Method for the detection of bacterial endotoxins of paliperidone palmitate

Abstract

The present application relates to the technical field of bacterial endotoxin detection, in particular to a detection method of bacterial endotoxin of paliperidone palmitate. The detection method comprises: mixing paliperidone palmitate to be tested, a solvent and bacterial endotoxin test water to form a test solution, and then performing detection, wherein the solvent is a mixed solution formed by mixing tetrahydrofuran and Tween. The detection method provided by the present application can completely dissolve the paliperidone palmitate to be tested, the selected solvent does not interfere with the detection of bacterial endotoxin, effectively solves the problem that endotoxin is wrapped by the sample and cannot be completely released, and has very important significance for the safety of poorly soluble injections.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacterial endotoxin detection, in particular to a method for detecting bacterial endotoxin of paliperidone palmitate. BACKGROUND

[0002] Bacterial endotoxin is a component of the cell wall of gram-negative bacteria, which is released after the bacteria die or autolysis. When endotoxin enters the blood through injection or other means, it can cause different diseases, and when a large amount of endotoxin enters the blood, it can cause fever reaction--"pyrogen reaction". Therefore, bacterial endotoxin test belongs to one of the safety test items of injection. In order to avoid the quality risk of paliperidone palmitate injection in the production process and ensure the safety of drug use, it is necessary to strictly control the bacterial endotoxin content of the raw material and the preparation. Paliperidone palmitate is insoluble in water, and it needs to be dissolved into a clear solution before bacterial endotoxin test, otherwise the insoluble particles may wrap bacterial endotoxin and cause false negative results.

[0003] However, there is no literature report on the method for detecting bacterial endotoxin of paliperidone palmitate. When detecting or quantifying bacterial endotoxin in a sample by conventional bacterial endotoxin test method, the sample needs to be first reconstituted and diluted, then the endotoxin limit value and the maximum valid dilution multiple (MVD) are determined according to the properties of the sample, and finally the gel method or photometric method is used to test the bacterial endotoxin of the sample. However, paliperidone palmitate is difficult to dissolve in water, and the above conventional bacterial endotoxin detection method cannot directly detect the bacterial endotoxin in the paliperidone palmitate sample.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The present application provides a method for detecting bacterial endotoxin of paliperidone palmitate. The detection method provided by the present application can accurately and quickly detect whether the content of bacterial endotoxin of paliperidone palmitate meets the requirements.

[0006] The present application is implemented as follows:

[0007] In a first aspect, the present application provides a method for detecting bacterial endotoxin of paliperidone palmitate, comprising: mixing paliperidone palmitate sample to be tested, solvent and water for bacterial endotoxin test to form a test solution, and then detecting, wherein the solvent is a mixed solution of tetrahydrofuran and Tween.

[0008] In an optional embodiment, the volume ratio of the tetrahydrofuran and the Tween is 1.5:1-15:5, preferably 2:1-8:1, and more preferably 4:1. This ratio can dissolve the paliperidone palmitate sample, and the solution is still clear after dilution with water for bacterial endotoxin test, and the solvent does not interfere with the detection of bacterial endotoxin.

[0009] In an optional embodiment, the Tween includes at least one of Tween 80 and Tween 20;

[0010] Preferably, the Tween is for injection, and more preferably, the Tween 80 is Tween 80(II);

[0011] Preferably, the tetrahydrofuran is chromatographic grade tetrahydrofuran.

[0012] In an optional embodiment, the concentration of paliperidone palmitate in the test solution is below 0.5 mg / ml;

[0013] Preferably, the concentration of paliperidone palmitate in the test solution is 0.0625-0.5 mg / ml; more preferably 0.125-0.5 mg / ml; and even more preferably 0.125-0.25 mg / ml.

[0014] In an optional embodiment, the method includes: dissolving the paliperidone palmitate test sample in the solvent to form a stock solution, and then diluting the stock solution with the bacterial endotoxin test water to form the test solution;

[0015] Preferably, the concentration of the mother liquor is 5-25 mg / ml; more preferably, it is 12.5 mg / ml.

[0016] In an optional embodiment, the method includes: dissolving the paliperidone palmitate test sample in the solvent to form a stock solution, and then diluting the stock solution with the bacterial endotoxin test water to form the test solution; wherein the concentration of the stock solution is 5-25 mg / ml; the concentration of the paliperidone palmitate test sample in the test solution is below 0.5 mg / ml; and the volume ratio of the tetrahydrofuran to the Tween is 1.5:1-15:1.

[0017] In an optional implementation, the method includes: preparing a bacterial endotoxin standard positive control and a test sample positive control;

[0018] Preferably, the method further includes testing the bacterial endotoxin standard positive control, the test sample positive control, and the water used for bacterial endotoxin detection.

[0019] In an optional implementation, the method includes: mixing water for bacterial endotoxin testing with bacterial endotoxin standards to form a positive control of bacterial endotoxin standards with a concentration of 2*LMS reagent sensitivity;

[0020] The paliperidone palmitate sample and the solvent are mixed to form a stock solution, and then bacterial endotoxin standard solution and water for bacterial endotoxin testing are added sequentially to form a positive control for the test sample.

[0021] Wherein, the concentration of the bacterial endotoxin standard in the positive control of the test sample is consistent with the concentration of the bacterial endotoxin standard in the positive control of the bacterial endotoxin standard; the concentration of the paliperidone palmitate test sample in the positive control of the test sample is consistent with the concentration of the paliperidone palmitate test sample in the test sample solution.

[0022] In optional implementations, detection may be performed using a gel electrophoresis or photometric method.

[0023] In an optional embodiment, the sensitivity of the Limulus amebocyte lysate (LAL) reagent used for detection is 0.0625-0.125 EU / ml; and / or, the paliperidone palmitate test sample includes a paliperidone palmitate raw material sample or a paliperidone palmitate preparation sample.

[0024] The present invention has the following beneficial effects: The embodiments of the present invention can effectively dissolve the paliperidone palmitate test sample by using a specific solvent, which can not only efficiently and rapidly detect bacterial endotoxins in the poorly soluble paliperidone palmitate test sample, but also effectively solve the problem that the endotoxins are encapsulated in the sample and cannot be completely released. Moreover, the solvent will not interfere with the detection of bacterial endotoxins. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a diagram showing the dissolution results provided in Comparative Example 2 of the present invention;

[0027] Figure 2 This is a diagram showing the dissolution results provided in Comparative Example 9 of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0029] Paliperidone palmitate is a medication used to treat acute and maintenance phases of schizophrenia. Clinically, it is commonly administered as an injectable form. However, as is well known to those skilled in the art, bacterial endotoxins are a major contaminant in injectable drugs. Therefore, to ensure patient safety, the endotoxin content in injectable products must be strictly controlled during development. Because paliperidone palmitate is poorly soluble in water, conventional water-soluble methods cannot be used for detection. Furthermore, there is currently no method for detecting bacterial endotoxins in paliperidone palmitate.

[0030] The present invention provides a method for detecting the bacterial endotoxin of paliperidone palmitate, which does not dissolve paliperidone palmitate in water, but uses a specific solvent to dissolve paliperidone palmitate, and then can quickly and efficiently detect the bacterial endotoxin of paliperidone palmitate, and the solvent does not affect the detection of the bacterial endotoxin of paliperidone palmitate.

[0031] It should be noted that the paliperidone palmitate for detecting bacterial endotoxins mentioned in the embodiments of the present invention includes paliperidone palmitate raw material, as well as finished paliperidone palmitate preparations, such as paliperidone palmitate injections, or other pharmaceutical preparations.

[0032] Specifically as follows:

[0033] S1. Preparation of the test sample:

[0034] Paliperidone palmitate sample and solvent are mixed to form a stock solution (also referred to herein as test stock solution or stock solution), wherein the concentration of the stock solution is 5-25 mg / ml, preferably 12.5 mg / ml; for example, any value between 5 mg / ml, 7.5 mg / ml, 9 mg / ml, 10 mg / ml, 12.5 mg / ml, 15 mg / ml, 16 mg / ml, 17.5 mg / ml, 19 mg / ml, 20 mg / ml, 21.5 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml and 25 mg / ml.

[0035] The solvent used is a mixed solution of tetrahydrofuran and Tween, with a volume ratio of 1.5:1 to 15:1. Preferably, the volume ratio of tetrahydrofuran to Tween is 2:1 to 8:1; more preferably, it is 4:1. For example, the volume ratio of tetrahydrofuran to Tween can be any value between 1.5:1 and 15:1, such as 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, and 15:1.

[0036] The inventors of this invention unexpectedly discovered that by mixing tetrahydrofuran and Tween in a specific volume ratio to form a mixed solution, the poorly soluble paliperidone palmitate sample can be fully dissolved, ensuring the detection of bacterial endotoxins in the paliperidone palmitate sample. Furthermore, the detection speed, efficiency, and accuracy are high. Simultaneously, this solvent does not interfere with the bacterial endotoxin detection results. Changing the ratio of tetrahydrofuran and Tween in the solvent of this embodiment, or altering the composition of substances in the solvent, may affect the detection of bacterial endotoxins in the paliperidone palmitate sample, or even prevent detection altogether.

[0037] Specifically, if the volume ratio of tetrahydrofuran to Tween is greater than 15:1, paliperidone palmitate will precipitate as a lumpy sample after dissolution and dilution with water, affecting endotoxin recovery. When the ratio is less than 1.5:1, the sample solubility is poor, requiring a large volume of mixed solution for dissolution. In this case, the initial sample concentration is low, the available dilution factor is small, and it interferes with the horseshoe crab reagent. When the volume ratio is optimal at 4:1, the sample remains clear after dilution with water, and the initial concentration is high, allowing for a large dilution factor, and the endotoxin limit can be less than 0.25 EU / mg. When the volume ratio of tetrahydrofuran to Tween is greater than or equal to 1.5:1 and less than 4:1, the dilution factor is small, leading to a larger endotoxin limit, but it still meets the specified 0.5 EU / mg. When the volume ratio of tetrahydrofuran to Tween is greater than 4:1 and less than or equal to 15:1, the clarity decreases after dilution, but it does not interfere with the endotoxin determination and meets the specified detection requirements.

[0038] Furthermore, Tween 80 or Tween 20 can be used. Tween is for injection. Tween 80(II) is preferred to further ensure the accuracy of the test results and reduce the influence of the solvent on the test results.

[0039] The mother liquor is then diluted with water used for bacterial endotoxin testing to form the test solution; the concentration of paliperidone palmitate in the test solution is 0.0625-0.5 mg / ml; preferably 0.125-0.5 mg / ml; more preferably 0.125-0.25 mg / ml. For example, the concentration of paliperidone palmitate in the test solution can be any value between 0.0625-0.5 mg / ml, such as 0.0625 mg / ml, 0.125 mg / ml, 0.25 mg / ml, and 0.5 mg / ml.

[0040] S2. Preparation of positive control for bacterial endotoxin standards;

[0041] A positive control of bacterial endotoxin standards is prepared by mixing water with bacterial endotoxin standards at a concentration of 2*LAL reagent sensitivity.

[0042] S3. Preparation of test sample and positive control.

[0043] The mother liquor prepared in S1 is added to the bacterial endotoxin standard solution and water for bacterial endotoxin testing to form a positive control for the test sample.

[0044] Wherein, the concentration of the bacterial endotoxin standard in the positive control of the test sample is consistent with the concentration of the bacterial endotoxin standard in the positive control of the bacterial endotoxin standard; the concentration of the paliperidone palmitate test sample in the positive control of the test sample is consistent with the concentration of the paliperidone palmitate test sample in the test sample solution.

[0045] It should be noted that although bacterial endotoxin standards are added during the formation of the positive control of the test sample, the amount used is very small and has almost no effect on the volume of the positive control of the test sample. Therefore, it is considered that the concentration of paliperidone palmitate test sample in the positive control of the test sample is consistent with the concentration of paliperidone palmitate test sample in the test sample solution.

[0046] S4, Detection

[0047] Water used for bacterial endotoxin testing was used as a negative control. The test solution, negative control, bacterial endotoxin standard positive control, and test solution positive control were then tested. The detection method adopted was the "Bacterial Endotoxin Test Method" of the 2020 edition of the Chinese Pharmacopoeia, Volume IV, using either the gel electrophoresis method or photometric method. The sensitivity of the Limulus amebocyte lysate (LAL) reagent used in the detection was 0.0625-0.125 EU / ml.

[0048] It should be noted that both the gelation method and the photometric method are described in the pharmacopoeia and will not be described in detail in the embodiments of this invention.

[0049] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0050] The paliperidone palmitate sample used in this invention can be a commercially available paliperidone palmitate raw material or paliperidone palmitate injection, or a paliperidone palmitate raw material or paliperidone palmitate injection synthesized or prepared according to existing methods.

[0051] Paliperidone palmitate used in the embodiments of the present invention is a substance synthesized by Livzon Pharmaceutical Co., Ltd., including three batches of paliperidone palmitate, namely 01, 02 and 03.

[0052] Example 1

[0053] This embodiment provides a method for detecting the bacterial endotoxin of paliperidone palmitate, comprising:

[0054] Preparation of bacterial endotoxin standards: Dissolve the bacterial endotoxin standards in water for bacterial endotoxin testing and mix well. After mixing, dilute the bacterial endotoxin standards with water for bacterial endotoxin testing to 0.125 EU / ml (2λ, where λ represents the sensitivity of the Limulus amebocyte lysate (LAL) reagent, and λ is in EU / ml) as a positive control, and use water for bacterial endotoxin testing as a negative control.

[0055] Preparation of test solution: Add paliperidone palmitate to a mixture of tetrahydrofuran and Tween 80(II) (volume ratio: 4:1) to prepare a stock solution with a concentration of 12.5 mg / ml (calculated as paliperidone). Then dilute the stock solution to 0.25 mg / ml with water for bacterial endotoxin test.

[0056] Preparation of the positive control for the test sample: Take 1 ml of the 12.5 mg / ml stock solution prepared above, add 10 μl of bacterial endotoxin standard solution with a concentration of 625 EU / ml; then dilute 50 times with water for bacterial endotoxin testing, mixing well for 30 seconds for each dilution step, and each dilution should not exceed 10 times. The resulting test sample solution containing 0.125 EU / ml standard endotoxin serves as the positive control for the test sample.

[0057] Perform bacterial endotoxin testing:

[0058] According to the Chinese Pharmacopoeia 2020 Edition, Volume IV, "Test Method for Bacterial Endotoxins", the gel electrophoresis method was used, and the test was performed with 0.0625 Eu / ml Limulus Amebocyte Lysate (LAL) reagent. Two tubes were prepared for each test solution. The test results are as follows:

[0059] Batch No. Test solution Test positive control Positive control Negative control 01 -- ++ ++ -- 02 -- ++ ++ -- 03 -- ++ ++ --

[0060] A positive result (+) is indicated by the formation of a firm, undeformed gel within the tube that does not slip off the tube wall; a negative result (-) is indicated by the absence of a gel, or by a weak, deformed gel that slips off the tube wall. Vibration should be avoided during the insulation and handling of the test tubes to prevent false negative results.

[0061] If all parallel tubes of the negative control solution are negative, and all parallel tubes of the positive control solution of the test sample are positive, the test is valid. If both parallel tubes of the test sample solution are negative, the test sample is deemed to meet the requirements. Therefore, all three batches of test samples meet the requirements, and the endotoxin limit is less than 0.25 EU / mg (calculated as paliperidone).

[0062] Example 2

[0063] This embodiment provides a method for detecting the bacterial endotoxin of paliperidone palmitate, comprising:

[0064] Preparation of bacterial endotoxin standards: Dissolve the bacterial endotoxin standards in water for bacterial endotoxin testing and mix well. After mixing, dilute the bacterial endotoxin standards to 2λ (0.125 EU / ml) with water for bacterial endotoxin testing as a positive control, and use water for bacterial endotoxin testing as a negative control.

[0065] Preparation of test solution: Prepare a stock solution with a concentration of 25 mg / ml (calculated as paliperidone) by adding paliperidone palmitate to a mixture of tetrahydrofuran and Tween 80(II) (volume ratio: 15:1). Then dilute the stock solution to 0.25 mg / ml with water for bacterial endotoxin test.

[0066] Preparation of the positive control for the test sample: Take 1 ml of the 25 mg / ml stock solution prepared above, add 10 μl of bacterial endotoxin standard solution with a concentration of 1250 EU / ml; then dilute 100 times with water for bacterial endotoxin testing, mixing well for 30 seconds for each dilution step, and each dilution should not exceed 10 times. The resulting test sample solution containing 0.125 EU / ml standard endotoxin serves as the positive control for the test sample.

[0067] Perform bacterial endotoxin testing:

[0068] According to the Chinese Pharmacopoeia 2020 Edition, Part IV, "Test Method for Bacterial Endotoxins", the gel electrophoresis method was used, and the test was performed with 0.0625 Eu / ml Limulus Amebocyte Lysate (LAL) reagent. Two tubes were prepared for each test solution. The results are as follows:

[0069] Batch No. Test solution Test positive control Positive control Negative control 01 -- ++ ++ -- 02 -- ++ ++ -- 03 -- ++ ++ --

[0070] If all parallel tubes of the negative control solution are negative, and all parallel tubes of the positive control solution of the test sample are positive, the test is valid. If both parallel tubes of the test sample solution are negative, the test sample is deemed to meet the requirements. Therefore, all three batches of test samples meet the requirements, and the endotoxin limit is less than 0.25 EU / mg (calculated as paliperidone).

[0071] Example 3

[0072] This embodiment provides a method for detecting the bacterial endotoxin of paliperidone palmitate, comprising:

[0073] Preparation of bacterial endotoxin standards: Dissolve the bacterial endotoxin standards in water for bacterial endotoxin testing and mix well. After mixing, dilute the bacterial endotoxin standards to 2λ (0.125 EU / ml) with water for bacterial endotoxin testing as a positive control, and use water for bacterial endotoxin testing as a negative control.

[0074] Preparation of test solution: Add paliperidone palmitate to a mixture of tetrahydrofuran and Tween 80 (volume ratio: 1.5:1) to prepare a stock solution with a concentration of 5 mg / ml (calculated as paliperidone). Then dilute the stock solution with water for bacterial endotoxin test to 0.125 mg / ml for later use.

[0075] Preparation of the positive control for the test sample: Take 1 ml of the 5 mg / ml stock solution prepared above, add 10 μl of bacterial endotoxin standard solution with a concentration of 500 EU / ml; then dilute 40 times with water for bacterial endotoxin testing, mixing well for 30 seconds for each dilution step, and each dilution should not exceed 10 times. The resulting test sample solution containing 0.125 EU / ml standard endotoxin is used as the positive control for the test sample.

[0076] Perform bacterial endotoxin testing:

[0077] According to the Chinese Pharmacopoeia 2020 Edition, Volume IV, "Test Method for Bacterial Endotoxins", the gel electrophoresis method was used, and the test was conducted with 0.0625 Eu / ml Limulus Amebocyte Lysate (LAL) reagent. Two parallel tubes were prepared for each test solution. The results are as follows:

[0078] Batch No. Test solution Test positive control Positive control Negative control 01 -- ++ ++ -- 02 -- ++ ++ -- 03 -- ++ ++ --

[0079] If all parallel tubes of the negative control solution are negative, and all parallel tubes of the positive control solution of the test sample are positive, the test is valid. If both parallel tubes of the test sample solution are negative, the test sample is deemed to meet the requirements. Therefore, all three batches of test samples meet the requirements, and the endotoxin limit is less than 0.5 EU / mg (calculated as paliperidone).

[0080] Example 4

[0081] This embodiment provides a method for detecting the bacterial endotoxin of paliperidone palmitate, comprising:

[0082] Preparation of bacterial endotoxin standards: Dissolve the bacterial endotoxin standards in water for bacterial endotoxin testing and mix well. After mixing, dilute the bacterial endotoxin standards to 2λ (0.25 EU / ml) with water for bacterial endotoxin testing as a positive control, and use water for bacterial endotoxin testing as a negative control.

[0083] Preparation of test solution: Add paliperidone palmitate to a mixture of tetrahydrofuran and Tween 80 (volume ratio: 4:1) to prepare a stock solution with a concentration of 12.5 mg / ml (calculated as paliperidone). Then dilute the stock solution to 0.5 mg / ml with water for bacterial endotoxin test.

[0084] Preparation of the positive control for the test sample: Take 1 ml of the 12.5 mg / ml stock solution prepared above, add 10 μl of bacterial endotoxin standard solution with a concentration of 625 EU / ml; then dilute 25 times with water for bacterial endotoxin testing, mixing well for 30 seconds for each dilution step, and each dilution should not exceed 10 times. The resulting test sample solution containing 0.25 EU / ml standard endotoxin serves as the positive control for the test sample.

[0085] Perform bacterial endotoxin testing:

[0086] According to the Chinese Pharmacopoeia 2020 Edition, Volume IV, "Test Method for Bacterial Endotoxins", the gel electrophoresis method was used with 0.125 Eu / ml Limulus Amebocyte Lysate (LAL) reagent. Two tubes were prepared for each test solution. The results are as follows:

[0087] Batch No. Test solution Test positive control Positive control Negative control 01 -- ++ ++ -- 02 -- ++ ++ -- 03 -- ++ ++ --

[0088] If all parallel tubes of the negative control solution are negative, and all parallel tubes of the positive control solution of the test sample are positive, the test is valid. If both parallel tubes of the test sample solution are negative, the test sample is deemed to meet the requirements. Therefore, all three batches of test samples meet the requirements, and the endotoxin limit is less than 0.25 EU / mg (calculated as paliperidone).

[0089] Example 5

[0090] This embodiment provides a method for detecting the bacterial endotoxin of paliperidone palmitate, comprising:

[0091] Preparation of bacterial endotoxin standards: Dissolve the bacterial endotoxin standards in water for bacterial endotoxin testing and mix well. After mixing, dilute the bacterial endotoxin standards to 2λ (0.25 EU / ml) with water for bacterial endotoxin testing as a positive control, and use water for bacterial endotoxin testing as a negative control.

[0092] Preparation of test solution: Prepare a stock solution with a concentration of 25 mg / ml (calculated as paliperidone) by adding paliperidone palmitate to a mixture of tetrahydrofuran and Tween 80 (volume ratio: 15:1). Then dilute the stock solution to 0.5 mg / ml with water for bacterial endotoxin test (the clarity of the test solution will decrease after dilution).

[0093] Preparation of the positive control for the test sample: Take 1 ml of the 25 mg / ml stock solution prepared above, add 10 μl of bacterial endotoxin standard solution with a concentration of 625 EU / ml; then dilute 50 times with water for bacterial endotoxin testing, mixing well for 30 seconds for each dilution step, and each dilution should not exceed 10 times. The resulting test sample solution containing 0.25 EU / ml standard endotoxin serves as the positive control for the test sample.

[0094] Perform bacterial endotoxin testing:

[0095] According to the Chinese Pharmacopoeia 2020 Edition, Volume IV, "Test Method for Bacterial Endotoxins", the gel electrophoresis method was used with 0.125 Eu / ml Limulus Amebocyte Lysate (LAL) reagent. Two tubes were prepared for each test solution. The results are as follows:

[0096] Batch No. Test solution Test positive control Positive control Negative control 01 -- ++ ++ -- 02 -- ++ ++ -- 03 -- ++ ++ --

[0097] If all parallel tubes of the negative control solution are negative, and all parallel tubes of the positive control solution of the test sample are positive, the test is valid. If both parallel tubes of the test sample solution are negative, the test sample is deemed to meet the requirements. Therefore, all three batches of test samples meet the requirements, and the endotoxin limit is less than 0.25 EU / mg (calculated as paliperidone).

[0098] Example 6

[0099] This embodiment provides a method for detecting the bacterial endotoxin of paliperidone palmitate, comprising:

[0100] Preparation of bacterial endotoxin standards: Dissolve the bacterial endotoxin standards in water for bacterial endotoxin testing and mix well. After mixing, dilute the bacterial endotoxin standards to 2λ (0.125 EU / ml) with water for bacterial endotoxin testing as a positive control, and use water for bacterial endotoxin testing as a negative control.

[0101] Preparation of test solution: Add paliperidone palmitate to a mixture of tetrahydrofuran and Tween 20 (volume ratio: 4:1) to prepare a stock solution with a concentration of 12.5 mg / ml (calculated as paliperidone). Then dilute the stock solution to 0.25 mg / ml with water for bacterial endotoxin test.

[0102] Preparation of the positive control for the test sample: Take 1 ml of the 12.5 mg / ml stock solution prepared above, add 10 μl of bacterial endotoxin standard solution with a concentration of 625 EU / ml; then dilute 50 times with water for bacterial endotoxin testing, mixing well for 30 seconds for each dilution step, and each dilution should not exceed 10 times. The resulting test sample solution containing 0.125 EU / ml standard endotoxin serves as the positive control for the test sample.

[0103] Perform bacterial endotoxin testing:

[0104] According to the Chinese Pharmacopoeia 2020 Edition, Part IV, "Test Method for Bacterial Endotoxins", the gel electrophoresis method was used, and the test was performed with 0.0625 Eu / ml Limulus Amebocyte Lysate (LAL) reagent. Two tubes were prepared for each test solution. The results are as follows:

[0105] Batch No. Test solution Test positive control Positive control Negative control 01 -- ++ ++ -- 02 -- ++ ++ -- 03 -- ++ ++ --

[0106] If all parallel tubes of the negative control solution are negative, and all parallel tubes of the positive control solution of the test sample are positive, the test is valid. If both parallel tubes of the test sample solution are negative, the test sample is deemed to meet the requirements. Therefore, all three batches of test samples meet the requirements, and the endotoxin limit is less than 0.25 EU / mg (calculated as paliperidone).

[0107] Comparative Example 1

[0108] A 30% acetic acid solution was used as the solvent, and the operation was consistent with that in Example 1. The test results are as follows:

[0109] Figure 1 Figure 1 Figure 1 Batch No. Test solution 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0110] It is evident that paliperidone palmitate can be dissolved using a 30% acetic acid solution, but the positive control result of the test sample was negative, indicating that it interferes with the horseshoe crab reagent and cannot be used to detect paliperidone palmitate.

[0111] Comparative Example 2

[0112] Tetrahydrofuran was used as the solvent, and the operation was consistent with that in Example 1. The results are shown in [Figure 1]. Test positive control ,in Positive control The left image is a photograph of the paliperidone palmitate sample dissolved in hydrogen furan. Negative control The right image shows the mother liquor formed after dissolving paliperidone palmitate in hydrogen furan. It was then diluted 10 times with water for bacterial endotoxin testing. As can be seen, there are obvious clumps. These clumps encapsulate the endotoxins, making it impossible to accurately detect them.

[0113] The test results are as follows:

[0114] Batch No. Test solution Test positive control Positive control Negative control 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0115] It is evident that tetrahydrofuran can dissolve paliperidone palmitate, but the positive control result of the test sample was negative, indicating that it interferes with the horseshoe crab reagent and cannot be used to detect paliperidone palmitate.

[0116] Comparative Example 3

[0117] An ethanol-Tween 80(II) mixture was used as the solvent. The mixture was dissolved at 50°C with a volume ratio of ethanol to Tween 80(II) of 1:1.08. The operation was the same as in Example 1. The test results are as follows:

[0118] Batch No. Test solution Test positive control Positive control Negative control 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0119] It can be seen that paliperidone palmitate can be dissolved using ethanol Tween 80(II), but the positive control of the test sample was negative, indicating that it interferes with the horseshoe crab reagent and cannot be used to detect paliperidone palmitate.

[0120] Comparative Example 4

[0121] DMF was used as the solvent (approximately 80 ml was required for a sample of 100 mg paliperidone). The procedure was the same as in Example 1, and the results are as follows:

[0122] Batch No. Test solution Test positive control Positive control Negative control 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0123] It is evident that DMF can dissolve paliperidone palmitate, but the positive control result of the test sample was negative, indicating that it interferes with the horseshoe crab reagent and cannot be used to detect paliperidone palmitate.

[0124] Comparative Example 5

[0125] Dissolved in anhydrous ethanol by heating at 50°C, the procedure was the same as in Example 1, and the test results are as follows:

[0126] Figure 2 Figure 2 Figure 2 Batch No. Test solution 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0127] It is evident that the test sample was negative despite being dissolved by heating with ethanol, indicating interference with the horseshoe crab reagent.

[0128] Comparative Example 6

[0129] Dissolving in Tween 80 (II) and glacial acetic acid: Weigh approximately 32 mg of sample (20 mg as paliperidone), add 4 ml of Tween 80 to disperse the sample, then add 15.5 ml of water for bacterial endotoxin testing, add 0.5 ml of glacial acetic acid, dissolve the sample in a 50°C water bath, and dilute with water for bacterial endotoxin testing to 0.125 mg / ml. The procedure is consistent with Example 1. The test results are as follows:

[0130] Test positive control Positive control Negative control Batch No. Test solution 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0131] It is evident that the positive result of the test sample was negative, indicating that it interfered with the Limulus amebocyte lysate (LAL) reagent.

[0132] Comparative Example 7

[0133] DMSO is used, but it cannot dissolve the sample, making detection impossible.

[0134] Comparative Example 8

[0135] Tween 80(II) was used, but it could not dissolve the sample, making detection impossible.

[0136] Comparative Example 9

[0137] The ratio of tetrahydrofuran to Tween was 16:1, and all other operations were consistent with Example 1. See the results below. Test positive control ,in, Positive controlThe left image shows a sample of paliperidone palmitate dissolved in tetrahydrofuran and Tween(II) at a ratio of 16:1 (v / v). Negative control The image on the right shows the mother liquor formed by dissolving paliperidone palmitate in tetrahydrofuran and Tween (II) at a ratio of 16:1 (V / V) and diluted 10 times with water for bacterial endotoxin testing. As can be seen, the sample is turbid and has a few clumps at the bottom. These clumps can encapsulate the endotoxins, making it impossible to accurately detect endotoxins.

[0138] Meanwhile, the test results are as follows:

[0139] Batch No. Test solution Test positive control Positive control Negative control 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0140] It is evident that the positive result of the test sample was negative, indicating that it interfered with the Limulus amebocyte lysate (LAL) reagent.

[0141] Comparative Example 10

[0142] The ratio of tetrahydrofuran to Tween was 1:1, and the operation was consistent with Example 1. The sample had poor solubility and required more than 30 ml of mixed solution for dissolution. At this point, the initial sample concentration was low, at 2.5 mg / ml. It was then diluted 20 times with water for bacterial endotoxin testing, resulting in a concentration of 0.125 mg / ml. The test results are as follows:

[0143] Batch No. Test solution Test positive control Positive control Negative control Figure 1 Figure 2 Figure 2 Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Figure 1 Figure 2 Figure 2 Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Figure 1 Figure 2 Figure 2 Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Figure 1 Figure 2 Figure 2 Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Figure 1 Figure 2 Figure 2 Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Figure 1 Figure 2 Figure 2 Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative control Batch No. Test solution Test positive control Positive control Negative 01 -- -- ++ -- 02 -- -- ++ -- 03 -- -- ++ --

[0144] It is evident that the positive result of the test sample was negative, indicating that it interfered with the Limulus amebocyte lysate (LAL) reagent.

[0145] verify

[0146] To ensure the reliability of the method for testing the bacterial endotoxins of paliperidone palmitate, it is necessary to verify, under actual testing conditions, whether it interferes with the bacterial endotoxin test, in order to confirm that the method is suitable for the bacterial endotoxin testing of paliperidone palmitate raw materials. Specifically:

[0147] The gel electrophoresis method was used to verify the interference of three batches of paliperidone palmitate raw materials with horseshoe crab reagents from two manufacturers (Xiamen Horseshoe Crab Reagent Biotechnology Co., Ltd. and Fuzhou Xinbei Biochemical Industry Co., Ltd.). The verification results are shown in the table below:

[0148]

[0149]

[0150] Specifically as follows:

[0151] The limit for paliperidone palmitate injection is determined as follows: L = K / M [K = 5 EU / (kg·h) for injections; for paliperidone palmitate injection with a maximum specification of 1.5 ml, the maximum dose M = 150 mg / (60 kg·h); the limit for paliperidone palmitate injection is 2.0 EU / mg (calculated as paliperidone). Due to differences in drugs and indications (such as anti-infectives, anti-tumor drugs, cardiovascular drugs for emergency use, drugs for children and the elderly, compound drugs, large-volume infusions, etc.), the limit can be appropriately tightened to 1 / 3 to 1 / 2 of the calculated value to ensure safe medication use. Therefore, the limit for paliperidone palmitate injection is set at 1.0 EU / mg. Considering the addition of excipients in the injection, the bacterial endotoxin limit for paliperidone palmitate raw material is set at L = 0.5 EU / mg (calculated as paliperidone).

[0152] Interference Preliminary Experiment 1

[0153] Paliperidone palmitate raw material was dissolved in a mixed solution of tetrahydrofuran and Tween 80(II) (volume ratio: 1.5:1) (V / V) to prepare a 5 mg / ml paliperidone solution. This solution was then diluted with water for bacterial endotoxin testing to a series of concentrations: 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml, denoted as NPC. Simultaneously, a positive control solution containing 2λ concentrations of standard endotoxin was prepared for each of the above concentrations, denoted as PPC. The negative control solution was water for bacterial endotoxin testing, denoted as NC. A bacterial endotoxin standard with a potency of 9000 EU / vial was dissolved in 1 ml of water for bacterial endotoxin testing according to the standard's instructions. This solution was then used to prepare the required bacterial endotoxin standard solution, which served as the positive control solution PC.

[0154] Two different manufacturers' horseshoe crab reagents were used to react with NPC, PPC, NC, and PC respectively, with two replicates for each concentration. Preliminary experimental results are shown in Tables 1 and 2.

[0155] Table 1. Limulus amebocyte lysate (LAL) reagent test results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0156]

[0157] Table 2. Limulus amebocyte lysate (LAL) test results from Xiamen Limulus amebocyte lysate Reagent Co., Ltd.

[0158]

[0159] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.125 EU / ml, and the batch number of the horseshoe crab reagent is 22120912.

[0160] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.125 EU / ml and its batch number is 22111043.

[0161] The results in Tables 1 and 2 preliminarily indicate that a paliperidone palmitate test solution at a concentration of 5 mg / ml, diluted to 0.125 mg / ml (40-fold dilution) with water for bacterial endotoxin testing, showed no interference with the Limulus Amebocyte Lysate (LAL) reagents from either manufacturer. A sample concentration of 0.125 mg / ml and a LAL reagent sensitivity of 0.0625 EU / ml were selected for the formal interference test.

[0162] Formal Interference Experiment 1:

[0163] Take 1 ml of the above-mentioned test sample stock solution (5 mg / ml) from four tubes, add 10 μl of an appropriate concentration of endotoxin solution to each, mix well, and then dilute to 40 times with water for bacterial endotoxin testing to obtain test sample solutions containing endotoxins of 2λ, λ, 0.5λ, and 0.25λ, respectively. Perform the experiment according to the "Bacterial Endotoxin Test Method - Gel Method Interference Test" section in the Chinese Pharmacopoeia, using a Limulus Amebocyte Lysate (LAL) reagent with a sensitivity of 0.0625 EU / ml. The results are shown in Tables 3 and 4.

[0164] Table 3. Limulus amebocyte lysate (LAL) test results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0165]

[0166] Table 4. Limulus amebocyte lysate (LAL) test results from Xiamen Limulus amebocyte lysate Reagent Co., Ltd.

[0167]

[0168] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.0625 EU / ml, and the batch number of the horseshoe crab reagent is 22122312.

[0169] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.0625 EU / ml and its batch number is 22111045.

[0170] Formal interference experiments showed that the E values ​​of the three batches of samples for the two manufacturers' Limulus amebocyte lysate (LAL) reagents were significantly affected. S and E t All values ​​were within the range of 0.5λ to 2.0λ (inclusive), meeting the requirements for Limulus amebocyte lysate (LAL) reagent sensitivity verification. It was confirmed that there was no interference at a 40-fold dilution (concentration of 0.125 mg / ml), and the endotoxin content was less than 0.5 EU / mg.

[0171] Interference Preliminary Experiment 2

[0172] Paliperidone palmitate raw material was dissolved in a mixed solution of tetrahydrofuran and Tween 80(II) (volume ratio 15:1) (v / v) to prepare a 25 mg / ml paliperidone solution. This solution was then diluted with water for bacterial endotoxin testing to a series of concentrations: 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml, denoted as NPC. Simultaneously, a positive control solution containing 2λ concentrations of standard endotoxin was prepared for each of the above concentrations, denoted as PPC. The negative control solution was water for bacterial endotoxin testing, denoted as NC. A bacterial endotoxin standard with a potency of 9000 EU / vial was dissolved in 1 ml of water for bacterial endotoxin testing according to the standard's instructions. This solution was then used to prepare the required bacterial endotoxin standard solution, which served as the positive control solution PC.

[0173] Two different manufacturers' horseshoe crab reagents were used to react with NPC, PPC, NC, and PC respectively, with two replicates for each concentration. The preliminary test results are shown in Tables 5 and 6 below.

[0174] Table 5. Limulus amebocyte lysate (LAL) test results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0175]

[0176] Table 6. Limulus amebocyte lysate (LAL) test results from Xiamen Limulus amebocyte lysate Reagent Co., Ltd.

[0177]

[0178] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.125 EU / ml, and the batch number of the horseshoe crab reagent is 22120912.

[0179] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.125 EU / ml and its batch number is 22111043.

[0180] The results in Tables 5 and 6 preliminarily indicate that a paliperidone palmitate test solution at a concentration of 25 mg / ml, diluted to 0.5 mg / ml (50-fold dilution) with water for bacterial endotoxin testing, showed no interference with the Limulus Amebocyte Lysate (LAL) reagents from either manufacturer. Considering a safety margin of 2, a sample concentration of 0.25 mg / ml and a LAL reagent sensitivity of 0.0625 EU / ml were selected for the formal interference test.

[0181] Formal Interference Experiment 2

[0182] Take 1 ml of the original test sample solution (25 mg / ml) from four tubes, add 10 μL of an appropriate concentration of endotoxin solution to each, mix well, and then dilute to 100 times with water for bacterial endotoxin testing to obtain test sample solutions containing endotoxins of 2λ, λ, 0.5λ, and 0.25λ, respectively. Perform the experiment according to the "Bacterial Endotoxin Test - Gel Method Interference Test" section in the Chinese Pharmacopoeia, using a Limulus Amebocyte Lysate (LAL) reagent with a sensitivity of 0.0625 EU / ml. The results are shown in Tables 7 and 8.

[0183] Table 7. Limulus amebocyte lysate (LAL) test results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0184]

[0185] Table 8. Limulus amebocyte lysate (LAL) test results from Xiamen Limulus amebocyte lysate Reagent Co., Ltd.

[0186]

[0187] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.0625 EU / ml, and the batch number of the horseshoe crab reagent is 22122312.

[0188] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.0625 EU / ml and its batch number is 22111045.

[0189] Formal interference experiments showed that the E values ​​of the three batches of samples for the two manufacturers' Limulus amebocyte lysate (LAL) reagents were significantly affected. S and E t All values ​​were within the range of 0.5λ to 2.0λ (inclusive), meeting the requirements for Limulus amebocyte lysate (LAL) reagent sensitivity verification. It was confirmed that there was no interference at a 100-fold dilution (concentration of 0.25 mg / ml), and the endotoxin content was less than 0.25 EU / mg.

[0190] Interference Preliminary Experiment 3

[0191] Paliperidone palmitate raw material was dissolved in a mixed solution of tetrahydrofuran and Tween 80(II) (volume ratio 4:1) (v / v) to prepare a 12.5 mg / ml paliperidone solution. This solution was then diluted with water for bacterial endotoxin testing to a series of concentrations: 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml, denoted as NPC. Simultaneously, a positive control solution containing 2λ concentrations of standard endotoxin was prepared for each of the above concentrations, denoted as PPC. The negative control solution was water for bacterial endotoxin testing, denoted as NC. A bacterial endotoxin standard with a potency of 9000 EU / vial was dissolved in 1 ml of water for bacterial endotoxin testing according to the standard's instructions. This solution was then used to prepare the required bacterial endotoxin standard solution, which served as the positive control solution PC.

[0192] Two different manufacturers' horseshoe crab reagents were used to react with NPC, PPC, NC, and PC respectively, with two replicates for each concentration. The preliminary test results are shown in Tables 9 and 10 below.

[0193] Table 9. Limulus Amebocyte Lysate (LAL) Reagent Test Results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0194]

[0195] Table 10. Limulus amebocyte lysate (LAL) test results from Xiamen Limulus amebocyte lysate Reagent Co., Ltd.

[0196]

[0197]

[0198] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.125 EU / ml, and the batch number of the horseshoe crab reagent is 22120912.

[0199] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.125 EU / ml and its batch number is 22111043.

[0200] The results in Tables 9 and 10 preliminarily indicate that a paliperidone palmitate test solution at a concentration of 12.5 mg / ml, diluted to 0.5 mg / ml (25-fold dilution) with water for bacterial endotoxin testing, showed no interference with the Limulus Amebocyte Lysate (LAL) reagents from either manufacturer. Considering a safety margin of 2, a sample concentration of 0.25 mg / ml and a LAL reagent sensitivity of 0.0625 EU / ml were selected for the formal interference test.

[0201] Formal Interference Experiment 3

[0202] Take 1 ml of the original test sample solution (12.5 mg / ml) from four tubes, add 10 μL of an appropriate concentration of endotoxin solution to each, mix well, and then dilute to 50 times with water for bacterial endotoxin testing to obtain test sample solutions containing endotoxins of 2λ, λ, 0.5λ, and 0.25λ, respectively. Perform the experiment according to the "Bacterial Endotoxin Test - Gel Method Interference Test" section in the Chinese Pharmacopoeia, using a Limulus Amebocyte Lysate (LAL) reagent with a sensitivity of 0.0625 EU / ml. The results are shown in Tables 11 and 12.

[0203] Table 11. Limulus Amebocyte Lysate (LIL) Reagent Test Results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0204]

[0205] Table 12. Limulus amebocyte lysate (LAL) test results from Xiamen Limulus amebocyte lysate Reagent Co., Ltd.

[0206]

[0207] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.0625 EU / ml, and the batch number of the horseshoe crab reagent is 22122312.

[0208] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.0625 EU / ml and its batch number is 22111045.

[0209] Formal interference experiments showed that the E values ​​of the three batches of samples for the two manufacturers' Limulus amebocyte lysate (LAL) reagents were significantly affected. S and E t All values ​​were within the range of 0.5λ to 2.0λ (inclusive), meeting the requirements for Limulus amebocyte lysate (LAL) reagent sensitivity verification. It was confirmed that there was no interference at a 50-fold dilution (concentration of 0.25 mg / ml), and the endotoxin content was less than 0.25 EU / mg.

[0210] Interference Preliminary Experiment 4

[0211] Paliperidone palmitate was dissolved in a mixture of tetrahydrofuran and Tween 20 (4:1, v / v) to prepare a 12.5 mg / ml paliperidone solution. This solution was then diluted with water for bacterial endotoxin testing to concentrations of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml, denoted as NPC. Simultaneously, a positive control solution containing 2λ concentrations of standard endotoxin was prepared for each of these concentrations, denoted as PPC. The negative control solution was water for bacterial endotoxin testing, denoted as NC. A bacterial endotoxin standard with a potency of 9000 EU / vial was dissolved in 1 ml of water for bacterial endotoxin testing according to the standard's instructions. This solution was then used to prepare the required bacterial endotoxin standard solution, which served as the positive control solution PC.

[0212] Two different manufacturers' horseshoe crab reagents were used to react with NPC, PPC, NC, and PC respectively, with two replicates for each concentration. The preliminary test results are shown in Tables 13 and 14 below.

[0213] Table 13. Limulus Amebocyte Lysate (LAL) Reagent Detection Results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0214]

[0215] Table 14. Limulus Amebocyte Lysate (LAL) Reagent Test Results from Xiamen Limulus Amebocyte Lysate Reagent Co., Ltd.

[0216]

[0217]

[0218] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.125 EU / ml, and the batch number of the horseshoe crab reagent is 22120912.

[0219] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.125 EU / ml and its batch number is 22111043.

[0220] The results in Tables 13 and 14 preliminarily indicate that a paliperidone palmitate test solution at a concentration of 12.5 mg / ml, diluted to 0.25 mg / ml (50-fold dilution) with water for bacterial endotoxin testing, showed no interference with the Limulus Amebocyte Lysate (LAL) reagents from either manufacturer. A sample concentration of 0.25 mg / ml and a LAL reagent sensitivity of 0.0625 EU / ml were selected for the formal interference test.

[0221] Formal Interference Experiment 4

[0222] Take 1 ml of the original test sample solution (12.5 mg / ml) from 4 tubes, add 10 μL of an appropriate concentration of endotoxin solution to each, mix well, and then dilute to 50 times with water for bacterial endotoxin testing to obtain test sample solutions containing endotoxins of 2λ, λ, 0.5λ, and 0.25λ, respectively. Perform the experiment according to the "Bacterial Endotoxin Test - Gel Method Interference Test" section in the Chinese Pharmacopoeia, using a Limulus Amebocyte Lysate (LAL) reagent with a sensitivity of 0.0625 EU / ml. The results are shown in Tables 15 and 16.

[0223] Table 15. Limulus Amebocyte Lysate (LIL) Reagent Detection Results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0224]

[0225] Table 16. Limulus Amebocyte Lysate (LAL) Reagent Test Results from Xiamen Limulus Amebocyte Lysate Reagent Co., Ltd.

[0226]

[0227]

[0228] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.0625 EU / ml, and the batch number of the horseshoe crab reagent is 22122312.

[0229] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.0625 EU / ml and its batch number is 22111045.

[0230] Formal interference experiments showed that the E values ​​of the three batches of samples for the two manufacturers' Limulus amebocyte lysate (LAL) reagents were significantly affected. S and E t All values ​​were within the range of 0.5λ to 2.0λ (inclusive), meeting the requirements for Limulus amebocyte lysate (LAL) reagent sensitivity verification. It was confirmed that there was no interference at a 50-fold dilution (concentration of 0.25 mg / ml), and the endotoxin content was less than 0.25 EU / mg.

[0231] Interference Preliminary Experiment 5

[0232] Paliperidone palmitate injection was dissolved in a mixture of tetrahydrofuran and Tween 80(II) (volume ratio 4:1) (v / v) to prepare a 10 mg / ml paliperidone solution. This solution was then diluted with water for bacterial endotoxin testing to concentrations of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, and 0.0625 mg / ml, denoted as NPC. Simultaneously, a positive control solution containing 2λ concentrations of standard endotoxin was prepared for each of these concentrations, denoted as PPC. The negative control solution was water for bacterial endotoxin testing, denoted as NC. A bacterial endotoxin standard with a potency of 9000 EU / vial was dissolved in 1 ml of water for bacterial endotoxin testing according to the standard's instructions. This solution was then used to prepare the required bacterial endotoxin standard solution, which served as the positive control solution PC.

[0233] Two different manufacturers' horseshoe crab reagents were used to react with NPC, PPC, NC, and PC respectively, with two replicates for each concentration. The preliminary test results are shown in Tables 17 and 18 below.

[0234] Table 17. Limulus Amebocyte Lysate (LIL) Reagent Detection Results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0235]

[0236]

[0237] Table 18. Limulus Amebocyte Lysate (LAL) Reagent Test Results from Xiamen Limulus Amebocyte Lysate Reagent Co., Ltd.

[0238]

[0239] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.125 EU / ml, and the batch number of the horseshoe crab reagent is 22120912.

[0240] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.125 EU / ml and its batch number is 22111043.

[0241] The results in Tables 17 and 18 preliminarily indicate that a 10 mg / ml paliperidone palmitate test solution, diluted to 0.25 mg / ml (40-fold dilution) with water for bacterial endotoxin testing, has no interference with the Limulus Amebocyte Lysate (LAL) reagents from either manufacturer. A sample concentration of 0.25 mg / ml and a LAL reagent sensitivity of 0.0625 EU / ml were selected for the formal interference test.

[0242] Formal Interference Experiment 5

[0243] Take 1 ml of the original test sample solution (10 mg / ml) from four tubes, add 10 μL of an appropriate concentration of endotoxin solution to each, mix well, and then dilute to 40 times with water for bacterial endotoxin testing to obtain test sample solutions containing endotoxins of 2λ, λ, 0.5λ, and 0.25λ, respectively. Perform the experiment according to the "Bacterial Endotoxin Test - Gel Method Interference Test" section in the Chinese Pharmacopoeia, using a Limulus Amebocyte Lysate (LAL) reagent with a sensitivity of 0.0625 EU / ml. The results are shown in Tables 19 and 20.

[0244] Table 19. Limulus Amebocyte Lysate (LIL) Reagent Detection Results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0245]

[0246]

[0247] Table 20. Limulus Amebocyte Lysate (LAL) Reagent Test Results from Xiamen Limulus Amebocyte Lysate Reagent Co., Ltd.

[0248]

[0249] It should be noted that the λ of the horseshoe crab reagent from Fuzhou Xinbei Biochemical Industry Co., Ltd. is 0.0625 EU / ml, and the batch number of the horseshoe crab reagent is 22122312.

[0250] The horseshoe crab reagent from Xiamen Horseshoe Crab Reagent Co., Ltd. has a λ of 0.0625 EU / ml and its batch number is 22111045.

[0251] Formal interference experiments showed that the E values ​​of the three batches of samples for the two manufacturers' Limulus amebocyte lysate (LAL) reagents were significantly affected. S and E t All values ​​were within the range of 0.5λ to 2.0λ (inclusive), meeting the requirements for Limulus amebocyte lysate (LAL) reagent sensitivity verification. It was confirmed that there was no interference at a 40-fold dilution (concentration of 0.25 mg / ml), and the endotoxin content was less than 0.25 EU / mg.

[0252] Recycling Experiment

[0253] The endotoxin recovery test was conducted by replacing the original test sample with a solvent and diluting it at the minimum dilution factor (40 times). If the endotoxin recovery was satisfactory at the minimum dilution factor, it was also satisfactory at a higher dilution factor. The results are shown in Tables 21 and 22.

[0254] Table 21. Limulus Amebocyte Lysate (LIL) Reagent Test Results from Fuzhou Xinbei Biochemical Industry Co., Ltd.

[0255]

[0256] Table 22. Limulus Amebocyte Lysate (LAL) Reagent Test Results from Xiamen Limulus Amebocyte Lysate Reagent Co., Ltd.

[0257]

[0258]

[0259] In summary, paliperidone palmitate is a non-water-soluble drug. For endotoxin detection, a mixed solution of tetrahydrofuran and Tween was used as the solvent to prepare a stock solution of 5–25 mg / ml. This stock solution was then diluted with water for bacterial endotoxin testing before reacting with Limulus Amebocyte Lysate (LAL) reagent. Using LAL reagents with a sensitivity of 0.0625 EU / ml produced by Xiamen Limulus Amebocyte Lysate Reagent Biotechnology Co., Ltd. and Fuzhou Xinbei Biochemical Industry Co., Ltd., no interference was observed in the results. Therefore, paliperidone palmitate samples are suitable for the bacterial endotoxin test, and the endotoxin content of the three batches of samples was less than 0.5 EU / mg, which meets the requirements.

[0260] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting the bacterial endotoxin of paliperidone palmitate, characterized in that, include: Paliperidone palmitate sample, solvent, and water for bacterial endotoxin testing are mixed to form a test solution, which is then used for detection. The solvent is a mixed solution of tetrahydrofuran and Tween; the volume ratio of tetrahydrofuran to Tween is 1.5:1-15:1; the concentration of paliperidone palmitate sample in the test solution is below 0.5 mg / ml. It also includes: dissolving the paliperidone palmitate test sample in the solvent to form a stock solution, and then diluting the stock solution with the bacterial endotoxin test water to form the test solution.

2. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The volume ratio of the tetrahydrofuran to the Tween is 2:1 to 8:

1.

3. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The volume ratio of the tetrahydrofuran to the Tween is 4:

1.

4. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The Tween includes at least one of Tween 80 and Tween 20.

5. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The Tween mentioned is injectable Tween.

6. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 4, characterized in that, The Tween 80 is Tween 80 (II).

7. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The tetrahydrofuran is chromatographic grade tetrahydrofuran.

8. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The concentration of paliperidone palmitate in the test solution is 0.0625-0.5 mg / ml.

9. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The concentration of paliperidone palmitate in the test solution is 0.125-0.5 mg / ml.

10. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The concentration of paliperidone palmitate in the test solution is 0.125-0.25 mg / ml.

11. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The concentration of the mother liquor is 5-25 mg / ml.

12. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, The concentration of the mother liquor was 12.5 mg / ml.

13. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, Also includes: Prepare positive control standards and positive control samples for bacterial endotoxins.

14. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 1, characterized in that, This includes testing for bacterial endotoxin standards, positive controls for test samples, and water used for bacterial endotoxin detection.

15. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 14, characterized in that, include: The water used for bacterial endotoxin testing was mixed with bacterial endotoxin standards to form a concentration of 2. Positive control for bacterial endotoxin standards to improve the sensitivity of horseshoe crab reagent; The paliperidone palmitate sample and the solvent were mixed to form a stock solution, and then bacterial endotoxin standard solution and water for bacterial endotoxin testing were added sequentially to form a positive control for the test sample. Wherein, the concentration of the bacterial endotoxin standard in the positive control of the test sample is consistent with the concentration of the bacterial endotoxin standard in the positive control of the bacterial endotoxin standard; the concentration of the paliperidone palmitate test sample in the positive control of the test sample is consistent with the concentration of the paliperidone palmitate test sample in the test sample solution.

16. The method for detecting bacterial endotoxins of paliperidone palmitate according to any one of claims 1-10 or 13-15, characterized in that, include: Detection can be performed using gel electrophoresis or photometry.

17. The method for detecting bacterial endotoxins of paliperidone palmitate according to claim 15, characterized in that, The sensitivity of the Limulus amebocyte lysate (LAL) reagent used for detection is 0.0625-0.125 EU / ml; and / or, the paliperidone palmitate test sample includes a paliperidone palmitate raw material sample or a paliperidone palmitate preparation sample.

Images