Method for detecting bacterial endotoxin in melphalan hydrochloride bulk drug

Abstract

The invention relates to a method for detecting bacterial endotoxin in a melphalan hydrochloride bulk drug, and belongs to the technical field of drug analysis. The invention provides a test solution for detecting bacterial endotoxin in a melphalan hydrochloride raw material medicine, which comprises a test product melphalan hydrochloride raw material medicine, a 2-8 DEG C sulfobutyl beta-cyclodextrin sodium water solution and calcium and / or magnesium ions. The detection method disclosed by the invention is simple to operate, the sample can be dissolved in BET water without reanalysis, and the detection result is accurate; no organic solvent is used for preparing a test solution, so that the safety to operators is good, and bacterial endotoxin interference experiments prove that no interference effect exists; and the detection limit of the bacterial endotoxin in the melphalan hydrochloride bulk drug can reach 0.2 EU / mg.

Description

Technical Field

[0001] The invention relates to a method for detecting bacterial endotoxins of melphalan hydrochloride raw material medicine, and belongs to the technical field of drug analysis. Background Art

[0002] Melphalan hydrochloride, chemical name is 4-bis(2-chloroethyl)-amino-L-phenylalanine hydrochloride, molecular formula C 13 H 18 Cl 2 N 2 O 2 HCl, usually prepared as an injection, is clinically used to treat multiple myeloma, breast cancer, ovarian cancer, chronic leukemia, malignant lymphoma, Waldenstrom's disease, etc. The structural formula is as follows:

[0003]

[0004] As a clinical drug, melphalan hydrochloride must meet the drug quality control standards for its bacterial endotoxins. Therefore, it is very necessary to test the bacterial endotoxins of both the raw materials and the preparations. However, there are few studies on the test methods for bacterial endotoxins in melphalan hydrochloride. Zhang Zhen et al. Study on the test method for bacterial endotoxins of melphalan hydrochloride for injection [J]. Strait Pharmacy, 2020, 32(10): 71-74. The disclosed test method is for injection (melphalan hydrochloride for injection, trade name Maiweining). According to the investigation of the inventors of the present invention, for the raw material of melphalan hydrochloride, since it is almost insoluble in water, the test sample cannot be completely dissolved in BET water (unlike the injection containing solubilizing excipients), and some bacterial endotoxins are wrapped, resulting in the inability to effectively detect using the method disclosed in the document. Chinese patent application CN116047076A discloses a method for detecting bacterial endotoxins in melphalan hydrochloride, which uses methanol as the test sample solvent. Not only is there a problem of toxicity to operators, but organic solvents are also prone to interfere with bacterial endotoxin detection. Although the patent application verifies that low concentration (0.0625 ml / ml) of methanol will not interfere with the test, pure methanol is required to dissolve the melphalan hydrochloride raw material test sample when the test sample solution is actually prepared. Under conditions of high concentration of methanol, bacterial endotoxins can easily be inactivated, affecting the accuracy of the test results.

[0005] Therefore, it is necessary to develop a method for detecting bacterial endotoxins in melphalan hydrochloride raw materials with better operator safety, less interference, and higher detection accuracy to meet the increasingly stringent drug safety regulatory requirements. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the object of the present invention is to provide a method for detecting bacterial endotoxins in melphalan hydrochloride bulk drug.

[0007] The invention provides a test solution for detecting bacterial endotoxins in a melphalan hydrochloride bulk drug, comprising a test sample melphalan hydrochloride bulk drug, an added 2-8° C. sodium sulfobutyl beta-cyclodextrin aqueous solution, and calcium and / or magnesium ions.

[0008] Furthermore, the test solution is an aqueous solution.

[0009] Furthermore, the concentration of melphalan hydrochloride is 0.25-0.64 mg / ml.

[0010] Preferably, the concentration of melphalan hydrochloride is 0.25-0.34 mg / ml.

[0011] Furthermore, the concentration of the sodium sulfobutyl-beta-cyclodextrin aqueous solution is 200-300 mg / ml, preferably 270 mg / ml.

[0012] Furthermore, the mass ratio of melphalan hydrochloride to sodium sulfobutyl beta-cyclodextrin is 1:20 to 1:60, preferably 1:54.

[0013] Furthermore, the calcium and / or magnesium ions are added in the form of calcium hydroxide and / or magnesium sulfate.

[0014] Preferably, magnesium ions are added via a magnesium ion buffer, which mainly consists of magnesium sulfate and Tris buffer.

[0015] Preferably, calcium ions are added via a calcium ion buffer, which is mainly composed of calcium hydroxide and Tris buffer.

[0016] The present invention provides a method for preparing the test solution, comprising the following steps: taking melphalan hydrochloride raw material, adding 2-8° C. sulfobutyl beta-cyclodextrin sodium aqueous solution to dissolve, optionally diluting with bacterial endotoxin test water, and adding calcium and / or magnesium ions.

[0017] Further, a 2-8° C. aqueous solution of sodium sulfobutyl-beta-cyclodextrin is added to a concentration of melphalan hydrochloride of 5-10 mg / ml, preferably 5 mg / ml.

[0018] Furthermore, the sample is diluted 8 to 16 times, preferably 8 times, with water for bacterial endotoxin test.

[0019] Furthermore, calcium ion buffer and / or magnesium ion buffer were added to dilute the mixture 2-fold.

[0020] The present invention provides a method for detecting bacterial endotoxins in melphalan hydrochloride raw material medicine, which uses a horseshoe crab reagent to detect any one of the test sample solutions.

[0021] Preferably, detection is by gel-based methods.

[0022] Furthermore, the sensitivity of the horseshoe crab reagent is 0.0625-0.125 EU / ml.

[0023] Preferably, the sensitivity of the horseshoe crab reagent is 0.0625EU / ml.

[0024] Furthermore, the detection limit of bacterial endotoxins of melphalan hydrochloride raw material is 0.4EU / mg.

[0025] Preferably, the detection limit of bacterial endotoxins of melphalan hydrochloride bulk drug substance is 0.2 EU / mg.

[0026] Among them, based on the bacterial endotoxin limit of 1EU / mg of melphalan hydrochloride injection, it is calculated that the bacterial endotoxin limit of melphalan hydrochloride bulk drug is preferably no more than 0.302EU / mg. To ensure quality, the limit of the bulk drug is more preferably 0.2EU / mg.

[0027] In the present invention, the role of calcium and / or magnesium ions is to maintain the normal sensitivity of the horseshoe crab reagent.

[0028] In some specific embodiments of the present invention, magnesium ions are added to the test solution via a magnesium ion buffer (manufacturer: Zhanjiang Andus Biological Co., Ltd.; main components are magnesium sulfate and Tris buffer).

[0029] In some specific embodiments of the present invention, calcium ions are added to the test solution via a calcium ion buffer (manufacturer: Zhanjiang Andus Biological Co., Ltd.; main components are calcium hydroxide and Tris buffer).

[0030] In some specific embodiments of the present invention, the mass volume ratio of melphalan hydrochloride: calcium ion buffer and / or magnesium ion buffer is 25:40, mg / ml.

[0031] The present invention provides a method for detecting bacterial endotoxins in melphalan hydrochloride raw material medicine, which has at least the following characteristics:

[0032] Beneficial effects:

[0033] 1. The detection method of the present invention is simple to operate, can dissolve the test sample in BET water without re-analysis, and the detection result is accurate.

[0034] 2. No organic solvent is used in the preparation of the test solution, which is safer for operators, and has been proven to have no interference effect through bacterial endotoxin interference experiments.

[0035] 3. The detection limit of bacterial endotoxins in melphalan hydrochloride raw materials can reach 0.2EU / mg. DETAILED DESCRIPTION

[0036] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0037] Example 1 Interference experiment of melphalan hydrochloride raw material and solvent

[0038] The experiment was conducted using a horseshoe crab reagent with a sensitivity of 0.125 EU / ml (λ).

[0039] Positive control solution (Solution C): Take a bacterial endotoxin working standard (9000EU / vial), tap the bottle wall to make the powder fall to the bottom of the bottle, disinfect the bottleneck with an alcohol cotton ball and open the ampoule, and prevent glass chips from falling into the bottle when opening. Take the opened bacterial endotoxin working standard, add 900μl of bacterial endotoxin test water, seal the bottle mouth with a sealing film; place it on a vortex mixer and mix for no less than 15 minutes, then gradually dilute it with bacterial endotoxin test water to make 0.25EU / ml (2λ), 0.125EU / ml (λ), 0.0625EU / ml (0.5λ), 0.03125EU / ml (0.25λ) bacterial endotoxin control solution, and each dilution step should be mixed on a vortex mixer for 1 minute.

[0040] Negative control solution (Solution D): water for bacterial endotoxin detection.

[0041] Test solution (A1 solution): Take about 25 mg of melphalan hydrochloride raw material test sample, place it in a pyrogen-free centrifuge tube, add 5 ml of sulfobutyl beta-cyclodextrin sodium aqueous solution (270 mg / ml, refrigerated at 2-8°C) to dissolve, dilute with bacterial endotoxin test water to a 0.625 mg / ml solution, and then dilute with calcium and magnesium ion additives (manufacturer: Xiamen Limulus Reagent Biotechnology Co., Ltd.; batch number: 21028106) to make a 0.3125 mg / ml test solution. Each dilution step should be mixed on a vortex mixer for 1 min.

[0042] Test sample positive control solution (B1 solution): Take about 25 mg of melphalan hydrochloride raw material test sample, place it in a pyrogen-free centrifuge tube, add 4.9 ml of sulfobutyl beta-cyclodextrin sodium aqueous solution (270 mg / ml, refrigerated at 2-8°C) to dissolve, add 100 μl of 200 EU / ml bacterial endotoxin standard solution, mix on a vortex mixer for 1 min, dilute with bacterial endotoxin test water to make a solution of 0.625 mg / ml melphalan hydrochloride solution containing 0.5 EU / ml of bacterial endotoxin, and then dilute with calcium and magnesium ion additives to make a solution of 0.3125 mg / ml melphalan hydrochloride solution containing 0.25 EU / ml of bacterial endotoxin. Each dilution step should be mixed on a vortex mixer for 1 min.

[0043] Solvent (A2 solution): Take an aqueous solution of sodium sulfobutyl beta-cyclodextrin (270 mg / ml, refrigerated at 2-8°C), dilute it 8 times with water for bacterial endotoxin test, and then dilute it 2 times with calcium and magnesium ion additives. Each dilution step should be mixed on a vortex mixer for 1 minute.

[0044] Solvent positive control (B2 solution): Take 4.9 ml of sodium sulfobutyl beta-cyclodextrin aqueous solution (270 mg / ml, refrigerated at 2-8°C), place it in a pyrogen-free centrifuge tube, add 100 μl of 200 EU / ml bacterial endotoxin standard solution, mix on a vortex mixer for 1 min, dilute with bacterial endotoxin test water to make a solution containing 0.5 EU / ml of bacterial endotoxin, and then dilute with calcium and magnesium ion additives to make a solution containing 0.25 EU / ml of bacterial endotoxin. Each dilution step should be mixed on a vortex mixer for 1 min.

[0045] Take 80 horseshoe crab reagents (40 from Zhanjiang Bokang Marine Biological Co., Ltd. and 40 from Zhanjiang Andus Biological Co., Ltd.), tap the bottle wall to make the powder fall to the bottom of the bottle, disinfect the bottleneck with an alcohol cotton ball and use a marker to number the position below the bottleneck of the horseshoe crab reagent, open the ampoule after the alcohol evaporates, and prevent glass chips from falling into the bottle when opening. Add 100 μl of bacterial endotoxin test water to each tube of horseshoe crab reagent for reconstitution, take 100 μl of each solution and place it in the horseshoe crab reagent. After the addition is completed, seal the ampoule with a sealing film, gently shake and mix to avoid bubbles, and put it in a thermostat at 37℃±1℃ for 60±2 minutes.

[0046] After the insulation time is up, take out the ampoule bottle, 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 recorded as positive and recorded as (+); if gel is not formed or the gel cannot remain intact and slides off the tube wall, it is recorded as negative and recorded as (-). The experimental results are as follows:

[0047]

[0048]

[0049] Example 2 Method Applicability Check Example

[0050] Standard solution Take a bacterial endotoxin working standard (90EU / vial), tap the bottle wall to make the powder fall to the bottom of the bottle, disinfect the bottleneck with an alcohol cotton ball and open the ampoule to prevent glass chips from falling into the bottle when opening. Take the opened bacterial endotoxin working standard, add 900μl of bacterial endotoxin test water, seal the bottle mouth with a sealing film, and mix on a vortex mixer for no less than 15min to obtain a bacterial endotoxin concentration of 100EU / ml standard solution; then gradually dilute with bacterial endotoxin test water to prepare 0.125EU / ml (2λ), 0.0625EU / ml (λ), 0.03125EU / ml (0.5λ), 0.015625EU / ml (0.25λ) bacterial endotoxin reference solution, and each dilution step should be mixed on a vortex mixer for 1min.

[0051] Test solution: Weigh about 25 mg of melphalan hydrochloride bulk drug into a pyrogen-free test tube, add 5 ml of sulfobutyl beta-cyclodextrin sodium aqueous solution (270 mg / ml, refrigerated at 2-8°C) to dissolve, and dilute 8 times with water for bacterial endotoxin test, and then dilute 2 times with magnesium ion buffer (manufacturer: Zhanjiang Andus Biological Co., Ltd.; batch number: 2104200; main components are magnesium sulfate and Tris buffer) to make a solution containing about 0.3125 mg of melphalan hydrochloride per 1 ml.

[0052] Spiked test solution Take 240 μl of the 100 EU / ml standard solution and add the test solution to gradually dilute it to make a solution containing about 0.125 EU / ml of bacterial endotoxin.

[0053] Take a horseshoe crab reagent solution with a sensitivity of 0.0625EU / ml (λ) and add 0.1ml of bacterial endotoxin test water to dissolve it.

[0054] Take 0.1 ml of each of the test solution, solvent (270 mg / ml, sodium sulfobutyl beta-cyclodextrin aqueous solution refrigerated at 2-8°C), spiked test solution, standard solution, and water for bacterial endotoxin test, add them to the horseshoe crab reagent solution, and use them as the test tube (A), solvent control tube (A), and so on. ＇ ), test sample positive control tube (B), positive control tube (C), and negative control tube (D) were placed in a 37°C water bath for reaction for 1.0 h.

[0055] The limit should not exceed 0.2EU / mg

[0056] The results of the method suitability check example are as follows:

[0057] Table 2

[0058]

[0059]

[0060]

[0061] The beneficial effects of the present invention are further illustrated by comparative examples below.

[0062] Comparative Example 1: Using sulfobutyl beta-cyclodextrin sodium aqueous solution as solvent

[0063] In this experiment, 270 mg / ml sodium sulfobutyl beta-cyclodextrin aqueous solution was used as a solvent to dissolve the melphalan hydrochloride raw material to a concentration of 5 mg / ml, and then diluted 16 times with bacterial endotoxin test water, and tested using 0.125 EU / ml sensitive horseshoe crab reagent.

[0064] Standard solution Take a bacterial endotoxin working standard (90EU / vial), tap the bottle wall to make the powder fall to the bottom of the bottle, disinfect the bottleneck with an alcohol cotton ball and open the ampoule, and prevent glass chips from falling into the bottle when opening. Take the opened bacterial endotoxin working standard, add 900μl of bacterial endotoxin test water, seal the bottle mouth with a sealing film, and mix on a vortex mixer for no less than 15 minutes to obtain a standard solution with a bacterial endotoxin concentration of 100EU / ml; then gradually dilute with bacterial endotoxin test water to make a bacterial endotoxin reference solution of 0.25EU / ml (2λ), and each dilution step should be mixed on a vortex mixer for 1 minute.

[0065] Test solution: Weigh about 25 mg of melphalan hydrochloride bulk drug into a pyrogen-free test tube, add 5 ml of sodium sulfobutyl beta-cyclodextrin aqueous solution (270 mg / ml) to dissolve, and dilute 8 times with bacterial endotoxin test water, and then dilute 2 times with bacterial endotoxin test water to make a solution containing about 0.3125 mg of melphalan hydrochloride per 1 ml.

[0066] Spiked test solution: Take about 25 mg of melphalan hydrochloride bulk drug test sample, place it in a pyrogen-free centrifuge tube, add 4.8 ml of sodium sulfobutyl beta-cyclodextrin aqueous solution (270 mg / ml) to dissolve it, add 200 μl of 100 EU / ml bacterial endotoxin standard solution, mix on a vortex mixer for 1 min, and then gradually dilute with bacterial endotoxin test water to make a solution of 0.3125 mg / ml melphalan hydrochloride solution containing 0.25 EU / ml bacterial endotoxin. Each dilution step should be mixed on a vortex mixer for 1 min.

[0067] Take a horseshoe crab reagent solution with a sensitivity of 0.125EU / ml (λ) and add 0.1ml of bacterial endotoxin test water to dissolve it.

[0068] For the determination, 0.1 ml each of the test sample solution, spiked test sample solution, standard solution, and water for bacterial endotoxin test were pipetted and added to the horseshoe crab reagent solution. The tubes were used as the test sample tube, test sample positive control tube, positive control tube, and negative control tube respectively. The tubes were placed in a 37°C water bath for reaction for 1.0 h.

[0069] The experimental results are as follows:

[0070]

[0071]

[0072] The experimental results show that when only sodium sulfobutyl beta-cyclodextrin aqueous solution is used as the solvent, there is interference in the detection of bacterial endotoxins in melphalan hydrochloride raw material.

[0073] Comparative Example 2: Using sulfobutyl beta-cyclodextrin sodium aqueous solution as solvent + adding calcium and magnesium ions

[0074] In this experiment, calcium and magnesium ions were added to prepare the test solution and the spiked test solution based on Comparative Example 1.

[0075] Test solution: Weigh about 25 mg of melphalan hydrochloride bulk drug into a pyrogen-free test tube, add 5 ml of sulfobutyl beta-cyclodextrin sodium aqueous solution (270 mg / ml) to dissolve, and dilute 8 times with bacterial endotoxin test water, and then dilute 2 times with calcium and magnesium ion additives (manufacturer: Xiamen Limulus Reagent Biotechnology Co., Ltd.; batch number: 21028106) to make a solution containing about 0.3125 mg of melphalan hydrochloride per 1 ml.

[0076] Spiked test solution: Take about 25 mg of melphalan hydrochloride bulk drug test sample, place it in a pyrogen-free centrifuge tube, add 4.8 ml of sodium sulfobutyl beta-cyclodextrin aqueous solution (270 mg / ml) to dissolve it, add 200 μl of 100 EU / ml bacterial endotoxin standard solution, mix on a vortex mixer for 1 min, dilute with bacterial endotoxin test water to make a solution of 0.625 mg / ml melphalan hydrochloride solution containing 0.5 EU / ml of bacterial endotoxin, and then dilute with calcium and magnesium ion additives to make a solution of 0.3125 mg / ml melphalan hydrochloride solution containing 0.25 EU / ml of bacterial endotoxin. Each dilution step should be mixed on a vortex mixer for 1 min.

[0077] The remaining operations are referred to Comparative Example 1.

[0078] The experimental results are as follows:

[0079]

[0080] After adding calcium and magnesium ions, the coagulation state of the test sample positive control tube was significantly improved. The test sample positive control tube with a result of "-" slipped when inverted, rather than being almost uncoagulated before adding calcium and magnesium ions. The above experimental results show that adding calcium and magnesium ions on the basis of sodium sulfobutyl beta-cyclodextrin aqueous solution as a solvent can reduce the interference of bacterial endotoxin detection of melphalan hydrochloride raw material, but it cannot completely eliminate the interference.

[0081] Comparative Example 3: Using sulfobutyl beta-cyclodextrin sodium aqueous solution as solvent + adding calcium and magnesium ions

[0082] This experiment adds a solvent positive control tube for comparison based on Comparative Example 2.

[0083] Solution preparation process:

[0084] Standard solution Take a bacterial endotoxin working standard (90EU / vial), tap the bottle wall to make the powder fall to the bottom of the bottle, disinfect the bottleneck with an alcohol cotton ball and open the ampoule, and prevent glass chips from falling into the bottle when opening. Take the opened bacterial endotoxin working standard, add 900μl of bacterial endotoxin test water, seal the bottle mouth with a sealing film, and mix on a vortex mixer for no less than 15 minutes to obtain a standard solution with a bacterial endotoxin concentration of 100EU / ml; then gradually dilute with bacterial endotoxin test water to make a bacterial endotoxin reference solution of 0.25EU / ml (2λ), and each dilution step should be mixed on a vortex mixer for 1 minute.

[0085] Test solution: Weigh about 25 mg of melphalan hydrochloride bulk drug into a pyrogen-free test tube, add 5 ml of sulfobutyl beta-cyclodextrin sodium aqueous solution (270 mg / ml) to dissolve, and dilute 8 times with bacterial endotoxin test water, and then dilute 2 times with calcium and magnesium ion additives (manufacturer: Xiamen Limulus Reagent Biotechnology Co., Ltd.; batch number: 21028106) to make a solution containing about 0.3125 mg of melphalan hydrochloride per 1 ml.

[0086] Spiked test solution: Take about 25 mg of melphalan hydrochloride bulk drug test sample, place it in a pyrogen-free centrifuge tube, add 4.8 ml of sodium sulfobutyl beta-cyclodextrin aqueous solution (270 mg / ml) to dissolve it, add 200 μl of 100 EU / ml bacterial endotoxin standard solution, mix on a vortex mixer for 1 min, dilute with bacterial endotoxin test water to make a solution of 0.625 mg / ml melphalan hydrochloride solution containing 0.5 EU / ml of bacterial endotoxin, and then dilute with calcium and magnesium ion additives to make a solution of 0.3125 mg / ml melphalan hydrochloride solution containing 0.25 EU / ml of bacterial endotoxin. Each dilution step should be mixed on a vortex mixer for 1 min.

[0087] Solvent positive solution: Take 240 μl of 100 EU / ml standard solution, add sulfobutyl beta-cyclodextrin sodium aqueous solution (270 mg / ml) and gradually dilute to make a solution containing about 0.25 EU / ml of bacterial endotoxin.

[0088] Take a horseshoe crab reagent solution with a sensitivity of 0.125EU / ml (λ) and add 0.1ml of bacterial endotoxin test water to dissolve it.

[0089] For the determination, 0.1 ml each of the test sample solution, spiked test sample solution, solvent positive solution, standard solution, and water for bacterial endotoxin test were pipetted and added to the horseshoe crab reagent solution. The tubes were used as the test sample tube, test sample positive control tube, solvent positive control tube, positive control tube, and negative control tube respectively. The tubes were placed in a 37°C water bath for reaction for 1.0 h.

[0090] The experimental results are as follows:

[0091]

[0092] The experimental results show that the solvent positive control tube is positive, indicating that the sodium sulfobutyl beta-cyclodextrin aqueous solution does not interfere with the horseshoe crab reagent reaction. The test sample positive control tube did not appear to be almost unsolidified before the addition of calcium and magnesium ions, but slipped when inverted and showed a negative reaction, indicating that the high concentration of melphalan hydrochloride solution itself will interfere with the bacterial endotoxin detection.

[0093] Comparative Example 4: Using refrigerated sodium sulfobutyl beta-cyclodextrin aqueous solution as solvent + adding calcium and magnesium ions

[0094] In this experiment, the sodium sulfobutyl beta-cyclodextrin aqueous solution was refrigerated at 2-8° C. on the basis of Comparative Example 2. The rest of the operations were all referred to Comparative Example 2.

[0095] The experimental results are as follows:

[0096]

[0097] The experimental results show that the aqueous solution of sodium sulfobutyl beta-cyclodextrin needs to be refrigerated at 2-8°C and calcium and magnesium ions should be added at the same time to eliminate the interference of bacterial endotoxin detection in melphalan hydrochloride raw material.

[0098] Comparative Example 5 Effect of diluent type on test results

[0099] In this experiment, the recovery rate and spike recovery rate were determined by dynamic turbidity method using diluents other than sodium sulfobutyl beta-cyclodextrin aqueous solution.

[0100] Standard solution: Take the bacterial endotoxin standard, tap the bottle wall to make the powder fall to the bottom of the bottle, add 1 ml of bacterial endotoxin test water after opening, seal the bottle mouth with sealing film, mix on a vortex mixer for 15 minutes, and then gradually dilute with bacterial endotoxin test water to 100 times the sensitivity of horseshoe crab reagent, 20 times the sensitivity of horseshoe crab reagent, 10 times the sensitivity of horseshoe crab reagent, and 1 times the sensitivity of horseshoe crab reagent. Each dilution step should be mixed on the vortex mixer for 1 minute.

[0101] Negative control solution: water for bacterial endotoxin test.

[0102] Test solution: Take melphalan hydrochloride bulk drug, place it in a pyrogen-free centrifuge tube, add diluent to dissolve it to the intended concentration, and gradually dilute it with bacterial endotoxin test water to the intended multiple. Each dilution step should be mixed on a vortex mixer for 1 minute.

[0103] Test sample positive control solution: Accurately measure 0.2 ml of test sample solution with 1 / 2 target dilution concentration and 0.2 ml of 2 times positive control standard endotoxin concentration solution, and mix them on a vortex mixer for 1 min.

[0104] Spiked test solution: Take melphalan hydrochloride bulk drug, place it in a pyrogen-free centrifuge tube, add diluent to dissolve, then add quantitative bacterial endotoxin standard solution, and gradually dilute it with bacterial endotoxin test water to the intended multiple. Each dilution step should be mixed on a vortex mixer for 1 minute.

[0105] Spiked test sample positive control solution: Accurately measure 0.2 ml of 1 / 2 target dilution concentration spiked test sample solution and 0.2 ml of 2 times positive control standard endotoxin concentration solution, and mix on a vortex mixer for 1 min.

[0106] Reconstitution of horseshoe crab reagent: Accurately measure 1.7 ml of bacterial endotoxin test water and reconstitute the horseshoe crab reagent.

[0107] Take 100 μl of the test solution, spiked test solution, test positive control solution, spiked test positive control solution, negative control solution, and each concentration standard solution, place them in an ELISA plate, take two portions of each solution, and then add 100 μl of the reconstituted horseshoe crab reagent to each plate. After the addition is completed, immediately put it into the endotoxin ELISA instrument for detection.

[0108] Calculation of recovery rate: (test sample positive control bacterial endotoxin detection concentration - test sample bacterial endotoxin detection concentration) / test sample positive control theoretical added bacterial endotoxin concentration.

[0109] Calculation of spike recovery rate: (bacterial endotoxin detection concentration of spiked test sample - bacterial endotoxin detection concentration of test sample) / theoretical bacterial endotoxin concentration of spiked test sample.

[0110] When using water or Tris buffer as diluent for bacterial endotoxin testing, melphalan hydrochloride API is almost insoluble in the diluent; the experimental results of other diluents that can dissolve the test sample are as follows:

[0111] Table 3

[0112]

[0113] According to the provisions of the General Rules 1143 of Part IV of the Chinese Pharmacopoeia (2020 Edition) on the bacterial endotoxin test method, the endotoxin recovery rate of the interference experiment of the photometric detection method (i.e., the spike recovery rate in this experiment) must be between 50% and 200% to determine that the test solution does not interfere. From the above experimental results, it can be seen that the spike recovery rates of experimental groups 1 and 2 are both less than 50%, and the spike recovery rate of experimental group 3 is less than 75% (because the bacterial endotoxin concentration is too small, the specific value cannot be obtained), indicating that sodium hydroxide aqueous solution, methanol, and DMSO, when used as diluents, will interfere with the detection of bacterial endotoxins in melphalan hydrochloride raw materials.

[0114] Comparative Example 6 Effect of diluent concentration on test results

[0115] In this experiment, the recovery rate was determined by the dynamic turbidity method using different concentrations of sodium sulfobutyl beta-cyclodextrin aqueous solution as a diluent. The rest of the operations were referred to Comparative Example 5.

[0116] The experimental results are as follows:

[0117] Table 4

[0118]

[0119] By comparing experimental group 1 and experimental group 2, it can be seen that the recovery rate measured using 270 mg / ml sulfobutyl beta-cyclodextrin sodium aqueous solution as a diluent is higher than that measured using 67.5 mg / ml sulfobutyl beta-cyclodextrin sodium aqueous solution as a diluent, indicating that using 270 mg / ml sulfobutyl beta-cyclodextrin sodium aqueous solution as a diluent is more ideal and the sample is easier to dissolve during preparation.

[0120] It should be noted that the specific features, structures, materials or characteristics described in this specification may be combined in any one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradicting each other.

Claims

1. A test solution for detecting bacterial endotoxins in melphalan hydrochloride raw material, characterized by: It contains the test substance melphalan hydrochloride, an aqueous solution of sodium sulfobutyl beta-cyclodextrin at 2-8°C, and calcium and / or magnesium ions.

2. The test solution according to claim 1, wherein: The test solution is an aqueous solution.

3. The test solution according to claim 1 or 2, characterized in that: Of which hydrochloric acid The concentration of melphalan is 0.25-0.64 mg / ml; preferably, the concentration of melphalan hydrochloride is 0.25-0.34 mg / ml.

4. The test solution as claimed in claim 1, characterized in that: The concentration of the sodium sulfobutyl beta-cyclodextrin aqueous solution is 200-300 mg / ml, preferably 270 mg / ml; and / or the mass ratio of melphalan hydrochloride to sodium sulfobutyl beta-cyclodextrin is 1:20-1:60, preferably 1:

54.

5. The test solution according to claim 1, wherein: The calcium and / or magnesium ions are added in the form of calcium hydroxide and / or magnesium sulfate; preferably, the magnesium ions are added via a magnesium ion buffer, which is mainly composed of magnesium sulfate and Tris buffer; preferably, the calcium ions are added via a calcium ion buffer, which is mainly composed of calcium hydroxide and Tris buffer.

6. The method for preparing the test solution according to any one of claims 1 to 5, characterized in that: The following steps are involved: Take melphalan hydrochloride raw material, add 2-8°C sulfobutyl beta-cyclodextrin sodium aqueous solution to dissolve, optionally dilute with bacterial endotoxin test water, and add calcium and / or magnesium ions.

7. The preparation method according to claim 6, characterized in that: Add 2-8°C sulfobutyl beta-cyclodextrin sodium aqueous solution to a melphalan hydrochloride concentration of 5-10 mg / ml, preferably 5 mg / ml; dilute 8-16 times, preferably 8 times, with bacterial endotoxin test water; and / or add calcium ion buffer and / or magnesium ion buffer to dilute 2 times.

8. A method for detecting bacterial endotoxins in melphalan hydrochloride raw material, characterized by: The test solution according to any one of claims 1 to 5 is detected by using a horseshoe crab reagent; preferably, the test is detected by a gel method.

9. The detection method according to claim 8, characterized in that: The sensitivity of the horseshoe crab reagent is 0.0625-0.125 EU / ml; preferably, the sensitivity of the horseshoe crab reagent is 0.0625 EU / ml.

10. The detection method as claimed in claim 8, characterized in that: hydrochloric acid The detection limit of bacterial endotoxins in melphalan bulk drug is 0.4EU / mg; preferably, the detection limit of bacterial endotoxins in melphalan hydrochloride bulk drug is 0.2EU / mg.

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