A detection method for Yinhuang medicinal composition

Abstract

The present invention relates to a method for detecting a Yinhuang medicinal composition. The detection method comprises: (1) thin layer chromatography identification of baicalin in a scutellaria baicalensis extract; (2) thin layer chromatography identification of chlorogenic acid in a honeysuckle extract; (3) determination of the content of baicalin in a scutellaria baicalensis extract; and (4) determination of the content of chlorogenic acid in a honeysuckle extract. Methodological investigations have shown that the detection method has the effects of strong specificity, good durability, and a good recovery rate, and the method is stable and feasible. It is used to detect the content of baicalin and chlorogenic acid in a Yinhuang medicinal composition. It can effectively detect the quality of the product and accurately control the product quality.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a detection method for a silver-yellow medicinal composition. Background Art

[0002] Yinhuang Hanhua Dropping Pills, a product of Guizhou Weilide Pharmaceutical Co., Ltd., are used to clear away heat and toxic substances, and reduce inflammation. They are used for acute and chronic tonsillitis, pharyngitis, and upper respiratory tract infections.

[0003] Prescription: 90g of honeysuckle extract and 90g of scutellaria baicalensis extract; Preparation method: Grind the above two ingredients into fine powder, add 812g of polyethylene glycol-4000 and mix evenly, heat (about 80-90℃) to melt into a uniform medicinal liquid, then add 4.5g of aspartame and 3.5g of menthol, mix evenly, maintain the temperature of the medicinal liquid at 70-80℃, add dropwise into dimethyl silicone oil (5-10℃), make into pills, and make 1000g.

[0004] In the existing technology, the 2020 edition of the "Pharmacopoeia of the People's Republic of China" includes Chinese medicine preparations such as Yinhuang Oral Liquid, Yinhuang Pills, and Yinhuang Tablets. Their preparation processes are different, and the detection standards only include the determination of baicalin and chlorogenic acid content. The determination of the content of each effective ingredient requires different determination methods, resulting in low detection efficiency; and without identification of the effective ingredients of the drug, the quality of the drug cannot be effectively and comprehensively controlled.

[0005] In order to improve detection efficiency, effectively control drug quality, and enhance the quality standards of Yinhuang pharmaceutical compositions, the present invention team conducted in-depth research and development and investigation on the detection of Yinhuang pharmaceutical compositions, and established a detection method for Yinhuang pharmaceutical compositions, which added thin layer identification of baicalin and chlorogenic acid in the extract, and used high performance liquid chromatography to simultaneously measure the contents of baicalin and chlorogenic acid under the same conditions. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for detecting a Yinhuang medicinal composition.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for detecting a Yinhuang medicinal composition, comprising: (1) thin layer chromatography identification of baicalin in a scutellaria baicalensis extract; (2) thin layer chromatography identification of chlorogenic acid in a honeysuckle flower extract; and (3) determination of the contents of baicalin and chlorogenic acid.

[0009] The thin layer chromatography identification method of baicalin of the present invention is:

[0010] Take 1-3 g of sample powder, add 10-30 mL of ether, sonicate for 10-20 min, filter, evaporate the ether from the filter residue, add 10-30 mL of methanol, sonicate for 10-20 min, filter, evaporate the filtrate to dryness, add 10-30 mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 2-3, add ethyl acetate and shake to extract 1-3 times, 10-30 mL each time, take the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 1-2 mL of methanol to prepare the test solution; take another baicalin reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the baicalin reference substance solution; take 2-4 μL of each of the above two solutions and spot them on the same silica gel G plate, use ethyl acetate-butanone-formic acid-water (15:4.5:1:2) as the developing solvent, develop, remove, dry, spray with 3-5% ferric chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0011] Preferably, the thin layer chromatography identification method of baicalin of the present invention is:

[0012] Take 2g of sample powder, add 20mL of ether, ultrasonically treat for 15min, filter, evaporate the ether from the residue, add 20mL of methanol, ultrasonically treat for 15min, filter, evaporate the filtrate to dryness, add 20mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 2.5, add ethyl acetate and shake to extract twice, 20mL each time, take ethyl acetate solution, evaporate to dryness, and dissolve the residue in 2mL of methanol as the test solution; take baicalin reference substance, add methanol to make a solution containing 1mg per 1mL, as baicalin reference substance solution; take 2μL of each of the above two solutions, respectively, spot them on the same silica gel G plate, use ethyl acetate-butanone-formic acid-water (15:4.5:1:2) as the developing agent, develop, take out, dry, spray with 4% ferric chloride ethanol solution, and inspect under ultraviolet light at 365nm.

[0013] The thin layer identification method of chlorogenic acid of the present invention is:

[0014] Take 2-4 g of the sample powder, add 40-60 mL of anhydrous ethanol, sonicate for 20-40 min, filter, evaporate the filtrate to dryness, dissolve the residue in 10-20 mL of water, pass it through a polyamide column, elute with 40-60 mL of water, discard the water, and elute with 20-40 mL of methanol in batches. Collect the eluate and evaporate it to dryness in a water bath. Dissolve the residue in 1-2 mL of methanol to prepare the test solution. Separately, take the chlorogenic acid reference substance and add methanol to prepare a solution containing 1 mg per 1 mL. This is the chlorogenic acid reference substance solution. Pipette 2-4 μL of each of the above two solutions and spot them on the same silica gel G plate. Use ethyl acetate-butanone-chloroform-formic acid-water (15:12:4:3:1) as the developing solvent. Develop, remove, dry, spray with 1-5% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0015] Preferably, the thin layer identification method of chlorogenic acid of the present invention is:

[0016] Take 3 g of sample powder, add 50 mL of anhydrous ethanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 15 mL of water to dissolve the residue, pass it through a polyamide column, elute with 50 mL of water, discard the water, elute with 30 mL of methanol in batches, collect the eluate, evaporate to dryness in a water bath, and dissolve the residue in 1 mL of methanol as the test solution; take another chlorogenic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the chlorogenic acid reference substance solution; take 2 μL of each of the above two solutions, respectively, and spot them on the same silica gel G plate, use ethyl acetate-butanone-chloroform-formic acid-water (15:12:4:3:1) as the developing solvent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0017] The method for determining the content of baicalin and chlorogenic acid of the present invention comprises the following steps:

[0018] (1) Preparation of reference solution: Accurately weigh appropriate amounts of baicalin and chlorogenic acid reference substances, add methanol to make the mass concentrations 40 μg·mL -1 Baicalin, 25 μg·mL -1 The mixed reference solution of chlorogenic acid is obtained;

[0019] (2) Preparation of test solution: Take 0.1 g of sample, accurately weigh it, place it in a 50 ml brown volumetric flask, add an appropriate amount of methanol, ultrasonicate for 0.5 h, let it cool to room temperature, dilute to the mark with methanol, shake well, filter to obtain the filtrate, accurately pipette 10 ml of the filtrate, place it in a 25 ml volumetric flask, dilute to the mark with methanol, shake well, and obtain the solution.

[0020] (3) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile solution containing 0.01% formic acid was used as mobile phase A, and aqueous solution containing 0.2% formic acid was used as mobile phase B. Gradient elution: 0-5 min, 15% A; 5-15 min, 85% A; 15-20 min, 15% A; detection wavelength was 315 nm; column temperature was 32°C; flow rate was 1.0 mL min -1 The injection volume was 10 μL. The theoretical plate number calculated based on the baicalin and chlorogenic acid peaks should be no less than 3000.

[0021] (4) Determination method: Accurately aspirate 10ul of each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0022] The concentration of methanol in steps (1) and (2) of the present invention is: 50-70% methanol solution.

[0023] Preferably, the concentration of methanol in steps (1) and (2) of the present invention is 60% methanol solution.

[0024] The conditions for the ultrasonic treatment in step (2) of the present invention are: ultrasonic power 300-400W, frequency 30-50kHz.

[0025] Preferably, the conditions for the ultrasonic treatment in step (2) of the present invention are: ultrasonic power 350W, frequency 40kHz.

[0026] Beneficial effects of the present invention:

[0027] 1. The present invention adds thin-layer chromatography identification of baicalin and chlorogenic acid. A large number of screening experiments are carried out on the sample weight, solvent selection, extraction method, and developing agent in thin-layer chromatography identification, and a preferred scheme for the thin-layer chromatography identification method of baicalin and chlorogenic acid is obtained. The methodological investigation is also carried out. The results show that in the specificity investigation, in the chromatogram of the test sample, the main spot of the same color appears at the corresponding position in the chromatogram of the reference sample, and the spots are well separated, and there is no interference in the negative, indicating that this method has strong specificity; in the reproducibility investigation, the test results of 10 batches of samples are consistent, indicating the stability of this method; in the durability investigation, the results are investigated under different thin-layer plates, temperatures, and humidities, and the spots are well separated and there is no interference, indicating that this method has good durability.

[0028] 2. This invention establishes a sensitive, accurate, and simple method for determining the contents of baicalin and chlorogenic acid in Yinhuang medicinal products using high-performance liquid chromatography. This method can be used as a quality control method for key active ingredients. Under the conditions of this method, baicalin and chlorogenic acid chromatographic peak shapes are ideal, baselines are stable, and separation is good. This method was applied to the determination of Yinhuang medicinal compositions and demonstrated good specificity, repeatability, intermediate precision, stability, and accuracy.

[0029] 3. The present invention conducted a screening experiment on the extraction solvent, extraction method, extraction time, mobile phase, etc. of the test sample in the content determination method, obtained the optimal solution for content determination, and conducted a methodological investigation on it. The results are as follows:

[0030] (1) In the linear study, baicalin showed a good linear relationship in the range of 0.0799μg-0.3995μg (R 2 =0.9994); chlorogenic acid showed a good linear relationship with its peak area in the range of 0.0498 μg-0.2492 μg (R 2 =0.9998);

[0031] (2) In the precision study, the peak areas of baicalin and chlorogenic acid were measured. The RSD of baicalin was 1.96%, and the RSD of chlorogenic acid was 1.81%, indicating that the precision of the instrument was good;

[0032] (3) In the stability study, the peak areas of baicalin and chlorogenic acid were measured. The RSD of baicalin was 1.94%, and the RSD of chlorogenic acid was 1.18%, indicating that the test solution had good stability within 12 hours.

[0033] (4) In repeated investigations, the contents of baicalin and chlorogenic acid were determined. The RSD of baicalin was 0.89%, and the RSD of chlorogenic acid was 1.14%, indicating that the method had good reproducibility.

[0034] (5) In the recovery rate investigation, the average recovery rate of baicalin was 98.73% with an RSD of 0.93%, and the average recovery rate of chlorogenic acid was 98.35% with an RSD of 1.32%, both meeting the requirements;

[0035] (6) The results of the specificity investigation showed that the sample solution chromatogram had corresponding chromatographic peaks at the corresponding positions of the reference solution chromatogram, and the negative results were without interference. The contents of baicalin and chlorogenic acid were determined for 10 batches of samples. The results showed that the average contents of baicalin and chlorogenic acid in the 10 batches of samples were 81.78 mg / g and 15.21 mg / g, respectively, which met the requirements. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0037] Example 1 Thin layer chromatography identification of baicalin

[0038] Take 2g of sample powder, add 20mL of ether, ultrasonically treat for 15min, filter, evaporate the ether from the residue, add 20mL of methanol, ultrasonically treat for 15min, filter, evaporate the filtrate to dryness, add 20mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 2.5, add ethyl acetate and shake to extract twice, 20mL each time, take ethyl acetate solution, evaporate to dryness, and dissolve the residue in 2mL of methanol as the test solution; take baicalin reference substance, add methanol to make a solution containing 1mg per 1mL, as baicalin reference substance solution; take 2μL of each of the above two solutions, respectively, spot them on the same silica gel G plate, use ethyl acetate-butanone-formic acid-water (15:4.5:1:2) as the developing agent, develop, take out, dry, spray with 4% ferric chloride ethanol solution, and inspect under ultraviolet light at 365nm.

[0039] Example 2 Thin layer identification of baicalin

[0040] Take 3g of sample powder, add 30mL of ether, ultrasonically treat for 20min, filter, evaporate the ether from the residue, add 30mL of methanol, ultrasonically treat for 20min, filter, evaporate the filtrate to dryness, add 30mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 3, add ethyl acetate and shake to extract 3 times, 30mL each time, take ethyl acetate solution, evaporate to dryness, add 2mL of methanol to dissolve the residue, as the test solution; take baicalin reference substance, add methanol to make a solution containing 1mg per 1mL, as baicalin reference substance solution; take 4μL of each of the above two solutions, respectively, spot them on the same silica gel G plate, use ethyl acetate-butanone-formic acid-water (15:4.5:1:2) as the developing agent, develop, take out, dry, spray with 5% ferric chloride ethanol solution, and inspect under ultraviolet light at 365nm.

[0041] Example 3 Thin layer identification of baicalin

[0042] Take 1 g of sample powder, add 10 mL of ether, ultrasonically treat for 10 minutes, filter, evaporate the ether from the residue, add 10 mL of methanol, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, add 10 mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 2, add ethyl acetate and shake to extract once, 10 mL each time, take ethyl acetate solution, evaporate to dryness, add 1 mL of methanol to dissolve the residue, as the test solution; take another baicalin reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the baicalin reference substance solution; take 2 μL of each of the above two solutions, respectively, spot them on the same silica gel G plate, use ethyl acetate-butanone-formic acid-water (15:4.5:1:2) as the developing agent, develop, take out, dry, spray with 3% ferric chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0043] Example 4 Thin-layer identification of chlorogenic acid

[0044] Take 3 g of sample powder, add 50 mL of anhydrous ethanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 15 mL of water to dissolve the residue, pass it through a polyamide column, elute with 50 mL of water, discard the water, elute with 30 mL of methanol in batches, collect the eluate, evaporate to dryness in a water bath, and dissolve the residue in 1 mL of methanol as the test solution; take another chlorogenic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the chlorogenic acid reference substance solution; take 2 μL of each of the above two solutions, respectively, and spot them on the same silica gel G plate, use ethyl acetate-butanone-chloroform-formic acid-water (15:12:4:3:1) as the developing solvent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0045] Example 5 Thin layer chromatography identification of chlorogenic acid II

[0046] Take 4 g of sample powder, add 60 mL of anhydrous ethanol, ultrasonically treat for 40 min, filter, evaporate the filtrate to dryness, add 20 mL of water to dissolve the residue, pass it through a polyamide column, elute with 60 mL of water, discard the water, elute with 40 mL of methanol in batches, collect the eluate, evaporate it to dryness in a water bath, and dissolve the residue in 2 mL of methanol as the test solution; take another chlorogenic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the chlorogenic acid reference substance solution; take 4 μL of each of the above two solutions, respectively, and spot them on the same silica gel G plate, use ethyl acetate-butanone-chloroform-formic acid-water (15:12:4:3:1) as the developing solvent, develop, take out, dry, spray with 5% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0047] Example 6 Thin-layer Identification of Chlorogenic Acid III

[0048] Take 2 g of sample powder, add 40 mL of anhydrous ethanol, ultrasonically treat for 20 min, filter, evaporate the filtrate to dryness, add 10 mL of water to dissolve the residue, pass it through a polyamide column, elute with 40 mL of water, discard the water, elute with 20 mL of methanol in batches, collect the eluate, evaporate it to dryness in a water bath, and dissolve the residue in 1 mL of methanol as the test solution; take another chlorogenic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the chlorogenic acid reference substance solution; take 2 μL of each of the above two solutions, respectively, and spot them on the same silica gel G plate, use ethyl acetate-butanone-chloroform-formic acid-water (15:12:4:3:1) as the developing solvent, develop, take out, dry, spray with 1% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0049] Example 7 Content Determination

[0050] (1) Preparation of reference solution: Accurately weigh appropriate amounts of baicalin and chlorogenic acid reference substances, add 60% methanol to make the mass concentration of each solution 40 μg·mL -1 Baicalin, 25 μg·mL -1 The mixed reference solution of chlorogenic acid is obtained;

[0051] (2) Preparation of test solution: Take 0.1 g of sample, accurately weigh it, place it in a 50 ml brown volumetric flask, add an appropriate amount of 60% methanol, and sonicate for 0.5 h (power 350 W, frequency 40 kHz). Let it cool to room temperature, dilute it to the mark with 60% methanol, shake it well, filter it, and obtain the filtrate. Accurately pipette 10 ml of the filtrate, place it in a 25 ml volumetric flask, dilute it to the mark with 60% methanol, and shake it well.

[0052] (3) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile solution containing 0.01% formic acid was used as mobile phase A, and aqueous solution containing 0.2% formic acid was used as mobile phase B. Gradient elution: 0-5 min, 15% A; 5-15 min, 85% A; 15-20 min, 15% A; detection wavelength was 315 nm; column temperature was 30°C; flow rate was 1.0 mL·min-1; injection volume was 10 uL; the number of theoretical plates calculated based on the baicalin and chlorogenic acid peaks should be no less than 3000;

[0053] (4) Determination method: Accurately aspirate 10ul of each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.

[0054] In order to further verify the feasibility of the present invention, the inventors conducted a series of experiments, the steps of which are as follows:

[0055] 1. Thin layer chromatography identification study of baicalin and chlorogenic acid in the Yinhuang medicinal composition

[0056] 1. Thin layer chromatography identification and screening of baicalin

[0057] 1.1 Method 1: Refer to the identification standard of Scutellaria baicalensis extract in the 2020 edition of the Chinese Pharmacopoeia

[0058] Dissolve 1g of the sample in 1ml of methanol to prepare the test solution. Separately, dissolve 1mg of baicalin in methanol to prepare a 1ml solution of baicalin as the reference solution. Perform the thin-layer chromatography (TLC) test (General Method 0502) by applying 2μl of each solution to the same polyamide film. Develop the film using acetic acid, remove the film, air dry, and examine under ultraviolet light (365nm). Fluorescent spots of the same color will appear in the chromatogram of the test sample at the corresponding positions in the chromatogram of the reference.

[0059] Result: At the position corresponding to the chromatogram of the reference substance, the spot was fuzzy and had a tail, and the separation did not meet the requirements.

[0060] 1.2 Method 2: Based on the analysis results of Method 1 and the properties of baicalin, the sample treatment method, developing agent, and color developing agent were adjusted. The tentative thin layer identification method is as follows:

[0061] Take 2g of sample powder, add 20mL of ether, ultrasonically treat for 15min, filter, evaporate the ether from the residue, add 20mL of methanol, ultrasonically treat for 15min, filter, evaporate the filtrate to dryness, add 20mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 2.5, add ethyl acetate and shake to extract twice, 20mL each time, take ethyl acetate solution, evaporate to dryness, and dissolve the residue in 2mL of methanol as the test solution; take another baicalin reference substance, add methanol to make a solution containing 1mg per 1mL, as the baicalin reference substance solution; take 2μL of each of the above two solutions, respectively, spot them on the same polyamide film, use ethyl acetate-butanone-formic acid-water (15:4.5:1:2) as the developing agent, develop, take out, dry, spray with 4% ferric chloride ethanol solution, and inspect under ultraviolet light at 365nm.

[0062] Results: Observed under 365nm ultraviolet light, the spots were clear, the separation met the requirements, the Rf value was moderate, and the reproducibility was good.

[0063] 1.3 Method Validation

[0064] 1.3.1 Determination of the sample size

[0065] Take samples and perform the experiment according to the method in "1.2" to screen the sample quantity. The results are shown in Table 1.

[0066] Table 1 Test sample sampling quantity screening table

[0067]

[0068] Results: As shown in Table 1, when the sample size was 2.0 g and 3.0 g, the thin layer showed clear spots, the separation met the requirements, the Rf value was moderate, and the reproducibility was good. Therefore, "2.0 g" is the optimal sample size.

[0069] 1.3.2 Screening of extraction solvents for test samples

[0070] According to the preferred method experiment under "1.3.1", the extraction solvents of the samples were screened. The results are shown in Table 2.

[0071] Table 2 Extraction solvent screening table

[0072]

[0073] As shown in Table 2, when methanol was used as the extraction solvent for the sample, the results showed clear spots, the separation met the requirements, the Rf value was moderate, there was no tailing phenomenon, and the reproducibility was good. Therefore, "methanol" is the preferred sample extraction solvent.

[0074] 1.3.3 Screening of extraction methods

[0075] Screen the sample extraction methods based on the preferred thin-layer method under "1.3.2". The results are shown in Table 3.

[0076] Table 3 Extraction method screening table

[0077]

[0078] As shown in Table 3, ultrasonic extraction results showed clear spots, satisfactory separation, moderate Rf values, and good reproducibility. Therefore, ultrasonic extraction was the preferred sample extraction method.

[0079] 1.3.4 Screening of extraction time

[0080] The extraction time of the samples was screened according to the preferred thin layer method under "1.3.3". The results are shown in Table 4.

[0081] Table 4 Extraction time screening table

[0082]

[0083] As shown in Table 4, when the ultrasonic extraction time was 15 minutes, the TLC results all showed clear spots, the separation met the requirements, the Rf value was moderate, and the reproducibility was good. Therefore, "15 minutes" is the preferred sample extraction time.

[0084] 1.3.5 Screening of pH value of test solution

[0085] The pH values ​​of the samples were screened according to the preferred thin layer chromatography method under "1.3.4". The results are shown in Table 5.

[0086] Table 5 Extraction time screening table

[0087]

[0088] As shown in Table 5, when the pH value is 2.5, the thin layer results show clear spots, the separation meets the requirements, the Rf value is moderate, there is no tailing, and the reproducibility is good. Therefore, the pH value of 2.5 is preferred.

[0089] 1.3.6 Screening of developing agent ratio

[0090] According to the preferred thin layer method under "1.3.5", the developing agent ratio was screened. The results are shown in Table 6.

[0091] Table 6 Developing agent ratio screening table

[0092]

[0093] Table 6 shows that when the developing solvent ratio of ethyl acetate-butanone-formic acid-water is 15:4.5:1:2, the TLC results show clear spots, satisfactory resolution, moderate Rf values, and good reproducibility. Therefore, 15:4.5:1:2 is the preferred developing solvent ratio.

[0094] 1.3.7 Determination of the amount of sample to be sampled

[0095] According to the preferred thin layer method under "1.3.6", the sample spotting amount was screened. The results are shown in Table 7.

[0096] Table 7 Screening table for sample spotting quantity

[0097]

[0098] As shown in Table 7, when the sample volume is 2 μL, the thin layer spots are clear, there is no tailing, the resolution meets the requirements, the Rf value is moderate, and the reproducibility is good. Therefore, "2 μL" is the preferred sample spot volume.

[0099] 1.3.8 Results

[0100] After the above screening experiments, the optimal thin layer chromatography identification method for baicalin was obtained as follows:

[0101] Take 2g of sample powder, add 20mL of ether, ultrasonically treat for 15min, filter, evaporate the ether from the residue, add 20mL of methanol, ultrasonically treat for 15min, filter, evaporate the filtrate to dryness, add 20mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 2.5, add ethyl acetate and shake to extract twice, 20mL each time, take ethyl acetate solution, evaporate to dryness, and dissolve the residue in 2mL of methanol as the test solution; take another baicalin reference substance, add methanol to make a solution containing 1mg per 1mL, as the baicalin reference substance solution; take 2μL of each of the above two solutions, respectively, spot them on the same polyamide film, use ethyl acetate-butanone-formic acid-water (15:4.5:1:2) as the developing agent, develop, take out, dry, spray with 4% ferric chloride ethanol solution, and inspect under ultraviolet light at 365nm.

[0102] 1.4 Methodological Validation

[0103] 1.4.1 Specificity

[0104] Take three batches of samples, and use Qingdao Ocean Chemical Plant silica gel G plate as the thin layer plate. At a temperature of 25℃ and a humidity of 60%, prepare the test solution according to the method under "1.3.8". Take the test solution, reference solution, and negative sample solution for spotting, development, and color development respectively. Inspect under 365nm ultraviolet light, in the test sample chromatogram, the same main spot is shown at the corresponding position of the reference sample chromatogram, the spot separation is good, and there is no interference with the negative.

[0105] 1.4.2 Reproducibility

[0106] Take 10 batches of samples and develop them according to the TLC identification method in “1.3.8”. The results are shown in Table 8.

[0107] Table 8 Test methods, conditions and reproducibility test results

[0108]

[0109] As shown in Table 8, the thin layer of 10 batches of Yinhuang drug samples showed clear spots, the separation met the requirements, the Rf value was moderate, and the reproducibility was good, indicating that this method has good reproducibility.

[0110] 1.4.3 Durability

[0111] 1.4.3.1 Comparison of different thin layer plates

[0112] Take 10 batches of samples and develop them according to the method of "1.3.8". Compare the thin layers of Qingdao Ocean Chemical Plant Branch Silica G commercial board and high-efficiency Silica G commercial board. The results are shown in Table 9.

[0113] Table 9 Durability results of different thin layer boards

[0114]

[0115] As shown in Table 9, the high-efficiency silica gel plate has clear spots, the separation meets the requirements, and the Rf value is moderate; the silica gel G plate has clear spots, the separation meets the requirements, and the Rf value is moderate, so this method has good durability.

[0116] 1.4.3.2 Comparison of different temperatures

[0117] Take sample (20211201), and use the silica gel G plate from Qingdao Ocean Chemical Plant as the thin layer plate. According to the thin layer identification method of "1.3.8", develop it at temperatures of 4°C and 24°C and humidity of 55%, respectively. The results are shown in Table 10.

[0118] Table 10 Test results at different temperatures

[0119]

[0120] As shown in Table 10, the thin layer spots are clear, the separation meets the requirements, the Rf value is moderate, and good identification chromatograms can be obtained under both temperature conditions. Therefore, this method has good durability.

[0121] 1.4.3.3 Comparison of different humidity levels

[0122] Take sample (20211201), and use the silica gel G plate from Qingdao Ocean Chemical Plant as the thin layer plate. According to the thin layer identification method of "1.3.8", develop it at a temperature of 24°C and a humidity of 30% and 65% respectively. The results are shown in Table 11.

[0123] Table 11 Test results at different humidity levels

[0124]

[0125] As shown in Table 11, the thin layer spots are clear, the separation meets the requirements, the Rf value is moderate, and good identification chromatograms can be obtained under two humidity conditions. Therefore, this method has good durability.

[0126] 2. Thin layer identification and screening of chlorogenic acid

[0127] 2.1 Method 1: Refer to the identification standard of honeysuckle in the 2020 edition of the Chinese Pharmacopoeia

[0128] Take 0.2g of this product powder, add 5ml of methanol, let it stand for 12 hours, filter, and take the filtrate as the test solution. Take another chlorogenic acid reference substance, add methanol to make a solution containing 1mg per 1ml, as the reference solution. According to the thin layer chromatography method (General Rule 0502), take 10-20μl of the test solution and 10μl of the reference solution, and spot them on the same silica gel H thin layer plate, use butyl acetate-formic acid-water (7:2.5:2.5) as the upper layer solution as the developing agent, develop, remove, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test product, at the corresponding position of the chromatogram of the reference substance, a fluorescent spot of the same color appears.

[0129] Result: At the position corresponding to the chromatogram of the reference substance, the spot was fuzzy and had a tail, and the separation did not meet the requirements.

[0130] 2.2 Method 2: Based on the analysis results of Method 1 and the properties of chlorogenic acid, the treatment of the sample, the developing agent and the color developing agent were adjusted. The tentative thin layer identification method is as follows:

[0131] Take 3 g of sample powder, add 50 mL of anhydrous ethanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 15 mL of water to dissolve the residue, pass it through a polyamide column, elute with 50 mL of water, discard the water, elute with 30 mL of methanol in batches, collect the eluate, evaporate it in a water bath, and dissolve the residue in 1 mL of methanol as the test solution; take another chlorogenic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the chlorogenic acid reference substance solution; take 2 μL of each of the above two solutions, respectively, and spot them on the same polyamide film, use ethyl acetate-butanone-chloroform-formic acid-water (15:12:4:3:1) as the developing agent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0132] Results: Observed under 365nm ultraviolet light, the spots were clear, the separation met the requirements, the Rf value was moderate, and the reproducibility was good.

[0133] 2.3 Method validation

[0134] 2.3.1 Determination of sample volume

[0135] Take samples and perform the test according to the method in "2.2" to screen the sample size. The results are shown in Table 12.

[0136] Table 12 Test sample sampling quantity screening table

[0137]

[0138] Results: As shown in Table 12, when the sample size was 3.0 g, the thin layer showed clear spots, the separation met the requirements, the Rf value was moderate, and the reproducibility was good. Therefore, "3.0 g" is the optimal sample size.

[0139] 2.3.2 Screening of extraction solvents for test samples

[0140] The extraction solvents for the samples were screened according to the preferred method experiment under "2.3.1". The results are shown in Table 13.

[0141] Table 13 Extraction solvent screening table

[0142]

[0143] As shown in Table 13, when anhydrous ethanol was used as the extraction solvent for the sample, the spots were clear, the separation met the requirements, the Rf value was moderate, there was no tailing phenomenon, and the reproducibility was good. Therefore, "anhydrous ethanol" is the preferred sample extraction solvent.

[0144] 2.3.3 Screening of extraction methods

[0145] Screen the sample extraction methods based on the preferred thin layer chromatography method under "2.3.2". The results are shown in Table 14.

[0146] Table 14 Extraction method screening table

[0147]

[0148] As shown in Table 14, ultrasonic extraction results showed clear spots, satisfactory separation, moderate Rf values, and good reproducibility. Therefore, ultrasonic extraction was the preferred sample extraction method.

[0149] 2.3.4 Screening of extraction time

[0150] The extraction time of the samples was screened according to the preferred thin layer method under "2.3.3". The results are shown in Table 15.

[0151] Table 15 Extraction time screening table

[0152]

[0153] As shown in Table 15, when the ultrasonic extraction time was 30 minutes, the TLC results all showed clear spots, met the required separation, had moderate Rf values, and good reproducibility. Therefore, "30 minutes" is the preferred sample extraction time.

[0154] 2.3.5 Screening of developing agents

[0155] Screen the developing agents for the samples according to the preferred thin layer method under "2.3.4". The results are shown in Table 16.

[0156] Table 16 Developing agent screening table

[0157]

[0158] Table 16 shows that when ethyl acetate-butanone-chloroform-formic acid-water was used as the developing solvent, the TLC results showed clear spots, satisfactory resolution, moderate Rf values, no tailing, and good reproducibility. Therefore, ethyl acetate-butanone-chloroform-formic acid-water was the preferred developing solvent.

[0159] 2.3.6 Screening of developing agent ratio

[0160] According to the preferred thin layer method under "2.3.5", the developing agent ratio was screened. The results are shown in Table 17.

[0161] Table 17 Developing agent ratio screening table

[0162]

[0163] Table 17 shows that when the developing solvent ratio of ethyl acetate-butanone-formic acid-water is "15:12:4:3:1," the TLC results show clear spots, satisfactory resolution, moderate Rf values, and good reproducibility. Therefore, "15:12:4:3:1" is the preferred developing solvent ratio.

[0164] 2.3.7 Determination of the amount of sample to be sampled

[0165] According to the preferred thin layer method under "2.3.6", the sample spotting amount was screened. The results are shown in Table 18.

[0166] Table 18 Screening table for sample spotting quantity

[0167]

[0168] As shown in Table 18, when the sample volume is 2 μL, the thin layer spots are clear, there is no tailing, the resolution meets the requirements, the Rf value is moderate, and the reproducibility is good. Therefore, "2 μL" is the preferred sample spot volume.

[0169] 2.3.8 Results

[0170] After the above screening experiments, the optimal thin layer chromatography identification method for chlorogenic acid is:

[0171] Take 3 g of sample powder, add 50 mL of anhydrous ethanol, ultrasonically treat for 30 min, filter, evaporate the filtrate to dryness, add 15 mL of water to dissolve the residue, pass it through a polyamide column, elute with 50 mL of water, discard the water, elute with 30 mL of methanol in batches, collect the eluate, evaporate to dryness in a water bath, and dissolve the residue in 1 mL of methanol as the test solution; take another chlorogenic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the chlorogenic acid reference substance solution; take 2 μL of each of the above two solutions, respectively, and spot them on the same silica gel G plate, use ethyl acetate-butanone-chloroform-formic acid-water (15:12:4:3:1) as the developing solvent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

[0172] 2.4 Methodological validation

[0173] 2.4.1 Specificity

[0174] Take three batches of samples and use silica gel G plates from Qingdao Ocean Chemical Plant as thin layer plates. At a temperature of 25°C and a humidity of 60%, prepare the test solution according to the method under "2.3.8". Spot the test solution, reference solution, and negative sample solution, develop the solution, and inspect them under 365nm ultraviolet light. In the test sample chromatogram, the same main spots appear at the corresponding positions in the reference sample chromatogram. The spots are well separated, and there is no interference with the negative sample.

[0175] 2.4.2 Reproducibility

[0176] Take 10 batches of samples and develop them according to the thin layer identification method in "2.3.8". The results are shown in Table 19.

[0177] Table 19 Test methods, conditions and reproducibility test results

[0178]

[0179] As shown in Table 19, the thin layer of 10 batches of Yinhuang drug samples showed clear spots, the separation met the requirements, the Rf value was moderate, and the reproducibility was good, indicating that this method has good reproducibility.

[0180] 2.4.3 Durability

[0181] 2.4.3.1 Comparison of different thin layer plates

[0182] Take 10 batches of samples and develop them according to the method of "2.3.8" to compare the thin layers of silica gel G commercial plates and high-efficiency silica gel G commercial plates produced by Qingdao Ocean Chemical Plant Branch. The results are shown in Table 20.

[0183] Table 20 Durability results of different thin layer boards

[0184]

[0185] As shown in Table 20, the high-efficiency silica gel plate has clear spots, the separation meets the requirements, and the Rf value is moderate; the silica gel G plate has clear spots, the separation meets the requirements, and the Rf value is moderate, so this method has good durability.

[0186] 2.4.3.2 Comparison of different temperatures

[0187] Take sample (20211201), and use the silica gel G plate from Qingdao Ocean Chemical Plant as the thin layer plate. According to the thin layer identification method in "2.3.8", develop it at temperatures of 4°C and 24°C and humidity of 55%, respectively. The results are shown in Table 21.

[0188] Table 21 Test results at different temperatures

[0189]

[0190] As shown in Table 21, the thin layer spots are clear, the separation meets the requirements, the Rf value is moderate, and good identification chromatograms can be obtained under both temperature conditions. Therefore, this method has good durability.

[0191] 2.4.3.3 Comparison of different humidity levels

[0192] Take sample (20211201), and use the silica gel G plate from Qingdao Ocean Chemical Plant as the thin layer plate. According to the thin layer identification method in "2.3.8", develop it in an environment with a temperature of 24°C and a humidity of 30% and 65% respectively. The results are shown in Table 22.

[0193] Table 22 Test results at different humidity levels

[0194]

[0195] As shown in Table 22, the thin layer spots are clear, the separation meets the requirements, the Rf value is moderate, and good identification chromatograms can be obtained under both humidity conditions. Therefore, this method has good durability.

[0196] 3. Experimental study on the determination method of baicalin and chlorogenic acid content

[0197] 3.1 Instruments and reagents

[0198] High-performance liquid chromatography: THERMO liquid chromatograph; baicalin reference substance (purity ≥99%, provided by the China Food and Drug Inspection Institutes); chlorogenic acid reference substance (purity ≥99%, provided by the China Food and Drug Inspection Institutes); reagents: methanol and acetonitrile are chromatographic alcohols, formic acid and phosphoric acid are of analytical grade, and water is double-distilled water.

[0199] 3.2 Content determination method design

[0200] Because the Yinhuang pharmaceutical composition of the present invention is prepared from two substances, Scutellaria baicalensis extract and Honeysuckle Flos extract, with the target components being baicalin and chlorogenic acid, respectively, the existing techniques for content determination mostly employ separate determinations of each indicator, with few reports on simultaneous determination of both indicators under the same conditions. The present research team employed high-performance liquid chromatography (HPLC) to simultaneously determine the contents of baicalin and chlorogenic acid using gradient elution and identical conditions, which is more conducive to controlling the quality of the Yinhuang pharmaceutical composition.

[0201] Method 1: Refer to the content determination method of Yinhuang Pills in the 2020 edition of the Chinese Pharmacopoeia

[0202] The chromatographic conditions and system suitability test used octadecylsilane bonded silica as the filler; acetonitrile as mobile phase A, and 0.4% phosphoric acid solution as mobile phase B. The gradient elution was as follows: 5% A to 20% (0-15 min); 20% A to 30% (15-30 min); and 30% A (30-40 min). The column temperature was 25°C; the flow rate was 1.0 ml / min; and the detection wavelength was 327 nm. The number of theoretical plates, calculated based on the chlorogenic acid peak, should be no less than 2000.

[0203] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, place it in a brown volumetric flask, and add 50% methanol to make a solution containing 40 μg per 1 ml.

[0204] Preparation of the test solution: Take about 0.25 g of sample, accurately weigh it, place it in a 100 ml brown volumetric flask, add 80 ml of 50% methanol, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, let it cool, add 50% methanol to the scale, shake well, filter, and take the filtrate to obtain the product.

[0205] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0206] Results: In the chromatogram of the sample sample from "Method 1," the main peaks of baicalin and chlorogenic acid were not well separated from the impurity peaks, resulting in tailing peaks, low peak response, and poor precision. To address these issues, we investigated sample processing, chromatographic conditions, and extraction solvents, as follows:

[0207] 3.2.1 Selection of detection wavelength

[0208] Preparation of test solution: Take 1 g of sample, accurately weigh it, place it in a 50 ml brown volumetric flask, add an appropriate amount of methanol, ultrasonically treat it for 0.5 h, let it cool to room temperature, add methanol to dilute to the scale, shake well, filter to obtain the filtrate, accurately aspirate 10 ml of the filtrate, place it in a 25 ml volumetric flask, add methanol to dilute to the scale, shake well, and obtain it.

[0209] Inject the sample according to the method under "Method 1" and record the absorption spectrum in the range of 190-400nm.

[0210] Results: Baicalin has the strongest absorption at 280nm, while chlorogenic acid has the strongest absorption at 315nm. The chlorogenic acid content in the Yinhuang drug preparation is much lower than that of baicalin. When simultaneously determining baicalin and chlorogenic acid at the same wavelength, the maximum absorption wavelength of 315nm, where the content of chlorogenic acid is relatively low, should be selected as the detection wavelength to reduce errors. This not only improves the sensitivity of chlorogenic acid detection, but also has no significant effect on the detection of baicalin. Therefore, the detection wavelength of 315nm was ultimately selected.

[0211] 3.2.2 Selection of mobile phase and gradient

[0212] Optimization method 1: THERMO liquid chromatograph; acetonitrile as mobile phase A, 0.2% formic acid in water as mobile phase B, gradient elution: 0-10 min, 5% A; 10-25 min, 95% A; 25-35 min, 5% A; detection wavelength, 315 nm; column temperature, 30°C; flow rate, 1.0 mL min -1 The injection volume was 10 μL. The theoretical plate number calculated based on the baicalin and chlorogenic acid peaks should be no less than 3000.

[0213] Results: When the mobile phase and mobile phase gradient ratio were changed, the baseline of the method was unstable, the peak shape was general, the separation of adjacent peaks was poor, and the stability was poor.

[0214] Optimization method 2: THERMO liquid chromatograph; acetonitrile solution containing 0.01% formic acid as mobile phase A, and aqueous solution containing 0.2% formic acid as mobile phase B, gradient elution: 0-5 min, 15% A; 5-15 min, 85% A; 15-20 min, 15% A; detection wavelength 315 nm; column temperature 30°C; flow rate 1.0 mL min -1 The injection volume was 10 μL. The theoretical plate number calculated based on the baicalin and chlorogenic acid peaks should be no less than 3000.

[0215] Results: The baseline was stable, and the peak separation, peak shape, and peak purity were good, indicating good stability. Therefore, "Optimization Method 2" was selected as the optimal chromatographic condition for content detection.

[0216] 3.2.3 Investigation of extraction solvent

[0217] Take 5 samples prepared according to the method in "3.2.1" and inject them according to the preferred chromatographic conditions in "3.2.2". The results are shown in Table 23 below:

[0218] Table 23 Screening of extraction solvents

[0219]

[0220] The results show that when 60% methanol is used as the extraction solvent, the contents of baicalin and chlorogenic acid are the highest, so 60% methanol is preferably used as the extraction solvent.

[0221] 3.2.4 Investigation of extraction methods

[0222] Three samples were prepared according to the method in 3.2.1 and injected according to the preferred chromatographic conditions in 3.2.3. The results are shown in Table 24 below:

[0223] Table 24 Effect of different extraction methods on content

[0224]

[0225]

[0226] The results show that the contents of baicalin and chlorogenic acid are the highest when ultrasonic extraction is used, so ultrasonic extraction is the preferred method.

[0227] 3.3.5 Investigation of extraction time

[0228] Three samples were prepared according to the method in 3.2.1 and injected according to the preferred chromatographic conditions in 3.2.4. The results are shown in Table 25 below:

[0229] Table 25 Effect of different extraction time on content

[0230]

[0231] The results show that the contents of baicalin and chlorogenic acid are the highest when ultrasonic extraction is performed for 45 minutes, but there is almost no difference with the extraction time of 30 minutes. Considering the cost factors and efficiency, the ultrasonic time is preferably 30 minutes.

[0232] 3.3.6 Final content determination method

[0233] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile solution containing 0.01% formic acid was used as mobile phase A, and aqueous solution containing 0.2% formic acid was used as mobile phase B. Gradient elution: 0-5 min, 15% A; 5-15 min, 85% A; 15-20 min, 15% A; detection wavelength was 315 nm; column temperature was 30°C; flow rate was 1.0 mL min -1 The injection volume was 10 μL. The theoretical plate number calculated based on the baicalin and chlorogenic acid peaks should be no less than 3000.

[0234] Preparation of reference solution: Accurately weigh appropriate amounts of baicalin and chlorogenic acid reference substances, add methanol to make the mass concentrations of baicalin and chlorogenic acid respectively 40 μg mL -1 Baicalin, 25 μg·mL -1 The mixed reference solution of chlorogenic acid is obtained;

[0235] Preparation of test solution: Take 0.1 g of sample, accurately weigh it, place it in a 50 ml brown volumetric flask, add an appropriate amount of methanol, ultrasonically treat it for 0.5 h, let it cool to room temperature, dilute it to the scale with methanol, shake it well, filter it to obtain the filtrate, accurately draw 10 ml of the filtrate, place it in a 25 ml volumetric flask, dilute it to the scale with methanol, shake it well, and obtain the solution;

[0236] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0237] 3.4 Method validation

[0238] 3.4.1 Linear Investigation

[0239] Accurately pipette the mixed reference solution (39.95 μg mL -1 Baicalin, 24.92 μg·mL -1 Chlorogenic acid) 2, 4, 6, 8, 10 μl injection, record the chromatogram, with the peak area as the horizontal axis and the injection volume as the vertical axis, the regression equations of baicalin and chlorogenic acid were Y = 623347x + 5631.8, R 2 =0.9994, Y=499609x+1231.8, R 2 =0.9998. The results showed that baicalin had a good linear relationship between 0.0799μg-0.3995μg, and chlorogenic acid had a good linear relationship between 0.0498μg-0.2492μg.

[0240] 3.4.2 Precision test

[0241] Take the mixed reference solution and inject it six times continuously according to the chromatographic conditions in "3.3.6". Determine the peak areas of baicalin and chlorogenic acid. The RSD for baicalin is 1.96%, and the RSD for chlorogenic acid is 1.81%, indicating good instrument precision. See Table 26.

[0242] Table 26 Precision test

[0243]

[0244] 3.4.3 Stability test

[0245] The test solution was injected six times at 0, 2, 4, 8, and 12 hours, with 10 μL injected each time. The peak areas of baicalin and chlorogenic acid were measured. The RSDs for baicalin and chlorogenic acid were 1.94% and 1.18%, respectively, indicating that the test solution had good stability over the 12-hour assay period. See Table 27.

[0246] Table 27 Stability test

[0247]

[0248] 3.4.4 Repeatability test

[0249] Prepare six test sample solutions according to the method in "3.3.6." Determine the contents of baicalin and chlorogenic acid using the chromatographic conditions in "3.3.6." The RSD for baicalin was 0.89%, and the RSD for chlorogenic acid was 1.14%, indicating good reproducibility. See Table 28.

[0250] Table 28 Repeatability test

[0251]

[0252]

[0253] 3.4.5 Recovery rate investigation

[0254] Accurately weigh six samples and add appropriate amounts of the corresponding baicalin and chlorogenic acid reference solutions. Determine the contents according to the chromatographic conditions in "3.3.6" and calculate the RSDs. The average recovery of baicalin was 98.73% with an RSD of 0.93%, and the average recovery of chlorogenic acid was 98.35% with an RSD of 1.32%, both meeting the requirements. See Table 29.

[0255] Table 29 Recovery test

[0256]

[0257] 3.4.6 Specificity

[0258] Prepare sample solution according to the test solution preparation method. According to the chromatographic conditions under "3.3.6", take the reference solution and sample solution and inject them into the liquid chromatograph respectively. The sample solution chromatogram has corresponding chromatographic peaks at the corresponding positions of the reference solution chromatogram, and the separation is good, and the negative is without interference.

[0259] 3.4.7 Content determination of samples

[0260] Ten batches of samples were taken and measured and calculated according to the method in "3.3.6". The results are shown in Table 30. This shows that the method of the present invention is stable and feasible.

[0261] Table 30 Ten batches of sample content determination results

[0262]

[0263]

[0264] 3.4.8 Sample determination

[0265] The contents of baicalin and chlorogenic acid in three batches of Yinhuang pharmaceutical preparations were determined according to the determination method under “3.3.6”, with two parallel determinations for each batch. The results are shown in Table 31.

[0266] Table 31 Sample content determination results (n=2)

[0267]

[0268] Although the present invention has been described in detail above using general explanations, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for detecting Yinhuang Containing Dropping Pills, characterized in that: The thin-layer identification method of baicalin is as follows: take 2 g of sample powder, add 20 mL of ether, sonicate for 15 minutes, filter, evaporate the ether from the filter residue, add 20 mL of methanol, sonicate for 15 minutes, filter, evaporate the filtrate to dryness, add 20 mL of water to the residue, heat to dissolve, add dilute hydrochloric acid to adjust the pH to 2.5, add ethyl acetate and shake to extract twice, 20 mL each time, take the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 2 mL of methanol to prepare the test solution; take another baicalin reference substance, add methanol to make a solution containing 1 mg per 1 mL, and use it as the baicalin reference substance solution; take 2 µL of each of the above two solutions and spot them on a silica gel G plate, develop with ethyl acetate-butanone-formic acid-water = 15:4.5:1:2 as the developing solvent, remove, dry, spray with 4% ferric chloride ethanol solution, and inspect under ultraviolet light at 365 nm; The thin layer identification method of chlorogenic acid is as follows: take 3 g of sample powder, add 50 mL of anhydrous ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 15 mL of water to dissolve the residue, pass it through a polyamide column, elute with 50 mL of water, discard the water, elute with 30 mL of methanol in batches, collect the eluate, evaporate it to dryness in a water bath, and dissolve the residue in 1 mL of methanol as the test solution; take another chlorogenic acid reference substance, add methanol to make a solution containing 1 mg per 1 mL, as the chlorogenic acid reference substance solution; take 2 µL of each of the above two solutions, spot them on the same silica gel G plate, use ethyl acetate-butanone-chloroform-formic acid-water = 15:12:4:3:1 as the developing solvent, develop, take out, dry, spray with 3% aluminum chloride ethanol solution, and inspect under ultraviolet light at 365 nm.

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