Analytical models for thin-layer chromatography, their establishment methods, and applications

Abstract

This invention discloses an analytical model for thin-layer chromatography, its establishment method, and its application, belonging to the field of traditional Chinese medicine identification technology. The analytical model obtained by this method uses the H-value range of standard hues and color system channels to digitally determine the presence or absence of spots from a dual perspective, enabling an objective and quantitative evaluation method that reduces the bias of subjective human evaluation results.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine identification technology, and in particular to an analytical model for thin-layer chromatography, its establishment method, and its application. Background Technology

[0002] Thin-layer chromatography (TLC) technology originated in the 1950s and was rapidly adopted in the 1980s. TLC is an important method for evaluating the authenticity of processed Chinese medicinal herbs and the integrity of raw materials used in prepared Chinese medicines. It has advantages such as ease of operation, cost-effectiveness, speed, and wide applicability, and plays an irreplaceable role in the methodology system of Chinese medicine quality standards.

[0003] Currently, the standard for evaluating thin-layer chromatography (TLC) results is defined as follows: the test sample chromatogram should show spots of the same color at the corresponding positions as the reference medicinal material or reference standard TLC chromatograms. However, in practical applications, due to the vague description of the standard and the lack of objective, quantifiable numerical values, testing personnel need to rely on visual observation and sensory evaluation of various information such as the color of the characteristic spots in TLC chromatograms. Since different testing personnel have different understandings and evaluation criteria of the standard, this can easily lead to discrepancies in test conclusions. Summary of the Invention

[0004] In view of the current problems that the evaluation standards for thin-layer chromatography are relatively vague, lack objective and quantifiable numerical specifications, and often result in differences in sensory evaluations among different testing personnel, this invention provides a method for establishing an analytical model for thin-layer chromatography. The analytical model obtained by this method can reduce the bias of subjective human evaluation results through objective and quantitative evaluation.

[0005] On one hand, the present invention provides a method for establishing an analytical model for thin-layer chromatography, including a method for establishing a spot presence / absence determination model, wherein the method for establishing the spot presence / absence determination model includes the following steps:

[0006] Thin-layer chromatogram collection: Collect thin-layer chromatograms of qualified and unqualified Chinese medicine products. The thin-layer chromatograms of unqualified products have missing characteristic spots. Record the presence and absence of characteristic spots. The thin-layer chromatograms include several thin-layer chromatograms under differential experimental conditions. The differential experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Chinese medicine products.

[0007] Standard hue determination: Collect the H value of each characteristic spot in the HSB system from the thin-layer chromatogram of all qualified products, and use the range of H values ​​of the corresponding characteristic spot in the thin-layer chromatogram of each qualified product as the standard H value range of that characteristic spot;

[0008] Color system and channel determination: The channel values ​​of the regions corresponding to each characteristic spot in all thin-layer chromatograms were collected in the R, G, and B channels of the RGB color space, the H, S, and B channels of the HSB color space, and the L, a, and b channels of the CIE-Lab color space. All thin-layer chromatograms were randomly divided into a test group and a validation group. The channel value ranges of missing characteristic spots and the channel value ranges of present characteristic spots in the test group were obtained. The minimum value of the channel value range of present characteristic spots in the test group was used as the analysis threshold. The channel values ​​of characteristic spots in the validation group were compared with the analysis threshold. When the channel value of a characteristic spot was greater than the analysis threshold, it was determined that the characteristic spot was present. This determination result was compared and verified with the records of missing and present characteristic spots. The channel with the higher determination accuracy was used as the analysis channel of the characteristic spot, and the analysis threshold of the analysis channel was used as the channel determination standard for the characteristic spot.

[0009] Establish a spot presence / absence judgment model: The channel judgment criteria and the standard H value range together constitute the spot presence / absence judgment criteria, which is obtained.

[0010] The inventors considered using a digital standard to regulate the identification process of spot presence / absence. Since different thin-layer chromatography spots exhibit different colors, and spots of varying shades are difficult to standardize using a unified digital standard, they initially considered comparing the values ​​of different channels in different color spaces to find a color space channel that could accurately identify the presence / absence of a specific spot. However, in practice, it was found that using only a single criterion could easily lead to spots with significantly different shades (potentially indicating changes in composition) being judged as acceptable. Therefore, after exploration and adjustment, the H value from the HSB system was added as a prerequisite for judgment. That is, the hue must first meet the requirements before using the channel values ​​of the color space to determine the presence / absence of a spot. If the H value is outside the standard range, indicating a hue different from the standard, it cannot be considered an acceptable spot and can be judged as a "missing" acceptable spot.

[0011] By using the channel judgment standard and the standard H value range together to form the spot presence or absence judgment standard, the digital model can accurately judge the spots and reduce the bias of the results of human subjective evaluation.

[0012] In some of these schemes, the characteristic spot is determined by the location of a control spot obtained by thin-layer chromatography using a reference standard or a reference medicinal material under the same conditions.

[0013] In some of these schemes, the characteristic spot is determined by a preset Rf value range. When a spot falls into the preset Rf value range, it is considered a characteristic spot of this range. The preset Rf value range is defined by the Rf value range of the corresponding characteristic spot in the thin-layer chromatograms of several qualified products.

[0014] Understandably, the above methods for determining the location of characteristic spots can be selected according to specific circumstances. For example, if the goal is to achieve fully digital evaluation and avoid using reference standards or reference medicinal materials to reduce testing costs or protect rare medicinal materials, the method of defining the Rf value range of the corresponding characteristic spot in the thin-layer chromatograms of several qualified samples has obvious advantages.

[0015] In some of these schemes, the channel values ​​are calibrated by subtracting the background value of the corresponding channel from the extracted channel values.

[0016] In some of these schemes, during the standard hue determination step, characteristic spots with the same hue are grouped into the same hue group. Each characteristic spot in this hue group shares a common standard H value range, which is the H value range of all characteristic spots in that group in the thin-layer chromatogram of the qualified product. Preferably, the method for determining whether hues are the same is as follows: the thin-layer chromatogram is distributed to at least three inspectors, who independently observe and judge the hue of the characteristic spots. If all inspectors judge the hues to be the same, then the hues are considered the same. If at least one inspector's judgment differs from that of the other inspectors, then the hues are considered different, and the groups are regrouped until all inspectors judge the characteristic spots in the same hue group to be the same.

[0017] In some of these schemes, the determination accuracy is calculated by dividing the number of spots in the validation group whose determination results match the records of missing and present feature spots by the total number of feature spots in the validation group.

[0018] In some of these schemes, the method for determining the presence or absence of characteristic spots is as follows: the thin-layer chromatogram is distributed to at least two inspectors, who independently observe and determine whether the characteristic spots are present or absent. If all inspectors determine that the spots are present, or if at least one inspector's judgment is inconsistent with that of the other inspectors, then the spots are considered present. If all inspectors determine that the spots are absent, then the spots are considered absent. Preferably, the presence of characteristic spots includes being clearly visible or faintly visible. The standard for being clearly visible is: if all inspectors determine that the spots are present, then the characteristic spot is considered clearly visible. The standard for being faintly visible is: if at least one inspector's judgment is inconsistent with that of the other inspectors, then the characteristic spot is considered faintly visible.

[0019] In some of these schemes, the methods for establishing the analytical models for thin-layer chromatography described above meet at least one of the following conditions:

[0020] (1) The Chinese medicine varieties mentioned include Chinese medicinal decoction pieces and / or Chinese medicine preparations;

[0021] (2) The differential experimental conditions also include at least one of the following conditions: at least two specifications of thin-layer plates, at least two experimental dates, at least two development times, and at least two operators;

[0022] (3) The characteristic spots meet the following requirements:

[0023] A: The characteristic spots are present in both the chromatogram of the sample of this Chinese herbal medicine and the chromatogram of the reference herb or reference standard, and their positions correspond;

[0024] B: The characteristic spots are regular in shape, have clear boundaries, and exist stably;

[0025] C: The specific displacement value of the characteristic spots is 0.05 to 0.8, preferably 0.1 to 0.8, more preferably 0.2 to 0.8; and

[0026] D: The characteristic spots are not affected by negative light and are specific.

[0027] In some of these schemes, the method for establishing the analytical model for thin-layer chromatography also includes a method for establishing a spacing ratio determination model, which includes the following steps:

[0028] Establishment of the Spacing Ratio Judgment Model: A standard chromatogram library is formed using thin-layer chromatograms of qualified Chinese herbal medicine products. At least two representative spots from the thin-layer chromatograms of the qualified products are selected as analytical spots. With or without the origin, characteristic spots are formed. The characteristic spots are grouped to obtain characteristic spot groups of three. In each characteristic spot group, one characteristic spot is used as the positioning spot. The distances between the other two characteristic spots and the positioning spot are calculated. The ratio between the minimum distance and the maximum distance is calculated and denoted as the spacing ratio RPf. The spacing ratio RPf is used as the evaluation index. Based on the spacing ratio RPf of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library, the evaluation standard range of the spacing ratio RPf is established, thus obtaining the spacing ratio judgment model.

[0029] In thin-layer chromatography (TLC), the retardation factor (Rf) is typically used to represent the relative distance a substance has traveled for compound identification. However, experimental temperature and humidity, silica gel G plates, and the specifications and batches of polyamide films can all affect the Rf value, leading to inaccuracies in Rf comparisons of the same component between different laboratories. The relative retardation factor (RRf) eliminates systematic errors to some extent, exhibiting better repeatability and reliability than the Rf value. However, both Rf and RRf values ​​use the origin as the positioning point and are calculated by measuring the distance between the thin-layer spot and the origin. Since the origin does not change position due to differences in the developing solvent or environment during TLC development, the reproducibility of retardation factors and relative retardation factors is poor.

[0030] The inventors considered that although thin-layer chromatography behavior is affected by a variety of factors, such as the thickness and density of the thin-layer plate, the accuracy of the developing solvent preparation, the temperature and humidity of the experimental environment, the adequacy of the colorimetric agent spraying, and the heating and color development time, the same component is prone to produce large differences in Rf values. However, since the components detected and developed in the same thin-layer system have a certain similarity, the degree of influence they are affected by environmental factors should be balanced.

[0031] Guided by this concept, the inventors proposed the above-mentioned spacing ratio judgment model, collected thin-layer chromatograms under different conditions, and based on their own long-term practical experience, proposed that the analytical spots can be grouped, and according to the characteristics of each group of characteristic spots, the relative spacing value theory was proposed for the first time. The spacing ratio RPf was calculated based on the relative spacing value to reduce the influence of different environmental conditions on thin-layer chromatography, thereby realizing digital thin-layer chromatography detection without reference standards or without reference medicinal materials.

[0032] In some of these schemes, the criterion for grouping feature spots by adding or not adding the origin in the spacing ratio judgment model establishment step is as follows:

[0033] Step 1 Judgment: When the number of analytical spots is 2, the origin is added as a feature spot for grouping. If the origin has an irregular shape or unclear boundaries, the thin-layer plate sampling point is used as the feature spot.

[0034] The second step of judgment: If the number of analyzed spots is ≥3, and the original spot is not clear and difficult to identify, then the original spot is not added to form the feature spot; if the original spot is clear and identifiable, then proceed to the third step of judgment.

[0035] The third step is to determine the origin and the first three spots of the analysis spot along the solvent spread direction, starting from the origin and moving towards the solvent front. These spots are numbered A, B, C, and D respectively. The distances L-ac and L-bd between AC and BD are calculated respectively. If L-ac ≥ L-bd, the origin is not included as a feature spot in the group. If L-ac < L-bd, the origin is included as a feature spot in the group.

[0036] In some of these schemes, the analytical spots meet the following requirements:

[0037] A: The spots were present in both the chromatogram of the sample of this Chinese herbal medicine and the chromatogram of the reference herb or standard. Corresponding position;

[0038] B: The analysis shows that the spots have regular shapes, clear boundaries, and are stable.

[0039] C: The specific displacement value of the analytical spot is 0.05 to 0.8, preferably 0.1 to 0.8, and more preferably 0.2 to 0.8;

[0040] D: The analysis of spots is not affected by negative samples and has specificity.

[0041] In some of these schemes, in the spacing ratio judgment model establishment step, the positioning spots are determined by the following method: within each feature spot group, three feature spots are selected as candidate positioning spots, the distances between the other two feature spots and the positioning spots are measured, the ratio of the minimum distance to the maximum distance is calculated, the relative standard deviation of each ratio is calculated, and the feature spot with the smallest relative standard deviation is selected as the positioning spot.

[0042] In some of these schemes, the spacing ratio judgment model establishment step is grouped according to the following rules:

[0043] A: Number the feature spots along the solvent spreading direction, and divide the first three spots and the last three spots into the first group and the last group, respectively.

[0044] B: Each group shares common characteristic spots with adjacent groups;

[0045] C: Except for the first or last group, the feature spots in the remaining groups are grouped according to the principle of proximity.

[0046] D: The grouping meets the requirements of items A and D above, and the total number of groups is the smallest.

[0047] In some of these schemes, during the step of establishing the spacing ratio judgment model, the feature spots are numbered along the solvent spreading direction and grouped according to the following rules:

[0048] When the total number of spots analyzed is 2, add the origin or thin-layer plate spot as a characteristic spot to form a group;

[0049] When the total number of feature spots is 3, these 3 feature spots are grouped together.

[0050] When the total number of feature spots is 4, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 2, 3, and 4 are divided into the last group.

[0051] When the total number of feature spots is 5, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 3, 4, and 5 are divided into the last group.

[0052] When the total number of feature spots is 6, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 4, 5, and 6 are divided into the last group. The distance between feature spots numbered 2 and 4 and the distance between feature spots numbered 3 and 5 are calculated, which are D-24 and D-35 respectively. When D-24 > D-35, feature spots numbered 3, 4, and 5 are selected as the middle group. When D-24 < D-35, feature spots numbered 2, 3, and 4 are selected as the middle group. When D-24 = D-35, feature spots numbered 3, 4, and 5 or feature spots numbered 2, 3, and 4 can be selected as the middle group.

[0053] When the total number of feature spots is 7, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 5, 6, and 7 are divided into the last group, and feature spots numbered 3, 4, and 5 are divided into the middle group.

[0054] When the total number of feature spots is 8, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 6, 7, and 8 are divided into the last group, feature spots numbered 3, 4, and 5 are divided into the first middle group, and feature spots numbered 4, 5, and 6 are divided into the second middle group.

[0055] On the other hand, the present invention also discloses a thin-layer chromatography analysis model, which is established using the above-described method for establishing an analysis model for thin-layer chromatography.

[0056] On the other hand, the present invention also discloses a thin-layer chromatography detection and analysis method, which involves taking the thin-layer chromatogram of the Chinese herbal medicine to be tested, collecting the H value and analysis channel value of each characteristic spot in the HSB system, substituting them into the spot presence or absence judgment model obtained by the above-mentioned method, and comparing it with the spot presence or absence judgment standard to obtain the spot presence or absence judgment result.

[0057] On the other hand, the present invention also discloses the above-mentioned thin-layer chromatography detection and analysis method, which involves taking the thin-layer chromatogram of the Chinese herbal medicine to be tested and, according to... The above-described method for establishing the spacing ratio judgment model calculates the corresponding spacing ratio RPf and compares it with the evaluation criteria. By comparing the standard ranges, the intervals are obtained. Distance ratio evaluation results.

[0058] On the other hand, the present invention also discloses a thin-layer chromatography analysis model for cinnamon, which is established by the following method:

[0059] Thin-layer chromatography detection: Take cinnamon sample, extract with ethanol as extraction solvent to obtain test solution, spot test solution on silica gel G thin-layer plate, develop with petroleum ether-ethyl acetate at 60-90℃ with a volume ratio of 17:2-4, spray with dinitrophenylhydrazine ethanol test solution, take picture at 254±5nm to obtain thin-layer chromatogram.

[0060] Thin-layer chromatogram collection: collect thin-layer chromatograms of qualified and unqualified cinnamon products. The thin-layer chromatograms of unqualified products have missing characteristic spots. The thin-layer chromatograms include thin-layer chromatograms under differential experimental conditions. The differential experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Chinese herbal medicine varieties. The thin-layer chromatograms of qualified cinnamon products constitute a cinnamon standard chromatogram library.

[0061] Characteristic spot determination: Along the solvent spread direction, number the origin and each spot sequentially from the origin to the solvent front, and designate the 2nd, 3rd and 4th spots as characteristic spots;

[0062] Establishment of a spot presence / absence judgment model: 1) Collect the H values ​​of each characteristic spot in the HSB system from the thin-layer chromatograms of all qualified products, and use the H value range of the corresponding characteristic spot in different thin-layer chromatograms as the standard H value range of the characteristic spot; 2) Collect the L channel of each characteristic spot in the HSB color space from all thin-layer chromatograms as the analysis channel, and obtain the B channel value range of the characteristic spot missing group and the B channel value range of the characteristic spot present group; 3) Determine the B channel judgment standard based on the above B channel value ranges of the characteristic spot missing group and the characteristic spot present group. The B channel judgment standard and the standard H value range together constitute the spot presence / absence judgment standard, which is obtained.

[0063] In some of the thin-layer chromatography detection procedures for cinnamon samples, the test solution is extracted by the following method: take cinnamon powder, add ethanol at a rate of 20±5 mL solvent per gram of medicinal material, soak for 20±10 min, filter, and the filtrate is the test solution.

[0064] In some protocols for the thin-layer chromatography detection of cinnamon as the sample, the developing solvent is petroleum ether-ethyl acetate at 60–90°C at a ratio of 17:3.

[0065] In some protocols for the thin-layer chromatography detection of cinnamon samples, the humidity conditions include relative humidity of 32±5%, 59±5%, and 88±5%.

[0066] In some protocols for the thin-layer chromatography detection of cinnamon as the sample, the temperature conditions include 24±5℃ and 50±5℃.

[0067] In some protocols for the thin-layer chromatography detection of cinnamon as the sample, the differential experimental conditions also include at least two thin-layer plates of different sizes.

[0068] In some protocols for the thin-layer chromatography detection of cinnamon as the sample, the thin-layer plate includes silica gel G thin-layer plates manufactured by Merck AG & Co., Ltd. and Qingdao Ocean Chemical Co., Ltd.

[0069] In some schemes for establishing the spacing ratio judgment model for the above-mentioned sample as cinnamon, the Rf value of the second spot is 0.11±0.01, the Rf value of the third spot is 0.27±0.03, and the Rf value of the fourth spot is 0.47±0.05.

[0070] In some of these schemes, the cinnamon thin-layer chromatography analysis model also includes a spacing ratio judgment model, which is established by the following method: Along the solvent development direction, the origin and each spot are numbered sequentially from the origin to the solvent front. Spots 1, 2, 3, and 4 are designated as analytical spots. Spots 1, 2, and 3 are divided into the first group, and spots 2, 3, and 4 are divided into the last group. In the first group, spot 3 is used as the positioning spot, and in the last group, spot 2 is used as the positioning spot. In the first group, the distances L-31 and L-32 between spot 3 and spots 1 and 2 are calculated, respectively. The ratio between the minimum distance L-32 and the maximum distance L-31 is calculated and recorded. Given the spacing ratio RPf1, in the last group, calculate the distance between the second spot and the third spot. The distances L-23 and L between the spot and the 4th spot -24, calculate the ratio between the minimum distance L-23 and the maximum distance L-24, and denot it as the spacing ratio RPf2. Using the spacing ratios RPf1 and RPf2 as evaluation indicators, establish the evaluation standard range of the spacing ratios RPf1 and RPf2 based on the spacing ratios RPf1 and RPf2 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library.

[0071] On the other hand, the present invention also discloses a thin-layer chromatography detection and analysis method for cinnamon, including the following steps: taking the thin-layer chromatogram of the cinnamon sample to be tested obtained by the same method in the above thin-layer chromatography detection steps, comparing the H value and B channel value of its characteristic spots with the standard H value range and B channel judgment standard to obtain the result of whether the spots are present or not;

[0072] Furthermore, the corresponding spacing ratios RPf1 and RPf2 are calculated according to the method in the spacing ratio judgment model establishment step of the cinnamon thin-layer chromatography analysis model described above, and compared with the evaluation standard range to obtain the spacing ratio detection results;

[0073] Preferably, the standard H value range is 120-129, the B channel judgment standard is >2.8, or the RPf1 is 0.54-0.71 and the RPf2 is 0.51-0.59.

[0074] On the other hand, this invention also discloses a thin-layer chromatography analysis model for Shuxiong tablets, which is established using Chuanxiong as an indicator herb through the following method:

[0075] Thin-layer chromatography detection: Take the Shuxiong tablet sample, extract it with ether as the extraction solvent to obtain the test solution, spot the test solution on a silica gel G thin-layer plate, develop it with petroleum ether-chloroform at 30-60℃ with a volume ratio of 1:7-11, take a picture at 365±5nm to obtain the thin-layer chromatogram.

[0076] Thin-layer chromatogram collection: collect thin-layer chromatograms of qualified and unqualified Shuxiong Pian products. The thin-layer chromatograms of unqualified products have missing characteristic spots. The thin-layer chromatograms include those under differential experimental conditions, including at least two humidity conditions, at least two temperature conditions, and at least two batches of traditional Chinese medicine varieties. The thin-layer chromatograms of qualified Shuxiong Pian products constitute the Shuxiong Pian standard chromatogram library.

[0077] Characteristic spot determination: Along the solvent spread direction, the origin and each spot are numbered sequentially from the origin to the solvent front, and the 4th, 5th and 6th spots are designated as characteristic spots;

[0078] Establishment of a spot presence / absence judgment model: 1) Collect the H values ​​of each characteristic spot in the HSB system from the thin-layer chromatograms of all qualified products, and use the H value range of the corresponding characteristic spot in different thin-layer chromatograms as the standard H value range of the characteristic spot; 2) Collect the B channel of each characteristic spot in the RGB color space from all thin-layer chromatograms as the analysis channel, and obtain the B channel value range of the characteristic spot missing group and the B channel value range of the characteristic spot present group; 3) Determine the B channel judgment standard based on the above B channel value ranges of the characteristic spot missing group and the characteristic spot present group. The B channel judgment standard and the standard H value range together constitute the spot presence / absence judgment standard, which is obtained.

[0079] In some procedures for the thin-layer chromatography detection of the sample as Shuxiong tablets (with Chuanxiong as the indicator herb), the test solution is extracted by the following method: Take Shuxiong tablets, remove the coating, grind them into a fine powder, add 20±5mL of solvent for every 20 Shuxiong tablets, heat and reflux for 60±30min, filter, evaporate the filtrate to dryness, and dissolve the residue in 2±0.5mL of ether to obtain the test solution.

[0080] In some procedures for the thin-layer chromatography detection of the sample as Shuxiong tablets (with Chuanxiong as the indicator herb), the developing solvent is petroleum ether-chloroform at 30-60°C at a ratio of 1:9.

[0081] In some procedures for the thin-layer chromatography detection of the above-mentioned sample as Shuxiong tablets (with Chuanxiong as the indicator herb), the humidity conditions include relative humidity of 32±5%, 56±5%, and 88±5%.

[0082] In some procedures for the thin-layer chromatography detection of the sample as Shuxiong tablets (with Chuanxiong as the indicator herb), the temperature conditions include 5±2℃ and 25±5℃.

[0083] In some procedures for the thin-layer chromatography detection of the above-mentioned sample (using Ligusticum chuanxiong as the indicator herb), the... Differential experimental conditions It also includes at least two different sizes of thin-layer plates.

[0084] In some procedures for the thin-layer chromatography detection of the sample as Shuxiong tablets (with Chuanxiong as the indicator herb), the thin-layer plate includes silica gel G thin-layer plates produced by Merck & Co., Ltd. and Qingdao Ocean Chemical Co., Ltd.

[0085] In some schemes of the above sample interval ratio judgment model establishment (Ligusticum striatum as indicator herb), the Rf value of the 4th spot is 0.39±0.04, the Rf value of the 5th spot is 0.44±0.04, and the Rf value of the 6th spot is 0.67±0.06.

[0086] In some of these schemes, the thin-layer chromatography analysis model for the chest-soothing tablets also includes the establishment of a spacing ratio judgment model. The establishment of the spacing ratio judgment model includes the following steps: along the solvent development direction, number the origin and each spot sequentially from the origin to the solvent front direction, taking the 4th, 5th, and 6th spots as analytical spots, and using these three analytical spots as characteristic spot groups, taking the 4th spot as the positioning spot, calculating the distances L-45 and L-46 between the 4th spot and the 5th and 6th spots respectively, calculating the ratio between the minimum distance L-45 and the maximum distance L-46, and recording it as the spacing ratio RPf3, using the spacing ratio RPf3 as the evaluation index, and establishing the evaluation standard range of the spacing ratio RPf3 based on the spacing ratio RPf3 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library.

[0087] On the other hand, the present invention also discloses a thin-layer chromatography detection and analysis method for chest-soothing tablets, including the following steps: taking the thin-layer chromatogram of the chest-soothing tablet sample to be tested obtained by the same method in the above-mentioned thin-layer chromatography detection steps, comparing the H value and B channel value of its characteristic spots with the standard H value range and B channel judgment standard to obtain the result of whether the spots are present or not;

[0088] Furthermore, the corresponding spacing ratio RPf3 is calculated according to the method in the spacing ratio judgment model establishment step of the above-mentioned chest-soothing tablet thin-layer chromatography analysis model, and compared with the evaluation standard range to obtain the spacing ratio detection result;

[0089] Preferably, the standard H value ranges from 175 to 237, the B channel judgment standard is >43, or the RPf3 is 0.77 to 0.88.

[0090] On the other hand, this invention also discloses a thin-layer chromatography analysis model for Xinning tablets, which is established using Panax notoginseng as an indicator herb through the following method:

[0091] Thin-layer chromatography detection: Take the Xinning tablet sample, grind it into a fine powder, add ether and sonicate, filter, take the residue, evaporate to dryness, add methanol and sonicate, filter, take the filtrate, evaporate the filtrate to dryness, add water to dissolve the residue, extract with water-saturated n-butanol, wash the n-butanol solution with ammonia test solution, take the n-butanol solution and evaporate to dryness, add methanol to dissolve the residue as the test solution, take the test solution and spot it on a silica gel G thin-layer plate, use the upper layer solution of n-butanol-acetic acid-water with a volume ratio of 2-5:0.5-1.5:5 as the developing solvent, develop, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly visible, take a picture, and obtain the thin-layer chromatogram;

[0092] Thin-layer chromatogram collection: collect thin-layer chromatograms of qualified and unqualified Xinning tablets. The thin-layer chromatograms of unqualified products have missing characteristic spots. The thin-layer chromatograms include those under differential experimental conditions, including at least two humidity conditions, at least two temperature conditions, and at least two batches of traditional Chinese medicine varieties. The thin-layer chromatograms of qualified Xinning tablets constitute the Xinning tablet standard chromatogram library.

[0093] Characteristic spot determination: Along the solvent spread direction, the origin and each spot are numbered sequentially from the origin to the solvent front, and the 2nd, 4th, 5th, 6th, 8th and 9th spots are designated as characteristic spots;

[0094] Establishment of a spot presence / absence judgment model: 1) Collect the H values ​​of each characteristic spot in the HSB system from the thin-layer chromatograms of all qualified products, and use the H value range of the corresponding characteristic spot in different thin-layer chromatograms as the standard H value range of the characteristic spot; 2) Collect the L channel of each characteristic spot in the CIE-Lab color space from all thin-layer chromatograms as the analysis channel, and obtain the L channel value range of the characteristic spot missing group and the L channel value range of the characteristic spot present group; 3) Determine the L channel judgment standard based on the above L channel value ranges of the characteristic spot missing group and the characteristic spot present group. The L channel judgment standard and the standard H value range together constitute the spot presence / absence judgment standard, which is obtained.

[0095] In some procedures for the thin-layer chromatography detection of the above-mentioned sample (with Panax notoginseng as the indicator herb), the... The developing solvent is by volume The upper layer of a solution of n-butanol-acetic acid-water in a ratio of 4:1:5.

[0096] In some procedures for the thin-layer chromatography detection of Xinning tablets (with Panax notoginseng as the indicator herb), the humidity conditions include relative humidity of 32%, 65%, and 88%.

[0097] In some procedures for the thin-layer chromatography detection of Xinning tablets (with Panax notoginseng as the indicator herb), the temperature conditions include 5.1±2℃ and 17.8±5℃.

[0098] In some procedures for the thin-layer chromatography detection of Xinning tablets (with Panax notoginseng as the indicator herb), the differential experimental conditions also include at least two different sizes of thin-layer plates.

[0099] In some of the above-mentioned thin-layer chromatography detection steps for Xinning tablets (with Panax notoginseng as the indicator herb), the thin-layer plate includes silica gel G thin-layer plates produced by Merck & Co., Ltd. and Yantai Chemical Industry Co., Ltd.

[0100] In some of the steps of establishing the interval ratio judgment model for the above-mentioned sample as Xinning tablets (with Panax notoginseng as the indicator herb), the Rf value of the second spot was 0.068±0.01, the Rf value of the fourth spot was 0.20±0.02, the Rf value of the fifth spot was 0.22±0.02, the Rf value of the sixth spot was 0.38±0.04, the Rf value of the eighth spot was 0.58±0.06, and the Rf value of the ninth spot was 0.68±0.07.

[0101] In some of these schemes, the thin-layer chromatography analysis model for Xinning tablets also includes a spacing ratio judgment model. This model is established as follows: Along the solvent development direction, the origin and each spot are numbered sequentially from the origin towards the solvent front. Spots 2, 4, 5, 6, 8, and 9 are designated as analytical spots. Spots 2, 4, and 5 are divided into the first group; spots 6, 8, and 9 are divided into the last group; and spots 4, 5, and 6 form the middle group. In the first group, spot 2 is used as the positioning spot; in the middle group, spot 6 is used as the positioning spot; and in the last group, spot 6 is used as the positioning spot. In the first group, the distances L-24 and L-25 between the second spot and the fourth and fifth spots are calculated, respectively. The minimum distance L-24 and the maximum distance L-25 are calculated. The ratio between 25 is denoted as the spacing ratio RPf4. In the middle group, the distances L-46 and L-56 between the 6th spot and the 4th and 5th spots are calculated respectively. The ratio between the minimum distance L-56 and the maximum distance L-46 is calculated and denoted as the spacing ratio RPf5. In the last group, the distances L-68 and L-69 between the 6th spot and the 8th and 9th spots are calculated respectively. The ratio between the minimum distance L-68 and the maximum distance L-69 is calculated and denoted as the spacing ratio RPf6. Using the spacing ratios RPf4, RPf5, and RPf6 as evaluation indicators, the evaluation standard range of the characteristic ratios is established based on the spacing ratios RPf4, RPf5, and RPf6 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library.

[0102] On the other hand, the present invention also discloses a thin-layer chromatography detection and analysis method for Xinning tablets, including the following steps: taking the thin-layer chromatogram of the sample of Shuxiong tablet to be tested obtained by the same method in the above thin-layer chromatography detection steps, comparing the H value and L channel value of its characteristic spots with the standard H value range and L channel judgment standard to obtain the result of whether the spots are present or not;

[0103] Furthermore, the corresponding interval ratios RPf4, RPf5, and RPf6 were calculated according to the method in the interval ratio judgment model establishment step of the thin-layer chromatography analysis model of Xinning tablets, and compared with the evaluation standard range to obtain the interval ratio detection results;

[0104] Preferably, the standard H value range is 280-360, the L channel judgment standard is >-2, or the RPf4 is 0.81-0.89, RPf5 is 0.64-0.76, and RPf6 is 0.74-0.91.

[0105] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0106] The reagents and raw materials used in this invention are all commercially available.

[0107] The positive and progressive effects of this invention are as follows:

[0108] The present invention provides an analytical model for thin-layer chromatography. By selecting channel criteria from each channel of the RGB color space, HSB color space, and CIE-Lab color space to determine the presence or absence of characteristic spots, and combining these criteria with the standard H value range in the HSB system that reflects hue, a spot presence or absence determination standard is formed. This digital model can accurately determine the presence or absence of spots, reducing the bias of subjective human evaluation results through an objective and quantitative evaluation method.

[0109] Furthermore, by combining the interval ratio judgment model and utilizing the relative interval value theory, the interval ratio RPf can be calculated based on the relative interval value to reduce the influence of different environmental conditions on thin-layer chromatography, thereby realizing digital thin-layer chromatography detection without reference standards or without reference medicinal materials. Attached Figure Description

[0110] Figure 1 This is a thin-layer chromatogram of cinnamon from Example 2.

[0111] Figure 2 The H values ​​and ranges of the three characteristic spots of cinnamon in Example 2 are shown.

[0112] Figure 3The range of RGB channel color values ​​for the strong, weak, and missing spots in the cinnamon thin-layer chromatogram in Example 2.

[0113] Figure 4 The range of HSB channel color values ​​for strong, weak, and missing spots in the cinnamon thin-layer chromatogram in Example 2.

[0114] Figure 5 The range of Lab channel color values ​​for the strong, weak, and missing spots in the cinnamon thin-layer chromatogram in Example 2.

[0115] Figure 6 This is a diagram illustrating the distance ratio in Example 2.

[0116] Figure 7 The values ​​and ranges of the spacing ratio RPf1 in different thin-layer chromatograms in Example 2 are shown.

[0117] Figure 8 The values ​​and ranges of the spacing ratio RPf2 in different thin-layer chromatograms in Example 2 are shown.

[0118] Figure 9 This is a thin-layer chromatogram of the Shuxiong tablets in Example 3, with Ligusticum chuanxiong as the indicator herb.

[0119] Figure 10 The H values ​​are the three characteristic spots of Ligusticum chuanxiong in the chest-soothing tablets in Example 3.

[0120] Figure 11 The range of RGB channel color values ​​for the clearly visible (Strong), faintly visible (weak), and missing spots in the thin-layer chromatogram of the chest-soothing tablets in Example 3.

[0121] Figure 12 The range of HSB channel color values ​​for strong, weak, and missing spots in the thin-layer chromatogram of the chest-soothing tablets in Example 3.

[0122] Figure 13 The range of Lab channel color values ​​for the clearly visible (Strong), faintly visible (weak), and missing spots in the thin-layer chromatogram of the chest-soothing tablets in Example 3.

[0123] Figure 14 The values ​​and ranges of the spacing ratio RPf3 in different thin-layer chromatograms in Example 3 are shown.

[0124] Figure 15The thin-layer chromatogram of Zhongxin Ning tablets in Example 4, with Panax notoginseng as the indicator herb.

[0125] Figure 16 The H values ​​and ranges of the six characteristic spots of Panax notoginseng in the central ginseng tablets of Example 4 are shown.

[0126] Figure 17 The range of RGB channel color values ​​for the clearly visible (Strong), faintly visible (weak), and missing spots in the thin-layer chromatogram of Panax notoginseng tablets in Example 4.

[0127] Figure 18 The HSB channel color values ​​for the strong, weak, and missing spots in the thin-layer chromatogram of Panax notoginseng tablets in Example 4 are shown.

[0128] Figure 19 The range of Lab channel color values ​​for the clearly visible (Strong), faintly visible (weak), and missing spots in the thin-layer chromatogram of Panax notoginseng tablets in Example 4.

[0129] Figure 20 The values ​​and ranges of the spacing ratio RPf3 in different thin-layer chromatograms in Example 4 are shown.

[0130] Figure 21 The values ​​and ranges of the spacing ratio RPf4 in different thin-layer chromatograms in Example 4 are shown.

[0131] Figure 22 The values ​​and ranges of the spacing ratio RPf5 in different thin-layer chromatograms in Example 4 are shown. Detailed Implementation

[0132] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0133] Example 1

[0134] A thin-layer chromatography detection and analysis method, comprising:

[0135] I. Model Establishment

[0136] 1. Establishment of a model for determining the presence or absence of spots

[0137] 1.1 Collection of thin-layer chromatograms:

[0138] Collect thin-layer chromatograms of qualified and unqualified Chinese medicine products. The thin-layer chromatograms of unqualified products have missing characteristic spots. Record the presence and absence of characteristic spots. The thin-layer chromatograms include those under differential experimental conditions. The differential experimental conditions include at least two humidity conditions, at least two temperature conditions, at least two batches of Chinese medicine products, at least two specifications of thin-layer plates, at least two experimental dates, at least two development times, and at least two operators.

[0139] The selection of the above-mentioned characteristic spots should meet the following requirements:

[0140] A: The characteristic spots are present in both the chromatogram of the sample of this Chinese herbal medicine and the chromatogram of the reference herb or reference standard, and their positions correspond;

[0141] B: The characteristic spots are regular in shape, have clear boundaries, and exist stably;

[0142] C: The specific displacement value of the characteristic spot is 0.05 to 0.8, preferably 0.1 to 0.8, more preferably 0.2 to 0.8;

[0143] D: The characteristic spots are not affected by negative light and are specific.

[0144] After selecting the characteristic spot, the location of the characteristic spot is determined by the thin-layer chromatography obtained by using a reference standard or a reference medicinal material under the same conditions.

[0145] Alternatively, the characteristic spot can be determined by a preset Rf value range. When a spot falls within the preset Rf value range, it is considered a characteristic spot of this range. The preset Rf value range is defined by the Rf value range of the corresponding characteristic spot in the thin-layer chromatograms of several qualified samples. If a method without a reference standard or reference medicinal material is considered for detection and analysis, the Rf value can be used for localization.

[0146] In this embodiment, the method for determining whether the characteristic spots are missing is as follows: the thin-layer chromatogram is distributed to 5 inspectors, who independently observe whether the characteristic spots exist.

[0147] If all five inspectors determine that the characteristic spot is present, the result for that characteristic spot is considered clearly visible. If one inspector's determination differs from the others (e.g., four inspectors determine the characteristic spot is present and one determines it is not present, or four inspectors determine it is not present and one determines it is present), the result for that characteristic spot is considered faintly visible. If all five inspectors determine that the characteristic spot is completely absent, the result for that characteristic spot is considered missing.

[0148] If the final determination result is that the feature spot is clearly visible or faintly visible, it is considered to exist; if the final determination result is that the feature spot is missing, it is considered to be missing.

[0149] 1.2 Determination of Standard Hue:

[0150] Collect the H values ​​of each characteristic spot in the thin-layer chromatogram of all qualified products in the HSB system, and use the range of H values ​​of the corresponding characteristic spots in different thin-layer chromatograms as the standard H value range of the characteristic spot.

[0151] Understandably, the standard H value range for each characteristic spot can be defined based on the H value range of the corresponding characteristic spots in different thin-layer chromatograms. Therefore, if there are 3 characteristic spots in a thin-layer chromatogram, there are 3 corresponding standard H value ranges.

[0152] However, considering that thin-layer chromatograms are usually examined under the same conditions, such as under sunlight, Or detect at 254nm Visual inspection, or examination at 365nm, allows for the grouping of characteristic spots according to hue. Spots exhibiting the same hue are grouped together for analysis, while spots exhibiting different hues are divided into different hue groups. Each characteristic spot within the same hue group shares a common standard H-value range, which is the range of H-values ​​for all characteristic spots in that group in the thin-layer chromatogram of a qualified sample. This method offers advantages such as ease of operation and high processing efficiency.

[0153] In this embodiment, the method for determining whether the hues are the same is as follows: the thin-layer chromatogram is distributed to at least 5 inspectors, who independently observe and judge the hues of the characteristic spots. When all inspectors judge that the hues are the same, the hues are determined to be the same. When at least one inspector's judgment is inconsistent with that of the other inspectors, the hues are determined to be different, and the groups are regrouped until all inspectors judge the characteristic spots of the same hue group to be the same.

[0154] 1.3 Determination of Color System and Channels:

[0155] The channel values ​​of each characteristic spot in all thin-layer chromatograms were collected in the R, G, and B channels of the RGB color space, the H, S, and B channels of the HSB color space, and the L, a, and b channels of the CIE-Lab color space. All thin-layer chromatograms were randomly divided into a test group and a validation group. The channel value ranges of missing characteristic spots and the channel value ranges of present characteristic spots in the test group were obtained. The minimum value of the channel value range of present characteristic spots in the test group was used as the analysis threshold. The channel values ​​of characteristic spots in the validation group were compared with the analysis threshold. When the channel value of a characteristic spot was greater than the analysis threshold, it was determined that the characteristic spot was present. This determination result was compared and verified with the records of missing and present characteristic spots. The channel with the higher determination accuracy was used as the analysis channel of the characteristic spot, and the analysis threshold of the analysis channel was used as the channel determination criterion for the characteristic spot.

[0156] Understandably, the above channel values ​​have been calibrated. The calibration method is to subtract the background value of the corresponding channel from the extracted channel value to eliminate background interference.

[0157] Specifically, the determination accuracy is calculated by dividing the number of spots in the verification group that match the determination results and the records of missing and present feature spots by the total number of feature spots in the verification group.

[0158] 1.4 Establish a model for determining the presence or absence of spots:

[0159] The channel judgment criteria and the standard H value range together constitute the spot presence or absence judgment criteria, which is obtained.

[0160] 2. Establishment of the Spacing Ratio Judgment Model

[0161] 2.1 Selecting analytical spots

[0162] Select at least two representative spots from the thin-layer chromatogram of the above-mentioned qualified products as analytical spots, and these analytical spots shall meet the following requirements:

[0163] (1) The spots were found in both the chromatogram of the sample of this Chinese medicine and the chromatogram of the reference herb or reference substance, and their positions corresponded.

[0164] It is understandable that although reference standards or reference medicinal materials are not necessarily used in the establishment of the standard spectral library and in subsequent analysis and evaluation, considering the spots of index reference standards or reference medicinal materials in the establishment of the standard spectral library and selecting characteristic spots based on these can better reflect the quality of the Chinese medicine varieties being analyzed and tested.

[0165] (2) The spots are regular in shape, have clear boundaries, and are stable.

[0166] This standard is consistent with the requirements for inspecting spots in the field, which can improve the stability of the method.

[0167] (3) The specific displacement value of the analytical spot is 0.05 to 0.8, preferably 0.1 to 0.8, and more preferably 0.2 to 0.8.

[0168] By limiting the specific flotation value of the analytical spots to 0.05–0.8, preferably 0.1–0.8, and more preferably 0.2–0.8, the specific flotation value of different spots can be further reduced. By comparing the effects of the conditional shift, the stability of this scheme can be improved.

[0169] (4) The analysis of spots is not affected by negative interference and has specificity.

[0170] 2.2 Consider whether to add the origin.

[0171] The criteria for determining whether to include the origin to form feature spots for grouping are as follows:

[0172] Step 1 Judgment: When there are 2 analytical spots, the origin is added as a characteristic spot for grouping. If the origin spot is not clear and difficult to identify, the thin-layer plate spot is used as the characteristic spot.

[0173] The second step of judgment: If the number of analyzed spots is ≥3, and the original spot is not clear and difficult to identify, then the original spot is not added to form the characteristic spot. If the original spot is clear and identifiable, then proceed to the third step of judgment.

[0174] The second step involves determining the origin and the first three analytical spots along the solvent spreading direction, sequentially numbering them A, B, C, and D from the origin towards the solvent front. The distances between AC and BD are L-ac and L-bd. If L-ac ≥ L-bd, the origin is not included as a characteristic spot in the group; if L-ac < L-bd, the origin is included as a characteristic spot in the group.

[0175] 2.3 Grouping

[0176] The feature spots are grouped into groups of three, according to the following grouping rules:

[0177] (1) Number the feature spots along the solvent spreading direction, and divide the first three spots and the last three spots into the first group and the last group, respectively.

[0178] (2) Each group shares common characteristic spots with its adjacent groups;

[0179] (3) Except for the first or last group, the feature spots in the remaining groups are grouped according to the principle of proximity;

[0180] (4) The grouping meets the requirements of (1)-(3) above, and the total number of groups is the smallest.

[0181] The following are examples of grouping methods when the number of feature spots is different.

[0182] The characteristic spots are numbered along the solvent spread direction and grouped according to the following rules:

[0183] When the total number of spots analyzed is 2, which is less than three spots, the origin or a thin-layer plate spot must be added as a characteristic spot (if the origin is difficult to identify, it can be manually identified by the spotting location and then added as a thin-layer plate spot) to form a group.

[0184] When the total number of feature spots is 3, these 3 feature spots are grouped together.

[0185] When the total number of feature spots is 4, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 2, 3, and 4 are divided into the last group.

[0186] When the total number of feature spots is 5, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 3, 4, and 5 are divided into the last group.

[0187] When the total number of feature spots is 6, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 4, 5, and 6 are divided into the last group. The distance between feature spots numbered 2 and 4 and the distance between feature spots numbered 3 and 5 are calculated, which are D-24 and D-35 respectively. When D-24 > D-35, feature spots numbered 3, 4, and 5 are selected as the middle group. When D-24 < D-35, feature spots numbered 2, 3, and 4 are selected as the middle group. When D-24 = D-35, feature spots numbered 3, 4, and 5 or feature spots numbered 2, 3, and 4 can be selected as the middle group.

[0188] When the total number of feature spots is 7, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 5, 6, and 7 are divided into the last group, and feature spots numbered 3, 4, and 5 are divided into the middle group.

[0189] When the total number of feature spots is 8, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 6, 7, and 8 are divided into the last group, feature spots numbered 3, 4, and 5 are divided into the first middle group, and feature spots numbered 4, 5, and 6 are divided into the second middle group.

[0190] 2.4 Selecting and Positioning Spots

[0191] After grouping, at least one feature spot group is obtained, and each feature spot group contains one feature spot. To locate the spot.The method for selecting positioning spots is as follows: within each feature spot group, three feature spots are selected as candidate positioning spots, the distances between the other two feature spots and the positioning spot are measured, the ratio of the minimum distance to the maximum distance is calculated, the relative standard deviation of each ratio is calculated, and the feature spot with the smallest relative standard deviation value is selected as the positioning spot.

[0192] 2.5 Calculate the spacing ratio RPf

[0193] In each group of feature spots, the distance between the other two feature spots and the localized spot is calculated, and the ratio between the minimum distance and the maximum distance is calculated and denoted as the spacing ratio RPf. The spacing ratio RPf is used as the evaluation index.

[0194] Understandably, each set of feature spots can yield a corresponding spacing ratio RPf. If there are two sets of feature spots, then the corresponding spacing ratios RPf1 and RPf2 can be obtained respectively; if there are three sets of feature spots, then the corresponding spacing ratios RPf1, RPf2 and RPf3 can be obtained respectively; and so on.

[0195] 2.6 Establish the scope of evaluation criteria

[0196] Based on the spacing ratio RPf of each characteristic spot group in the standard chromatogram library composed of qualified products, an evaluation standard range for the spacing ratio RPf is established, and the evaluation standard range of the spacing ratio judgment model is compared.

[0197] Understandably, the evaluation criteria range for the above-mentioned interval ratio RPf is defined by the values ​​that may be obtained under different experimental conditions of a large sample size of qualified Chinese medicine varieties.

[0198] The specific requirements depend on the testing requirements and the data from different Chinese medicine varieties.

[0199] II. Analytical Methods

[0200] 1. Determining the presence or absence of spots

[0201] Take the thin-layer chromatogram of the Chinese herbal medicine to be tested, collect the H value and analytical channel value of each characteristic spot in the HSB system, substitute them into the spot presence or absence judgment model obtained above, and compare them with the spot presence or absence judgment standard. If the H value falls into the standard H value and the channel value of the characteristic spot is greater than the channel judgment standard, then the characteristic spot is judged to be present.

[0202] 2. Spacing ratio evaluation

[0203] Take the thin-layer chromatogram of the Chinese herbal medicine to be tested, calculate the corresponding interval ratio RPf according to the above-mentioned method for establishing the interval ratio judgment model, and compare it with the evaluation standard range to obtain the interval ratio evaluation result.

[0204] Example 2

[0205] A thin-layer chromatography detection and analysis method for cinnamon, referring to the method in Example 1, is applied to the thin-layer chromatography detection and analysis of cinnamon.

[0206] I. Thin-layer chromatography detection

[0207] Take 0.5g of cinnamon powder, add 10mL of ethanol, and soak in cold water for 20 minutes, shaking occasionally. Filter and use the filtrate as the test solution. Separately, prepare a reference solution of cinnamaldehyde by dissolving it in ethanol to a concentration of 1μL per mL. Apply 5μL of the test solution and 2μL of the reference solution to a silica gel G thin-layer plate. Develop the plate using petroleum ether (60–90℃)-ethyl acetate (17:3) as the developing solvent. Remove the plate, air dry, spray with dinitrophenylhydrazine ethanol solution, examine at 254nm, and photograph to obtain the thin-layer chromatogram (e.g., [image of chromatogram]). Figure 1 ).

[0208] II. Collection of Thin-Layer Chromatograms

[0209] Thin-layer chromatograms of qualified and unqualified cinnamon samples were collected. The unqualified samples showed a lack of characteristic spots in their thin-layer chromatograms, and the presence and absence of these characteristic spots were recorded. The thin-layer chromatograms of the qualified samples were used to form a standard chromatogram library. The thin-layer chromatograms included those obtained under differential experimental conditions.

[0210] Referring to the judgment criteria of Example 1, the thin-layer chromatograms were distributed to 5 inspectors who independently observed the presence of characteristic spots and determined the final result according to the criteria of Example 1.

[0211] In this embodiment, cinnamon thin-layer chromatograms were collected from different silica gel thin-layer chromatography plates (Merck Corporation, Qingdao Ocean Chemical Co., Ltd.) at different temperatures (24℃, 50℃) and different relative humidityes (32%, 59%, 88%), using the method described above.

[0212] The thin-layer chromatograms of cinnamon were divided into 54 batches of cinnamon samples for establishing the method (test group) and 35 batches of cinnamon samples for validating the method (validation group). The samples for establishing the method uniformly covered various silica gel G plates, various temperatures and various relative humidity.

[0213] III. Establishment of Analytical Model

[0214] 1. Establishment of a model for determining the presence or absence of spots

[0215] 1.1 Selection of Feature Spots

[0216] Based on the selection principle of characteristic spots in Example 1, select Figure 1The second, third, and fourth spots are characteristic spots, with Rf values ​​of 0.11, 0.27, and 0.47 for spots 2, 3, and 4, respectively.

[0217] 1.2 Extraction of color parameters of feature spots

[0218] The values ​​of the R, G, and B channels of the thin-layer chromatography characteristic spots in the RGB color space, the H, S, and B channels in the HSB color space, and the L, a, and b channels in the CIE-Lab color space were read using TLC Measure software and Photoshop software.

[0219] 1.3 Hue Determination

[0220] In color theory, a color wheel is a circular diagram of a rainbow, dividing hues into 12 hues according to the periodicity of colors. Each hue represents a basic color (such as red, green, blue, etc.) and a mixture of other colors. Hue (H) is a fundamental attribute of color, measuring its position on the color wheel and used to characterize its hue. The H value is a parameter describing the hue of a color, used to measure its position on the color wheel. H values ​​are usually expressed in degrees, ranging from 0 to 360 degrees. For example, 0 degrees represents red, 120 degrees represents green, and 240 degrees represents blue. Therefore, the background color of characteristic spots in thin-layer chromatography can be determined by the H value, achieving an accurate description of the spot color.

[0221] The H values ​​of all cinnamon characteristic spots were collected using TLC Measure software, and the minimum and maximum values ​​were set as the range of values, such as... Figure 2 As shown in the figure, the horizontal axis represents three characteristic spots, and the vertical axis represents the H value. As described in Example 1, considering that all three cinnamon characteristic spots were examined at 254 nm and that the three characteristic spots had the same color hue, the common H value range of each characteristic spot in the thin-layer chromatography of cinnamon was finally determined to be 120 to 129.

[0222] 1.4 Background Calibration

[0223] In each color system, the background value of each channel is extracted, and the channel color values ​​are calibrated by subtracting the background value.

[0224] 1.5 Preliminary Screening of Color System and Channels

[0225] The values ​​of characteristic spots in various color systems and channels were analyzed. All thin-layer chromatograms were randomly divided into a test group (54 batches) and a validation group (35 batches). The range of channel values ​​for missing characteristic spots in the test group and the range of channel values ​​for present characteristic spots in the test group were obtained. The minimum value of the channel value range for present characteristic spots in the test group was used as the analysis threshold. The channel values ​​of characteristic spots in the validation group were compared with the analysis threshold. When the channel value of a characteristic spot was greater than the analysis threshold, it was determined that the characteristic spot was present. This determination result was compared and verified with the records of missing and present characteristic spots. The channel with the higher determination accuracy was used as the analysis channel for the characteristic spot, and the analysis threshold of the analysis channel was used as the channel determination criterion for the characteristic spot.

[0226] The test groups (54 batches in total) were divided into two groups according to the above method: clearly visible (strong spots) group and faintly visible (weak spots) group. and missing spots As mentioned above, since all three cinnamon-related feature spots were examined at 254nm and exhibited the same color hue, the channel values ​​of the three feature spots were analyzed together without distinguishing between them. However, if it is confirmed that the feature spots being analyzed have different hues, they should be analyzed separately.

[0227] Using the group classification of the three data sets as the x-axis and the calibrated channel values ​​as the y-axis, a box plot is drawn. In the figure, the midline of the box plot represents the median value of the group, and the upper and lower quartiles are the upper and lower quartiles, respectively. If outliers are identified according to the Turkey's test standard, these data are removed. The resulting box plot is shown below. Figure 3 As shown.

[0228] Figure 3 The results are in the RGB color space system, where A, B, and C are the results for the R, G, and B channels, respectively. As can be clearly seen from the figure, the calibrated G channel provides the best differentiation between clearly visible spots, faintly visible spots, and missing spots in the cinnamon thin-layer chromatogram. Figure 3 In the B part), the minimum channel value of the faintly visible spot group in the G channel is 13. This is used as the analysis threshold of the G channel. That is, in the RGB color space system, the G value after correction of the cinnamon feature spots is defined as a spot with a value greater than 13, and a spot with a value less than or equal to 13 is a missing spot.

[0229] Figure 4 The results are for the HSB color space system, where A, B, and C are the H, S, and B channels, respectively. As can be clearly seen from the figure, the calibrated B channel provides the best differentiation between clearly visible spots, faintly visible spots, and missing spots in the cinnamon thin-layer chromatogram. Figure 4In the C section, the minimum channel value of the faintly visible spots in the B channel is 2.8. This is used as the analysis threshold for the B channel. That is, in the HSB color space system, the B value after correction of the cinnamon characteristic spots is defined as having spots if it is greater than 2.8, and having spots less than or equal to 2.8 if it is missing.

[0230] Figure 5 The results are from the CIE-Lab color space system, where A, B, and C represent the L, a, and b channels, respectively. As can be clearly seen from the figure, the calibrated L channel provides the best differentiation between clearly visible spots, faintly visible spots, and missing spots in the cinnamon thin-layer chromatogram. Figure 5 In Part A of the color space, the minimum channel value of the faintly visible spots in the L channel is 5. This value is used as the analysis threshold for the L channel. In the CIE-Lab color space system, after correction of the cinnamon characteristic spots, an L value greater than 5 is considered a present spot, and a value less than or equal to 5 is considered a missing spot.

[0231] 1.7 Objective Validation of the Method and Further Screening of the Color System and Channels

[0232] The presence or absence of feature spots is determined according to the analysis threshold and judgment method obtained from the above test group. The judgment accuracy is calculated by dividing the number of spots in the verification group whose judgment results match the records of missing and present feature spots by the total number of feature spots in the verification group. The judgment accuracy is obtained by filtering the selected channels in each color system.

[0233] In this embodiment, 54 batches of cinnamon samples were used to establish the method, with a total of 162 thin-layer chromatographic characteristic spots; 35 batches of cinnamon samples were used to validate the method, with a total of 105 thin-layer chromatographic characteristic spots.

[0234] The data from 105 characteristic spots in the thin-layer chromatography verification sample of cinnamon showed that, in the G channel of the RGB color system, 2 missing spots fell within the range of faintly visible spots, 4 faintly visible spots fell within the range of missing spots, and 1 clearly visible spot was in the area of ​​missing spots. A total of 7 characteristic spots were incorrectly identified, and 98 characteristic spots were correctly identified, with an accuracy rate of 98 / 105, or 93.33%.

[0235] In the B channel of the HSB color system, clearly visible, faintly visible, and missing spots are all within the corresponding defined range. All 105 feature spots were correctly identified, with an accuracy rate of 100%.

[0236] In the L channel of the CIE-Lab color system, both clearly visible spots and missing spots are within the specified range. There are 2 misjudgments of faintly visible spots and 103 correct judgments of feature spots. The accuracy rate is 103 / 105, or 98.10%.

[0237] Therefore, the HSB system B channel, which has the highest accuracy, was selected to determine the color of cinnamon.

[0238] The proposed digital standard for cinnamon thin-layer chromatography color is as follows: In the HSB color system, the H value of the three characteristic spots should be in the range of 120-129. Using the thin-layer background as a blank to correct the characteristic spots, the B value of the three characteristic spots in the HSB system should be greater than 2.8.

[0239] 2. Establishment of the Spacing Ratio Judgment Model

[0240] 2.1 Selecting analytical spots

[0241] Following the principles of Example 1, analytical spots were selected. Along the solvent development direction, the origin and each spot were numbered sequentially from the origin to the solvent front. Based on the characteristics of the reference spots in the cinnamon identification chromatogram, the differences in the test sample spots, and combined with the experience of manual judgment, the relatively stable 2nd, 3rd, and 4th spots were finally selected as analytical spots. The Rf values ​​of these spots 2, 3, and 4 were 0.11, 0.27, and 0.47, respectively.

[0242] 2.2 Consider whether to add the origin.

[0243] In this embodiment, there are 3 analytical spots. Along the solvent spreading direction, the origin and the first three analytical spots are numbered A, B, C, and D sequentially from the origin to the solvent front. The distances L-ac (L13) and L-bd (L24) between AC and BD are calculated, which are 3.52 and 4.69, respectively. That is, L-ac < L-bd, so the origin is added as a feature spot for grouping.

[0244] 2.3 Grouping

[0245] This embodiment has a total of 4 feature spots, which are divided into 2 groups. The first group (first group) includes the 1st, 2nd and 3rd feature spots, and the second group (last group) includes the 2nd, 3rd and 4th feature spots.

[0246] 2.4 Selecting and Positioning Spots

[0247] Within the aforementioned feature spot group, three feature spots were selected as candidate positioning spots. The distances between the other two feature spots and the positioning spots were measured, the ratio of the minimum distance to the maximum distance was calculated, and the relative standard deviation of each ratio was calculated. The results are shown in the table below.

[0248] Table 1. Analysis results of location data of different spots in the first group.

[0249]

[0250] The results show that the relative standard deviations of the three different positioning points in the cinnamon thin-layer chromatogram are 0.12, 0.18 and 0.07, respectively. The third spot has the smallest relative standard deviation, so the third spot with the smallest relative standard deviation is selected as the positioning spot.

[0251] Table 2. Analysis results of location data of different spots in the second group.

[0252]

[0253] The results show that the relative standard deviations of the three different positioning points in the cinnamon thin-layer chromatogram are 0.04, 0.07 and 0.05, respectively. The second spot has the smallest relative standard deviation, so the second spot with the smallest relative standard deviation is selected as the positioning spot.

[0254] 2.5 Calculation Rules for Feature Spot Locations

[0255] To determine the relative positional relationship of multiple target components in thin-layer chromatography, this invention proposes for the first time the concepts and theories of ratio distance and relative ratio distance, and applies this algorithm to digital standards without physical controls.

[0256] The position factor (Pf) is the ratio of the distance from the target component to the location component to the distance from the origin to the location component. Figure 6 For example, if thin-layer spot D is used as the positioning spot, then the distance ratio of thin-layer spot C is... The relative position factor (RPf) is the ratio of the distance from the target fraction to the location fraction to the distance from the reference fraction to the location fraction. Figure 6 For example, if thin-layer spot D is used as the localization spot and thin-layer spot B is used as the reference component spot, then the relative distance value of thin-layer spot C is... The relative distance value positioning method can effectively reflect the relative positional relationship of the three thin-layer spots, while the developing solvent front and origin can be excluded from the calculation.

[0257] This embodiment compares three calculation methods to evaluate the locations of three spots simultaneously.

[0258] The first calculation method: Referring to the method for calculating relative distance, three thin-layer spots are marked as positioning points, and the other two thin-layer spots are marked as reference spots, resulting in the following three different calculation formulas:

[0259] ① If spot D is the localizing spot and spot B is the reference spot, then the relative distance value of spot C is...

[0260] ②Spot C is the localization spot, and spot D is the reference spot. Then the relative distance value of spot B is...

[0261] ③ If spot B is the localization spot and spot D is the reference spot, then the relative distance value of spot C is...

[0262] The distances from different feature spots to the localized spot are obtained. L1 is defined as the minimum distance, and L3 as the maximum distance, resulting in the following general formula:

[0263]

[0264] The second calculation method: Using the origin as the positioning point, measure the distance from each feature spot to the positioning point, and divide the smaller distances by the larger distances in turn to obtain the following general formula:

[0265]

[0266] The third calculation method: Calculate the distance ratio between feature spots. There are two intervals between three feature spots. Divide the minimum interval by the maximum interval to obtain the following general formula:

[0267]

[0268] The results of calculating X1, X2, and X3 using the three different methods described above are shown in the table below. In the first group, location point 1 is the origin, therefore calculation method one is the same as calculation method two.

[0269] Table 3. Analysis results of location data for the first group using different calculation methods.

[0270]

[0271] The above results show that the relative standard deviations of the two different calculation methods in the first group of cinnamon samples were 0.07 and 0.18, respectively. This indicates that the relative standard deviation of the first calculation method is smaller, which means that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for spot localization in thin-layer chromatography of cinnamon.

[0272] Table 4. Analysis results of location data for the second group of different calculation methods.

[0273]

[0274] The results above show that the relative standard deviations of the three different calculation methods in cinnamon samples are 0.04, 0.07 and 0.05, respectively. The first calculation method has the smallest relative standard deviation, which also indicates that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for spot localization in thin-layer chromatography of cinnamon.

[0275] 2.6 Calculate the spacing ratio RPf

[0276] Following the first method described above, the third spot in the first group is used as the positioning spot. The distances L-32 and L-31 between the third spot and the second and first spots are calculated, respectively. The ratio between the minimum distance L-32 and the maximum distance L-31 is calculated and denoted as the spacing ratio RPf1. This spacing ratio RPf1 is used as the evaluation index. Based on the spacing ratio RPf1 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library (54 batches of samples) (e.g., ...), Figure 7 (The black circle in the middle) The evaluation criteria for the spacing ratio RPf1 is set in the range of 0.54 to 0.71.

[0277] In the second group, the second spot is used as the positioning spot. The distances L-23 and L-24 between the second spot and the third and fourth spots are calculated respectively. The ratio between the minimum distance L-23 and the maximum distance L-24 is calculated and denoted as the spacing ratio RPf2. This spacing ratio RPf2 is used as the evaluation index. Based on the spacing ratio RPf2 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library (54 batches of samples) (e.g., Figure 8 (The black circle in the middle) The evaluation criteria for the spacing ratio RPf2 are set in the range of 0.51 to 0.59.

[0278] IV. Comparative Analysis

[0279] 1. Determining the presence or absence of spots

[0280] The thin-layer chromatograms of 35 batches of cinnamon samples were obtained using the above verification method. The H values ​​and analytical channel values ​​of each characteristic spot in the HSB system were collected and substituted into the spot presence / absence judgment model obtained above. The results were compared with the spot presence / absence judgment criteria. The results showed that in the B channel of the HSB color system, the clearly visible, faintly visible, and missing spots were all within the corresponding proposed range.

[0281] 2. Spacing ratio evaluation

[0282] The thin-layer chromatograms of 35 batches of cinnamon samples were obtained using the above verification method, and the corresponding spacing ratios RPf1 and RPf2 were calculated according to the first method determined in the spacing ratio judgment model establishment step (e.g., ...). Figure 7 and Figure 8 The results of digital thin-layer chromatography detection of cinnamon were obtained by comparing the blue circled area with the above evaluation standard range.

[0283] The data results show that in the first group, the RPf values ​​of all samples were within the proposed evaluation criteria range, but in the second group, the RPf1 values ​​of samples 67 and 68 were both 0.49 (e.g., ...). Figure 8The part circled in red is outside the scope of the established evaluation criteria. After tracing the sample chromatograms, it was found that spot 2 was missing in the thin-layer chromatograms of these two batches of samples, which were judged as unqualified samples and needed to be retested. This further proved the high feasibility of the digital evaluation method for the thin-layer chromatography of cinnamon.

[0284] Example 3

[0285] A method for detecting and analyzing the thin-layer chromatography of Ligusticum chuanxiong in Shuxiong tablets was applied to the detection and analysis of the thin-layer chromatography of Ligusticum chuanxiong in Shuxiong tablets according to the method of Example 1.

[0286] I. Thin-layer chromatography detection

[0287] Take 20 tablets of Shuxiong tablets, remove the coating, grind them finely, add 20 mL of diethyl ether, reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of diethyl ether to obtain the test solution. Take another 1 g of the reference medicinal material of Ligusticum chuanxiong, soak it in 10 mL of diethyl ether for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of diethyl ether to obtain the reference medicinal material solution.吸取上述两种溶液各8μL,点于硅胶G薄层板上,以石油醚(30~60℃)-三氯甲烷(1∶9)为展开剂,展开,取出,晾干,置紫外光灯(365nm)下检视,拍照,得到薄层色谱图(如 Figure 9 )。

[0288] II. Collection of thin-layer chromatograms

[0289] Thin-layer chromatograms of qualified and unqualified Chuanxiong (Ligusticum striatum) products in Shuxiong tablets were collected. The thin-layer chromatogram of the unqualified product was... Characteristic spots exist Missing, and record the missing and existing characteristic spots. The thin-layer chromatograms of the qualified products form a standard chromatogram library. The thin-layer chromatograms include those under different experimental conditions.

[0290] According to the judgment criteria of Example 1, distribute the thin-layer chromatograms to 5 inspectors, independently observe the presence or absence of characteristic spots, and determine the final result according to the criteria of Example 1.

[0291] In this example, according to the above method, collect the thin-layer chromatograms of Shuxiong tablets on different silica gel thin-layer plates (Merck KGaA, Qingdao Ocean Chemical Co., Ltd.), at different temperatures (5 °C, 25 °C), and different relative humidities (32%, 56%, 88%).

[0292] Divide the thin-layer chromatogram of Shuxiong tablets into 50 batches of Shuxiong tablet samples (test group) for establishing the method and 27 batches of Shuxiong tablet samples (verification group) for verifying the method. Among them, the samples for establishing the method evenly cover various silica gel G plates, various temperatures, and various relative humidities.

[0293] III. Establishment of the analysis model

[0294] 1. Establishment of the model for judging the presence or absence of spots

[0295] 1.1 Selection of Feature Spots

[0296] Based on the selection principle of characteristic spots in Example 1, select Figure 9 The 4th, 5th, and 6th spots are characteristic spots, with Rf values ​​of 0.39, 0.44, and 0.67 for spots 4, 5, and 6, respectively.

[0297] 1.2 Extraction of color parameters of feature spots

[0298] The values ​​of the R, G, and B channels of the thin-layer chromatography characteristic spots in the RGB color space, the H, S, and B channels in the HSB color space, and the L, a, and b channels in the CIE-Lab color space were read using TLC Measure software and Photoshop software.

[0299] 1.3 Hue Determination

[0300] In color theory, a color wheel is a circular diagram of a rainbow, dividing hues into 12 hues according to the periodicity of colors. Each hue represents a basic color (such as red, green, blue, etc.) and a mixture of other colors. Hue (H) is a fundamental attribute of color, measuring its position on the color wheel and used to characterize its hue. The H value is a parameter describing the hue of a color, used to measure its position on the color wheel. H values ​​are usually expressed in degrees, ranging from 0 to 360 degrees. For example, 0 degrees represents red, 120 degrees represents green, and 240 degrees represents blue. Therefore, the background color of characteristic spots in thin-layer chromatography can be determined by the H value, achieving an accurate description of the spot color.

[0301] The H values ​​of all characteristic spots on the chest-soothing films were collected using TLC Measure software. The minimum and maximum values ​​were set as the range of values. Figure 10 As shown in the figure, the horizontal axis represents three characteristic spots, and the vertical axis represents the H value. As described in Example 1, considering that the characteristic spots of Ligusticum chuanxiong in these three chest-soothing tablets were all examined at 365nm, and that the colors of the three characteristic spots showed the same hue, the common H value range of each characteristic spot of Ligusticum chuanxiong in the chest-soothing tablets was finally determined to be 175-237.

[0302] 1.4 Background Calibration

[0303] In each color system, the background value of each channel is extracted, and the channel color values ​​are calibrated by subtracting the background value.

[0304] 1.5 Preliminary Screening of Color System and Channels

[0305] The numerical values ​​of characteristic spots in various color systems and channels were analyzed. All thin-layer chromatograms were randomly divided into a test group (50 batches) and a validation group (27 batches). The channel value ranges for missing characteristic spots and the channel value ranges for present characteristic spots in the test group were obtained. The minimum value of the channel value range for present characteristic spots in the test group was used as the analytical threshold. The channel values ​​of characteristic spots in the validation group were compared with the analytical threshold. When the channel value of a characteristic spot was greater than the analytical threshold, it was determined that the characteristic spot was present. Compare this determination result with the records of the absence and presence of the aforementioned feature spots. Comparative verification was performed, and the channel with the higher accuracy rate was used as the feature patch. The analysis channel of the point is used, and the analysis threshold of the analysis channel is used as the channel determination criterion for the feature spot.

[0306] The test group (50 batches in total) was divided into three groups according to the above method: clearly visible (strong spots), faintly visible (weak spots), and missing spots. As mentioned above, since the characteristic spots in the three chest X-ray films were all examined at 365nm and the three characteristic spots showed the same color hue, the channel values ​​of the three characteristic spots were analyzed together without distinguishing between different characteristic spots. However, if it is confirmed that the characteristic spots to be analyzed have different hues, they need to be analyzed separately.

[0307] Using the group classification of the three data sets as the x-axis and the calibrated channel values ​​as the y-axis, a box plot is drawn. In the figure, the midline of the box plot represents the median value of the group, and the upper and lower quartiles are the upper and lower quartiles, respectively. If outliers are identified according to the Turkey's test standard, these data are removed. The resulting box plot is shown below. Figures 11-13 As shown.

[0308] Figure 11 The results are in the RGB color space system, where A, B, and C are the results for the R, G, and B channels, respectively. As can be clearly seen from the figure, the calibrated B channel has the best effect in distinguishing between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Ligusticum chuanxiong in Shuxiong tablets. Figure 11 In the C section, the minimum channel value of the faintly visible spots in the B channel is 43. This value is used as the analysis threshold for the B channel. That is, in the RGB color space system, the B value of the corrected Chuanxiong characteristic spots in the chest tablet is defined as having a presence if it is greater than 43, and having a missing spot if it is less than or equal to 43.

[0309] Figure 12 The results are for the HSB color space system, where A, B, and C are the results for the H, S, and B channels, respectively. As can be clearly seen from the figure, the calibrated B channel has the best effect in distinguishing between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Chuanxiong in Shuxiong tablets. Figure 12In the C section, the minimum channel value of the faintly visible spots in the B channel is 7.9. This value is used as the analysis threshold for the B channel. That is, in the HSB color space system, the B value of the Chuanxiong characteristic spots in the chest relief tablets after correction is defined as having a presence of spots if it is greater than 7.9, and less than or equal to 7.9 if it is missing spots.

[0310] Figure 13 The results are from the CIE-Lab color space system, where A, B, and C are the results for channels L, a, and b, respectively. It can be clearly seen from the figure that the calibrated L channel provides the best differentiation between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Ligusticum chuanxiong in Shuxiong tablets. Figure 13 In Part A of the color space, the minimum channel value of the faintly visible spots in the L channel is 9. This value is used as the analysis threshold for the L channel. In the CIE-Lab color space system, the L value of the corrected Chuanxiong characteristic spots in the chest relief tablets is defined as a present spot if it is greater than 9, and a missing spot if it is less than or equal to 9.

[0311] 1.7 Objective Validation of the Method and Further Screening of the Color System and Channels

[0312] The presence or absence of feature spots is determined according to the analysis threshold and judgment method obtained from the above test group. The judgment accuracy is calculated by dividing the number of spots in the verification group whose judgment results match the records of missing and present feature spots by the total number of feature spots in the verification group. The judgment accuracy is obtained by filtering the selected channels in each color system.

[0313] In this embodiment, 50 batches of Shuxiong Pian samples were used to establish the method, with a total of 150 thin-layer chromatographic characteristic spots; 27 batches of Shuxiong Pian samples were used to validate the method, with a total of 81 thin-layer chromatographic characteristic spots.

[0314] The thin-layer chromatography characteristic spot verification data of chuanxiong in Shuxiong tablets showed that, in the B channel of the RGB color system, 2 missing spots fell within the range of faintly visible spots, and 1 faintly visible spot fell within the range of missing spots. A total of 3 characteristic spots were incorrectly identified, and 78 characteristic spots were correctly identified. The accuracy rate was 78 / 81, or 96.30%.

[0315] In the B channel of the HSB color system, 3 missing spots fall within the range of faintly visible spots, and 3 faintly visible spots fall within the range of missing spots. All 75 feature spots were correctly identified, with an accuracy rate of 75 / 81, or 92.59%.

[0316] In the L channel of the CIE-Lab color system, both clearly visible spots and missing spots are within the specified range. There are 6 incorrect judgments for faintly visible spots and 4 incorrect judgments for missing spots. A total of 71 feature spots are correctly judged, with an accuracy rate of 71 / 81, or 87.65%.

[0317] Therefore, the G channel of the RGB system, which has the highest accuracy, was selected to determine the color of Chuanxiong in the chest-soothing tablets.

[0318] The proposed digital standard for the thin-layer chromatography color of Chuanxiong in Shuxiong tablets is as follows: In the RGB color system, the H value of the three characteristic spots should be in the range of 175-237. Using the thin-layer background as a blank to correct the characteristic spots, the B value of the three characteristic spots in the RGB system should be greater than 43.

[0319] 2. Establishment of the Spacing Ratio Judgment Model

[0320] 1. Select and analyze spots

[0321] Following the principles of Example 1, analytical spots were selected. Along the solvent development direction, the origin and each spot were numbered sequentially from the origin to the solvent front. Based on the characteristics of the reference herb spots in the identification chromatogram of Shuxiong tablets, the differences in the test sample spots, and combined with the experience of manual judgment, the 4th, 5th, and 6th spots, which were relatively stable, were finally selected as analytical spots. The Rf values ​​of spots 4, 5, and 6 were 0.39, 0.44, and 0.67, respectively.

[0322] 2. Consider whether to add the origin.

[0323] In this embodiment, there are three analytical spots. Along the solvent spread direction, the origin and the first three analytical spots are sequentially numbered A, B, C, and D from the origin towards the solvent front (e.g., ...). Figure 10 As shown in the figure, calculate the distances L-ac and L-bd between AC and BD respectively. Since L-ac≥L-bd, the origin is not included as a feature spot for grouping.

[0324] 3. Grouping

[0325] This embodiment has a total of 3 feature spots, which are grouped together.

[0326] 4. Selecting and positioning spots

[0327] Within the aforementioned feature spot group, three feature spots were selected as candidate positioning spots. The distances between the other two feature spots and the positioning spots were measured, the ratio of the minimum distance to the maximum distance was calculated, and the relative standard deviation of each ratio was calculated. The results are shown in the table below.

[0328] Table 5. Analysis results of location data for different locating spots

[0329]

[0330] The results show that the relative standard deviations of the three different positioning points in the thin-layer chromatogram of Shuxiong tablets are 0.04, 0.22 and 0.18, respectively. The relative standard deviation obtained by using the fourth spot as the positioning spot is the smallest. Therefore, the fourth spot with the smallest relative standard deviation value is selected as the positioning spot.

[0331] 5. Establishment of calculation rules for feature spot positions

[0332] Referring to the method in Example 2, this example simultaneously compares three calculation methods to evaluate the location of three spots in the chest-relieving tablet.

[0333] The results of calculating X1, X2, and X3 using the three different calculation methods described above are shown in the table below.

[0334] Table 6. Analysis results of location data using different calculation methods

[0335]

[0336] The above results show that the relative standard deviations of the three different calculation methods in the Shuxiong tablet samples are 0.04, 0.05 and 0.21, respectively. The first calculation method has the smallest relative standard deviation, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for spot localization in thin-layer chromatography of Shuxiong tablets.

[0337] 6. Calculate the spacing ratio RPf

[0338] Following the first method described above, using the fourth spot as the positioning spot, calculate the distances L-45 and L-46 between the fourth spot and the fifth and sixth spots, respectively. Calculate the ratio between the minimum distance L-45 and the maximum distance L-46, denoted as the spacing ratio RPf3. Use this spacing ratio RPf3 as the evaluation index, based on the spacing ratio RPf3 of each characteristic spot group in different thin-layer chromatograms of the standard chromatogram library (50 batches of samples) (e.g., ...). Figure 14 (The black circle in the middle) The evaluation criteria for the spacing ratio RPf3 is set in the range of 0.77 to 0.88.

[0339] IV. Comparative Analysis

[0340] 1. Determining the presence or absence of spots

[0341] The thin-layer chromatograms of 27 batches of Shuxiong tablets were obtained using the above verification method. The H values ​​and analytical channel values ​​of each characteristic spot in the HSB system were collected and substituted into the spot presence / absence judgment model obtained above. The results were compared with the spot presence / absence judgment criteria. The results showed that in the B channel of the RGB color system, 3 missing spots fell within the range of faintly visible spots, and 3 faintly visible spots fell within the range of missing spots. A total of 75 characteristic spots were correctly judged, with a judgment accuracy rate of 92.59%.

[0342] 2. Spacing ratio evaluation

[0343] Take the thin-layer chromatograms of the above 27 batches of Shuxiong tablets, and calculate the corresponding spacing ratio RPf3 according to the first method determined in the spacing ratio judgment model establishment step (e.g., Figure 14 The results of digital thin-layer chromatography detection of the chest-soothing tablets were obtained by comparing the blue circled area with the above evaluation criteria range.

[0344] The data results show that the RPf3 values ​​of the 27 batches of Shuxiong tablets used for the validation method were all within the intended range, indicating that the Shuxiong tablets thin-layer chromatography digital evaluation method based on RPf3 values ​​has good performance.

[0345] Example 4

[0346] A thin-layer chromatography method for detecting and analyzing Panax notoginseng in Xinning tablets, referring to the method in Example 1, is applied to the thin-layer chromatography detection and analysis of Panax notoginseng in Xinning tablets.

[0347] I. Thin-layer chromatography detection

[0348] Take 10 tablets of Xinning, remove the coating, grind into a fine powder, add 50 ml of ether, sonicate for 20 minutes, filter, evaporate the ether from the residue, add 50 ml of methanol, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 50 ml of water. Extract twice with water-saturated n-butanol, 30 ml each time, combine the n-butanol extracts, wash twice with ammonia solution, 60 ml each time, take the n-butanol extract, evaporate to dryness, and dissolve the residue in 1 ml of methanol to obtain the test solution. Spot the test solution onto a silica gel G thin-layer plate, using the upper layer of n-butanol-acetic acid-water (4:1:5) as the developing solvent, develop, remove, and air dry. Spray with 10% sulfuric acid ethanol, heat at 105℃ until the spots are clearly visible, and examine under sunlight to obtain the thin-layer chromatogram (e.g., ...). Figure 15 ).

[0349] II. Collection of Thin-Layer Chromatograms

[0350] Thin-layer chromatograms of qualified and unqualified Panax notoginseng tablets were collected. The unqualified products showed missing characteristic spots in their thin-layer chromatograms, and the presence and absence of characteristic spots were recorded. The thin-layer chromatograms of the qualified products were used to form a standard chromatogram library. The thin-layer chromatograms included those obtained under different experimental conditions.

[0351] Referring to the judgment criteria in Example 1, the thin-layer chromatograms were distributed to five inspectors who independently observed the characteristic spots. Does it exist, and The final result is determined according to the standard of Example 1.

[0352] In this embodiment, the thin-layer chromatograms of Panax notoginseng tablets in Xinning tablets were collected using the above method on different silica gel thin-layer chromatography plates (Merck Corporation and Yantai Chemical Industry Research Institute), at different temperatures (5.1℃, 17.8℃), and at different relative humidityes (32%, 65%, 88%).

[0353] The thin-layer chromatograms of Panax notoginseng in Xinning tablets were divided into 29 batches of Xinning tablets samples for establishing the method (test group) and 48 batches of Panax notoginseng samples for validating the method (validation group). The samples for establishing the method uniformly covered various silica gel G plates, various temperatures and various relative humidity.

[0354] III. Establishment of Analytical Model

[0355] 1. Establishment of a model for determining the presence or absence of spots

[0356] 1.1 Selection of Feature Spots

[0357] Based on the selection principle of characteristic spots in Example 1, select Figure 15 Spots 2, 4, 5, 6, 8, and 9 are characteristic spots, with Rf values ​​of 0.068, 0.20, 0.22, 0.38, 0.58, and 0.68 for spots 2, 4, 5, 6, 8, and 9, respectively.

[0358] 1.2 Extraction of color parameters of feature spots

[0359] The values ​​of the R, G, and B channels of the thin-layer chromatography characteristic spots in the RGB color space, the H, S, and B channels in the HSB color space, and the L, a, and b channels in the CIE-Lab color space were read using TLC Measure software and Photoshop software.

[0360] 1.3 Hue Determination

[0361] In color theory, a color wheel is a circular diagram of a rainbow, dividing hues into 12 hues according to the periodicity of colors. Each hue represents a basic color (such as red, green, blue, etc.) and a mixture of other colors. Hue (H) is a fundamental attribute of color, measuring its position on the color wheel and used to characterize its hue. The H value is a parameter describing the hue of a color, used to measure its position on the color wheel. H values ​​are usually expressed in degrees, ranging from 0 to 360 degrees. For example, 0 degrees represents red, 120 degrees represents green, and 240 degrees represents blue. Therefore, the background color of characteristic spots in thin-layer chromatography can be determined by the H value, achieving an accurate description of the spot color.

[0362] The H values ​​of all characteristic spots of Panax notoginseng in Xinning tablets were collected using TLC Measure software. The minimum and maximum values ​​were set as the range of values, such as... Figure 16As shown in the figure, the horizontal axis represents 6 characteristic spots, and the vertical axis represents the H value. As described in Example 1, considering that the characteristic spots of Panax notoginseng in these three Xinning tablets were all examined at 254nm, and the colors of the three characteristic spots showed the same hue, the common H value range of each characteristic spot in the thin-layer chromatography of Panax notoginseng in Xinning tablets was finally determined to be 280-360.

[0363] 1.4 Background Calibration

[0364] In each color system, the background value of each channel is extracted, and the channel color values ​​are calibrated by subtracting the background value.

[0365] 1.5 Preliminary Screening of Color System and Channels

[0366] The values ​​of characteristic spots in various color systems and channels were analyzed. All thin-layer chromatograms were randomly divided into a test group (29 batches) and a validation group (48 batches). The range of channel values ​​for missing characteristic spots in the test group and the range of channel values ​​for present characteristic spots in the test group were obtained. The minimum value of the channel value range for present characteristic spots in the test group was used as the analysis threshold. The channel values ​​of characteristic spots in the validation group were compared with the analysis threshold. When the channel value of a characteristic spot was greater than the analysis threshold, it was determined that the characteristic spot was present. This determination result was compared and verified with the records of missing and present characteristic spots. The channel with the higher determination accuracy was used as the analysis channel for the characteristic spot, and the analysis threshold of the analysis channel was used as the channel determination criterion for the characteristic spot.

[0367] The test group (a total of 29 batches) was divided into two groups according to the above method: a clearly visible (strong spot) group and a faintly visible (weak spot) group. and missing spots As mentioned above, since all six characteristic spots of Panax notoginseng in the Xinning tablets were examined under sunlight and all six characteristic spots exhibited the same color hue, the channel values ​​of the six characteristic spots were analyzed together without distinguishing between different characteristic spots. However, if it is confirmed that the characteristic spots being analyzed have different color hues, they need to be analyzed separately.

[0368] Using the group classification of the three data sets as the x-axis and the calibrated channel values ​​as the y-axis, a box plot is drawn. In the figure, the midline of the box plot represents the median value of the group, and the upper and lower quartiles are the upper and lower quartiles, respectively. If outliers are identified according to the Turkey's test standard, these data are removed. The resulting box plot is shown below. Figure 17-19 As shown.

[0369] Figure 17 The results are in the RGB color space system, where A, B, and C are the results for the R, G, and B channels, respectively. As can be clearly seen from the figure, the calibrated B channel has the best effect in distinguishing between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Panax notoginseng in Xinning tablets. Figure 17In the C section, the minimum channel value of the faintly visible spots in the B channel is 2. This is used as the analysis threshold for the B channel. That is, in the RGB color space system, the B value of the corrected Panax notoginseng feature spots in Xinning tablets is greater than 2 to indicate the presence of spots, and less than or equal to 2 to indicate missing spots.

[0370] Figure 18 The results are for the HSB color space system, where A, B, and C are the H, S, and B channel results, respectively. It can be clearly seen from the figure that the calibrated S channel has the best effect in distinguishing between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Panax notoginseng in Xinning tablets. Figure 18 In the B part), the minimum channel value of the faintly visible spots in the S channel is -0.3. This is used as the analysis threshold of the S channel. That is, in the HSB color space system, the S value of the Panax notoginseng characteristic spots in Xinning tablets after correction is greater than -0.3 as the presence of spots, and less than or equal to -0.3 as the absence of spots.

[0371] Figure 19 The results are from the CIE-Lab color space system, where A, B, and C represent the L, a, and b channels, respectively. As can be clearly seen from the figure, the calibrated L channel provides the best differentiation between clearly visible spots, faintly visible spots, and missing spots in the thin-layer chromatogram of Panax notoginseng in Xinning tablets. Figure 19 In Part A of the color space, the maximum channel value of the faintly visible spots in the L channel is -2. This value is used as the analysis threshold for the L channel. In the CIE-Lab color space system, the L value of the corrected Panax notoginseng feature spots in Xinning tablets is less than -2 to indicate the presence of spots, and greater than or equal to -2 to indicate missing spots.

[0372] 1.7 Objective Validation of the Method and Further Screening of the Color System and Channels

[0373] The presence or absence of feature spots is determined according to the analysis threshold and judgment method obtained from the above test group. The judgment accuracy is calculated by dividing the number of spots in the verification group whose judgment results match the records of missing and present feature spots by the total number of feature spots in the verification group. The judgment accuracy is obtained by filtering the selected channels in each color system.

[0374] In this embodiment, 29 batches of Xining tablets were used to establish the method, with a total of 174 thin-layer chromatographic characteristic spots; 48 batches of Xining tablets were used to validate the method, with a total of 288 thin-layer chromatographic characteristic spots.

[0375] The data from 288 characteristic spots in the thin-layer chromatography verification sample of Panax notoginseng in Xinning tablets showed that, in the B channel of the RGB color system, 3 missing spots fell within the range of faintly visible spots, 8 faintly visible spots fell within the range of missing spots, and 1 clearly visible spot was in the area of ​​missing spots. A total of 12 characteristic spots were incorrectly identified, and 98 characteristic spots were correctly identified, with an accuracy rate of 276 / 288, or 95.83%.

[0376] In the B channel of the HSB color system, 5 missing spots fall within the range of faintly visible spots, and 6 faintly visible spots fall within the range of missing spots. All 277 feature spots were correctly identified, with an accuracy rate of 277 / 288, or 96.21%.

[0377] In the L channel of the CIE-Lab color system, both clearly visible spots and missing spots are within the specified range. There are 6 misjudged spots and 282 correctly judged feature spots, with an accuracy rate of 282 / 288, or 98.06%.

[0378] Therefore, the L channel of the CIE-Lab system, which has the highest accuracy, was selected to determine the color of Panax notoginseng in Xinning tablets.

[0379] The proposed digital standard for the thin-layer chromatography color of Panax notoginseng in Xinning tablets is as follows: In the CIE-Lab color system, the H value of the 6 characteristic spots should be in the range of 280-360. Using the thin-layer background as a blank to correct the characteristic spots, the L value of the 6 characteristic spots in the CIE-Lab system should be less than -2.

[0380] 2. Establishment of the Spacing Ratio Judgment Model

[0381] 1. Select and analyze spots

[0382] Following the principles of Example 1, analytical spots were selected. Along the solvent development direction, the origin and each spot were numbered sequentially from the origin towards the solvent front. Based on the characteristics of the reference herb spots in the identification chromatography of Panax notoginseng tablets, the differences in the test sample spots, and combined with experience gained from manual judgment, the relatively stable spots 2, 4, 5, 6, 8, and 9 were ultimately selected as analytical spots. The Rf values ​​of these spots 2, 4, 5, 6, 8, and 9 were 0.068, 0.20, 0.22, 0.38, 0.58, and 0.68, respectively (e.g., ...). Figure 15 (As shown).

[0383] 2. Consider whether to add the origin.

[0384] In this embodiment, there are 6 analytical spots. Along the solvent spread direction, the origin and the first three analytical spots are numbered A, B, C, and D (corresponding to the 1st, 2nd, 4th, and 5th spots) sequentially from the origin to the solvent front. The distances L-ac (L14) and L-bd (L25) between AC and BD are calculated respectively. Since L-ac > L-bd, the origin is not included as a feature spot for grouping.

[0385] 3. Grouping

[0386] This embodiment has a total of 6 characteristic spots, which are divided into 3 groups. Spots 2, 4, and 5 are divided into the first group, and spots 6, 8, and 9 are divided into the second group. The distances between spots 4 and 6 and between spots 5 and 8 are calculated to be D-46 and D-58, respectively. Since D-46 < D-58, spots 4, 5, and 6 are divided into the third group.

[0387] 4. Selecting and positioning spots

[0388] Within the aforementioned feature spot group, three feature spots were selected as candidate positioning spots. The distances between the other two feature spots and the positioning spots were measured, the ratio of the minimum distance to the maximum distance was calculated, and the relative standard deviation of each ratio was calculated. The results are shown in the table below.

[0389] Table 7. Analysis results of location data of different spots in the first group.

[0390]

[0391] The results show that in the thin-layer chromatogram of Panax notoginseng in Xinning tablets, the relative standard deviations of the three different positioning points are 0.02, 0.16 and 0.14, respectively. The second spot has the smallest relative standard deviation, so the second spot with the smallest relative standard deviation is selected as the positioning spot.

[0392] Table 8. Analysis results of location data of different spots in the second group.

[0393]

[0394] The results above show that in the thin-layer chromatogram of Panax notoginseng in Xinning tablets, the relative standard deviations of the three different positioning points are 0.04, 0.11 and 0.08, respectively. The relative standard deviation obtained by using the 6th spot as the positioning spot is the smallest. Therefore, the 6th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0395] Table 17. Analysis results of location data of different spots in the third group.

[0396]

[0397] The results show that in the thin-layer chromatogram of Panax notoginseng in Xinning tablets, the relative standard deviations of the three different positioning points are 0.28, 0.34 and 0.05, respectively. The relative standard deviation obtained by using the 6th spot as the positioning spot is the smallest. Therefore, the 6th spot with the smallest relative standard deviation value is selected as the positioning spot.

[0398] 5. Establishment of calculation rules for feature spot positions

[0399] Referring to the method in Example 2, this example simultaneously compares three calculation methods to evaluate the location of six Panax notoginseng spots in Xinning tablets. X1, X2, and X3 were calculated according to the two different calculation methods described above, and the results are shown in the table below.

[0400] Table 18 Analysis results of location data for the first group of different calculation methods

[0401]

[0402] The above results show that the relative standard deviations of the two different calculation methods in the Panax notoginseng samples of Xinning tablets are 0.02, 0.12 and 0.16, respectively. The first calculation method has the smallest relative standard deviation, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the localization of Panax notoginseng spots in thin-layer chromatography in Xinning tablets.

[0403] Table 19 Analysis results of location data for the second group of different calculation methods

[0404]

[0405] The above results show that the relative standard deviations of the three different calculation methods in the second group of Xinning tablets were 0.04, 0.07 and 0.11, respectively. The first calculation method has the smallest relative standard deviation, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the localization of Panax notoginseng spots in Xinning tablets using thin-layer chromatography.

[0406] Table 20: Analysis Results of Location Data Using Different Calculation Methods in Group 3

[0407]

[0408] The above results show that the relative standard deviations of the three different calculation methods in the second group of Xinning tablets were 0.05, 0.14 and 0.34, respectively. The first calculation method has the smallest relative standard deviation, indicating that the first calculation method is least affected by various factors. Therefore, the RPf value is selected as the evaluation method for the localization of Panax notoginseng spots in Xinning tablets using thin-layer chromatography.

[0409] 6. Calculate the spacing ratio RPf

[0410] Following the first method described above, in the first group, the second spot is used as the positioning spot. The distances L-24 and L-25 between the second spot and the fourth and fifth spots are calculated, respectively. The ratio between the minimum distance L-24 and the maximum distance L-25 is calculated and denoted as the spacing ratio RPf4. In the second group, the sixth spot is used as the positioning spot. The distances L-68 and L-69 between the sixth spot and the eighth and ninth spots are calculated, respectively. The ratio between the minimum distance L-68 and the maximum distance L-69 is calculated. The ratio between 69 and 69 is denoted as the spacing ratio RPf5; in the third group, the 6th spot is used as the positioning spot, and the distances L-65 and L-64 between the 6th spot and the 5th and 4th spots are calculated respectively. The ratio between the minimum distance L-65 and the maximum distance L-64 is calculated and denoted as the spacing ratio RPf6; using the spacing ratios RPf4, RPf5, and RPf6 as evaluation indicators, based on the spacing ratio RPf4 of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library (29 batches of samples) (e.g., ... Figure 20 (The black circle in the middle), the evaluation criteria for the spacing ratio RPf4 is established to be within the range of 0.81 to 0.89; the spacing ratio RPf5 of each feature spot group (such as...) Figure 21 (The black circle in the middle), the evaluation criteria for the spacing ratio RPf5 is established to be within the range of 0.64–0.76; the spacing ratio RPf6 of each feature spot group (such as...) Figure 22 (The black circle in the middle) The evaluation criteria for the spacing ratio RPf6 is set in the range of 0.74 to 0.91.

[0411] IV. Comparative Analysis

[0412] 1. Determining the presence or absence of spots

[0413] The thin-layer chromatograms of 48 batches of Xinning tablets were used for the above verification method. The H values ​​and analytical channel values ​​of each characteristic spot in the HSB system were collected and substituted into the spot presence and absence judgment model obtained above. The results showed that in the B channel of the RGB color system, 3 missing spots fell within the range of faintly visible spots, and 3 faintly visible spots fell within the range of missing spots. A total of 75 characteristic spots were correctly judged, and the judgment accuracy rate was 92.59%.

[0414] 2. Spacing ratio evaluation

[0415] Thin-layer chromatograms of Panax notoginseng samples from the above 48 batches of Xinning tablets were obtained, and the corresponding interval ratios RPf4, RPf5, and RPf6 were calculated according to the first method determined in the interval ratio judgment model establishment step (e.g., ...). Figure 20 , Figure 21 , Figure 22 The results of digital thin-layer chromatography detection of Panax notoginseng in Xinning tablets were obtained by comparing the blue circled area with the above evaluation standard range.

[0416] The data results show that the RPf8, RPf9, and RPf10 values ​​of Panax notoginseng samples in Xinning tablets used for the validation method were all within the intended range, indicating that the thin-layer chromatography digital evaluation method for Panax notoginseng in Xinning tablets based on RPf values ​​has good performance.

Claims

1. A method for establishing an analytical model for thin-layer chromatography, characterized in that, The method includes a method for establishing a spot presence / absence determination model and a method for establishing a spacing ratio determination model. The method for establishing the spot presence / absence determination model includes the following steps: Thin-layer chromatogram collection: Collect thin-layer chromatograms of qualified and unqualified Chinese medicine products. The thin-layer chromatograms of unqualified products have missing characteristic spots. Record the presence and absence of characteristic spots. The thin-layer chromatograms include several thin-layer chromatograms under differential experimental conditions. The differential experimental conditions include at least two humidity conditions, at least two temperature conditions, and at least two batches of Chinese medicine products. Standard hue determination: Collect the H values ​​of each characteristic spot in the HSB system from the thin-layer chromatograms of all qualified products, and use the range of H values ​​of the corresponding characteristic spots in the thin-layer chromatograms of each qualified product as the standard H value range of that characteristic spot; in the standard hue determination step, characteristic spots with the same hue are grouped into the same hue group, and each characteristic spot in the same hue group has a common standard H value range, which is the range of H values ​​of all characteristic spots in that group in the thin-layer chromatograms of qualified products; Color system and channel determination: Channel values ​​were collected for each characteristic spot in all thin-layer chromatograms, corresponding to the R, G, and B channels in the RGB color space; the H, S, and B channels in the HSB color space; and the L, a, and b channels in the CIE-Lab color space. All thin-layer chromatograms were randomly divided into a test group and a validation group. The channel value ranges for missing characteristic spots and those for present characteristic spots in the test group were obtained. The minimum channel value range for present characteristic spots in the test group was used as the analysis threshold. The channel values ​​for characteristic spots in the validation group were compared with this analysis threshold. When the channel value of a feature spot is greater than the analysis threshold, it is determined that the feature spot exists. This determination result is compared and verified with the records of missing and present feature spots. The channel with the higher determination accuracy is used as the analysis channel of the feature spot, and the analysis threshold of the analysis channel is used as the channel determination criterion for the feature spot. The channel value is calibrated by subtracting the background value of the corresponding channel from the extracted channel value. The determination accuracy is calculated by dividing the number of spots in the verification group whose determination results match the records of missing and present feature spots by the total number of feature spots in the verification group. Establish a spot presence / absence judgment model: The channel judgment criteria and the standard H value range together constitute the spot presence / absence judgment criteria, i.e.; The method for establishing the spacing ratio judgment model includes the following steps: Establishment of the Spacing Ratio Judgment Model: A standard chromatogram library is formed using thin-layer chromatograms of qualified Chinese herbal medicine products. At least two representative spots from the thin-layer chromatograms of the qualified products are selected as analytical spots. With or without the origin, characteristic spots are formed. The characteristic spots are grouped to obtain characteristic spot groups of three. In each characteristic spot group, one characteristic spot is used as the positioning spot. The distances between the other two characteristic spots and the positioning spot are calculated. The ratio between the minimum distance and the maximum distance is calculated and denoted as the spacing ratio RPf. The spacing ratio RPf is used as the evaluation index. Based on the spacing ratio RPf of each characteristic spot group in different thin-layer chromatograms in the standard chromatogram library, the evaluation standard range of the spacing ratio RPf is established.

2. The method for establishing according to claim 1, characterized in that, The characteristic spots were determined by the location of control spots obtained by thin-layer chromatography using a reference standard or a reference medicinal material under the same conditions. Alternatively, the characteristic spot is determined by a preset Rf value range. When a spot falls into the preset Rf value range, it is considered a characteristic spot of this range. The preset Rf value range is defined by the Rf value range of the corresponding characteristic spot in the thin-layer chromatograms of several qualified products.

3. The method for establishing according to claim 1, characterized in that, The method for determining whether the hues are the same is as follows: distribute the thin-layer chromatogram to at least 3 inspectors, who independently observe and judge the hues of the characteristic spots. If all inspectors judge that the hues are the same, then the hues are considered to be the same. If at least one inspector's judgment is inconsistent with that of the other inspectors, then the hues are considered to be different, and the groups are regrouped until all inspectors judge the characteristic spots of the same hue group to be the same. And / or, the method for determining the presence or absence of the characteristic spots is as follows: distribute the thin-layer chromatogram to at least two inspectors, who independently observe and determine whether the characteristic spots are present or absent. If all inspectors determine that the spots are present, or if at least one inspector's judgment is inconsistent with that of the other inspectors, then the spots are considered to be present. If all inspectors determine that the spots are absent, then the spots are considered to be absent.

4. The method for establishing according to claim 3, characterized in that, The presence of the characteristic spot includes being clearly visible or faintly visible. The standard for being clearly visible is that when all inspectors determine that the feature spot is present, the result is determined to be clearly visible. The standard for being faintly visible is that when at least one inspector's judgment is inconsistent with that of other inspectors, the result is determined to be faintly visible.

5. The method for establishing according to claim 1, characterized in that, Meets at least one of the following conditions: (1) The differential experimental conditions also include at least one of the following conditions: at least two specifications of thin-layer plates, at least two experimental dates, at least two development times, and at least two operators; (2) The characteristic spots meet the following requirements: A: The characteristic spot is present in the chromatogram of the sample of this Chinese herbal medicine, and the characteristic spot is also present in the chromatogram of the reference herb or reference standard, and the positions are corresponding. B: The characteristic spots are regular in shape, have clear boundaries, and exist stably; C: The shift value of the characteristic spots is 0.05–0.8; and D The characteristic spots are not affected by negative light and are specific.

6. The method for establishing according to claim 5, characterized in that, The specific displacement value of the characteristic spot is 0.1 to 0.

8.

7. The method for establishing according to claim 6, characterized in that, The specific displacement value of the characteristic spot is 0.2 to 0.

8.

8. The method for establishing according to any one of claims 1-7, characterized in that, In the step of establishing the spacing ratio judgment model, the criterion for grouping feature spots by adding or not adding the origin is as follows: Step 1 Judgment: When there are 2 analytical spots, the origin is added as a feature spot for grouping. If the origin has an irregular shape or unclear boundaries, the thin-layer plate sampling point is used as the feature spot. The second step of judgment: When the number of analytical spots is ≥3, if the original spot is not clear and difficult to identify, then the original spot is not added to form the feature spot; if the original spot is clear and identifiable, then proceed to the third step of judgment. The third step is to determine the origin and the first three spots of the analytical spot along the solvent spreading direction, starting from the origin and moving towards the solvent front. These spots are numbered A, B, C, and D respectively. The distances L-ac and L-bd between AC and BD are calculated. If L-ac ≥ L-bd, the origin is not included as a feature spot in the group. If L-ac < L-bd, the origin is included as a feature spot in the group. The analytical spots meet the following requirements: A: The analytical spot is present in the chromatogram of the sample of this Chinese herbal medicine, and the analytical spot is also present in the chromatogram of the reference herb or reference standard, and the positions are corresponding. B: The analysis shows that the spots have regular shapes, clear boundaries, and are stable. C: The relative shift value of the analytical spots is 0.05–0.8; D: The analysis of spots is not affected by negative samples and has specificity; The positioning spots are determined by the following method: within each group of feature spots, three feature spots are selected as candidate positioning spots, the distances between the other two feature spots and the positioning spots are measured, the ratio of the minimum distance to the maximum distance is calculated, the relative standard deviation of each ratio is calculated, and the feature spot with the smallest relative standard deviation is selected as the positioning spot. In the step of establishing the spacing ratio judgment model, the following grouping rules are followed: A: Number the feature spots along the solvent spreading direction, and divide the first three spots and the last three spots into the first group and the last group, respectively. B: Each group shares common characteristic spots with adjacent groups; C: Except for the first or last group, the feature spots in the remaining groups are grouped according to the principle of proximity. D: The grouping meets the requirements of items A and C in the above grouping rules, and the total number of groups is the smallest.

9. The method for establishing according to claim 1, characterized in that, The specific displacement value of the analytical spot is 0.1 to 0.

8.

10. The method for establishing according to claim 9, characterized in that, In the step of establishing the spacing ratio judgment model, the feature spots are numbered along the solvent spreading direction and grouped according to the following rules: When the total number of spots analyzed is 2, add the origin or thin-layer plate spot as a characteristic spot to form a group; When the total number of feature spots is 3, these 3 feature spots are grouped together. When the total number of feature spots is 4, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 2, 3, and 4 are divided into the last group. When the total number of feature spots is 5, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 3, 4, and 5 are divided into the last group. When the total number of feature spots is 6, feature spots numbered 1, 2, and 3 are divided into the first group, and feature spots numbered 4, 5, and 6 are divided into the last group. The distance between feature spots numbered 2 and 4 and the distance between feature spots numbered 3 and 5 are calculated, which are D-24 and D-35 respectively. When D-24 > D-35, feature spots numbered 3, 4, and 5 are selected as the middle group. When D-24 < D-35, feature spots numbered 2, 3, and 4 are selected as the middle group. When D-24 = D-35, feature spots numbered 3, 4, and 5 or feature spots numbered 2, 3, and 4 can be selected as the middle group. When the total number of feature spots is 7, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 5, 6, and 7 are divided into the last group, and feature spots numbered 3, 4, and 5 are divided into the middle group. When the total number of feature spots is 8, feature spots numbered 1, 2, and 3 are divided into the first group, feature spots numbered 6, 7, and 8 are divided into the last group, feature spots numbered 3, 4, and 5 are divided into the first middle group, and feature spots numbered 4, 5, and 6 are divided into the second middle group.

11. A thin-layer chromatography detection and analysis method, characterized in that, Take the thin-layer chromatogram of the Chinese herbal medicine to be tested, collect the H value and analysis channel value of each characteristic spot in the HSB system, and compare the spot presence or absence judgment model obtained by the method described in any one of claims 1-10 with the spot presence or absence judgment standard to obtain the spot presence or absence judgment result. Take the thin-layer chromatogram of the Chinese herbal medicine to be tested, calculate the corresponding interval ratio RPf according to the method of establishing the interval ratio judgment model as described in any one of claims 1-10, and compare it with the evaluation standard range to obtain the interval ratio evaluation result.

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