Keywords Establishment of TLC method for related substances
Related substances are impurities other than the main components in the drugs studied, which may be raw materials, intermediates, reagents, degradants, by-products, polymers, isomers and substances with different crystal forms and optical isomers brought in during the synthesis of APIs, and may also be degradants produced during the preparation process or during the storage, transportation and use [1]. The existence of these impurities directly reflects the effectiveness and safety of drugs, so it is necessary to study them, especially to establish their detection methods in the quality research data of drug declaration, and consider whether to formulate this inspection item in the quality standard and stipulate its limit according to the actual situation of production and stability assessment. At present, the commonly used methods for the determination of related substances are high performance liquid chromatography and thin layer chromatography.
Thin-layer chromatography is fast and simple, especially for the determination of impurities without ultraviolet absorption, which has more application value. If TLC and HPLC can be combined organically, or compared with each other, more and more accurate impurity information can be obtained, so that the two methods can complement each other and complement each other! This paper will focus on how to establish a method for the determination of related substances by TLC.
1. Determination method type
Commonly used methods include impurity reference method (applicable to known impurities) and self-dilution reference method (applicable to general impurity inspection, with few impurity components and no impurity reference substance at present). At present, it is difficult to obtain the impurity reference substance in China, and the self-reference method is generally used.
2. Determination of developing agent (i.e. specificity test)
Specificity study is to provide proof that analytes can be distinguished when impurities and excipients exist, and it is the key to establish chromatographic conditions. Usually, a certain amount of impurities or auxiliary materials are added to the reference substance or refined product of the analyte, which proves that impurities can be separated from the analyte under chromatographic conditions [1]. The key here is: how many main components plus how many impurities. The correct method is to add each impurity with a concentration of 1%(w/w) to the main component with a concentration of 100% to prepare such a solution.
Verify the system applicability. The purpose of this preparation is to imitate the possible state in the sample, that is, whether a small amount of impurities (about 1%) can be completely separated from the main components, and only in this way can the actual situation in the sample be objectively and scientifically reflected (see figure1); Solutions should not be formulated such that the main components and intermediates have the same concentration. Because this situation is impossible to exist in the sample when actually detecting one; It is not easy to determine the concentration of the two. At present, the general practice in domestic application materials is to prepare a low uniform concentration, so that the spots are of course easy to completely separate (see Figure 2). However, in the actual measurement, due to the sharp increase of the main spots, it is easy to contain adjacent impurities in the main component spots. Similarly, the preparation method of solution for system suitability test in quality standard is the same.
(1, 3,4 are impurities and 2 is the main component)
Figure 1 Figure 2 (impurity concentrations are all 1% of the concentration of the test solution)
3. Determination of detection conditions
The basic starting point is that the main components and related impurities should be colored under this condition, and the spot size is basically the same at the same concentration. Select the type of thin-layer plate according to the nature of the substance to be detected, and usually use GF254 or GF365 plate to determine the substance with ultraviolet absorption; Silica gel G plate or H plate is often used to ensure that there is no ultraviolet absorption and color reagent needs to be sprayed. When selecting this thin-layer plate, the color development method is selected according to the structural formula of the tested substance. However, when there are a variety of color development methods, experiments should be carried out separately to choose the color development method with the highest sensitivity. For example, the determination of hydrocortisone acetate related substances, China Pharmacopoeia in 2000 with alkaline tetrazolium blue test solution, 26 years in the United States Pharmacopoeia with sulfuric acid-ethanol (10:90) solution color development, both of which are hormone drugs color development methods. Hydrocortisone acetate belongs to adrenocortical hormone, and tetrazolium salt method is an important chromogenic method for adrenocortical hormone. However, the sulfuric acid-ethanol chromogenic rule is mainly aimed at the chromogenic reaction of estrogen in hormones, but it is not active for hydrocortisone acetate, which belongs to adrenocortical hormone. As a result, the sensitivity difference between the two methods is more than 10 times. Therefore, the determination of detection conditions must be based on literature review and comprehensive consideration according to the test results.
4. Determination of test solution concentration (sensitivity test-determination of minimum detection limit)
It is very important to set the concentration of test solution in the detection of related substances. The higher the concentration, the more it can reflect the existence of impurities in the sample. However, if the setting is too high, it will lead to serious tailing of the main spot, "broken waist" and other overload phenomena, leading to wrong conclusions; If the setting is too low, the purpose of detecting impurities can not be achieved, and the change of impurity quality can not be observed. Its setting is based on the minimum sample size and the maximum sample size.
Although the minimum detection limit is an absolute value, its real meaning is its relative value, that is, relative to the concentration of the test solution, so the determination result should list not only its absolute value, but also its relative value, so that the minimum detection limit is meaningful! The maximum sampling amount is obtained by increasing the concentration of the test solution until the main spot is seriously tailed and "broken waist" appears. Then, according to the lowest detection limit, the "up-push method" is adopted to determine: if the impurity spots are generally set to be less than 1.0% of the control spots, and the concentration of the control solution should be at least 20-50 times of the lowest detection concentration (i.e. the lowest detection limit), then the concentration of the test solution is 2000-5000 times of the lowest detection concentration; On the contrary, the minimum detection concentration should be at least 0.02%~0.05% of the concentration of the test solution. It should be noted that the minimum detection amount and the maximum sampling amount change with the test environment, thin-layer plate quality and other factors (i.e. tolerance factors), so the concentration of test solution can be set higher on the basis of ensuring that it is less than the maximum sampling amount, so as to ensure that the concentration can be applied to various conditions. See table 1 for an example (impurity limit is 1.0%).
Table 1 Proportional relationship among minimum detection amount, maximum sampling amount, concentration of test solution and control solution
Maximum sample size
test solution
Checking fluid
The minimum detection concentration is 8 mg/ml 3 mg/ml 30 μ g/ml1μ g/ml sample amount10 μ l absolute amount 30μg 0.3μg 10ng relative to the measured concentration of the sample.
The concentration of the test solution can also be set higher, but it cannot exceed the maximum sample volume.
5. Sample recovery test (i.e. accuracy test)
After the determination of related substances, the accuracy test can be evaluated by adding a known amount of impurities to the sample. Accurately weigh the impurities, add the impurities with the concentration of 65438 0% (w/w) into the test solution, and verify whether the corresponding impurities can be separated and detected under the condition of thin-layer determination, whether the existing impurities in the sample have accumulated and whether the spots have deepened. This principle is consistent with the specificity study mentioned above.
6. Strong failure test
The purpose of this study is to reveal the inherent stability characteristics of APIs, which is part of the development research. These tests are carried out under more stringent conditions than accelerated tests, which can include strict conditions encountered in the process of drug sales. A batch of samples can be tested under strong light, high temperature, high humidity, oxidation damage and acid-base damage, which proves that the developing conditions can separate and detect impurities.
7. Deployment distance
When measuring, a thin plate with a length of 25cm must be used, and the spreading distance should be as long as possible, so that impurities and main components can be separated as much as possible. If a short board is used, it is easy to cause impurity spots near the main spot to be "hidden" in the main spot. But at the same time, it should be noted that when the distance becomes larger and the light spot becomes scattered, the minimum detection limit will be lost and the sensitivity will be reduced, which should be considered comprehensively.
8. Other factors
The blending temperature should be controlled between 20 ~ 25℃ as far as possible, especially in winter, and attention should be paid to the ambient temperature. If it is too low, the deployment effect will be seriously affected. In addition, the cover of the chromatographic cylinder should be coated with vaseline oil to ensure the sealing of the chromatographic cylinder in the whole test process and avoid the evaporation of the developing agent; Before developing, the developing agent should be poured in advance to saturate the air in the chromatographic column to achieve the best developing effect. Thin-layer board is self-made and sold in the market, so we should pay attention to different quality.
Two. discuss
1. For the system suitability test in the quality standard, it is best to prepare the most difficult-to-separate impurity into solution for the system suitability test, and prepare the concentration of the impurity to 65438 0%, or 0.5%, or 0.2% (depending on the impurity limit) for the test, and then determine the sample after verifying the separation degree to ensure the accuracy of the test.
2. A series of concentration control solutions (gradient control) should be prepared in the quality standard, which has the concept of "semi-quantitative" for impurities and can better evaluate the existence of impurities; The quantity of impurities and the limit of maximum impurity spots should be specified to make the quality standard more perfect and scientific. After consulting, there are 70 varieties of related substances determined by TLC in China Pharmacopoeia, and only 2 varieties adopt gradient control. Most varieties have only formulated the control solution, without specifying the quantity of impurities and the maximum limit of impurity spots. If there are several impurity points, you can't be sure. However, British Pharmacopoeia and American Pharmacopoeia adopt gradient control for almost every variety, and stipulate the maximum number of impurities and the maximum limit of impurity spots, which is worth learning and popularizing.
3. The mistake is that HPLC completely replaced TLC, which is incorrect. It is the most objective and scientific to complement, demonstrate and refer to each other!
This paper is based on the reference to Japan's "Verification of Analytical Methods" and a large number of Japanese domestic new drug application materials, and the Quality Research Department.
The content written is divided into two parts.