Detection method of gentisic acid bacterial endotoxin

Abstract

The invention discloses a method for detecting gentisic acid bacterial endotoxin, which comprises the following steps: (1) preparing a test solution: taking a gentisic acid sample to be detected, adding BET water, and then adding a sodium hydroxide solution to prepare the test solution; a to-be-detected product mixed solution: adding the gentisic acid bacterial endotoxin standard solution into the obtained to-be-detected product solution to obtain the to-be-detected product mixed solution; (2) interference pre-screening experiment: respectively adding tachypleus amebocyte lysate into the test solution and the mixed solution of the to-be-detected product for reaction to obtain the concentration of the test solution corresponding to a second positive result; and (3) formal interference experiment: taking the concentration of the test solution corresponding to the second positive result obtained by the interference pre-screening experiment as a standard, and carrying out the interference experiment according to the description in the general rule 1143 of the four parts of the Chinese pharmacopoeia 2020 edition. The method provided by the invention can effectively solve the problem that the sample cannot be dissolved in water, and meanwhile, the sample is directly adjusted from an acid solution to neutral and slightly alkaline, so that the interference of the sample on the tachypleus amebocyte lysate can be eliminated.

Description

Technical Field

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

[0002] Gentisic acid, chemical name 2,5-dihydroxybenzoic acid (also known as 5-hydroxysalicylic acid), was originally isolated from the roots of Gentiana plants. It is a polyhydroxycarboxylic acid and one of the metabolites of aspirin. After the ingestion of salicylate, gentisic acid is excreted into the urine. In nature, gentisic acid is found in the roots of Gentiana plants and is also widely found in a variety of vegetables and fruits.

[0003] Gentisic acid is a white needle-shaped crystal, soluble in hot water, ethanol and ether, and insoluble in chloroform, benzene and carbon disulfide. It decomposes into carbon dioxide and hydroquinone when heated for a long time at above 150°C. It is unstable in the air and turns blue when exposed to ferric chloride. Gentisic acid belongs to the hydroquinone class of compounds and is easily oxidized. It is often used as an antioxidant in the pharmaceutical industry. Gentisic acid is also an antipyretic and analgesic, and an important pharmaceutical intermediate. Gentisic acid is also a commonly used matrix in laser desorption ionization mass spectrometry, and its application is very wide.

[0004] At present, the detection of gentisic acid bacterial endotoxins has low accuracy and is easily affected by pH interference due to the poor solubility of the test samples in aqueous solution and the acidity of the solution itself. Summary of the invention

[0005] The primary purpose of the present invention is to provide a method for detecting gentisic acid bacterial endotoxins, which can improve the detection accuracy and simplify the experimental steps by selecting a suitable dissolution reagent.

[0006] To this end, the present invention provides the following technical solutions.

[0007] One aspect of the present invention provides a method for detecting gentisic acid bacterial endotoxin, the method comprising the following steps:

[0008] (1) Test solution: Take a gentisic acid sample to be tested, add BET water, and then add sodium hydroxide solution to prepare a test solution;

[0009] Mixed solution of the test product: add the gentisic acid bacterial endotoxin standard solution to the obtained test product solution to obtain a mixed solution of the test product;

[0010] (2) Interference pre-screening experiment: Add horseshoe crab reagent to the above-mentioned test solution and the mixed solution of the test product to react and obtain the test solution concentration corresponding to the second positive result;

[0011] (3) Formal interference experiment: Using the test sample solution concentration corresponding to the second positive result obtained in the above interference pre-screening experiment as the standard, the interference experiment was carried out in accordance with the description in 1143 of the General Rules of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0012] As a preferred embodiment of the present invention, the concentration of the test solution in step (1) is 50 mg / ml.

[0013] As a preferred embodiment of the present invention, the concentration of the sodium hydroxide solution in step (1) is 1 mol / L, and the sodium hydroxide solution is added in an amount equal to the volume of BET water.

[0014] As a preferred embodiment of the present invention, the concentration of bacterial endotoxin in the mixed solution of the test sample in step (1) is 2λ.

[0015] As a preferred embodiment of the present invention, the concentration of the horseshoe crab reagent λ in step (2) is 0.25, 0.125, 0.06, or 0.03 Eu / ml.

[0016] By means of the above technical solution, the present invention has at least the following advantages:

[0017] 1. The present invention provides a method for detecting gentisic acid bacterial endotoxins, which successfully solves the problem that the sample to be tested cannot be completely dissolved in the aqueous solution by adding sodium hydroxide solution in the preparation of the sample solution to be tested, and at the same time adjusts the pH value of the aqueous solution from acidic to neutral to alkaline. This method effectively solves the problem of the insolubility of the raw material in the aqueous solution.

[0018] 2. The present invention provides a method for detecting gentisic acid bacterial endotoxin. Due to the sodium hydroxide solution, the original aqueous solution is adjusted from acidic to neutral and alkaline (pH is about 7 to 8). This step is crucial to eliminating the interference of the test sample on the horseshoe crab reagent.

[0019] 3. The present invention provides a method for detecting gentisic acid bacterial endotoxin. Since the test sample itself is acidic, the pH value must be adjusted to make the detection solution added to the horseshoe crab reagent neutral or alkaline in order to eliminate the interference of the test sample on the horseshoe crab reagent. However, after the sample is added with sodium hydroxide solution, the color of the solution gradually changes from green to brown-red. The use of the gel method to detect bacterial endotoxin can avoid the drawback of the enzyme labeling method that the color change affects the absorbance, thereby affecting the accuracy of the data, showing that the method has good adaptability to the detection of gentisic acid bacterial endotoxin.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a flow chart of the method for detecting gentisic acid bacterial endotoxin. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0024] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0025] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] 1. If Figure 1 As shown, the detection method of the present invention specifically comprises the following steps:

[0029] Step S1: test solution: take a gentisic acid sample to be tested, add BET water, and then add sodium hydroxide solution to prepare a test solution; the concentration of the obtained test solution is 50 mg / ml; the concentration of the sodium hydroxide solution is 1 mol / L; the sodium hydroxide solution is added in an amount equal to the volume of BET water.

[0030] Mixed solution of the test product: add the gentisic acid bacterial endotoxin standard solution to the obtained test product solution to obtain a mixed solution of the test product; the concentration of bacterial endotoxin in the mixed solution of the test product is 2λ.

[0031] Step S2: Interference pre-screening experiment: add horseshoe crab reagent to the above-mentioned test solution and the mixed solution of the test product to react, and obtain the test solution concentration corresponding to the second positive result; the horseshoe crab reagent λ is 0.25, 0.125, 0.06, and 0.03Eu / ml.

[0032] Step S3: Formal interference experiment: Using the test sample solution concentration corresponding to the second positive result obtained in the above interference pre-screening experiment as the standard, the interference experiment was performed according to the description in 1143 of the General Rules of Part IV of the Chinese Pharmacopoeia 2020 Edition.

[0033] 2. The instruments and test materials designed by the present invention are as follows:

[0034] 2.1 The instruments used are shown in Table 1 below:

[0035] Table 1 Instrument name and model source

[0036]

[0037] 2.2 Reagents and drugs used

[0038] 2.2.1 Test products

[0039] Product Name: Gentisic Acid

[0040] Batch number: 20230401K, 20230501K, 20230502K

[0041] 2.2.2 Detection reagents are shown in Table 2:

[0042] Table 2 Detection reagents and model sources

[0043]

[0044] 2.2.3 Experimental equipment

[0045] Pipette guns, disposable non-pyrogen tips, non-pyrogen test tubes, test tube racks, sealing films, marker pens, etc. are all commercially available products.

[0046] Example 1: Preparation of test solution

[0047] According to current knowledge, the L value of gentisic acid is 0.1Eu / mg, and MVC = λ / L. In addition, the λ of the currently available horseshoe crab reagent is generally 0.25, 0.125, 0.06, and 0.03Eu / ml. Therefore, the corresponding effective dilution concentrations (MVC) of gentisic acid are:

[0048] C 0.5 =(0.5Eu / ml) / (0.1Eu / mg)=5mg / ml;

[0049] C0.25 =(0.25Eu / ml) / (0.1Eu / mg)=2.5mg / ml;

[0050] C 0.125 =(0.125Eu / ml) / (0.1Eu / mg)=1.25mg / ml;

[0051] C 0.06 =(0.0625Eu / ml) / (0.1Eu / mg)=0.625mg / ml;

[0052] C 0.03 =(0.0312Eu / ml) / (0.1Eu / mg)=0.3125mg / ml;

[0053] Weigh 0.1g of the gentisic acid sample to be tested, add 1ml of BET water, then add 1ml of 1mol / L sodium hydroxide solution, shake thoroughly until dissolved, and prepare a 50mg / ml test solution. During the preparation process, it can be observed that the color of the test solution gradually changes from green to brown-red.

[0054] Example 2: Interference pre-screening test

[0055] The test solution prepared in Example 1 was diluted to concentrations of 5, 2.5, 1.25, 0.625, and 0.3125 mg / ml, respectively, and this series of solutions was recorded as NPC. At the same time, bacterial endotoxin working standard solutions were added to the above NPC concentrations, and this series of solutions was recorded as PPC, so that each concentration of PPC contained 2λ concentration of bacterial endotoxin.

[0056] According to Table 2, take two different manufacturers' horseshoe crab reagents with a sensitivity of 0.125EU / ml, tap the bottle wall lightly to make the powder fall to the bottom of the bottle, scratch the bottleneck lightly with a grinding wheel, wipe it with 75% ethanol cotton and open it, and dissolve it with 0.1ml endotoxin test water respectively. Take 0.1ml of the above NPC and PPC respectively and add them to the horseshoe crab reagent. Repeat two tubes for each concentration, and set up a negative control (NC, BET water) and a positive control (PC, bacterial endotoxin working standard solution). All tubes are placed in parallel in a bacterial endotoxin analyzer at 37°C for 60 minutes. The results are shown in Table 3:

[0057] Table 3: Statistics of preliminary test results of gentisic acid interference test (BET water)

[0058]

[0059]

[0060] Note: “+” means positive, i.e. the horseshoe crab reagent coagulates; “-” means negative, i.e. the horseshoe crab reagent does not coagulate.

[0061] According to the results in Table 3, solution NPC and solution NC were both negative, solution PC was positive, and the experiment was valid.

[0062] The above pre-interference test results show that gentisic acid has no obvious interference inhibitory effect on the reaction between horseshoe crab reagent and bacterial endotoxin at 1.25, 0.625, and 0.3125 mg / ml. Based on the fact that the first test sample concentration with a positive result (corresponding to sensitivity λ = 0.125Eu / ml) is generally not selected for interference test when doing interference experiments, the concentration of 0.625 mg / ml (corresponding to sensitivity λ = 0.06Eu / ml) selected for interference experiment in this experiment is used for subsequent interference test.

[0063] Example 3: Formal interference test

[0064] Weigh 0.1 g of the gentisic acid sample to be tested, add 1 ml of BET water, then add 1 ml of 1 mol / L sodium hydroxide solution, shake until dissolved, and dilute to a concentration of 0.625 mg / ml as the test solution.

[0065] According to Table 2, two different manufacturers' horseshoe crab reagents with a sensitivity of 0.06EU / ml were used to test the test sample interference test item in the 2020 edition of the Chinese Pharmacopoeia, General Rules 1143 of Part IV. The specific operation is as follows: prepare bacterial endotoxin working standard solution, dilute to concentrations of 0.125, 0.06, 0.03, and 0.0156Eu / ml, and mix the mixed solution on the mixer for at least 30 seconds at each step. At the same time, add the test sample solution to the above-mentioned bacterial endotoxin working standard solution at each concentration, so that each concentration of the bacterial endotoxin working standard solution contains a test sample at a concentration of 0.625mg / ml.

[0066] The results are shown in Table 4:

[0067] Table 4 Statistics of gentisic acid interference test results

[0068]

[0069] Note: “+” means positive, i.e. the horseshoe crab reagent coagulates; “-” means negative, i.e. the horseshoe crab reagent does not coagulate.

[0070] From the results in Table 4, it can be seen that Es (the interference test sensitivity measurement value) is within the range of 0.5λ~2.0λ (including 0.5λ and 2.0λ), and the test sample Et (the indicated sensitivity measurement value of the horseshoe crab reagent) is within the range of 0.5Es~2.0Es (including 0.5Es and 2.0Es). It is confirmed that gentisic acid has no obvious interference and inhibition effect on the reaction of horseshoe crab reagent and bacterial endotoxin at a concentration of 0.625mg / ml. The limit value is 0.1Eu / mg, and gentisic acid can be detected by bacterial endotoxin test method (gel method).

[0071] The present invention utilizes the addition of sodium hydroxide solution to successfully solve the problem that the sample cannot be completely dissolved in the aqueous solution, and at the same time adjusts the pH value of the aqueous solution from acidic to neutral and alkaline, effectively solving the problem of the insolubility of the test sample in the aqueous solution. At the same time, this step is crucial to eliminate the interference of the test sample on the horseshoe crab reagent. After the sample is added with sodium hydroxide solution, the color of the solution gradually changes from green to brownish red. The use of the gel method to detect bacterial endotoxins can avoid the drawback that the color change affects the absorbance in the enzyme labeling method, thereby affecting the accuracy of the data, showing that the method has good adaptability to the detection of gentisic acid bacterial endotoxins.

[0072] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for detecting gentisic acid bacterial endotoxin, characterized in that: The method comprises the following steps: (1) Test solution: Take a gentisic acid sample to be tested, add BET water, and then add sodium hydroxide solution to prepare a test solution; Mixed solution of the test product: add the gentisic acid bacterial endotoxin standard solution to the obtained test product solution to obtain a mixed solution of the test product; (2) Interference pre-screening experiment: Add horseshoe crab reagent to the above-mentioned test solution and the mixed solution of the test product to react and obtain the test solution concentration corresponding to the second positive result; (3) Formal interference experiment: Using the test sample solution concentration corresponding to the second positive result obtained in the above interference pre-screening experiment as the standard, the interference experiment was carried out in accordance with the description in 1143 of the General Rules of Part IV of the Chinese Pharmacopoeia 2020 Edition.

2. The detection method according to claim 1, characterized in that: The concentration of the test solution in step (1) is 50 mg / ml.

3. The detection method according to claim 1, characterized in that: The concentration of the sodium hydroxide solution in step (1) is 1 mol / L, and the sodium hydroxide solution is added in an amount equal to the volume of BET water.

4. The detection method according to claim 1, characterized in that: The concentration of bacterial endotoxin in the mixed solution of the test sample in step (1) is 2λ.

5. The detection method according to claim 1, characterized in that: The concentration of the horseshoe crab reagent λ in step (2) is 0.25, 0.125, 0.06, and 0.03Eu / ml.

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