A method for detecting bacterial endotoxins in melphalan hydrochloride

Abstract

The present application provides a method for detecting bacterial endotoxins in melphalan hydrochloride, by using methanol as a pretreatment reagent for the melphalan hydrochloride test sample, and preparing the test sample solution by gradually dripping the test sample stock solution into the Tris buffer and mixing it evenly, so that the horseshoe crab reagent in the test solution matrix has good interference resistance to methanol and melphalan hydrochloride, and the sensitivity requirement for the horseshoe crab reagent is low. The detection limit of bacterial endotoxins in melphalan hydrochloride in the detection method of the present application is 0.4EU / mg, which effectively reduces the detection limit of the bacterial endotoxin gel detection method in melphalan hydrochloride, and has good stability and accuracy.

Description

Technical Field

[0001] The present application belongs to the technical field of drug analysis, and in particular relates to a method for detecting bacterial endotoxins in melphalan hydrochloride. Background Art

[0002] Bacterial endotoxins are products of the cell walls of Gram-negative bacteria. They are not released when the bacteria are alive. They only show their toxicity when the bacteria die, autolyze, or adhere to other cells. When endotoxins enter the human body through the digestive tract, they do not cause harm, but when endotoxins enter the blood through injection and other methods, they can cause different diseases. Therefore, biological products, injectable drugs, chemicals, radiopharmaceuticals, antibiotics, vaccines, dialysis fluids and other preparations, as well as medical devices (such as disposable syringes, implantable biomaterials) must pass bacterial endotoxin testing before they can be used.

[0003] At present, the bacterial endotoxin test methods include gel method and photometry. The former uses the principle of agglutination reaction between horseshoe crab reagent and bacterial endotoxin to qualitatively detect or semi-quantitatively detect endotoxin, while the latter includes turbidity method and colorimetric matrix (colorimetric) method, which respectively use the turbidity change during the reaction between horseshoe crab reagent and endotoxin and the amount of chromophore released by the generated coagulase to quantitatively determine endotoxin. Among them, the photometry method has a low popularity rate due to its reliance on professional equipment and complex operation.

[0004] The main component of melphalan hydrochloride is 4-[bis(2-chloroethyl)amino]-L-phenylalanine hydrochloride. The limit of bacterial endotoxin in melphalan hydrochloride is generally not more than 0.4EU / mg. However, for some drug varieties, the bacterial endotoxin test method (gel method) of the Chinese Pharmacopoeia cannot meet the detection limit requirements. Therefore, it is necessary to develop a bacterial endotoxin detection method with higher detection accuracy to meet the increasingly stringent drug safety regulatory requirements. Summary of the invention

[0005] In view of this, the present application provides a method for detecting bacterial endotoxins in melphalan hydrochloride, which can effectively reduce the detection limit of the bacterial endotoxin gel detection method in melphalan hydrochloride and has good stability and accuracy.

[0006] The specific technical solutions of this application are as follows:

[0007] The present application provides a method for detecting bacterial endotoxins in melphalan hydrochloride, comprising the following steps:

[0008] Prepare bacterial endotoxin standard solutions of different concentrations, use the bacterial endotoxin standard solutions to conduct a sensitivity verification test of the horseshoe crab reagent, and determine the labeled sensitivity of the horseshoe crab reagent;

[0009] Prepare bacterial endotoxin reference substance solutions of different concentrations, use bacterial endotoxin standard solution and bacterial endotoxin reference substance solution to carry out methanol interference pre-test and methanol interference test in sequence, and determine the maximum non-interfering concentration of methanol at the labeled sensitivity of the horseshoe crab reagent;

[0010] Prepare different concentrations of bacterial endotoxin spiked test solution, use bacterial endotoxin standard solution and bacterial endotoxin spiked test solution to conduct melphalan hydrochloride interference test, and determine the maximum non-interfering concentration of melphalan hydrochloride at the labeled sensitivity of limulus amebocyte lysate;

[0011] Melphalan hydrochloride test sample was dissolved in methanol to prepare a test sample stock solution, which was then gradually added dropwise to the Tris buffer solution and mixed to obtain a test sample solution that met the maximum non-interference concentration. Horseshoe crab reagent that met the indicated sensitivity was added to the test sample solution to perform a gel-based detection test for bacterial endotoxins.

[0012] Furthermore, the volume ratio of the test sample stock solution and Tris buffer was 1:(10~30).

[0013] Furthermore, the concentration of the test sample in the test sample stock solution is 10-20 mg / ml.

[0014] Furthermore, the preparation method of the bacterial endotoxin spiked test solution is:

[0015] Take the melphalan hydrochloride test sample, add a small amount of methanol and mix well, then gradually add it dropwise to the bacterial endotoxin standard solution diluted with Tris buffer and mix well.

[0016] Further, the preparation method of the bacterial endotoxin reference solution is:

[0017] Methanol was serially diluted with endotoxin test water, and bacterial endotoxin standards with concentrations of 2λ, λ, 0.5λ and 0.25λ were added to prepare bacterial endotoxin reference solutions.

[0018] Furthermore, the maximum non-interfering concentration of methanol at the labeled sensitivity of the horseshoe crab reagent is 0.0625 ml / ml.

[0019] Furthermore, the maximum non-interfering concentration of melphalan hydrochloride at the labeled sensitivity of the Limulus amebocyte lysate is 0.625 ml / ml.

[0020] Furthermore, the indicated sensitivity of the horseshoe crab reagent is 0.125EU / ml.

[0021] Furthermore, the detection limit of bacterial endotoxin in melphalan hydrochloride is 0.4EU / mg.

[0022] Furthermore, melphalan hydrochloride is the API or preparation of melphalan hydrochloride.

[0023] In summary, the present application provides a method for detecting bacterial endotoxins in melphalan hydrochloride, by using methanol as a pretreatment reagent for the melphalan hydrochloride test sample, and preparing the test sample solution by gradually dropping the test sample stock solution into the Tris buffer and mixing it evenly, so that the horseshoe crab reagent in the test solution matrix has good interference resistance to methanol and melphalan hydrochloride, and the sensitivity requirements for the horseshoe crab reagent are low. The detection limit of bacterial endotoxins in melphalan hydrochloride in the detection method of the present application is 0.4EU / mg, which effectively reduces the detection limit of the bacterial endotoxin gel detection method in melphalan hydrochloride, and has good stability and accuracy. DETAILED DESCRIPTION

[0024] In order to make the purpose, features, and advantages of this application more obvious and easy to understand, the technical solutions in the embodiments of this application are clearly and completely described. Obviously, the embodiments described below are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0025] The reagents and raw materials used in the examples of this application are all commercially available or homemade.

[0026] Example

[0027] 1. Solution preparation:

[0028] Preparation of bacterial endotoxin standard solution: Take 1 10EU bacterial endotoxin standard, tap the bottle wall to make the powder fall to the bottom of the bottle, wipe it with a 75% alcohol cotton ball and open it. Prevent glass chips from falling into the bottle during the opening process. According to the standard product instructions, add 1ml of bacterial endotoxin test water to dissolve its contents, seal the bottle mouth with a sealing film, and mix it on a micro vortex shaker for 15 minutes. Then dilute it to prepare 6 concentrations of bacterial endotoxin standard solutions, namely 4λ (1EU / ml), 2λ (0.5EU / ml), λ (0.25EU / ml), 0.5λ (0.125EU / ml), 0.25λ (0.0625EU / ml), 0.125λ (0.03125EU / ml), and each dilution step should be mixed on a micro vortex shaker for 30 seconds.

[0029] Preparation of bacterial endotoxin reference solution: dilute methanol in series with endotoxin test water (concentration of 0.0625~0.5ml / ml), and add bacterial endotoxin standards with concentrations of 2λ (0.5EU / ml), λ (0.25EU / ml), 0.5λ (0.125EU / ml), and 0.25λ (0.0625EU / ml) to prepare the bacterial endotoxin reference solution.

[0030] Preparation of test solution: Take 30 mg of melphalan hydrochloride API test sample, place it in a pyrogen-free dilution tube, add 2.4 ml of methanol and mix well to prepare a 12.5 mg / ml test sample solution. Take 0.8 ml of the above 12.5 mg / ml test sample solution, gradually add it dropwise to 15.2 ml of Tris buffer, mix well, and prepare a 0.625 mg / ml test sample solution for use.

[0031] Preparation of bacterial endotoxin spiked test solution: Take 0.8 ml of the above 12.5 mg / ml test solution and gradually add it dropwise to 15.2 ml of bacterial endotoxin standard solution diluted with Tris buffer, mix well, and prepare a bacterial endotoxin spiked test solution containing 0.25 EU / ml bacterial endotoxin standard solution in 0.625 mg / ml test solution.

[0032] 2. Re-examination test of sensitivity of horseshoe crab reagent:

[0033] Take 18 horseshoe crab reagents and add 0.1ml of bacterial endotoxin test water to each of them to reconstitute, and divide them into pyrogen-free empty ampoules. Take 16 of them and add 0.1ml of bacterial endotoxin standard solution with concentrations of 2λ, λ, 0.5λ, and 0.25λ respectively, and make 4 parallels for each concentration. Take another 2 and add 0.1ml of bacterial endotoxin test water as a negative control. After the addition of the sample, seal the ampoule with a sealing film, gently shake and mix, and place it vertically in a test tube thermostat at 37℃±1℃, and keep warm for 60min±2min.

[0034] Take the ampoule out of the test tube thermostat to avoid vibration, slowly turn each tube upside down 180°, and observe; if gel is formed in the tube, and the gel does not deform or slide off the tube wall, it is positive and recorded as (+); if gel is not formed or the formed gel is not solid, deforms, and slides off the tube wall, it is negative and recorded as (-). The test results are shown in Table 1, and λc is the sensitivity test value of the horseshoe crab reagent.

[0035] Table 1

[0036]

[0037] As shown in Table 1, the sensitivity review results of the horseshoe crab reagent meet the requirements, and its labeled sensitivity is 0.125EU / ml, which can be used for endotoxin testing. The sensitivity review of the horseshoe crab reagent of 0.25EU / ml was carried out in the same way, and the sensitivity review results of the horseshoe crab reagent met the requirements.

[0038] 3. Methanol interference test:

[0039] Interference pre-test: Take 8 tubes of horseshoe crab reagent with a sensitivity of 0.25EU / ml and reconstitute them, add 0.1ml of methanol solution with concentrations of 0.5 ml / ml, 0.25 ml / ml, 0.125 ml / ml and 0.0625ml / ml to 2 tubes each as test tubes, take 8 tubes and add 0.1ml of 2λ bacterial endotoxin standard methanol solution with concentrations of 0.5 ml / ml, 0.25 ml / ml, 0.125 ml / ml and 0.0625ml / ml to 2 tubes each as test positive control tubes, take 2 tubes and add 0.1ml of endotoxin test water as negative control tubes, take 2 tubes and add 0.1ml of 2λ bacterial endotoxin standard solution as positive control tubes. After adding the sample, seal the ampoule with sealing film, gently shake and mix to avoid bubbles, put it in a thermostat, keep it at 37℃±1℃ for 60±2 minutes, observe and record the results.

[0040] The results of the methanol interference pre-test showed that the negative control was negative and the positive control was positive, proving that the test results were valid. Methanol diluted to a concentration of 0.0625 ml / ml had no interference with the 0.25 EU / ml horseshoe crab reagent.

[0041] Formal interference test: Take 10 reconstituted horseshoe crab reagents and add 0.1 ml of bacterial endotoxin standard solutions with concentrations of 0.5 EU / ml, 0.25 EU / ml, 0.125 EU / ml and 0.0625 EU / ml to each of 2 tubes, and add 0.1 ml of endotoxin test water to 2 tubes as negative controls. Take another 18 tubes and add 0.1 ml of endotoxin standard endotoxin reference solutions containing methanol (concentration of 0.0625 ml / ml) to each of 4 tubes, and add 0.1 ml of diluent containing 0.0625 ml / ml of methanol to 2 tubes as sample negative controls. After adding the sample, seal the ampoule with a sealing film, gently shake and mix to avoid bubbles, and put it in a thermostat and keep it warm at 37℃±1℃ for 60±2 minutes.

[0042] After the insulation time is up, take out the ampoule to avoid vibration. Take out each tube and slowly turn it upside down 180° for observation. If gel is formed in the tube and the gel does not deform or slide off the tube wall, it is positive and recorded as (+). If no gel is formed or the gel cannot remain intact and slides off the tube wall, it is negative and recorded as (-).

[0043] The test results are shown in Table 2, where C is the geometric mean of the reaction endpoint concentration of the bacterial endotoxin standard solution made with water for endotoxin testing, and B is the geometric mean of the reaction endpoint concentration of the endotoxin solution prepared by diluting the test solution.

[0044] Table 2

[0045]

[0046] From the above table, it can be seen that the methanol solution meets the requirements when the interference test is performed using the horseshoe crab reagent with a sensitivity of 0.25EU / ml at a concentration of 0.0625ml / ml.

[0047] 4. Melphalan hydrochloride bacterial endotoxin interference test:

[0048] Take a horseshoe crab reagent with a sensitivity of 0.125EU / ml, tap the bottle wall to make the powder fall to the bottom of the bottle, wipe it with a 75% alcohol cotton ball and open it for use to prevent glass chips from falling into the bottle. Add 0.1ml of endotoxin test water to each bottle and dissolve it. Gently rotate the bottle wall to fully dissolve the contents and avoid bubbles.

[0049] Take 10 tubes of the reconstituted horseshoe crab reagent and add 0.1 ml of 0.25 EU / ml, 0.125 EU / ml, 0.0625 EU / ml and 0.0312 EU / ml bacterial endotoxin standard solutions to each of the 10 tubes. Take another 2 tubes and add 0.1 ml of endotoxin test water as a negative control.

[0050] For the other 18 tubes, add 0.1 ml of the test solution spiked with bacterial endotoxins at concentrations of 0.25 EU / ml, 0.125 EU / ml, 0.0625 EU / ml, 0.0312 EU / ml and 0.625 mg / ml of melphalan hydrochloride to each of the 4 tubes, and add 0.1 ml of the test solution of 0.625 mg / ml of melphalan hydrochloride to the other 2 tubes as negative controls.

[0051] After the sample is added, seal the ampoule with a sealing film, shake gently to mix, avoid bubbles, put it in a thermostat, and keep it at 37℃±1℃ for 60±2 minutes. After the insulation time is up, take out the ampoule to avoid vibration, take out each tube and slowly turn it upside down 180° for observation. If gel is formed in the tube, and the gel does not deform or slide off the tube wall, it is positive and recorded as (+); if no gel is formed or the gel cannot remain intact and slides off the tube wall, it is negative and recorded as (-).

[0052] The test results are shown in Table 3 below. C is the geometric mean of the reaction endpoint concentration of the endotoxin standard solution prepared with endotoxin test water, and B is the geometric mean of the reaction endpoint concentration of the endotoxin solution prepared by diluting the test solution.

[0053] Table 3

[0054]

[0055] From the above data, it can be seen that the test samples met the requirements when the spike recovery interference test was carried out at a concentration of 0.625 mg / ml using the horseshoe crab reagent with a sensitivity of 0.125 EU / ml.

[0056] 5. Melphalan hydrochloride bacterial endotoxin test:

[0057] Take the horseshoe crab reagent with a marked sensitivity of 0.125EU / ml, tap the bottle wall to make the powder fall to the bottom of the bottle, wipe it with a 75% alcohol cotton ball and open it for use to prevent glass chips from falling into the bottle. Add 0.1ml of bacterial endotoxin test water to each bottle and dissolve it. Gently rotate the bottle wall to fully dissolve the contents and avoid bubbles.

[0058] Take 0.1 ml of each of the test solution spiked with bacterial endotoxin (test positive control), bacterial endotoxin reference solution (positive control), test solution and negative control (water for bacterial endotoxin test), and add them to the reconstituted horseshoe crab reagent. After adding the sample, seal the ampoule with a sealing film, shake it gently to mix it to avoid bubbles, and put it in a test tube thermostat at 37℃±1℃ for 60±2 minutes. Avoid vibration during the insulation and removal of the test tube to cause false negative results.

[0059] After the holding time is up, take the ampoule out of the thermostat to avoid vibration, slowly turn each tube upside down 180°, and observe; if gel is formed in the tube, and the gel does not deform or slip off the tube wall, it is positive and recorded as (+); if gel is not formed or the formed gel is not solid, deforms, and slips off the tube wall, it is negative and recorded as (-). The test results are shown in Table 4.

[0060] Table 4

[0061]

[0062] As can be seen from the table above, the test samples were tested for bacterial endotoxins at a concentration of 0.625 mg / ml using a horseshoe crab reagent with a sensitivity of 0.125 EU / ml, and the results were in compliance with regulations. Among them, the detection limit of bacterial endotoxins in melphalan hydrochloride is 0.4 EU / mg.

[0063] Comparative Example 1

[0064] Take 10.0 mg of the test sample, place it in a pyrogen-free dilution tube, add 0.2 ml of methanol to dissolve, vortex vigorously for 3-5 minutes, add 0.1 ml of 75 EU / ml bacterial endotoxin standard solution, and then add 1.7 ml of bacterial endotoxin test water to obtain a test solution containing 3.75 EU / ml bacterial endotoxin and a concentration of 5 mg / ml. Take 1 ml of the above test solution, place it in a pyrogen-free dilution tube, adjust the pH to 7.0-8.0 with 0.1N sodium hydroxide solution, and make up the volume to 3.75 ml with bacterial endotoxin test water to obtain a 1.333 mg / ml test solution (containing 1 EU / ml bacterial endotoxin). Dilute the test solution to 0.333 mg / ml (containing 0.25 EU / ml bacterial endotoxin) with bacterial endotoxin test water.

[0065] Take 4 reconstituted horseshoe crab reagents and add 0.1ml 0.333 mg / ml test solution (containing 0.25EU / ml bacterial endotoxin) as the test sample positive control tube, take 2 horseshoe crab reagents and add 0.1ml endotoxin test water as the negative control tube, take 2 horseshoe crab reagents and add 0.1ml 2λ bacterial endotoxin standard solution as the positive control tube.

[0066] After adding the sample, seal the ampoule with sealing film, shake gently to mix, avoid bubbles, put it in a thermostat, keep it at 37℃±1℃ for 60±2 minutes, observe and record the results.

[0067] The test results are shown in Table 5. The test sample was diluted with methanol solution and then directly added to the bacterial endotoxin standard solution. The pH was adjusted with sodium hydroxide solution to prepare the test solution. The test sample did not meet the requirements when the spike recovery interference test was performed at a concentration of 0.333 mg / ml using a horseshoe crab reagent with a sensitivity of 0.125 EU / ml.

[0068] Table 5

[0069]

[0070] Comparative Example 2

[0071] (1) Take 10.0 mg of the test sample and place it in a pyrogen-free dilution tube. Add 0.2 ml of methanol to dissolve it, vortex vigorously for 3 to 5 minutes, add 0.4 ml of 20 EU / ml bacterial endotoxin standard solution, and then add 1.4 ml of bacterial endotoxin test water to obtain a 5 mg / ml test solution containing 4 EU / ml of bacterial endotoxin. Take 1 ml of the above test solution and place it in a pyrogen-free dilution tube. Adjust the pH to 7.0-8.0 with 0.1N sodium hydroxide solution, and make up the volume to 2 ml with bacterial endotoxin test water to obtain a 2.5 mg / ml test solution (containing 2 EU / ml bacterial endotoxin). Dilute it with bacterial endotoxin test water to a test solution of 0.156 mg / ml (containing 0.125 EU / ml bacterial endotoxin).

[0072] (2) Take 10.0 mg of the test sample and place it in a pyrogen-free dilution tube. Add 0.2 ml of methanol to dissolve it. Vortex vigorously for 3 to 5 minutes. Add 340 μl of 0.1 N sodium hydroxide solution and mix well. Then add 0.4 ml of 20 EU / ml bacterial endotoxin standard solution and 1060 μl of bacterial endotoxin injection water. Mix well to obtain a 5 mg / ml test solution containing 4 EU / ml of bacterial endotoxin. Dilute it to 0.156 mg / ml test solution (containing 0.125 EU / ml bacterial endotoxin) with bacterial endotoxin test water.

[0073] Take 2 tubes each with 0.1 ml of 0.156 mg / ml test solution (containing 0.125 EU / ml bacterial endotoxin) of the horseshoe crab reagent with a sensitivity of 0.06 EU / ml after reconstitution as the positive control tubes for the test sample, 2 tubes with 0.1 ml of endotoxin test water as the negative control tubes, and 2 tubes with 0.1 ml of 2λ bacterial endotoxin standard solution as the positive control tubes.

[0074] After adding the sample, seal the ampoule with sealing film, shake gently to mix, avoid bubbles, put it in a thermostat, keep it at 37℃±1℃ for 60±2 minutes, observe and record the results.

[0075] The test results are shown in Table 6. The results show that the spike recovery test in step (1) by sequentially adding bacterial endotoxin standard solution, bacterial endotoxin test water and sodium hydroxide regulator to prepare the test solution, or in step (2) by sequentially adding sodium hydroxide regulator, bacterial endotoxin standard solution and bacterial endotoxin test water to prepare the test solution, failed. The reason may be that the test sample was dissolved in methanol and then dissolved in water, and its acidity was too strong (about pH 2~3), which destroyed the bacterial endotoxin standard and caused errors in the test results. In addition, the high concentration of the test sample can easily cause the sensitivity of the horseshoe crab reagent to fail.

[0076] Table 6

[0077]

[0078] Comparative Example 3

[0079] (1) Take 10.0 mg of the test sample and place it in a pyrogen-free dilution tube. Add 0.8 ml of methanol to dissolve it, add 0.4 ml of 10 EU / ml bacterial endotoxin standard solution, add 340 μl of 0.1N sodium hydroxide solution and mix well. Then add 14.46 ml of bacterial endotoxin injection water and mix well to obtain a 0.625 mg / ml test solution (containing 0.25 EU / ml bacterial endotoxin).

[0080] (2) Take 10.0 mg of the test sample and place it in a pyrogen-free dilution tube. Add 0.8 ml of methanol to dissolve it, add 340 ul of 0.1 N sodium hydroxide solution to mix well, add 0.4 ml of 10 EU / ml bacterial endotoxin standard solution, and then add 14.46 ml of bacterial endotoxin injection water to mix well to obtain 0.625 mg / ml test solution (containing 0.25 EU / ml bacterial endotoxin).

[0081] Take two tubes each as positive control tubes for the test sample after reconstitution of the horseshoe crab reagent with a sensitivity of 0.125EU / ml and add 0.1ml of 0.625mg / ml test solution (containing 0.25EU / ml bacterial endotoxin), add 0.1ml of endotoxin test water to two tubes as negative control tubes, and add 0.1ml of 2λ bacterial endotoxin standard solution to two tubes as positive control tubes.

[0082] After adding the sample, seal the ampoule with sealing film, shake gently to mix, avoid bubbles, put it in a thermostat, keep it at 37℃±1℃ for 60±2 minutes, observe and record the results.

[0083] The test results are shown in Table 7. The results show that after changing the concentration of the test solution and the indicated sensitivity of the horseshoe crab reagent, in step (1), the test solution is prepared by sequentially adding the bacterial endotoxin standard solution, the sodium hydroxide regulator and the bacterial endotoxin test water, or in step (2), the test solution is prepared by sequentially adding the sodium hydroxide regulator, the bacterial endotoxin standard solution and the bacterial endotoxin test water. The spike recovery is unqualified, and the problem of the too high acidity of the test solution matrix and the incompatible concentration is still not solved.

[0084] Table 7

[0085]

[0086] Comparative Example 4

[0087] Take 10.0 mg of the test sample, place it in a pyrogen-free dilution tube, add 8 ml of methanol to dissolve, add 0.8 ml of 10 EU / ml bacterial endotoxin standard solution, and then add 1.2 ml of bacterial endotoxin test water to obtain a 1 mg / ml test solution containing 0.8 EU / ml bacterial endotoxin. Take 0.3 ml of the above test solution, place it in a pyrogen-free dilution tube, adjust the pH to 7.0-8.0 with 0.1 N sodium hydroxide solution, and make up the volume to 3 ml with bacterial endotoxin test water to obtain a 0.1 mg / ml test solution (containing 0.08 EU / ml bacterial endotoxin). Dilute with bacterial endotoxin test water to a test solution of 0.078 mg / ml (containing 0.0625 EU / ml bacterial endotoxin).

[0088] Take 2 tubes each of which are reconstituted with 0.03EU / ml horseshoe crab reagent and added with 0.1ml of 0.078mg / ml test solution (containing 0.0625EU / ml bacterial endotoxin) as positive control tubes for the test sample, 2 tubes are added with 0.1ml of endotoxin test water as negative control tubes, and 2 tubes are added with 0.1ml of 2λ bacterial endotoxin standard solution as positive control tubes.

[0089] After adding the sample, seal the ampoule with sealing film, shake gently to mix, avoid bubbles, put it in a thermostat, keep it at 37℃±1℃ for 60±2 minutes, observe and record the results.

[0090] The test results are shown in Table 8. After changing the concentration of the test solution and the indicated sensitivity of the horseshoe crab reagent, a spike recovery pre-experiment was carried out by sequentially adding bacterial endotoxin standard solution, bacterial endotoxin test water and sodium hydroxide regulator to prepare the test solution. The spike recovery was unqualified, and the problems of excessive acidity of the test solution matrix and incompatible concentrations were still not solved.

[0091] Table 8

[0092]

[0093] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting bacterial endotoxins in melphalan hydrochloride, characterized in that: The steps include: Prepare bacterial endotoxin standard solutions of different concentrations, use the bacterial endotoxin standard solutions to conduct a sensitivity verification test of the horseshoe crab reagent, and determine the labeled sensitivity of the horseshoe crab reagent; Prepare bacterial endotoxin reference substance solutions of different concentrations, use bacterial endotoxin standard solution and bacterial endotoxin reference substance solution to carry out methanol interference pre-test and methanol interference test in sequence, and determine the maximum non-interfering concentration of methanol at the labeled sensitivity of the horseshoe crab reagent; Prepare different concentrations of bacterial endotoxin spiked test solution, use bacterial endotoxin standard solution and bacterial endotoxin spiked test solution to conduct melphalan hydrochloride interference test, and determine the maximum non-interfering concentration of melphalan hydrochloride at the labeled sensitivity of limulus amebocyte lysate; Melphalan hydrochloride test sample was dissolved in methanol to prepare a test sample stock solution, which was then gradually added dropwise to the Tris buffer solution and mixed to obtain a test sample solution that met the maximum non-interference concentration. Horseshoe crab reagent that met the indicated sensitivity was added to the test sample solution to perform a gel-based detection test for bacterial endotoxins.

2. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The volume ratio of the test sample solution and Tris buffer is 1: (10~30).

3. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The concentration of the test sample in the test sample stock solution is 10~20mg / ml.

4. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The preparation method of bacterial endotoxin spiked test solution is as follows: Take the melphalan hydrochloride test sample, add a small amount of methanol and mix well, then gradually add it dropwise to the bacterial endotoxin standard solution diluted with Tris buffer and mix well.

5. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The preparation method of bacterial endotoxin reference solution is as follows: Methanol was serially diluted with endotoxin test water, and bacterial endotoxin standards with concentrations of 2λ, λ, 0.5λ and 0.25λ were added to prepare bacterial endotoxin reference solutions.

6. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The maximum non-interfering concentration of methanol at the labeled sensitivity of the horseshoe crab reagent is 0.0625ml / ml.

7. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The maximum non-interfering concentration of melphalan hydrochloride at the labeled sensitivity of the Limulus amebocyte lysate is 0.625 ml / ml.

8. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The indicated sensitivity of the horseshoe crab reagent is 0.125EU / ml.

9. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: The detection limit of bacterial endotoxin in melphalan hydrochloride is 0.4EU / mg.

10. The method for detecting bacterial endotoxins in melphalan hydrochloride according to claim 1, characterized in that: Melphalan hydrochloride is the raw material or preparation of melphalan hydrochloride.

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