CN115420565A - Method for detecting bacterial endotoxin in triamcinolone acetonide injection - Google Patents
Method for detecting bacterial endotoxin in triamcinolone acetonide injection Download PDFInfo
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- CN115420565A CN115420565A CN202211008004.5A CN202211008004A CN115420565A CN 115420565 A CN115420565 A CN 115420565A CN 202211008004 A CN202211008004 A CN 202211008004A CN 115420565 A CN115420565 A CN 115420565A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Abstract
The invention relates to a method for detecting bacterial endotoxin in triamcinolone acetonide injection, which comprises the following steps of (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; 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 an organic solvent aqueous solution with determined organic solvent concentration, and detecting bacterial endotoxin in the dissolved triamcinolone acetonide injection. By the method, the triamcinolone acetonide injection can be completely dissolved, the endotoxin can be completely released, the phenomenon that the endotoxin cannot be detected because of being wrapped is avoided, and the detection accuracy is improved.
Description
Technical Field
The invention belongs to the technical field of endotoxin detection, and particularly relates to a method for detecting bacterial endotoxin in triamcinolone acetonide injection.
Background
Bacterial endotoxin is a common pyrogen impurity in injection, and if the content of bacterial endotoxin exceeds a certain limit, the body temperature of a user can be raised, and symptoms such as fever can appear. As a key quality attribute in injection solutions, strict control is required. The limulus reagent method, particularly the gel method, for detecting bacterial endotoxin has been the mainstream method for detecting bacterial endotoxin recorded in pharmacopoeias of various countries since the advent.
As indicated in the Chinese pharmacopoeia (2020 edition, four departments) 9251 guidelines for the use of bacterial endotoxin test methods, bacterial endotoxin tests should generally be carried out using samples dissolved in water for endotoxin tests. The sample with low solubility in water can be improved in solubility by adopting methods such as ultrasonic wave, heating for assisting dissolution, adding a cosolvent, adjusting pH and the like. The new preparation adopting the inclusion technology, such as microspheres, liposome and other test articles, adopts a proper method to destroy the inclusion body, so that the bacterial endotoxin wrapped inside is completely released, and then the detection is carried out.
Among them, the water for endotoxin test must not interfere with the test. That is, the endotoxin test water used should meet the standard for sterile water for injection, and the endotoxin content thereof is less than 0.015EU/ml (for gel method). The vessels used in the assay are treated to remove any exogenous endotoxin that may be present. The ovenware are typically removed by dry heat sterilization (250 ℃ for at least 30 minutes), and other suitable methods that do not interfere with the detection of bacterial endotoxins may be used. If plastic instruments such as a micro-porous plate and a suction head matched with a micro-sample applicator are used, instruments which are marked to be free from endotoxin and have no interference to the test are selected.
Regarding to triamcinolone acetonide injection, when the bacterial endotoxin is checked, the main drug triamcinolone acetonide is insoluble in water, and in addition, the triamcinolone acetonide injection also contains auxiliary materials such as a suspension, a surfactant, a preservative, an osmotic pressure regulator and the like, and national pharmacopoeias and related documents do not provide a proper solvent to completely dissolve the triamcinolone acetonide injection, but the injection is directly diluted with checking water for checking the bacterial endotoxin, but the main drug triamcinolone acetonide and the auxiliary materials (especially sodium carboxymethylcellulose as the suspension) are difficult to dissolve simultaneously, so that the method cannot accurately detect the endotoxin wrapped in the insoluble triamcinolone acetonide, and a great risk is brought to patients.
Disclosure of Invention
The present invention has been made in view of the above problems in the prior art, and an object of the present invention is to provide a method for detecting bacterial endotoxin in a triamcinolone acetonide injection, and to provide a dissolution kit for a triamcinolone acetonide injection and use thereof.
In one aspect, the invention provides a method for detecting bacterial endotoxin in triamcinolone acetonide injection, which comprises 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 bacterial endotoxin test;
and (3): dissolving the triamcinolone acetonide injection by adopting an organic solvent water solution with determined organic solvent concentration, and detecting bacterial endotoxin in the dissolved triamcinolone acetonide injection.
Preferably, in the step (1), every 1ml of the triamcinolone acetonide injection comprises 40mg of triamcinolone acetonide and 0.63% of sodium carboxymethyl cellulose;
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.
Preferably, in the step (1), the dissolving of the triamcinolone acetonide injection solution with the organic solvent and the check water comprises:
dissolving the triamcinolone acetonide injection by adopting a mixed solvent of an organic solvent and water for examination;
alternatively, the first and second electrodes may be,
dissolving the triamcinolone acetonide injection by adopting an alternative dropwise adding mode; wherein, in the alternating dripping process, part of the inspection water is firstly dripped, and then the organic solvent is dripped.
In another aspect, the present invention provides a kit for dissolving triamcinolone acetonide injection, 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.
In the dissolution kit, it is preferable that the triamcinolone acetonide injection comprises 40mg of triamcinolone acetonide and 0.63% of sodium carboxymethylcellulose per 1ml of the triamcinolone acetonide injection,
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.
On the other hand, the invention provides the application of the dissolving kit in detecting bacterial endotoxin in the triamcinolone acetonide injection.
In another aspect, the present invention also provides a kit for detecting bacterial endotoxin in triamcinolone acetonide injection, comprising: the dissolution kit as described above; and a limulus reagent.
Effects of the invention
According to the present invention, by dissolving triamcinolone acetonide injection with an organic solvent and test water, triamcinolone acetonide as a main drug in a triamcinolone acetonide injection suspension and an auxiliary material (particularly sodium carboxymethylcellulose as a suspension) can be completely dissolved, and a clear triamcinolone acetonide injection solution can be obtained. Compared with the prior art, the method can completely dissolve the triamcinolone acetonide injection, so that the endotoxin in the triamcinolone acetonide can be completely released, and further the phenomenon that the endotoxin is wrapped and cannot be detected out can be avoided, so that the detection accuracy is improved.
Detailed Description
The following describes in detail specific embodiments of the present invention, but the present invention is not limited thereto.
In the process of researching the difficult problem that the liquid medicine can not be completely dissolved when the limulus reagent gel method is adopted to detect the bacterial endotoxin in the triamcinolone acetonide injection, the inventor successively tries methods of ultrasonic assisted dissolution, heating assisted dissolution, pH value adjustment assisted dissolution and the like. Wherein, the triamcinolone acetonide injection with the main component of triamcinolone acetonide has a plurality of specifications (1ml.
In the above specifications, the bacterial endotoxin test water is added in a large amount and then the bacterial endotoxin test water is not dissolved by the above method.
In the case of adding a co-solvent, the inventors tried a commonly used solvent including methanol, ethanol, n-propanol, isopropanol, ethylene glycol, glycerol, diethylene glycol, dimethyl sulfoxide, dimethylformamide, acetone, chloroform, etc. As a result, it was found that: when the organic solvent is directly added, the triamcinolone acetonide injection is still in a suspension or emulsion state and cannot be completely dissolved. The inventors have not been able to solve the problem by adding surfactant solutions, such as polysorbate 80, sodium lauryl sulfate solution, etc.
Through a large number of experimental researches, the inventor finds that the aqueous solution containing methanol is added into the triamcinolone acetonide injection; or adding an aqueous solution containing ethanol; or adding an aqueous solution containing n-propanol; or adding an aqueous solution containing isopropanol; or adding water solution containing dimethyl sulfoxide to dissolve triamcinolone acetonide injection completely to clear state, and optionally ultrasonic wave or shaking to assist dissolution. Based on the test sample stock solution which is dissolved to be clear, the bacterial endotoxin in the test sample stock solution can be detected by adopting a gel method after the test sample stock solution is diluted to the required concentration by endotoxin detection water, so that the bacterial endotoxin in the triamcinolone acetonide injection can be accurately detected.
Based on the research, the invention provides a method for detecting bacterial endotoxin in triamcinolone acetonide injection, which comprises 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 an organic solvent aqueous solution with the determined concentration of the organic solvent, and detecting bacterial endotoxin in the dissolved triamcinolone acetonide injection.
The sample stock solution is a solution that can be diluted into a series of sample dilutions for testing in a preliminary interference test for endotoxin detection.
In the embodiment of the present invention, the sample solution refers to a solution obtained by dissolving triamcinolone acetonide injection with an organic solvent and test water until the solution is clear and is not diluted with test water.
The stock solution and the diluent of the sample are collectively called as sample solution.
Here, it is understood that, before detecting bacterial endotoxin in the dissolved triamcinolone acetonide injection, the method may further include: the test sample stock solution is diluted with test water, and the maximum concentration of an organic solvent for bacterial endotoxin detection is determined by a pre-interference test, so that the bacterial endotoxin detection is performed under the dissolving action of an aqueous organic solvent solution having the maximum concentration or less.
For example, if the maximum concentration of the organic solvent used for the detection of bacterial endotoxin is m as determined by the pre-interference test, the detection of bacterial endotoxin can be performed by dissolving the triamcinolone acetonide injection by using a combination of any organic solvent having a concentration of less than m and capable of dissolving the triamcinolone acetonide injection to a clear state and the water for examination.
For another example, bacterial endotoxin in the dissolved triamcinolone acetonide injection can be detected by a gel method.
The maximum concentration of the organic solvent is dependent on factors such as the sensitivity of the limulus reagent and the endotoxin limit.
After determining the limit of endotoxin in the test solution, selecting a limulus reagent with certain sensitivity, and performing a pre-interference test, wherein the pre-interference test is to test dilutions of a series of samples (such as a test solution stock solution), at each dilution concentration, a group of endotoxin-added samples and a group of endotoxin-not-added samples are provided, the endotoxin-added concentration is kept unchanged, the sample concentrations are gradually diluted, and the dilution multiple of the final sample does not exceed the maximum available dilution Multiple (MVD).
Maximum effective dilution factor (MVD) refers to: the test solution is allowed to reach the maximum fold dilution under the selected limulus reagent sensitivity conditions. During detection, endotoxin limit detection needs to be carried out at a concentration not exceeding the dilution factor, and the endotoxin limit can still be accurately detected at the maximum effective dilution factor.
Wherein, the concentration of the added endotoxin can be determined according to the limit of endotoxin. As a result of the gel method, the sample to which no endotoxin has been added should not be agglutinated, and if agglutination occurs, it indicates that endotoxin or endotoxinlike material is contained. The sample to which endotoxin has been added should be agglutinated, and the absence of agglutination indicates that the agglutination reaction of the sample is inhibited.
In some embodiments, after the limulus reagent sensitivity is obtained by the pre-interference test, the interference test can be performed on the aqueous solutions of the organic solvents with different dilution times, so as to obtain the maximum concentration of the organic solvent in the aqueous solutions of the organic solvents, which does not interfere with the test result, and the concentration of the organic solvent in the test solution is determined by taking the maximum concentration as a reference, so as to detect the bacterial endotoxin in the test solution. The concentration of the organic solvent in the test solution should be less than the maximum concentration of the organic solvent that does not interfere with the test result, and the test solution with the concentration of the organic solvent can be used for detecting bacterial endotoxin without interfering with the detection of bacterial endotoxin.
Wherein the limit of endotoxin is calculated by the formula "L = K/M", wherein L is the limit of bacterial endotoxin of the test sample (i.e. the test sample solution) expressed in EU/mg or EU/u, K is the maximum acceptable endotoxin dose per kilogram body weight per hour for human beings according to the prescribed administration route, and the unit is EU/kg. The endotoxin limit is generally determined in relation to the maximum dose administered in a clinical human, the higher the dose, the lower the endotoxin limit per weight or volume.
In some embodiments, the triamcinolone acetonide injection comprises 40mg of triamcinolone acetonide and 0.63% sodium carboxymethylcellulose per 1ml of triamcinolone acetonide injection. 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 each 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.
In these examples, it was found through experiments that in the triamcinolone acetonide injection of the above specification, the triamcinolone acetonide injection can be dissolved to be clear by the above correspondence between the addition amount of each 1mg of triamcinolone acetonide and the corresponding organic solvent.
Even more preferably, in the case where the organic solvent is methanol, 8ml of methanol is added per 1ml of triamcinolone acetonide injection. In the case where the organic solvent is ethanol, 5.5ml of ethanol is added per 1ml of triamcinolone acetonide injection. In the case where the organic solvent is n-propanol, 5.5ml of n-propanol per 1ml of triamcinolone acetonide injection is added. In the case where the organic solvent is isopropanol, 6ml of isopropanol is added per 1ml of triamcinolone acetonide injection. In the case where the organic solvent is dimethyl sulfoxide, 7ml of dimethyl sulfoxide is added per 1ml of triamcinolone acetonide injection.
In one embodiment of the present invention, the dissolving of triamcinolone acetonide injection with an organic solvent and test water in step (1) 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, and dripping part of inspection water and then dripping the organic solvent in the alternative dripping process.
In the case of the alternate dropwise addition, the triamcinolone acetonide injection can be dissolved to be clear by adding part of the examination water dropwise first and then adding the organic solvent dropwise.
In the experiment, 1ml of triamcinolone acetonide injection is accurately measured as an example, and the volume of the part of the inspection water which is dripped first can be 1ml.
In contrast, the inventors found through experiments that in the course of the alternate dropping, it is sometimes difficult to dissolve the triamcinolone acetonide injection until it is clear by dropping the organic solvent first and then dropping the inspection water.
In addition, the present invention provides a dissolution kit for triamcinolone acetonide injection, comprising: triamcinolone acetonide injection and dissolving reagent. The triamcinolone acetonide injection comprises triamcinolone acetonide serving as a main drug and can also comprise: suspending agent (sodium carboxymethylcellulose), surfactant, antiseptic, osmotic pressure regulator, etc. The dissolving reagent comprises 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. The inspection water, namely the endotoxin inspection water, has extremely low endotoxin content and does not influence the subsequent endotoxin detection. Of course, in some embodiments, the water for examination may be replaced by purified water having an endotoxin content of less than 0.015EU/ml.
In the dissolving kit provided by the invention, the triamcinolone acetonide injection is dissolved by adopting the organic solvent and the checking water, so that the triamcinolone acetonide injection can be dissolved to be clear, and compared with the related technology (namely, when the bacterial endotoxin detection is carried out on the suspension solution of the triamcinolone acetonide injection, the suspension solution cannot be completely dissolved, so that the endotoxin cannot be completely detected due to the fact that the undissolved triamcinolone acetonide main component and/or auxiliary material is wrapped by the endotoxin), the triamcinolone acetonide main component and auxiliary material can be completely dissolved, so that the endotoxin in the triamcinolone acetonide main component and auxiliary material can be completely released, the endotoxin can be prevented from being wrapped and not detected, the detection accuracy can be improved, and the risk brought to a patient can be reduced.
Further, in the case where the triamcinolone acetonide injection includes 40mg of triamcinolone acetonide and 0.63% of sodium carboxymethylcellulose per 1ml of the triamcinolone acetonide injection, when the organic solvent is methanol, the amount of methanol added per 1mg of triamcinolone acetonide is 0.196ml to 0.204ml. 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 each 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 0.147ml to 0.153ml. 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.
Experiments show that under the condition of the corresponding addition amount of the organic solvent, the corresponding amount of the triamcinolone acetonide injection can be dissolved to be clear, so that preparation can be made for accurately detecting bacterial endotoxin in the triamcinolone acetonide injection.
Even more preferably, in the case where the organic solvent is methanol, 8ml of methanol is added per 1ml of triamcinolone acetonide injection. In the case that the organic solvent is ethanol, 5.5ml of ethanol is added per 1ml of triamcinolone acetonide injection. In the case where the organic solvent is n-propanol, 5.5ml of n-propanol per 1ml of triamcinolone acetonide injection is added. In the case where the organic solvent is isopropanol, 6ml of isopropanol is added per 1ml of triamcinolone acetonide injection. In the case where the organic solvent is dimethyl sulfoxide, 7ml of dimethyl sulfoxide is added per 1ml of triamcinolone acetonide injection.
In another aspect, the present invention further provides a kit for detecting bacterial endotoxin in a triamcinolone acetonide injection, comprising: the dissolution kit as described above; and a limulus reagent.
The limulus reagent is prepared with the blood deformed cell lysate of sea arthropod limulus and contains coagulating zymogen and coagulating proteinogen capable of being activated by trace amount of bacterial endotoxin and fungal glucan, and through low temperature freeze drying, it can detect qualitatively and quantitatively whether the sample contains bacterial endotoxin and (1, 3) -beta-glucan accurately and fast.
The gel method is a method for detecting or semi-quantifying endotoxin by the principle of agglutination reaction between limulus reagent and endotoxin. The gel method is a method in which the end point of the reaction is observed as the presence or absence of gel formation. The method is simple and economical to operate, does not need special measuring equipment, and can carry out qualitative or semi-quantitative measurement.
Of course, in practical application, in order to determine the sensitivity of the limulus reagent so as to accurately detect the bacterial endotoxin in the triamcinolone acetonide injection, the kit may optionally further include an endotoxin standard solution and the like. So as to determine the sensitivity of the limulus reagent and the maximum effective dilution factor of the test sample stock solution by interference experiments.
Alternatively, in practical use, in the case where the limulus reagent is determined, the sensitivity of the limulus reagent may be determined. Thus, the bacterial endotoxin in the triamcinolone acetonide injection can be directly detected according to the sensitivity of the limulus reagent during detection.
In another aspect, the invention provides an application of the kit in detecting bacterial endotoxin in the triamcinolone acetonide injection.
The present invention will be described in more detail below by way of specific examples. It should be noted that these examples are only for illustrating the present invention, and the present invention is not limited thereto.
In the following examples, the preparation method of the test solution in the pre-interference test may be a formal interference test according to the method defined in the "Chinese pharmacopoeia" (1143 bacterial endotoxin test) or other pharmacopoeias of various countries. And is not particularly limited herein.
In the following examples, the preparation of a sample solution is described by a method specified in "Chinese pharmacopoeia" (2020 edition). Wherein the limit of bacterial endotoxin in the triamcinolone acetonide injection is not more than 3.0EU/mg. The concentration of triamcinolone acetonide in the test solution was subjected to pre-interference tests using 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
In the following examples, the organic solvents exemplified above, such as methanol, ethanol, n-propanol, isopropanol, and dimethyl sulfoxide, may be of analytical grade, chromatographic grade, or ultra-pure grade. And is not particularly limited herein. Of course, the organic solvent may be an anhydrous reagent dried according to the above specification.
Example 1
Dissolution test of triamcinolone acetonide injection (specification: 1ml, wherein, 40mg triamcinolone acetonide and 0.63% sodium carboxymethyl cellulose; batch number: ABU 8750) in different solvents:
(1) Methanol
Precisely measuring 1ml of triamcinolone acetonide injection by using a pipette, placing the triamcinolone acetonide injection into a measuring flask, adding 1ml of purified water, carrying out vortex oscillation for 5 seconds, dropwise adding 8ml of methanol, dropwise adding the purified water to a scale mark, and clarifying and transparent the solution. The volume of the solution at this time was 10ml (containing 1ml of injection solution +8ml of methanol +1ml of water, i.e., 0.2ml of methanol per 1mg of triamcinolone acetonide).
It should be noted that the volume ratios of the triamcinolone acetonide injection, the purified water and the methanol are all regarded as being accurately measured, and it can be understood by those skilled in the art that the volume ratios may have certain fluctuation along with the difference of experimental conditions, environmental temperature and the like, and the fluctuation caused by the fluctuation is also within the protection scope of the embodiments of the present disclosure.
In addition, according to the national standards for volume instruments (GBT 12806-2011 and GBT 12807-2021), the maximum allowable error of the mechanical pipettor system can be at least 0.8%, and the maximum tolerance of the commonly used grade-B volume bottle of 10ml can reach 0.4%. It is estimated that, when preparing a sample stock solution, the liquid medicine needs to be removed at least once, the organic solvent needs to be removed once, and the volumetric flask needs to be used once, so that an error of 0.8% +0.8% +0.4% =2% is introduced. From this, it was found that a clear solution was obtained in this example by performing the operation in the range of 0.196ml to 0.204ml of methanol per 1mg of triamcinolone acetonide, based on an error of. + -. 2%.
(2) Ethanol
Precisely measuring 1ml of triamcinolone acetonide injection into a 10ml measuring flask by using a pipette, adding 1ml of purified water, dropwise adding 5.5ml of ethanol, adding the purified water to constant volume of 10ml, and clarifying and transparent the solution (comprising 1ml of injection, 5.5ml of ethanol and 3.5ml of water, namely the addition of 0.1375ml of ethanol for each 1mg of triamcinolone acetonide).
For the same reason as in (1) above, it was found that a clear solution was obtained in this example in the range of 0.135ml to 0.140ml of ethanol per 1mg of triamcinolone acetonide, based on an error of. + -. 2%.
(3) N-propanol
Precisely measuring 1ml of triamcinolone acetonide injection by using a liquid transfer gun, placing the triamcinolone acetonide injection into a 10ml measuring flask, adding 1ml of purified water, carrying out vortex oscillation for 5 seconds, then dropwise adding 5.5ml of n-propanol, wherein floccules appear, dropwise adding the purified water to a constant volume to a scale line, shaking up, carrying out ultrasound for 1 minute, fading the floccules, and clarifying the solution (comprising 1ml of injection, 5.5ml of n-propanol and 3.5ml of water, namely, the addition of the n-propanol corresponding to each 1mg of triamcinolone acetonide is 0.1375 ml).
For the same reason as in (1) above, it was determined that, in this example, a clear solution was obtained by performing the operation in the range of 0.135ml to 0.140ml of n-propanol per 1mg of triamcinolone acetonide, based on an error of ± 2%.
(4) Isopropanol (I-propanol)
1ml of triamcinolone acetonide injection is precisely measured by a pipette and placed in a 10ml measuring flask, 1ml of purified water is added, vortex oscillation is carried out for 5 seconds, and after 3ml of isopropanol is dripped, the liquid is in a suspension state. After dropwise adding 3ml of isopropanol, no main medicine granular precipitate but flocculent precipitate appears. Subsequently, 2ml of purified water was added dropwise to the above solution, and the flocculent precipitate began to disappear. Purified water was then added dropwise to the scale and the solution was clear (containing 1ml of injection +6ml of isopropanol +3ml of water, i.e. 0.15ml of isopropanol per 1mg of triamcinolone acetonide).
For the same reason as in (1) above, it was found that a clear solution was obtained in this example in which 0.147ml to 0.153ml of isopropyl alcohol was added to 1mg of triamcinolone acetonide, based on an error of. + -. 2%.
(5) Dimethyl sulfoxide
Precisely measuring 1ml of triamcinolone acetonide injection by using a liquid transfer gun, placing the triamcinolone acetonide injection into a 10ml measuring flask, adding 1ml of purified water, carrying out vortex oscillation for 5 seconds, and dropwise adding 4ml of dimethyl sulfoxide, wherein no main granular medicine exists, and the solution presents opalescence. 3ml of dimethyl sulfoxide was added dropwise and purified water was added dropwise to the scale, at which time the solution was clear and transparent (containing 1ml of injection solution +7ml of dimethyl sulfoxide +2ml of water, i.e., 0.175ml of dimethyl sulfoxide per 1mg of triamcinolone acetonide).
For the same reason as in (1) above, it was found that a clear solution was obtained in this example in the range of 0.172ml to 0.178ml of dimethyl sulfoxide per 1mg of triamcinolone acetonide, based on an error of. + -. 2%.
(6) Propylene glycol
1ml of triamcinolone acetonide injection is precisely measured by a pipette and placed in a test tube, 1ml of purified water is added, vortex oscillation is carried out for 5 seconds, 2ml of propylene glycol is dripped, the propylene glycol is precipitated at the bottom of the test tube, and the solution state is not improved after vortex oscillation. After further dropwise addition of 1ml of purified water, no improvement in the dissolution state was observed. After further dropwise addition of 3ml of propylene glycol, no improvement in the dissolution state was observed and the solution had a high viscosity. After further dropwise addition of 1ml of purified water, no improvement in the dissolution state was observed. After further dropwise addition of 2ml of propylene glycol, no improvement in the dissolution state was observed. After further dropwise addition of 2ml of purified water, no improvement in the dissolution state was observed.
(7) Glycerol
1ml of triamcinolone acetonide injection is precisely measured by a pipette and placed in a test tube, 1ml of purified water is added, vortex oscillation is carried out for 5 seconds, 1ml of glycerol is dropwise added, vortex oscillation is carried out, and the solution state is not improved. After further dropwise addition of 1ml of purified water, no improvement in the dissolution state was observed. After 2ml of glycerol was added dropwise, no change in the dissolved state was observed, and the solution had a high viscosity.
(8) Ethylene glycol
1ml of triamcinolone acetonide injection is precisely measured by a pipette gun, placed in a test tube, added with 1ml of purified water, vortexed and shaken for 5 seconds, and after 2ml of ethylene glycol is dripped, vortexed and shaken, and the solution state is not improved. After further dropwise addition of 1ml of purified water, no improvement in the dissolution state was observed. After further dropwise addition of 1ml of ethylene glycol, no change in the dissolved state was observed. After further dropwise addition of 2ml of ethylene glycol, no change in the dissolved state was observed.
(9) Diethylene glycol
1ml of triamcinolone acetonide injection is precisely measured by a pipette and placed in a test tube, 1ml of purified water is added, vortex oscillation is carried out for 5 seconds, and after 2ml of diethylene glycol is dropwise added, vortex oscillation is carried out, so that the solution state is not improved. After further dropwise addition of 1ml of purified water, no improvement in the dissolution state was observed. After further dropwise addition of 2ml of ethylene glycol, no change in the dissolved state was observed. After further dropwise addition of 2ml of ethylene glycol, no change in the dissolution state was observed.
And (4) conclusion: the triamcinolone acetonide injection can be dissolved to be clear by using a specific organic solvent selected from any one of methanol, ethanol, n-propanol, isopropanol and dimethyl sulfoxide, and water for examination.
Example 2 Pre-disturb test using aqueous methanol as solvent
1ml to 10ml of pyrogen-free bottle of triamcinolone acetonide injection (same specification and batch number as example 1) is taken, 1ml of BET examination water is added, 8.0ml of methanol is added, the BET examination water is added to a constant volume of 10ml, and the solution is shaken to obtain a clear solution (namely a test sample stock solution) with the triamcinolone acetonide content of 4 mg/ml. The sample stock solution was gradually diluted with BET examination water to prepare a sample solution (NPC) containing triamcinolone acetonide at 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
And adding bacterial endotoxin solution into the test solution of triamcinolone acetonide injection with corresponding concentration to prepare a test positive control solution (PPC) containing the triamcinolone acetonide with the endotoxin concentration of 2 lambda, 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
Two limulus reagents which have been rechecked by different manufacturers according to the requirement of Chinese pharmacopoeia and have the sensitivity of 0.25EU/ml are taken to react with the series of solutions, 2 tubes are repeated at each concentration, 2 tubes of Negative Control (NC) and Positive Control (PC) are arranged respectively, and the result is shown in table 1.
Table 1: bacterial endotoxin examination pre-interference test result of triamcinolone acetonide injection
As can be seen from the above table, the limulus reagent Negative Control (NC) with a sensitivity of 0.25EU/ml was negative and the Positive Control (PC) was positive for both manufacturers, and the test was effective. When the concentration of triamcinolone acetonide in the test solution is below 0.08334mg/ml, the agglutination reaction of limulus reagent and bacterial endotoxin is not interfered, and limulus reagent with sensitivity not lower than 0.25EU/ml can be selected for the subsequent test.
Example 3 methanol interference test
From example 2, it is understood that the concentration of triamcinolone acetonide in the sample solution is 0.0834mg/ml (the volume ratio of methanol is 1.67%) or less, since the agglutination reaction between the limulus reagent and bacterial endotoxin is not interfered with, the test was performed using the limulus reagent with a sensitivity λ of 0.25 EU/ml.
The volume ratio of methanol in the test solution at this time was calculated to be 1.67%, i.e., 1.67ml of methanol was contained in 100ml of the test solution.
The methanol interference test was designed accordingly as follows:
preparing a stock solution of methanol by using water for detecting bacterial endotoxin and a series of 2,4,8, 16, 32 and 64 times of diluent as a solution A, and adding a bacterial endotoxin solution into the methanol diluent with the corresponding concentration to prepare a series of positive control solutions B containing the test sample with the bacterial endotoxin concentration of 2 lambda and the methanol dilution of 2,4,8, 16, 32 and 64 times; another 2 tubes were used as a negative control D by adding BET examination water, and another 2 tubes were used as a positive control C by adding a bacterial endotoxin solution at a concentration of 2. Lambda. Two limulus reagents with sensitivity of 0.25EU/ml and 0.125EU/ml from different manufacturers were reacted with the above series of solutions, and 2 tubes were repeated for each concentration. The results are shown in Table 2 below.
Table 2: bacterial endotoxin examination methanol interference test results:
as can be seen from the above table, the methanol interference test is effective when the limulus reagent Negative Control (NC) with the sensitivity of 0.25EU/ml is used by two manufacturers, and the test is positive when the Positive Control (PC) is used by the manufacturers. The results showed that no interference was observed when methanol was diluted 16-fold or more (the volume ratio of methanol in the methanol diluent was 6.25%), and therefore, no interference was observed when the volume ratio of methanol in the sample solution was 1.67% in the bacterial endotoxin test of triamcinolone acetonide injection.
Example 4 a formal interference test was performed with an aqueous solution of methanol:
test solutions were prepared in example 3 according to the interference test requirements in the general rules of the four parts of the "chinese pharmacopoeia" 2020 edition (1143 bacterial endotoxin test method) using 3 batches of triamcinolone acetonide injections, and the results are shown in table 3 below, where the concentrations of the positive control/test solution B and the positive control/internal test aqueous solution C were tested in parallel in 4 tubes, and the concentrations of the test solution a and the negative control solution D were tested in parallel in 2 tubes.
Table 3: triamcinolone acetonide injection (batch: ABU 8750) bacterial endotoxin test interference confirmation test:
as can be seen from the table, in the formal interference experiment of the triamcinolone acetonide injection, A and D are negative, and the marked sensitivity control series result lambdac of the limulus reagent of two manufacturers is in the range of 0.5 lambda to 2 lambda, so that the requirement of the sensitivity rechecking experiment of the limulus reagent is met, and the experiment is effective. The results Et of the interference test series of the limulus reagents of the two manufacturers are between 0.5 lambda c and 2 lambda c, which are in accordance with the regulations. In addition, the results of two batches of triamcinolone acetonide injection also meet the requirements. The method for checking the bacterial endotoxin of the triamcinolone acetonide injection has no interference when the volume percentage of the methanol in the test solution is 1.67 percent. (in the above table, N/A indicates that the cell has no contents.)
Example 5 Pre-disturb test with aqueous ethanol as solvent
Taking 1ml to 10ml pyrogen-free bottle of triamcinolone acetonide injection (batch No. ABU 8750), adding 1ml of BET inspection water, adding 5.5ml of ethanol, adding the BET inspection water to a constant volume of 10ml, shaking to obtain a clarified solution (namely a sample solution) with the triamcinolone acetonide content of 4mg/ml, and gradually diluting the sample solution with the BET inspection water to prepare a sample solution (NPC) with the triamcinolone acetonide content of 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
And adding bacterial endotoxin solution into the test solution of triamcinolone acetonide injection with corresponding concentration to prepare a test positive control solution (PPC) containing the triamcinolone acetonide with the endotoxin concentration of 2 lambda, 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
Two limulus reagents which have been rechecked by different manufacturers according to the requirement of Chinese pharmacopoeia and have the sensitivity of 0.25EU/ml are taken to react with the series of solutions, 2 tubes are repeated at each concentration, and 2 tubes of Negative Control (NC) and Positive Control (PC) are arranged respectively, and the results are shown in the following table 4.
Table 4: the water solution pre-interference test result of ethanol for checking bacterial endotoxin of triamcinolone acetonide injection comprises the following steps:
as can be seen from the above table, the limulus reagent Negative Control (NC) with a sensitivity of 0.25EU/ml was negative and the Positive Control (PC) was positive for both manufacturers, and the test was effective. When the concentration of triamcinolone acetonide in the test solution is below 0.08334mg/ml, the agglutination reaction of the limulus reagent and bacterial endotoxin is not interfered, and the limulus reagent with the sensitivity of not less than 0.25EU/ml can be selected for the subsequent test.
Example 6 interference test of ethanol:
from example 5, it is understood that the concentration of triamcinolone acetonide in the sample solution is 0.0834mg/ml or less (e.g., ethanol is 1.15% by volume) and therefore the agglutination reaction between limulus reagent and bacterial endotoxin is not disturbed, and therefore the test is carried out using limulus reagent having a sensitivity λ of 0.25 EU/ml.
The volume ratio of ethanol in the test solution is 1.15%, i.e. 1.15ml ethanol is contained in 100ml test solution.
The ethanol interference test was designed accordingly as follows:
preparing stock solution of ethanol and 2,4,8, 16, 32, 64 times of diluent as solution A series, adding ethanol diluent with corresponding concentration into bacterial endotoxin solution to prepare positive control solution B series containing 2 lambda of bacterial endotoxin concentration and 2,4,8, 16, 32, 64 times of ethanol dilution; another 2 tubes were filled with BET examination water as a negative control D, and 2 tubes were filled with a bacterial endotoxin solution at a concentration of 2. Lambda. As a positive control C. Limulus reagents with a sensitivity of 0.25EU/ml and 0.125EU/ml from two different manufacturers were reacted with the above series of solutions, repeating 2 tubes at each concentration. The results are shown in Table 5 below.
Table 5: bacterial endotoxin examination ethanol interference test results:
as can be seen from the above table, the ethanol interference test is effective when the limulus reagent Negative Control (NC) with the sensitivity of 0.25EU/ml and the Positive Control (PC) are negative and 0.25 EU/ml. The results showed that no interference was observed when ethanol was diluted 32-fold or more (the volume ratio of ethanol in the ethanol diluent was 3.125%), and therefore, no interference was observed in the bacterial endotoxin test of triamcinolone acetonide injection when the volume ratio of ethanol in the test solution was 1.15%.
Example 7 aqueous ethanol solution formal interference test
Test solutions were prepared in example 6 according to the interference test requirements in the general rules of the four parts (1143 bacterial endotoxin test method) of the "chinese pharmacopoeia" 2020 edition using 3 batches of triamcinolone acetonide injections, and the results are shown in table 6 below, where the concentrations of the positive control/test solution B and the positive control/internal test aqueous solution C were tested in parallel with 4 tubes, and the concentrations of the test solution a and the negative control solution D were tested in parallel with 2 tubes.
Table 6: interference confirmation test for bacterial endotoxin examination of triamcinolone acetonide injection:
as can be seen from the table above, A and D in the formal interference experiment of the triamcinolone acetonide injection are negative, and the limulus reagent marked sensitivity control series result lambada c of the two manufacturers is in the range of 0.5 lambada-2 lambada, so that the requirements of the limulus reagent sensitivity rechecking experiment are met, and the experiment is effective. The results Et of the interference test series of the limulus reagents of the two manufacturers are between 0.5 lambda c and 2 lambda c, which are in accordance with the regulations. The results of two batches of triamcinolone acetonide injection also meet the requirements. The test solution has no interference to the check of bacterial endotoxin in triamcinolone acetonide injection when the volume of ethanol accounts for 1.15%.
Example 8 Pre-disturb test with aqueous n-propanol as solvent
Taking 1ml to 10ml pyrogen-free bottle of triamcinolone acetonide injection (batch number ABU 8750), adding 1ml of BET inspection water, adding 5.5ml of n-propanol, adding the BET inspection water to a constant volume of 10ml, and shaking to obtain a clear solution (namely the test sample stock solution) with the triamcinolone acetonide content of 4 mg/ml. The sample stock solution was gradually diluted with BET examination water to prepare a sample solution (NPC) containing triamcinolone acetonide at 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
And adding bacterial endotoxin solution into the test solution of triamcinolone acetonide injection with corresponding concentration to prepare a test positive control solution (PPC) containing the triamcinolone acetonide with the endotoxin concentration of 2 lambda, 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
Two limulus reagents which have been rechecked by different manufacturers according to the requirement of Chinese pharmacopoeia and have the sensitivity of 0.25EU/ml are taken to react with the series of solutions, 2 tubes are repeated at each concentration, and 2 tubes of Negative Control (NC) and Positive Control (PC) are arranged respectively, and the results are shown in the following table 7.
Table 7: bacterial endotoxin examination pre-interference test result of triamcinolone acetonide injection
As can be seen from the above table, the limulus reagent Negative Control (NC) with a sensitivity of 0.25EU/ml was negative and the Positive Control (PC) was positive for both manufacturers, and the test was effective. When the concentration of triamcinolone acetonide in the test solution is below 0.08334mg/ml, the agglutination reaction of the limulus reagent and bacterial endotoxin is not interfered, and the limulus reagent with the sensitivity of not less than 0.25EU/ml can be selected for the subsequent test.
Example 9 interference test of n-propanol
From example 8, it is understood that the concentration of triamcinolone acetonide in the sample solution is 0.0834mg/ml or less (the volume ratio of n-propanol is 1.15%) and the limulus reagent with a sensitivity λ of 0.25EU/ml is used for the test because the agglutination reaction between the limulus reagent and bacterial endotoxin is not disturbed.
The volume ratio of n-propanol in the test solution was calculated to be 1.15%, i.e. 1.15ml of n-propanol was contained in 100ml of the test solution.
The n-propanol interference test was designed accordingly as follows:
preparing a stock solution of n-propanol by using bacterial endotoxin detection water and a 2,4,8, 16, 32 and 64-time diluent as a solution A series, taking the n-propanol diluent with corresponding concentration and adding a bacterial endotoxin solution to prepare a positive control solution B series of a test sample with the bacterial endotoxin concentration of 2 lambda and the n-propanol dilution of 2,4,8, 16, 32 and 64 times; another 2 tubes were used as a negative control D by adding BET examination water, and another 2 tubes were used as a positive control C by adding a bacterial endotoxin solution at a concentration of 2. Lambda. Two limulus reagents with sensitivity of 0.25EU/ml and 0.125EU/ml from different manufacturers were reacted with the above series of solutions, and 2 tubes were repeated for each concentration. The results are shown in Table 8 below.
Table 8: test result of n-propanol interference in bacterial endotoxin test
As can be seen from the above table, the n-propanol interference test is effective when the limulus reagent Negative Control (NC) with the sensitivity of 0.25EU/ml is used by two manufacturers, and the Positive Control (PC) is positive. The results showed that the test solution was not affected when diluted 32-fold or more with n-propanol (the volume ratio of n-propanol in the n-propanol diluent was 3.125%), and therefore, when the volume ratio of n-propanol in the test solution was 1.15%, the test solution was not affected when the bacterial endotoxin of the triamcinolone acetonide injection was checked.
Example 10 a formal interference test was performed using an aqueous solution of n-propanol:
test solutions were prepared in example 9 using 3 batches of triamcinolone acetonide injections according to the interference test requirements in the general rule of the four departments of the 2020 edition of the Chinese pharmacopoeia (1143 bacterial endotoxin test method), and the results are shown in table 9 below, where each concentration of the positive control/test solution B and the positive control/internal test aqueous solution C was tested in 4 tubes, and each concentration of the test solution A and the negative control solution D was tested in 2 tubes.
Table 9: interference confirmation test for bacterial endotoxin examination of triamcinolone acetonide injection
As can be seen from the table above, A and D in the formal interference experiment of the triamcinolone acetonide injection are negative, and the limulus reagent marked sensitivity control series result lambada c of the two manufacturers is in the range of 0.5 lambada-2 lambada, so that the requirements of the limulus reagent sensitivity rechecking experiment are met, and the experiment is effective. The results Et of the interference test series of the two manufacturer limulus reagents are between 0.5 lambda c and 2 lambda c, which meet the requirements. The results of two batches of triamcinolone acetonide injection also meet the requirements. The test solution has no interference to the check of bacterial endotoxin in triamcinolone acetonide injection when the volume percentage of the n-propanol is 1.15%.
Example 11 pre-disturb test using an aqueous solution of isopropanol as solvent:
1ml to 10ml of pyrogen-free bottle of triamcinolone acetonide injection (batch number: ABU 8750) is taken, 1ml of BET examination water is added, 6.0ml of isopropanol is added, the BET examination water is added to the solution to be determined to 10ml, and the solution is shaken to obtain a clear solution (namely the test sample stock solution) with the triamcinolone acetonide content of 4 mg/ml. The sample stock solution was gradually diluted with BET examination water to prepare a sample solution (NPC) containing triamcinolone acetonide at 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
And adding bacterial endotoxin solution into the test solution of triamcinolone acetonide injection with corresponding concentration to prepare a test positive control solution (PPC) containing the triamcinolone acetonide with the endotoxin concentration of 2 lambda, 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
Two limulus reagents which have been re-checked by different manufacturers according to the requirement of Chinese pharmacopoeia and have the sensitivity of 0.25EU/ml are taken to react with the series of solutions, 2 tubes are repeated at each concentration, and 2 tubes of Negative Control (NC) and Positive Control (PC) are arranged respectively, and the results are shown in the following table 10.
Table 10: bacterial endotoxin examination pre-interference test result of triamcinolone acetonide injection
As can be seen from the above table, the limulus reagent Negative Control (NC) with a sensitivity of 0.25EU/ml was negative and the Positive Control (PC) was positive for both manufacturers, and the test was effective. When the concentration of triamcinolone acetonide in the test solution is below 0.08334mg/ml, the agglutination reaction of limulus reagent and bacterial endotoxin is not interfered, and limulus reagent with sensitivity not lower than 0.25EU/ml can be selected for the subsequent test.
EXAMPLE 12 interference test of isopropyl alcohol
From example 11, it is understood that the concentration of triamcinolone acetonide in the sample solution is 0.0834mg/ml or less (the volume ratio of isopropyl alcohol is 1.25%) and the limulus reagent with a sensitivity λ of 0.25EU/ml is used for the test since the agglutination reaction between the limulus reagent and bacterial endotoxin is not disturbed.
The volume of isopropanol in the test solution at this point was calculated to be 1.25%, i.e., 100ml of test solution contained 1.25ml of isopropanol.
The isopropanol interference test was designed accordingly as follows:
preparing isopropanol into stock solution and 2,4,8, 16, 32, 64 times of diluent as solution A series by using bacterial endotoxin detection water, and adding the isopropanol diluent with corresponding concentration into the bacterial endotoxin solution to prepare a positive control solution B series containing the test sample with the bacterial endotoxin concentration of 2 lambda and 2,4,8, 16, 32, 64 times of the isopropanol diluent; another 2 tubes were used as a negative control D by adding BET examination water, and another 2 tubes were used as a positive control C by adding a bacterial endotoxin solution at a concentration of 2. Lambda. Limulus reagents with a sensitivity of 0.25EU/ml and 0.125EU/ml from two different manufacturers were reacted with the above series of solutions, repeating 2 tubes at each concentration. The results are shown in table 11 below.
Table 11: isopropanol bacterial endotoxin inspection interference test result
As can be seen from the above table, two limulus reagent Negative Controls (NC) with a sensitivity of 0.25EU/ml for the isopropanol interference test were negative, the Positive Control (PC) was positive, and the test was effective. The results show that no interference occurs when isopropanol is diluted by more than 32 times (the volume ratio of isopropanol in the isopropanol diluent is 3.125%), so that no interference occurs when the volume ratio of isopropanol in the test solution is 1.25% in the bacterial endotoxin test of the triamcinolone acetonide injection.
Example 13 aqueous solution of isopropanol formal interference test:
test solutions were prepared and examined by the method of example 12 according to the interference test requirements in the general rules of the four parts (1143 bacterial endotoxin test method) of the "chinese pharmacopoeia" 2020 edition using 3 batches of triamcinolone acetonide injections, each concentration of the positive control/test solution B and the positive control/internal test aqueous solution C was tested in parallel with 4 tubes, and each concentration of the test solution a and the negative control solution D was tested in parallel with 2 tubes, and the results are shown in table 12.
Table 12: interference confirmation test for bacterial endotoxin examination of triamcinolone acetonide injection
As can be seen from the table above, A and D in the formal interference experiment of the triamcinolone acetonide injection are negative, and the limulus reagent marked sensitivity control series result lambada c of the two manufacturers is in the range of 0.5 lambada-2 lambada, so that the requirements of the limulus reagent sensitivity rechecking experiment are met, and the experiment is effective. The results Et of the interference test series of the limulus reagents of the two manufacturers are between 0.5 lambda c and 2 lambda c, which are in accordance with the regulations. The results of two batches of triamcinolone acetonide injection also meet the requirements. The test solution has no interference to the check of bacterial endotoxin in triamcinolone acetonide injection when the volume ratio of isopropanol in the test solution is 1.25%.
Example 14 pre-perturbation test with dimethyl sulfoxide as solvent:
1ml to 10ml of pyrogen-free bottle of triamcinolone acetonide injection (batch number: ABU 8750) is taken, 1ml of BET examination water is added, 7.0ml of dimethyl sulfoxide is added, the volume of the BET examination water is kept to 10ml, and the solution is shaken to obtain a clear solution (namely the test sample stock solution) with the triamcinolone acetonide content of 4 mg/ml. The sample stock solution was gradually diluted with BET examination water to prepare a sample solution (NPC) containing triamcinolone acetonide at 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
And adding bacterial endotoxin solution into the test sample solution of the triamcinolone acetonide injection with corresponding concentration to prepare a test sample positive control solution (PPC) with endotoxin concentration of 2 lambda, 0.08334mg/ml, 0.04167mg/ml, 0.02084mg/ml and 0.01042 mg/ml.
Two limulus reagents which have been re-checked by different manufacturers according to the requirement of Chinese pharmacopoeia and have the sensitivity of 0.25EU/ml are taken to react with the series of solutions, 2 tubes are repeated at each concentration, and 2 tubes of Negative Control (NC) and Positive Control (PC) are arranged respectively, and the results are shown in the following table 13.
Table 13: bacterial endotoxin examination pre-interference test result of triamcinolone acetonide injection
As can be seen from the above table, the limulus reagent Negative Control (NC) with a sensitivity of 0.25EU/ml was negative and the Positive Control (PC) was positive for both manufacturers, and the test was effective. When the concentration of triamcinolone acetonide in the test solution is below 0.08334mg/ml, the agglutination reaction between limulus reagent and bacterial endotoxin is not interfered, and limulus reagent with sensitivity not lower than 0.25EU/ml can be selected for the subsequent test.
Example 15 interference test of dimethyl sulfoxide
From example 14, it is understood that the test was carried out using a limulus reagent having a sensitivity λ of 0.25EU/ml because triamcinolone acetonide at a concentration of 0.0834mg/ml or less (volume ratio of dimethyl sulfoxide of 1.46%) does not interfere with the agglutination reaction between the limulus reagent and bacterial endotoxin in the test solution.
The volume ratio of the dimethyl sulfoxide in the test solution at this time was calculated to be 1.46%, i.e., 1.46ml of dimethyl sulfoxide was contained in 100ml of the test solution.
The dmso interference test was designed accordingly as follows:
preparing dimethyl sulfoxide into stock solution and 2,4,8, 16, 32, 64-fold and 128-fold diluent by using bacterial endotoxin detection water as a solution A series, and adding bacterial endotoxin solution into the dimethyl sulfoxide diluent with corresponding concentration to prepare a test article positive control solution B series containing 2 lambda of bacterial endotoxin and 2,4,8, 16, 32 and 64-fold dilution of the dimethyl sulfoxide; another 2 tubes were used as a negative control D by adding BET examination water, and another 2 tubes were used as a positive control C by adding a bacterial endotoxin solution at a concentration of 2. Lambda. Two limulus reagents with sensitivity of 0.25EU/ml and 0.125EU/ml from different manufacturers were reacted with the above series of solutions, and 2 tubes were repeated for each concentration. The results are shown in table 14 below.
Table 14: test results of interference of endotoxin examination in dimethyl sulfoxide bacteria
As can be seen from the above table, two limulus reagent Negative Controls (NC) with the manufacturer sensitivity of 0.25EU/ml adopted in the DMSO interference test are negative, the Positive Control (PC) is positive, and the test is effective. The results showed that the test solution was not affected when diluted 16-fold or more (volume ratio of dimethyl sulfoxide in dimethyl sulfoxide diluted solution was 6.25%), and therefore, when the volume ratio of dimethyl sulfoxide in the test solution was 1.46%, the test solution was not affected in bacterial endotoxin examination of triamcinolone acetonide injection.
Example 16 aqueous solution formal interference test of dimethyl sulfoxide
Test solutions were prepared and examined by the method in example 15 according to the interference test requirements in the general rule of the four departments of the 2020 edition of the Chinese pharmacopoeia (1143 bacterial endotoxin test method) using 3 batches of triamcinolone acetonide injections, each concentration of the positive control/test solution B and the positive control/internal test aqueous solution C was tested in parallel with 4 tubes, and each concentration of the test solution A and the negative control solution D was tested in parallel with 2 tubes, and the results are shown in the following table 15.
Table 15: interference confirmation test for bacterial endotoxin examination of triamcinolone acetonide injection
As can be seen from the table above, A and D in the formal interference experiment of the triamcinolone acetonide injection are negative, and the limulus reagent marked sensitivity control series result lambada c of the two manufacturers is in the range of 0.5 lambada-2 lambada, so that the requirements of the limulus reagent sensitivity rechecking experiment are met, and the experiment is effective. The results Et of the interference test series of the limulus reagents of the two manufacturers are between 0.5 lambda c and 2 lambda c, which are in accordance with the regulations. The results of two batches of triamcinolone acetonide injection also meet the requirements. The test solution adopted has no interference to the check of bacterial endotoxin in the triamcinolone acetonide injection when the volume percentage of the dimethyl sulfoxide in the test solution is 1.46 percent.
In summary, it can be seen from the above embodiments that, by dissolving triamcinolone acetonide injection with an organic solvent and water, a clear solution can be obtained, and diluting the clear solution into a test sample solution for performing a pre-interference test, the concentration of the organic solvent used in the bacterial endotoxin detection of the dissolved triamcinolone acetonide injection can be determined, and at this concentration, the organic solvent does not interfere with the endotoxin detection of the triamcinolone acetonide injection. Thus, the triamcinolone acetonide injection can be dissolved by using the aqueous solution with the determined concentration of the organic solvent, and the endotoxin of the triamcinolone acetonide injection can be detected. On one hand, the triamcinolone acetonide injection can be dissolved to be clear, so that accurate detection of endotoxin in the triamcinolone acetonide injection is facilitated. On the other hand, when the water solution with the determined organic solvent concentration is used for detecting endotoxin of the triamcinolone acetonide injection, the endotoxin detection cannot be interfered, and the accuracy of endotoxin detection is further improved.
The invention being thus described by way of example, it should be understood that any simple alterations, modifications or other equivalent alterations as would be within the skill of the art without the exercise of inventive faculty, are within the 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;
alternatively, the first and second liquid crystal display panels may be,
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.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211008004.5A CN115420565A (en) | 2022-08-22 | 2022-08-22 | Method for detecting bacterial endotoxin in triamcinolone acetonide injection |
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| CN202211008004.5A CN115420565A (en) | 2022-08-22 | 2022-08-22 | Method for detecting bacterial endotoxin in triamcinolone acetonide injection |
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