A method for simultaneously identifying ephedra, licorice and bitter almond in Ma Xing Shi Gan oral liquid

Abstract

This invention belongs to the technical field of qualitative determination of ephedra, licorice, and bitter almond in traditional Chinese medicine compound preparations. This invention discloses a method for simultaneously identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid. The test sample is placed in a Soxhlet extractor and treated with reagents of different properties to prepare a fractional extraction solution. Ephedra and licorice reference materials are then processed to obtain a reference material solution. Amygdalin reference standard is prepared by adding methanol to prepare a reference solution. The above test sample solution, reference material solution, and reference solution are used in a thin-layer chromatography system to simultaneously identify ephedra, licorice, and bitter almond. The results show that the chromatographic spots are well separated and clear, with no interference from the negative control. This method has simple pretreatment, is environmentally friendly, has high sensitivity, and its repeatability and robustness meet the requirements of the Chinese Veterinary Pharmacopoeia, thus enabling better quality control of Ma Xing Shi Gan oral liquid.

Description

Technical Field

[0001] This invention relates to the technical field of qualitative determination of ephedra, licorice and bitter almond in traditional Chinese medicine compound preparations, and particularly to a method for simultaneously identifying ephedra, licorice and bitter almond in Ma Xing Shi Gan oral liquid. Background Technology

[0002] Ma Xing Shi Gan Oral Solution is a traditional Chinese veterinary medicine preparation, included in the 2020 edition (Part II) of the *Pharmacopoeia of Veterinary Medicine of the People's Republic of China*. The original standard required three thin-layer chromatography identification methods to distinguish ephedra (using ephedra as a reference material), licorice (using licorice as a reference material), and bitter almond (using amygdalin as a reference). This required three pretreatment processes to prepare the test solution. The pretreatment for licorice and bitter almond identification was particularly cumbersome, requiring three different developing systems to complete the entire test. In other words, the existing methods for identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan Oral Solution are cumbersome, with low sensitivity, poor accuracy, poor repeatability, and low specificity. Furthermore, the existing methods require a large amount of reagents, which is not only costly but also time-consuming, hindering the improvement of identification efficiency.

[0003] Therefore, there is an urgent need in this field to develop a rapid, sensitive, accurate, repeatable, and specific method for simultaneously identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid. Summary of the Invention

[0004] The purpose of this invention is to provide a method for simultaneously identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid, thereby solving the problems of existing methods for identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid, which are cumbersome, have low sensitivity, poor accuracy, poor repeatability, and low specificity. Furthermore, it addresses the issue that existing methods for identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid require a large amount of reagents, resulting in high costs and long identification times, which is detrimental to improving identification efficiency.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides a method for simultaneously identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid, comprising the following steps:

[0007] (1) Preparation of the test solution;

[0008] (2) Preparation of reference solutions: Take ephedra reference material, add methanol, extract by ultrasonication, take the supernatant, evaporate to dryness, dissolve the residue in methanol to obtain ephedra reference material solution; take licorice reference material, add water, heat under reflux, filter, concentrate the filtrate, extract with water-saturated n-butanol, take the n-butanol solution, wash with water-saturated n-butanol, evaporate the n-butanol solution to dryness, dissolve the residue in methanol to obtain licorice reference material solution; take amygdalin reference standard, dissolve in methanol to obtain amygdalin reference standard solution;

[0009] (3) Thin-layer chromatography test: Sample volume: 3-8 μL, silica gel G thin-layer plate, developing system: the upper layer of a mixed solution of ethyl acetate, methanol, water and formic acid that has been left at 10°C overnight;

[0010] Result Interpretation: Under ultraviolet light, if a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the test sample and the ephedra reference material, it indicates that ephedra can be detected; under ultraviolet light, if two black spots of the same color appear at the corresponding position in the chromatogram of the test sample and the licorice reference material, it indicates that licorice can be detected; after spraying α-naphthol solution and heating until the spots are clearly visible, and then examining under sunlight, if a pink spot of the same color appears at the corresponding position in the chromatogram of the test sample and the amygdalin reference standard, it indicates that amygdalin can be detected.

[0011] Preferably, the preparation of the test solution includes the following steps:

[0012] S1: Take the test sample, extract with chloroform, take the chloroform layer, evaporate to dryness, dissolve the residue in methanol, and set aside.

[0013] S2: Take the solution obtained from dissolving in S1 and place it in a Soxhlet extractor. Add ethanol, heat to reflux, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, concentrate, and use it as the test solution.

[0014] Preferably, the preparation process of the test solution includes:

[0015] S1: Take 3-10 mL of the test sample, extract with 20 mL of chloroform 3 times each time, combine the chloroform layers, evaporate to dryness, dissolve the residue in 2-5 mL of methanol, and set aside.

[0016] S2: Take the solution obtained from dissolving in S1 and place it in a Soxhlet extractor. Add 30-50 mL of 50% ethanol, heat under reflux for 30 min, cool at room temperature, filter, evaporate the filtrate to dryness, add 2-5 mL of methanol to dissolve the residue, concentrate to 1-2 mL, and use as the test solution.

[0017] Preferably, in the preparation of the ephedra reference material solution, 0.5-1g of ephedra reference material is taken, 10-20mL of methanol is added, and the mixture is extracted by ultrasonication for 10min. The supernatant is taken, evaporated to dryness, and the residue is dissolved in 2-4mL of methanol to obtain the ephedra reference material solution.

[0018] Preferably, in the preparation of the licorice reference material solution, 0.5-2g of licorice reference material is taken, 30-120mL of water is added, the mixture is heated under reflux for 30min, cooled at room temperature, filtered, and the filtrate is concentrated to 20-80mL. The mixture is extracted twice with 20-80mL of water-saturated n-butanol each time. The n-butanol solutions are combined and washed twice with 10-40mL of water-saturated n-butanol each time. The n-butanol solutions are combined, evaporated to dryness, and the residue is dissolved in 1-4mL of methanol to obtain the licorice reference material solution.

[0019] Preferably, in the preparation of the amygdalin reference solution, amygdalin reference standard is taken, dissolved in methanol, and prepared into a solution containing 1-3 mg of amygdalin reference standard per 1 mL, which is used as the amygdalin reference solution.

[0020] Preferably, the wavelength of the ultraviolet lamp is 365nm.

[0021] Preferably, in the mixed solution of ethyl acetate, methanol, water and formic acid, the volume ratio of ethyl acetate, methanol, water and formic acid is 18-22:4.5-5.8:5-6:0.2-0.4.

[0022] Preferably, the heating temperature for heating until the spots show clear color is 85–95°C.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The method of the present invention is superior to the disclosed technology. It can simultaneously identify ephedra, licorice and bitter almond through a thin layer development system. The method is simple and fast, with good separation effect, high identification accuracy, good reproducibility, easy observation, strong specificity and clear spots. It meets the requirements of the Chinese Veterinary Pharmacopoeia and saves time and reagents.

[0025] (2) The method of the present invention is simple to process, saves reagents and time; the original standard of Ma Xing Shi Gan oral liquid requires three sample processing steps to identify ephedra, licorice and bitter almond separately; the method of the present invention only requires one sample processing step, which can reduce the use of 20ml of ether and 45ml of n-butanol, and there is no need to use "D101 type macroporous adsorption resin column" for elution, which greatly saves reagents and operation time.

[0026] (3) The developing agent of the present invention uses only 3 chemical reagents: ethyl acetate, methanol and formic acid. The original method requires 6 chemical reagents: chloroform, n-butanol, ethyl acetate, methanol, glacial acetic acid and concentrated ammonia solution. Moreover, chloroform is a Class II precursor chemical. Therefore, the method described in the present invention is more environmentally friendly and safer. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 For Comparative Example 1, the method (ephedra identification) recorded in the 2020 edition (Part II) of the Veterinary Pharmacopoeia of the People's Republic of China was used.

[0029] Figure 2 For Comparative Example 1, the method (licorice identification) recorded in the 2020 edition (Part II) of the "Pharmacopoeia of the People's Republic of China" was used.

[0030] Figure 3 For Comparative Example 1, the method (identification of bitter almond) recorded in the 2020 edition (Part II) of the Pharmacopoeia of the People's Republic of China was used.

[0031] Figure 4 The image shows a thin-layer chromatogram of Ma Xing Shi Gan oral liquid prepared using the method described in this invention in Example 1, which was examined under ultraviolet light (365 nm); 1 is negative for ephedra deficiency; 2 is negative for bitter apricot kernel deficiency; 3 is negative for licorice deficiency; 4 is ephedra reference material; 5 is amygdalin reference standard; 6 is licorice reference material; 7-9 are self-made standards.

[0032] Figure 5 The image shows a thin-layer chromatogram of the Ma Xing Shi Gan oral liquid prepared using the method described in this invention in Example 1. The sample was sprayed with α-naphthol reagent, heated to 85°C until the spots were clearly visible, and then examined under sunlight. 1 represents a negative result for ephedra deficiency; 2 represents a negative result for bitter almond deficiency; 3 represents a negative result for licorice deficiency; 4 represents ephedra reference material; 5 represents amygdalin reference standard; 6 represents licorice reference material; and 7–9 represent self-prepared standards. Detailed Implementation

[0033] This invention provides a method for simultaneously identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid, comprising the following steps:

[0034] (1) Preparation of the test solution;

[0035] (2) Preparation of reference solutions: Take ephedra reference material, add methanol, extract by ultrasonication, take the supernatant, evaporate to dryness, dissolve the residue in methanol to obtain ephedra reference material solution; take licorice reference material, add water, heat under reflux, filter, concentrate the filtrate, extract with water-saturated n-butanol, take the n-butanol solution, wash with water-saturated n-butanol, evaporate the n-butanol solution to dryness, dissolve the residue in methanol to obtain licorice reference material solution; take amygdalin reference standard, dissolve in methanol to obtain amygdalin reference standard solution;

[0036] (3) Thin-layer chromatography test: Sample volume: 3-8 μL, silica gel G thin-layer plate, developing system: the upper layer of a mixed solution of ethyl acetate, methanol, water and formic acid that has been left at 10°C overnight;

[0037] Result Interpretation: Under ultraviolet light, if a fluorescent spot of the same color appears at the corresponding position in the chromatogram of the test sample and the ephedra reference material, it indicates that ephedra can be detected; under ultraviolet light, if two black spots of the same color appear at the corresponding position in the chromatogram of the test sample and the licorice reference material, it indicates that licorice can be detected; after spraying α-naphthol solution and heating until the spots are clearly visible, and then examining under sunlight, if a pink spot of the same color appears at the corresponding position in the chromatogram of the test sample and the amygdalin reference standard, it indicates that amygdalin can be detected.

[0038] This invention uses a Soxhlet extractor to extract samples in stages with reagents of different properties, and prepares the test solution in one step, thereby simultaneously identifying ephedra, licorice and bitter almond; in the Ephedra, Licorice and Glycyrrhiza Oral Solution, the three medicinal materials are identified by a single sample pretreatment and a thin-layer chromatography system.

[0039] The present invention performs result determination in two steps: first, the fluorescent spots of ephedra and licorice are examined under an ultraviolet lamp (365nm), then a color developer is sprayed on and heated until the spots are clearly visible, and the pink spots of amygdalin are examined under sunlight.

[0040] In this invention, the preparation of the test solution includes the following steps:

[0041] S1: Take the test sample, extract with chloroform, take the chloroform layer, evaporate to dryness, dissolve the residue in methanol, and set aside.

[0042] S2: Take the solution obtained from dissolving in S1 and place it in a Soxhlet extractor. Add ethanol, heat to reflux, filter, evaporate the filtrate to dryness, dissolve the residue in methanol, concentrate, and use it as the test solution.

[0043] In this invention, the preparation process of the test solution is as follows:

[0044] S1: Take 3-10 mL of the test sample, extract with 20 mL of chloroform 3 times each time, combine the chloroform layers, evaporate to dryness, dissolve the residue in 2-5 mL of methanol, and set aside.

[0045] S2: Take the solution obtained from dissolving in S1 and place it in a Soxhlet extractor. Add 30-50 mL of 50% ethanol, heat under reflux for 30 min, cool at room temperature, filter, evaporate the filtrate to dryness, add 2-5 mL of methanol to dissolve the residue, concentrate to 1-2 mL, and use as the test solution.

[0046] In this invention, the preparation of the ephedra reference herb solution involves taking 0.5-1g of ephedra reference herb, adding 10-20mL of methanol, ultrasonically extracting for 10min, taking the supernatant, evaporating to dryness, and dissolving the residue in 2-4mL of methanol to obtain the ephedra reference herb solution.

[0047] In this invention, the preparation of the licorice reference material solution involves taking 0.5–2 g of licorice reference material, adding 30–120 mL of water, heating under reflux for 30 min, cooling at room temperature, filtering, concentrating the filtrate to 20–80 mL, extracting twice with 20–80 mL of water-saturated n-butanol each time, combining the n-butanol solutions, washing twice with 10–40 mL of water-saturated n-butanol each time, combining the n-butanol solutions, evaporating to dryness, dissolving the residue in 1–4 mL of methanol to obtain the licorice reference material solution.

[0048] In this invention, the preparation of the amygdalin reference solution involves dissolving the amygdalin reference standard in methanol to prepare a solution containing 1-3 mg of amygdalin reference standard per 1 mL, which is then used as the amygdalin reference solution.

[0049] In this invention, the wavelength of the ultraviolet lamp is 365nm.

[0050] In this invention, the volume ratio of ethyl acetate, methanol, water and formic acid in the mixed solution is preferably 18-22:4.5-5.8:5-6:0.2-0.4, and more preferably 20-21:5.1-5.5:5.5-5.8:0.3.

[0051] In this invention, the heating temperature at which the spots show clear color is preferably 85-95°C, and more preferably 88-92°C.

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] The following examples use the following reagents and instruments:

[0054] Test drug and reagents: Licorice reference material, China National Institutes for Food and Drug Control, batch number 120904-202021;

[0055] Ephedra reference material, China National Institutes for Food and Drug Control, batch number 121051-201606;

[0056] Amygdalin reference standard, China National Institutes for Food and Drug Control, batch number 110820-202109.

[0057] All other reagents used were of analytical grade.

[0058] The instruments used included: BP211D analytical balance (Sartorius, Germany); Wright CAMAG automatic thin-layer sampler (model LINOMAT5); and CAMAG VISUALIZER fully automated thin-layer chromatography imaging system.

[0059] Example 1

[0060] This embodiment presents a method for simultaneously identifying ephedra, licorice, and bitter almond in Ma Xing Shi Gan oral liquid:

[0061] S1: Take 5 mL of the test sample and extract it three times with 20 mL of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 4 mL of methanol for later use.

[0062] S2: Take the aqueous solution from S1 and place it in a Soxhlet extractor. Add 30 mL of 50% ethanol, heat under reflux for 30 min, cool at room temperature, filter, evaporate the filtrate to dryness, add 4 mL of methanol to dissolve the residue, and concentrate to 2 mL as the test solution.

[0063] S3: Take 0.5g of ephedra reference material, add 10mL of methanol, sonicate for 10min, centrifuge, evaporate the supernatant to dryness, dissolve the residue in 2mL of methanol to prepare the ephedra reference material solution; Take 0.5g of licorice reference material, add 30mL of water, heat under reflux for 30min, remove, cool at room temperature, filter, concentrate the filtrate to 20mL, extract twice with 20mL of water-saturated n-butanol each time, combine the n-butanol solutions, wash twice with 10mL of water-saturated n-butanol each time, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in 1mL of methanol to prepare the licorice reference material solution; Take amygdalin reference standard, add methanol to prepare a solution containing 2mg of amygdalin reference standard per 1mL to prepare the amygdalin reference standard solution.

[0064] S4: Thin-layer chromatography:

[0065] Sample volume: 5 μL;

[0066] Silicone G thin film (10cm×20cm), (batch number: 20230329, purchased from Qingdao Haiyang Chemical Co., Ltd.);

[0067] Development system: The upper layer of a mixed solution of ethyl acetate, methanol, water and formic acid (volume ratio of ethyl acetate, methanol, water and formic acid is 20:5.1:5.8:0.4) left overnight at 10°C;

[0068] Result determination:

[0069] 4.1: Examine under a UV lamp (365nm). In the chromatogram of the test sample, a single fluorescent spot of the same color at the corresponding position as the chromatogram of the ephedra reference material indicates the detection of ephedra; two black spots of the same color at the corresponding position as the chromatogram of the licorice reference material indicate the detection of licorice. Negative samples lacking ephedra or licorice do not show spots of the same color at the corresponding positions as the chromatograms of the ephedra and licorice reference materials, indicating no interference from the negative samples. This demonstrates the specificity and feasibility of the method, and that the chromatographic spots are well separated, clear, and free from background interference. The results are as follows. Figure 4 As shown.

[0070] 4.2: Spray with α-naphthol reagent again, heat at 85℃ until the spots are clearly visible, and examine under sunlight. In the chromatogram of the test sample, a pink spot of the same color as the amygdalin reference standard appears at the corresponding position, indicating that bitter almonds can be detected. The chromatographic spots are well separated and clear, with no background interference. After comparing with a negative sample without bitter almonds, there was no chromatographic interference. The results are as follows... Figure 5 As shown.

[0071] Example 2

[0072] S1: Take 3 mL of the test sample and extract it 3 times with 20 mL of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 2 mL of methanol for later use.

[0073] S2: Take the aqueous solution from S1 and place it in a Soxhlet extractor. Add 40 mL of 50% ethanol, heat under reflux for 30 min, cool at room temperature, filter, evaporate the filtrate to dryness, add 2 mL of methanol to dissolve the residue, and concentrate to 1 mL as the test solution.

[0074] S3: Take 1g of ephedra reference material, add 20mL of methanol, sonicate for 10min, centrifuge, take the supernatant and evaporate to dryness, dissolve the residue in 4mL of methanol to prepare the ephedra reference material solution; Take 1g of licorice reference material, add 60mL of water, heat under reflux for 30min, remove, cool at room temperature, filter, concentrate the filtrate to 40mL, extract twice with 40mL of water-saturated n-butanol each time, combine the n-butanol solutions, wash twice with 20mL of water-saturated n-butanol each time, discard the aqueous solution, take the n-butanol solution and evaporate to dryness, dissolve the residue in 3mL of methanol to prepare the licorice reference material solution; Take amygdalin reference standard, add methanol to prepare a solution containing 1mg of amygdalin reference standard per 1mL to prepare the amygdalin reference standard solution.

[0075] S4: Thin-layer chromatography:

[0076] Sample volume: 7 μL;

[0077] Silicone G thin film (10cm×20cm), (batch number: 20230329, purchased from Qingdao Haiyang Chemical Co., Ltd.);

[0078] Development system: The upper layer of a mixed solution of ethyl acetate, methanol, water and formic acid (volume ratio of ethyl acetate, methanol, water and formic acid is 18:5.0:5.5:0.3) left overnight at 10°C;

[0079] Result determination:

[0080] 4.1 Examine under a UV lamp (365nm). In the chromatogram of the test sample, a single fluorescent spot of the same color appears at the corresponding position as in the chromatogram of the ephedra reference material, indicating the detection of ephedra; two black spots of the same color appear at the corresponding positions as in the chromatogram of the licorice reference material, indicating the detection of licorice. The chromatographic spots are well separated, clear, and without background interference.

[0081] 4.2 Spray with α-naphthol reagent again, heat at 85℃ until the spots are clearly visible, and examine under sunlight. In the chromatogram of the test sample, a pink spot of the same color as the amygdalin reference standard should appear at the corresponding position, indicating that bitter amygdalin can be detected. The chromatographic spots are well separated and clear, with no background interference.

[0082] Example 3

[0083] S1: Take 10 mL of the test sample and extract it three times with 20 mL of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 5 mL of methanol for later use.

[0084] S2: Take the aqueous solution from S1 and place it in a Soxhlet extractor. Add 50 mL of 50% ethanol and heat under reflux for 30 min. Cool at room temperature, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol, and concentrate to 2 mL as the test solution.

[0085] S3: Take 0.8g of ephedra reference material, add 20mL of methanol, sonicate for 10min, centrifuge, take the supernatant and evaporate to dryness, dissolve the residue in 3mL of methanol to prepare the ephedra reference material solution; Take 2g of licorice reference material, add 120mL of water, heat under reflux for 30min, remove, cool at room temperature, filter, concentrate the filtrate to 80mL, extract twice with 40mL of water-saturated n-butanol each time, combine the n-butanol solutions, wash twice with 40mL of water-saturated n-butanol each time, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in 4mL of methanol to prepare the licorice reference material solution; Take amygdalin reference standard, add methanol to prepare a solution containing 3mg of amygdalin reference standard per 1mL to prepare the amygdalin reference standard solution.

[0086] S4: Thin-layer chromatography:

[0087] Sample volume: 6 μL;

[0088] Silicone G thin film (10cm×20cm), (batch number: 20230329, purchased from Qingdao Haiyang Chemical Co., Ltd.);

[0089] Development system: The upper layer of a mixed solution of ethyl acetate, methanol, water and formic acid (volume ratio of ethyl acetate, methanol, water and formic acid is 22:5.8:6:0.4) left overnight at 10°C;

[0090] Result determination:

[0091] 4.1 Examine under a UV lamp (365nm). In the chromatogram of the test sample, a single fluorescent spot of the same color appears at the corresponding position as in the chromatogram of the ephedra reference material, indicating the detection of ephedra; two black spots of the same color appear at the corresponding positions as in the chromatogram of the licorice reference material, indicating the detection of licorice. The chromatographic spots are well separated, clear, and without background interference.

[0092] 4.2 Spray with α-naphthol reagent again, heat at 85℃ until the spots are clearly visible, and examine under sunlight. In the chromatogram of the test sample, a pink spot of the same color as the amygdalin reference standard should appear at the corresponding position, indicating that bitter amygdalin can be detected. The chromatographic spots are well separated and clear, with no background interference.

[0093] Comparative Example 1

[0094] The method adopted from the 2020 edition (Part II) of the "Veterinary Pharmacopoeia of the People's Republic of China" is as follows:

[0095] (1) Take 10 mL of the test sample, add 1 mL of concentrated ammonia solution, and extract three times with 20 mL of chloroform each time. Combine the chloroform extracts, evaporate to dryness, and dissolve the residue in 2 mL of methanol to obtain the test sample solution. Separately, take 0.5 g of ephedra reference material, add 10 mL of methanol, sonicate for 10 min, centrifuge, take the supernatant, evaporate to dryness, and dissolve the residue in 2 mL of methanol to obtain the reference material solution. Perform thin-layer chromatography (Appendix 0502), apply 5 μL of each of the above two solutions to the same silica gel G thin-layer plate, and develop using a mixture of chloroform, methanol, and concentrated ammonia (chloroform, methanol, and concentrated ammonia volume ratio of 20:5:0.5). Remove the plate, air dry, spray with ninhydrin solution, and heat at 105 °C until the spots are clearly visible. In the chromatogram of the test sample, a purple-red main spot appears at the same position as in the chromatogram of the reference material.

[0096] (2) Take 10 mL of the test sample, add 20 mL of diethyl ether, heat under reflux for 30 min, discard the ether solution, add 20 mL of methanol to the aqueous solution, heat under reflux for 1 h, filter, evaporate the filtrate to dryness, dissolve the residue in 30 mL of water, and extract three times with 15 mL of water-saturated n-butanol each time. Combine the n-butanol extracts, wash three times with water-saturated n-butanol each time, discard the aqueous solution, evaporate the n-butanol extract to dryness, dissolve the residue in 5 mL of methanol to obtain the test solution. Separately, take 1 g of licorice reference material and prepare a reference material solution using the same method. Perform the thin-layer chromatography test (Appendix 0502). Apply 5 μL of each of the two solutions to the same silica gel GF254 thin-layer plate. Develop the plate using the upper layer of a mixture of n-butanol, glacial acetic acid, and water (volume ratio of n-butanol, glacial acetic acid, and water 6:1:3) as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (254 nm). The chromatogram of the test sample should show spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material.

[0097] (3) Take 5 mL of the test sample and place it on a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 8 cm, pre-washed with water until clean and alcohol-free). Elute with 30 mL of ammonia solution (elution gradient 4→100), discard the ammonia solution, then elute with 20 mL of water, discard the aqueous solution, and then elute with 30 mL of 50% ethanol. Collect the eluent, evaporate to dryness, and dissolve the residue in 2 mL of methanol to obtain the test sample solution. Separately, take amygdalin reference standard and prepare a solution containing 2 mg of amygdalin reference standard per 1 mL with methanol to obtain the reference solution. Separately, take 0.5 g each of ephedra reference material and licorice reference material, add 10 mL of methanol to each, sonicate for 10 min, centrifuge, take the supernatant, evaporate to dryness, dissolve in 5 mL of water, and prepare ephedra reference material solution and licorice reference material solution respectively using the same method starting from "placed on D101 macroporous adsorption resin column". According to the thin-layer chromatography method (Appendix 0502), take 3 μL of the above two licorice reference material solutions and 5 μL of the remaining solutions, and spot them separately on the same silica gel G thin-layer plate. Use the upper layer of a mixture of ethyl acetate, methanol, water, and formic acid (volume ratio of ethyl acetate, methanol, water, and formic acid 20:5.1:5.8:0.4) that has been left overnight at 10°C as the developing solvent. Develop, remove, air dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position as in the chromatogram of the ephedra reference material; and two black spots of the same color appear at the corresponding position as in the chromatogram of the licorice reference material. Spray with α-naphthol reagent and heat at 85°C until the spots are clearly visible. Examine under sunlight. In the chromatogram of the test sample, a pink spot of the same color appears at the corresponding position as in the chromatogram of the amygdalin reference standard.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneously identifying Ephedra, Glycyrrhiza and Bitter Almond in Maxingshigan oral liquid, characterized in that, It comprises the following steps: (1) Preparation of test sample solution; In the process of preparation of test sample solution: S1: Take 3-10 mL of test sample, extract with chloroform for 3 times, 20 mL of chloroform is added each time, combine the chloroform layers, evaporate to dryness, add 2-5 mL of methanol to dissolve, and reserve for use; S2: Take the solution obtained by dissolving in S1, place it in a Soxhlet extractor, add 30-50 mL of 50% ethanol, heat for 30 min, cool at room temperature, filter, evaporate the filtrate to dryness, add 2-5 mL of methanol to dissolve, concentrate to 1-2 mL, and use as test sample solution; (2) Preparation of control solution: In the preparation of ephedra control drug solution, take 0.5-1 g of ephedra control drug, add 10-20 mL of methanol, ultrasonic extraction for 10 min, take the supernatant, evaporate to dryness, add 2-4 mL of methanol to dissolve, and use as ephedra control drug solution; In the preparation of licorice control drug solution, take 0.5-2 g of licorice control drug, add 30-120 mL of water, heat for 30 min, cool at room temperature, filter, concentrate the filtrate to 20-80 mL, extract with water-saturated n-butanol for 2 times, 20-80 mL of water-saturated n-butanol is used each time, wash the combined n-butanol liquid with water-saturated n-butanol for 2 times, 10-40 mL of water-saturated n-butanol is used each time, take the n-butanol liquid, evaporate to dryness, add 1-4 mL of methanol to dissolve, and use as licorice control drug solution; In the preparation of amygdalin control solution, take amygdalin control, add methanol to dissolve, prepare a solution containing 1-3 mg of amygdalin control per 1 mL, and use as amygdalin control solution; (3) Thin layer chromatography test: sample size: 3-8 μL, silica gel G thin layer plate, developing system: the upper solution of the mixture of ethyl acetate, methanol, water and formic acid after standing overnight at 10℃; Determination of results: observe under ultraviolet lamp, the wavelength of ultraviolet lamp is 365 nm, in the test sample chromatogram, at the position corresponding to the ephedra control drug chromatogram, one fluorescent spot of the same color is shown, indicating that ephedra can be detected; observe under ultraviolet lamp, in the test sample chromatogram, at the position corresponding to the licorice control drug chromatogram, two black spots of the same color are shown, indicating that licorice can be detected; spray α-naphthol test solution, heat to 85-95℃ until the spots develop clearly, observe under sunlight, in the test sample chromatogram, at the position corresponding to the amygdalin control chromatogram, one pink spot of the same color is shown, indicating that amygdalin can be detected; In the mixture of ethyl acetate, methanol, water and formic acid, the volume ratio of ethyl acetate, methanol, water and formic acid is 18-22:4.5-5.8:5-6:0.2-0.

4.

2. The method for simultaneously identifying Ephedra, Glycyrrhiza and Bitter Almond in Maxingshigan Oral Liquid according to claim 1, characterized in that, The heating temperature for heating to 85-95℃ until the spots develop clearly is 85-95℃.

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