constant temperature water bath pot with precision ±0.1℃ sterilized by dry heat at 250℃ for 1 hour; Take the edaravone raw material sample, dilute it to 1.0 mg / mL-5.0 mg / mL with pyrogen-free water, and filter the filtrate when it is turbid through a 0.22 μm pyrogen-free filter membrane; Prepare 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL series of test sample dilutions, each adding bacterial endotoxin to make the concentration 2λ (λ is the sensitivity of limulus reagent), to obtain test sample spiked solution; At the same time, set up positive control (2λ concentration standard solution) and negative control (pyrogen-free water), incubate the test sample spiked solution with positive and negative controls synchronously, and the recovery rate of 50%-200% is non-interference; Take the non-interference concentration test sample solution, set up positive control (containing λ concentration standard solution, 2λ concentration standard solution) and negative control (pyrogen-free water) at the same time, and react the test sample solution, positive control and negative control with limulus reagent respectively, incubate at 37℃±0.5℃ for 60 min±2 min, and determine according to the gel integrity; If the first determination does not meet the requirements, retest is needed.

2. The method of claim 1, wherein, The test sample test sample dilution is preferably diluted to a concentration of 2.0 mg / mL.

3. The method of claim 1, wherein, The test sample dilution is preferably a concentration gradient of 0.2 mg / mL, 0.5 mg / mL and 1.0 mg / mL.

4. The method of claim 1, wherein, The positive control in the interference test is 2λ concentration standard solution, and in addition to λ and 2λ concentration standard solution, test sample spiked solution (non-interference concentration test sample solution added with bacterial endotoxin to make the concentration 2λ) is additionally required in the positive control of endotoxin test.

5. The method of claim 1, wherein, In addition to dry heat sterilization, the pyrogen-free device is sterilized by ethylene oxide and then rinsed with pyrogen-free water for 3 times.

6. The method of claim 1, wherein, The bacterial endotoxin working standard is diluted on a 100-grade clean pyrogen-free operating table, and stored at 4℃ for no more than 24 hours after dilution.

7. The method of claim 1, wherein, During the incubation process, use pyrogen-free test tubes with sealed lids, and the test tube opening is 3 cm lower than the water bath liquid surface and does not touch the water surface.

8. The method of claim 1, wherein, The inclination angle when observing the results is 180° slow inversion.

9. The method of claim 1, wherein, The filter membrane needs to be soaked in pyrogen-free water for 30 min before use, and the initial filtrate of 5 mL is discarded, and then the filtrate is collected.

10. The method of claim 1, wherein, The sampling amount during retest is twice of the first time, and the limulus reagent and standard are replaced, and if the results of both times are unqualified, it is determined that the requirements are not met.

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