Method for detecting bacterial endotoxin of rafenasin and application of method for detecting bacterial endotoxin of rafenasin
By combining refenacin with polysorbate 80 and N-methylpyrrolidone, the problem of refenacin in bacterial endotoxin detection was solved, and more accurate and reliable test results were achieved, ensuring the evaluation of drug safety.
Patent Information
- Application Number
- CN202510042413.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-10
- Publication Date
- 2025-03-18
AI Technical Summary
The insoluble nature of refenacin leads to uneven dispersion in bacterial endotoxin detection, resulting in inaccurate test results and may interfere with the detection process and affect the evaluation of drug safety.
Refinac was prepared with water containing polysorbate 80, and was diluted and mixed and dissolved by N-methylpyrrolidone, then diluted with water to an appropriate concentration, and then bacterial endotoxin examination was performed using lilium reagent.
This method effectively solves the dissolution problem of ravenacin test products, improves its dispersion and solubility in the detection, ensures the accuracy and reliability of the test results, and avoids the occurrence of false positive or false negative results.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of drug quality control, and in particular to a method for detecting refenacin bacterial endotoxin and an application thereof. Background Art
[0002] In the field of drug development and production, ensuring the safety of drugs is of vital importance. As a compound with specific pharmacological activity, revenacin is increasingly used in the pharmaceutical field, but due to the particularity of its physical properties, that is, it is almost insoluble in water, it brings challenges to the quality control of drugs, especially in the key link of bacterial endotoxin inspection.
[0003] Bacterial endotoxin testing is an important indicator for evaluating drug safety. It is used to detect endotoxins produced by Gram-negative bacteria that may be introduced during the drug production process. These endotoxins have potential toxic effects on the human body and can cause fever, shock, and even death. Therefore, accurate and sensitive detection of bacterial endotoxin content in drugs is crucial to ensure patient safety.
[0004] However, the poor solubility of refenacin has become a technical problem in bacterial endotoxin testing. Since refenacin is not evenly dispersed in water, direct bacterial endotoxin testing will lead to inaccurate test results, which may mask or exaggerate the actual endotoxin content, thereby affecting the correct assessment of drug safety. In addition, the solubility problem of the test sample may also interfere with the chemical reaction during the test process, further affecting the reliability of the test results.
[0005] In order to solve this technical problem, researchers need to develop a method that can effectively dissolve revenacin or at least disperse it evenly in water, while ensuring that the method does not interfere with the subsequent bacterial endotoxin detection process. This requires that the new method not only solves the solubility problem of the test article, but also eliminates or minimizes any potential interference with bacterial endotoxin detection, thereby ensuring the accuracy and reliability of the test results.
[0006] In summary, there is an urgent need for a new method for detecting bacterial endotoxins in refenacin, which should be able to effectively solve the dissolution problem of the test sample and eliminate the interference with bacterial endotoxin detection, provide strong support for the quality control of refenacin and its related drugs, and ensure the safety of patients' medication. Summary of the invention
[0007] The technical problem to be solved by the present invention is that the refenacin test sample is difficult to dissolve and has an interfering effect on bacterial endotoxins. The purpose is to provide a refenacin bacterial endotoxin detection method and application, which solves the dissolution problem of the test sample and eliminates the interfering effect on bacterial endotoxin detection.
[0008] The present invention is achieved through the following technical solutions:
[0009] In a first aspect, the present invention provides a method for detecting bacterial endotoxins of revenacin, comprising preparing a suspension of revenacin with water containing polysorbate 80, diluting the suspension with N-methylpyrrolidone and mixing and dissolving it, and then diluting it with water, and then performing a bacterial endotoxin test with a horseshoe crab reagent.
[0010] As a possible design, the concentration of polysorbate 80 in the bacterial endotoxin test water containing polysorbate 80 is 0.5-1.5%.
[0011] As a possible design, the above water is water for bacterial endotoxin inspection.
[0012] As a possible design, the concentration of refenacin in the above suspension is 18-22 mg / mL.
[0013] As a possible design, after the suspension is diluted with the above-mentioned N-methylpyrrolidone, the concentration of refenacin is 3 to 5 mg / mL.
[0014] As a possible design, the dilution with water is specifically diluted to a refenacin concentration of 0.02-0.04 mg / mL.
[0015] As a possible design, the above-mentioned horseshoe crab reagent is dissolved in a magnesium ion buffer solution before use.
[0016] As a possible design, the λ of the above-mentioned horseshoe crab reagent is 0.1-0.15EU / ml.
[0017] As a possible design, the suspension is diluted with the above-mentioned N-methylpyrrolidone and mixed to dissolve, and then diluted with water, and horseshoe crab reagent is added, mixed and sealed, and kept warm at 37°C±1°C for 60±2min, and then bacterial endotoxin detection is performed.
[0018] In a second aspect, the present invention also provides application of the above method in quality control of revenacin suspension.
[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0020] The present invention uses water containing polysorbate 80 to prepare a suspension of refenacin. This treatment effectively improves the dispersion state of the test sample in the solution, so that refenacin can be better dispersed and dissolved in the subsequent treatment process; the addition of polysorbate 80 not only improves the stability of the suspension, but also enhances its uniformity, which is convenient for subsequent detection. The suspension is then diluted and mixed with N-methylpyrrolidone to further improve the solubility and dispersibility of the test sample. This treatment ensures that the solution has stronger representativeness when sampling and can more truly reflect the content of bacterial endotoxins in the test sample. Finally, the treated solution is tested for bacterial endotoxins using horseshoe crab reagent. Since the previous treatment steps have ensured the good solubility and uniformity of the test sample, the horseshoe crab reagent can accurately detect the bacterial endotoxin content in the test sample; at the same time, the method has no effect on the background value of bacterial endotoxins in the test sample, avoiding false positive or false negative results caused by improper treatment.
[0021] The refenacin bacterial endotoxin detection method of the present invention has significant effects and advantages. It solves the dissolution problem of the test sample and the interference effect on bacterial endotoxin, and improves the accuracy and reliability of the detection. At the same time, the method has good applicability and reproducibility, can be widely used in the bacterial endotoxin detection of refenacin and its related products, and provides strong technical support for drug quality control and safety assessment. DETAILED DESCRIPTION
[0022] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with Examples. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0023] Hereinafter, the embodiments of the present invention are specifically disclosed with appropriate reference to the detailed description. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there may be cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structure are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the following description is provided for those skilled in the art to fully understand the present invention and is not intended to limit the subject matter described in the claims.
[0024] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present invention, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.
[0025] If not otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution. If not otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution. If not otherwise specified, all steps of the present invention can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, it is mentioned that the method may also include step (c), which means that step (c) can be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.
[0026] If there is no special explanation, the "include" and "comprising" mentioned in the present invention represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or only the listed components may be included or only the listed components may be included.
[0027] If not specifically stated, in the present invention, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).
[0028] A method for detecting bacterial endotoxin of refenacin comprises preparing a suspension of refenacin with water containing polysorbate 80, diluting the suspension with N-methylpyrrolidone and mixing and dissolving it, and then diluting it with water and performing a bacterial endotoxin test with a horseshoe crab reagent.
[0029] As a possible design, the concentration of polysorbate 80 in the bacterial endotoxin test water containing polysorbate 80 is 0.5-1.5%. The concentration of polysorbate 80 can be any value within 0.5-1.5%, including but not limited to 0.6%, 0.8%, 1.3% and 1.4%.
[0030] Preferably, the concentration of the polysorbate 80 is 1%.
[0031] As a possible design, the above water is water for bacterial endotoxin inspection.
[0032] As a possible design, in the above suspension, the concentration of refenacin is 18-22 mg / mL. The concentration of refenacin can be any value within the range of 18-22 mg / mL, including but not limited to 19 mg / mL, 20 mg / mL, 21 mg / mL and 21.5 mg / mL.
[0033] Preferably, in the above suspension, the concentration of revenacin is 20 mg / mL.
[0034] As a possible design, after the suspension is diluted with the above-mentioned N-methylpyrrolidone, the concentration of refenacin is 3 to 5 mg / mL.
[0035] As a possible design, the dilution with water is specifically diluted to a refenacin concentration of 0.02-0.04 mg / mL. The refenacin concentration after dilution with water can be any value within the range of 0.02-0.04 mg / mL, including but not limited to 0.03 mg / mL, 0.025 mg / mL and 0.035 mg / mL.
[0036] Preferably, the concentration of revenacin after dilution with water may be 0.03 mg / mL.
[0037] As a possible design, the above-mentioned horseshoe crab reagent is dissolved in a magnesium ion buffer solution before use.
[0038] As a possible design, the λ of the above horseshoe crab reagent is 0.1-0.15EU / ml. The λ of the horseshoe crab reagent can be any value within 0.1-0.15EU / ml, including but not limited to 0.11EU / ml, 0.12EU / ml, 0.14EU / ml and 0.135EU / ml. The above λ is the indicated value of the sensitivity of the horseshoe crab reagent.
[0039] Preferably, the λ of the horseshoe crab reagent is 0.125 EU / ml.
[0040] As a possible design, the suspension is diluted with the above-mentioned N-methylpyrrolidone and mixed to dissolve, and then diluted with water, and horseshoe crab reagent is added, mixed and sealed, and kept warm at 37°C±1°C for 60±2min, and then bacterial endotoxin detection is performed.
[0041] Preferably, the method of the present invention for detecting bacterial endotoxins using horseshoe crab reagent can be based on the gel method in the art.
[0042] In a second aspect, the present invention also provides application of the above method in quality control of revenacin suspension.
[0043] Example 1
[0044] Interference experiment:
[0045] (I) Preparation of experimental solution
[0046] 1. Test sample group (Solution A): Weigh 0.1 g of revenacin test sample and add 5 ml of bacterial endotoxin test water containing 1% polysorbate 80 to make a test solution with a concentration of 20 mg / ml (S 20 ), S was treated with N-methylpyrrolidone 20 Dilute 5 times, mix and dissolve to make S4 with a concentration of 4 mg / ml, and then dilute 64 times and 128 times with bacterial endotoxin test water to make a concentration of 0.06 mg / ml (S 0.06 ) and 0.03mg / ml(S 0.03 ) of the test solution, where S 0.03 Solution A is used for the test.
[0047] 2. Test sample positive group (Solution B): Take S 0.06 The test solution was mixed with 4λ(E 0.5 , bacterial endotoxin standard concentration 0.5EU / mL), 2λ(E 0.25 ),λ(E 0.125 )、0.5λ(E 0.06 ) bacterial endotoxin solution was mixed in equal volumes to prepare a solution containing 2λ(E 0.25 ),λ(E 0.125 )、0.5λ(E 0.06 )、0.25λ(E0.03 ) The positive solution B of the test sample of the bacterial endotoxin standard. Wherein, λ is the labeled amount of the sensitivity of the horseshoe crab reagent, 1λ = 0.125EU / ml.
[0048] 3. Positive control group (Solution C): Take one bacterial endotoxin standard (80EU / tube), add 1ml of bacterial endotoxin test water to dissolve, make a bacterial endotoxin standard solution with a concentration of 80EU / ml, and dilute it with bacterial endotoxin test water to 4λ (E 0.5 )、2λ(E 0.25 ),λ(E 0.125 )、0.5λ(E 0.06 )、0.25λ(E 0.03 ) bacterial endotoxin standard solution, where 2λ(E 0.25 ),λ(E 0.125 )、0.5λ(E 0.06 )、0.25λ(E 0.03 ) is C solution.
[0049] 4. Negative control group (Solution D): water for bacterial endotoxin test.
[0050] (II) Sample addition and heat preservation test
[0051] After re-dissolving the horseshoe crab reagent with a sensitivity of 0.125EU / ml with magnesium ion buffer, dispense it into reaction tubes (50μl / tube), add the above test solution in equal volume with the horseshoe crab reagent, prepare 2 tubes of A, C, and D solutions in parallel, and prepare 4 tubes of B solution in parallel. After gently mixing the solution in the reaction tube, seal the tube mouth, vertically place it in a thermostat at 37℃±1℃, keep it warm for 60±2min, and then take it out for detection. The results are shown in Table 1.
[0052] Table 1
[0053]
[0054]
[0055] Observe Table 1, the maximum concentration of solution C 2λ tubes are all positive, the minimum concentration of solution C 0.25λ tubes are all negative, and the negative control tubes and all parallel tubes of solution A are all negative, which shows that the test is effective. The results of solution B in the interference test meet the requirements of the sensitivity verification test of the horseshoe crab reagent, and the test sample has no interference at this concentration. Therefore, the above method can be used for the bacterial endotoxin test of revenacin.
[0056] Example 2
[0057] Spike interference experiment:
[0058] (I) Preparation of experimental solution
[0059] 1. Test sample group (Solution A): Weigh 0.1 g of revenacin test sample and add 5 ml of bacterial endotoxin test water containing 1% polysorbate 80 to make a test solution with a concentration of 20 mg / ml (S 20 ), S was treated with N-methylpyrrolidone 20 Dilute 5 times, dissolve and mix to make S4 with a concentration of 4 mg / ml, and then dilute 128 times with bacterial endotoxin test water to make a test solution A (S4) with a concentration of 0.03 mg / ml. 0.03 ).
[0060] 2. Test sample positive group (Solution B): Reconstitute the bacterial endotoxin standard with bacterial endotoxin test water containing 1% polysorbate 80 to prepare a bacterial endotoxin standard solution with a concentration of 160EU / ml. Take 0.5ml of the reconstituted bacterial standard solution and add it to 10mg of the test sample, mix well, and prepare a 20mg / ml test solution (S solution) containing 160EU / ml bacterial endotoxin standard. 20 E 160 ), using N-methylpyrrolidone as a cosolvent, S 20 E 160 Dilute 5 times, mix and dissolve, dilute 128 times with bacterial endotoxin test water to prepare a 2λ test solution (S 0.03 E 0.25 ), then use solution A (S 0.03 ) 0.03 E 0.25 Dilute 2 times, 4 times, 8 times to make λ(S 0.03 E 0.125 )、0.5λ(S 0.03 E 0.06 )、0.25λ(S 0.03 E 0.03 ) Test sample positive B solution of bacterial endotoxin standard.
[0061] 3. Positive control group (Solution C): Take bacterial endotoxin standard, reconstitute it with bacterial endotoxin test water, and dilute it into 2λ, λ, 0.5λ, and 0.25λ bacterial endotoxin standard solutions, which are Solution C.
[0062] 4. Negative control group (Solution D): Bacterial endotoxin test water.
[0063] (II) Sample addition and heat preservation test
[0064] After re-dissolving the horseshoe crab reagent with a sensitivity of 0.125EU / ml with magnesium ion buffer, dispense it into reaction tubes (50μl / tube), add the above test solution in equal volume with the horseshoe crab reagent, prepare 2 tubes of A, C, and D solutions in parallel, and prepare 4 tubes of B solution in parallel. After gently mixing the solution in the reaction tube, seal the tube mouth, vertically place it in a thermostat at 37℃±1℃, keep it warm for 60±2min, and then take it out for detection. The results are shown in Table 2.
[0065] Table 2
[0066]
[0067] According to Table 2, the 2λ tubes of solution C were all positive, the 0.25λ tubes were all negative, and the negative control tubes and all parallel tubes of solution A were negative, which shows that the test is effective. The results of solution B in the interference test were between 2λ and 0.5λ. After the test sample was treated with this method, the interference could be effectively eliminated without inactivating the endotoxin. This method can be used for the interference test of refenacin and the bacterial endotoxin test.
[0068] The present invention uses water containing polysorbate 80 to prepare a suspension of refenacin. This treatment effectively improves the dispersion state of the test sample in the solution, so that refenacin can be better dispersed and dissolved in the subsequent treatment process; the addition of polysorbate 80 not only improves the stability of the suspension, but also enhances its uniformity, which is convenient for subsequent detection. The suspension is then diluted and mixed with N-methylpyrrolidone to further improve the solubility and dispersibility of the test sample. This treatment ensures that the solution has stronger representativeness when sampling and can more truly reflect the content of bacterial endotoxins in the test sample. Finally, the treated solution is tested for bacterial endotoxins using horseshoe crab reagent. Since the previous treatment steps have ensured the good solubility and uniformity of the test sample, the horseshoe crab reagent can accurately detect the bacterial endotoxin content in the test sample; at the same time, the method has no effect on the background value of bacterial endotoxins in the test sample, avoiding false positive or false negative results caused by improper treatment.
[0069] The refenacin test sample suspension of the present invention has good uniformity and strong representativeness when the solution is sampled; the background value of bacterial endotoxins in the test sample is not affected during the dissolution treatment process, the bacterial endotoxins in the test sample can be effectively detected, and the method has good applicability and reproducibility, thereby solving the dissolution problem of the test sample and the interference effect on the bacterial endotoxins.
[0070] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for detecting bacterial endotoxin of refenacin, characterized in that: The method comprises preparing a suspension of revenacin with water containing polysorbate 80, diluting the suspension with N-methylpyrrolidone and mixing and dissolving the suspension, and then diluting the suspension with water and performing a bacterial endotoxin test with a horseshoe crab reagent.
2. A method for detecting bacterial endotoxin of refenacin according to claim 1, characterized in that: The concentration of polysorbate 80 in the bacterial endotoxin test water containing polysorbate 80 is 0.5-1.5%.
3. A method for detecting bacterial endotoxin of refenacin according to claim 1, characterized in that: The water is water for bacterial endotoxin testing.
4. A method for detecting bacterial endotoxin of refenacin according to claim 1, characterized in that: In the suspension, the concentration of refenacin is 18-22 mg / mL.
5. A method for detecting bacterial endotoxin of refenacin according to claim 1, characterized in that: After the suspension is diluted by N-methylpyrrolidone, the concentration of refenacin is 3-5 mg / mL.
6. A method for detecting bacterial endotoxin of refenacin according to claim 1, characterized in that: The dilution with water is specifically to dilute to a refenacin concentration of 0.02-0.04 mg / mL.
7. A method for detecting bacterial endotoxin of revenacin according to claim 1, characterized in that: The horseshoe crab reagent is dissolved with a magnesium ion buffer solution before use.
8. A method for detecting bacterial endotoxin of refenacin according to claim 1, characterized in that: The λ of the horseshoe crab reagent is 0.1 to 0.15 EU / ml.
9. A method for detecting bacterial endotoxin of refenacin according to claim 1, characterized in that: The suspension is diluted with N-methylpyrrolidone and mixed to dissolve, and then diluted with water, and then the horseshoe crab reagent is added, mixed and sealed, and kept warm at 37°C±1°C for 60±2min, and then the bacterial endotoxin test is performed.
10. Use of the method according to any one of claims 1 to 9 in the quality control of revenacin suspension.