CN115902087A - Method for simultaneously detecting six components of qingkailing preparation - Google Patents

Abstract

The invention discloses a method for simultaneously detecting six components of a qingkailing preparation, which comprises the following steps: preparing a test solution and a reference solution; adding the test solution and the reference solution to the thin-layer plate respectively; the volume ratio of the raw materials is (22-28): (12 to 18): (1-5): (2-7) taking ethyl acetate, acetone, formic acid and water as developing agents to fully react with the thin-layer plate obtained in the last step; the reacted thin layer plate is developed under 250-260 nm ultraviolet light. The method can simultaneously and qualitatively detect the active ingredients of six traditional Chinese medicines, namely geniposide, chlorogenic acid, baicalin, luteoloside, isochlorogenic acid A and isochlorogenic acid C, has the advantages of simple and quick operation, clear color development result, easy identification, good specificity and reproducibility, realization of the quality control of the compound traditional Chinese medicine preparation, standardized medicine production, improved medicine safety, stable production process and controllability, and wide application prospect in component identification and quality control of the compound traditional Chinese medicine preparation.

Description

Method for simultaneously detecting six components of qingkailing preparation

Technical Field

The invention relates to the technical field of identification of traditional Chinese medicine compound preparations, in particular to a method for simultaneously detecting six components of a qingkailing preparation.

Background

The traditional Chinese medicine fructus Gardeniae is dry mature fruit of Gardenia jasminoides Ellis (Gardenia jasminoides Ellis) belonging to Rubiaceae, has cold and bitter taste, enters heart and lung triple energizer meridian, and has effects of clearing pathogenic fire, relieving restlessness, clearing heat, promoting diuresis, cooling blood and removing toxic substance. It is indicated for feverish dysphoria, jaundice with reddish urine, stranguria with blood, pain with astringents, conjunctival congestion with swelling and pain, and sores and ulcers due to fire toxin by oral administration, and for sprain, contusion and pain by external application. The Chinese medicine cape jasmine contains over 40 kinds of bioactive matter, mainly iridoid glycoside and volatile oil, and has effective component of iridoid glycoside with highest content.

The flos Lonicerae is dried bud or flower with initial bloom of Lonicera japonica Thunb of Caprifoliaceae, and has antibacterial, antiviral, antitumor, immunity enhancing, fertility resisting, antioxidant, liver protecting, and gallbladder promoting effects. Caffeoylquinic acid components (caffeoylquinic acids) are important active ingredients in honeysuckle, and mainly exist in the form of chlorogenic acid (5-O-caffeoylquinic acid), isochlorogenic acid A and isochlorogenic acid C in honeysuckle.

Baicalin (baicailin) is a flavonoid compound extracted from dried root of Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi) belonging to the family of dicotyledonous Labiatae, and is in form of yellowish powder at room temperature and bitter in taste. Has remarkable biological activity, has the functions of bacteriostasis, diuresis, anti-inflammation, cholesterol reduction, thrombosis resistance, asthma relief, fire purging, detoxification, hemostasis, miscarriage prevention, allergy resistance and spasmolysis, is a specific inhibitor of liver sialidase of mammals, has the function of regulating certain diseases, and also has stronger physiological efficiency of anticancer reaction.

In the existing traditional Chinese medicine composition with the compatibility of baicalin, gardenia and honeysuckle, single components or medicinal materials are often identified. At present, because the components of a traditional Chinese medicine compound preparation are complex, if a traditional thin-layer identification method is adopted, namely if a thin-layer identification method is established for each medicinal material or each effective component, the operation process is complicated and complicated, the time consumption is long, the efficiency is low, and the retrieval result accuracy is poor.

Although the theoretical basis of simultaneously measuring multiple components in the traditional Chinese medicine by adopting thin-layer chromatography and the detection of the effective components in the traditional Chinese medicine are disclosed in the prior art, no method capable of simultaneously and rapidly identifying multiple effective components of baicalin, gardenia and honeysuckle exists for different detection objects and traditional Chinese medicine compositions with more complex components.

Disclosure of Invention

In order to solve the problem that a method for rapidly and simultaneously identifying multiple effective components of a traditional Chinese medicine is lacked in the prior art, the invention provides a method for simultaneously detecting multiple components of a qingkailing preparation.

The first purpose of the invention is to provide a developing agent for thin layer chromatography.

The second purpose of the invention is to provide the application of the developing agent in detecting and/or analyzing the components of the qingkailing preparation.

The third purpose of the invention is to provide a method for simultaneously detecting six components of a qingkailing preparation.

A fourth object of the present invention is to provide the use of the above method in the analysis of the components and/or quality control of qingkailing preparation.

In order to achieve the purpose, the invention is realized by the following scheme:

thin layer chromatography, also known as thin layer chromatography, is one type of chromatography. The basic principle of thin layer chromatography is: the components of the mixture are repeatedly adsorbed and distributed by utilizing the difference of adsorption performance and solubility performance of the components of the mixture in a certain substance or the difference of other affinity action performances, so that the components are distinguished. The key point of successfully separating the multi-component sample by the thin layer chromatography lies in the selection of the developing solvent, and the developing solvent is correspondingly adjusted according to the polarity of the component to be detected.

The invention aims to simultaneously detect 6 chemical components of 3 traditional Chinese medicinal materials of baicalin, gardenia and honeysuckle in a qingkailing preparation by adopting a thin-layer chromatography, wherein the geniposide belongs to iridoid glycosides, the baicalin and the luteolin belong to flavonoids, and chlorogenic acid, isochlorogenic acid A and isochlorogenic acid C belong to phenylpropanoids and are compounds with higher polarity. For the detection purpose of the present invention, it is necessary to separate 6 chemical components in the same developing solvent system, and therefore, it is important for the formulation of the developing solvent.

The invention provides a developing agent for thin-layer chromatography, which comprises the following components in percentage by volume (22-28): (12 to 18): (1-5): (2-7).

Preferably, the volume ratio of the ethyl acetate to the acetone to the formic acid to the water is (25-28): (12-15): (1.5-3): (4.5-6).

More preferably, the volume ratio of ethyl acetate, acetone, formic acid and water is 25:15:3:4.5 or 28:12:1.5:6.

further preferably, the volume ratio of ethyl acetate, acetone, formic acid and water is 25:15:3:4.5.

ethyl acetate is a weak polar solvent, acetone, formic acid and water are strong polar solvents, 4 different solvents form the developing solvent to play a synergistic effect, and the change of the proportion of a certain solvent can influence the separation effect of the whole developing solvent on the components to be detected.

The use of the above developing agent in the detection and/or analysis of the components of a qingkailing preparation is also intended to be within the scope of the present invention.

A method for simultaneously detecting six components of a qingkailing preparation comprises the following steps:

s1, removing a solvent from an ethyl acetate extracting solution of a sample to be tested to obtain a precipitate, and dissolving the precipitate with acetonitrile to obtain a test solution; methanol solution of geniposide, chlorogenic acid, baicalin, luteoloside, isochlorogenic acid A and isochlorogenic acid C as control solution;

s2, adding a test solution and a reference solution to the thin-layer plate;

s3, fully reacting the developing solvent with the thin-layer plate obtained in the step S2;

and S4, developing the thin-layer plate obtained in the step S3 under 250-260 nm ultraviolet light.

In the step S1, ethyl acetate is used as a solvent, and according to a similar compatibility principle, effective components in a Chinese medicinal compound preparation with complex components are greatly extracted, so that a good separation effect is obtained, and the accuracy and precision of later-stage detection and judgment are facilitated. Acetonitrile is added into filter residues, the polarity of the acetonitrile is greater than that of ethyl acetate, the acetonitrile can effectively ensure that the extracted components of the ethyl acetate are dissolved to a greater extent, and the problem that part of the components are insoluble in methanol is solved.

Preferably, the qingkailing preparation is one of oral liquid, granules, injection, dry powder, capsules, tablets or paste.

More preferably, the qingkailing preparation is oral liquid or granules.

Preferably, in step S1, if the sample to be tested is qingkailing oral liquid, the preparation method of the ethyl acetate extract of the sample to be tested is as follows: the qingkailing oral liquid, the dilute hydrochloric acid and the ethyl acetate are mixed according to the volume ratio (10-20): (0.5-1): (20-60) mixing and extracting.

More preferably, in step S1, the qingkailing oral liquid contains baicalin more than or equal to 3.5mg/mL and geniposide more than or equal to 0.2mg/mL.

More preferably, in step S1, the volume ratio of the qingkailing oral liquid to the diluted hydrochloric acid to the ethyl acetate is 40:1:40.

more preferably, in step S1, the dilute hydrochloric acid has a mass concentration of 9.5% to 10.5% by weight in g/mL.

Preferably, in step S1, if the sample to be detected is qingkailing granule, the preparation method of the ethyl acetate extract of the sample is as follows: mixing qingkailing granules, water and ethyl acetate according to the mass volume ratio (7-11) g: (35-45) mL: (35-45) mL, and mixing and extracting.

More preferably, in step S1, each 3g of qingkailing granule contains 18-22 mg/mL of baicalin and more than or equal to 1mg of geniposide.

More preferably, in step S1, the mass-to-volume ratio of the qingkailing granules to the water to the ethyl acetate is 9g:40mL of: 40mL.

Preferably, in step S1, the solvent is removed by heating evaporation.

Preferably, in step S1, the extraction method of the ethyl acetate extract of the sample to be detected is shaking extraction with a separating funnel.

More preferably, in step S1, the shaking extraction of the separatory funnel is repeated 3 times.

Preferably, in step S1, the methanol solution has a volume concentration of 65 to 75%.

More preferably, in step S1, the methanol solution has a concentration of 70% by volume.

Preferably, in step S1, the working concentration of the reference substance is 0.5-2.5 mg/mL.

More preferably, in step S1, the working concentration of the reference substance is 1-2 mg/mL.

Further preferably, in step S1, the working concentration of the geniposide control solution is 1mg/mL, the working concentration of the chlorogenic acid control solution is 1mg/mL, the working concentration of the baicalin control solution is 1mg/mL, the working concentration of the luteolin control solution is 2mg/mL, the working concentration of the isochlorogenic acid a control solution is 2mg/mL, and the working concentration of the isochlorogenic acid C control solution is 2mg/mL.

Preferably, in step S2, a control solution is also added to the thin-layer plate; the reference medicinal material solution comprises flos Lonicerae reference medicinal material solution and fructus Gardeniae reference medicinal material solution.

The reference medicinal material solution is used for determining the sources of different spots in different test samples, and if the spots of the test samples have corresponding spots at the same positions in the honeysuckle reference medicinal material solution, the test samples contain the components of the honeysuckle medicinal material; if the spots of the test sample have corresponding spots at the same positions in the fructus Gardeniae reference medicinal material solution, it is indicated that the test sample contains fructus Gardeniae components.

More preferably, in step S2, the preparation method of the honeysuckle control drug solution includes the following steps: ultrasonic extracting with methanol and flos Lonicerae as reference materials, filtering, and concentrating; mixing the concentrated solution with dilute hydrochloric acid and ethyl acetate, and shaking and extracting by using a separating funnel; evaporating; dissolving the evaporated precipitate with methanol to obtain flos Lonicerae control solution.

More preferably, in step S2, the preparation method of the honeysuckle control drug solution includes the following steps: the honeysuckle flower reference medicinal materials in mass-volume ratio: methanol 1=0.512g:30mL, fully mixing the honeysuckle reference medicinal material and methanol 1, performing ultrasonic extraction for 30min by using an ultrasonic cleaner with the ultrasonic frequency of 250W and 40KHz, filtering, collecting filtrate, heating the filtrate in water bath, and concentrating to obtain a concentrated solution, wherein the volume of the concentrated solution is one third of that of the methanol 1; the honeysuckle flower reference medicinal materials in mass-volume ratio: dilute hydrochloric acid: ethyl acetate =0.512g:0.5mL:20mL, fully mixing dilute hydrochloric acid, ethyl acetate and the concentrated solution, and extracting for 2 times by using ethyl acetate to obtain honeysuckle reference medicinal material extracting solution; heating the honeysuckle reference medicinal material extracting solution until the solvent is completely evaporated, and collecting the solid remained after evaporation; dissolving the solid with methanol 2 of the same volume as the concentrated solution to obtain flos Lonicerae control solution.

More preferably, in step S2, the preparation method of the gardenia control drug solution comprises the following steps: extracting with methanol and fructus Gardeniae control material by ultrasonic extraction, and filtering to obtain fructus Gardeniae control solution.

More preferably, in step S2, the preparation method of the gardenia control drug solution comprises the following steps: according to the mass-volume ratio, the gardenia contrast medicinal materials: volume concentration 50% methanol =1g:10mL, mixing the gardenia contrast medicinal material with 50% methanol by volume, extracting for 40min by using an ultrasonic cleaner with 250W and 40KHz ultrasonic waves, and filtering to obtain a gardenia contrast medicinal material solution.

Preferably, in step S2, a negative control solution is also added to the thin layer plate; the negative control solution comprises a honeysuckle medicinal material negative control solution and a gardenia medicinal material negative control solution.

The negative control solution is used for indicating whether the detection system of the thin layer chromatography has interference. The honeysuckle medicine negative control solution is a qingkailing preparation lacking honeysuckle medicine, the honeysuckle medicine negative control solution should lack spots of honeysuckle medicine sources, but if corresponding spots exist in the detection result, the spots at the position are not single components, interference exists in the detection system of thin-layer chromatography, and the detection result is not reliable. The negative control solution of fructus Gardeniae is QINGKAILING preparation lacking fructus Gardeniae, and has the same effect.

More preferably, in step S2, the preparation method of the negative control solution for honeysuckle flower comprises the following steps:

s21, radix isatidis in mass-volume ratio: gardenia: water 1=20g:2.5g:50mL, mixing isatis root, gardenia and water, decocting twice for 1 hour each time, combining decoctions, filtering, and collecting filtrate 1; heating the filtrate 1 at 50 ℃, concentrating until the relative density is 1.15-1.20, and cooling to obtain a concentrated solution 1; the isatis root is mixed according to the mass-volume ratio: ethanol 1=20g:50mL, fully mixing the concentrated solution 1 with ethanol 1, standing, filtering, and recovering the ethanol 1 to obtain a filtrate 2; the isatis root is mixed according to the mass-volume ratio: water 2=20g:50mL, mixing the filtrate 2 with water 2, and standing to obtain an isatis root-gardenia extracting solution;

s22, according to the mass-volume ratio of the buffalo horn powder: mother-of-pearl: 98% sulfuric acid mass =2.5g:5g:10g, mixing, and fully hydrolyzing to obtain hydrolysate 1; the isatis root is prepared from the following raw materials in percentage by mass: water 3=2.5g:50mL, fully mixing the hydrolysate 1 with water 3, and filtering to obtain a filtrate 3; heating the filtrate 3 at 50 ℃, concentrating until the relative density is 1.05-1.10, and cooling to obtain a concentrated solution 2; the buffalo horn powder comprises the following components in percentage by mass and volume: ethanol 2=2.5g:100mL, fully mixing the concentrated solution 2 with ethanol 2, standing, filtering, and recovering ethanol 2 to obtain filtrate 4; the buffalo horn powder comprises the following components in percentage by mass and volume: water 4=2.5g:100mL, fully mixing the filtrate 4 with water 4, and standing to obtain cornu bubali-mother-of-pearl hydrolysate;

s23, cholic acid according to mass-volume ratio: hyodeoxycholic acid: ethanol 3=3.25g:3.75g:50mL, mixing and fully dissolving to obtain cholic acid-hyodeoxycholic acid ethanol solution;

s24, combining the radix isatidis-cape jasmine fruit extract and the buffalo horn-mother-of-pearl hydrolysate, uniformly mixing, adding into cholic acid-hyodesoxycholic acid ethanol solution, and mixing the cholic acid: ethanol 4=3.25g: adding 50mL of ethanol 4 into the cholic acid-hyodesoxycholic acid ethanol solution, fully mixing, standing, filtering, and recovering the ethanol 4 to obtain a filtrate 5; according to the mass-to-volume ratio of cholic acid: water 5=3.25g:100mL, fully mixing the filtrate 5 with water 5, and standing; adding baicalin with the mass 2 times of that of the buffalo horn powder, and adjusting the pH value to 8.2-9.4 to dissolve the baicalin; the isatis root is mixed according to the mass-volume ratio: honeysuckle medicinal material negative medicinal material solution =20g:1000mL, adding water 6 to complement the residual volume; adjusting the pH value to 7.2-7.5 by using sodium hydroxide, uniformly stirring, standing and filtering to obtain filtrate, namely the honeysuckle medicinal material negative medicinal material solution.

More preferably, in step S2, the preparation method of the negative control solution for gardenia is similar to that of the negative control solution for honeysuckle, and the difference is only that: in the step S21, honeysuckle is used for replacing gardenia, and the mass volume ratio of the isatis root is as follows: honeysuckle flower: water 7=20g:6g:50mL, mixing isatis root, honeysuckle and water.

Preferably, in step S2, the volume ratio of the test solution to the control solution is: geniposide control solution: chlorogenic acid control solution: baicalin control solution: luteolin control solution: isochlorogenic acid a control solution: isochlorogenic acid C control solution = 0.8-1.2: 0.8 to 1.2: 1.8-2.2: 1.8-2.2: 0.8 to 1.2:0.8 to 1.2:0.8 to 1.2:0.8 to 1.2.

More preferably, in step S2, the volume ratio of the test solution to the reference solution is: jasminoidin control solution: chlorogenic acid control solution: baicalin control solution: luteolin control solution: isochlorogenic acid a control solution: isochlorogenic acid C control solution =1:1:2:2:1:1:1.

more preferably, in step S2, the volume ratio of the test solution to the honeysuckle control solution to the gardenia control solution is: honeysuckle flower reference medicinal material solution: gardenia contrast medicinal material solution = 0.8-1.2: 0.8 to 1.2:0.8 to 1.2.

Further preferably, in step S2, the volume ratio of the test solution to the honeysuckle control solution to the gardenia control solution is: honeysuckle flower reference medicinal material solution: gardenia control solution =1:1:1.

more preferably, in step S2, the volume ratio of the test solution to the negative control solution of honeysuckle flower and the negative control solution of gardenia jasminoides is: negative control solution of honeysuckle flower: negative control solution = 0.8-1.2 of gardenia medicinal material: 0.8 to 1.2:0.8 to 1.2.

Further preferably, in the step S2, the volume ratio of the test solution to the negative control solution of honeysuckle flower and the negative control solution of gardenia jasminoides is: negative control solution of honeysuckle medicinal material: negative control solution of gardenia drug =1:1:1.

preferably, in step S2, the sample solution, the reference solution and the negative reference solution are dotted on the same horizontal line 150-200 mm away from the bottom edge of the thin-layer plate to form a plurality of dot-shaped spots with diameters of 1-5 mm, the distance between the spots at two ends and the edge of the side edge of the thin-layer plate is 5-10 mm, and every two spots are spaced at equal intervals.

Preferably, in the steps S2 to S4, the thin layer plate is a silica gel GF254 thin layer plate.

Preferably, in step S4, the thin layer plate obtained in step S3 is developed under 245nm ultraviolet light.

Selecting a silica gel GF254 thin layer plate, adding a specific fluorescent agent into the silica gel GF254 thin layer plate, wherein the fluorescent agent emits green fluorescence under the wavelength of 254nm, and most compounds with aromatic rings or conjugated systems can absorb ultraviolet under the wavelength of 254nm, so that the green fluorescence is covered to form dark spots; baicalin, chlorogenic acid, geniposide, luteolin, isochlorogenic acid A and isochlorogenic acid C can form obvious dark spots at the wavelength of 254nm, and if other thin-layer plates such as a silica gel G plate and the like are replaced, the effect is not good, so that the identification cannot be better realized.

The application of the above method in the component analysis and/or quality control of qingkailing preparation also falls within the scope of the present invention.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, 6 traditional Chinese medicine components including jasminoidin, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A and isochlorogenic acid C in 3 traditional Chinese medicine flavors of gardenia, scutellaria and honeysuckle can be detected through a silica gel GF254 thin-layer plate under an inspection condition without using a color developing agent, so that the solvent and labor cost are effectively saved, the detection time is shortened by about 70%, the detection method can be used for detecting other components containing the six components, meanwhile, the interference of other complex components in the traditional Chinese medicine is overcome, and the detection accuracy is ensured.

The invention successfully realizes the simultaneous qualitative detection of six traditional Chinese medicine effective components of geniposide, chlorogenic acid, baicalin, luteoloside, isochlorogenic acid A and isochlorogenic acid C in the qingkailing preparation by preparing a test solution, preparing a reference solution, detecting by thin-layer chromatography and each specific control condition. The detection method provided by the invention has the advantages of simple and rapid operation, clear color development result, good separation degree, effective elimination of negative interference, clear inspection, easy interpretation, good specificity and reproducibility, effective realization of quality control of the compound traditional Chinese medicine preparation, realization of drug production standardization, improvement of drug safety, production process stability and controllability, and wide application prospect in component identification and quality control of the compound traditional Chinese medicine preparation.

Drawings

FIG. 1 shows the results of the test in example 1;1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is flos Lonicerae reference material; 8 is fructus Gardeniae reference material; 9-11 are qingkailing oral liquid sample points, corresponding to batch numbers: 1220701, 1220702 and 1220703;12 is a negative control of gardenia; 13 is negative control of flos Lonicerae.

FIG. 2 shows the results of detection in example 2;1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is flos Lonicerae reference material; 8 is fructus Gardeniae reference material; 9-11 are test points of qingkailing granules, corresponding to the batch numbers 5220801, 5220802 and 5220803;12 is a negative control of gardenia; 13 is negative control of flos Lonicerae.

FIG. 3 shows the results of the test in example 3;1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle flower reference medicinal material; 8 is fructus Gardeniae reference material; 9-11 are test points of qingkailing granules, corresponding to the batch numbers 5220801, 5220802 and 5220803;12 is a negative control of gardenia; 13 is negative control of flos Lonicerae.

FIG. 4 is the result of the test of comparative example 1;1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle flower reference medicinal material; 8 is fructus Gardeniae reference material; 9-11 are qingkailing oral liquid sample points corresponding to the batch numbers 1220701, 1220702 and 1220703;12 is a negative control of gardenia; 13 is negative control of flos Lonicerae.

FIG. 5 shows the results of the test of comparative example 2; a is a 254nm ultraviolet light color development result; b is a daylight color development result; 1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is flos Lonicerae reference material; 8 is fructus Gardeniae reference material; 9 is qingkailing oral liquid sample number 1220701;10 is the negative control of gardenia; 11 is negative control of honeysuckle.

FIG. 6 is the result of the test of comparative example 3; a is a 254nm ultraviolet light color development result; b is a daylight color development result; 1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle flower reference medicinal material; 8 is fructus Gardeniae reference material; 9 is qingkailing oral liquid sample number 1220701;10 is the negative control of gardenia; 11 is negative control of honeysuckle.

FIG. 7 shows the results of the test of comparative example 4; 1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle flower reference medicinal material; 8 is fructus Gardeniae reference material; 9 is QINGKAILING oral liquid sample number 1220701.

FIG. 8 is a graph showing the results of the measurement of comparative example 5; 1 is baicalin reference substance; 2 is chlorogenic acid reference substance; 3 is geniposide reference substance; 4 is luteolin control; 5 is isochlorogenic acid A reference substance; 6 is isochlorogenic acid C reference substance; 7 is honeysuckle flower reference medicinal material; 8 is fructus Gardeniae reference material; 9 is QINGKAILING oral liquid sample number 1220701.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

In the following examples, the instruments and consumables involved include: ultrasonic cleaner (KQ-250 DE model, 250W,40KHz; ultrasonic instruments Co., ltd., kunshan city), silica gel GF254 thin-layer plate (Qingdao oceanic chemical plant division, tianjin Si Lida science & technology Co., ltd., qingdao Yu Min chemical plant).

In the following examples, all organic solvents such as methanol, ethyl acetate, chloroform, acetone and formic acid were analytically pure.

In the following examples, reference controls include: baicalin (Chinese food and drug inspection research institute, batch number: 110715-202122); chlorogenic acid (Chinese institute for testing food and drug, batch No. 110753-202119); geniposide (Chinese institute for testing food and drug, batch number: 110749-201919); luteolin (Chinese institute for food and drug assay, batch No. 111720-202111); isochlorogenic acid A (ChemFaces, lot: CFN 99118); isochlorogenic acid C (NATURE STANDARD, batch No.: RS 06601020).

In the following examples, reference drug materials include: honeysuckle control drug (China institute for testing food and drug substance, batch number: 121060-201608); gardenia jasminoides control (Chinese food and drug testing research institute, batch No. 120986-201610).

In the following examples, the diluted hydrochloric acid used was prepared according to pharmacopoeia 2020 edition, and its mass concentration was 9.5% to 10.5% (g/mL).

Example 1A method for simultaneously detecting six ingredients of qingkailing oral liquid

1. Preparation of test solution

(1) Taking 20mL of a sample to be detected (the sample to be detected contains baicalin which is more than or equal to 3.5mg/mL and jasminoidin which is more than or equal to 0.2 mg/mL), adding 0.5mL of dilute hydrochloric acid and 20mL of ethyl acetate into the sample to be detected, shaking and extracting the mixture by a separating funnel, and collecting an organic phase extracting solution; adding 20mL of ethyl acetate again, repeatedly extracting for 2 times, and collecting an organic phase to obtain a sample extracting solution.

(2) And putting the sample extracting solution into a container, heating until the solvent of the sample extracting solution is completely evaporated, and collecting the solid remained after evaporation, wherein the solid is marked as solid 1.

(3) And dissolving the solid 1 in 2mL of acetonitrile to obtain a test solution.

2. Preparation of control solutions

Dissolving 10.82mg of geniposide, 10.61mg of chlorogenic acid, 10.17mg of baicalin, 20.81mg of luteolin, 20.12mg of isochlorogenic acid A and 20.34mg of isochlorogenic acid C in 70% methanol solution by volume concentration to prepare geniposide reference solution with the concentration of 1mg/mL, chlorogenic acid reference solution with the concentration of 1mg/mL, baicalin reference solution with the concentration of 1mg/mL, luteolin reference solution with the concentration of 2mg/mL, isochlorogenic acid A reference solution with the concentration of 2mg/mL and isochlorogenic acid C reference solution with the concentration of 2mg/mL.

3. Preparation of reference drug solution

(1) Taking 0.512g of flos Lonicerae as reference material, adding 30mL of methanol, ultrasonically extracting with ultrasonic cleaner 250W and 40KHz for 30min, filtering, and collecting filtrate as filtrate 1.

Heating the filtrate 1 in a water bath until the filtrate 1 is concentrated to 10mL, adding 0.5mL of dilute hydrochloric acid and 20mL of ethyl acetate, shaking and extracting by using a separating funnel, and collecting an organic phase extracting solution; adding ethyl acetate 20mL again, extracting for 2 times, and collecting organic phase to obtain flos Lonicerae control medicinal material extractive solution.

Heating the honeysuckle reference medicinal material extracting solution in a container until the solvent is completely evaporated, collecting the solid remained after evaporation, and marking as solid 2; dissolving the solid substance 2 with 2mL of methanol to obtain the flos Lonicerae control solution.

(2) Taking 1g of the gardenia control medicinal material, adding 10mL of 50% methanol by volume concentration, performing ultrasonic extraction for 40min by using an ultrasonic cleaner with 250W and 40KHz, and filtering to obtain filtrate 2, wherein the filtrate 2 is the gardenia control medicinal material solution.

4. Preparation of negative control solution

(1) Negative control solution of flos Lonicerae (i.e. qingkailing preparation without flos Lonicerae)

Weighing 20g of isatis root and 2.5g of gardenia, adding 50mL of water, decocting twice for 1 hour each time, combining decoctions, filtering, and collecting filtrate; heating the filtrate at 50 ℃, concentrating the filtrate until the relative density is 1.15-1.20, and cooling the filtrate; adding 50mL of ethanol, standing, filtering, recovering ethanol, adding 50mL of water, and standing to obtain the radix Isatidis-fructus Gardeniae extract.

Weighing 2.5g of buffalo horn powder and 5g of nacre, adding 10mL of sulfuric acid with the mass concentration of 98%, fully hydrolyzing, adding 50mL of water, and filtering; adjusting the pH value of the filtrate to 4 by using a calcium hydroxide solution with the mass concentration of 15%, filtering, and collecting the filtrate; heating the filtrate at 50 ℃, concentrating the filtrate until the relative density is 1.05-1.10, and cooling the filtrate; adding 100mL of ethanol, standing, filtering, recovering ethanol, adding 100mL of water, and standing to obtain cornu Bubali-Concha Margaritifera hydrolysate.

3.25g of cholic acid and 3.75g of hyodeoxycholic acid are weighed, 50mL of ethanol is added, and the cholic acid-hyodeoxycholic acid ethanol solution is obtained after full dissolution.

Mixing the radix isatidis-cape jasmine fruit extract and the buffalo horn-mother-of-pearl hydrolysate, uniformly mixing, adding 50mL of ethanol into cholic acid-hyodesoxycholic acid ethanol solution, and standing; filtering, recovering ethanol from the filtrate, adding 100mL of water, and standing; adding 5g of baicalin, and adjusting the pH value to 8.2-9.4 to dissolve the baicalin; adding water to complement the volume to 1000mL; adjusting the pH value to 7.2-7.5 by using sodium hydroxide, uniformly stirring, standing and filtering to obtain filtrate, namely the honeysuckle medicinal material negative medicinal material solution.

(2) Negative control solution of fructus Gardeniae (QINGKAILING preparation without fructus Gardeniae)

Weighing 20g of isatis root and 6g of honeysuckle, adding 50mL of water, decocting twice for 1 hour each time, combining decoctions, filtering, and collecting filtrate; heating the filtrate at 50 ℃, concentrating the filtrate until the relative density is 1.15-1.20, and cooling the filtrate; adding 50mL of ethanol, standing, filtering, recovering ethanol, adding 50mL of water, and standing to obtain the radix isatidis-honeysuckle extracting solution.

Weighing 2.5g of buffalo horn powder and 5g of mother-of-pearl, adding 10mL of sulfuric acid with the mass concentration of 98%, fully hydrolyzing, adding 50mL of water, and filtering; adjusting the pH value of the filtrate to 4 by using a calcium hydroxide solution with the mass concentration of 15%, filtering and collecting the filtrate; heating the filtrate at 50 ℃, concentrating the filtrate until the relative density is 1.05-1.10, and cooling the filtrate; adding 100mL of ethanol, standing, filtering, recovering ethanol, adding 100mL of water, and standing to obtain cornu Bubali-Concha Margaritifera hydrolysate.

3.25g of cholic acid and 3.75g of hyodeoxycholic acid are weighed, 50mL of ethanol is added, and the cholic acid-hyodeoxycholic acid ethanol solution is obtained after full dissolution.

Mixing the radix isatidis-honeysuckle extracting solution and the buffalo horn-mother-of-pearl hydrolysate, uniformly mixing, adding into cholic acid-hyodesoxycholic acid ethanol solution, adding 50mL of ethanol, and standing; filtering, recovering ethanol from the filtrate, adding 100mL of water, and standing; adding 5g of baicalin, and adjusting the pH value to 8.2-9.4 to dissolve the baicalin; adding water to complement the volume to 1000mL; adjusting the pH value to 7.2-7.5 by using sodium hydroxide, uniformly stirring, standing, and filtering to obtain filtrate, namely the gardenia medicine negative medicine solution.

5. Detection by thin layer chromatography

(1) Spotting is carried out

Using a quantitative capillary tube to respectively take 10 mu L of a test sample solution, 10 mu L of 1mg/mL geniposide reference substance solution, 20 mu L of 1mg/mL chlorogenic acid reference substance solution, 20 mu L of 1mg/mL baicalin reference substance solution, 10 mu L of 2mg/mL luteolin reference substance solution, 10 mu L of 2mg/mL isochlorogenic acid A reference substance solution, 10 mu L of 2mg/mL isochlorogenic acid C reference substance solution, 10 mu L of honeysuckle flower reference substance solution, 10 mu L of gardenia jasminoides reference substance solution, 10 mu L of honeysuckle flower negative reference substance solution and 10 mu L of gardenia jasminoides negative reference substance solution, and point on the same horizontal line 150-200 mm away from the bottom edge of a GF254 thin layer plate to form a plurality of round point-shaped spots with the diameter of 1-5 mm, the distance between the spots at two ends and the side edge of the adjacent thin layer plate is 5-10 mm, and the spots are spaced at equal intervals in pairs. Thus obtaining the point sample plate.

(2) Is unfolded

Ethyl acetate by volume ratio: acetone: formic acid: water =25:15:3: and 4.5, preparing the developing agent.

Adding a developing agent into a chromatographic cylinder, standing for 30min for pre-saturation, then putting the sample plate into the chromatographic cylinder, and developing until the spots extend to 15cm from the bottom edge of the sample plate.

And (5) drying the sample application plate until the developing agent is completely volatilized.

(3) Inspection of

And placing the dried spot template under 254nm ultraviolet light for color development and inspection.

Example 2 method for simultaneously detecting six ingredients of qingkailing oral liquid

The preparation of the test solution, the preparation of the reference solution, the preparation of the negative reference solution and the thin-layer chromatography detection are the same as those in example 1, except that in the step of 'inspection' of the thin-layer chromatography detection, the components are mixed according to the volume ratio of ethyl acetate: acetone: formic acid: water =28:12:1.5: and 6, preparing the developing agent.

Example 3 method for simultaneously detecting six ingredients of qingkailing granule

1. Preparation of test solution

(1) Taking 3 bags of qingkailing granules (each bag of qingkailing granules is 3g and contains 18-22 mg/mL of baicalin and more than or equal to 1mg of jasminoidin) as a sample to be detected, grinding, adding 40mL of hot water, sufficiently shaking for dissolving, cooling, shaking and extracting by using a 40mL ethyl acetate separating funnel for 2 times, and combining extracting solutions to obtain a sample extracting solution.

(2) And (3) putting the sample extracting solution into a container, heating in a water bath until the solvent of the sample extracting solution is completely evaporated, and collecting the solid remained after evaporation, wherein the solid is marked as solid 1.

(3) And dissolving the solid 1 in 2mL of acetonitrile to obtain a test solution.

2. The preparation of the control solution, the preparation of the negative control solution and the detection by thin layer chromatography are the same as those in example 1.

Comparative example 1 method for detecting components of qingkailing preparation

The preparation of the test solution, the preparation of the reference solution, the preparation of the negative reference solution and the detection by thin-layer chromatography are the same as those in example 1, except that in the step of 'development' of the detection by thin-layer chromatography, the dried spot-on plate is placed under 365nm ultraviolet light for color development.

Comparative example 2 method for detecting components of qingkailing preparation

1. The preparation of the test solution, the preparation of the control solution and the preparation of the negative control solution are the same as those in example 1.

2. Detection by thin layer chromatography

(1) Spotting is carried out

Respectively taking 10 mu L of a test solution, 10 mu L of a 1mg/mL geniposide reference solution, 20 mu L of a 1mg/mL chlorogenic acid reference solution, 20 mu L of a 1mg/mL baicalin reference solution, 10 mu L of a 2mg/mL luteolin reference solution, 10 mu L of a 2mg/mL isochlorogenic acid A reference solution, 10 mu L of a 2mg/mL isochlorogenic acid C reference solution, 10 mu L of a honeysuckle flower reference solution and 10 mu L of a gardenia reference solution by using a quantitative capillary tube, and dotting the spots on the same horizontal line 150-200 mm away from the bottom edge of a silica gel GF254 thin-layer plate to form a plurality of round-point-shaped spots with the diameter of 1-5 mm, wherein the distance between the spots at two ends and the edge close to the side edge of the thin-layer plate is 5-10 mm, and the spots are spaced at equal intervals. Thus obtaining the point sample plate.

(2) Is unfolded

According to the volume ratio of ethyl acetate: acetone: formic acid: water =2:10:1:0.5, preparing the developing agent.

Adding a developing agent into a chromatographic cylinder, standing for 30min for presaturation, then placing the spot plate into the chromatographic cylinder, and developing until the spots extend to 15cm from the bottom edge of the spot plate.

And drying the sample application plate until the developing solvent is completely volatilized.

(3) Color development

57mL of sulfuric acid with the mass concentration of 98% is taken, and ethanol is added to dilute the sulfuric acid to 1000mL, so that 10% sulfuric acid ethanol solution is obtained and is used as a color developing agent.

Spraying the color developing agent on the thin-layer plate, air-drying the color developing agent, and heating in an oven at 105 deg.C for 3min until the spots are clear.

(4) Inspection of

And placing the dried sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

Comparative example 3 method for detecting components of qingkailing preparation

1. The preparation of the test solution, the preparation of the control solution and the preparation of the negative control solution are the same as those in example 1.

2. Detection by thin layer chromatography

(1) Spotting is carried out

Respectively taking 10 mu L of a test solution, 10 mu L of a 1mg/mL geniposide reference solution, 20 mu L of a 1mg/mL chlorogenic acid reference solution, 20 mu L of a 1mg/mL baicalin reference solution, 10 mu L of a 2mg/mL luteolin reference solution, 10 mu L of a 2mg/mL isochlorogenic acid A reference solution, 10 mu L of a 2mg/mL isochlorogenic acid C reference solution, 10 mu L of a honeysuckle flower reference solution and 10 mu L of a gardenia reference solution by using a quantitative capillary tube, and dotting the spots on the same horizontal line 150-200 mm away from the bottom edge of a silica gel GF254 thin-layer plate to form a plurality of round-point-shaped spots with the diameter of 1-5 mm, wherein the distance between the spots at two ends and the edge close to the side edge of the thin-layer plate is 5-10 mm, and the spots are spaced at equal intervals. And obtaining the point sample plate.

(2) Is unfolded

Ethyl acetate by volume ratio: acetone: formic acid: water =5:5:1:1, preparing the developing agent.

Adding a developing agent into a chromatographic cylinder, standing for 30min for pre-saturation, then putting the sample plate into the chromatographic cylinder, and developing until the spots extend to 15cm from the bottom edge of the sample plate.

And (5) drying the sample application plate until the developing agent is completely volatilized.

(3) Color development

57mL of sulfuric acid with the volume concentration of 98% is taken and diluted to 1000mL by adding ethanol to obtain 10% sulfuric acid ethanol solution as a color developing agent.

Spraying the color developing agent on the thin-layer plate, air-drying the color developing agent, and heating in an oven at 105 deg.C for 3min until the spots are clear.

(4) Inspection of

And placing the dried sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

Comparative example 4 method for detecting components of qingkailing preparation

1. The preparation of the test solution, the preparation of the control solution and the preparation of the control solution were the same as in example 1.

2. Detection by thin layer chromatography

(1) Spotting is carried out

Using a quantitative capillary tube to respectively take 10 mu L of a test sample solution, 10 mu L of 1mg/mL geniposide reference substance solution, 20 mu L of 1mg/mL chlorogenic acid reference substance solution, 20 mu L of 1mg/mL baicalin reference substance solution, 10 mu L of 2mg/mL luteolin reference substance solution, 10 mu L of 2mg/mL isochlorogenic acid A reference substance solution, 10 mu L of 2mg/mL isochlorogenic acid C reference substance solution, 10 mu L of honeysuckle flower reference substance solution, 10 mu L of gardenia jasminoides reference substance solution, 10 mu L of honeysuckle flower negative reference substance solution and 10 mu L of gardenia jasminoides negative reference substance solution, and point on the same horizontal line 150-200 mm away from the bottom edge of a GF254 thin layer plate to form a plurality of round point-shaped spots with the diameter of 1-5 mm, the distance between the spots at two ends and the side edge of the adjacent thin layer plate is 5-10 mm, and the spots are spaced at equal intervals in pairs. Thus obtaining the point sample plate.

(2) Is unfolded

Ethyl acetate according to volume ratio: formic acid: water =8:3: and 2, preparing the developing agent.

Adding a developing agent into a chromatographic cylinder, standing for 30min for pre-saturation, then putting the sample plate into the chromatographic cylinder, and developing until the spots extend to 15cm from the bottom edge of the sample plate.

And drying the sample application plate until the developing solvent is completely volatilized.

(3) Color development

57mL of sulfuric acid with the volume concentration of 98% is taken and diluted to 1000mL by adding ethanol to obtain 10% sulfuric acid ethanol solution as a color developing agent.

Spraying the color developing agent on the thin-layer plate, air-drying the color developing agent, and heating in an oven at 105 deg.C for 3min until the spots are clear.

(4) Inspection of

And placing the dried sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

Comparative example 5 method for detecting components of qingkailing preparation

1. The preparation of the test solution, the preparation of the control solution and the preparation of the control solution were the same as in example 1.

2. Detection by thin layer chromatography

(1) Spotting is carried out

Using a quantitative capillary tube to respectively take 10 mu L of a test sample solution, 10 mu L of 1mg/mL geniposide reference substance solution, 20 mu L of 1mg/mL chlorogenic acid reference substance solution, 20 mu L of 1mg/mL baicalin reference substance solution, 10 mu L of 2mg/mL luteolin reference substance solution, 10 mu L of 2mg/mL isochlorogenic acid A reference substance solution, 10 mu L of 2mg/mL isochlorogenic acid C reference substance solution, 10 mu L of honeysuckle flower reference substance solution, 10 mu L of gardenia jasminoides reference substance solution, 10 mu L of honeysuckle flower negative reference substance solution and 10 mu L of gardenia jasminoides negative reference substance solution, and point on the same horizontal line 150-200 mm away from the bottom edge of a GF254 thin layer plate to form a plurality of round point-shaped spots with the diameter of 1-5 mm, the distance between the spots at two ends and the side edge of the adjacent thin layer plate is 5-10 mm, and the spots are spaced at equal intervals in pairs. Thus obtaining the point sample plate.

(2) Is unfolded

Chloroform according to volume ratio: ethyl acetate: methanol: water =15:40:22:10, preparing the developing agent.

Adding a developing agent into a chromatographic cylinder, standing for 30min for pre-saturation, then putting the sample plate into the chromatographic cylinder, and developing until the spots extend to 15cm from the bottom edge of the sample plate.

And (5) drying the sample application plate until the developing agent is completely volatilized.

(3) Color development

57mL of sulfuric acid with the volume concentration of 98% is taken and diluted to 1000mL by adding ethanol to obtain 10% sulfuric acid ethanol solution as a color developing agent.

Spraying the color developing agent on the thin-layer plate, air-drying the color developing agent, and heating in an oven at 105 deg.C for 3min until the spots are clear.

(4) Inspection of

And placing the dried sample plate under sunlight or 254nm ultraviolet light for color development and inspection.

Application example

1. Experimental methods

Using the methods of examples 1 to 3 and comparative examples 1 to 5, jasminoidin, chlorogenic acid, baicalin, luteolin, isochlorogenic acid a and isochlorogenic acid C of qingkailing oral liquid (lot No. 1220701 1221220703) and qingkailing granules (lot No. 5220801 525220820802.

2. Results of the experiment

As shown in fig. 1 and fig. 2, in the chromatogram of the test sample under the irradiation of 254nm ultraviolet light, the same spots are displayed on the corresponding positions of the chlorogenic acid control solution, the geniposide control solution, the baicalin control solution, the luteolin control solution, the isochlorogenic acid a control solution, the isochlorogenic acid C control solution, the gardenia control medicinal material and the honeysuckle control medicinal material in the chromatogram, and the honeysuckle negative medicinal material solution and the gardenia negative medicinal material solution lack the spots of the source components of the corresponding medicinal materials in the corresponding positions. The method in example 1 and example 2 is shown to be capable of detecting geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A and isochlorogenic acid C in the oral liquid of qingkailing simultaneously.

As shown in fig. 3, in the chromatogram of the test sample under the irradiation of 254nm ultraviolet light, the same spots are displayed on the corresponding positions of the chlorogenic acid reference solution, the geniposide reference solution, the baicalin reference solution, the luteolin reference solution, the isochlorogenic acid a reference solution, the isochlorogenic acid C reference solution, the gardenia reference medicinal material and the honeysuckle reference medicinal material in the chromatogram, and the honeysuckle negative medicinal material solution and the gardenia negative medicinal material solution lack the spots of the source components of the corresponding medicinal materials in the corresponding positions. The method of example 3 is shown to be capable of simultaneously detecting geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A and isochlorogenic acid C in qingkailing granules.

As shown in fig. 4, in the chromatogram of the test sample of comparative example 1 under 365nm ultraviolet irradiation, the same spots were shown in the corresponding positions of the chromatogram of the chlorogenic acid control solution, the galuteolin control solution, the isochlorogenic acid a control solution, the isochlorogenic acid C control solution and the honeysuckle flower control drug, but there was no obvious spot in the baicalin control solution and the geniposide control solution. It is shown that baicalin and geniposide in the qingkailing oral liquid could not be detected by the method of comparative example 1.

As shown in fig. 5 and 6, the developing solutions of comparative example 2 and comparative example 3 could not separate the components of the sample, and even though the 10% ethanol sulfate solution was used as the color developing agent, the sample could not be observed to have obvious spots on the corresponding positions of the chromatogram of the baicalin control solution, the chlorogenic acid control solution, the geniposide control solution, the honeysuckle control material and the gardenia control material under ultraviolet light and sunlight.

As shown in fig. 7 and fig. 8, the developing agent of comparative example 4 (chloroform: ethyl acetate: methanol: water = 15).

In summary, the method of embodiments 1 to 3 can detect geniposide, chlorogenic acid, baicalin, galuteolin, isochlorogenic acid a and isochlorogenic acid C in qingkailing oral liquid and qingkailing granules simultaneously, can complete detection without using a color developing agent, is simple and rapid to operate, has a clear color developing result, good separation degree, can effectively eliminate negative interference, is clear to inspect and easy to interpret, has good specificity and reproducibility, can effectively realize quality control of a compound preparation, realizes drug production standardization, and can better satisfy drug safety, production process stability and controllability.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. The developing agent for the thin layer chromatography is characterized by comprising the following components in percentage by volume (22-28): (12 to 18): (1-5): (2-7).

2. Use of a developer according to claim 1 for the detection and/or analysis of the components of a qingkailing preparation.

3. A method for simultaneously detecting six components of a qingkailing preparation is characterized by comprising the following steps:

s1, removing a solvent from an ethyl acetate extracting solution of a sample to be tested to obtain a precipitate, and dissolving the precipitate with acetonitrile to obtain a test solution; methanol solution of geniposide, chlorogenic acid, baicalin, luteolin, isochlorogenic acid A and isochlorogenic acid C as control solution;

s2, respectively adding the test solution and the reference solution to the thin-layer plate;

s3, fully reacting the developing agent in the claim 1 with the thin-layer plate obtained in the step S2;

and S4, developing the reacted thin-layer plate obtained in the step S3 under 250-260 nm ultraviolet light.

4. The method according to claim 3, wherein in step S1, if the sample to be tested is qingkailing oral liquid, the preparation method of the ethyl acetate extract of the sample to be tested is as follows: the qingkailing oral liquid, the dilute hydrochloric acid and the ethyl acetate are mixed according to the volume ratio (10-20): (0.5-1): (20-60) mixing and extracting.

5. The method according to claim 3, wherein in step S1, if the sample to be tested is qingkailing granule, the ethyl acetate extracting solution of the sample is prepared by: mixing qingkailing granules, water and ethyl acetate according to the mass volume ratio (7-11) g: (35-45) mL: (35-45) mL, and extracting the mixture.

6. The method according to claim 3, wherein the methanol solution has a concentration of 65 to 75% by volume in step S1.

7. The method of claim 3, wherein in step S1, the working concentration of the control is 0.5-2.5 mg/mL.

8. The method according to claim 3, wherein in step S2, a control solution is also added to the lamella plate; the reference medicinal material solution comprises flos Lonicerae reference medicinal material solution and fructus Gardeniae reference medicinal material solution.

9. The method according to claim 3, wherein the thin layer plate is a silica gel GF254 thin layer plate.

10. Use of a method according to any one of claims 3 to 9 for the analysis of the components and/or quality control of a qingkailing preparation.

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