Quality detection method for compound pear syrup

Abstract

The invention discloses a quality detection method of compound pear syrup, the compound pear syrup is mainly prepared from 31 medicinal effective components by weight: pear clear paste, adenophora tetraphylla, radix asteris, ephedra, tussilago farfara, fried fructus perillae, platycodon grandiflorum and the like. The detection method comprises a method for identifying a loquat leaf reference medicinal material and ursolic acid, a method for identifying a radix peucedani reference medicinal material, a method for identifying hesperidin and a method for determining the content of ammonium glycyrrhizinate, and the quality of the compound pear syrup is evaluated through multi-aspect measurement, so that a solid foundation is laid for the stable quality of a product, and the method is suitable for popularization and application. According to the present invention, the feasible quality standard of the compound pear syrup can be established, the effective control of the quality of the compound pear syrup can be achieved, and the liquid phase analysis is performed by using the chromatographic condition so as to obtain the clearer chromatogram with the good separation degree.

Description

Technical Field

[0001] This invention relates to a method for quality testing of traditional Chinese medicine preparations, and in particular to a method for quality testing of compound pear syrup. Background Technology

[0002] Modern medicines need to possess three major characteristics: stability and uniformity, safety, and effectiveness. Traditional Chinese medicines are difficult to match Western medicines in these aspects. Therefore, it is even more necessary to use multiple methods for testing to ensure the reliability and stability of the test results.

[0003] Compound pear syrup is a traditional Chinese medicine preparation composed of over thirty herbs, including pear syrup extract, Adenophora stricta, Aster tataricus, Ephedra sinica, Tussilago farfara, Perilla frutescens (fried), Platycodon grandiflorus, Perilla frutescens leaf, Peucedanum praeruptorum, Morus alba leaf, Citrus aurantium, Glycyrrhiza uralensis, Poria cocos, Imperata cylindrica root, Stemona japonica, Bletilla striata, Pinellia ternata (processed), Sinapis alba seed, Fritillaria thunbergii, Charred lotus leaf, Polygonatum odoratum, Trichosanthes kirilowii seed (fried), Citrus reticulata peel, Asparagus cochinchinensis, Paeonia lactiflora, Nelumbo nucifera rhizome, Bitter almond, Charred palm fiber, Colla corii asini, Eriobotrya japonica leaf, and Gardenia jasminoides (fried). It is primarily used to nourish yin and moisten the lungs, increase body fluids and promote saliva production, resolve phlegm and relieve cough. It is indicated for chronic cough due to yin deficiency, dry cough due to lung dryness, post-illness cough, asthma due to phlegm obstruction, hemoptysis, and dry lips and mouth.

[0004] Currently, compound pear syrup is only tested for its physical properties such as appearance and quantity; this cannot meet the quality control requirements of traditional Chinese medicine. Due to its complex composition, it is very necessary to establish a quality testing method for compound pear syrup. Summary of the Invention

[0005] The purpose of this invention is to disclose a method for quality testing of compound pear syrup, so as to better control the quality of compound pear syrup, characterize drug quality, and improve drug stability.

[0006] The technical solution of this invention: A quality testing method for compound pear syrup, wherein the effective medicinal components of the compound pear syrup, calculated by weight, mainly consist of 1600 parts pear extract, 40 parts Adenophora stricta, 30 parts Aster tataricus, 20 parts Ephedra sinica, 20 parts Tussilago farfara, 30 parts stir-fried Perilla frutescens, 40 parts Platycodon grandiflorus, 20 parts Perilla frutescens leaf, 40 parts Peucedanum praeruptorum, 30 parts Morus alba leaf, 20 parts Citrus aurantium, 30 parts Glycyrrhiza uralensis, 30 parts Poria cocos, 30 parts Imperata cylindrica, 30 parts Stemona japonica, 30 parts Bletilla striata, 30 parts Pinellia ternata, 30 parts Sinapis alba, 30 parts Fritillaria thunbergii, 30 parts charred Nelumbo nucifera leaf, 30 parts Polygonatum odoratum, 20 parts stir-fried Trichosanthes kirilowii seed, 20 parts Citrus reticulata peel, 20 parts Asparagus cochinchinensis, and 20 parts Paeonia lactiflora. This formula is prepared from 20 parts of pear extract, 20 parts of lotus root nodes, 20 parts of bitter almonds, 40 parts of palm charcoal, 40 parts of donkey-hide gelatin, 160 parts of loquat leaves, and 10 parts of stir-fried gardenia. All the ingredients in the formula are weighed according to the proportions. Except for the pear extract, the donkey-hide gelatin is melted. The remaining 29 ingredients are decocted three times with water, the first time for 2 hours, and each subsequent time for 1 hour. The decoctions are combined, filtered, and the filtrate is concentrated to a relative density of 1.20–1.18 at 80°C. Separately, 4000g of honey and 8000g of maltose are heated to boiling, filtered, and then the pear extract and melted donkey-hide gelatin are added and dissolved. This is filtered, mixed with the above extract, and concentrated to a relative density of 1.35–1.38 at 80°C. The mixture is then bottled into 500 bottles. The method for preparing the pear extract is as follows: take fresh pears, wash them with water, remove the core and rotten parts, cut them into pieces, put them in a container, add water and boil them, filter the supernatant and concentrate it to a relative density of 1.20 at 80℃, and then collect the extract. The detection methods include methods for identifying loquat leaf reference material and ursolic acid, methods for identifying Peucedanum praeruptorum reference material, methods for identifying hesperidin, and methods for determining the content of glycyrrhizic acid ammonium. (1) The identification method for the loquat leaf reference material and ursolic acid shall be carried out according to the following steps: Preparation of test sample a: Take 20-40g of compound pear paste, add 20-40mL of water, shake well, place in a separatory funnel, extract with chloroform 2-4 times, 20-40mL each time, collect and combine the chloroform layers, wash the chloroform layers with water 2-3 times, 320-40mL each time, discard the aqueous layer, dehydrate the collected chloroform layer with 15-25g of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5-1.5mL of methanol, and use as test sample a; Preparation of loquat leaf reference material solution: Take 0.4-0.6g of loquat leaf reference material, add 40-60mL of water, simmer gently until the solution volume is 20-30mL, then filter while hot. Add 40-60mL of water to the residue, stir thoroughly, and simmer again until the solution volume is 20-30mL. Filter while hot, combine the two filtrates, concentrate the filtrate to 15-25mL, let cool, place in a separatory funnel, and extract with chloroform 2-4 times, 15-25mL each time. Combine the chloroform layers, add 8-12g of anhydrous sodium sulfate to dehydrate, filter, evaporate the filtrate to dryness, and dissolve the residue in 0.5-1.5mL of methanol to obtain the loquat leaf reference material solution. Preparation of ursolic acid reference solution: Take ursolic acid reference standard and add methanol to prepare a solution containing 0.8-1.2 mg per 1 mL. Thin-layer chromatography analysis: The thin-layer chromatographic conditions were as follows: silica gel G thin-layer plate; sample volume: test sample a: 6-8 μL, loquat leaf reference solution: 6-8 μL, ursolic acid reference solution: 9-11 μL; developing solvent: cyclohexane:chloroform:ethyl acetate = 18-22:4-6:10-14 (v / v); colorimetric reagent: 10% sulfuric acid in ethanol; drying at 105℃ until spots are clear, and observation under sunlight. (2) The identification method for the aforementioned Peucedanum praeruptorum reference material shall be carried out according to the following steps: Preparation of test sample b: Take 80-120g of compound pear paste, add 20-40mL of ethyl acetate, shake thoroughly and sonicate for 8-12 minutes, collect the ethyl acetate layer, add another 20-40mL of ethyl acetate to the sample, shake thoroughly and sonicate for 8-12 minutes, collect the ethyl acetate layer again, combine the two ethyl acetate layers, evaporate to dryness, and dissolve the residue in 1mL of methanol as test sample b. Preparation of the Peucedanum praeruptorum reference herb solution: Take 0.8-1.2g of Peucedanum praeruptorum powder, add 8-12mL of chloroform, sonicate for 8-12 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 0.5-1.5mL of methanol to obtain the Peucedanum praeruptorum reference herb solution. Thin-layer chromatography analysis was performed under the following conditions: silica gel G thin-layer plate; sample loading: test sample b: 2-4 μL, Peucedanum praeruptorum reference solution: 2-4 μL; developing solvent: chloroform: ethyl acetate = 1.8-2.2: 0.8-1.2 (v / v); colorimetric reagent: observed under 365 nm ultraviolet light. (3) The identification method for hesperidin shall be carried out according to the following steps: Preparation of test sample c: Take 80-120g of compound pear paste, add 80-120mL of water and mix thoroughly. Adjust the pH to 11-13 with NaOH aqueous solution, extract three times with 20-40mL of ethyl acetate each time, collect the ethyl acetate layer, evaporate to dryness, and dissolve the residue with 0.5-1.5mL of methanol to obtain test sample c. Preparation of hesperidin reference solution: Take hesperidin reference standard, add methanol to prepare a saturated solution, and use it as the hesperidin reference solution; Thin-layer chromatography (TLC) analysis was performed under the following conditions: the chromatographic plate was a silica gel G TLC plate prepared with 1% sodium hydroxide solution; the sample loading was: c: 2-4 μL for the test sample and c: 2-4 μL for the reference standard; the developing solvent was ethyl acetate-methanol-water at a volume ratio of 90-110:16-18:12-14; the colorimetric reagent was aluminum trichloride solution. The sample was dried with hot air and examined under a 365 nm ultraviolet lamp. (4) The method for determining the content of glycyrrhizic acid ammonium shall be carried out according to the following steps: Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile:0.05% phosphoric acid = 37-39:61-63 was used as the mobile phase; the detection wavelength was 252 nm, the flow rate was 1 mL / min, the column temperature was 25 ℃, the injection volume was 10 μL, and the theoretical plate number calculated based on the glycyrrhizic acid peak should not be less than 3000; Preparation of test solution d: Take 8-12g of compound pear paste, place it in a stoppered conical flask, add 25-35mL of 70% ethanol solution, heat and reflux for 25-35 minutes, transfer it to a volumetric flask while hot, take 70% ethanol solution, rinse the conical flask several times while hot, transfer the rinsing solution to the volumetric flask, after the solution in the volumetric flask has cooled, make up to volume with 70% ethanol solution, shake well, filter, and take the filtrate to obtain test solution d; Preparation of ammonium glycyrrhizate reference solution: Weigh the ammonium glycyrrhizate reference standard accurately, add 70% ethanol to prepare a solution containing 10-20 μg of glycyrrhizic acid per 1 mL, and the ammonium glycyrrhizate reference solution is obtained. Determination method: Accurately pipette 10-20 μL each of the ammonium glycyrrhizate reference solution and the test solution into the liquid chromatograph and determine the result.

[0007] The identification method for the aforementioned loquat leaf reference material and ursolic acid shall be carried out according to the following steps: Preparation of test sample a: Take 30.0g of compound pear paste, add 30mL of water, shake well, place in a separatory funnel, extract with chloroform 3 times, 30mL each time, collect and combine the chloroform layers, wash the chloroform layers with water 2 times, 30mL each time, discard the aqueous layer, dehydrate the collected chloroform layer with 20g of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol, and use as test sample a; Preparation of loquat leaf reference material solution: Take 0.5g of loquat leaf reference material, add 50mL of water, boil gently until the solution volume is 25mL, filter while hot, add 50mL of water to the residue, stir thoroughly, boil gently again until the solution volume is 25mL, filter while hot, combine the two filtrates, concentrate the filtrate to 20mL, let cool, place in a separatory funnel, extract with 20mL of chloroform 3 times, combine the chloroform layers, add 10g of anhydrous sodium sulfate to dehydrate, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to obtain loquat leaf reference material solution; Preparation of ursolic acid reference solution: Take ursolic acid reference standard, add methanol to prepare a solution containing 1 mg per 1 mL, and that is, ursolic acid reference solution. Thin-layer chromatography analysis: The thin-layer chromatography conditions were as follows: silica gel G thin-layer plate; sample volume: test sample a: 7 μL, loquat leaf reference material solution: 7 μL, ursolic acid reference solution: 10 μL; developing solvent: cyclohexane:chloroform:ethyl acetate = 20:5:12 (v / v); colorimetric reagent: 10% sulfuric acid ethanol; dried at 105℃ until the spots were clear, and observed under sunlight.

[0008] The identification method for the aforementioned Peucedanum praeruptorum reference material shall be carried out according to the following steps: Preparation of test sample b: Take 100g of compound pear paste, add 30mL of ethyl acetate, shake thoroughly and sonicate for 10 minutes, collect the ethyl acetate layer, add another 30mL of ethyl acetate to the sample, shake thoroughly and sonicate for 10 minutes, collect the ethyl acetate layer again, combine the two ethyl acetate layers, evaporate to dryness, and dissolve the residue in 1mL of methanol as test sample b. Preparation of Peucedanum praeruptorum control herb solution: Take 1g of Peucedanum praeruptorum powder, add 10mL of chloroform, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue to obtain Peucedanum praeruptorum control herb solution. Thin-layer chromatography analysis was performed under the following conditions: silica gel G thin-layer plate; sample volume: test sample b: 3 μL, Peucedanum praeruptorum reference herb solution: 3 μL; developing solvent: chloroform: ethyl acetate = 2:1 (v / v); colorimetric reagent: observed under 365 nm ultraviolet light.

[0009] The aforementioned method for identifying hesperidin is carried out according to the following steps: Preparation of test sample c: Take 100.0g of compound pear paste, add 100mL of water and mix thoroughly. Adjust the pH to 12 with NaOH aqueous solution, extract three times with 30mL of ethyl acetate each time, collect the ethyl acetate layer, evaporate to dryness, and dissolve the residue with 1mL of methanol to obtain test sample c. Preparation of hesperidin reference solution: Take hesperidin reference standard, add methanol to prepare a saturated solution, and use it as the hesperidin reference solution; Thin-layer chromatography analysis was performed under the following conditions: the chromatographic plate was a silica gel G thin-layer plate prepared with 1% sodium hydroxide solution; the sample loading was: c: 3 μL for the test sample and c: 3 μL for the reference sample; the developing solvent was ethyl acetate-methanol-water at a volume ratio of 100:17:13; the colorimetric reagent was aluminum trichloride solution. The sample was dried with hot air and examined under a 365 nm ultraviolet lamp.

[0010] In the aforementioned method for determining the content of ammonium glycyrrhizate, the chromatographic conditions and system suitability test are as follows: octadecylsilane-bonded silica gel is used as the packing material; acetonitrile:0.05% phosphoric acid = 38:62 is used as the mobile phase; the detection wavelength is 252 nm, the flow rate is 1 mL / min, the column temperature is 25 ℃, the injection volume is 10 μL, and the theoretical plate number calculated based on the glycyrrhizic acid peak should not be less than 3000.

[0011] In the aforementioned method for determining the content of ammonium glycyrrhizate, the preparation of test solution d is as follows: Take 9-11g of compound pear paste, place it in a stoppered conical flask, add 28-32mL of 70% ethanol solution, heat and reflux for 28-32 minutes, transfer it to a 50mL volumetric flask while hot, take 70% ethanol solution, rinse the conical flask several times while hot, transfer the rinsing solution to the 50mL volumetric flask, after the solution in the volumetric flask has cooled, make up to volume with 70% ethanol solution, shake well, filter, and take the filtrate to obtain test solution d.

[0012] Specifically, in the aforementioned method for determining the content of ammonium glycyrrhizate, the preparation of test solution d is as follows: Take 10g of compound pear paste, place it in a stoppered conical flask, add 30mL of 70% ethanol solution, heat and reflux for 30 minutes, transfer it to a 50mL volumetric flask while hot, take 70% ethanol solution, rinse the conical flask several times while hot, transfer the rinsing solution to the 50mL volumetric flask, after the solution in the volumetric flask has cooled, make up to volume with 70% ethanol solution, shake well, filter, and take the filtrate to obtain test solution d.

[0013] In the aforementioned method for determining the content of ammonium glycyrrhizate, the preparation of the ammonium glycyrrhizate reference solution is as follows: accurately weigh the ammonium glycyrrhizate reference standard, add 70% ethanol to prepare a solution containing 12-17 μg of glycyrrhizic acid per 1 mL, thus obtaining the ammonium glycyrrhizate reference solution.

[0014] Specifically, in the aforementioned method for determining the content of ammonium glycyrrhizate, the preparation of the ammonium glycyrrhizate reference solution is as follows: accurately weigh the ammonium glycyrrhizate reference standard, add 70% ethanol to prepare a solution containing 15 μg of glycyrrhizic acid per 1 mL, thus obtaining the ammonium glycyrrhizate reference solution.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention studies the thin-layer chromatography identification and content determination of compound pear syrup, and evaluates the quality of compound pear syrup through multiple measurements, laying a solid foundation for product quality stability, establishing feasible quality standards for compound pear syrup, and achieving effective quality control of compound pear syrup. Furthermore, using the chromatographic conditions of this application for liquid phase analysis can obtain chromatograms with better separation and clearer clarity. Attached Figure Description

[0016] Figure 1 Thin-layer chromatograms of loquat leaf reference material and ursolic acid extracted by different extraction methods were used to identify the test samples (1 for ursolic acid; 2 for condition 1; 3 for condition 2; 4 for reference material; 5 for condition 3; 6 for condition 4; 7 for condition 5). Figure 2 Thin-layer chromatograms of loquat leaf reference material and ursolic acid thin-layer identification developing solvents 1-3 (1 is negative for loquat leaf deficiency; 2 is the test sample (batch number 56722013); 3 is the test sample (batch number 56722020); 4 is the test sample (batch number 56723022); 5 is loquat leaf reference material; 6 is ursolic acid reference standard). Figure 3 Thin-layer chromatograms of loquat leaf reference material and ursolic acid for identification by sample volume (1: loquat leaf reference material; 2: 3 μL of test sample; 3: 7 μL of test sample; 4: 15 μL of test sample). Figure 4 : Thin-layer chromatograms of loquat leaf reference material and ursolic acid for specific identification (1 is the negative control without loquat leaf; 2 is the test sample (batch number 56722013); 3 is the ursolic acid reference standard). Figure 5 Thin-layer chromatography identification of Peucedanum praeruptorum reference material: thin-layer chromatograms of the test samples extracted by different methods (1 is condition 1; 2 is condition 2; 3 is condition 3; 4 is condition 4; 5 is Peucedanum praeruptorum reference material). Figure 6 Thin-layer chromatograms of the developing solvents 1-3 for the thin-layer identification of Peucedanum praeruptorum reference material (1 is the test sample (batch number 56722021); 2 is the test sample (batch number 56722012); 3 is the test sample (batch number 56723004); 4 is the Peucedanum praeruptorum reference material; 5 is the negative control without Peucedanum praeruptorum). Figure 7 Thin-layer chromatograms of sample loading for identification of Peucedanum praeruptorum reference material (1 is 3 μL of test sample; 2 is 7 μL of test sample; 3 is 10 μL of test sample; 4 is 15 μL of test sample; 5 is Peucedanum praeruptorum reference material). Figure 8 Thin-layer chromatograms of specific identification of Peucedanum praeruptorum reference material (1 is the test sample (batch number: 567022012); 2 is the negative sample lacking Peucedanum praeruptorum; 3 is the Peucedanum praeruptorum reference material). Figure 9Thin-layer chromatogram of hesperidin identification reference standard (1 is dried tangerine peel; 2 is bitter orange peel; 3 is hesperidin; 4 is neohesperidin). Figure 10 Thin-layer chromatography identification of hesperidin: thin-layer chromatograms of the test sample extracted by different methods (1 is hesperidin reference standard; 2 is condition 1; 3 is condition 2; 4 is condition 3). Figure 11 Thin-layer chromatograms of hesperidin identification developing solvents 1-3 (1 is the test sample (batch number 56722024); 2 is the test sample (batch number 56722012); 3 is the test sample (batch number 56723003); 4 is the hesperidin reference standard; 5 is the hesperidin-deficient negative control). Figure 12 Thin-layer chromatograms for hesperidin identification by sample volume (1: 3 μL of test sample; 2: 7 μL of test sample; 3: 10 μL of test sample; 4: 15 μL of test sample; 5: hesperidin reference standard). Figure 13 Thin-layer chromatography chromatograms for the identification of hesperidin (1 is the test sample (batch number 56722024); 2 is the test sample (batch number 56722012); 3 is the test sample (56722022); 4 is the hesperidin reference standard; 5 is the negative sample lacking tangerine peel and bitter orange peel). Figure 14 Linear equation for glycyrrhizic acid. Detailed Implementation

[0017] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically described in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0018] Example 1: Preparation method of compound pear syrup (1) The method for preparing pear extract is as follows: take fresh pears, wash them with water, remove the core and rotten parts, cut them into pieces, put them in a container, add water and boil them, filter the supernatant and concentrate it to a relative density of 1.20 at 80℃, and then collect the extract to obtain the final product. (2) Prescription: 1600g of pear extract, 40g of Adenophora stricta, 30g of Aster tataricus, 20g of Ephedra sinica, 20g of Tussilago farfara, 30g of stir-fried Perilla frutescens, 40g of Platycodon grandiflorus, 20g of Perilla frutescens leaf, 40g of Peucedanum praeruptorum, 30g of Morus alba leaf, 20g of Citrus aurantium, 30g of Glycyrrhiza uralensis, 30g of Poria cocos, 30g of Imperata cylindrica root, 30g of Stemona japonica, 30g of Bletilla striata, 30g of Pinellia ternata, 30g of Sinapis alba, 30g of Fritillaria thunbergii, 30g of charred lotus leaf, 30g of Polygonatum odoratum, 20g of stir-fried Trichosanthes kirilowii seed, 20g of Citrus reticulata peel, 20g of Asparagus cochinchinensis, 20g of Paeonia lactiflora, 20g of Nelumbo nucifera rhizome, 20g of bitter almond, 40g of charred palm, 40g of donkey-hide gelatin, 160g of Eriobotrya japonica leaf, 10g of stir-fried Gardenia jasminoides. (3) Preparation method of compound pear paste: Weigh all the medicinal materials in the prescription according to the proportions. Except for the pear paste, melt the donkey-hide gelatin. Add water to the remaining 29 ingredients and decoct three times. The first time is 2 hours and the subsequent times is 1 hour. Combine the decoctions, filter, and concentrate the filtrate to a relative density of 1.20 to 1.18 at 80°C. Separately, take 4000g of honey and 8000g of maltose, heat to boiling, filter, add pear paste to dissolve, filter, mix with the above paste, heat and concentrate to a relative density of 1.35 to 1.38 at 80°C, and divide into 500 bottles to obtain the final product. The method for preparing the pear extract is as follows: take fresh pears, wash them with water, remove the core and rotten parts, cut them into pieces, put them in a container, add water and boil them, filter the supernatant and concentrate it to a relative density of 1.20 at 80℃, and then collect the extract. Example 2: Identification method of loquat leaf reference material and ursolic acid, carried out according to the following steps: Preparation of test sample a: Take 30.0g of compound pear paste, add 30mL of water, shake well, place in a separatory funnel, extract with chloroform 3 times, 30mL each time, collect and combine the chloroform layers, wash the chloroform layers with water 2 times, 30mL each time, discard the aqueous layer, dehydrate the collected chloroform layer with 20g of anhydrous sodium sulfate, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol, and use as test sample a; Preparation of loquat leaf reference material solution: Take 0.5g of loquat leaf reference material, add 50mL of water, boil gently until the solution volume is 25mL, filter while hot, add 50mL of water to the residue, stir thoroughly, boil gently again until the solution volume is 25mL, filter while hot, combine the two filtrates, concentrate the filtrate to 20mL, let cool, place in a separatory funnel, extract with 20mL of chloroform 3 times, combine the chloroform layers, add 10g of anhydrous sodium sulfate to dehydrate, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol to obtain loquat leaf reference material solution; Preparation of ursolic acid reference solution: Take ursolic acid reference standard, add methanol to prepare a solution containing 1 mg per 1 mL, and that is, ursolic acid reference solution. Thin-layer chromatography analysis: The thin-layer chromatography conditions were as follows: silica gel G thin-layer plate; sample volume: test sample a: 7 μL, loquat leaf reference material solution: 7 μL, ursolic acid reference solution: 10 μL; developing solvent: cyclohexane:chloroform:ethyl acetate = 20:5:12 (v / v); colorimetric reagent: 10% sulfuric acid ethanol; dried at 105℃ until the spots were clear, and observed under sunlight.

[0019] Example 3: Identification method of Peucedanum praeruptorum (Qianhu) as a control herb, which is carried out according to the following steps: Preparation of test sample b: Take 100g of compound pear paste, add 30mL of ethyl acetate, shake thoroughly and sonicate for 10 minutes, collect the ethyl acetate layer, add another 30mL of ethyl acetate to the sample, shake thoroughly and sonicate for 10 minutes, collect the ethyl acetate layer again, combine the two ethyl acetate layers, evaporate to dryness, and dissolve the residue in 1mL of methanol as test sample b. Preparation of Peucedanum praeruptorum control herb solution: Take 1g of Peucedanum praeruptorum powder, add 10mL of chloroform, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, add 1mL of methanol to dissolve the residue to obtain Peucedanum praeruptorum control herb solution. Thin-layer chromatography analysis was performed under the following conditions: silica gel G thin-layer plate; sample volume: test sample b: 3 μL, Peucedanum praeruptorum reference herb solution: 3 μL; developing solvent: chloroform: ethyl acetate = 2:1 (v / v); colorimetric reagent: observed under 365 nm ultraviolet light.

[0020] Example 4: Identification method for hesperidin, performed according to the following steps: Preparation of test sample c: Take 100.0g of compound pear paste, add 100mL of water and mix thoroughly. Adjust the pH to 12 with NaOH aqueous solution, extract three times with 30mL of ethyl acetate each time, collect the ethyl acetate layer, evaporate to dryness, and dissolve the residue with 1mL of methanol to obtain test sample c. Preparation of hesperidin reference solution: Take hesperidin reference standard, add methanol to prepare a saturated solution, and use it as the hesperidin reference solution; Thin-layer chromatography analysis was performed under the following conditions: the chromatographic plate was a silica gel G thin-layer plate prepared with 1% sodium hydroxide solution; the sample loading was: c: 3 μL for the test sample and c: 3 μL for the reference sample; the developing solvent was ethyl acetate-methanol-water at a volume ratio of 100:17:13; the colorimetric reagent was aluminum trichloride solution. The sample was dried with hot air and examined under a 365 nm ultraviolet lamp.

[0021] Example 5: Method for determining ammonium glycyrrhizate content, performed according to the following steps: Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile:0.05% phosphoric acid = 38:62 was used as the mobile phase; the detection wavelength was 252 nm, the flow rate was 1 mL / min, the column temperature was 25 ℃, the injection volume was 10 μL, and the theoretical plate number calculated based on the glycyrrhizic acid peak should not be less than 3000. Preparation of test solution d: Take 10g of compound pear paste, place it in a stoppered conical flask, add 30mL of 70% ethanol solution, heat and reflux for 30 minutes, transfer it to a 50mL volumetric flask while hot, take 70% ethanol solution, rinse the conical flask several times while hot, transfer the rinsing solution to the 50mL volumetric flask, after the solution in the volumetric flask has cooled, make up to volume with 70% ethanol solution, shake well, filter, and take the filtrate to obtain test solution d; Preparation of ammonium glycyrrhizate reference solution: Weigh the ammonium glycyrrhizate reference standard accurately, add 70% ethanol to prepare a solution containing 15 μg of glycyrrhizic acid per 1 mL, and the ammonium glycyrrhizate reference solution is obtained. Determination method: Accurately pipette 10-20 μL each of the ammonium glycyrrhizate reference solution and the test solution into the liquid chromatograph and determine the result.

[0022] This invention has undergone extensive experimental research, and the results of this experimental research are as follows: 1. Thin-layer identification of loquat leaves 1.1 Reagents and reagents Compound pear syrup (batch numbers: 56722023, 56722013, 56722020, 56723022, 56722007), loquat leaf medicinal material (batch number: 121261-201804, manufacturer: China National Institutes for Food and Drug Control), ursolic acid reference standard (batch number: 20190708, manufacturer: China National Institutes for Food and Drug Control).

[0023] Preparation of loquat leaf reference material solution: Take about 0.5g of loquat leaf reference material, add 50mL of water, simmer gently until the solution volume is about 25mL, then filter while hot. Add 50mL of water to the residue, stir thoroughly, and simmer again until the solution volume is about 25mL. Filter while hot, combine the two filtrates, concentrate the filtrate to about 20mL, let it cool, place it in a separatory funnel, and extract it three times with 20mL of chloroform each time. Combine the chloroform layers, add 10g of anhydrous sodium sulfate to dehydrate, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of methanol to obtain the loquat leaf reference material solution.

[0024] Preparation of ursolic acid reference solution: ursolic acid reference standard is prepared by adding methanol to a solution containing 1 mg per 1 mL, which is used as the ursolic acid reference solution.

[0025] 1.2 Investigation of thin-layer chromatography identification experimental conditions 1.2.1 Investigation of solution preparation conditions The following compound pear extract used in the preparation of the test solution has batch number 56722023.

[0026] Condition 1: Take 30g of compound pear syrup, add 30mL of water, shake well, place in a separatory funnel, extract with chloroform 3 times, 30mL each time, collect and combine the chloroform layers, wash with chloroform 2 times with water, 20mL each time, discard the aqueous layer, dehydrate the chloroform layer with 20g of anhydrous sodium sulfate, evaporate the chloroform layer to dryness after dehydration, dissolve the residue in 1mL of methanol as the test solution.

[0027] Condition 2: Take 30 g of compound pear paste, add 30 mL of methanol, shake well, sonicate for 30 minutes, cool and filter, collect the methanol layer, add an equal volume of petroleum ether (60-90℃) for extraction twice, retain the methanol layer, evaporate to dryness, add 30 mL of water to dilute, and then extract twice with an equal volume of chloroform, 30 mL each time, retain the chloroform layer, remove water with 20 g of anhydrous sodium sulfate, evaporate to dryness, add 1 mL of methanol to dissolve the residue, and use it as the test solution.

[0028] Condition 3: Take 30 g of compound pear paste, add 30 mL of anhydrous ethanol, shake well, sonicate for 30 minutes, cool and filter, collect the ethanol layer, add an equal volume of petroleum ether (60-90℃) for extraction twice, retain the ethanol layer, evaporate to dryness, add 30 mL of water to dilute, and then extract twice with an equal volume of chloroform, 30 mL each time, retain the chloroform layer, remove water with 20 g of anhydrous sodium sulfate, evaporate to dryness, dissolve the residue in 1 mL of methanol, and use it as the test solution.

[0029] Condition 4: Take 30g of compound pear syrup, add 30mL of chloroform, shake well, sonicate for 30 minutes, cool and filter, collect the chloroform layer, evaporate to dryness, add 1mL of methanol to make up to volume, and use as the test solution.

[0030] Condition 5: Take 30g of compound pear syrup, add 30mL of chloroform, shake well, heat under reflux for 30 minutes, cool and filter, collect the chloroform layer, evaporate to dryness, add 1mL of methanol to make up to volume, and use as the test solution.

[0031] Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Take 7 μL of the test solution under conditions 1-5, 7 μL of the loquat leaf reference material solution, and 10 μL of the ursolic acid reference solution, and spot them as strips onto the same silica gel G thin-layer plate. Use cyclohexane-chloroform-ethyl acetate (20:5:12) as the developing solvent. Develop, remove, and air-dry. Spray with 10% sulfuric acid ethanol solution and heat at 105℃ until the spots are clearly visible (results as shown in the figure). Figure 1 ).

[0032] Conclusion: Using condition 1 as the sample processing method resulted in clearer spots, higher separation, and Rf values ​​that met the testing requirements.

[0033] 1.2.2 Development of the developing solvent: Developing solvent 1: dichloromethane-ethyl acetate-methanol-acetic acid 15:3:1:0.3 (6 drops); Developing solvent 2: petroleum ether-dichloromethane-acetic acid 10:15:1; Developing solvent 3: Cyclohexane-chloroform-ethyl acetate 20:5:12; Preparation of negative sample solution lacking loquat leaves: Weigh all the medicinal materials in the compound pear syrup prescription except loquat leaves, and prepare them according to the method in Example 1 to obtain a negative sample lacking loquat leaves. Then, prepare the negative sample solution lacking loquat leaves according to the method in condition 1 under "1.2.1".

[0034] According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 7 μL of the test solution (prepared under condition 1), 7 μL of loquat leaf reference material solution, 10 μL of ursolic acid reference solution, and 7 μL of a negative sample lacking loquat leaves were spotted in strips onto the same silica gel G thin-layer plate. The plates were developed in different developing solvents, removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible (results as shown in Figure 1). Figure 2 Conclusion: Using developing solvent 3 resulted in clear spots, high separation, and Rf values ​​that met the testing requirements.

[0035] 1.2.3 Investigation of Sample Dosage Take compound pear syrup (batch number: 56722007) and prepare the test solution according to the method of condition 1 under "1.2.1".

[0036] According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 7 μL of loquat leaf reference material and 3 μL, 7 μL, and 15 μL of the test solution were respectively spotted as strips onto the same silica gel G thin-layer plate. Cyclohexane-chloroform-ethyl acetate (20:5:12) was used as the developing solvent. After development, the plate was removed, air-dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105℃ until the spots were clearly visible (results as shown in the figure). Figure 3 ).

[0037] Conclusion: A sample volume of 7 μL is sufficient to meet the experimental requirements and achieve the expected experimental results.

[0038] 1.2.4 Method Validation Based on the above experiments, the thin-layer chromatography method for identifying loquat leaves in compound pear syrup is determined as follows: Preparation of the test solution: Take 30g of compound pear paste, add 30mL of water, shake well, place in a separatory funnel, extract with chloroform 3 times, 30mL each time, collect and combine the chloroform layers, wash with chloroform 2 times with water, 20mL each time, discard the aqueous layer, dehydrate the collected chloroform layer with 20g of anhydrous sodium sulfate, filter, evaporate to dryness, dissolve the residue in 1mL of methanol to obtain the test solution.

[0039] Preparation of loquat leaf reference material solution: Take about 0.5g of loquat leaf reference material, add 50mL of water, simmer gently until the solution volume is about 25mL, then filter while hot. Add 50mL of water to the residue, stir thoroughly, and simmer again until the solution volume is about 25mL. Filter while hot, combine the two filtrates, concentrate the filtrate to about 20mL, let it cool, place it in a separatory funnel, and extract it three times with 20mL of chloroform each time. Combine the chloroform layers, add 10g of anhydrous sodium sulfate to dehydrate, filter, evaporate to dryness, and dissolve the residue in 1mL of methanol to obtain the loquat leaf reference material solution.

[0040] Preparation of ursolic acid reference solution: Take ursolic acid reference standard, add methanol to prepare a solution containing 1 mg per 1 mL, which is used as ursolic acid reference solution.

[0041] The test was performed according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). 7 μL of the test solution, 7 μL of the reference medicinal material, 10 μL of the reference solution, and 7 μL of the negative sample lacking loquat leaves were spotted as strips onto the same silica gel G thin-layer plate. The plate was developed using cyclohexane-chloroform-ethyl acetate (20:5:12) as the developing solvent. The plate was then removed, air-dried, sprayed with 10% sulfuric acid in ethanol, and heated at 105℃ until the spots were clearly visible. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatogram of the reference solution. The spots showed high resolution and clear color development, with no interference from the negative control.

[0042] 1.3 Specificity According to the thin-layer chromatography identification method for loquat leaves in compound pear syrup obtained in section "1.2.4", negative samples lacking loquat leaves were compared with normal test samples to determine whether ursolic acid-specific spots could be obtained. Figure 4 ).

[0043] Results: The separation met the requirements, the Rf value was moderate, there was no interference from the negative control, and the specificity met the test standards.

[0044] 2. Thin-layer identification of Peucedanum praeruptorum 2.1 Reagents and reagents Compound pear syrup (batch numbers: 56722021, 56722012, 56723004, 56722022, 56722015, 56722013, 56722020), and Peucedanum praeruptorum reference material (prepared in the laboratory).

[0045] Preparation of Peucedanum praeruptorum control herb solution: Take 0.5g of Peucedanum praeruptorum powder, add 10mL of chloroform, sonicate for 10 minutes, filter, evaporate to dryness, add 1mL of methanol to dissolve the residue, and use it as the Peucedanum praeruptorum control herb solution.

[0046] 2.2 Investigation of thin-layer chromatography identification experimental conditions 2.2.1 Investigation of preparation method (batch number of compound pear syrup: 56722022) Condition 1: Take about 100.0g of compound pear paste, add 50mL of chloroform, shake thoroughly, sonicate for 10 minutes, collect the chloroform layer, evaporate to dryness, dissolve the residue in 1mL of methanol, and use it as the test solution.

[0047] Condition 2: Take about 100.0g of compound pear paste, add 50mL of dichloromethane, shake thoroughly, sonicate for 10 minutes, collect the trichloromethane layer, evaporate to dryness, dissolve the residue in 1mL of methanol, and use it as the test solution.

[0048] Condition 3: Take about 100.0g of compound pear extract, add 30mL of ethyl acetate and shake thoroughly. Sonicate for 10 minutes, collect the ethyl acetate layer, add another 30mL of ethyl acetate to the sample, repeat the above steps, combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in 1mL of methanol to obtain the test solution.

[0049] Condition 4: Take about 100.0g of compound pear extract, add 50mL of ethyl acetate, heat under reflux for 30 minutes, remove and let stand to cool, collect the ethyl acetate layer, evaporate to dryness, dissolve the residue in 1mL of methanol, and use it as the test solution.

[0050] Thin-layer chromatography analysis: Perform the thin-layer chromatography test (General Rule 0502). Apply 3 μL each of the test solution and the Peucedanum praeruptorum reference material solution to the same activated silica gel G thin-layer plate. Develop using chloroform:ethyl acetate 2:1 as the developing solvent. Remove the plate, air dry, and examine under ultraviolet light (365 nm). The results are as follows: Figure 5 As shown.

[0051] Conclusion: Using condition 3 as the sample processing method resulted in clearer spots, higher separation, and Rf values ​​that met the testing requirements.

[0052] 2.2.2 Investigation of the developing solvent Developing solvent 1: dichloromethane: ethyl acetate: methanol 8:2:0.1; Developing solvent 2: dichloromethane: ethyl acetate 2:1; Developing solvent 3: chloroform: ethyl acetate 2:1; Preparation of negative sample solution without Peucedanum: Weigh all the medicinal materials in the compound pear paste prescription except Peucedanum, and prepare them according to the method in Example 1 to obtain a negative sample without Peucedanum. Then, prepare the negative sample solution without Peucedanum according to the method in condition 3 under "2.2.1".

[0053] Perform the thin-layer chromatography test (General Rule 0502). Take 3 μL each of the test solution (prepared according to method 1), the Peucedanum praeruptorum reference solution, and the negative sample lacking Peucedanum praeruptorum, and spot them separately onto the same activated silica gel G thin-layer plate. Use different developing solvents, develop, remove, air dry, and examine under ultraviolet light (365 nm). The results are as follows. Figure 6 As shown.

[0054] Conclusion: Using developing solvent 3 resulted in clearer spots, higher separation, and Rf values ​​that met the testing requirements.

[0055] 2.2.3 Sample dosage (Compound pear syrup batch number: 56722007) Perform thin-layer chromatography (General Rule 0502). Apply 3 μL, 7 μL, 10 μL, and 15 μL of the test solution (prepared under condition 1) and 3 μL of the reference herb solution to the same activated silica gel G thin-layer plate. Develop the plate using chloroform:ethyl acetate in a 2:1 ratio as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (365 nm). The results are as follows. Figure 7 As shown: Conclusion: A sample volume of 3 μL is sufficient to meet the experimental requirements and achieve the expected experimental results.

[0056] 2.2.4 Method Validation Based on the above experiments, the thin-layer chromatography method for identifying Peucedanum praeruptorum in compound pear syrup is determined as follows: Preparation of the test solution: Take 100g of compound pear paste, add 30mL of ethyl acetate, shake thoroughly and sonicate for 10 minutes, collect the ethyl acetate layer, add another 30mL of ethyl acetate to the sample, repeat the above steps, combine the ethyl acetate layers, evaporate to dryness, and dissolve the residue in 1mL of methanol to obtain the test solution.

[0057] Preparation of negative sample solution for sample lacking Peucedanum: Take another 100g of negative sample lacking Peucedanum and prepare negative sample solution according to the method for preparing test sample solution.

[0058] Preparation of the Peucedanum praeruptorum control solution: Take 1g of Peucedanum praeruptorum powder, add 10mL of chloroform, sonicate for 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1mL of methanol, and use it as the Peucedanum praeruptorum control solution.

[0059] The test was performed according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). 3 μL of each of the two solutions were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using chloroform-ethyl acetate (2:1) as the developing solvent. The plate was then removed, dried, and examined under ultraviolet light (365 nm). The test sample chromatogram showed fluorescent spots of the same color at the corresponding positions as the reference sample chromatogram. The spots showed high resolution and clear color development, with no interference from the negative control. Therefore, it was selected for inclusion in the main text of the quality standard.

[0060] 2.3 Specificity The thin-layer chromatography method for identifying Peucedanum praeruptorum in compound pear paste, as determined in "2.2.4", was followed. A negative sample lacking Peucedanum praeruptorum was compared with a normal test sample to confirm whether the corresponding spots of the Peucedanum praeruptorum control material could be obtained. Figure 8 The separation results met the requirements, the Rf value was moderate, there was no interference from the negative control, and the specificity met the testing standards.

[0061] 3. Identification of hesperidin 3.1 Reagents and reagents ZF-20D UV analyzer, KQ-500V DB dual-frequency CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 1% NaOH silica gel G plate (100*100mm, batch number: 20230620, manufacturer: Qingdao Haiyang Chemical Co., Ltd.), 1% NaOH silica gel G plate (100*100mm, batch number: 20230916, manufacturer: Yantai Jiangyou Silica Gel Development Co., Ltd.).

[0062] Compound pear syrup (batch numbers: 56722024, 56722012, 56723003, 56722022), hesperidin reference standard (batch number: 110721-202019, manufacturer: China National Institutes for Food and Drug Control), neohesperidin reference standard (batch number: 111857-202305, manufacturer: China National Institutes for Food and Drug Control).

[0063] 3.2 Investigation of thin-layer chromatography identification experimental conditions In the 2020 edition of the Chinese Pharmacopoeia, the identification item for dried tangerine peel is hesperidin, and the thin-layer chromatography identification item for immature bitter orange peel is neohesperidin. Upon verification, hesperidin and neohesperidin are isomers, and their positions in the thin-layer chromatography are as follows: Figure 9In the chromatogram, tangerine peel, immature bitter orange peel, hesperidin reference standard, and neohesperidin reference standard showed fluorescent spots of the same color at the corresponding positions. Since tangerine peel and immature bitter orange peel are medicinal materials in compound pear paste, and their medicinal effects are similar to the main functions of the compound pear paste, and the prescription quantity is large but the content of each individual medicinal material is small, the thin-layer chromatography identification results are not ideal. Therefore, a saturated methanol solution of hesperidin was chosen as a control, and the compound pear paste, lacking tangerine peel and immature bitter orange peel, was chosen as a negative sample.

[0064] Preparation of hesperidin reference solution: Take hesperidin reference standard, add methanol to prepare a saturated solution, and use it as the hesperidin reference solution.

[0065] 3.2.1 Investigation of preparation method (batch number of compound pear syrup: 56722022) Condition 1: Take about 100.0g of compound pear paste, add 100mL of water and mix thoroughly. Adjust the pH to 12 with NaOH aqueous solution, extract three times with 30mL of ethyl acetate each time, collect the ethyl acetate layer, evaporate to dryness, and dissolve the residue with 1mL of methanol to obtain the test solution. Condition 2: Take about 100.0g of compound pear paste and add it to diatomaceous earth in batches while stirring until the sample after stirring is fine sand-like. Then add 150mL of methanol to cover the sample. After sealing, sonicate for 30 minutes, remove and let stand to cool. Evaporate the supernatant to an appropriate volume and pass it through a macroporous resin D101 column (10g macroporous resin D101, 2cm chromatography column). Wash the column with water several times, drain the water in the column, and then elute the column with 15mL of methanol. Collect the eluent, evaporate it to dryness, and dilute to 1mL of methanol as the test solution. Condition 3: Take about 100.0g of compound pear paste, add 100mL of anhydrous ethanol, mix well, seal, sonicate for 30 minutes, remove and let stand, cool, and then pass the supernatant through a macroporous resin D101 column (10g macroporous resin D101, 2cm chromatography column). Wash the column with water several times, drain the water in the column, then elute the column with 15mL of ethanol, collect the eluent, evaporate to dryness, and dilute to volume with 1mL of methanol as the test solution.

[0066] Perform the thin-layer chromatography test (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Apply 3 μL of the test sample and 3 μL of the hesperidin reference solution separately to the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. Develop the plate using ethyl acetate-methanol-water (100:17:13) as the developing solvent. Remove the plate, air dry, spray with aluminum trichloride solution, dry with hot air, and examine under ultraviolet light (365 nm). The results are as follows. Figure 10 .

[0067] Conclusion: Using condition 1 as the sample processing method resulted in clearer spots, higher separation, and Rf values ​​that met the testing requirements.

[0068] 3.2.2 Development of the developing solvent: Developing solvent 1: ethyl acetate-methanol-water 10:1:0.5; Developing solvent 2: ethyl acetate-methanol-water 10:1.7:1.3; Developing solvent 3: Ethyl acetate - Methanol 10:1.5; Preparation of negative sample solution lacking tangerine peel and immature bitter orange peel: Weigh all the medicinal materials in the compound pear paste prescription except tangerine peel and immature bitter orange peel, and prepare them according to the method of Example 1 to obtain a negative sample lacking tangerine peel and immature bitter orange peel. Prepare the negative sample solution lacking tangerine peel and immature bitter orange peel according to the test sample solution preparation method in condition 1 under "3.2.1".

[0069] The thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502) was used. 3 μL each of the test sample, reference solution, and negative sample lacking tangerine peel and immature bitter orange peel were spotted onto the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. The plates were developed using the corresponding developing solvents, removed, air-dried, sprayed with aluminum trichloride solution, dried with hot air, and examined under ultraviolet light (365 nm). The results are as follows: Figure 11 As shown: Conclusion: Using developing solvent 2 resulted in clear spots, high separation, and Rf values ​​that met the testing requirements.

[0070] 3.2.3 Sample dosage (Compound pear syrup batch number: 56722007) According to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502), 3 μL of hesperidin reference solution, 3 μL, 7 μL, 10 μL, and 15 μL of the test solution were spotted separately onto the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. The plate was developed using ethyl acetate-methanol-water (100:17:13) as the developing solvent. After development, the plate was removed, air-dried, sprayed with aluminum trichloride reagent, dried with hot air, and examined under ultraviolet light (365 nm). The results are as follows. Figure 12 As shown.

[0071] Conclusion: A sample volume of 3 μL is sufficient to meet the experimental requirements and achieve the expected experimental results.

[0072] 3.2.4 Method Validation Based on the above experiments, the thin-layer chromatography method for identifying hesperidin in compound pear paste is determined as follows: Preparation of test solution: Take about 100.0g of the contents of this product, add 100mL of water and mix thoroughly. Adjust the pH to 12 with NaOH aqueous solution, extract three times with 30mL of ethyl acetate each time, collect the ethyl acetate layer, evaporate to dryness, and dissolve the residue with 1mL of methanol to obtain the test solution. Preparation of negative sample solution for samples lacking tangerine peel and immature bitter orange peel: Take a negative sample lacking tangerine peel and immature bitter orange peel and prepare a negative sample solution according to the preparation method of the test sample solution.

[0073] Preparation of hesperidin reference solution: Take hesperidin reference standard, add methanol to prepare a saturated solution, and use it as the hesperidin reference solution.

[0074] The test was performed according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). 3 μL of each of the two solutions were applied separately to the same silica gel G thin-layer plate prepared with 1% sodium hydroxide solution. The plate was developed using ethyl acetate-methanol-water (100:17:13) as the developing solvent. After development, the plate was removed, air-dried, sprayed with aluminum trichloride reagent, dried with hot air, and examined under ultraviolet light (365 nm). The test sample chromatogram showed fluorescent spots of the same color at the corresponding positions as the reference sample chromatogram. The spot resolution was high, the color development was clear, and there was no interference from the negative control. Therefore, it was selected for inclusion in the main text of the quality standard.

[0075] 3.3 Specificity The thin-layer chromatography method for identifying Peucedanum praeruptorum in compound pear paste, as determined in "3.3", was followed. Negative samples lacking Citrus reticulata peel and Citrus aurantium were compared with normal test samples to confirm whether hesperidin-specific spots could be detected. Figure 13 ).

[0076] The separation met the requirements, the Rf value was moderate, the negative control showed no interference, and the specificity met the testing standards.

[0077] 4. Determination of glycyrrhizic acid content 4.1 Reagents and reagents Agilent 1200 high performance liquid chromatograph; Thermo Fisher UltiMate 3000 standard dual-system liquid chromatograph; KQ-500V DB dual-frequency CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0078] The compound pear syrup was provided by Guizhou Dechangxiang Pharmaceutical Co., Ltd., and ammonium glycyrrhizate reference standard (purity: 96.2%, China National Institutes for Food and Drug Control, batch number: 110805-201808, for content determination).

[0079] 4.2 Determination Method 4.2.1 Selection of Chromatographic Column Chromatographic column: An octadecylsilane-bonded silica gel column was used as the packing material. Mobile phase: Mobile phase A was acetonitrile, and mobile phase B was 0.05% phosphoric acid solution, with an acetonitrile:0.05% phosphoric acid ratio of 38:62, using isocratic elution. Detection wavelength: 252 nm. Theoretical plate number: Based on actual experimental data and considering factors such as the purity of the sample and reference standards, the theoretical plate number was set at 3000.

[0080] 4.2.2 Investigation of Sample Extraction Methods 4.2.2.1 Analysis of the extract Method 1: Take about 10g of compound pear paste (batch number: 56818021) sample, place it in a stoppered conical flask, add 20mL of 70% ethanol, sonicate for 30 minutes, remove, and transfer it to a 50mL volumetric flask while hot. Rinse the conical flask several times with 70% methanol solution while hot, transfer the washings to a 50mL volumetric flask, add 70% methanol to the 50mL mark, shake well, filter, and collect the filtrate.

[0081] Method 2: Take about 10g of compound pear paste (batch number: 56818021) sample, place it in a stoppered conical flask, add 20mL of 70% methanol, sonicate for 30 minutes, remove, and transfer to a 50mL volumetric flask while hot. Rinse the conical flask several times with 70% ethanol solution while hot, transfer the rinsing solution to a 50mL volumetric flask, add 70% ethanol to the 50mL mark, shake well, filter, and collect the filtrate to obtain the final product.

[0082] Method 3: Take about 10g of compound pear paste (batch number: 56818021) sample, place it in a stoppered conical flask, add 20mL of acetonitrile-0.05% phosphoric acid (38:62) mobile phase, sonicate for 30 minutes, remove, transfer to a 50mL volumetric flask while hot, rinse the conical flask several times with the mobile phase while hot, transfer the washings to the 50mL volumetric flask, add the mobile phase to the 50mL mark, shake well, filter, and collect the filtrate to obtain the final product.

[0083] Table 1. Investigation of different extracts Conclusion: The extraction effect of the extract using method 1 is better.

[0084] 4.2.2.2 Investigation of Ultrasonic and Reflux Extraction Take approximately 10g of compound pear syrup (batch number: 56722009) sample, weigh five portions in parallel, and place them in stoppered conical flasks. Add 20mL of 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol to each flask in sequence. Sonicate the flasks (power 250W, frequency 35kHz) for 30 minutes. Remove the flasks and transfer them to 50mL volumetric flasks. Rinse the conical flasks multiple times with the corresponding solutions. Transfer the rinsing solution to the 50mL volumetric flasks and fill the flasks to the 50mL mark with the corresponding solutions. Shake well, filter, and collect the filtrate to obtain the final product.

[0085] Take approximately 10g of compound pear extract (batch number: 56722009) as a separate sample, weigh five portions in parallel, and place them in stoppered conical flasks. Add 20mL of 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 90% ethanol to each flask in sequence. Heat under reflux for 30 minutes. Remove the flasks and transfer them to 50mL volumetric flasks while hot. Rinse the conical flasks several times with the corresponding extracts while hot. Transfer the rinsing solution to the 50mL volumetric flasks and fill the flasks to the 50mL mark with the corresponding solutions. Shake well, filter, and collect the filtrate to obtain the final product.

[0086] Table 2. Investigation of ultrasonic and reflux extraction of extracts at different concentrations Conclusion: The extraction method using 70% ethanol under reflux is better.

[0087] 4.2.2.3 Investigation of Extraction Liquid Volume Take approximately 5g of compound pear syrup (batch number: 56722003) sample, weigh three portions in parallel, and place them in stoppered conical flasks respectively. Add 10mL, 30mL, and 80mL of 70% ethanol sequentially, heat under reflux for 30 minutes, remove, and transfer while hot to the corresponding 20mL, 50mL, and 100mL volumetric flasks. Rinse the conical flasks several times with 70% ethanol solution while hot, transfer the rinsing solution to the 50mL volumetric flask, add 70% ethanol to the mark, shake well, filter, and collect the filtrate to obtain the final product.

[0088] Table 3. Analysis of Extraction Liquid Volume Conclusion: The reflux method with a 50 mL system is better for sample extraction.

[0089] 4.2.2.4 Investigation of Hot Reflow Time Take approximately 10g of compound pear syrup (batch number: 56722023) sample, weigh three portions in parallel, and place each portion in a stoppered conical flask. Add 30mL of 70% ethanol, heat under reflux for 15 minutes, 30 minutes, and 45 minutes, respectively. Remove the flasks and transfer them to 50mL volumetric flasks while hot. Rinse the conical flasks several times with 70% ethanol solution while hot. Transfer the rinsing solution to the 50mL volumetric flasks, and fill the flasks to the 50mL mark with 70% ethanol. Shake well, filter, and collect the filtrate to obtain the final product.

[0090] Table 4 Comparison of Heating and Reflux Times Conclusion: Heating under reflux for 30 minutes is the better method.

[0091] 4.3 Method Validation Based on the above experiments, the sample preparation method for determining the glycyrrhizic acid content in compound pear syrup is as follows: Take 10g of the contents of this product and place them in a stoppered conical flask. Add 30mL of 70% ethanol solution and heat under reflux for 30 minutes. While hot, transfer the extract to a 50mL volumetric flask. Take 70% ethanol solution and rinse the conical flask several times while hot. Transfer the rinsing solution to the 50mL volumetric flask. After the solution in the volumetric flask cools, add 70% ethanol solution to the mark of the volumetric flask. Shake well, filter, and take the filtrate to obtain the test solution. Accurately weigh an appropriate amount of ammonium glycyrrhizate reference standard and add 70% ethanol to prepare solutions containing 15 μg of glycyrrhizic acid per 1 mL. The sample extracted by this method can be completely separated from other peaks, and all indicators meet the standards. Therefore, this extraction method for the sample is selected and included in the main text of the quality standard.

[0092] 4.4 Method Validation 4.4.1 Accuracy Take approximately 10g of the compound pear paste (batch number: 5622015) with the determined content, accurately weigh it, and weigh it in 9 parallel batches, dividing it into three groups: high, medium, and low concentrations, with 3 samples in each group. Place the samples in stoppered conical flasks and prepare the sample solutions according to the test solution preparation method. Accurately add 150ug of glycyrrhizic acid reference standard to the low concentration group, 300ug of glycyrrhizic acid reference standard to the medium concentration group, and 600ug of glycyrrhizic acid reference standard to the high concentration group. Prepare the sample solutions according to the test solution preparation method, determine the glycyrrhizic acid content under the above chromatographic conditions, and calculate the recovery rate. The results are shown in Table 5.

[0093] Table 5. Recovery rate test data (n=9) As shown in the table above, glycyrrhizic acid in the samples showed good recovery rates under low, medium, and high conditions, with an average recovery rate of 98.85% and an RSD of 8.86%. 4.4.2 Repeatability Test Seven samples of the same batch of compound pear paste (batch number: 56723004) were prepared in parallel according to the preparation method of the test solution. The glycyrrhizic acid content was determined under the above chromatographic conditions. The results are shown in Table 6.

[0094] Table 6. Repeatable experiments (n=7) As shown in the table above, the glycyrrhizic acid content in the sample is relatively uniform, and the method has good repeatability with an RSD of 5.31%.

[0095] 4.4.3 Precision Test Accurately weigh the same batch of compound pear syrup (batch number: 56722009), prepare one sample according to the preparation method of the test sample, inject the sample 6 times consecutively, record the chromatogram, and the results are shown in Table 7.

[0096] Table 7 Precision test (n=6) As shown in the figure above, the method has good precision, with an RSD of 0.46%.

[0097] 4.4.4 Specificity Glycyrrhizic acid reference standard was prepared to a concentration of 7.5 μg / mL. Compound pear extract (batch number: 56722012) and negative sample (the prescription lacks licorice) were prepared into test solutions according to the test solution preparation method. 10 μL of the reference solution, sample test solution, and negative sample test solution were injected into the liquid chromatograph under the above chromatographic conditions. The retention time of glycyrrhizic acid in the chromatograms of the licorice extract and sample solution should be similar to that in the chromatogram of the reference solution, and corresponding chromatographic peaks should appear. There should be no interference from the negative sample, as shown in the dedicated chromatogram.

[0098] 4.4.5 Linearity and Range Under the chromatographic conditions described above, precisely inject 1 μL, 2.5 μL, 5 μL, 10 μL, 15 μL, 20 μL, and 25 μL of glycyrrhizic acid reference solution (15 µg / mL) sequentially into the liquid chromatograph, and determine the peak area. Plot the injection volume X as the abscissa and the peak area Y as the ordinate, and construct the working curve (Figure). The linear regression equation is: Y = 12.484x + 0.008, R² = 1. The results are shown below. Figure 14 For a single injection, within the range of 0.015–0.375 μg of glycyrrhizic acid, the injection amount of the reference standard showed a good linear relationship with the peak area.

[0099] Table 8. Linearity of glycyrrhizic acid reference standard 4.4.6 Durability 4.4.6.1 Sample solution stability test Compound pear extract (batch number: 56722019) was prepared according to the method for preparing the test solution, and the glycyrrhizic acid content was determined under the above chromatographic conditions. The results are shown in Table 9.

[0100] Table 9. Stability Test of Sample Solutions Conclusion: The solution is relatively stable within 72 hours.

[0101] 4.4.6.2 Effect of column temperature on glycyrrhizic acid content determination Without changing the composition of the mobile phase, the column oven temperature was changed to investigate the effect of different column temperatures. The results showed that the separation of the samples was better at column oven temperatures of 25℃, 30℃ and 35℃. The content determination results are shown in Table 10.

[0102] Table 10. Measurement results under different column oven conditions As shown in the table above, the content of glycyrrhizic acid did not change significantly when the column temperature changed by ±5℃.

[0103] 4.4.6.3 Effect of changing flow rate on the determination of glycyrrhizic acid content Without changing the composition of the mobile phase, the flow rate was varied to investigate the effect of different flow rates. The results showed that the separation of the sample was better at flow rates of 0.8 mL / min, 1.0 mL / min and 1.2 mL / min. The content determination results are shown in Table 11.

[0104] Table 11. Measurement results under different column oven conditions As shown in the table above, the content of glycyrrhizic acid did not change significantly when the flow rate varied by ±0.2 mL / min.

[0105] 4.4.6.4 The impact of different brand pillars The glycyrrhizic acid content in compound pear extract (batch number: 56722023) was determined using columns packed with octadecylsilane-bonded silica gel from different brands, to investigate the effect of different brands of chromatographic columns. Chromatograms are shown in the robustness chromatogram, and the content determination results are shown in Table 12. The results indicate that chromatographic columns packed with octadecylsilane-bonded silica gel from different brands have no effect on the content determination.

[0106] Table 12 Measurement results of columns from different brands 4.5 Sample Determination Fifteen batches of samples were tested under the above conditions, and the results are shown in Table 13.

[0107] Table 13 Sample Measurement Results The licorice in this product is extracted by boiling with water; therefore, the content limit for each gram of the extract, calculated as glycyrrhizic acid (C42H62O16), should be: average total content of 15 batches of samples × 70% = 0.03332 × 70% = 0.023324 mg / g ≈ 0.02 mg / g Therefore, it is tentatively determined that each 1g of this product contains not less than 0.02mg of licorice, calculated as glycyrrhizic acid (C42H62O16).

Claims

1. A quality detection method of compound pear syrup, characterized in that: The medicinal effective components of the compound pear syrup are prepared from 1600 parts of pear extract, 40 parts of south radix adenosmae, 30 parts of radix pini, 20 parts of radix eupatii, 20 parts of flos forsythiae, 30 parts of torrefactus perillae fructus, 40 parts of platycodonis radix, 20 parts of perillae folium, 40 parts of radix peucedani, 30 parts of mori folium, 20 parts of citri fructus, 30 parts of glycyrrhizae radix, 30 parts of poriae fructus, 30 parts of baiibu, 30 parts of bletillae rhizoma, 30 parts of radix bupleuri, 30 parts of radix trichosanthis, 20 parts of semen ziziphi spinosae torrefactus, 20 parts of pericarpium citri reticulatae, 20 parts of radix asparagi, 20 parts of radix paeoniae alba, 20 parts of lotus node, 20 parts of amygdule armeniaceae amara, 40 parts of carbonis palmitae, 40 parts of mella, 160 parts of folium eriobotryae, 10 parts of semen ziziphi spinosae torrefactus, and 4000 parts of honey and 8000 parts of treacle are heated to boiling, filtered, dissolved with the pear extract and the carbonis palmitae, mixed with the above extract, heated and concentrated to the relative density of 1.35-1.38 at 80 DEG C, and then filled into 500 bottles to obtain the compound pear syrup. ​ The preparation method of the pear extract is as follows: fresh pears are washed with water, the cores and rotten parts are removed, and then the pears are cut into pieces, which are boiled in water, and the supernatant is collected and concentrated to the relative density of 1.20 at 80 DEG C to obtain the pear extract. The detection method comprises the identification method of the control medicine of folium eriobotryae and ursolic acid, the identification method of radix peucedani control medicine, the identification method of hesperidin, and the content determination method of ammonium glycyrrhizate. (1) The identification method of the control medicine of folium eriobotryae and ursolic acid is carried out according to the following steps: Preparation of the test sample a: 20-40 g of the compound pear syrup is added into 20-40 mL of water, fully shaken, and then placed in a separating funnel, and extracted with chloroform for 2-4 times, each time with 20-40 mL of chloroform, and the chloroform layers are collected and combined, washed with water for 2-3 times, each time with 32-40 mL of water, and the water layer is discarded, the collected chloroform layer is dehydrated with 15-25 g of anhydrous sodium sulfate, filtered, and the filtrate is evaporated to dryness, the residue is dissolved with 0.5-1.5 mL of methanol to obtain the test sample a; Preparation of the control medicine solution of folium eriobotryae: 0.4-0.6 g of the control medicine of folium eriobotryae is added into 40-60 mL of water, and boiled to obtain a solution, and then the solution is filtered while hot, the residue is fully stirred with 40-60 mL of water, boiled again to obtain a solution, and then the solution is filtered while hot, the two filtrates are combined, concentrated to 15-25 mL, cooled, and then placed in a separating funnel, and extracted with chloroform for 2-4 times, each time with 15-25 mL of chloroform, and the chloroform layers are combined, dehydrated with 8-12 g of anhydrous sodium sulfate, filtered, and the filtrate is evaporated to dryness, and the residue is dissolved with 0.5-1.5 mL of methanol to obtain the control medicine solution of folium eriobotryae; Preparation of the ursolic acid control solution: ursolic acid control is added into methanol to prepare a solution containing 0.8-1.2 mg per 1 mL to obtain the ursolic acid control solution. Thin layer chromatography analysis: thin layer chromatography conditions are, chromatographic plate is silica gel G thin layer plate; sample size: test product a: 6-8 μL, loquat leaf control drug solution preparation: 6-8 μL, ursolic acid control solution: 9-11 μL; developing agent is cyclohexane: chloroform: ethyl acetate = 18-22: 4-6: 10-14 by volume ratio; color reagent is 10% sulfuric acid ethanol; Place 105 DEG C to the spot clear, observe under daylight; (2) The identification method of the preceding radix peucidi, according to the following steps: Test product b preparation: take compound pear syrup 80-120g, add ethyl acetate 20-40mL, shake well, then ultrasonic 8-12 minutes, collect ethyl acetate layer, add ethyl acetate 20-40mL to the sample, shake well, then ultrasonic 8-12 minutes, collect ethyl acetate layer again, combine the two ethyl acetate layers, evaporate to dryness, dissolve the residue with 1mL methanol as test product b; Radix peucidi control drug solution preparation: take radix peucidi medicinal powder 0.8-1.2g, add chloroform 8-12mL, ultrasonic treatment 8-12 minutes, filter, evaporate the filtrate to dryness, add methanol 0.5-1.5mL to dissolve, obtain radix peucidi control drug solution; Thin layer chromatography analysis: thin layer chromatography conditions are, chromatographic plate is silica gel G thin layer plate; sample size: test product b: 2-4 μL, radix peucidi control drug solution: 2-4 μL; developing agent is chloroform: ethyl acetate = 1.8-2.2: 0.8-1.2 by volume ratio; color reagent: 365nm ultraviolet light observation; (3) The identification method of the hesperidin, according to the following steps: Prepare test product c: take compound pear syrup 80-120g, add 80-120mL water to mix well, adjust PH to 11-13 with NaOH aqueous solution, extract with ethyl acetate three times, 20-40mL each time, collect ethyl acetate layer, evaporate to dryness, dissolve the residue with 0.5-1.5mL methanol, obtain test product c; Hesperidin control solution preparation: take hesperidin control, add methanol to prepare saturated solution as hesperidin control solution; Thin layer chromatography analysis: thin layer chromatography conditions are, chromatographic plate is silica gel G thin layer plate prepared with 1% sodium hydroxide solution; sample size: test product c: 2-4 μL, control c: 2-4 μL; developing agent: ethyl acetate-methanol-water = 90-110: 16-18: 12-14 by volume ratio; color reagent: aluminum chloride test solution, hot air dry, place under 365nm ultraviolet light to observe; (4) The ammonium glycyrrhizinate content determination method, according to the following steps: Chromatographic conditions and system suitability test: octadecyl silane bonded silica gel as filler; acetonitrile: 0.05% phosphoric acid = 37-39: 61-63 as mobile phase; detection wavelength is 252nm, flow rate is 1mL / min, column temperature is 25 DEG C, 10 μL injection volume, theoretical plate number should not be less than 3000 according to glycyrrhizic acid peak calculation; Preparation of the test solution d: Take 8-12 g of the compound pear syrup, and put it into a conical flask with a stopper. Add 25-35 mL of 70% ethanol solution, and heat it to reflux for 25-35 minutes. Then, transfer it into a volumetric flask while it is hot. Take 70% ethanol solution, and rinse the conical flask several times while it is hot. Transfer the rinsing solution into the volumetric flask. After the solution in the volumetric flask cools down, add 70% ethanol solution to make it to the volume. Shake it well, filter it, and take the filtrate. Then, the test solution d is obtained. Preparation of the ammonium glycyrrhizinate control solution: Take the ammonium glycyrrhizinate control product, and accurately weigh it. Add 70% ethanol to make a solution containing 10-20 μg of glycyrrhizic acid per 1 mL. Then, the ammonium glycyrrhizinate control solution is obtained. Determination method: Accurately take 10-20 μL of the ammonium glycyrrhizinate control solution and the test solution, respectively, and inject them into the liquid chromatograph. Then, the determination is performed.

2. The quality detection method of the compound pear paste according to claim 1, characterized in that: The identification method of the loquat leaf control medicinal material and ursolic acid is performed according to the following steps: Preparation of the test solution a: Take 30.0 g of the compound pear syrup, add 30 mL of water, shake it well, and put it into a separatory funnel. Extract it with chloroform for three times, 30 mL each time. Collect and combine the chloroform layers. Wash the chloroform layer with water for two times, 30 mL each time, and discard the water layer. Dehydrate the collected chloroform layer with 20 g of anhydrous sodium sulfate, filter it, evaporate the filtrate, and dissolve the residue in 1 mL of methanol as the test solution a. Preparation of the loquat leaf control medicinal material solution: Take 0.5 g of the loquat leaf control medicinal material, add 50 mL of water, and boil it to extract. When the solution volume is 25 mL, filter it while it is hot. Stir the filter residue with 50 mL of water, and boil it to extract again. When the solution volume is 25 mL, filter it while it is hot. Combine the two filtrates, concentrate the combined filtrate to 20 mL, cool it, and put it into a separatory funnel. Extract it with chloroform for three times, 20 mL each time. Combine the chloroform layers, add 10 g of anhydrous sodium sulfate to dehydrate, filter it, evaporate the filtrate, and dissolve the residue in 1 mL of methanol as the loquat leaf control medicinal material solution. Preparation of the ursolic acid control solution: Take the ursolic acid control product, and add methanol to make a solution containing 1 mg per 1 mL. Then, the ursolic acid control solution is obtained. Thin layer chromatography analysis: The thin layer chromatography conditions are as follows: the chromatographic plate is a silica gel G thin layer plate; the sample amount is 7 μL for the test solution a, 7 μL for the loquat leaf control medicinal material solution, and 10 μL for the ursolic acid control solution; the developing agent is cyclohexane: chloroform: ethyl acetate = 20:5:12 by volume; the color developing agent is 10% sulfuric acid ethanol; and the plate is baked at 105°C until the spots are clear, and observed under sunlight.

3. The quality detection method of the compound pear paste according to claim 1, characterized in that: The identification method of the peony root control medicinal material is performed according to the following steps: Preparation of the test solution b: Take 100 g of the compound pear syrup, add 30 mL of ethyl acetate, shake it well, and ultrasonic it for 10 minutes. Collect the ethyl acetate layer. Add 30 mL of ethyl acetate to the sample, shake it well, and ultrasonic it for 10 minutes. Collect the ethyl acetate layer again. Combine the two ethyl acetate layers, evaporate them, dissolve the residue in 1 mL of methanol as the test solution b. Preparation of the peony root control medicinal material solution: Take 1 g of the peony root medicinal material powder, add 10 mL of chloroform, and ultrasonic it for 10 minutes. Filter it, evaporate the filtrate, and dissolve the residue in 1 mL of methanol as the peony root control medicinal material solution. Carry out thin layer chromatography analysis: thin layer chromatography conditions are that chromatographic plate is silica gel G thin layer plate; sample volume: test sample b: 3 μL, radix peucedani reference substance solution: 3 μL; developing agent is chloroform: ethyl acetate = 2:1 by volume; Color reagent: observe under 365nm ultraviolet light.

4. The quality detection method of the compound pear paste according to claim 1, characterized in that: The identification method of the hesperidin is carried out according to the following steps: Prepare test sample c: take 100.0g of compound pear syrup, add 100mL of water, mix well, adjust the pH to 12 with NaOH aqueous solution, extract with ethyl acetate for three times, 30mL each time, collect the ethyl acetate layer, evaporate to dryness, dissolve the residue with 1mL of methanol to obtain test sample c; Prepare hesperidin reference substance solution: take hesperidin reference substance, add methanol to prepare a saturated solution as hesperidin reference substance solution; Carry out thin layer chromatography analysis: thin layer chromatography conditions are that chromatographic plate is 1% sodium hydroxide solution prepared silica gel G thin layer plate; sample volume: test sample c: 3 μL, reference substance c: 3 μL; developing agent: ethyl acetate-methanol-water = 100:17:13 by volume; color reagent: aluminum chloride test solution, hot air dry, place under 365nm ultraviolet light to observe.

5. The quality detection method of the compound pear paste according to claim 1, characterized in that: In the content determination method of the ammonium glycyrrhizate, the chromatography conditions and system suitability test: octadecylsilane-bonded silica gel is used as the filler; acetonitrile: 0.05% phosphoric acid = 38:62 is used as the mobile phase; the detection wavelength is 252nm, the flow rate is 1mL / min, the column temperature is 25℃, the sample injection volume is 10μL, and the theoretical plate number calculated according to the glycyrrhizic acid peak should not be less than 3000.

6. The quality detection method of the compound pear paste according to claim 1, characterized in that: In the content determination method of the ammonium glycyrrhizate, the preparation of test sample solution d: take 9-11g of compound pear syrup, place it in a conical flask with a plug, add 70% ethanol solution 28-32mL, heat and reflux extract for 28-32 minutes, transfer to a 50mL volumetric flask while hot, take 70% ethanol solution, rinse the conical flask several times while hot, transfer the rinsing liquid to the 50mL volumetric flask, after the solution in the volumetric flask cools, add 70% ethanol solution to constant volume, shake well, filter, take the filtrate to obtain test sample solution d.

7. The quality detection method of the compound pear paste according to claim 6, characterized in that: In the content determination method of the ammonium glycyrrhizate, the preparation of test sample solution d: take 10g of compound pear syrup, place it in a conical flask with a plug, add 70% ethanol solution 30mL, heat and reflux extract for 30 minutes, transfer to a 50mL volumetric flask while hot, take 70% ethanol solution, rinse the conical flask several times while hot, transfer the rinsing liquid to the 50mL volumetric flask, after the solution in the volumetric flask cools, add 70% ethanol solution to constant volume, shake well, filter, take the filtrate to obtain test sample solution d.

8. The method for quality detection of the compound pear paste according to claim 1, characterized in that: In the content determination method of the ammonium glycyrrhizate, the preparation of ammonium glycyrrhizate reference substance solution: take ammonium glycyrrhizate reference substance, accurately weigh, add 70% ethanol to prepare a solution containing 12-17μg of glycyrrhizic acid per 1mL to obtain ammonium glycyrrhizate reference substance solution.

9. The quality detection method of the compound pear paste according to claim 8, characterized in that: In the content determination method of the ammonium glycyrrhizate, the preparation of ammonium glycyrrhizate reference substance solution: take ammonium glycyrrhizate reference substance, accurately weigh, add 70% ethanol to prepare a solution containing 15μg of glycyrrhizic acid per 1mL to obtain ammonium glycyrrhizate reference substance solution.

Images