CN115420565A - Method for detecting bacterial endotoxin in triamcinolone acetonide injection - Google Patents

Abstract

Translated fromChinese

本发明涉及一种检测曲安奈德注射液中细菌内毒素的方法，包括步骤(1)：采用有机溶剂和检查用水对曲安奈德注射液进行溶解，所述有机溶剂选自甲醇、乙醇、正丙醇、异丙醇和二甲基亚砜中的任一种；步骤(2)：将溶解后的曲安奈德注射液作为供试品原液，用检查用水对该供试品原液进行稀释，确定所述溶解后的曲安奈德注射液在进行细菌内毒素检测时所采用的有机溶剂浓度；步骤(3)：采用确定的有机溶剂浓度的有机溶剂水溶液对所述曲安奈德注射液进行溶解，并对溶解后的曲安奈德注射液中细菌内毒素进行检测。通过本发明，能够将曲安奈德注射液完全溶解，能够将内毒素完全释放出来，避免了内毒素被包裹而无法被检出，从而提高了检测的准确度。The invention relates to a method for detecting bacterial endotoxin in triamcinolone acetonide injection, comprising step (1): dissolving triamcinolone acetonide injection with organic solvent and inspection water, wherein the organic solvent is selected from methanol, ethanol, normal Any one of propanol, isopropanol and dimethyl sulfoxide; step (2): use the dissolved triamcinolone acetonide injection as the stock solution of the test sample, dilute the stock solution of the test sample with inspection water, and determine The organic solvent concentration used for the dissolved triamcinolone acetonide injection when carrying out bacterial endotoxin detection; step (3): the triamcinolone acetonide injection is dissolved by using an aqueous organic solvent solution with a determined organic solvent concentration, And the bacterial endotoxin in the dissolved triamcinolone acetonide injection was detected. Through the present invention, the triamcinolone acetonide injection can be completely dissolved, the endotoxin can be completely released, and the endotoxin is prevented from being wrapped and cannot be detected, thereby improving the accuracy of detection.

Description

Translated fromChinese

检测曲安奈德注射液中细菌内毒素的方法Method for detecting bacterial endotoxin in triamcinolone acetonide injection

技术领域technical field

本发明属于内毒素检测技术领域，具体而言，本发明涉及一种检测曲安奈德注射液中细菌内毒素的方法。The invention belongs to the technical field of endotoxin detection, in particular, the invention relates to a method for detecting bacterial endotoxin in triamcinolone acetonide injection.

背景技术Background technique

细菌内毒素是注射液中常见的热原性杂质，若超过一定限度，会引发用药者体温升高，出现发热等症状。作为注射液中的关键质量属性，需要进行严格控制。鲎试剂法，特别是凝胶法检测细菌内毒素，自问世后一直为各国药典收载的细菌内毒素检查的主流方法。Bacterial endotoxin is a common pyrogenic impurity in injections. If it exceeds a certain limit, it will cause the user's body temperature to rise and symptoms such as fever to appear. As a critical quality attribute in injections, it needs to be strictly controlled. The limulus reagent method, especially the gel method for detecting bacterial endotoxin, has been the mainstream method for bacterial endotoxin detection recorded in pharmacopoeias of various countries since its inception.

《中国药典》(2020版，四部)《9251细菌内毒素检查法应用指导原则》中指出，一般应使用内毒素检查用水溶解样品进行细菌内毒素检查。在水中溶解度低的样品可以采取超声波、加热助溶、添加助溶剂、调节pH等方法提高其溶解度。采用包合技术的新型制剂如微球、脂质体等供试品，应采取适宜方法将包合体破坏，使包裹在内部的细菌内毒素完全释放，再进行检测。"Chinese Pharmacopoeia" (2020 Edition, Volume 4) "9251 Guidelines for the Application of Bacterial Endotoxin Test Method" states that in general, endotoxin test water should be used to dissolve samples for bacterial endotoxin test. For samples with low solubility in water, methods such as ultrasonication, heating to aid dissolution, addition of auxiliary solvents, and pH adjustment can be used to improve their solubility. For new preparations using inclusion technology, such as microspheres, liposomes and other test products, suitable methods should be adopted to destroy the inclusion body, so that the bacterial endotoxin encapsulated inside can be completely released before testing.

其中，内毒素检查用水必须对试验不得有干扰。也就是说，内毒素检查用水是应符合灭菌注射用水标准，其内毒素含量小于0.015EU/ml(用于凝胶法)。试验所用的器皿需经处理，以去除可能存在的外源性内毒素。耐热器皿常用干热灭菌法(250℃、至少30分钟)去除，也可采用其他确证不干扰细菌内毒素检查的适宜方法。若使用塑料器具，如微孔板和与微量加样器配套的吸头等，应选用标明无内毒素并且对试验无干扰的器具。Among them, the water for endotoxin inspection must not interfere with the test. That is to say, the water for endotoxin inspection should meet the standard of sterile water for injection, and its endotoxin content is less than 0.015EU/ml (for gel method). The utensils used in the test need to be treated to remove possible exogenous endotoxins. Heat-resistant utensils are usually removed by dry heat sterilization (250°C, at least 30 minutes), and other appropriate methods can also be used to confirm that they do not interfere with the bacterial endotoxin test. If plastic utensils are used, such as microwell plates and matching tips for micro-samplers, etc., utensils marked as endotoxin-free and non-interfering to the test should be selected.

针对曲安奈德注射液而言，在进行细菌内毒素检查时，由于主药曲安奈德不溶于水，另外曲安奈德注射液中还含有混悬剂、表面活性剂、防腐剂、渗透压调节剂等辅料，各国药典及相关文献都没有提供合适的溶剂使曲安奈德注射液完全溶解，而是直接使用检查用水稀释注射液后进行细菌内毒素的检查，但是，由于主药曲安奈德与辅料(特别是作为混悬剂的羧甲基纤维素钠)难以同时溶解，因此，上述方法无法准确检测难溶的曲安奈德中包裹的内毒素，从而给患者带来了较大的风险。For triamcinolone acetonide injection, when performing bacterial endotoxin inspection, because the main drug triamcinolone acetonide is insoluble in water, in addition, triamcinolone acetonide injection also contains suspending agent, surfactant, preservative, osmotic pressure regulator However, the pharmacopoeias and related documents of various countries do not provide suitable solvents to completely dissolve the triamcinolone acetonide injection, but directly use the inspection water to dilute the injection solution and carry out bacterial endotoxin inspection. However, due to the main drug triamcinolone acetonide and Excipients (especially sodium carboxymethylcellulose as a suspension) are difficult to dissolve at the same time. Therefore, the above method cannot accurately detect the endotoxin encapsulated in the insoluble triamcinolone acetonide, thereby bringing greater risks to patients.

发明内容Contents of the invention

本发明正是鉴于上述现有技术中存在的问题而提出的，目的在于提供一种用于检测曲安奈德注射液中细菌内毒素的方法，并提供一种曲安奈德注射液的溶解用试剂盒及其应用。The present invention is proposed in view of the above-mentioned problems in the prior art, and the purpose is to provide a method for detecting bacterial endotoxin in triamcinolone acetonide injection, and provide a reagent for dissolving triamcinolone acetonide injection boxes and their applications.

一方面，本发明提供一种检测曲安奈德注射液中细菌内毒素的方法，包括如下步骤：In one aspect, the present invention provides a method for detecting bacterial endotoxin in triamcinolone acetonide injection, comprising the steps of:

步骤(1)：采用有机溶剂和检查用水对曲安奈德注射液进行溶解，所述有机溶剂选自甲醇、乙醇、正丙醇、异丙醇和二甲基亚砜中的任一种；以及Step (1): dissolving the triamcinolone acetonide injection with an organic solvent and inspection water, the organic solvent being selected from any one of methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide; and

步骤(2)：将溶解后的曲安奈德注射液作为供试品原液，用检查用水对该供试品原液进行稀释，确定溶解后的曲安奈德注射液在进行细菌内毒素检测时所采用的有机溶剂的浓度；Step (2): Use the dissolved triamcinolone acetonide injection as the stock solution of the test sample, dilute the stock solution of the test sample with test water, and determine the solution used for the bacterial endotoxin detection of the dissolved triamcinolone acetonide injection. The concentration of the organic solvent;

步骤(3)：采用确定的有机溶剂的浓度的有机溶剂水溶液对曲安奈德注射液进行溶解，对溶解后的曲安奈德注射液中细菌内毒素进行检测。Step (3): dissolving the triamcinolone acetonide injection with an organic solvent aqueous solution with a determined concentration of the organic solvent, and detecting the bacterial endotoxin in the dissolved triamcinolone acetonide injection.

优选地，在步骤(1)中，所述曲安奈德注射液中，每1ml所述曲安奈德注射液包括40mg的曲安奈德以及0.63％的羧甲基纤维素钠；Preferably, in step (1), in the triamcinolone acetonide injection, every 1 ml of the triamcinolone acetonide injection includes 40 mg of triamcinolone acetonide and 0.63% sodium carboxymethylcellulose;

所述有机溶剂为甲醇时，每1mg曲安奈德对应的甲醇的加入量为0.196ml～0.204ml；When the organic solvent is methanol, the amount of methanol added per 1 mg of triamcinolone acetonide is 0.196ml to 0.204ml;

所述有机溶剂为乙醇时，每1mg曲安奈德对应的乙醇的加入量为0.135ml～0.140ml；When the organic solvent is ethanol, the amount of ethanol added per 1 mg of triamcinolone acetonide is 0.135ml to 0.140ml;

所述有机溶剂为正丙醇时，每1mg曲安奈德对应的正丙醇的加入量为0.135ml～0.140ml；When the organic solvent is n-propanol, the amount of n-propanol added per 1 mg of triamcinolone acetonide is 0.135ml to 0.140ml;

所述有机溶剂为异丙醇时，每1mg曲安奈德对应的异丙醇的加入量为0.147ml～0.153ml；When the organic solvent is isopropanol, the amount of isopropanol added per 1 mg of triamcinolone acetonide is 0.147ml to 0.153ml;

所述有机溶剂为二甲基亚砜时，每1mg曲安奈德对应的二甲基亚砜的加入量为0.172ml～0.178ml。When the organic solvent is dimethyl sulfoxide, the amount of dimethyl sulfoxide added per 1 mg of triamcinolone acetonide is 0.172ml-0.178ml.

优选地，在步骤(1)中，所述采用有机溶剂和检查用水对曲安奈德注射液进行溶解，包括：Preferably, in step (1), the triamcinolone acetonide injection is dissolved with organic solvent and inspection water, including:

采用有机溶剂和检查用水的混合溶剂对所述曲安奈德注射液进行溶解；The triamcinolone acetonide injection is dissolved by using a mixed solvent of an organic solvent and inspection water;

或者，or,

采用交替滴加的方式对所述曲安奈德注射液进行溶解；其中，在所述交替滴加过程中，先滴加部分检查用水再滴加有机溶剂。The triamcinolone acetonide injection is dissolved by alternately dropping; wherein, in the alternately dropping process, part of the water for inspection is firstly added dropwise, and then the organic solvent is added dropwise.

另一方面，本发明提供一种曲安奈德注射液的溶解用试剂盒，包括：In another aspect, the present invention provides a kit for dissolving triamcinolone acetonide injection, comprising:

曲安奈德注射液；以及triamcinolone acetonide injection; and

溶解用试剂，所述溶解用试剂包括有机溶剂和检查用水，所述有机溶剂选自甲醇、乙醇、正丙醇、异丙醇和二甲基亚砜中的任一种。A reagent for dissolving, the reagent for dissolving includes an organic solvent and water for inspection, and the organic solvent is selected from any one of methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide.

对于上述溶解用试剂盒，优选地，所述曲安奈德注射液中，每1ml所述曲安奈德注射液包括40mg的曲安奈德以及0.63％的羧甲基纤维素钠，For the above dissolving kit, preferably, in the triamcinolone acetonide injection, every 1 ml of the triamcinolone acetonide injection includes 40 mg of triamcinolone acetonide and 0.63% sodium carboxymethylcellulose,

所述有机溶剂为甲醇时，每1mg曲安奈德对应的甲醇的加入量为0.196ml～0.204ml；When the organic solvent is methanol, the amount of methanol added per 1 mg of triamcinolone acetonide is 0.196ml to 0.204ml;

所述有机溶剂为乙醇时，每1mg曲安奈德对应的乙醇的加入量为0.135ml～0.140ml；When the organic solvent is ethanol, the amount of ethanol added per 1 mg of triamcinolone acetonide is 0.135ml to 0.140ml;

所述有机溶剂为正丙醇时，每1mg曲安奈德对应的正丙醇的加入量为0.135ml～0.140ml；When the organic solvent is n-propanol, the amount of n-propanol added per 1 mg of triamcinolone acetonide is 0.135ml to 0.140ml;

所述有机溶剂为异丙醇时，每1mg曲安奈德对应的异丙醇的加入量为0.147ml～0.153ml；When the organic solvent is isopropanol, the amount of isopropanol added per 1 mg of triamcinolone acetonide is 0.147ml to 0.153ml;

所述有机溶剂为二甲基亚砜时，每1mg曲安奈德对应的二甲基亚砜的加入量为0.172ml～0.178ml。When the organic solvent is dimethyl sulfoxide, the amount of dimethyl sulfoxide added per 1 mg of triamcinolone acetonide is 0.172ml-0.178ml.

另一方面，本发明提供上述的溶解用试剂盒在检测曲安奈德注射液中的细菌内毒素中的应用。In another aspect, the present invention provides the application of the above dissolving kit in detecting bacterial endotoxin in triamcinolone acetonide injection.

另一方面，本发明还提供一种用于检测曲安奈德注射液中细菌内毒素的试剂盒，包括：如上述所述的溶解用试剂盒；以及鲎试剂。On the other hand, the present invention also provides a kit for detecting bacterial endotoxin in triamcinolone acetonide injection, comprising: the above-mentioned dissolving kit; and Limulus reagent.

发明效果Invention effect

根据本发明，通过采用有机溶剂和检查用水对曲安奈德注射液进行溶解，能够使曲安奈德注射液的混悬溶液中的主药曲安奈德与辅料(特别是作为混悬剂的羧甲基纤维素钠)完全溶解，能够得到曲安奈德注射液的澄清溶液。在相关技术中，对曲安奈德注射液的混悬溶液进行细菌内毒素检测时，混悬溶液无法完全溶解导致未溶解的曲安奈德主成分中包裹有内毒素，从而无法被完全检出，与之相比，本发明能够将曲安奈德注射液完全溶解，从而能够将曲安奈德中的内毒素完全释放出来，进而能够避免内毒素被包裹而无法被检出，从而提高了检测的准确度。According to the present invention, by adopting organic solvent and examination water to dissolve triamcinolone acetonide injection, can make the main drug triamcinolone acetonide in the suspension solution of triamcinolone acetonide injection and adjuvant (particularly carboxymethyl carboxymethyl as suspending agent) Sodium cellulose) is completely dissolved, and a clear solution of triamcinolone acetonide injection can be obtained. In related technologies, when the suspension solution of triamcinolone acetonide injection is tested for bacterial endotoxin, the suspension solution cannot be completely dissolved, resulting in the undissolved main component of triamcinolone acetonide being coated with endotoxin, which cannot be completely detected. In contrast, the present invention can completely dissolve the triamcinolone acetonide injection, so that the endotoxin in the triamcinolone acetonide can be completely released, thereby preventing the endotoxin from being wrapped and unable to be detected, thereby improving the accuracy of detection Spend.

具体实施方式detailed description

下面，对本发明的具体实施方式进行详细的说明，但本发明不限于此。Hereinafter, specific embodiments of the present invention will be described in detail, but the present invention is not limited thereto.

在针对采用鲎试剂凝胶法检查曲安奈德注射液细菌内毒素时药液不能完全溶解的难题进行研究的过程中，发明人先后试用了超声波助溶、加热助溶、调节pH值助溶等方法。其中，主成分为曲安奈德的曲安奈德注射液有多个规格(1ml:10mg、1ml:40mg、1ml:80mg)，1ml中含有主成分曲安奈德至少为10mg。In the course of research on the problem that the drug solution cannot be completely dissolved when the bacterial endotoxin of triamcinolone acetonide injection is tested by the limulus reagent gel method, the inventor has successively tried ultrasonic aiding, heating aiding, adjusting pH value, etc. method. Among them, triamcinolone acetonide injection, whose main component is triamcinolone acetonide, has multiple specifications (1ml: 10mg, 1ml: 40mg, 1ml: 80mg), and 1ml contains at least 10mg of triamcinolone acetonide as the main component.

在以上多个规格中，在大量加入细菌内毒素检查用水后采用上述方法均不溶解。In the above multiple specifications, after adding a large amount of water for bacterial endotoxin inspection, the above methods are not dissolved.

在添加助溶剂时，发明人尝试了常用的溶剂，包括甲醇、乙醇、正丙醇、异丙醇、乙二醇、丙三醇、二甘醇、二甲基亚砜、二甲基甲酰胺、丙酮、三氯甲烷等。结果发现：直接加入上述有机溶剂时，曲安奈德注射液仍呈混悬或乳浊状态，无法完全溶解。发明人又通过添加表面活性剂溶液，如聚山梨酯80、十二烷基硫酸钠溶液等，仍无法解决问题。When adding co-solvents, the inventors tried common solvents, including methanol, ethanol, n-propanol, isopropanol, ethylene glycol, glycerol, diethylene glycol, dimethyl sulfoxide, dimethylformamide , acetone, chloroform, etc. It was found that: when the above-mentioned organic solvent was directly added, the triamcinolone acetonide injection was still in a suspended or emulsified state and could not be completely dissolved. The inventor has added a surfactant solution, such as polysorbate 80, sodium lauryl sulfate solution, etc., but still cannot solve the problem.

经过大量试验研究，发明人发现在曲安奈德注射液中，加入含甲醇的水溶液；或者加入含乙醇的水溶液；或者加入含正丙醇的水溶液；或者加入含异丙醇的水溶液；或者加入含二甲基亚砜的水溶液，均能使曲安奈德注射液完全溶解至澄清状态，必要时可采用超声波或振摇助溶。基于以上溶解至澄清的供试品原液，后续通过内毒素检查用水将上述供试品原液稀释至所需浓度后，可采用凝胶法检测其中的细菌内毒素，从而实现对曲安奈德注射液中细菌内毒素进行精确检测。After a large number of experimental studies, the inventors found that in the triamcinolone acetonide injection, add an aqueous solution containing methanol; or add an aqueous solution containing ethanol; or add an aqueous solution containing n-propanol; or add an aqueous solution containing isopropanol; The aqueous solution of dimethyl sulfoxide can completely dissolve the triamcinolone acetonide injection to a clear state. If necessary, ultrasonic waves or shaking can be used to assist the dissolution. Based on the above dissolved to clear stock solution of the test product, after diluting the above stock solution of the test product to the required concentration with water for endotoxin testing, the bacterial endotoxin in it can be detected by the gel method, so as to realize the detection of triamcinolone acetonide injection. Accurate detection of bacterial endotoxins.

基于上述研究，本发明提供一种检测曲安奈德注射液中细菌内毒素的方法，包括如下步骤：Based on the above research, the present invention provides a method for detecting bacterial endotoxin in triamcinolone acetonide injection, comprising the steps of:

步骤(1)：采用有机溶剂和检查用水对曲安奈德注射液进行溶解，有机溶剂选自甲醇、乙醇、正丙醇、异丙醇和二甲基亚砜中的任一种；以及Step (1): dissolving triamcinolone acetonide injection with organic solvent and test water, the organic solvent is selected from any one of methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide; and

步骤(2)：将溶解后的曲安奈德注射液作为供试品原液，用检查用水对供试品原液进行稀释，确定溶解后的曲安奈德注射液在进行细菌内毒素检测时所采用的有机溶剂的浓度；Step (2): Use the dissolved triamcinolone acetonide injection as the stock solution of the test sample, dilute the stock solution of the test sample with test water, and determine the concentration of the triamcinolone acetonide injection after the dissolution that is used for bacterial endotoxin detection. The concentration of organic solvents;

步骤(3)：采用确定的有机溶剂的浓度的有机溶剂水溶液对曲安奈德注射液进行溶解，并对溶解后的曲安奈德注射液中细菌内毒素进行检测。Step (3): dissolving the triamcinolone acetonide injection with an organic solvent aqueous solution with a determined concentration of the organic solvent, and detecting the bacterial endotoxin in the dissolved triamcinolone acetonide injection.

需要说明的是，上述供试品原液是指在内毒素检测的预干扰试验中，可以被稀释成一系列供试品稀释液进行试验的溶液。It should be noted that the above-mentioned stock solution of the test substance refers to a solution that can be diluted into a series of dilutions of the test substance for testing in the pre-interference test of endotoxin detection.

在本发明的实施例中，供试品原液是指采用有机溶剂和检查用水将曲安奈德注射液溶解至澄清，且未采用检查用水进行稀释时的溶液。In the embodiments of the present invention, the stock solution of the test product refers to the solution in which the triamcinolone acetonide injection is dissolved until clear with an organic solvent and test water, and is not diluted with test water.

供试品原液和供试品稀释液统称为供试品溶液。The stock solution of the test product and the dilution of the test product are collectively referred to as the test solution.

在此，不难理解，在对溶解后的曲安奈德注射液中细菌内毒素进行检测之前，还可以包括：采用检查用水对供试品原液进行稀释，并通过预干扰试验确定用于细菌内毒素检测的有机溶剂的最大浓度，以在该最大浓度以下的有机溶剂水溶液的溶解作用下进行上述细菌内毒素检测。Here, it is not difficult to understand that before the detection of bacterial endotoxin in the dissolved triamcinolone acetonide injection, it may also include: dilute the stock solution of the test product with inspection water, and determine the bacterial endotoxin used for the bacterial endotoxin through the pre-interference test. The maximum concentration of the organic solvent for toxin detection is to perform the above-mentioned bacterial endotoxin detection under the dissolution of the organic solvent aqueous solution below the maximum concentration.

例如，若通过预干扰试验确定用于细菌内毒素检测的有机溶剂的最大浓度为m，则可以采用有机溶剂的浓度小于m，且能够对曲安奈德注射液溶解至澄清的任意有机溶剂和检查用水的组合，对曲安奈德注射液进行溶解并进行细菌内毒素检测。For example, if the maximum concentration of the organic solvent used for bacterial endotoxin detection is determined to be m through the pre-interference test, any organic solvent with a concentration of the organic solvent less than m and capable of dissolving triamcinolone acetonide injection to clarity can be used and tested A combination of water was used to dissolve triamcinolone acetonide injection and perform bacterial endotoxin detection.

再例如，可以采用凝胶法对溶解后的曲安奈德注射液中细菌内毒素进行检测。For another example, the bacterial endotoxin in the dissolved triamcinolone acetonide injection can be detected by the gel method.

这里的有机溶剂的最大浓度与鲎试剂的灵敏度、内毒素限值等因素有关。The maximum concentration of the organic solvent here is related to factors such as the sensitivity of the LAL reagent and the limit value of endotoxin.

在确定了供试品溶液的内毒素限值，选定了一定灵敏度的鲎试剂，就可以进行预干扰试验了，预干扰试验是对一系列样品(如供试品原液)的稀释液进行试验，在每一个稀释浓度，都有一组添加内毒素的样品和一组不添加内毒素的样品，添加内毒素的浓度保持不变，而样品浓度会逐级稀释，但最终样品的稀释倍数不超过最大有效稀释倍数(MVD)。After determining the endotoxin limit of the test solution and selecting a certain sensitivity of the limulus reagent, the pre-interference test can be carried out. The pre-interference test is to test a series of dilutions of samples (such as the original solution of the test) , at each dilution concentration, there is a group of samples with added endotoxin and a group of samples without added endotoxin, the concentration of added endotoxin remains unchanged, and the sample concentration will be diluted step by step, but the dilution factor of the final sample does not exceed Maximum Effective Dilution (MVD).

最大有效稀释倍数(MVD)是指：在所选择的鲎试剂灵敏度条件下，供试品溶液被允许达到稀释的最大倍数。在检测时，需在不超过此稀释倍数的浓度下进行内毒素限值检测，而在此最大有效稀释倍数下，仍然能够对内毒素限值进行准确检测。The maximum effective dilution (MVD) refers to: under the selected LAL reagent sensitivity conditions, the test solution is allowed to reach the maximum dilution. When testing, it is necessary to perform endotoxin limit detection at a concentration that does not exceed this dilution factor, and under this maximum effective dilution factor, the endotoxin limit value can still be accurately detected.

其中，添加内毒素的浓度可以根据内毒素的限值进行确定。对于凝胶法的判断结果，不添加内毒素的样品不应该凝集，如果发生凝集说明含有内毒素或内毒素样物质。添加有内毒素的样品应该出现凝集，若没有发生凝集表明样品凝集反应被抑制。Wherein, the concentration of added endotoxin can be determined according to the limit value of endotoxin. For the judgment result of the gel method, the sample without adding endotoxin should not agglutinate, and if agglutination occurs, it means that it contains endotoxin or endotoxin-like substances. The sample added with endotoxin should agglutinate, if agglutination does not occur, it indicates that the agglutination reaction of the sample is inhibited.

在一些实施方式中，采用上述预干扰试验获得鲎试剂灵敏度后，可以对不同稀释倍数的有机溶剂的水溶液进行干扰试验，即可得出不干扰试验结果的有机溶剂的水溶液中有机溶剂的最大浓度，以此浓度为参考确定供试品溶液中有机溶剂的浓度，以对供试品溶液中细菌内毒素进行检测。其中，上述供试品溶液中有机溶剂的浓度应该小于上述不干扰试验结果的有机溶剂的最大浓度，而具有该有机溶剂的浓度的供试品溶液可以用于细菌内毒素检测，且不会对细菌内毒素检测产生干扰。In some embodiments, after the above-mentioned pre-interference test is used to obtain the sensitivity of the Limulus reagent, the interference test can be performed on aqueous solutions of organic solvents with different dilution factors, and the maximum concentration of the organic solvent in the aqueous solution of the organic solvent that does not interfere with the test results can be obtained. , take this concentration as a reference to determine the concentration of the organic solvent in the test solution to detect the bacterial endotoxin in the test solution. Wherein, the concentration of the organic solvent in the above-mentioned need testing solution should be less than the maximum concentration of the above-mentioned organic solvent that does not interfere with the test results, and the need testing solution with the concentration of the organic solvent can be used for bacterial endotoxin detection, and will not affect Bacterial endotoxin detection interferes.

其中，上述内毒素的限值由公式“L＝K/M”计算得到，其中，L为供试品(也即供试品溶液)的细菌内毒素限值，以EU/mg或EU/u表示，K为按规定的给药途径，人用每千克体重每小时最大可接受的内毒素剂量，单位为EU/kg.u，M为人用每公斤体重每小时最大剂量。内毒素限值的确定一般与临床人体最大给药剂量有关，剂量越大，单位重量或体积的内毒素限值越低。Wherein, the limit value of the above-mentioned endotoxin is calculated by the formula "L=K/M", wherein, L is the bacterial endotoxin limit value of the test product (that is, the test product solution), expressed in EU/mg or EU/u Indicates that K is the maximum acceptable endotoxin dose per kilogram of body weight per hour for humans according to the prescribed route of administration, and the unit is EU/kg.u, and M is the maximum dose per kilogram of body weight for humans per hour. The determination of the endotoxin limit is generally related to the maximum clinical human dose, the greater the dose, the lower the endotoxin limit per unit weight or volume.

在一些实施方式中，曲安奈德注射液中，每1ml曲安奈德注射液包括40mg的曲安奈德以及0.63％的羧甲基纤维素钠。在有机溶剂为甲醇时，每1mg曲安奈德对应的甲醇的加入量为0.196ml～0.204ml。在有机溶剂为乙醇时，每1mg曲安奈德对应的乙醇的加入量为0.135ml～0.140ml。在有机溶剂为正丙醇时，每1mg曲安奈德对应的正丙醇的加入量为0.135ml～0.140ml。在有机溶剂为异丙醇时，每1mg曲安奈德对应的异丙醇的加入量为0.147ml～0.153ml。在有机溶剂为二甲基亚砜时，每1mg曲安奈德对应的二甲基亚砜的加入量为0.172ml～0.178ml。In some embodiments, in the triamcinolone acetonide injection, every 1 ml of the triamcinolone acetonide injection includes 40 mg of triamcinolone acetonide and 0.63% sodium carboxymethylcellulose. When the organic solvent is methanol, the amount of methanol added per 1 mg of triamcinolone acetonide is 0.196ml-0.204ml. When the organic solvent is ethanol, the amount of ethanol added per 1 mg of triamcinolone acetonide is 0.135ml-0.140ml. When the organic solvent is n-propanol, the amount of n-propanol added per 1 mg of triamcinolone acetonide is 0.135ml-0.140ml. When the organic solvent is isopropanol, the amount of isopropanol added per 1 mg of triamcinolone acetonide is 0.147ml-0.153ml. When the organic solvent is dimethyl sulfoxide, the amount of dimethyl sulfoxide added per 1 mg of triamcinolone acetonide is 0.172ml-0.178ml.

在这些实施例中，通过实验发现，在上述规格的曲安奈德注射液中，每1mg的曲安奈德和对应的有机溶剂的加入量之间存在上述对应关系，可以将曲安奈德注射液溶解至澄清。In these embodiments, it is found through experiments that in the triamcinolone acetonide injection of the above specifications, the above-mentioned corresponding relationship exists between the addition amount of every 1 mg of triamcinolone acetonide and the corresponding organic solvent, and the triamcinolone acetonide injection can be dissolved to clarify.

进而更优选，在有机溶剂为甲醇的情况下，每1ml曲安奈德注射液加入8ml甲醇。在有机溶剂为乙醇的情况下，每1ml曲安奈德注射液加入5.5ml乙醇。在有机溶剂为正丙醇的情况下，每1ml曲安奈德注射液加入5.5ml正丙醇。在有机溶剂为异丙醇的情况下，每1ml曲安奈德注射液加入6ml异丙醇。在有机溶剂为二甲基亚砜的情况下，每1ml曲安奈德注射液加入7ml二甲基亚砜。Even more preferably, when the organic solvent is methanol, 8 ml of methanol is added to every 1 ml of triamcinolone acetonide injection. When the organic solvent is ethanol, add 5.5ml of ethanol to every 1ml of triamcinolone acetonide injection. In the case that the organic solvent is n-propanol, add 5.5ml of n-propanol per 1ml of triamcinolone acetonide injection. In the case that the organic solvent is isopropanol, add 6ml of isopropanol to every 1ml of triamcinolone acetonide injection. In the case that the organic solvent is dimethyl sulfoxide, add 7 ml of dimethyl sulfoxide to every 1 ml of triamcinolone acetonide injection.

在本发明一实施方式中，在步骤(1)中，采用有机溶剂和检查用水对曲安奈德注射液进行溶解，包括：采用有机溶剂和检查用水的混合溶剂对上述曲安奈德注射液进行溶解；或者，采用交替滴加的方式对上述曲安奈德注射液进行溶解，在交替滴加过程中，先滴加部分检查用水再滴加有机溶剂。In one embodiment of the present invention, in step (1), the triamcinolone acetonide injection is dissolved with an organic solvent and water for inspection, including: dissolving the above triamcinolone acetonide injection with a mixed solvent of an organic solvent and water for inspection ; Or, the above-mentioned triamcinolone acetonide injection is dissolved by means of alternate dripping, and in the process of alternate dripping, part of the inspection water is first added dropwise and then the organic solvent is added dropwise.

在采用交替滴加的方式时，通过先滴加部分检查用水后滴加有机溶剂，能够将曲安奈德注射液溶解至澄清。When using the method of alternating drops, the triamcinolone acetonide injection can be dissolved until clear by adding part of the test water first and then the organic solvent.

其中，以精确量取1ml曲安奈德注射液为例进行实验，先滴加的部分检查用水的体积可以为1ml。Wherein, taking the accurate measurement of 1ml of triamcinolone acetonide injection as an example to carry out the experiment, the volume of the part of the test water added dropwise may be 1ml.

相反地，发明人通过实验发现，在交替滴加过程中，先滴加有机溶剂后滴加检查用水，有时难以将曲安奈德注射液溶解至澄清。On the contrary, the inventors have found through experiments that in the process of alternate dropping, it is sometimes difficult to dissolve triamcinolone acetonide injection until it becomes clear when the organic solvent is added dropwise first and then the test water is added dropwise.

此外，本发明还提供一种曲安奈德注射液的溶解用试剂盒，包括：曲安奈德注射液以及溶解用试剂。上述曲安奈德注射液包括作为主药的曲安奈德，还可以包括：混悬剂(羧甲基纤维素钠)、表面活性剂、防腐剂、渗透压调节剂等辅料。上述溶解用试剂包括有机溶剂和检查用水，有机溶剂选自甲醇、乙醇、正丙醇、异丙醇和二甲基亚砜中的任一种。检查用水，也即内毒素检查用水，其内毒素含量极低，不会对后续内毒素检测产生影响。当然，在一些实施例中，检查用水也可以用内毒素含量符合要求的纯化水来代替，如纯化水中内毒素含量小于0.015EU/ml。In addition, the present invention also provides a kit for dissolving triamcinolone acetonide injection, comprising: triamcinolone acetonide injection and a dissolving reagent. The above-mentioned triamcinolone acetonide injection includes triamcinolone acetonide as the main drug, and may also include: suspending agent (sodium carboxymethylcellulose), surfactant, preservative, osmotic pressure regulator and other auxiliary materials. The reagent for dissolving includes an organic solvent and inspection water, and the organic solvent is selected from any one of methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide. The water for inspection, that is, the water for endotoxin inspection, has extremely low endotoxin content and will not affect the subsequent endotoxin detection. Of course, in some embodiments, the inspection water can also be replaced by purified water with endotoxin content meeting the requirements, for example, the endotoxin content in purified water is less than 0.015 EU/ml.

在本发明提供的溶解用试剂盒中，通过采用有机溶剂和检查用水对曲安奈德注射液进行溶解，能够将曲安奈德注射液溶解至澄清，与相关技术(即，对曲安奈德注射液的混悬溶液进行细菌内毒素检测时，混悬溶液不能被完全溶解，导致未溶解的曲安奈德主成分和/或辅料中包裹内毒素，从而导致内毒素无法被完全检出)相比，本发明能够将曲安奈德主成分和辅料完全溶解，从而能够将曲安奈德主成分和辅料中的内毒素完全释放出来，进而能够避免内毒素被包裹而不被检出，从而能够提高检测的准确度，能够减小给患者带来的风险。In the dissolving kit provided by the present invention, triamcinolone acetonide injection can be dissolved to clarification by adopting organic solvent and inspection water to dissolve triamcinolone acetonide injection. When the suspension solution is used for bacterial endotoxin detection, the suspension solution cannot be completely dissolved, resulting in the undissolved triamcinolone acetonide main component and/or encapsulating endotoxin in the auxiliary materials, thereby causing the endotoxin to be unable to be completely detected) compared to, The present invention can completely dissolve the main component of triamcinolone acetonide and the auxiliary materials, so that the endotoxin in the main component of triamcinolone acetonide and the auxiliary materials can be completely released, thereby preventing the endotoxin from being wrapped and not detected, thereby improving the detection efficiency Accuracy can reduce the risk to patients.

进一步地，在上述曲安奈德注射液中，每1ml曲安奈德注射液包括40mg的曲安奈德以及0.63％的羧甲基纤维素钠的情况下，在有机溶剂为甲醇时，每1mg曲安奈德对应的甲醇的加入量为0.196ml～0.204ml。在有机溶剂为乙醇时，每1mg曲安奈德对应的乙醇的加入量为0.135ml～0.140ml。在有机溶剂为正丙醇时，每1mg曲安奈德对应的正丙醇的加入量为0.135ml～0.140ml。在有机溶剂为异丙醇时，每1mg曲安奈德对应的异丙醇的加入量为0.147ml～0.153ml。在有机溶剂为二甲基亚砜时，每1mg曲安奈德对应的二甲基亚砜的加入量为0.172ml～0.178ml。Further, in the above triamcinolone acetonide injection, every 1ml of triamcinolone acetonide injection includes 40mg of triamcinolone acetonide and 0.63% sodium carboxymethylcellulose, when the organic solvent is methanol, every 1mg of triamcinolone acetonide The amount of methanol corresponding to Germany is 0.196ml to 0.204ml. When the organic solvent is ethanol, the amount of ethanol added per 1 mg of triamcinolone acetonide is 0.135ml-0.140ml. When the organic solvent is n-propanol, the amount of n-propanol added per 1 mg of triamcinolone acetonide is 0.135ml-0.140ml. When the organic solvent is isopropanol, the amount of isopropanol added per 1 mg of triamcinolone acetonide is 0.147ml-0.153ml. When the organic solvent is dimethyl sulfoxide, the amount of dimethyl sulfoxide added per 1 mg of triamcinolone acetonide is 0.172ml-0.178ml.

通过实验发现，在上述对应的有机溶剂的加入量的情况下，均能够将相应量的曲安奈德注射液溶解至澄清，从而能够为后续准确检测曲安奈德注射液中细菌内毒素做准备。It has been found through experiments that, in the case of the addition of the above-mentioned corresponding organic solvents, the corresponding amount of triamcinolone acetonide injection can be dissolved until clear, so as to prepare for the subsequent accurate detection of bacterial endotoxin in triamcinolone acetonide injection.

进而更优选，在有机溶剂为甲醇的情况下，每1ml曲安奈德注射液加入8ml甲醇。在有机溶剂为乙醇的情况下，每1ml曲安奈德注射液加入5.5ml乙醇。在有机溶剂为正丙醇的情况下，每1ml曲安奈德注射液加入5.5ml正丙醇。在有机溶剂为异丙醇的情况下，每1ml曲安奈德注射液加入6ml异丙醇。在有机溶剂为二甲基亚砜的情况下，每1ml曲安奈德注射液加入7ml二甲基亚砜。Even more preferably, when the organic solvent is methanol, 8 ml of methanol is added to every 1 ml of triamcinolone acetonide injection. When the organic solvent is ethanol, add 5.5ml of ethanol to every 1ml of triamcinolone acetonide injection. In the case that the organic solvent is n-propanol, add 5.5ml of n-propanol per 1ml of triamcinolone acetonide injection. In the case that the organic solvent is isopropanol, add 6ml of isopropanol to every 1ml of triamcinolone acetonide injection. In the case that the organic solvent is dimethyl sulfoxide, add 7 ml of dimethyl sulfoxide to every 1 ml of triamcinolone acetonide injection.

另一方面，本发明还提供一种用于检测曲安奈德注射液中细菌内毒素的试剂盒，包括：如上述所述的溶解用试剂盒；以及鲎试剂。On the other hand, the present invention also provides a kit for detecting bacterial endotoxin in triamcinolone acetonide injection, comprising: the above-mentioned dissolving kit; and Limulus reagent.

鲎试剂是由海洋节肢动物鲎的血液变形细胞溶解物制成的无菌冷冻干燥品，含有能被微量细菌内毒素和真菌葡聚糖激活的凝固酶原、凝固蛋白原，经低温冷冻干燥而成，能够准确、快速地定性或定量检测样品中是否含有细菌内毒素和(1,3)-β-葡聚糖。Limulus reagent is a sterile freeze-dried product made from the blood amebocyte lysate of the marine arthropod Limulus limulus. It can accurately and quickly detect whether the sample contains bacterial endotoxin and (1,3)-β-glucan qualitatively or quantitatively.

凝胶法是通过鲎试剂与内毒素产生凝集反应的原理来检测或半定量内毒素的方法。凝胶法是通过观察有无凝胶形成作为反应的终点。此法操作比较简单，经济，不需要专用测定设备，可以进行定性或半定量测定。The gel method is a method for detecting or semi-quantitating endotoxin through the principle of agglutination reaction between Limulus reagent and endotoxin. The gel method is to observe whether the gel is formed as the end point of the reaction. This method is relatively simple to operate, economical, does not require special measuring equipment, and can be used for qualitative or semi-quantitative determination.

当然，在实际应用中，为了确定鲎试剂的灵敏度，以便于对曲安奈德注射液中细菌内毒素进行准确检测，可选的，该试剂盒还可以包括内毒素标准溶液等。以便于做干扰实验确定鲎试剂的灵敏度和供试品原液的最大有效稀释倍数。Of course, in practical applications, in order to determine the sensitivity of the LAL reagent so as to accurately detect the bacterial endotoxin in the triamcinolone acetonide injection, optionally, the kit may also include an endotoxin standard solution and the like. In order to facilitate the interference experiment to determine the sensitivity of the LAL reagent and the maximum effective dilution factor of the stock solution of the test substance.

或者，在实际应用中，在上述鲎试剂确定的情况下，该鲎试剂的灵敏度也可以是确定的。如此，在检测时，可以直接按照鲎试剂的灵敏度对曲安奈德注射液中细菌内毒素进行检测。Alternatively, in practical applications, when the above-mentioned LAL reagent is determined, the sensitivity of the LAL reagent can also be determined. In this way, during the detection, the bacterial endotoxin in the triamcinolone acetonide injection can be detected directly according to the sensitivity of the LAL reagent.

再一方面，本发明提供一种如上所述的试剂盒在检测曲安奈德注射液中细菌内毒素中的应用。In another aspect, the present invention provides an application of the above-mentioned kit in detecting bacterial endotoxin in triamcinolone acetonide injection.

以下将通过具体实施例对本发明进行更详细地说明。需要说明的是，这些实施例只用于对本发明进行举例，本发明并不限制于此。The present invention will be described in more detail through specific examples below. It should be noted that these examples are only used to illustrate the present invention, and the present invention is not limited thereto.

在以下的实施例中，预干扰试验中供试品溶液的配制方法，可按照《中国药典》(1143细菌内毒素检查法)或者其它各国药典中规定的方法进行正式干扰试验。在此不做具体限定。In the following examples, the preparation method of the test solution in the pre-interference test can be carried out in a formal interference test according to the methods stipulated in the "Chinese Pharmacopoeia" (1143 Bacterial Endotoxin Test Method) or other national pharmacopoeias. No specific limitation is made here.

在以下的实施例中，以《中国药典》(2020年版)规定的方法进行供试品溶液的配制为例进行说明。其中，下述的曲安奈德注射液中细菌内毒素的限度为不超过3.0EU/mg。供试品溶液中曲安奈德的浓度采用0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml进行预干扰试验。In the following examples, the preparation of the test solution by the method stipulated in the "Chinese Pharmacopoeia" (2020 edition) is taken as an example for illustration. Among them, the limit of bacterial endotoxin in the following triamcinolone acetonide injection is not more than 3.0 EU/mg. The concentration of triamcinolone acetonide in the test solution was 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml for the pre-interference test.

需要说明的是，在以下的实施例中，上述所列举的甲醇、乙醇、正丙醇、异丙醇和二甲基亚砜等有机溶剂，可以为分析纯、色谱纯、超级纯等规格。在此不做具体限定。当然，上述有机溶剂也可以为上述规格经干燥的无水试剂。It should be noted that, in the following examples, the organic solvents such as methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide listed above can be analytically pure, chromatographically pure, super pure and other specifications. No specific limitation is made here. Certainly, the above-mentioned organic solvent may also be a dried anhydrous reagent of the above-mentioned specification.

实施例1Example 1

曲安奈德注射液(规格：1ml，其中，40mg曲安奈德和0.63％的羧甲基纤维素钠；批号：ABU8750)在不同溶剂中的溶解试验：Dissolution test of triamcinolone acetonide injection (specification: 1ml, wherein, 40mg triamcinolone acetonide and 0.63% sodium carboxymethylcellulose; batch number: ABU8750) in different solvents:

(1)甲醇(1) Methanol

用移液枪精密量取曲安奈德注射液1ml，置于量瓶中，加入1ml纯化水，涡旋振荡5秒，滴加8ml甲醇后，再逐滴加入纯化水至刻度线，溶液澄清透明。此时溶液体积为10ml(包含1ml注射液+8ml甲醇+1ml水，即，每1mg曲安奈德对应的甲醇的加入量为0.2ml)。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, put it in a measuring bottle, add 1ml of purified water, vortex for 5 seconds, add 8ml of methanol dropwise, then add purified water drop by drop to the mark line, the solution is clear and transparent . At this time, the volume of the solution is 10 ml (including 1 ml injection + 8 ml methanol + 1 ml water, that is, the amount of methanol added per 1 mg of triamcinolone acetonide is 0.2 ml).

这里，需要说明的是，上述曲安奈德注射液、纯化水和甲醇的体积比均看做是精确测量得到，本领域技术人员能够理解的是，随着实验条件、环境温度等的差异，上述体积比可以具有一定的波动，由此所带来的波动也均在本公开实施例的保护范围之内。Here, it should be noted that the volume ratios of the above-mentioned triamcinolone acetonide injection, purified water and methanol are all regarded as accurately measured, and those skilled in the art can understand that, with the differences in experimental conditions, ambient temperatures, etc., the above-mentioned The volume ratio may have certain fluctuations, and the fluctuations caused by this are also within the protection scope of the embodiments of the present disclosure.

此外，根据国家对容量器具的标准(GBT12806-2011以及GBT12807-2021)，机械移液器最大系统允许误差至少可为0.8％，常用的10ml的B级容量瓶的最大容量允差可达0.4％。由此估算，配制供试品原液时，至少需要移取药液一次，有机溶剂一次，使用容量瓶一次，这样至少会引入0.8％+0.8％+0.4％＝2％的误差。由此可以判定，基于±2％的误差，在该例中，以每1mg曲安奈德加入0.196ml～0.204ml甲醇的范围进行操作均可得到澄清溶液。In addition, according to the national standards for volumetric appliances (GBT12806-2011 and GBT12807-2021), the maximum allowable error of the mechanical pipette system can be at least 0.8%, and the maximum capacity of the commonly used 10ml B-grade volumetric flask can reach 0.4%. . It is estimated from this that when preparing the stock solution of the test sample, it is necessary to pipette the medicinal solution once, the organic solvent once, and use the volumetric flask once at least, which will at least introduce an error of 0.8%+0.8%+0.4%=2%. It can be judged from this that, based on the error of ±2%, in this example, a clear solution can be obtained by adding 0.196ml-0.204ml of methanol per 1mg of triamcinolone acetonide.

(2)乙醇(2) Ethanol

用移液枪精密量取曲安奈德注射液1ml至10ml量瓶中，加入1ml纯化水，再逐滴加入5.5ml乙醇，再加纯化水定容至10ml，溶液澄清透明(包含1ml注射液+5.5ml乙醇+3.5ml水，即，每1mg曲安奈德对应的乙醇的加入量为0.1375ml)。Precisely measure 1ml of triamcinolone acetonide injection into a 10ml measuring bottle with a pipette gun, add 1ml of purified water, then add 5.5ml of ethanol drop by drop, add purified water to make up to 10ml, the solution is clear and transparent (including 1ml of injection + 5.5ml of ethanol + 3.5ml of water, that is, the amount of ethanol added per 1mg of triamcinolone acetonide is 0.1375ml).

基于与上述(1)相同的理由，可以判定，基于±2％的误差，在该例中，以每1mg曲安奈德加入0.135ml～0.140ml乙醇的范围进行操作均可得到澄清溶液。Based on the same reason as (1) above, it can be judged that based on the error of ±2%, in this example, a clear solution can be obtained by adding 0.135ml to 0.140ml of ethanol per 1mg of triamcinolone acetonide.

(3)正丙醇(3) n-propanol

用移液枪精密量取曲安奈德注射液1ml，置于10ml量瓶中，加入1ml纯化水，涡旋振荡5秒，再逐滴加入5.5ml正丙醇，此时絮状物出现，滴加纯化水定容至刻度线，摇匀，超声1分钟，絮状物消褪，溶液澄清(包含1ml注射液+5.5ml正丙醇+3.5ml水，即，每1mg曲安奈德对应的正丙醇的加入量为0.1375ml)。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, put it in a 10ml measuring bottle, add 1ml of purified water, vortex for 5 seconds, then add 5.5ml of n-propanol drop by drop, at this time flocs appear, drop Add purified water to make up to the scale line, shake well, and ultrasonic for 1 minute, the flocs fade, and the solution is clarified (contains 1ml injection + 5.5ml n-propanol + 3.5ml water, that is, every 1mg of triamcinolone acetonide corresponds to n The add-on of propanol is 0.1375ml).

基于与上述(1)相同的理由，可以判定，基于±2％的误差，在该例中，以每1mg曲安奈德加入0.135ml～0.140ml正丙醇的范围进行操作均可得到澄清溶液。Based on the same reason as (1) above, it can be judged that based on the error of ±2%, in this example, a clear solution can be obtained by adding 0.135ml to 0.140ml of n-propanol per 1mg of triamcinolone acetonide.

(4)异丙醇(4) Isopropanol

用移液枪精密量取曲安奈德注射液1ml，置于10ml量瓶中，加入1ml纯化水，涡旋振荡5秒，滴加3ml异丙醇后，液体呈混悬状态。再逐滴加入3ml异丙醇后，无颗粒状主药沉淀，但絮状沉淀出现。接着，在上述溶液中逐滴加入2ml纯化水，絮状沉淀开始消褪。再逐滴加入纯化水至刻度线，溶液澄清(包含1ml注射液+6ml异丙醇+3ml水，即，每1mg曲安奈德对应的异丙醇的加入量为0.15ml)。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, place it in a 10ml measuring bottle, add 1ml of purified water, vortex for 5 seconds, add 3ml of isopropanol dropwise, and the liquid is in a suspension state. After adding 3ml of isopropanol dropwise, there was no precipitation of the main drug in granular form, but flocculent precipitation appeared. Then, 2 ml of purified water was added dropwise to the above solution, and the flocculent precipitates began to fade. Purified water was then added dropwise to the mark, and the solution was clarified (comprising 1ml of injection solution + 6ml of isopropanol + 3ml of water, that is, the amount of isopropanol added per 1mg of triamcinolone acetonide was 0.15ml).

基于与上述(1)相同的理由，可以判定，基于±2％的误差，在该例中，以每1mg曲安奈德加入0.147ml～0.153ml异丙醇的范围进行操作均可得到澄清溶液。Based on the same reason as (1) above, it can be judged that based on the error of ±2%, in this example, a clear solution can be obtained by adding 0.147ml to 0.153ml of isopropanol per 1mg of triamcinolone acetonide.

(5)二甲基亚砜(5) Dimethyl sulfoxide

用移液枪精密量取曲安奈德注射液1ml，置于10ml量瓶中，加入1ml纯化水，涡旋振荡5秒，滴加4ml二甲基亚砜后，无颗粒状主药，溶液呈现乳光。再逐滴加入3ml二甲亚砜，再逐滴加入纯化水至刻度线，此时溶液澄清透明(包含1ml注射液+7ml二甲基亚砜+2ml水，即，每1mg曲安奈德对应的二甲基亚砜的加入量为0.175ml)。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, put it in a 10ml measuring bottle, add 1ml of purified water, vortex and shake for 5 seconds, after adding 4ml of dimethyl sulfoxide dropwise, there is no granular main drug, and the solution appears Opalescence. Then add 3ml dimethyl sulfoxide dropwise, and then add purified water dropwise to the scale line. At this time, the solution is clear and transparent (including 1ml injection + 7ml dimethyl sulfoxide + 2ml water, that is, every 1mg triamcinolone acetonide corresponding The addition amount of dimethyl sulfoxide is 0.175ml).

基于与上述(1)相同的理由，可以判定，基于±2％的误差，在该例中，以每1mg曲安奈德加入0.172ml～0.178ml二甲基亚砜的范围进行操作均可得到澄清溶液。Based on the same reason as above (1), it can be judged that based on the error of ±2%, in this example, adding 0.172ml to 0.178ml dimethyl sulfoxide per 1mg of triamcinolone acetonide can be clarified. solution.

(6)丙二醇(6) Propylene glycol

用移液枪精密量取曲安奈德注射液1ml，置于试管中，加入1ml纯化水，涡旋振荡5秒，滴加2ml丙二醇后，丙二醇沉在试管底部，涡旋振荡后，溶液状态未有改善。再逐滴加入1ml纯化水后，未看到溶解状态改善。再逐滴加入3ml丙二醇后，未看到溶解状态改善，且溶液黏度较大。再逐滴加入1ml纯化水后，未看到溶解状态改善。再逐滴加入2ml丙二醇后，未看到溶解状态改善。再逐滴加入2ml纯化水后，未看到溶解状态改善。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, put it in a test tube, add 1ml of purified water, and vortex for 5 seconds. After adding 2ml of propylene glycol dropwise, the propylene glycol settles at the bottom of the test tube. After vortexing, the solution state is not has improved. After 1 ml of purified water was added dropwise, no improvement in the dissolved state was observed. After adding 3ml of propylene glycol dropwise, no improvement in the dissolution state was seen, and the solution had a relatively high viscosity. After 1 ml of purified water was added dropwise, no improvement in the dissolved state was observed. After another 2 ml of propylene glycol was added dropwise, no improvement in the dissolution state was observed. After adding 2 ml of purified water dropwise, no improvement in the dissolved state was observed.

(7)丙三醇(7) Glycerol

用移液枪精密量取曲安奈德注射液1ml，置于试管中，加入1ml纯化水，涡旋振荡5秒，滴加1ml丙三醇后，涡旋振荡，溶液状态未有改善。再逐滴加入1ml纯化水后，未看到溶解状态改善。再逐滴加入2ml丙三醇后，未看到溶解状态发生变化，且溶液黏度较大。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, put it in a test tube, add 1ml of purified water, vortex for 5 seconds, add 1ml of glycerol dropwise, vortex and oscillate, the solution state does not improve. After 1 ml of purified water was added dropwise, no improvement in the dissolved state was observed. After adding 2ml of glycerol dropwise, no change in the dissolved state was observed, and the solution had a relatively high viscosity.

(8)乙二醇(8) Ethylene glycol

用移液枪精密量取曲安奈德注射液1ml，置于试管中，加入1ml纯化水，涡旋振荡5秒，滴加2ml乙二醇后，涡旋振荡，溶液状态未有改善。再逐滴加入1ml纯化水后，未看到溶解状态改善。再逐滴加入1ml乙二醇后，未看到溶解状态发生变化。再逐滴加入2ml乙二醇后，未看到溶解状态发生变化。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, put it in a test tube, add 1ml of purified water, vortex for 5 seconds, add 2ml of ethylene glycol dropwise, and vortex, the solution state does not improve. After 1 ml of purified water was added dropwise, no improvement in the dissolved state was observed. After adding 1 ml of ethylene glycol dropwise, no change in the dissolved state was observed. After adding 2 ml of ethylene glycol dropwise, no change in the dissolved state was observed.

(9)二甘醇(9) Diethylene glycol

用移液枪精密量取曲安奈德注射液1ml，置于试管中，加入1ml纯化水，涡旋振荡5秒，滴加2ml二甘醇后，涡旋振荡，溶液状态未有改善。再逐滴加入1ml纯化水后，未看到溶解状态改善。再逐滴加入2ml乙二醇后，未看到溶解状态发生变化。再逐滴加入2ml乙二醇后，未看到溶解状态发生变化。Precisely measure 1ml of triamcinolone acetonide injection with a pipette gun, put it in a test tube, add 1ml of purified water, vortex for 5 seconds, add 2ml of diethylene glycol dropwise, vortex and oscillate, the solution state does not improve. After 1 ml of purified water was added dropwise, no improvement in the dissolved state was observed. After adding 2 ml of ethylene glycol dropwise, no change in the dissolved state was observed. After adding 2 ml of ethylene glycol dropwise, no change in the dissolved state was observed.

结论：采用特定有机溶剂和检查用水可以将曲安奈德注射液溶解至澄清，该特定有机溶剂选自甲醇、乙醇、正丙醇、异丙醇和二甲基亚砜中的任一种。Conclusion: Triamcinolone acetonide injection can be dissolved to clear by using specific organic solvent and examination water, the specific organic solvent is selected from methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide.

实施例2采用甲醇的水溶液作为溶剂的预干扰试验Embodiment 2 adopts the aqueous solution of methanol as the pre-interference test of solvent

取曲安奈德注射液(与实施例1同规格，同批号)1ml至10ml无热原量瓶中，加入1mlBET检查用水，再加入8.0ml甲醇，加BET检查用水定容至10ml，振摇即得曲安奈德含量为4mg/ml的澄清溶液(也即供试品原液)。采用BET检查用水对供试品原液逐级稀释，制成曲安奈德含量为0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品溶液(NPC)。Take 1ml of triamcinolone acetonide injection (same specification and batch number as in Example 1) into a 10ml pyrogen-free measuring bottle, add 1ml of BET test water, then add 8.0ml of methanol, add BET test water to dilute to 10ml, shake Obtained triamcinolone acetonide content is the clarification solution (that is need testing stock solution) of 4mg/ml. The stock solution of the test sample was diluted step by step with BET water to prepare the test solution (NPC) with triamcinolone acetonide content of 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml.

另取相应浓度曲安奈德注射液的供试品溶液，加入细菌内毒素溶液制成含内毒素浓度为2λ，曲安奈德0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品阳性对照溶液(PPC)。Get the need testing solution of triamcinolone acetonide injection of corresponding concentration separately, add bacterial endotoxin solution and make containing endotoxin concentration and be 2λ, triamcinolone acetonide 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml ml of the positive control solution (PPC) of the test product.

取两个不同厂家已按《中国药典》要求复核过灵敏度为0.25EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管，并设阴性对照(NC)和阳性对照(PC)各2管，结果见表1。Take two limulus reagents from different manufacturers that have been checked according to the requirements of "Chinese Pharmacopoeia" and have a sensitivity of 0.25EU/ml to react with the above series of solutions. Repeat 2 tubes for each concentration, and set a negative control (NC) and a positive control (PC) 2 tubes each, the results are shown in Table 1.

表1：曲安奈德注射液细菌内毒素检查预干扰试验结果Table 1: Triamcinolone acetonide injection bacterial endotoxin inspection pre-interference test results

由上表可知，两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。供试品溶液中曲安奈德的浓度在0.08334mg/ml以下时对鲎试剂与细菌内毒素的凝集反应无干扰，后续可选用灵敏度不低于0.25EU/ml的鲎试剂进行试验。It can be seen from the above table that the negative control (NC) of the two manufacturers of Limulus reagent with a sensitivity of 0.25 EU/ml is negative, the positive control (PC) is positive, and the test is valid. When the concentration of triamcinolone acetonide in the test solution is below 0.08334 mg/ml, it will not interfere with the agglutination reaction between the LAL and bacterial endotoxin, and the LAL reagent with a sensitivity of not less than 0.25 EU/ml can be used for subsequent tests.

实施例3甲醇的干扰试验The interference test of embodiment 3 methanol

由实施例2可知，供试品溶液中曲安奈德的浓度在0.0834mg/ml(甲醇的体积占比为1.67％)以下时对鲎试剂与细菌内毒素的凝集反应无干扰，故使用灵敏度λ为0.25EU/ml的鲎试剂进行试验。As can be seen from Example 2, when the concentration of triamcinolone acetonide in the test solution is below 0.0834mg/ml (the volume ratio of methanol is 1.67%), there is no interference to the agglutination reaction of Limulus reagent and bacterial endotoxin, so the sensitivity λ is used. The test was performed for 0.25 EU/ml LAL reagent.

经计算，此时供试品溶液中甲醇的体积占比为1.67％，也即100ml供试品溶液中含有1.67ml甲醇。After calculation, the volume ratio of methanol in the test solution is 1.67%, that is, 100ml of the test solution contains 1.67ml of methanol.

据此设计甲醇干扰试验如下：Accordingly, the methanol interference test was designed as follows:

将甲醇用细菌内毒素检查用水制成原液和2，4，8，16，32，64倍稀释液作溶液A系列，取相应浓度甲醇稀释液加细菌内毒素溶液制成含细菌内毒素浓度为2λ，甲醇稀释2，4，8，16，32，64倍的供试品阳性对照溶液B系列；另取2管加BET检查用水作阴性对照D，2管加2λ浓度的细菌内毒素溶液作阳性对照C。取两个不同厂家灵敏度分别为0.25EU/ml和0.125EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管。结果如下述表2所示。Use methanol to test water for bacterial endotoxin to make stock solution and 2, 4, 8, 16, 32, 64 times dilutions as solution A series, take corresponding concentration of methanol dilution and add bacterial endotoxin solution to make bacterial endotoxin concentration of 2λ, methanol diluted 2, 4, 8, 16, 32, 64 times the positive control solution B series of the test sample; another 2 tubes plus BET water for negative control D, 2 tubes plus 2λ concentration of bacterial endotoxin solution as Positive control C. Take two limulus reagents with sensitivities of 0.25EU/ml and 0.125EU/ml from different manufacturers to react with the above series of solutions, and repeat 2 tubes for each concentration. The results are shown in Table 2 below.

表2：细菌内毒素检查甲醇干扰试验结果：Table 2: Bacterial endotoxin inspection Methanol interference test results:

由上表可知，甲醇干扰试验采用的两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。结果显示甲醇稀释16倍以上(甲醇在甲醇稀释液中的体积占比为6.25％)时无干扰，所以，在供试品溶液中，甲醇的体积占比为1.67％时，对曲安奈德注射液细菌内毒素检查无干扰。It can be seen from the above table that the negative control (NC) of the limulus reagent of two manufacturers with a sensitivity of 0.25 EU/ml used in the methanol interference test is negative, and the positive control (PC) is positive, and the test is effective. The result shows that there is no interference when methanol is diluted more than 16 times (the volume ratio of methanol in the methanol diluent is 6.25%), so, in the test solution, when the volume ratio of methanol is 1.67%, the triamcinolone acetonide injection There is no interference in the liquid bacterial endotoxin test.

实施例4采用甲醇的水溶液做正式干扰试验：Embodiment 4 adopts the aqueous solution of methanol to do formal interference test:

分别用3批曲安奈德注射液，按《中国药典》2020年版四部通则(1143细菌内毒素检查法)中干扰试验要求以实施例3中方法制备供试品溶液进行考察，阳性对照/供试品溶液B、阳性对照/内检水溶液C每个浓度平行4管，供试品溶液A、阴性对照溶液D每个浓度平行2管进行试验，结果如下述表3所示。Use 3 batches of triamcinolone acetonide injection respectively, according to the requirements of the interference test in "Chinese Pharmacopoeia" 2020 Edition Four General Rules (1143 Bacterial Endotoxin Inspection Method), prepare the test solution with the method in Example 3 for investigation, positive control/for test Each concentration of product solution B, positive control/internal inspection solution C is parallel 4 tubes, and each concentration of test product solution A and negative control solution D is tested in parallel 2 tubes. The results are shown in the following table 3.

表3：曲安奈德注射液(批号：ABU8750)细菌内毒素检查干扰确认试验：Table 3: Triamcinolone acetonide injection (batch number: ABU8750) Bacterial endotoxin inspection interference confirmation test:

由上表可知，曲安奈德注射液正式干扰实验中A、D为阴性，两个厂家鲎试剂标示灵敏度对照系列结果λc在0.5λ～2λ范围，符合鲎试剂灵敏度复核试验要求，试验有效。两个厂家鲎试剂干扰试验系列结果Et在0.5λc～2λc之间，符合规定。另外两批曲安奈德注射液结果也符合要求。说明采用上述供试品溶液中，甲醇的体积占比为1.67％时，对检查曲安奈德注射液细菌内毒素方法无干扰。(上表中，N/A表示本单元格无内容。)It can be seen from the above table that in the official interference test of triamcinolone acetonide injection, A and D are negative, and the results of the two manufacturers' LAL reagent sensitivity control series results λc are in the range of 0.5λ-2λ, which meets the requirements of the LAL reagent sensitivity recheck test, and the test is valid. The results of the TAL interference test series of two manufacturers' Et ranged from 0.5λc to 2λc, which met the regulations. The results of the other two batches of triamcinolone acetonide injection also met the requirements. Explain that in the above-mentioned test solution, when the volume ratio of methanol is 1.67%, there is no interference to the method for checking the bacterial endotoxin of triamcinolone acetonide injection. (In the above table, N/A means there is no content in this cell.)

实施例5采用乙醇的水溶液作为溶剂的预干扰试验Embodiment 5 adopts the aqueous solution of ethanol as the pre-interference test of solvent

取曲安奈德注射液(批号ABU8750)1ml至10ml无热原量瓶中，加入1ml BET检查用水，再加入5.5ml乙醇，加BET检查用水定容至10ml，振摇即得曲安奈德含量为4mg/ml的澄清溶液(也即供试品原液)，采用BET检查用水将供试品原液逐级稀释，制成曲安奈德含量为0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品溶液(NPC)。Take 1ml of triamcinolone acetonide injection (lot number ABU8750) into a 10ml pyrogen-free measuring bottle, add 1ml of BET test water, then add 5.5ml of ethanol, add BET test water to make the volume to 10ml, and shake to get the content of triamcinolone acetonide. 4mg/ml clear solution (that is, the stock solution of the test sample), the stock solution of the test sample is diluted step by step with BET inspection water, and the content of triamcinolone acetonide is 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.02084mg/ml. The need testing solution (NPC) of 0.01042mg/ml.

另取相应浓度曲安奈德注射液的供试品溶液，加入细菌内毒素溶液制成含内毒素浓度为2λ，曲安奈德0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品阳性对照溶液(PPC)。Get the need testing solution of triamcinolone acetonide injection of corresponding concentration separately, add bacterial endotoxin solution and make containing endotoxin concentration and be 2λ, triamcinolone acetonide 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml ml of the positive control solution (PPC) of the test product.

取两个不同厂家已按《中国药典》要求复核过灵敏度为0.25EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管，并设阴性对照(NC)和阳性对照(PC)各2管，结果见下述表4。Take two limulus reagents from different manufacturers that have been checked according to the requirements of "Chinese Pharmacopoeia" and have a sensitivity of 0.25EU/ml to react with the above series of solutions. Repeat 2 tubes for each concentration, and set a negative control (NC) and a positive control (PC) 2 tubes each, and the results are shown in Table 4 below.

表4：曲安奈德注射液细菌内毒素检查乙醇的水溶液预干扰试验结果：Table 4: Triamcinolone acetonide injection bacterial endotoxin test results of ethanol aqueous solution pre-interference test:

由上表可知，两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。供试品溶液中曲安奈德的浓度在0.08334mg/ml以下时对鲎试剂与细菌内毒素的凝集反应无干扰，后续可选用灵敏度不低于0.25EU/ml的鲎试剂进行试验。It can be seen from the above table that the negative control (NC) of the two manufacturers of Limulus reagent with a sensitivity of 0.25 EU/ml is negative, the positive control (PC) is positive, and the test is valid. When the concentration of triamcinolone acetonide in the test solution is below 0.08334 mg/ml, it will not interfere with the agglutination reaction between the LAL and bacterial endotoxin, and the LAL reagent with a sensitivity of not less than 0.25 EU/ml can be used for subsequent tests.

实施例6乙醇的干扰试验：The interference test of embodiment 6 ethanol:

由实施例5可知，供试品溶液中曲安奈德的浓度在0.0834mg/ml(如乙醇的体积占比为1.15％)以下时对鲎试剂与细菌内毒素的凝集反应无干扰，故使用灵敏度λ为0.25EU/ml的鲎试剂进行试验。As can be seen from Example 5, when the concentration of triamcinolone acetonide in the test solution is below 0.0834mg/ml (as the volume ratio of ethanol is 1.15%), there is no interference to the agglutination reaction of Limulus reagent and bacterial endotoxin, so the use sensitivity λ is 0.25EU/ml Limulus reagent for the test.

经计算，此时供试品溶液中乙醇的体积占比为1.15％，也即100ml供试品溶液中含有1.15ml乙醇。After calculation, the volume ratio of ethanol in the test solution is 1.15%, that is, 100ml of the test solution contains 1.15ml of ethanol.

据此设计乙醇干扰试验如下：Accordingly, the ethanol interference test was designed as follows:

将乙醇用细菌内毒素检查用水制成原液和2，4，8，16，32，64倍稀释液作溶液A系列，取相应浓度乙醇稀释液加细菌内毒素溶液制成含细菌内毒素浓度为2λ，乙醇稀释2，4，8，16，32，64倍的供试品阳性对照溶液B系列；另取2管加BET检查用水作阴性对照D，2管加2λ浓度的细菌内毒素溶液作阳性对照C。取两个不同厂家灵敏度分别为0.25EU/ml和0.125EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管。结果如下述表5所示。Use ethanol with bacterial endotoxin test water to make stock solution and 2, 4, 8, 16, 32, 64 times dilutions as solution A series, take corresponding concentration of ethanol dilution and add bacterial endotoxin solution to make bacterial endotoxin containing concentration of 2λ, dilute ethanol 2, 4, 8, 16, 32, 64 times the positive control solution B series of the test sample; take another 2 tubes plus BET inspection water as negative control D, and 2 tubes plus 2λ concentration of bacterial endotoxin solution as Positive control C. Take two limulus reagents with sensitivities of 0.25EU/ml and 0.125EU/ml from different manufacturers to react with the above series of solutions, and repeat 2 tubes for each concentration. The results are shown in Table 5 below.

表5：细菌内毒素检查乙醇干扰试验结果：Table 5: Bacterial endotoxin inspection ethanol interference test results:

由上表可知，乙醇干扰试验采用的两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。结果显示乙醇稀释32倍以上(乙醇在乙醇稀释液中的体积占比为3.125％)时无干扰，所以，在供试品溶液中，乙醇的体积占比为1.15％时，对曲安奈德注射液细菌内毒素检查无干扰。It can be seen from the above table that the two manufacturers' limulus reagent with a sensitivity of 0.25EU/ml used in the ethanol interference test was negative (NC) and the positive control (PC) was positive, and the test was effective. The result shows that there is no interference when ethanol is diluted more than 32 times (the volume ratio of ethanol in the ethanol diluent is 3.125%), so, in the test solution, when the volume ratio of ethanol is 1.15%, the triamcinolone acetonide injection There is no interference in the liquid bacterial endotoxin test.

实施例7乙醇的水溶液正式干扰试验The formal interference test of the aqueous solution of embodiment 7 ethanol

分别用3批曲安奈德注射液，按《中国药典》2020年版四部通则(1143细菌内毒素检查法)中干扰试验要求以实施例6中方法制备供试品溶液进行考察，阳性对照/供试品溶液B、阳性对照/内检水溶液C每个浓度平行4管，供试品溶液A、阴性对照溶液D每个浓度平行2管进行试验，结果如下述表6所示。Use 3 batches of triamcinolone acetonide injection respectively, according to the requirements of the interference test in "Chinese Pharmacopoeia" 2020 Edition Four General Rules (1143 Bacterial Endotoxin Inspection Method), prepare the test solution with the method in Example 6 for investigation, positive control/for test Each concentration of product solution B, positive control/internal inspection solution C is parallel 4 tubes, each concentration of test product solution A, negative control solution D is tested in parallel 2 tubes, and the results are shown in the following table 6.

表6：曲安奈德注射液细菌内毒素检查干扰确认试验：Table 6: Triamcinolone Acetonide Injection Bacterial Endotoxin Inspection Interference Confirmation Test:

由上表可知，曲安奈德注射液正式干扰实验中A、D为阴性，两个厂家鲎试剂标示灵敏度对照系列结果λc在0.5λ～2λ范围，符合鲎试剂灵敏度复核试验要求，试验有效。两个厂家鲎试剂干扰试验系列结果Et在0.5λc～2λc之间，符合规定。另外两批曲安奈德注射液结果也符合要求。说明采用上述供试品溶液中，乙醇的体积占比为1.15％时，对检查曲安奈德注射液细菌内毒素无干扰。It can be seen from the above table that in the official interference test of triamcinolone acetonide injection, A and D are negative, and the results of the two manufacturers' LAL reagent sensitivity control series results λc are in the range of 0.5λ-2λ, which meets the requirements of the LAL reagent sensitivity recheck test, and the test is valid. The results of the TAL interference test series of two manufacturers' Et ranged from 0.5λc to 2λc, which met the regulations. The results of the other two batches of triamcinolone acetonide injection also met the requirements. Explain that in the above test solution, when the volume ratio of ethanol is 1.15%, there is no interference to the bacterial endotoxin of triamcinolone acetonide injection.

实施例8采用正丙醇的水溶液作为溶剂的预干扰试验Embodiment 8 adopts the aqueous solution of n-propanol as the pre-interference test of solvent

取曲安奈德注射液(批号ABU8750)1ml至10ml无热原量瓶中，加入1ml BET检查用水，再加入5.5ml正丙醇，加BET检查用水定容至10ml，振摇即得曲安奈德含量为4mg/ml的澄清溶液(也即供试品原液)。采用BET检查用水对供试品原液逐级稀释，制成曲安奈德含量为0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品溶液(NPC)。Take 1ml of triamcinolone acetonide injection (batch number ABU8750) into a 10ml pyrogen-free measuring bottle, add 1ml of BET test water, then add 5.5ml of n-propanol, add BET test water to make up to 10ml, and shake to obtain triamcinolone acetonide Content is the clarification solution of 4mg/ml (that is need testing sample stoste). The stock solution of the test sample was diluted step by step with BET water to prepare the test solution (NPC) with triamcinolone acetonide content of 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml.

另取相应浓度曲安奈德注射液的供试品溶液，加入细菌内毒素溶液制成含内毒素浓度为2λ，曲安奈德0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品阳性对照溶液(PPC)。Get the need testing solution of triamcinolone acetonide injection of corresponding concentration separately, add bacterial endotoxin solution and make containing endotoxin concentration and be 2λ, triamcinolone acetonide 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml ml of the positive control solution (PPC) of the test product.

取两个不同厂家已按《中国药典》要求复核过灵敏度为0.25EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管，并设阴性对照(NC)和阳性对照(PC)各2管，结果见下述表7。Take two limulus reagents from different manufacturers that have been checked according to the requirements of "Chinese Pharmacopoeia" and have a sensitivity of 0.25EU/ml to react with the above series of solutions. Repeat 2 tubes for each concentration, and set a negative control (NC) and a positive control (PC) 2 tubes each, and the results are shown in Table 7 below.

表7：曲安奈德注射液细菌内毒素检查预干扰试验结果Table 7: Triamcinolone acetonide injection bacterial endotoxin inspection pre-interference test results

由上表可知，两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。供试品溶液中曲安奈德的浓度在0.08334mg/ml以下时对鲎试剂与细菌内毒素的凝集反应无干扰，后续可选用灵敏度不低于0.25EU/ml的鲎试剂进行试验。It can be seen from the above table that the negative control (NC) of the two manufacturers of Limulus reagent with a sensitivity of 0.25 EU/ml is negative, the positive control (PC) is positive, and the test is valid. When the concentration of triamcinolone acetonide in the test solution is below 0.08334 mg/ml, it will not interfere with the agglutination reaction between the LAL and bacterial endotoxin, and the LAL reagent with a sensitivity of not less than 0.25 EU/ml can be used for subsequent tests.

实施例9正丙醇的干扰试验The interference test of embodiment 9 n-propanol

由实施例8可知，供试品溶液中曲安奈德的浓度在0.0834mg/ml以下(正丙醇的体积占比为1.15％)时对鲎试剂与细菌内毒素的凝集反应无干扰，故使用灵敏度λ为0.25EU/ml的鲎试剂进行试验。As can be seen from Example 8, when the concentration of triamcinolone acetonide in the test solution is below 0.0834mg/ml (the volume ratio of n-propanol is 1.15%), there is no interference to the agglutination reaction of Limulus reagent and bacterial endotoxin, so use The limulus reagent with a sensitivity λ of 0.25 EU/ml was tested.

经计算，此时供试品溶液中正丙醇的体积占比为1.15％，也即100ml供试品溶液中含有1.15ml正丙醇。After calculation, the volume ratio of n-propanol in the test solution is 1.15%, that is, 100ml of the test solution contains 1.15ml of n-propanol.

据此设计正丙醇干扰试验如下：Based on this, the n-propanol interference test was designed as follows:

将正丙醇用细菌内毒素检查用水制成原液和2，4，8，16，32，64倍稀释液作溶液A系列，取相应浓度正丙醇稀释液加细菌内毒素溶液制成含细菌内毒素浓度为2λ，正丙醇稀释2，4，8，16，32，64倍的供试品阳性对照溶液B系列；另取2管加BET检查用水作阴性对照D，2管加2λ浓度的细菌内毒素溶液作阳性对照C。取两个不同厂家灵敏度分别为0.25EU/ml和0.125EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管。结果如下述表8所示。Use n-propanol with bacterial endotoxin test water to make stock solution and 2, 4, 8, 16, 32, 64 times dilutions as solution A series, take corresponding concentration of n-propanol dilution and add bacterial endotoxin solution to make bacteria-containing solution The endotoxin concentration is 2λ, the positive control solution B series of the test sample diluted 2, 4, 8, 16, 32, and 64 times with n-propanol; another 2 tubes plus BET water for negative control D, 2 tubes plus 2λ concentration The bacterial endotoxin solution was used as positive control C. Take two limulus reagents with sensitivities of 0.25EU/ml and 0.125EU/ml from different manufacturers to react with the above series of solutions, and repeat 2 tubes for each concentration. The results are shown in Table 8 below.

表8：细菌内毒素检查正丙醇干扰试验结果Table 8: Bacterial endotoxin inspection n-propanol interference test results

由上表可知，正丙醇干扰试验采用的两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。结果显示正丙醇稀释32倍以上(正丙醇在正丙醇稀释液中的体积占比为3.125％)时无干扰，所以，在供试品溶液中，正丙醇的体积占比为1.15％时，对曲安奈德注射液细菌内毒素检查无干扰。It can be seen from the above table that the negative control (NC) of the two manufacturers with a sensitivity of 0.25EU/ml used in the n-propanol interference test is negative, the positive control (PC) is positive, and the test is effective. The result shows that there is no interference when n-propanol is diluted more than 32 times (the volume ratio of n-propanol in n-propanol dilution is 3.125%), so, in need testing solution, the volume ratio of n-propanol is 1.15% %, there is no interference to the bacterial endotoxin test of triamcinolone acetonide injection.

实施例10采用正丙醇的水溶液做正式干扰试验：Embodiment 10 adopts the aqueous solution of n-propanol to do formal interference test:

分别用3批曲安奈德注射液，按《中国药典》2020年版四部通则(1143细菌内毒素检查法)中干扰试验要求以实施例9中方法制备供试品溶液进行考察，阳性对照/供试品溶液B、阳性对照/内检水溶液C每个浓度平行4管，供试品溶液A、阴性对照溶液D每个浓度平行2管进行试验，结果如下述表9所示。Use 3 batches of triamcinolone acetonide injection respectively, according to the requirements of the interference test in "Chinese Pharmacopoeia" 2020 Edition Four General Rules (1143 Bacterial Endotoxin Inspection Method), prepare the test solution with the method in Example 9 for investigation, positive control/test Each concentration of product solution B, positive control/internal inspection solution C is parallel 4 tubes, each concentration of test product solution A, negative control solution D is tested in parallel 2 tubes, and the results are as shown in the following table 9.

表9：曲安奈德注射液细菌内毒素检查干扰确认试验Table 9: Triamcinolone Acetonide Injection Bacterial Endotoxin Inspection Interference Confirmation Test

由上表可知，曲安奈德注射液正式干扰实验中A、D为阴性，两个厂家鲎试剂标示灵敏度对照系列结果λc在0.5λ～2λ范围，符合鲎试剂灵敏度复核试验要求，试验有效。两个厂家鲎试剂干扰试验系列结果Et在0.5λc～2λc之间，符合规定。另外两批曲安奈德注射液结果也符合要求。说明采用上述供试品溶液中，正丙醇的体积占比为1.15％时，对检查曲安奈德注射液细菌内毒素无干扰。It can be seen from the above table that in the official interference test of triamcinolone acetonide injection, A and D are negative, and the results of the two manufacturers' LAL reagent sensitivity control series results λc are in the range of 0.5λ-2λ, which meets the requirements of the LAL reagent sensitivity recheck test, and the test is valid. The results of the TAL interference test series of two manufacturers' Et ranged from 0.5λc to 2λc, which met the regulations. The results of the other two batches of triamcinolone acetonide injection also met the requirements. Explain that in the above-mentioned test solution, when the volume ratio of n-propanol is 1.15%, there is no interference to the bacterial endotoxin of triamcinolone acetonide injection.

实施例11采用异丙醇的水溶液作为溶剂的预干扰试验：Embodiment 11 adopts the aqueous solution of isopropanol as the pre-interference test of solvent:

取曲安奈德注射液(批号：ABU8750)1ml至10ml无热原量瓶中，加入1ml BET检查用水，再加入6.0ml异丙醇，加BET检查用水定容至10ml，振摇即得曲安奈德含量为4mg/ml的澄清溶液(也即供试品原液)。采用BET检查用水对供试品原液逐级稀释，制成曲安奈德含量为0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品溶液(NPC)。Take 1ml of triamcinolone acetonide injection (batch number: ABU8750) into a 10ml pyrogen-free measuring bottle, add 1ml of BET test water, then add 6.0ml of isopropanol, add BET test water to make up to 10ml, shake to get triamcinolone De content is the clarification solution of 4mg/ml (that is need testing stock solution). The stock solution of the test sample was diluted step by step with BET water to prepare the test solution (NPC) with triamcinolone acetonide content of 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml.

另取相应浓度曲安奈德注射液的供试品溶液，加入细菌内毒素溶液制成含内毒素浓度为2λ，曲安奈德0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品阳性对照溶液(PPC)。Get the need testing solution of triamcinolone acetonide injection of corresponding concentration separately, add bacterial endotoxin solution and make containing endotoxin concentration and be 2λ, triamcinolone acetonide 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml ml of the positive control solution (PPC) of the test product.

取两个不同厂家已按《中国药典》要求复核过灵敏度为0.25EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管，并设阴性对照(NC)和阳性对照(PC)各2管，结果见下述表10。Take two limulus reagents from different manufacturers that have been checked according to the requirements of "Chinese Pharmacopoeia" and have a sensitivity of 0.25EU/ml to react with the above series of solutions. Repeat 2 tubes for each concentration, and set a negative control (NC) and a positive control (PC) 2 tubes each, and the results are shown in Table 10 below.

表10：曲安奈德注射液细菌内毒素检查预干扰试验结果Table 10: Triamcinolone acetonide injection bacterial endotoxin inspection pre-interference test results

由上表可知，两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。供试品溶液中曲安奈德的浓度在0.08334mg/ml以下时对鲎试剂与细菌内毒素的凝集反应无干扰，后续可选用灵敏度不低于0.25EU/ml的鲎试剂进行试验。It can be seen from the above table that the negative control (NC) of the two manufacturers of Limulus reagent with a sensitivity of 0.25 EU/ml is negative, the positive control (PC) is positive, and the test is valid. When the concentration of triamcinolone acetonide in the test solution is below 0.08334 mg/ml, it will not interfere with the agglutination reaction between the LAL and bacterial endotoxin, and the LAL reagent with a sensitivity of not less than 0.25 EU/ml can be used for subsequent tests.

实施例12异丙醇的干扰试验The interference test of embodiment 12 isopropanol

由实施例11可知，供试品溶液中曲安奈德的浓度为0.0834mg/ml以下(异丙醇的体积占比为1.25％)时对鲎试剂与细菌内毒素的凝集反应无干扰，故使用灵敏度λ为0.25EU/ml的鲎试剂进行试验。As can be seen from Example 11, when the concentration of triamcinolone acetonide in the test solution is below 0.0834mg/ml (the volume ratio of isopropanol is 1.25%), there is no interference to the agglutination reaction of Limulus reagent and bacterial endotoxin, so use The limulus reagent with a sensitivity λ of 0.25 EU/ml was tested.

经计算，此时供试品溶液中异丙醇的体积占比为1.25％，也即100ml供试品溶液中含有1.25ml异丙醇。After calculation, the volume ratio of isopropanol in the test solution is 1.25%, that is, 100ml of the test solution contains 1.25ml of isopropanol.

据此设计异丙醇干扰试验如下：Accordingly, the isopropanol interference test was designed as follows:

将异丙醇用细菌内毒素检查用水制成原液和2，4，8，16，32，64倍稀释液作溶液A系列，取相应浓度异丙醇稀释液加细菌内毒素溶液制成含细菌内毒素浓度为2λ，异丙醇稀释2，4，8，16，32，64倍的供试品阳性对照溶液B系列；另取2管加BET检查用水作阴性对照D，2管加2λ浓度的细菌内毒素溶液作阳性对照C。取两个不同厂家灵敏度分别为0.25EU/ml和0.125EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管。结果如下述表11所示。Use isopropanol with bacterial endotoxin test water to make stock solution and 2, 4, 8, 16, 32, 64 times dilutions as solution A series, take corresponding concentration of isopropanol dilution and add bacterial endotoxin solution to make bacteria-containing solution Endotoxin concentration is 2λ, isopropanol diluted 2, 4, 8, 16, 32, 64 times of the positive control solution B series of the test sample; another 2 tubes plus BET water for negative control D, 2 tubes plus 2λ concentration The bacterial endotoxin solution was used as positive control C. Take two limulus reagents with sensitivities of 0.25EU/ml and 0.125EU/ml from different manufacturers to react with the above series of solutions, and repeat 2 tubes for each concentration. The results are shown in Table 11 below.

表11：异丙醇细菌内毒素检查干扰试验结果Table 11: Interference test results of isopropanol bacterial endotoxin inspection

由上表可知，异丙醇干扰试验采用的两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。结果显示异丙醇稀释32倍以上(异丙醇在异丙醇稀释液中的体积占比为3.125％)时无干扰，所以在供试品溶液中，异丙醇的体积占比为1.25％时，对曲安奈德注射液细菌内毒素检查无干扰。It can be seen from the above table that the negative control (NC) of the limulus reagent of two manufacturers with a sensitivity of 0.25 EU/ml used in the isopropanol interference test is negative, and the positive control (PC) is positive, and the test is effective. The result shows that there is no interference when the isopropanol is diluted more than 32 times (the volume ratio of isopropanol in the isopropanol diluent is 3.125%), so in the test solution, the volume ratio of isopropanol is 1.25% There was no interference to the bacterial endotoxin test of triamcinolone acetonide injection.

实施例13异丙醇的水溶液正式干扰试验：The formal interference test of the aqueous solution of embodiment 13 isopropanols:

分别用3批曲安奈德注射液，按《中国药典》2020年版四部通则(1143细菌内毒素检查法)中干扰试验要求以实施例12中方法制备供试品溶液进行考察进行考察，阳性对照/供试品溶液B、阳性对照/内检水溶液C每个浓度平行4管，供试品溶液A、阴性对照溶液D每个浓度平行2管进行试验，结果如表12所示。With 3 batches of triamcinolone acetonide injection respectively, according to " Chinese Pharmacopoeia " 2020 edition four general rules (1143 bacterial endotoxin examination method) in the interference test requirement, prepare the need testing solution with the method in embodiment 12 and investigate and investigate, positive control/ Need testing solution B, positive control/internal inspection solution C each concentration parallel 4 tubes, need testing solution A, negative control solution D each concentration parallel 2 tubes to carry out the test, the results are shown in Table 12.

表12：曲安奈德注射液细菌内毒素检查干扰确认试验Table 12: Triamcinolone Acetonide Injection Bacterial Endotoxin Inspection Interference Confirmation Test

由上表可知，曲安奈德注射液正式干扰实验中A、D为阴性，两个厂家鲎试剂标示灵敏度对照系列结果λc在0.5λ～2λ范围，符合鲎试剂灵敏度复核试验要求，试验有效。两个厂家鲎试剂干扰试验系列结果Et在0.5λc～2λc之间，符合规定。另外两批曲安奈德注射液结果也符合要求。说明采用上述供试品溶液中，异丙醇的体积占比为1.25％时，对检查曲安奈德注射液细菌内毒素无干扰。It can be seen from the above table that in the official interference test of triamcinolone acetonide injection, A and D are negative, and the results of the two manufacturers' LAL reagent sensitivity control series results λc are in the range of 0.5λ-2λ, which meets the requirements of the LAL reagent sensitivity recheck test, and the test is valid. The results of the TAL interference test series of two manufacturers' Et ranged from 0.5λc to 2λc, which met the regulations. The results of the other two batches of triamcinolone acetonide injection also met the requirements. Illustrate adopting above-mentioned need testing solution, when the volume ratio of isopropanol is 1.25%, there is no interference to checking bacterial endotoxin of triamcinolone acetonide injection.

实施例14二甲基亚砜作为溶剂的预干扰试验：Embodiment 14 dimethyl sulfoxide is used as the pre-interference test of solvent:

取曲安奈德注射液(批号：ABU8750)1ml至10ml无热原量瓶中，加入1ml BET检查用水，再加入7.0ml二甲基亚砜，加BET检查用水定容至10ml，振摇即得曲安奈德含量为4mg/ml的澄清溶液(也即供试品原液)。采用BET检查用水对供试品原液逐级稀释，制成曲安奈德含量为0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品溶液(NPC)。Take 1ml of triamcinolone acetonide injection (batch number: ABU8750) into a 10ml pyrogen-free measuring bottle, add 1ml of BET test water, then add 7.0ml of dimethyl sulfoxide, add BET test water to make up to 10ml, and shake to get ready Triamcinolone acetonide content is the clarification solution of 4mg/ml (that is need testing sample stoste). The stock solution of the test sample was diluted step by step with BET water to prepare the test solution (NPC) with triamcinolone acetonide content of 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml.

另取相应浓度曲安奈德注射液的供试品溶液，加入细菌内毒素溶液制成含内毒素浓度为2λ，曲安奈德0.08334mg/ml、0.04167mg/ml、0.02084mg/ml和0.01042mg/ml的供试品阳性对照溶液(PPC)。Get the need testing solution of triamcinolone acetonide injection of corresponding concentration separately, add bacterial endotoxin solution and make containing endotoxin concentration and be 2λ, triamcinolone acetonide 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042mg/ml ml of the positive control solution (PPC) of the test product.

取两个不同厂家已按《中国药典》要求复核过灵敏度为0.25EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管，并设阴性对照(NC)和阳性对照(PC)各2管，结果见下述表13。Take two limulus reagents from different manufacturers that have been checked according to the requirements of "Chinese Pharmacopoeia" and have a sensitivity of 0.25EU/ml to react with the above series of solutions. Repeat 2 tubes for each concentration, and set a negative control (NC) and a positive control (PC) 2 tubes each, and the results are shown in Table 13 below.

表13：曲安奈德注射液细菌内毒素检查预干扰试验结果Table 13: Triamcinolone Acetonide Injection Bacterial Endotoxin Inspection Pre-Interference Test Results

由上表可知，两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。供试品溶液中，曲安奈德的浓度在0.08334mg/ml以下时对鲎试剂与细菌内毒素的凝集反应无干扰，后续可选用灵敏度不低于0.25EU/ml的鲎试剂进行试验。It can be seen from the above table that the negative control (NC) of the two manufacturers of Limulus reagent with a sensitivity of 0.25 EU/ml is negative, the positive control (PC) is positive, and the test is valid. In the test solution, when the concentration of triamcinolone acetonide is below 0.08334mg/ml, it will not interfere with the agglutination reaction of the LAL reagent and bacterial endotoxin, and the LAL reagent with a sensitivity of not less than 0.25 EU/ml can be used for subsequent tests.

实施例15二甲基亚砜的干扰试验The interference test of embodiment 15 dimethyl sulfoxide

由实施例14可知，供试品溶液中，曲安奈德的浓度在0.0834mg/ml以下(二甲基亚砜的体积占比为1.46％)时对鲎试剂与细菌内毒素的凝集反应无干扰，故使用灵敏度λ为0.25EU/ml的鲎试剂进行试验。As can be seen from Example 14, in the test solution, when the concentration of triamcinolone acetonide is below 0.0834 mg/ml (the volume ratio of dimethyl sulfoxide is 1.46%), there is no interference with the agglutination reaction of Limulus reagent and bacterial endotoxin , so the test was carried out using the limulus reagent with a sensitivity λ of 0.25 EU/ml.

经计算，此时供试品溶液中，二甲基亚砜的体积占比为1.46％，也即100ml供试品溶液中含有1.46ml二甲基亚砜。After calculation, at this time, in the test solution, the volume ratio of dimethyl sulfoxide is 1.46%, that is, 100 ml of the test solution contains 1.46 ml of dimethyl sulfoxide.

据此设计二甲基亚砜干扰试验如下：Accordingly, the DMSO interference test was designed as follows:

将二甲基亚砜用细菌内毒素检查用水制成原液和2，4，8，16，32，64倍，128倍稀释液作溶液A系列，取相应浓度二甲基亚砜稀释液加细菌内毒素溶液制成含细菌内毒素浓度为2λ，二甲基亚砜稀释2，4，8，16，32，64倍的供试品阳性对照溶液B系列；另取2管加BET检查用水作阴性对照D，2管加2λ浓度的细菌内毒素溶液作阳性对照C。取两个不同厂家灵敏度分别为0.25EU/ml和0.125EU/ml的鲎试剂与上述系列溶液进行反应，每一浓度重复2管。结果如下述表14所示。Use dimethyl sulfoxide to test water for bacterial endotoxin to make the original solution and 2, 4, 8, 16, 32, 64 times, 128 times dilutions as solution A series, take the corresponding concentration of dimethyl sulfoxide dilution and add bacteria The endotoxin solution was prepared to contain bacterial endotoxin concentration of 2λ, dimethyl sulfoxide was diluted 2, 4, 8, 16, 32, 64 times the positive control solution B series of the test sample; another 2 tubes were added with BET water for inspection. Negative control D, 2 tubes plus 2λ concentration of bacterial endotoxin solution as positive control C. Take two limulus reagents with sensitivities of 0.25EU/ml and 0.125EU/ml from different manufacturers to react with the above series of solutions, and repeat 2 tubes for each concentration. The results are shown in Table 14 below.

表14：二甲基亚砜细菌内毒素检查干扰试验结果Table 14: DMSO Bacterial Endotoxin Interference Test Results

由上表可知，二甲基亚砜干扰试验采用的两个厂家灵敏度为0.25EU/ml的鲎试剂阴性对照(NC)为阴性，阳性对照(PC)为阳性，试验有效。结果显示二甲基亚砜稀释16倍以上(二甲基亚砜在二甲基亚砜稀释液中的体积占比为6.25％)时无干扰，所以在供试品溶液中，二甲基亚砜的体积占比为1.46％时，对曲安奈德注射液细菌内毒素检查无干扰。It can be seen from the above table that the negative control (NC) of the two manufacturers with a sensitivity of 0.25 EU/ml used in the dimethyl sulfoxide interference test is negative, the positive control (PC) is positive, and the test is effective. The result shows that there is no interference when dimethyl sulfoxide is diluted more than 16 times (the volume ratio of dimethyl sulfoxide in dimethyl sulfoxide dilution is 6.25%), so in need testing solution, dimethyl sulfoxide When the volume ratio of sulfone is 1.46%, there is no interference to the bacterial endotoxin test of triamcinolone acetonide injection.

实施例16二甲基亚砜的水溶液正式干扰试验The formal interference test of the aqueous solution of embodiment 16 dimethyl sulfoxide

分别用3批曲安奈德注射液，按《中国药典》2020年版四部通则(1143细菌内毒素检查法)中干扰试验要求以实施例15中方法制备供试品溶液进行考察进行考察，阳性对照/供试品溶液B、阳性对照/内检水溶液C每个浓度平行4管，供试品溶液A、阴性对照溶液D每个浓度平行2管进行试验，结果如下述表15所示。With 3 batches of triamcinolone acetonide injection respectively, according to " Chinese Pharmacopoeia " 2020 edition four general rules (1143 bacterial endotoxin examination method) in interference test requirement, prepare need testing solution with the method in embodiment 15 and investigate and investigate, positive control/ Need testing solution B, positive control/internal inspection solution C each concentration parallel 4 tubes, need testing solution A, negative control solution D each concentration parallel 2 tubes to carry out the test, the results are shown in the following table 15.

表15：曲安奈德注射液细菌内毒素检查干扰确认试验Table 15: Triamcinolone Acetonide Injection Bacterial Endotoxin Inspection Interference Confirmation Test

由上表可知，曲安奈德注射液正式干扰实验中A、D为阴性，两个厂家鲎试剂标示灵敏度对照系列结果λc在0.5λ～2λ范围，符合鲎试剂灵敏度复核试验要求，试验有效。两个厂家鲎试剂干扰试验系列结果Et在0.5λc～2λc之间，符合规定。另外两批曲安奈德注射液结果也符合要求。说明采用供试品溶液中，二甲基亚砜的体积占比为1.46％时，对检查曲安奈德注射液细菌内毒素无干扰。It can be seen from the above table that in the official interference test of triamcinolone acetonide injection, A and D are negative, and the results of the two manufacturers' LAL reagent sensitivity control series results λc are in the range of 0.5λ-2λ, which meets the requirements of the LAL reagent sensitivity recheck test, and the test is valid. The results of the TAL interference test series of two manufacturers' Et ranged from 0.5λc to 2λc, which met the regulations. The results of the other two batches of triamcinolone acetonide injection also met the requirements. Explain that in the test solution, when the volume ratio of dimethyl sulfoxide is 1.46%, there is no interference in checking the bacterial endotoxin of triamcinolone acetonide injection.

综上所述，由上述实施例可知，通过采用有机溶剂和水对曲安奈德注射液进行溶解，能够得到澄清溶液，将该澄清溶液稀释成供试品溶液进行预干扰试验，可以确定溶解后的曲安奈德注射液在进行细菌内毒素检测时所采用的有机溶剂的浓度，在该浓度下，该有机溶剂不会对曲安奈德注射液内毒素检测产生干扰。由此可以利用确定的有机溶剂的浓度的水溶液对曲安奈德注射液进行溶解，来对曲安奈德注射液内毒素进行检测。一方面，能够将该曲安奈德注射液溶解至澄清，便于后续曲安奈德注射液中内毒素的准确检测。另一方面，在采用上述确定的有机溶剂的浓度的水溶液对曲安奈德注射液进行内毒素检测时，不会对内毒素检测造成干扰，进一步提高内毒素检测准确性。In summary, it can be seen from the above examples that a clear solution can be obtained by dissolving triamcinolone acetonide injection with organic solvent and water, and the clear solution can be diluted into the test solution for pre-interference test, and it can be determined that after dissolution The concentration of the organic solvent used in the bacterial endotoxin detection of the triamcinolone acetonide injection, at this concentration, the organic solvent will not interfere with the endotoxin detection of the triamcinolone acetonide injection. Thus, the triamcinolone acetonide injection can be dissolved in an aqueous solution with a determined concentration of an organic solvent to detect the endotoxin of the triamcinolone acetonide injection. On the one hand, the triamcinolone acetonide injection can be dissolved until clear, which facilitates the accurate detection of endotoxin in the subsequent triamcinolone acetonide injection. On the other hand, when the aqueous solution with the concentration of the organic solvent determined above is used for the endotoxin detection of the triamcinolone acetonide injection, it will not interfere with the endotoxin detection, further improving the accuracy of the endotoxin detection.

以上对本发明做了示例性的描述，应该说明的是，在不脱离本发明的核心的情况下，任何简单的变形、修改或者其他本领域技术人员能够不花费创造性劳动的等同替换均落入本发明的保护范围。The present invention has been described as an example above, and it should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacements that can be made by those skilled in the art without creative labor all fall within the scope of this invention. protection scope of the invention.

Claims (7)

1. A method for detecting bacterial endotoxin in triamcinolone acetonide injection is characterized by comprising the following steps:

step (1): dissolving triamcinolone acetonide injection by using an organic solvent and inspection water, wherein the organic solvent is selected from any one of methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide; and

step (2): taking the dissolved triamcinolone acetonide injection as a test sample stock solution, diluting the test sample stock solution with test water, and determining the concentration of an organic solvent adopted by the dissolved triamcinolone acetonide injection in the detection of bacterial endotoxin;

and (3): and dissolving the triamcinolone acetonide injection by adopting the organic solvent water solution with the determined organic solvent concentration, and detecting bacterial endotoxin in the dissolved triamcinolone acetonide injection.

2. The method of claim 1,

in the triamcinolone acetonide injection, each 1ml of the triamcinolone acetonide injection comprises 40mg of triamcinolone acetonide and 0.63% of sodium carboxymethylcellulose;

when the organic solvent is methanol, the addition amount of the methanol corresponding to each 1mg of triamcinolone acetonide is 0.196 ml-0.204 ml;

when the organic solvent is ethanol, the addition amount of the ethanol corresponding to each 1mg of triamcinolone acetonide is 0.135 ml-0.140 ml;

when the organic solvent is n-propanol, the addition amount of the n-propanol corresponding to every 1mg of triamcinolone acetonide is 0.135 ml-0.140 ml;

when the organic solvent is isopropanol, the addition amount of the isopropanol corresponding to each 1mg of triamcinolone acetonide is 0.147 ml-0.153 ml;

when the organic solvent is dimethyl sulfoxide, the addition amount of the dimethyl sulfoxide corresponding to each 1mg of triamcinolone acetonide is 0.172 ml-0.178 ml.

3. The method of claim 1,

in the step (1), dissolving the triamcinolone acetonide injection by using the organic solvent and the water for examination comprises:

dissolving the triamcinolone acetonide injection by adopting a mixed solvent of an organic solvent and water for examination;

or,

dissolving the triamcinolone acetonide injection by adopting an alternative dripping mode, wherein in the alternative dripping process, part of inspection water is firstly dripped, and then the organic solvent is dripped.

4. A dissolution kit for a triamcinolone acetonide injection, characterized by comprising:

triamcinolone acetonide injection; and

a dissolving reagent comprising an organic solvent and water for examination, wherein the organic solvent is selected from any one of methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide.

5. The dissolution kit according to claim 4,

in the triamcinolone acetonide injection, each 1ml of the triamcinolone acetonide injection comprises 40mg of triamcinolone acetonide and 0.63% of sodium carboxymethylcellulose;

when the organic solvent is methanol, the addition amount of the methanol corresponding to each 1mg of triamcinolone acetonide is 0.196 ml-0.204 ml;

when the organic solvent is ethanol, the addition amount of the ethanol corresponding to each 1mg of triamcinolone acetonide is 0.135 ml-0.140 ml;

when the organic solvent is n-propanol, the addition amount of the n-propanol corresponding to every 1mg of triamcinolone acetonide is 0.135 ml-0.140 ml;

when the organic solvent is isopropanol, the addition amount of the isopropanol corresponding to each 1mg of triamcinolone acetonide is 0.147 ml-0.153 ml;

when the organic solvent is dimethyl sulfoxide, the addition amount of the dimethyl sulfoxide corresponding to each 1mg of triamcinolone acetonide is 0.172 ml-0.178 ml.

6. Use of the dissolution kit according to claim 4 or 5 for detecting bacterial endotoxin in triamcinolone acetonide injection.

7. A kit for detecting bacterial endotoxin in triamcinolone acetonide injection is characterized by comprising:

the dissolution kit according to claim 4 or 5; and

a limulus reagent.