Malt thin layer identification method

Abstract

The invention discloses a malt thin layer identification method, and relates to the technical field of traditional Chinese medicine detection. The detection method comprises the following steps: taking a malt reference medicinal material, mixing the malt reference medicinal material with an extraction solvent, performing ultrasonic treatment, filtering, evaporating filtrate to dryness, and redissolving to obtain a malt reference medicinal material solution; preparing a malt negative sample, mixing the malt negative sample with an extraction solvent, performing ultrasonic treatment, filtering, evaporating filtrate to dryness, and redissolving to obtain a malt negative control solution; the method comprises the following steps: taking a test product digestion-promoting tablet, mixing with an extraction solvent, carrying out ultrasonic treatment, filtering, evaporating filtrate to dryness, and redissolving to obtain a digestion-promoting tablet test product solution; sucking 10-15 microliters of the negative control solution, 10-15 microliters of the malt control medicinal material solution and 10-15 microliters of the test sample digestion-promoting tablet solution, respectively dripping the solutions on the same thin-layer plate, developing by using ethyl acetate-methanol-water as a developing solvent, taking out, spraying an anisaldehyde-sulfuric acid solution, heating at 100-110 DEG C until the color of spots is clear, and inspecting under a fluorescent lamp. The detection method disclosed by the invention is good in specificity, good in durability and high in stability.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically to a thin-layer chromatography method for identifying malt. Background Technology

[0002] Malt plays an important role as one of the raw materials for digestive tablets. Generally, the methods for identifying malt are relatively simple, but they are not easy to operate and have poor reproducibility. The thin-layer chromatographic information obtained is not comprehensive enough, thus failing to meet people's identification requirements.

[0003] Chinese patent application CN106018663A provides a method for identifying malt in digestive tablets. The method involves preparing a malt test solution A and a malt reference solution B. Using thin-layer chromatography, 10 μL of each solution (A and B) is applied separately to the same silica gel G thin-layer plate using sodium carboxymethyl cellulose as a binder. The plate is developed using benzene-chloroform as the developing solvent. After development, the plate is removed, dried, and sprayed with a 50% ethanol solution containing 15% nitric acid. The plate is then heated at 100°C until the spots are clearly visible. Upon examination under ultraviolet light, the chromatogram of the test sample shows fluorescent spots of the same color at the corresponding positions as the chromatogram of the reference material.

[0004] Chinese patent application CN110596105A provides a quality standard detection method for wheat germ medicinal materials, including microscopic identification, thin-layer chromatography identification, and determination of moisture, total ash, and extractives. The thin-layer chromatography identification steps are as follows: Take the powder of this product, add ethanol, sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in ethanol to obtain the test solution; separately take wheat germ reference medicinal materials and prepare a reference medicinal material solution using the same method; according to the thin-layer chromatography method, take the above two solutions and spot them separately on the same silica gel G thin-layer plate, use chloroform-ethyl acetate at a ratio of 10:1 as the developing solvent, develop, remove, dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible; examine under ultraviolet light; in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0005] Based on the existing technologies described above, malt thin-layer chromatography (TLC) identification primarily utilizes this technique, comparing the chromatographic bands of a reference standard and the test sample to achieve qualitative identification of malt using fluorescent spots. However, the extraction process described in Chinese patent application CN106018663A is extremely cumbersome, and the technique described in Chinese patent application CN110596105A lacks a negative solution, both of which are insufficient. Therefore, there is an incentive to optimize these methods, determine the optimal measurement parameters, and validate the malt TLC identification method to obtain stable and reliable results. Summary of the Invention

[0006] This invention provides a method for identifying malt, specifically for thin-layer chromatography (TLC) identification of malt in Jiangzhong brand digestive tablets. Jiangzhong brand digestive tablets contain the following ingredients: Codonopsis pilosula, malt, hawthorn, yam, tangerine peel, and Poria cocos. This method improves upon previous methods by optimizing the sample pretreatment, developing solvent ratio, colorimetric reagent, TLC plate, and sample application amount, thus resolving the issues of hazardous developing solvent components and complex operation. Method validation shows that the detection method of this invention has no impact on the identification of other medicinal materials in the product, exhibits good specificity, and meets the identification requirements for temperature, humidity, developing solvent ratio, and stability. It can more accurately identify malt in this traditional Chinese medicine compound preparation using TLC, providing a reference for its quality control.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On one hand, the present invention provides a method for identifying malt using thin-layer chromatography, comprising the following steps: (1) Preparation of reference medicinal material solution: Take malt reference medicinal material, mix with extraction solvent, sonicate, filter, evaporate the filtrate to dryness, add extraction solvent to reconstitute, and use as malt reference medicinal material solution; (2) Preparation of negative control solution: Prepare malt negative sample, mix with extraction solvent, sonicate, filter, evaporate the filtrate to dryness, add extraction solvent to reconstitute, and use as malt negative control solution; (3) Preparation of test solution: Take the test sample digestive tablets, mix with the extraction solvent, sonicate, filter, evaporate the filtrate to dryness, add the extraction solvent to redissolve, and use as the digestive tablets test solution; (4) Take 10-15 μL each of the negative control solution, malt control medicinal material solution and test sample digestive tablet solution, spot them on the same thin layer plate, develop with ethyl acetate-methanol-water as the developing solvent, remove the plate, spray with anisaldehyde-sulfuric acid solution and heat at 100-110℃ until the spots are clearly visible, and examine under a fluorescent lamp.

[0008] Preferably, in step (1), the extraction solvent is a mixture of chloroform and methanol.

[0009] Preferably, the volume ratio of chloroform to methanol is 0.9-1.1:0.9-1.1.

[0010] Any point value or sub-range value within the range of 0.9-1.1:0.9-1.1 is applicable to this invention, including but not limited to 0.9:1.1, 0.9:1, 0.9:0.9, 1:0.9, 1:1, 1:1.1, 1.1:0.9, and 1.1:1.

[0011] Preferably, in step (1), the ultrasound time is 25-35 minutes.

[0012] Any point value or sub-range value within the range of 25-35 min is applicable to this invention, including but not limited to 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, and 35 min.

[0013] Preferably, in step (1), the mass-to-volume ratio of the malt reference material to the extraction solvent is 1:28-32, in g:mL.

[0014] Any point value or sub-range value within the range of 1:28-32 (unit is g:mL) is applicable to this invention, including but not limited to 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, and 1:35.

[0015] Preferably, in step (1), the temperature of evaporation is 80-90°C.

[0016] Any point value or sub-range value within the range of 80-90℃ is applicable to this invention, including but not limited to 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, and 90℃.

[0017] Preferably, in step (2), the extraction solvent is a mixture of chloroform and methanol.

[0018] Preferably, the volume ratio of chloroform to methanol is 0.9-1.1:0.9-1.1.

[0019] Any point value or sub-range value within the range of 0.9-1.1:0.9-1.1 is applicable to this invention, including but not limited to 0.9:1.1, 0.9:1, 0.9:0.9, 1:0.9, 1:1, 1:1.1, 1.1:0.9, and 1.1:1.

[0020] Preferably, in step (2), the ultrasound time is 25-35 minutes.

[0021] Any point value or sub-range value within the range of 25-35 min is applicable to this invention, including but not limited to 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, and 35 min.

[0022] Preferably, in step (2), the mass-to-volume ratio of the malt negative sample to the extraction solvent is 10:28-32, in g:mL.

[0023] Any point value or sub-range value within the range of 10:28-32 (unit: g:mL) is applicable to this invention, including but not limited to 10:28, 10:29, 10:30, 10:31, 10:32, 10:33, 10:34, and 10:35.

[0024] Preferably, in step (2), the preparation of the malt-negative sample includes the following steps: Codonopsis pilosula, Citrus reticulata peel, Poria cocos, and hawthorn are decocted 1-3 times with 8-12 times their weight of water for 2.5-3.5 hours each time. The decoction is filtered, the filtrates are combined, concentrated into a clear extract, and spray-dried. Dioscorea opposita powder, dextrin, sucrose, and citric acid are added, mixed well, granulated, magnesium stearate is added, and the mixture is compressed into tablets and coated with a film to obtain a malt-negative sample.

[0025] More preferably, in step (2), the preparation of the malt-negative sample includes the following steps: 27g of Codonopsis pilosula, 27g of dried tangerine peel, 27g of Poria cocos, and 27g of hawthorn were decocted twice with 10 times their weight of water for 3 hours each time. The decoction was filtered, the filtrates were combined, concentrated into a clear extract, and spray-dried. 27g of yam powder, 10g of dextrin, 10g of sucrose, and 10g of citric acid were added, mixed well, granulated, and 1g of magnesium stearate were added. The mixture was then compressed into tablets and coated with a film to obtain a malt-negative sample.

[0026] Preferably, in step (2), the temperature of evaporation is 80-90℃.

[0027] Any point value or sub-range value within the range of 80-90℃ is applicable to this invention, including but not limited to 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, and 90℃.

[0028] Preferably, in step (3), the extraction solvent is a mixture of chloroform and methanol.

[0029] Preferably, the volume ratio of chloroform to methanol is 0.9-1.1:0.9-1.1.

[0030] Any point value or sub-range value within the range of 0.9-1.1:0.9-1.1 is applicable to this invention, including but not limited to 0.9:1.1, 0.9:1, 0.9:0.9, 1:0.9, 1:1, 1:1.1, 1.1:0.9, and 1.1:1.

[0031] Preferably, in step (3), the ultrasound time is 25-35 minutes.

[0032] Any point value or sub-range value within the range of 25-35 min is applicable to this invention, including but not limited to 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, and 35 min.

[0033] Preferably, in step (3), the mass-to-volume ratio of the test sample digestive tablets to the extraction solvent is 1:28-32, in g:mL.

[0034] Any point value or sub-range value within the range of 1:28-32 (unit is g:mL) is applicable to this invention, including but not limited to 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, and 1:35.

[0035] Preferably, in step (3), the temperature of evaporation is 80-90℃.

[0036] Any point value or sub-range value within the range of 80-90℃ is applicable to this invention, including but not limited to 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, and 90℃.

[0037] In step (4), any point value or sub-range value within the range of 10-15μL is applicable to the present invention, including but not limited to 10μL, 11μL, 12μL, 13μL, 14μL, and 15μL.

[0038] In step (4), any point value or sub-range value within the range of 100-110℃ is applicable to the present invention, including but not limited to 100℃, 101℃, 102℃, 103℃, 104℃, 105℃, 106℃, 107℃, 108℃, 109℃, and 110℃.

[0039] Preferably, in step (4), the thin film is a high-efficiency silicone G thin film.

[0040] Preferably, in step (4), the volume ratio of ethyl acetate-methanol-water is 10:2:0.7-1.3.

[0041] Any point value or sub-range value within the range of 8-12:1-3:1 is applicable to this invention, including but not limited to 10:2:0.7, 10:2:0.8, 10:2:0.9, 10:2:1, 10:2:1.1, 10:2:1.2, and 10:2:1.3.

[0042] Preferably, the detection method includes the following steps: (1) Preparation of reference medicinal material solution: Weigh 1g of malt reference medicinal material, add 30mL of a 1:1 mixture of chloroform and methanol, sonicate for 30 minutes, filter, take the filtrate and evaporate to dryness in a water bath at 85℃, add 1mL of a 1:1 mixture of chloroform and methanol to reconstitute, and use as malt reference medicinal material solution. (2) Preparation of negative control solution: 27g of Codonopsis pilosula, 27g of Citrus reticulata peel, 27g of Poria cocos, and 27g of Crataegus pinnatifida. The above four herbs are decocted twice with 10 times their weight of water, each time for 3 hours. After filtration, the filtrates are combined and concentrated into a clear paste, which is then spray-dried. 27g of Dioscorea opposita powder, 10g of dextrin, 10g of sucrose, and 10g of citric acid are added and mixed well. The mixture is then granulated, 1g of magnesium stearate is added, and the mixture is compressed into tablets and coated with a film to obtain the malt negative sample. 10g of the malt negative sample is weighed and added to 30mL of a 1:1 mixture of chloroform and methanol. After ultrasonic treatment for 30 minutes, the sample is filtered, and the filtrate is evaporated to dryness in a water bath at 85℃. 1mL of a 1:1 mixture of chloroform and methanol is added to redissolve the filtrate to obtain the malt negative control solution. (3) Preparation of test solution: Weigh 10g of the digestive tablets and add 30mL of a 1:1 mixture of chloroform and methanol. After sonication for 30 minutes, filter the solution and evaporate the filtrate to dryness in a water bath at 85℃. Add 1mL of a 1:1 mixture of chloroform and methanol to redissolve the solution and use it as the digestive tablets test solution. (4) Take 15 μL each of the negative control solution, malt control medicinal material solution and test sample digestive tablet solution and spot them on the same high-performance silica gel G thin layer plate. Develop with ethyl acetate-methanol-water in a volume ratio of 10:2:1. Remove the plate, spray with anisaldehyde-sulfuric acid solution and heat at 105℃ until the spots are clearly visible. Examine under a fluorescent lamp.

[0043] The beneficial effects of this invention are: This invention provides a thin-layer chromatography (TLC) detection method for Jiangzhong brand digestive tablets. By specifying the sample pretreatment method, developing solvent ratio, colorimetric reagent, TLC plate, and sample application amount, it solves the problems of hazardous developing solvent components and complex operation. Methodological validation shows that the detection method provided by this invention has no impact on the identification of other medicinal materials in the product, exhibits good specificity, and meets the identification requirements for temperature, humidity, developing solvent ratio, and stability. It can more accurately identify malt in Jiangzhong brand digestive tablets using TLC, providing a reference for its quality control. Attached Figure Description

[0044] Figure 1 This is a thin-layer chromatogram of Example 1.

[0045] Figure 2 This is a thin-layer chromatogram of Example 2.

[0046] Figure 3Thin-layer chromatograms for specificity investigation.

[0047] Figure 4 Thin-layer chromatograms for durability-temperature assessment.

[0048] Figure 5 Thin-layer chromatogram for durability-humidity assessment.

[0049] Figure 6 Thin-layer chromatograms for durability-developing solvent ratio investigation.

[0050] Figure 7 Thin-layer chromatograms for stability assessment.

[0051] Figure 8 This is the thin-layer chromatogram of Comparative Example 1.

[0052] Figure 9 This is the thin-layer chromatogram of Comparative Example 2.

[0053] Figure 10 This is the thin-layer chromatogram of Comparative Example 3.

[0054] Figure 11 This is the thin-layer chromatogram of Comparative Example 4.

[0055] Figure 12 This is the thin-layer chromatogram of Comparative Example 5.

[0056] Figure 13 This is the thin-layer chromatogram of Comparative Example 6.

[0057] Figure 14 This is the thin-layer chromatogram of Comparative Example 7.

[0058] Figure 15 This is the thin-layer chromatogram of Comparative Example 8. Detailed Implementation

[0059] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0060] Some raw materials, reagents, and instruments / equipment: 1. Raw materials and reagents (1) Samples: Jiangzhong brand digestive tablets, batch number: 25060004; malt negative sample (self-made: negative sample prepared according to the product formula and process flow of Jiangzhong brand digestive tablets, batch number: 250620); (2) Reference medicinal material: malt, batch number: 121105-202309, manufacturer: China National Institutes for Food and Drug Control, ID: 121105-202309; (3) Thin film: Yinlong brand silicone G thin film (Yantai Huayang New Material Technology Co., Ltd., 10×10cm, 20250611-high efficiency board, 20250605-ordinary board), silicone G thin film (Qingdao Ocean Chemical Plant, 10×10cm, 20250818), silicone GF254 thin film (Qingdao Ocean Chemical Plant, 10×10cm, 20250806); (4) Reagents: Water: Meets the Class I water standard of GB / T6682; Chloroform: AR, 2024040101, Chengdu Kelong Chemical Co., Ltd.; Methanol: AR, 20240509, Guangdong Guanghua Technology Co., Ltd.; Ethyl acetate: AR, 240724A1, Xilong Scientific Co., Ltd.; Sulfuric acid: AR, 20210301, Tianjin Damao Chemical Reagent Factory; Anhydrous ethanol: AR, 20250805, Tianjin Damao Chemical Reagent Factory; α-Naphthol: AR, P3011942, Shanghai Titan Technology Co., Ltd.; Glacial acetic acid: AR, 20250703, Tianjin Damao Chemical Reagent Factory; Anisaldehyde: AR, 20250617, Tianjin Damao Chemical Reagent Factory; Potassium hydroxide: AR, 20250103, Tianjin Damao Chemical Reagent Factory; Toluene: AR, 2024112701, Chengdu Kelong Chemical Co., Ltd.; Nitric acid: AR, 2021031101, Chengdu Kelong Chemical Co., Ltd.; Petroleum ether (30-60℃): AR, 20231201, Tianjin Damao Chemical Reagent Factory; Diethyl ether: AR, 2024051301, Chengdu Kelong Chemical Co., Ltd.

[0061] (5) Test solution: 32% relative humidity - sulfuric acid solution: Add 68 mL of sulfuric acid to 100 mL of water and mix well; 65% relative humidity - sulfuric acid solution: Add 34 mL of sulfuric acid to 100 mL of water and mix well; 88% relative humidity - sulfuric acid solution: Add 10.8 mL of sulfuric acid to 100 mL of water and mix well; Anisaldehyde-sulfuric acid solution: Take 0.5 mL anisaldehyde, 85 mL methanol, and 10 mL glacial acetic acid, mix well, then add 5 mL concentrated sulfuric acid and mix well to obtain the solution. 10% sulfuric acid ethanol solution: Slowly add 25 mL of sulfuric acid to anhydrous ethanol, and then dilute the ethanol to 250 mL. 15% α-naphthol ethanol test solution: Preparation method: Take 15g of α-naphthol, add 100mL of ethanol to dissolve it, and you will get the solution. α-Naphthol solution: Take 10.5 mL of 15% α-naphthol ethanol solution, slowly add 6.5 mL of sulfuric acid, mix well, then add 40.5 mL of ethanol and 4 mL of water, mix well, and the solution is ready. 15% nitric acid ethanol solution: Take 15 mL of nitric acid, slowly add it to ethanol, and dilute with ethanol to 100 mL to obtain the solution; 50% ethanol solution of 15% nitric acid: Take 58.8 mL of anhydrous ethanol and dilute to 100 mL to prepare a 58.8% ethanol solution; take 85 mL of the 58.8% ethanol solution, slowly add 15 mL of nitric acid, mix well, and you have the solution.

[0062] 2. Experimental instruments and equipment Electronic balance (0.01%): Mettler, ML204T, 12002715; Digital display constant temperature water bath: Guohua (Changzhou) Instrument Manufacturing Co., Ltd., HH-4; Ultrasonic cleaning machine: Xinzhi, SB-500DY, 12001792; Drying oven: being, BO-200FL(02), 12002650; Thin-layer spotting system: CAMAG LINOMAT5, 14000065, Switzerland; Thin-layer imaging system: CAMAG, REPROSTAR3, 99000028.

[0063] Example 1 A method for identifying malt using thin-layer chromatography includes the following steps: (1) Preparation of reference medicinal material solution: Weigh 1g of malt reference medicinal material, add 30mL of a 1:1 mixture of chloroform and methanol, sonicate for 30 minutes, filter, take the filtrate and evaporate to dryness in a water bath at 85℃, add 1mL of a 1:1 mixture of chloroform and methanol to reconstitute, and use as malt reference medicinal material solution.

[0064] (2) Preparation of negative control solution: 27g of Codonopsis pilosula, 27g of Citrus reticulata peel, 27g of Poria cocos, and 27g of Crataegus pinnatifida. The above four herbs are decocted twice with 10 times their weight of water, 3 hours each time. The decoction is filtered, the filtrates are combined, concentrated into a clear paste, and spray-dried. 27g of Dioscorea opposita powder, 10g of dextrin, 10g of sucrose, and 10g of citric acid are added, mixed well, granulated, 1g of magnesium stearate is added, tableted, and coated with a film to obtain the malt negative sample. 10g of the malt negative sample is weighed and added to 30mL of a 1:1 mixture of chloroform and methanol. After ultrasonic treatment for 30 minutes, the sample is filtered, and the filtrate is evaporated to dryness in an 85℃ water bath. 1mL of a 1:1 mixture of chloroform and methanol is added to redissolve the filtrate to obtain the malt negative control solution.

[0065] (3) Preparation of test solution: Weigh 10g of the test sample digestive tablets (Jiangzhong brand digestive tablets), add 30mL of a 1:1 mixture of chloroform and methanol, sonicate for 30 minutes, filter, take the filtrate and evaporate to dryness in a water bath at 85℃, add 1mL of a 1:1 mixture of chloroform and methanol to reconstitute, and use as the test solution of digestive tablets.

[0066] (4) Take 15 μL each of the negative control solution, malt reference medicinal material solution, and test sample digestive tablet solution, and spot them separately on the same Yinlong brand high-performance silica gel G thin-layer plate. Develop with ethyl acetate-methanol-water (v / v) as the developing solvent. Remove the plate, spray with anisaldehyde-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine under fluorescent light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0067] The thin-layer chromatogram of Example 1 is as follows: Figure 1 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0068] Example 2 Investigation of the sample loading amount: Following the procedure of Example 1, only the effect of the sample loading amount of the digestive tablet solution in step (4) was examined: 10 μL, 12 μL, 15 μL, and 20 μL of the sample solution were respectively taken, and the rest was the same as in Example 1. The thin-layer chromatogram is shown below. Figure 2 As shown, malt characteristic spots were visible when the sample loading volume was 10 μL, 12 μL, and 15 μL. However, the malt characteristic spots were slightly lighter in color when the sample loading volume was 10 μL and 12 μL. When the sample loading volume was 20 μL, the band separation was poor, possibly due to sample overload. Considering all factors, 15 μL of sample was the optimal loading volume.

[0069] Example 3 Methodological Validation As described in Example 1, a control herbal solution, a test solution, and a negative sample solution were prepared for use in the methodological verification of Example 3.

[0070] 3.1 Exclusivity 3.1.1 Spotting, unfolding, and inspection methods: Take 15 μL each of the negative control solution, reference herb solution, and test solution, and spot them separately on the same Yinlong brand high-performance silica gel G thin-layer plate. Develop the plate using ethyl acetate-methanol-water (v / v) as the developing solvent. Remove the plate, spray with anisaldehyde-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine the plate under fluorescent light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0071] 3.1.2 Results Thin-layer chromatogram as follows Figure 3 As shown, the digestive tablet solution exhibits characteristic spots of the same color at the corresponding positions in the malt reference material, and the negative controls show no interference (Rf values ​​of 0.4 and 0.6). The test solution shows spots of the same color at the corresponding positions as the reference solution, with clear spots and good separation. The negative control solution shows no interference. This indicates that the thin-layer chromatography identification method has high specificity.

[0072] 3.2 Durability 3.2.1 Temperature Investigation 3.2.1.1 Spotting, unfolding, and inspection methods: Take 15 μL each of the negative control solution, reference medicinal material solution, and test solution, and spot them separately on the same Yinlong brand high-performance silica gel G thin-layer plate at 4℃, 25℃, and 40℃. Develop with ethyl acetate-methanol-water (v / v) as the developing solvent. Remove the plate, spray with anisaldehyde-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine under fluorescent light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0073] 3.2.1.2 Results Thin-layer chromatogram as shown Figure 4 As shown, the results indicate that under various temperature conditions, the test sample chromatogram showed the same color spots at the corresponding positions as the malt reference material chromatogram. This suggests that the thin-layer chromatography identification method for malt can be performed at temperatures ranging from 4℃ to 40℃. However, it was found that at temperatures of 4℃ and 40℃, the development time was longer and the target spots were closer to other impurity spots, with a separation degree slightly lower than 25℃. Therefore, considering all factors, the experiment can be performed at room temperature.

[0074] 3.2.2 Humidity Assessment 3.2.2.1 Spotting, unfolding, and inspection methods: Take 15 μL each of the negative control solution, reference medicinal material solution, and test solution, and spot them separately on the same Yinlong brand high-performance silica gel G thin-layer plate at humidity levels of 32%, 65%, and 88%. Develop the plate using ethyl acetate-methanol-water (10:2:1) as the developing solvent. Remove the plate, spray with anisaldehyde-sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Examine the plate under fluorescent light. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0075] 3.2.2.2 Results Thin-layer chromatogram as shown Figure 5 As shown, the results indicate that the malt control solution has obvious characteristic spots under different humidity levels, and the digestive tablet solutions have spots of the same color at the same positions as the malt control spots. Although the Rf value varies under different humidity levels, it is within the range of 0.2-0.8, indicating that the malt thin-layer chromatography identification method can be performed at humidity levels of 32%-88%.

[0076] 3.2.3 Investigation of the composition ratio of the developing solvent 3.2.3.1 Spotting, unfolding, and inspection methods: The samples were spotted according to the proposed method. The thin-layer plates were developed in ethyl acetate-methanol-water (10:2:1.3, v / v / v) and ethyl acetate-methanol-water (10:2:0.7, v / v / v) developing solvents, respectively. The plates were then removed, dried, sprayed with anisaldehyde sulfuric acid solution, heated at 105℃ for color development, and examined under fluorescent light.

[0077] 3.2.3.2 Results Thin-layer chromatogram as shown Figure 6 As shown, the results indicate that the malt control solution had obvious purple spots under both developing solvent ratios, and the digestive tablet solution had purple spots of the same color at the same positions as the malt control solution. Although the Rf values ​​varied, they were all within the range of 0.2-0.8. This shows that even with slight adjustments to the proportions of the developing solvent components in the digestive tablet thin-layer chromatography method (adjusting the proportion of components with a smaller developing solvent ratio by ±30% relative to the relative value), the malt in the digestive tablet can still be successfully identified.

[0078] 3.3 Stability 3.3.1 Spotting, unfolding, and inspection methods: Malt control solution, digestive tablets test solution, and negative test solution were prepared according to the above-mentioned proposed method. After preparation, they were stored at 2-8℃. The malt control solution, digestive tablets test solution, and negative test solution stored for 0 days and 7 days were selected for spotting, development, and inspection.

[0079] 3.3.2 Results Thin-layer chromatogram as shown Figure 7As shown, the results indicate that the malt control solution and the digestive tablet test solution, after being stored at 2-8℃ for 7 days, exhibited characteristic spots. The size and depth of these spots were not significantly different from those of the samples prepared on the same day, indicating that the malt control solution and the digestive tablet test solution had good stability after being stored at 2-8℃ for 7 days and could still be used.

[0080] Comparative Example 1 Compared with Example 1, the preparation methods of the test solution, negative sample solution, and control herbal solution were changed, while the rest remained the same as in Example 1: Take 28g of the test sample Xiaoshi tablet powder, 28g of the malt negative sample, and 5g of the malt reference material. Add 60mL of anhydrous ethanol, sonicate for 40min, take the filtrate, add 1.5mL of 50% KOH, heat under reflux for 15min, cool in an ice-water bath for 5min, add 2mL of water, and extract three times with 20mL of petroleum ether (30-60℃). Combine the petroleum ether extracts, evaporate to dryness, and redissolve in 1mL of ethyl acetate to obtain the test sample solution, negative sample solution, and reference material solution.

[0081] Thin-layer chromatogram as shown Figure 8 As shown, fluorescent spots appeared at the same positions in the test sample Xiaoshi tablets, the malt control material, and the negative control. This method is not suitable for thin-layer identification experiments of malt characteristic spots in the test sample Xiaoshi tablets.

[0082] Comparative Example 2 Compared with Example 1, the only difference was that the 1:1 mixture of chloroform and methanol was replaced with anhydrous ethanol, while the rest remained the same as in Example 1.

[0083] Thin-layer chromatogram as shown Figure 9 As shown, the digestive tablets test sample and the malt control sample have corresponding spots at an Rf value of 0.9, and the malt is negative and has no interference, but the shape and size are inconsistent and the Rf is larger.

[0084] Comparative Example 3 Compared with Example 1, the only difference was that the mixed solution of chloroform and methanol with an extraction solvent volume ratio of 1:1 was changed to 70 wt% ethanol, and the rest was the same as Example 1.

[0085] Thin-layer chromatogram as shown Figure 10 As shown, spots appeared in the same positions as the test sample Xiaoshi tablets, the malt control material, and the negative control. This method is not suitable for thin-layer chromatography identification of malt in the test sample Xiaoshi tablets.

[0086] Comparative Example 4 Compared with Example 1, only the developing solvent was changed to petroleum ether (30℃-60℃)-diethyl ether (9:4, v / v), and the rest was the same as Example 1.

[0087] Thin-layer chromatogram as shown Figure 11 As shown, neither the tested digestive tablets nor the malt control material had corresponding negative and interference-free characteristic spots.

[0088] Comparative Example 5 Compared to Example 1, only the developing solvent was changed to ethyl acetate:methanol:water (10:2:1, v / v / v).

[0089] Thin-layer chromatogram as shown Figure 12 As shown, the test sample of digestive tablets and the malt control sample both have a light-colored, corresponding, negative, and non-interfering characteristic spot at an Rf of 0.7.

[0090] Comparative Example 6 Compared with Example 1, only the colorimetric reagent was changed to a 50% ethanol solution of 15% nitric acid; the rest was the same as Example 1.

[0091] Thin-layer chromatogram as shown Figure 13 As shown, the test sample of digestive tablets and the malt control sample do not have corresponding and negative characteristic spots that do not interfere with each other.

[0092] Comparative Example 7 Compared with Example 1, only the colorimetric agent was changed to α-naphthol solution, and the rest was the same as Example 1.

[0093] Thin-layer chromatogram as shown Figure 14 As shown, the test sample (digestive tablets) and the malt control sample both have a corresponding characteristic spot that is negative and interference-free. In Example 1, when anisaldehyde-sulfuric acid reagent was used as the colorimetric reagent, the chromatogram of the test sample clearly showed two characteristic spots corresponding to the chromatogram of the malt control sample. The spot boundaries were clear, the color development was stable, and the negative control chromatogram showed no spot interference at the corresponding positions.

[0094] Comparative Example 8 Compared with Example 1, the thin-layer plates were changed to Yinlong ordinary silicone G thin-layer plates, Qingdao Ocean silicone G thin-layer plates, and Qingdao Ocean silicone GF254 thin-layer plates, respectively, while the rest were the same as in Example 1.

[0095] Thin-layer chromatogram as shown Figure 15 As shown, in the comparative experiments of different types of thin-layer plates, when the Xiaoshi Pian (digestive tablet) test solution was analyzed using the Yinlong brand high-efficiency silica gel G thin-layer plate (Example 1), consistent thin-layer spots could be observed at the corresponding positions of the chromatographic spots of the malt control herb; however, no spots appeared at the same positions in the negative control sample, indicating a negative result. When using the Yinlong ordinary silica gel G thin-layer plate, although the chromatographic spots of the Xiaoshi Pian test solution were distinguishable, the overall separation effect was significantly different from that of the high-efficiency thin-layer plate. When the experiments were conducted using the Qingdao Ocean silica gel G thin-layer plate and the GF254 thin-layer plate, the chromatographic spots of the Xiaoshi Pian test solution were blurred, and the separation was poor.

[0096] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for the identification of malt lamella, characterized in that, It comprises the following steps: (1) Preparation of the control medicinal material solution: Take the control medicinal material of malt, mix with the extraction solvent, ultrasonic, filter, evaporate the filtrate to dryness, add the extraction solvent to dissolve, as the control medicinal material solution of malt; (2) Preparation of the negative control solution: Prepare the negative sample of malt, mix with the extraction solvent, ultrasonic, filter, evaporate the filtrate to dryness, add the extraction solvent to dissolve, as the negative control solution of malt; (3) Preparation of the test solution: Take the test product of Xiaoshi tablet, mix with the extraction solvent, ultrasonic, filter, evaporate the filtrate to dryness, add the extraction solvent to dissolve, as the test solution of Xiaoshi tablet; (4) Take 10-15 μL of the negative control solution, the control medicinal material solution of malt and the test solution of Xiaoshi tablet respectively, and point on the same thin layer plate, develop with ethyl acetate-methanol-water as the developing agent, take out, spray with anisaldehyde-sulfuric acid solution, heat at 100-110 ℃ until the spots develop clear color, and observe under the daylight lamp.

2. The malt thin layer identification method according to claim 1, characterized by, In step (1), step (2) and step (3), the extraction solvent is a mixture of chloroform and methanol with a volume ratio of 0.9-1.1:0.9-1.

1.

3. The malt thin layer identification method according to claim 1, characterized by, In step (1), step (2) and step (3), the ultrasonic time is 25-35 min.

4. The malt thin layer identification method according to claim 1, characterized by, In step (1), step (2) and step (3), the evaporation temperature is 80-90 ℃.

5. The malt thin layer identification method according to claim 1, characterized by, In step (1), the mass-volume ratio of the control medicinal material of malt to the extraction solvent is 1:28-32, the unit is g:mL.

6. The malt thin layer identification method according to claim 1, characterized by, In step (2), the mass-volume ratio of the negative sample of malt to the extraction solvent is 10:28-32, the unit is g:mL.

7. The malt thin layer identification method according to claim 1, characterized by, In step (2), the preparation of the negative sample of malt comprises the following steps: Taizishen, Chenpi, Fuling and Shanhua are decocted with 8-12 times the weight of water for 1-3 times, each time for 2.5-3.5 hours, filtered, the filtrates are combined and concentrated into a clear paste, which is spray dried; add yam powder, dextrin, sucrose and citric acid, mix well, make into granules, add magnesium stearate, press into tablets, and coat with film to obtain the negative sample of malt.

8. The malt thin layer identification method according to claim 1, characterized by, In step (3), the mass-volume ratio of the test product of Xiaoshi tablet to the extraction solvent is 1:28-32, the unit is g:mL.

9. The malt thin layer identification method according to claim 1, characterized by, In step (4), the thin layer plate is a high-performance silica gel G thin layer plate.

10. The malt thin layer identification method according to claim 1, characterized by, In step (4), the volume ratio of ethyl acetate-methanol-water is 10:2:0.7-1.3.

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