Arginine hydrochloride bacterial endotoxin detection method

Abstract

The invention discloses a method for detecting bacterial endotoxin in arginine hydrochloride. The method comprises the following steps: rechecking sensitivity of tachypleus amebocyte lysate; preparing a test solution; gradient dilution; interference test; and detecting by a gel method. According to the arginine hydrochloride bacterial endotoxin detection method, interference is effectively eliminated through pH value adjustment and a gradient dilution method, and an effective and reliable detection method is provided for arginine hydrochloride bacterial endotoxin detection.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial endotoxin detection, in particular to a method for detecting arginine hydrochloride bacterial endotoxin. Background Art

[0002] Endotoxins, lipopolysaccharides (LPS) found in the cell walls of Gram-negative bacteria, are a key indicator of drug safety. Excessive levels of endotoxins in drugs, medical devices, or biological products can cause fever, shock, or even death. Consequently, pharmacopoeias around the world strictly stipulate bacterial endotoxin limits for sterile preparations such as injections and infusions, and mandate testing.

[0003] The existing method for determining whether bacterial endotoxins exceed the standard is mainly the Limulus reagent method (gel method), which uses the procoagulant system in Limulus blood cell lysate to react with endotoxins to form a gel.

[0004] Arginine hydrochloride is a basic amino acid drug, and its properties may interfere with the accuracy of Limulus amebocyte lysate (LAL) assays. Existing technologies for bacterial endotoxin detection using arginine hydrochloride lack targeted interference elimination solutions, resulting in unstable detection sensitivity. Therefore, a method suitable for detecting bacterial endotoxins using arginine hydrochloride is needed. Summary of the Invention

[0005] The present invention provides a method for detecting bacterial endotoxins of arginine hydrochloride.

[0006] In order to solve the above technical problems, the present invention provides a method for detecting bacterial endotoxins of arginine hydrochloride, comprising the following steps: (1) Verification of the sensitivity of the limulus amebocyte lysate: The sensitivity of the limulus amebocyte lysate was verified by reacting the bacterial endotoxin standard solution with the concentration of 2λ, λ, 0.5λ and 0.25λ with the limulus amebocyte lysate. c In the range of 0.5λ~2λ; (2) Prepare the test solution: Weigh the arginine hydrochloride sample, dissolve it in 0.1-0.5 mol / L sodium hydroxide solution, and adjust the pH to 6.5-7.5; add bacterial endotoxin test water to make the volume to 0.1 g / ml; (3) Gradient dilution: The test solution is diluted to multiple concentrations (e.g., 0.02 g / ml, 0.01 g / ml, 0.005 g / ml), and the lowest concentration is not lower than the concentration corresponding to the MVD; (4) Interference test: Take the test solution of multiple concentrations in step (3) and react with equal volume of limulus amebocyte lysate, and set the following: Negative control group: bacterial endotoxin test water + Limulus amebocyte lysate; Positive control group: 2λ endotoxin standard solution + Limulus amebocyte lysate; Test sample positive control group: test sample solution + 2λ endotoxin standard solution + Limulus amebocyte lysate; Determine the non-interfering concentration through interference testing; (5) Gel method detection: After the test solution with the non-interference concentration determined in step (4) is mixed evenly with an equal volume of limulus amebocyte lysate, the tube mouth is sealed and placed in a thermostat for reaction. After taking out, invert 180° to determine the formation of gel.

[0007] In a preferred embodiment of the present invention, the maximum dilution factor calculation formula in step (3) is: , Where c is the initial concentration of the test sample, 0.08-0.12 g / ml; L is the endotoxin limit, 6 EU / g; λ is the sensitivity of the limulus amebocyte lysate, 0.03 EU / ml.

[0008] In a preferred embodiment of the present invention, in step (4), at least two limulus amebocyte lysates from different sources are used to verify the non-interference, and the sensitivity of the limulus amebocyte lysate is 0.03 EU / ml.

[0009] In a preferred embodiment of the present invention, the Et value of the positive control group of the test article is in the range of 0.5λ to 2λ.

[0010] In a preferred embodiment of the present invention, the reaction conditions in steps (1), (4) and (5) are: incubation at 37±1°C for 60±2 minutes in a pyrogen-free test tube.

[0011] In a preferred embodiment of the present invention, the concentration of the sodium hydroxide solution is 0.1 mol / L.

[0012] The beneficial effects of the present invention are as follows: the present invention provides an arginine hydrochloride bacterial endotoxin inspection method, which effectively eliminates interference through pH value adjustment and gradient dilution method, and provides an effective and reliable inspection method for arginine hydrochloride bacterial endotoxin inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The present invention is a schematic diagram of the process of a method for detecting bacterial endotoxins of arginine hydrochloride. DETAILED DESCRIPTION

[0014] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0015] 1. Experimental Materials Limulus amebocyte lysate reagent: Limulus amebocyte lysate reagent batch number 2208291, specification 0.1ml, sensitivity 0.03EU / ml, produced by Zhanjiang Andus Biological Co., Ltd.; Limulus amebocyte lysate reagent batch number 2109273, specification 0.1ml, sensitivity 0.03EU / ml, produced by Xiamen Limulus Amebocyte lysate Biotechnology Co., Ltd. (BIOENDO).

[0016] Bacterial endotoxin working standard: potency 90EU / vial, batch number 150601-202191, China Food and Drug Inspection Institutes.

[0017] Water for bacterial endotoxin test: specification 50mL / bottle, batch number 2205260, produced by Zhanjiang Andus Biological Co., Ltd.

[0018] Drug: Arginine hydrochloride, batch numbers 20230201, 20230202, 20230203, produced by Jiangsu Hanston Pharmaceutical Co., Ltd.

[0019] 2. The method for testing bacterial endotoxins of arginine hydrochloride includes the following steps: (1) Review of the sensitivity of the limulus amebocyte lysate reagent: Take 1 ml of bacterial endotoxin working standard and add 1 ml of endotoxin test water. Mix well in a vortex mixer. Then prepare endotoxin standard solutions with concentrations of 4λ, 2λ, λ, 0.5λ, and 0.25λ, namely 0.12 EU / ml, 0.06 EU / ml, 0.03 EU / ml, 0.015 EU / ml, and 0.0 075 EU / ml.

[0020] Take the above endotoxin standard solution and mix it with an equal volume (e.g., 0.1 ml) of Limulus Amebocyte Lysate (LAL) solution. Prepare four parallel tubes for each endotoxin concentration. In addition, take two tubes and add an equal volume of bacterial endotoxin test water as a negative control. After gently mixing the solution in the test tube, seal the tube and place it vertically in a thermostat at 37°C ± 1°C for 60 minutes ± 2 minutes.

[0021] Gently remove the test tube from the incubator and slowly invert it 180°. If a gel forms within the tube and does not deform or slide off the tube wall, the test is positive. If no gel forms, or if the gel is weak, deforms, and slides off the tube wall, the test is negative. The test is considered valid if all tubes with the maximum concentration of 2λ are positive, all tubes with the minimum concentration of 0.25λ are negative, and the negative control tube is negative. Calculate the geometric mean of the endpoint concentrations as follows; this is the sensitivity of the limulus amebocyte lysate (λc).

[0022] Where X is the logarithm of the endpoint concentration (log). The endpoint concentration is the last concentration in a series of decreasing endotoxin concentrations that gives a positive result. n is the number of parallel tubes for each concentration.

[0023] When the rechecked sensitivity λc of the limulus amebocyte lysate is between 0.5 and 2λ (inclusive), it can be used for bacterial endotoxin testing, and the labeled sensitivity λ is used as the sensitivity of the limulus amebocyte lysate for that batch. The results are shown in Table 2. The sensitivity of all limulus amebocyte lysate used meets the requirements. (2) Prepare the test solution: According to the provisions of General Chapter 1143 Bacterial Endotoxin Test Method in Part IV of the 2020 edition of the Chinese Pharmacopoeia, the bacterial endotoxin limit standard for arginine hydrochloride is 6EU / g.

[0024] In order to ensure that the test results can meet the sensitivity requirements of the pharmacopoeia and truly reflect the endotoxin risk of the drug at the clinical use concentration, the initial concentration of arginine hydrochloride is set to 0.1g / ml.

[0025] The maximum dilution factor of arginine hydrochloride is calculated according to the following formula: MVD=cL / λ=0.1g / ml×6.0EU / g÷0.03EU / ml=20 times.

[0026] Weigh 0.1g of the test sample and put it into an ampoule. Add an appropriate amount of 0.1mol / L sodium hydroxide solution to dissolve it, and adjust the pH of the sample solution to 6.5-7.5. Then add bacterial endotoxin test water (BET) to 5ml to prepare a test sample solution with a concentration of 0.02g / ml. Then use BET water to gradually dilute the sample to 0.01g / ml and 0.005g / ml, among which 0.005g / ml is a 20-fold sample dilution solution.

[0027] The present invention discovered that the degree of amino protonation in arginine hydrochloride at pH 6.5-7.5 matches the isoelectric point of the limulus amebocyte lysate (LAL), eliminating charge adsorption interference. A gradient dilution to 0.005 g / ml can achieve an endotoxin-sample molar ratio greater than 10:1, breaking the matrix inhibition threshold. False negatives can be caused by insufficient amino protonation at pH < 6.5, while limulus amebocyte lysate activity decreases at pH > 7.5.

[0028] (3) Prepare the control solution: Accurately measure 0.5 ml of 0.01 g / ml test sample dilution and 0.5 ml of (4λ) standard solution into a 2 ml ampoule to prepare 1 ml of dilution containing 0.06 EU endotoxin and 0.005 g / ml test sample as the test sample positive control.

[0029] Take an appropriate amount of test water as a negative control.

[0030] (4) Gel method interference pre-test Take 0.02g / ml, 0.01g / ml, and 0.005g / ml test solutions respectively and react with equal volumes of limulus amebocyte lysate. At the same time, set: Negative control group (NC): bacterial endotoxin test water + Limulus amebocyte lysate; Positive control group (PC): 2λ endotoxin standard solution + limulus amebocyte lysate; Test sample positive control group: test sample solution + 2λ endotoxin standard solution + Limulus amebocyte lysate; When the test sample positive control group is positive and the test sample solution group is negative, the concentration is determined to be the non-interference concentration; Two tubes were prepared in parallel for each concentration, and the results are shown in Table 1 below.

[0031] Table 1 Results of the preliminary test on the gel method interference Note: NPC is the sample tube, PPC is the test sample positive control, and each group is done in parallel; "+" represents positive, "-" represents negative, and ≥50% positive is considered positive.

[0032] According to the data in Table 1, NPC and NC are negative, PC is positive, and PPC is positive when the sample concentration is 0.005g / ml. This concentration of sample can be selected for interference test.

[0033] (5) Gel method interference test Interference testing was performed using the test solution (0.005 g / ml) and the corresponding solvents at the positive concentration determined by the above experimental results. Limulus amebocyte lysate (LA) reagents with a sensitivity of 0.03 EU / ml from two manufacturers (Zhanjiang Andus and BIOENDO) were used. Four replicates were performed for the test solution group, and two replicates were performed for the other groups. The results are shown in Table 2.

[0034] Table 2 Gel method interference test results Note: 4 parallel assays were performed for the test solution group and 2 parallel assays were performed for the other groups.

[0035] Take the Andus Limulus Amebocyte Lysate (Batch No. 2208291) for testing the 20230201 batch of samples as an example: End point concentration: 0.015 EU / ml (final positive concentration) → lgX = lg0.015 ≈ -1.824 Et=10^(ΣlgX / n)=10^{-1.824}=0.015 EU / ml (within 0.5λ~2λ).

[0036] According to the results in Table 2, the sensitivity of the limulus amebocyte lysate reagents with batch numbers 2208291 and 2109273 is 0.03EU / ml, and the λc values ​​of the endotoxin standard series solutions obtained using test water as the solvent are between (0.5 and 2.0)λ, which meets the requirements of the limulus amebocyte lysate sensitivity verification test.

[0037] According to the results in Table 2, the Et values ​​of the three test sample interference test series solutions were all between (0.5 and 2.0)λ, indicating that arginine hydrochloride had no interference effect on bacterial endotoxin detection when the concentration was below 0.005g / ml.

[0038] (6) Gel test of test sample: Mix 0.005 g / ml test sample solution with an equal volume of 0.03 EU / ml Limulus amebocyte lysate (Zhanjiang Andus Biological Co., Ltd., batch number: 2208291) in a pyrogen-free test tube according to the requirements of Table 3 below. Seal the tube mouth and place it in a 37±1℃ thermostat for 60±2 minutes. After taking it out, invert it 180° to determine the formation of gel. The test results are shown in Table 4 below.

[0039] Table 3 Gel limit test Table 4 Inspection results The test results in Table 4 show that the arginine hydrochloride bacterial endotoxin test method of the present invention has high detection accuracy, a detection coefficient of variation (CV) of ≤4.2%, and a recovery rate of higher than 98.5%, and can be used as a test method for arginine hydrochloride bacterial endotoxin.

[0040] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention's description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for testing arginine hydrochloride bacterial endotoxin, characterized in that: The steps include: (1) Verification of the sensitivity of the limulus amebocyte lysate: The sensitivity of the limulus amebocyte lysate was verified by reacting the bacterial endotoxin standard solution with the concentration of 2λ, λ, 0.5λ and 0.25λ with the limulus amebocyte lysate. c In the range of 0.5λ~2λ; (2) Prepare the test solution: Weigh the arginine hydrochloride sample, dissolve it in 0.1-0.5 mol / L sodium hydroxide solution, and adjust the pH to 6.5-7.5; add bacterial endotoxin test water to make the volume to 0.1 g / ml; (3) Gradient dilution: The test solution is diluted to multiple concentrations, and the lowest concentration is not lower than the concentration corresponding to the MVD; (4) Interference test: Take the test solution of multiple concentrations in step (3) and react with equal volume of limulus amebocyte lysate, and set the following: Negative control group: bacterial endotoxin test water + Limulus amebocyte lysate; Positive control group: 2λ endotoxin standard solution + Limulus amebocyte lysate; Test sample positive control group: test sample solution + 2λ endotoxin standard solution + Limulus amebocyte lysate; Determine the non-interfering concentration through interference testing; (5) Gel method detection: After the test solution with the non-interference concentration determined in step (4) is mixed evenly with an equal volume of limulus amebocyte lysate, the tube mouth is sealed and placed in a thermostat for reaction. After taking out, invert 180° to determine the formation of gel.

2. The method according to claim 1, characterized in that The formula for calculating the maximum dilution factor in step (3) is: ， Where c is the initial concentration of the test sample, 0.08-0.12 g / ml; L is the endotoxin limit, 6 EU / g; λ is the sensitivity of the limulus amebocyte lysate, 0.03 EU / ml.

3. The method according to claim 1, characterized in that In step (4), at least two horseshoe crab reagents from different sources are used to verify the non-interference, and the sensitivity of the horseshoe crab reagents is 0.03 EU / ml.

4. The method according to claim 3, characterized in that The Et value of the positive control group of the test article is in the range of 0.5λ to 2λ.

5. The method according to claim 1, wherein The reaction conditions in steps (1), (4) and (5) are all: incubation at 37±1°C for 60±2 minutes in a pyrogen-free test tube.

6. The method according to claim 1, characterized in that The concentration of the sodium hydroxide solution is 0.1 mol / L.

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