Detection method of sulpiride bacterial endotoxin

Abstract

The invention belongs to the technical field of bacterial endotoxin detection, and particularly discloses a sulpiride bacterial endotoxin detection method which comprises the following steps: sequentially preparing a series of standard substance solutions, a test substance solution, a test substance positive control solution and an interference test solution; adding the series of standard substance solutions into tachypleus amebocyte lysate, detecting by adopting a dynamic nephelometry, and drawing a standard curve; respectively adding the test sample solution, the test sample positive control solution and the interference test solution into a limulus reagent, detecting by adopting a dynamic nephelometry, and calculating the bacterial endotoxin content in the sulpiride sample according to a standard curve linear regression equation. Compared with the prior art, the sulpiride sample is dissolved by using thionyl chloride, and then the sulpiride sample is further diluted by using the bacterial endotoxin inspection water to prepare the test solution, so that the problems that sulpiride is difficult to dissolve in the bacterial endotoxin inspection water and cannot effectively release bacterial endotoxin can be solved.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial endotoxin detection, and particularly relates to a method for detecting sulpiride bacterial endotoxin. Background Art

[0002] Bacterial endotoxin is the main contaminant in injection drugs. It is a unique structure on the cell wall of Gram-negative bacteria, and its main chemical substance is lipopolysaccharide. Bacterial endotoxin is an exogenous pyrogen, which can activate neutrophils, etc., causing them to release an endogenous pyrogen, which acts on the body temperature regulation center to cause fever, leukocyte reaction, endotoxin shock and diffuse intravascular coagulation, etc. Therefore, it is very necessary to detect and control bacterial endotoxin in injections and injection APIs.

[0003] At present, the methods for detecting bacterial endotoxins are: gel method and photometry. Among them, photometry can quantitatively detect the content of bacterial endotoxins in drugs and has gradually been widely used.

[0004] Sulpiride is a benzamide antipsychotic drug that has an emotional activation effect and a strong central antiemetic effect. Sulpiride has a good effect on apathy, withdrawal, stupor, depression, hallucinations and delusions. It is suitable for the loneliness, withdrawal and apathy of simple schizophrenia, paranoid and catatonic schizophrenia and chronic schizophrenia; it has a certain effect on depression symptoms; it can also be used to treat vomiting, alcoholic psychosis, intellectual disability with personality disorder, gastric and duodenal ulcers, etc.

[0005] Sulpiride can be injected, and the bacterial endotoxin content in its raw materials must be strictly controlled. In the bacterial endotoxin test of raw materials, the raw materials are usually first dissolved and diluted with bacterial endotoxin test water to prepare a test solution. Since Sulpiride powder is insoluble in bacterial endotoxin test water, endotoxins cannot be effectively released, which can easily cause deviations in test results. Therefore, it is necessary to research and develop an accurate and appropriate detection method to determine the bacterial endotoxin content of Sulpiride. Summary of the invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for detecting bacterial endotoxins in sulpiride, which can effectively solve the problem that sulpiride is difficult to dissolve in water for bacterial endotoxin testing and cannot effectively release bacterial endotoxins.

[0007] The present invention is achieved through the following technical solutions: A method for detecting sulpiride bacterial endotoxins comprises the following steps: S1. Dissolve the bacterial endotoxin standard in bacterial endotoxin test water (BET water), and dilute it in a gradient to obtain a series of standard solutions; S2. Dissolve the sulpiride sample in an organic solvent, add water for bacterial endotoxin test to dilute and fix the volume to obtain a test sample stock solution; further dilute the test sample stock solution with water for bacterial endotoxin test to obtain a test sample solution; S3, dissolving the sulpiride sample in the organic solvent, adding a bacterial endotoxin standard solution, and then diluting with bacterial endotoxin test water to obtain a test sample positive control solution; S4, dissolving the organic solvent in bacterial endotoxin test water, and diluting to obtain an interference test solution; S5, adding the series of standard solutions to the horseshoe crab reagent, detecting by kinetic turbidity method, and drawing a standard curve; then adding the test solution, the test positive control solution, and the interference test solution to the horseshoe crab reagent, respectively, detecting by kinetic turbidity method, and calculating the bacterial endotoxin content in the sulpiride sample according to the linear regression equation of the standard curve; Wherein, the organic solvent is dichlorothionyl (DMSO).

[0008] A further improvement of the present invention is: In S1, the bacterial endotoxin standard is dissolved in bacterial endotoxin test water, and a series of standard solutions with concentrations of 1 EU / mL, 0.1 EU / mL, and 0.01 EU / mL are prepared by gradient dilution.

[0009] Furthermore, in S2, 50 mg of sulpiride sample was accurately weighed and dissolved in 2 mL of dichlorothionyl, and water for bacterial endotoxin test was added to dilute the volume to prepare a test solution with a sulpiride concentration of 1 mg / mL; the test solution was further diluted 10 times with water for bacterial endotoxin test to prepare a test solution with a sulpiride concentration of 0.1 mg / mL.

[0010] Furthermore, in S3, 50 mg of sulpiride sample was accurately weighed and dissolved in 2 mL of dichlorothionyl, and 2 vials of bacterial endotoxin standard solution with a concentration of 10 EU / mL were added and mixed, and then diluted 50 times with water for bacterial endotoxin test to prepare a test sample positive control solution with a bacterial endotoxin concentration of 0.1 EU / mL.

[0011] Furthermore, in S4, thionyl chloride is dissolved in bacterial endotoxin test water to prepare a solution with a concentration of 4%; and then further diluted with bacterial endotoxin test water to prepare an interference test solution with a concentration of 0.4%-2%.

[0012] Furthermore, in S5, the sensitivity of the horseshoe crab reagent is 10-0.01EU / mL. Beneficial Effects

[0013] Compared with the prior art, the present invention has the following obvious advantages: 1. The present invention uses thionyl chloride to dissolve sulpiride samples, thereby solving the problem that sulpiride is difficult to dissolve in water for bacterial endotoxin testing and cannot effectively release bacterial endotoxins; and then uses water for bacterial endotoxin testing to further dilute the sample to prepare a test solution, thereby reducing the interference of thionyl chloride on bacterial endotoxins and avoiding its influence on the bacterial endotoxin detection results.

[0014] Second, the present invention adopts the dynamic turbidity method in the photometric method to quantitatively detect bacterial endotoxins in sulpiride. Compared with the gel method, the photometric method has the advantages of higher sensitivity, better repeatability, large-scale quantification, and data traceability. It can avoid subjective factors affecting the judgment of the results, and is more suitable for monitoring the quality of test products during the production process, and better meet the quality control requirements of bacterial endotoxins.

[0015] 3. The method provided by the present invention is simple and easy to operate, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the standard curve diagram of the standard curve reliability test in Example 1. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with embodiments.

[0018] Example 1: Reliability test of standard curve (1) Preparation of bacterial endotoxin standard solution: Take a bacterial endotoxin standard with a specification of 10EU / tube, add 1mL BET water to dissolve it, and obtain a bacterial endotoxin standard solution with a concentration of 10EU / mL. Seal the bottle mouth with sealing film, place it on a vortex mixer to mix for 15 minutes, and then dilute it with BET water in each test tube to make three concentrations of 1EU / mL, 0.1EU / mL, and 0.01EU / mL. Place the vortex mixer to mix for 30 seconds during each dilution step. Prepare 3 parallel tubes for each concentration.

[0019] (2) Preparation of negative control solution (NC): Take the water used for bacterial endotoxin test and prepare 2 parallel tubes as negative control solution.

[0020] (3) Acceptable criteria: The absolute value of the correlation coefficient of the standard curve is greater than or equal to 0.980; the coefficient of variation (CV) of the parallel tubes is less than 10%; the endotoxin reaction time of the negative control tube is greater than the reaction time of the lowest concentration of the standard curve, indicating that the standard curve is reliable.

[0021] (4) Experimental results Standard curve see Figure 1 The bacterial endotoxin standard curve and reliability analysis results are shown in Table 1 below.

[0022] Table 1 Bacterial endotoxin standard curve and reliability analysis

[0023] Conclusion: According to the bacterial endotoxin test result report, the regression equation of the standard curve is logT=2.8259-0.2745LgC (where T: test sample reaction time, C: test sample endotoxin content), the correlation coefficient |r|= 0.9981>0.980, the coefficient of variation (CV) of each parallel tube is <10%, and the reaction time of the negative control (NC) is greater than the prescribed detection time. Therefore, the standard curve is established and the reliability test of the standard curve is passed.

[0024] Example 2: Determination of the maximum effective dilution factor (MVD) of the test sample According to the General Chapter 1143 Bacterial Endotoxin Test Method of the 2020 Edition of the "Chinese Pharmacopoeia", the maximum multiple MVD = cL / λ.

[0025] Wherein, L is the bacterial endotoxin limit of the test sample. In the present invention, the bacterial endotoxin limit of sulpiride is specified to be 0.44EU / mg; c is the concentration of the test solution, and the initial concentration is 1 mg / mL; λ is the lowest endotoxin concentration on the standard curve used in the photometric assay, which is 0.01 EU / mL.

[0026] According to the above conditions, the maximum dilution factor MVD was calculated to be 1 mg / mL × 0.44 EU / mg / 0.01 EU / mL = 44 times.

[0027] Example 3: Test sample and solvent interference test Solutions A, B, C, and D were prepared according to Table 2 below.

[0028] Table 2 Preparation of dynamic turbidity method interference pre-test solution

[0029] (1) Prepare the test solution (Solution A) Weigh 50 mg of sulpiride sample, add 2 mL of DMSO and mix on a vortex mixer for 30 seconds to dissolve, then add 48 mL of bacterial endotoxin test water, mix on a vortex mixer for another 30 seconds to obtain the test sample stock solution with a concentration of 1 mg / mL.

[0030] Use BET water to dilute the test sample stock solution to prepare 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL concentrations as 2-fold test sample solutions. Then dilute with BET water to 0.1 mg / mL, 0.05 mg / mL, and 0.025 mg / mL (i.e., 10-fold, 20-fold, and 40-fold dilutions, respectively) as test sample solutions, and prepare 2 tubes in parallel.

[0031] (2) Preparation of positive solution of test sample (solution B) First add 2 mL DMSO to 50 mg of sulpiride to dissolve it, then add 2 vials of 10 EU / mL bacterial endotoxin standard solution and mix well, then dilute 50 times with BET water to obtain a test sample positive solution with a bacterial endotoxin concentration of 0.1 EU / mL.

[0032] (3) Preparation of bacterial endotoxin standard solution (Solution C) Take a bacterial endotoxin standard with a specification of 10EU / tube, add 1mL BET water to dissolve it, and obtain a 10EU / mL bacterial endotoxin standard solution. Seal the bottle mouth with a sealing film, place it on a vortex mixer and mix for 15 minutes, then dilute it with BET water in each test tube to make 1EU / mL, 0.1EU / mL, 0.01EU / mL, 3 concentrations, and place it on a vortex mixer for 30 seconds during each dilution step.

[0033] (4) Preparation of negative control: DMSO aqueous solution (Solution D) 48 mL of BET water was added to 2 mL of DMSO to prepare a 4% DMSO aqueous solution, and then further diluted with BET water to obtain DMSO aqueous solutions with concentrations of 0.4%, 0.5%, 0.8%, 1%, and 2%, respectively.

[0034] (5) Implementation and results of interference pre-test Take a 10-0.01EU / mL horseshoe crab reagent, add 1.25mL BET water to dissolve it, then add it to the test tubes for photometric detection, add 0.1mL to each tube, and then add 0.1mL of the above-mentioned test solution, the test positive solution and the standard curve solution of each concentration, and set a negative control at the same time, and place it in a bacterial endotoxin detector for detection at a temperature of 37℃±1℃. The test results are shown in Table 3 below.

[0035] Table 3 Dynamic turbidity interference test results

[0036] Conclusion: When the concentration of the test solution was 0.1 mg / mL, 0.05 mg / mL, and 0.025 mg / mL (i.e., diluted 10 times, 20 times, and 40 times, respectively, and the concentration of the DMSO aqueous solution was between 0.4% and 2%), the bacterial endotoxin recoveries were 101%, 119%, and 123%, respectively, all within the range of 50%-200%. The bacterial endotoxins were detected by the kinetic turbidity method, and there was no interference between the sample and the DMSO aqueous solution.

[0037] In summary, a sulpiride concentration of 0.1 mg / mL (diluted 10 times, DMSO concentration of 0.4%) was selected as the test concentration of the test solution in the sample detection test of Example 4.

[0038] Example 4: Bacterial endotoxin detection test of sulpiride samples The horseshoe crab reagent produced by Zhanjiang Andus Biological Co., Ltd. was used, batch number 2206290.

[0039] Three batches of sulpiride samples (20221203, 20221204, and 20221105) were taken and test solutions with a sulpiride concentration of 0.1 mg / mL were prepared respectively. The tests were carried out according to the methods and steps of Example 3 above. The results are shown in Table 4 below.

[0040] Table 4 Quantitative detection results of samples by dynamic turbidity method

[0041] Conclusion: Three batches of sulpiride samples were tested by this method, and the recovery rates of added bacterial endotoxins in the test solutions were all in the range of 50%-200%, and the test results were in compliance with the regulations.

[0042] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting sulpiride bacterial endotoxin, characterized in that: The steps include: S1. Dissolve the bacterial endotoxin standard in bacterial endotoxin test water, and dilute it in a gradient to obtain a series of standard solutions; S2. Dissolve the sulpiride sample in an organic solvent, add water for bacterial endotoxin test to dilute and fix the volume to obtain a test sample stock solution; further dilute the test sample stock solution with water for bacterial endotoxin test to obtain a test sample solution; S3, dissolving the sulpiride sample in the organic solvent, adding a bacterial endotoxin standard solution, and then diluting with bacterial endotoxin test water to obtain a test sample positive control solution; S4, dissolving the organic solvent in water for bacterial endotoxin testing, and diluting to obtain an interference test solution; S5, adding the series of standard solutions to the horseshoe crab reagent, detecting by kinetic turbidity method, and drawing a standard curve; Then, the test solution, the test positive control solution, and the interference test solution were added to the horseshoe crab reagent, respectively, and the kinetic turbidity method was used for detection, and the bacterial endotoxin content in the sulpiride sample was calculated according to the linear regression equation of the standard curve; Wherein, the organic solvent is thionyl chloride.

2. The method for detecting bacterial endotoxin of sulpiride according to claim 1, characterized in that: In S1, the bacterial endotoxin standard is dissolved in bacterial endotoxin test water, and a series of standard solutions with concentrations of 1 EU / mL, 0.1 EU / mL, and 0.01 EU / mL are prepared by gradient dilution.

3. The method for detecting sulpiride bacterial endotoxin according to claim 1, characterized in that: In S2, 50 mg of sulpiride sample was accurately weighed and dissolved in 2 mL of dichlorothionyl, and water for bacterial endotoxin test was added to dilute the volume to prepare a test solution with a sulpiride concentration of 1 mg / mL; the test solution was further diluted 10 times with water for bacterial endotoxin test to prepare a test solution with a sulpiride concentration of 0.1 mg / mL.

4. The method for detecting sulpiride bacterial endotoxin according to claim 1, characterized in that: In S3, 50 mg of sulpiride sample was accurately weighed and dissolved in 2 mL of dichlorothionyl, and two tubes of bacterial endotoxin standard solution with a concentration of 10 EU / mL were added and mixed, and then diluted 50 times with water for bacterial endotoxin test to prepare a test sample positive control solution with a bacterial endotoxin concentration of 0.1 EU / mL.

5. The method for detecting sulpiride bacterial endotoxin according to claim 1, characterized in that: In S4, thionyl chloride is dissolved in bacterial endotoxin test water to prepare a solution with a concentration of 4%; and then further diluted with bacterial endotoxin test water to prepare an interference test solution with a concentration of 0.4%-2%.

6. The method for detecting sulpiride bacterial endotoxin according to claim 1, characterized in that: In S5, the sensitivity of the horseshoe crab reagent is 10-0.01EU / mL.

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