Method for detecting active ingredients in traditional chinese medicine preparations containing concha margaritiferae

Abstract

The present application relates to the technical field of pharmaceutical analysis, and in particular to a method for detecting active ingredients in a traditional Chinese medicine preparation containing Concha Murex. The present application first establishes a method for simultaneously detecting aurantio-olive yellow saponin, syringidin and ammonium glycyrrhizinate. The method is simple to operate, has good specificity, solution stability, linearity, repeatability and accuracy. The method provided by the present application provides a more reliable quality control method for traditional Chinese medicine preparations containing Concha Murex, and ensures the clinical efficacy of drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly to a method for detecting active ingredients in traditional Chinese medicine preparations containing abalone shell Background Art

[0002] The ancient classic prescription list (the first batch) announced by the National Medical Products Administration includes abalone shell powder, whose source is: "Puji Prescription" (by Zhu Su in the Ming Dynasty) "Abalone shell powder is used to treat wind toxin attacking the head and eyes, resulting in dim vision and discomfort in the head and eyes." The prescription is: abalone shell, notopterygium (removing the reed head), cassia seed, and chrysanthemum, each one tael, and licorice (roasted and cut into pieces), half tael. The preparation method and usage are: make the above ingredients into a powder, take two qian each time, add one cup of water, decoct to six fen, dissolve, and take warm after meals and before going to bed. It is a decocted powder. This prescription is widely used in ophthalmology, but it is always related to the liver and gallbladder meridians, and is mostly used for liver heat and eye diseases such as nebula. Modern ophthalmology clinical practice also follows this principle.

[0003] After that, based on the improvement of abalone shell powder, notopterygium was replaced by schizonepeta, and its prescription is abalone shell, cassia seed, chrysanthemum, schizonepeta, and licorice, to make a traditional Chinese medicine preparation containing abalone shell, where:

[0004] Abalone shell, a Chinese medicinal material. This product is the shell of abalone haliotis diversicolor supertexta or haliotis gigantea discus hannai in the family Haliotidae. It is captured in summer and autumn. After capture, the meat is removed, the shell is taken, washed, impurities are removed, and then dried. The shell of abalone haliotis diversicolor supertexta is called smooth-bottom sea abalone shell; the shell of haliotis gigantea discus hannai is called rough-bottom sea abalone shell. Its functions and indications are: suppressing the hyperactive liver and subsiding yang, clearing the liver and improving eyesight. It is used for headache and dizziness, redness and nebula of the eyes, blurred vision, and blindness.

[0005] Cassia seed, a Chinese medicinal material. It is the dried ripe seed of cassia obtusifolia or cassia tora in the family Leguminosae, named for its eyesight-improving effect. The mature fruits are harvested in autumn, dried, the seeds are beaten, and impurities are removed. Cassia seed is bitter, sweet, salty, and slightly cold in nature, and enters the liver, kidney, and large intestine meridians; it moistens the intestines and promotes defecation, reduces lipid and improves eyesight, treats constipation, hyperlipidemia, and hypertension. It clears the liver and improves eyesight, promotes diuresis and defecation, has a laxative effect, and reduces blood pressure and blood lipid.

[0006] Chrysanthemum, a Chinese medicinal material. This product is the dried capitulum of chrysanthemum morifolium ramat in the family Compositae. It is harvested in batches when the flowers are in full bloom from September to November, and dried in the shade or by baking, or by fumigation or steaming and then drying. According to different origins and processing methods, the medicinal materials are divided into "Boju", "Chuju", "Gongju", "Hangju", and "Huaiju". Chrysanthemum is sweet and bitter, and slightly cold in nature. It belongs to the lung and liver meridians. It dispels wind and clears heat, suppresses the hyperactive liver and improves eyesight, and clears heat and detoxifies. It is used for wind-heat cold, headache and dizziness, redness and swelling of the eyes, blurred vision, and sores and carbuncles.

[0007] *Schizonepeta tenuifolia* Briq., a plant in the Lamiaceae family, is a traditional Chinese medicine. It is the dried aerial part of the plant. Harvested in summer and autumn when the flowers are at their peak and the spikelets are green, impurities are removed, and it is then sun-dried. It is distributed in Hebei, Jiangsu, Zhejiang, Jiangxi, Hubei, and the three northeastern provinces. It has the effects of relieving exterior syndromes, dispelling wind, promoting rash eruption, and eliminating sores. It is used for colds, headaches, measles, rubella, and early-stage sores.

[0008] Licorice is a traditional Chinese medicine. It refers to the dried root and rhizome of *Glycyrrhiza uralensis* Fisch., *Glycyrrhiza inflata* Bat., or *Glycyrrhiza glabra* L., all belonging to the legume family. It is distributed in Northeast China, North China, Shaanxi, Gansu, Qinghai, Xinjiang, Shandong, and other regions. It has the effects of tonifying the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, relieving spasms and pain, and harmonizing other herbs. It is commonly used for spleen and stomach weakness, fatigue, palpitations, shortness of breath, cough with excessive phlegm, abdominal and limb spasms and pain, carbuncles and boils, and to alleviate the toxicity and harshness of other medications.

[0009] The aforementioned traditional Chinese medicine preparation containing abalone shell is used clinically as granules. The prescription is as follows: 250 parts abalone shell, 250 parts cassia seed, 250 parts schizonepeta, 250 parts chrysanthemum, 125 parts licorice, and an appropriate amount of maltodextrin, to make 1000g of granules. The preparation method is to decoct the above five medicinal materials with purified water, repeat twice, concentrate the filtrate, add maltodextrin, granulate, and dry to obtain the final product.

[0010] Currently, the aforementioned improved traditional Chinese medicine preparations containing abalone shell are not included in any standards or reported in the literature, and their quality control is lacking. In these improved abalone shell-containing traditional Chinese medicine preparations, cassia seed is the principal ingredient, and cassia seed extract is one of the indicative components of cassia seed; chrysanthemum and schizonepeta are the assistant and adjuvant ingredients, respectively, and luteolin is one of the indicative components shared by chrysanthemum and schizonepeta; licorice is the guiding ingredient, and ammonium glycyrrhizate is one of the indicative components of licorice.

[0011] Therefore, there is an urgent need to develop a method that can simultaneously detect the content of cassia seed extract, luteolin, and glycyrrhizic acid in abalone shell traditional Chinese medicine preparations, so as to provide a more reliable quality control method for abalone shell traditional Chinese medicine preparations and ensure the clinical efficacy of the drugs. Summary of the Invention

[0012] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting the active ingredients cassia seed extract, luteolin, and ammonium glycyrrhizate in traditional Chinese medicine preparations containing abalone shell.

[0013] This invention provides a method for detecting active ingredients in traditional Chinese medicine preparations containing abalone shell, comprising:

[0014] After solvent extraction to obtain the test solution of the traditional Chinese medicine preparation containing abalone shell, the test solution was detected by high performance liquid chromatography.

[0015] Chromatographic parameters include:

[0016] The chromatographic column was a C18 column; the column temperature was 25℃~40℃; the mobile phase was gradient elution.

[0017] Mobile phase A is acetonitrile, and mobile phase B is a 0.05%–2% phosphoric acid solution.

[0018] In this invention, the preparation of the test solution includes: mixing a traditional Chinese medicine preparation containing abalone shell with a solvent, heating under reflux for 15 to 60 minutes, and filtering to obtain the test solution.

[0019] In some embodiments, the solvent used in the preparation of the test solution is a 50 vol% to 70 vol% aqueous methanol solution. Specifically, the solvent is a 50 vol% aqueous methanol solution, a 60 vol% aqueous methanol solution, or a 70 vol% aqueous methanol solution. Preferably, the solvent is a 70 vol% aqueous methanol solution.

[0020] In this invention, in the preparation of the test solution, the ratio of the mass (g) of the traditional Chinese medicine preparation containing abalone shell and the volume (mL) of the solvent is 0.5g:(25-50)mL.

[0021] In some embodiments, during the preparation of the test solution, the mass ratio (g) to volume (mL) of the herbal preparation containing abalone shell and the solvent is 0.5g:25mL, 0.5g:30mL, 0.5g:35mL, 0.5g:40mL, 0.5g:45mL, or 0.5g:50mL. Preferably, the mass ratio (g) to volume (mL) of the herbal preparation containing abalone shell and the solvent is 0.5g:25mL.

[0022] In some embodiments, the reflux heating time is 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min. Preferably, the reflux heating time is 30 min.

[0023] In this invention, the preparation of the test solution includes a step of cooling and then replenishing the lost mass with solvent before filtration, wherein the cooling is to cool to room temperature. The filtrate after filtration is then used as the test solution.

[0024] In the detection method of the present invention, the active ingredient includes at least one of luteolin, cassia seed extract, or ammonium glycyrrhizate.

[0025] The detection method of the present invention further includes:

[0026] A reference solution was prepared, and the reference solution was analyzed by high performance liquid chromatography to obtain a chromatogram of the reference. The components of the traditional Chinese medicine preparation containing abalone shell were qualitatively and quantitatively analyzed based on the chromatogram of the reference.

[0027] The preparation of the reference solution includes: dissolving one or more of luteolin, cassia aurantium, or ammonium glycyrrhizate to obtain reference solutions; the solvent for dissolution is a 50 vol% to 70 vol% aqueous methanol solution. In some embodiments, the solvent for dissolution is a 50 vol% aqueous methanol solution, a 60 vol% aqueous methanol solution, or a 70 vol% aqueous methanol solution. Preferably, the solvent for dissolution is a 70 vol% aqueous methanol solution.

[0028] In this invention, the reference solution includes any one of luteolin, cassia aurantium, or ammonium glycyrrhizate; or includes luteolin and cassia aurantium, or includes ammonium glycyrrhizate and luteolin; or includes luteolin, cassia aurantium, and ammonium glycyrrhizate.

[0029] In a reference solution containing luteolin, cassia aurantium, and ammonium glycyrrhizate, the concentration of luteolin was 40 μg / mL, the concentration of cassia aurantium was 20 μg / mL, and the concentration of ammonium glycyrrhizate was 80 μg / mL.

[0030] In this invention, the gradient elution procedure includes:

[0031] From 0 to 6 minutes, the volume fraction of mobile phase A was 16%.

[0032] Over 6 to 8 minutes, the volume fraction of mobile phase A increased from 16% to 33%.

[0033] From 8 min to 20 min, the volume fraction of mobile phase A increased from 33% to 36%.

[0034] Over 20 to 24 minutes, the volume fraction of mobile phase A increased from 36% to 70%.

[0035] Over 24 to 25 minutes, the volume fraction of mobile phase A decreased from 70% to 16%.

[0036] The volume fraction of mobile phase A was 16% for 25-30 minutes.

[0037] In this invention, the detector in the detection method is a diode array detector; the detection wavelength is 348 nm for 0-12 min; the detection wavelength is 284 nm for 12 min-14.5 min; and the detection wavelength is 252 nm for 14.5 min-30 min.

[0038] In this invention, the chromatographic column used in the detection method is an Agilent Poroshell EC C column. 18 , 100mm×4.6mm, 2.7μm.

[0039] In this invention, the column temperature in the detection method is 25°C, 30°C, or 40°C, with 30°C being preferred.

[0040] In this invention, the mobile phase flow rate in the detection method is 1.4–1.6 mL / min. In some embodiments, the mobile phase flow rate is 1.4 mL / min, 1.5 mL / min, or 1.6 mL / min, preferably 1.5 mL / min.

[0041] In this invention, the injection volume in the detection method is 1–10 μL. In some embodiments, the injection volume is 1 μL, 5 μL, or 10 μL, preferably 5 μL.

[0042] In this invention, the separation degree of luteolin and cassia seed extract is 44.07-49.24, and the separation degree of cassia seed extract and ammonium glycyrrhizate is 9.16-12.23.

[0043] In this invention, the linear range of luteolin is 19.88–795.00 ng, the linear range of cassia aurantium is 11.33–566.5 ng, and the linear range of ammonium glycyrrhizate is 40.03–2001.25 ng.

[0044] In this invention, the raw materials for preparing the traditional Chinese medicine preparation containing abalone shell include: 250 parts by weight of abalone shell, 250 parts by weight of cassia seed, 250 parts by weight of schizonepeta, 250 parts by weight of chrysanthemum and 125 parts by weight of licorice.

[0045] In this invention, the preparation method of the traditional Chinese medicine preparation containing abalone shell includes: adding abalone shell, cassia seed, schizonepeta, chrysanthemum and licorice to water and decocting twice, with the amount of water added and the decoction time being 12 times the amount of water for 2 hours and 10 times the amount of water for 1.5 hours, respectively, filtering, combining the filtrates, taking the filtrate, concentrating it, adding an appropriate amount of maltodextrin, granulating and drying to obtain the product.

[0046] The detection method for traditional Chinese medicine preparations containing abalone shell provided by this invention has at least the following beneficial effects: Cassia seed is the principal ingredient in abalone shell preparations; cassia seed extract is one of the indicative components of cassia seed; chrysanthemum and schizonepeta are the assistant and adjuvant ingredients, respectively; luteolin is one of the indicative components shared by chrysanthemum and schizonepeta; licorice is the guiding ingredient in abalone shell preparations; and ammonium glycyrrhizate is one of the indicative components of licorice. This invention establishes for the first time a method that can simultaneously detect cassia seed extract, luteolin, and ammonium glycyrrhizate. Through reasonable optimization of the step parameters in the method, the method provided by this invention is simple to operate and has good specificity, solution stability, linearity, repeatability, and accuracy. The method provided by this invention provides a more reliable quality control method for traditional Chinese medicine preparations containing abalone shell, ensuring the clinical efficacy of the drugs. Attached Figure Description

[0047] Figure 1 The high-performance liquid chromatogram is for extraction with 70% methanol as the solvent;

[0048] Figure 2 High-performance liquid chromatography (HPLC) chromatogram for extraction solvent in 50% methanol;

[0049] Figure 3 High-performance liquid chromatography (HPLC) chromatogram for extraction solvent of 30% methanol;

[0050] Figure 4 This is a high-performance liquid chromatogram of the methanol extraction solvent;

[0051] Figure 5 The chromatogram is a high-performance liquid chromatogram at a column temperature of 20℃.

[0052] Figure 6 The chromatogram is a high-performance liquid chromatography (HPLC) chromatogram at a column temperature of 25°C.

[0053] Figure 7 The chromatogram is a high-performance liquid chromatography (HPLC) chromatogram at a column temperature of 30℃.

[0054] Figure 8 The chromatogram is a high-performance liquid chromatogram at a column temperature of 40℃.

[0055] Figure 9 The chromatogram is for a 1 μL injection volume high-performance liquid chromatography (HPLC) sample.

[0056] Figure 10 The chromatogram is for a 5 μL injection volume high-performance liquid chromatography (HPLC) sample.

[0057] Figure 11 The chromatogram is for a 10 μL injection volume high-performance liquid chromatography (HPLC) sample.

[0058] Figure 12 The chromatogram is for a 20 μL injection volume high-performance liquid chromatography (HPLC) sample.

[0059] Figure 13The chromatogram is for a 25 μL injection volume high-performance liquid chromatography (HPLC) sample.

[0060] Figure 14 The chromatogram is a high-performance liquid chromatogram at a flow rate of 1.0 mL / min.

[0061] Figure 15 The chromatogram is a high-performance liquid chromatogram at a flow rate of 1.2 mL / min.

[0062] Figure 16 The chromatogram is a high-performance liquid chromatogram at a flow rate of 1.4 mL / min.

[0063] Figure 17 The chromatogram is a high-performance liquid chromatogram at a flow rate of 1.5 mL / min.

[0064] Figure 18 The chromatogram is a high-performance liquid chromatogram at a flow rate of 1.6 mL / min.

[0065] Figure 19 The high-performance liquid chromatogram for the detection of the reference standard is shown below. 1 represents luteolin, 2 represents cassia seed extract, and 3 represents ammonium glycyrrhizate.

[0066] Figure 20 High performance liquid chromatograms for the detection of negative samples of chrysanthemum and catnip; 2 represents cassia aurantium; 3 represents ammonium glycyrrhizate.

[0067] Figure 21 The high-performance liquid chromatogram for the detection of cassia seed negative test sample is shown in Figure 1, where luteolin is 1 and glycyrrhizic acid ammonium is 3.

[0068] Figure 22 The high performance liquid chromatograms for the detection of licorice negative test samples are shown. 1 represents luteolin, and 2 represents cassia seed extract.

[0069] Figure 23 The linear relationship of luteolin is shown in the regression equation Y = 0.8532X + 1.2254, r = 0.9999, which shows a good linear relationship in the range of 19.88 to 795.00 ng.

[0070] Figure 24 The linear relationship of cassia seed extract is shown in the graph. The regression equation is Y = 2.9589X - 2.5973, r = 0.9999. The linear relationship is good in the range of 11.33 to 566.5 ng.

[0071] Figure 25 The linear relationship of ammonium glycyrrhizate is shown in the regression equation Y = 0.3945X + 0.9724, r = 0.9999, which shows a good linear relationship in the range of 40.03 to 2001.25 ng. Detailed Implementation

[0072] This invention provides a method for detecting the active ingredients in traditional Chinese medicine preparations containing abalone shell. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0073] The purpose of this invention is to provide a method for detecting traditional Chinese medicine preparations containing abalone shell. The method involves extracting the traditional Chinese medicine preparation containing abalone shell with a solvent to obtain a test solution, and then using high performance liquid chromatography to detect the test solution.

[0074] This invention optimizes the pretreatment process and HPLC detection conditions. The optimized scheme exhibits good specificity, solution stability, linearity, repeatability, and accuracy. Experiments show that, compared to inappropriate pretreatment schemes, column types, mobile phases, elution programs, injection volumes, column temperatures, and flow rates, the method provided by this invention can more effectively separate the active ingredients in traditional Chinese medicine preparations containing abalone shell, achieving better detection results.

[0075] This invention employs 70% methanol to extract the target components via reflux heating, followed by detection of the target components using liquid chromatography. This technology provides a method for simultaneously and accurately determining the content of multiple active ingredients in a traditional Chinese medicine preparation made from abalone shell. More specifically:

[0076] The present invention provides a method for detecting the content of a traditional Chinese medicine preparation containing abalone shell, comprising the following steps:

[0077] A. Preparation of the reference solution:

[0078] Take appropriate amounts of luteolin, cassia seed extract, and ammonium glycyrrhizate reference standards respectively, and dilute them with 70% methanol solution to prepare a mixed standard solution;

[0079] B. Preparation of the test solution:

[0080] Take an appropriate amount of traditional Chinese medicine preparation containing abalone shell, grind it into a fine powder, take about 0.5g, accurately weigh it, add 25-50mL of methanol solution of different proportions, weigh it, heat under reflux for 15-60 minutes, cool it, weigh it again, and make up the weight loss with solvent, shake it well, filter it, and take the filtrate to obtain the product.

[0081] C. Detection

[0082] High-performance liquid chromatography (HPLC) was used under the following chromatographic conditions: octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, with gradient elution; detection wavelengths were 348 nm (0–12 min), 284 nm (12–14.5 min), and 252 nm (14.5–30 min); flow rate was 1.4–1.6 mL / min; column temperature was 25–40 °C; and the theoretical plate number, calculated based on the cassia aurantium peak, should not be less than 20,000. 5 μL of both the reference solution and the test solution were injected into the HPLC instrument and analyzed.

[0083] Preferably, the mixed standard solution in step A consists of 40 μg / mL luteolin, 20 μg / mL cassia seed extract and 80 μg / mL ammonium glycyrrhizate.

[0084] The results are as follows Figures 1-4 The results showed that 70% methanol was the optimal extraction solvent in step B. Extraction solvents of methanol, 70% methanol, 50% methanol, and 30% methanol were used. The samples extracted with 70% and 50% methanol showed higher contents of luteolin, aurantium, and ammonium glycyrrhizate; the samples extracted with 100% and 30% methanol showed lower contents of these compounds. Therefore, 70% methanol was the preferred extraction solvent.

[0085] Preferably, the extraction volume in step B is 25 mL of 70% methanol solution.

[0086] Preferably, the extraction method in step B is heating and reflux.

[0087] Preferably, the extraction time in step B is reflux for 30 minutes.

[0088] Preferably, the chromatographic column used in step C is an Agilent Poroshell EC C column. 18 It is a chromatographic column with a length of 100 mm, an inner diameter of 4.6 mm, and a packing particle size of 2.7 μm.

[0089] Preferably, the column temperature is 25–40°C, more preferably 30°C. The chromatograms of samples at column temperatures of 20°C, 25°C, 30°C, and 40°C were examined respectively. Figures 5-8 At 20℃, no peak of cassia aurantium was detected in the chromatogram of the sample. At 25℃, 30℃ and 40℃, peaks of luteolin, cassia aurantium and ammonium glycyrrhizate were detected in the chromatogram of the sample. Therefore, the preferred column temperature is 30℃.

[0090] Preferably, the injection volume in this invention is 5 μL. Chromatograms of samples with injection volumes of 1 μL, 5 μL, 10 μL, 20 μL, and 25 μL were examined, with the injection volumes of 1 μL, 5 μL, and 10 μL being the most accurate. Figures 9-13 In the chromatograms of luteolin, cassia aurantium, and ammonium glycyrrhizate, the peak areas showed a linear relationship. The peak shape of luteolin in the chromatograms of samples with injection volumes of 20 μL and 25 μL deteriorated and did not meet the separation requirements. Therefore, the preferred injection volume was 5 μL.

[0091] Preferably, the flow rate is 1.0–1.6 mL / min, more preferably 1.5 mL / min. Chromatograms were collected at flow rates of 1.0 mL / min, 1.2 mL / min, 1.4 mL / min, 1.5 mL / min, and 1.6 mL / min, with particular attention to the chromatograms obtained at 1.0 mL / min and 1.2 mL / min. Figures 14-15 The separation rate of ammonium glycyrrhizate from adjacent impurities in the sample did not meet the standard requirements: 1.4 mL / min, 1.5 mL / min, and 1.6 mL / min. Figures 16-18 The chromatographic peaks of luteolin, cassia aurantium, and ammonium glycyrrhizate collected under the conditions all met the standard requirements for separation from adjacent impurities. Therefore, the preferred flow rate is 1.5 mL / min.

[0092] The method for detecting the content of abalone shell-containing traditional Chinese medicine preparations provided by this invention has the following beneficial effects:

[0093] Cassia seed is the principal ingredient in abalone shell traditional Chinese medicine preparations. Aurantioside is one of the indicator components of cassia seed. Chrysanthemum and schizonepeta are the assistant and adjuvant ingredients, respectively. Luteolin is one of the indicator components shared by chrysanthemum and schizonepeta. Licorice is the guiding ingredient in abalone shell traditional Chinese medicine preparations, and ammonium glycyrrhizate is one of the indicator components of licorice. This patent application establishes for the first time a method for simultaneously detecting the content of aurantioside, luteolin, and ammonium glycyrrhizate in abalone shell traditional Chinese medicine preparations, providing a more reliable quality control method for abalone shell traditional Chinese medicine preparations and ensuring the clinical efficacy of the drug.

[0094] The detection method provided by this invention is applicable to the detection of the contents of cassia seed extract, luteolin, and ammonium glycyrrhizate in traditional Chinese medicine preparations made from abalone shell. The method is simple to operate, exhibits good specificity, solution stability, and linearity, and meets the requirements for repeatability and accuracy. In the specificity test, the chromatogram of the negative control solution showed no interfering peaks at the same retention time as the chromatogram of the reference solution, and cassia seed extract, luteolin, and ammonium glycyrrhizate were completely separated from adjacent chromatographic peaks, demonstrating good specificity. The RSD of the content determination results of luteolin, cassia seed extract, and ammonium glycyrrhizate after being placed at room temperature for 24 hours was less than 2%, indicating that the sample obtained by this method has good stability after being placed at room temperature for 24 hours. Standard curves were plotted with injection volume on the x-axis and peak area on the y-axis. The regression equations were as follows: luteolin: Y = 0.8532X + 1.2254, r = 0.9999; cassia seed extract: Y = 2.9589X - 2.5973, r = 0.9999; ammonium glycyrrhizate: Y = 0.3945X + 0.9724, r = 0.9999. The results showed that luteolin exhibited good linearity in the injection volume range of 19.88–795.00 ng, cassia seed extract in the range of 11.33–566.5 ng, and ammonium glycyrrhizate in the range of 40.03–2001.25 ng. When known amounts of cassia aurantium, luteolin, and ammonium glycyrrhizate reference standards were added to the sample, the average recoveries (n=9) of the three components were 95.63%, 100.92%, and 102.29%, respectively, with RSDs of 1.24%, 3.28%, and 2.70%, respectively, indicating that the method has good accuracy.

[0095] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0096] The prescription for a traditional Chinese medicine preparation containing abalone shell is as follows: 250 parts abalone shell, 250 parts cassia seed, 250 parts schizonepeta, 250 parts chrysanthemum, 125 parts licorice, and an appropriate amount of maltodextrin, to be granulated to 1000g. The preparation method is to decoct the above five medicinal materials with purified water, repeat twice, concentrate the filtrate, add maltodextrin, granulate, and dry to obtain the final product.

[0097] The present invention will be further illustrated below with reference to the embodiments:

[0098] Example 1: Construction of Detection Method

[0099] I. Reagent Preparation

[0100] A. Preparation of the reference solution:

[0101] Take appropriate amounts of luteolin, cassia aurantium, and ammonium glycyrrhizate reference standards respectively, and dilute them with 70% methanol solution to prepare a mixed standard solution, wherein the concentrations are 40 μg / mL luteolin, 20 μg / mL cassia aurantium, and 80 μg / mL ammonium glycyrrhizate.

[0102] B. Preparation of the test solution:

[0103] Take an appropriate amount of traditional Chinese medicine preparation containing abalone shell, grind it into a fine powder, take about 0.5g, accurately weigh it, add 25mL of methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent, shake it well, filter it, and take the filtrate to obtain the final product.

[0104] II. Chromatographic Conditions

[0105] Using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, the gradient elution program is shown in Table 1:

[0106] Table 1 Gradient elution program

[0107]

[0108]

[0109] Detector: Diode array detector;

[0110] The detection wavelengths are 348nm (0–12 min), 284nm (12–14.5 min), and 252nm (14.5–30 min).

[0111] III. Examination of Testing Conditions:

[0112] 1. Methanol concentration

[0113] Other chromatographic conditions remained the same as before, with a column temperature of 30℃, injection volume of 5 μL, flow rate of 1.5 mL / min, and an Agilent Poroshell EC C column. 18 The effect of different concentrations of methanol extraction solvent on the detection effect under the conditions of (100mm×4.6mm, 2.7μm):

[0114] 100% methanol, 70% methanol, 50% methanol, and 30% methanol were used as extraction solvents, respectively. An appropriate amount of traditional Chinese medicine preparation containing abalone shell was taken, ground into a fine powder, and 0.5g of each sample was accurately weighed. 25mL of the above extraction solvents of different concentrations were added, the weight was measured, the mixture was heated under reflux for 30 minutes, cooled, weighed again, and the weight was replenished with solvent to make up the weight loss. The mixture was shaken well, filtered, and the filtrate was used as the test solution.

[0115] Accurately measure 5 μl of each test solution and inject it into the liquid chromatograph. Record the chromatographic peaks and examine the peak areas of luteolin, cassia aurantium, and ammonium glycyrrhizate. The results are shown in the table below. Figures 1-4 :

[0116] Table 2 Content of each component

[0117] condition 100% methanol 70% methanol 50% methanol 30% methanol Luteolin glycoside (mg / g) 0.420 0.510 0.467 0.381 Orange-yellow cassia extract (mg / g) 0.311 0.342 0.297 0.223 Ammonium glycyrrhizate (mg / g) 2.416 4.949 4.663 4.095

[0118] Among them, the contents of luteolin, cassia aurantium, and ammonium glycyrrhizate were relatively high in the 70% and 50% methanol extracts; while the contents of luteolin, cassia aurantium, and ammonium glycyrrhizate were relatively low in the 100% and 30% methanol extracts; therefore, the preferred extraction solvent is 70% methanol.

[0119] 2. Column temperature

[0120] Other chromatographic conditions remained the same as before. The methanol extraction solvent concentration was 70%, the injection volume was 5 μL, the flow rate was 1.5 mL / min, and the column was an Agilent Poroshell EC C10. 18 The effect of different column temperatures on detection results under conditions of (100mm × 4.6mm, 2.7μm):

[0121] The reference solution was tested at column temperatures of 20℃, 25℃, 30℃, and 40℃, and the chromatograms are shown in Figures 5-8. The results showed that no peak of cassia aurantium was detected in the chromatogram of the sample at 20℃, while peaks of luteolin, cassia aurantium, and ammonium glycyrrhizate were detected in the chromatograms of the sample at 25℃, 30℃, and 40℃. Therefore, a column temperature of 30℃ is preferred.

[0122] 3. Injection volume

[0123] Other chromatographic conditions remained the same as before. The methanol extraction solvent concentration was 70%, column temperature was 30℃, flow rate was 1.5 ml / min, and the column was an Agilent Poroshell EC C10. 18 The effect of different injection volumes on detection performance under conditions of (100mm × 4.6mm, 2.7μm):

[0124] The sample was analyzed with injection volumes of 1 μL, 5 μL, 10 μL, 20 μL, and 25 μL, and the resulting chromatograms are shown below. Figures 9-13 In the chromatograms of samples with injection volumes of 1 μL, 5 μL, and 10 μL, the peak areas of luteolin, cassia aurantium, and ammonium glycyrrhizate showed a linear relationship. In the chromatograms of samples with injection volumes of 20 μL and 25 μL, the peak shape of luteolin deteriorated and did not meet the separation requirements. Therefore, the preferred injection volume was 5 μL.

[0125] 4. Flow rate

[0126] Other chromatographic conditions remained the same as before. The methanol solution concentration was 70%, the column temperature was 30℃, the injection volume was 5 μL, and the chromatographic column was an Agilent Poroshell EC C10. 18 The effect of different flow rates on the detection effect was investigated under the conditions of (100mm × 4.6mm, 2.7μm):

[0127] Step (1): Take an appropriate amount of traditional Chinese medicine preparation containing abalone shell, grind it into a fine powder, take about 0.5g, weigh it accurately, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again and make up the weight lost with solvent, shake it well, filter it, and take the filtrate as the test solution.

[0128] Under the conditions of flow rates of 1.0 ml / min, 1.2 ml / min, 1.4 ml / min, 1.5 ml / min, and 1.6 ml / min respectively, 5 μl of each solution from step (1) was accurately measured and injected into the liquid chromatograph. The chromatographic peaks were recorded, and the separation of luteolin, cassia aurantium, and ammonium glycyrrhizate was investigated. The results are shown in the table below:

[0129] Table 3 Resolution of each group

[0130] condition 1.0 ml / min 1.2 ml / min 1.4 ml / min 1.5ml / min 1.6 ml / min Luteolin - - - - - Orange-yellow cassia seed extract - - 45.08 47.39 49.24 ammonium glycyrrhizate <1.5 <1.5 9.16 9.75 9.97

[0131] The results showed that the chromatograms collected under the conditions of 1.0 mL / min and 1.2 mL / min were... Figures 14-15 The separation rate of ammonium glycyrrhizate from adjacent impurities in the sample did not meet the standard requirements: 1.4 mL / min, 1.5 mL / min, and 1.6 mL / min. Figures 16-18 The chromatographic peaks of luteolin, cassia aurantium, and ammonium glycyrrhizate collected under the conditions all met the standard requirements for separation from adjacent impurities. Therefore, the preferred flow rate is 1.5 mL / min.

[0132] 5. Column type

[0133] Under the same chromatographic conditions as before, the effect of different chromatographic columns on the detection effect was investigated at a methanol solution concentration of 70%, a column temperature of 30℃, an injection volume of 5 μL, and a flow rate of 1.5 mL / min.

[0134] Step (1): Take an appropriate amount of Chinese medicine preparation containing abalone shell, grind it into a fine powder, take about 0.5g, weigh it accurately, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again and make up the weight lost with solvent, shake it well, filter it, and take the filtrate to obtain the product.

[0135] Under different column types and conditions, 5 μl of each solution from step (1) was precisely measured and injected into the liquid chromatograph. Chromatograms were recorded to examine the separation of luteolin, cassia aurantium, and ammonium glycyrrhizate. The results are shown in the table below:

[0136] Table 4 Resolution of each group

[0137] condition Agilent SB poro C18 Agilent Poroshell EC C18 Luteolin - - Orange-yellow cassia seed extract 44.07 47.39 ammonium glycyrrhizate 12.23 9.75

[0138] The results showed that the separation of luteolin, cassia aurantium, and ammonium glycyrrhizate all met the standard requirements using Agilent Poroshell EC C18 (100 mm × 4.6 mm, 2.7 μm) and Agilent SBporo C18 (100 mm × 4.6 mm, 2.7 μm) columns, and the columns demonstrated good robustness.

[0139] Example 2 Specificity Test

[0140] I. Reagent Preparation

[0141] 1. Chrysanthemum and catnip negative test solution

[0142] According to the prescription dosage in the preparation process of Shi Jue Ming traditional Chinese medicine preparation, take the other ingredients except for chrysanthemum and schizonepeta to prepare chrysanthemum and schizonepeta negative test sample. Take about 0.5g, accurately weigh it, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent. Shake well, filter it, and take the filtrate to obtain the chrysanthemum and schizonepeta negative test sample solution.

[0143] 2. Cassia seed negative test solution

[0144] According to the prescription amount in the preparation process of the traditional Chinese medicine preparation of Abalone Seed, take the other components except for Abalone Seed to make a negative test sample of Abalone Seed. Take about 0.5g, accurately weigh it, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent. Shake well, filter it, and take the filtrate to obtain the negative test sample solution of Abalone Seed.

[0145] 3. Licorice negative test solution

[0146] According to the prescription dosage in the preparation process of Shi Jue Ming traditional Chinese medicine preparation, take the other components except licorice to prepare licorice negative test sample. Take about 0.5g, accurately weigh it, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent. Shake well, filter it, and take the filtrate to obtain the licorice negative test sample solution.

[0147] 4. Reference solution

[0148] Take appropriate amounts of luteolin, cassia aurantium, and ammonium glycyrrhizate reference standards, accurately weigh them, and add 70% methanol solution to prepare a mixed solution containing 40 μg of luteolin, 20 μg of cassia aurantium, and 80 μg of ammonium glycyrrhizate per 1 mL, as the reference solution.

[0149] 5. Test solution

[0150] Take an appropriate amount of traditional Chinese medicine preparation containing abalone shell, grind it into a fine powder, take 0.5g, accurately weigh it, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent, shake it well, filter it, and take the filtrate as the test solution.

[0151] II. HPLC Detection

[0152] Accurately measure 5 μL of each of the solutions prepared in steps 1 to 5 above and inject them into the high-performance liquid chromatograph. Record the chromatograms as shown below. Figures 19-22 .

[0153] Liquid chromatography conditions:

[0154] The chromatographic column used was an Agilent Poroshell EC C10 column. 18 (100mm × 4.6mm, 2.7μm) chromatographic column;

[0155] Column temperature: 30℃;

[0156] Flow rate: 1.5 ml / min;

[0157] Mobile phase: Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. The gradient elution program is shown in Table 5.

[0158] Table 5 Gradient elution program

[0159]

[0160]

[0161] Detector: Diode array detector;

[0162] Injection volume: 5 μL

[0163] The detection wavelengths are 348nm (0–12 min), 284nm (12–14.5 min), and 252nm (14.5–30 min).

[0164] The results showed that the negative control solutions of chrysanthemum and schizonepeta or cassia seed or licorice had no interfering peaks at the same retention time as the chromatograms of the reference solutions.

[0165] The resolution of cassia aurantium, luteolin, and ammonium glycyrrhizate in the test solution was greater than 1.5, indicating complete separation and no interference with the determination of the content of cassia aurantium, luteolin, and ammonium glycyrrhizate.

[0166] The retention times of the chromatographic peaks of luteolin, cassia aurantium, and ammonium glycyrrhizate in step (5) are consistent with those in step (4).

[0167] Example 3 Solution Stability Test

[0168] Take an appropriate amount of a traditional Chinese medicine preparation containing abalone shell, grind it into a fine powder, accurately weigh 0.5g, add 25mL of 70% methanol solution, weigh again, heat under reflux for 30 minutes, cool, weigh again, and replenish the lost weight with solvent, shake well, filter, and take the filtrate as the test solution. Let it stand at room temperature for 24 hours, and then accurately inject 5μL of the test solution into the high-performance liquid chromatograph at 0, 6, 12, 18, and 24 hours. The chromatographic conditions are the same as in Example 2, and the chromatograms are recorded.

[0169] The results showed that the RSDs of the contents of luteolin, cassia seed extract, and ammonium glycyrrhizate after 24 hours at room temperature were 0.57%, 0.13%, and 0.18%, respectively, indicating that the samples obtained by this method had good stability after 24 hours at room temperature.

[0170] Example 4: Standard Curve Plotting

[0171] I. Reagent Preparation

[0172] 1. Accurately weigh 10 mg of luteolin reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with 70% methanol, shake well, and this is reference solution 1;

[0173] 2. Accurately weigh 11 mg of cassia aurantium reference standard and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with 70% methanol, and shake well to obtain reference solution 2.

[0174] 3. Accurately weigh 10 mg of ammonium glycyrrhizate reference standard and place it in a 50 ml volumetric flask. Dissolve and dilute to the mark with 70% methanol, and shake well to obtain reference solution 3.

[0175] 4. Accurately measure 1.2 ml of reference solution, 2.1 ml of reference solution, and 3.4 ml of reference solution, place them in the same 10 ml volumetric flask, dilute to the mark with 70% methanol, and shake well to obtain the linear solution.

[0176] II. HPLC Detection

[0177] Accurately measure 0.5 μl, 1 μl, 2 μl, 4 μl, 5 μl, 8 μl, 10 μl, 15 μl, 20 μl, and 25 μl of the linear solution respectively, inject them into the liquid chromatograph, and perform the liquid chromatography under the same conditions as in Example 2. Record the chromatograms.

[0178] Measure the chromatographic peak area; plot a standard curve with the injection volume on the x-axis and the peak area on the y-axis. The luteolin standard curve is shown below. Figure 23 Its regression equation is Y = 0.8532X + 1.2254, r = 0.9999; the standard curve of cassia seed extract is as follows: Figure 24 The regression equation is Y = 2.9589X - 2.5973, r = 0.9999; the standard curve for ammonium glycyrrhizate is as follows: Figure 25 The regression equation is Y = 0.3945X + 0.9724, r = 0.9999.

[0179] Example 5 Instrument Precision Test

[0180] Take an appropriate amount of traditional Chinese medicine preparation containing abalone shell, grind it into a fine powder, take 0.5g, accurately weigh it, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent, shake it well, filter it, and take the filtrate as the test solution.

[0181] Accurately measure 5 μl of the test solution and inject it into the liquid chromatograph. The liquid chromatography conditions are the same as in Example 2. Inject the sample 6 times consecutively and measure the peak area. The results show that the RSD values ​​of the peak areas of luteolin, cassia aurantium and ammonium glycyrrhizate are all less than 2.0%.

[0182] Example 6 Repeatability Test

[0183] I. Reagent Preparation

[0184] 1. Accurately weigh appropriate amounts of luteolin, cassia seed extract, and ammonium glycyrrhizate reference standards, and add 70% methanol solution to prepare a mixed solution containing 40 μg luteolin, 20 μg cassia seed extract, and 80 μg ammonium glycyrrhizate per mL. This solution is used as the reference solution. Prepare two aliquots using the same method.

[0185] 2. Take an appropriate amount of the traditional Chinese medicine preparation of abalone shell, grind it into a fine powder, take 0.5g, accurately weigh it, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent, shake well, filter it, and take the filtrate as the test solution. Prepare 6 portions in the same way.

[0186] II. HPLC Detection

[0187] 5 μl of the reference solution and the test solution were precisely measured and injected into the liquid chromatograph. The liquid chromatographic conditions were the same as in Example 2. The peak areas were measured. The results showed that the RSD values ​​of luteolin, cassia aurantium and ammonium glycyrrhizate were all less than 2.0%, indicating that the method had good repeatability.

[0188] Example 7 Accuracy Test

[0189] I. Reagent Preparation

[0190] 1. Accurately weigh 10 mg of luteolin reference standard and place it in a 100 ml volumetric flask. Dissolve and dilute to the mark with 70% methanol, shake well, and obtain the stock solution of luteolin recovery test reference standard.

[0191] 2. Accurately weigh 12 mg of cassia aurantium reference standard and place it in a 100 ml volumetric flask. Add 70% methanol to dissolve and dilute to the mark. Shake well to obtain the reference standard stock solution for the recovery test of cassia aurantium.

[0192] 3. Accurately weigh 27 mg of ammonium glycyrrhizate reference standard and place it in a 25 ml volumetric flask. Dissolve and dilute to the mark with 70% methanol, shake well, and obtain the stock solution of ammonium glycyrrhizate reference standard for recovery test.

[0193] 4. Accurately weigh 0.25g of the traditional Chinese medicine preparation containing abalone shell, totaling 9 portions.

[0194] Three of the samples were respectively supplemented with 0.5 ml of the reference stock solution for the recovery rate test of luteolin, 0.5 ml of the reference stock solution for the recovery rate test of cassia aurantium, and 0.5 ml of the reference stock solution for the recovery rate test of ammonium glycyrrhizate.

[0195] The other three samples were respectively added with 1 ml of luteolin recovery rate test reference stock solution, 1 ml of cassia aurantium recovery rate test reference stock solution, and 1 ml of glycyrrhizic acid ammonium recovery rate test reference stock solution;

[0196] The other three samples were prepared by adding 1.5 ml of the stock solution for the recovery rate test of luteolin, 1.5 ml of the stock solution for the recovery rate test of cassia aurantium, and 1.5 ml of the stock solution for the recovery rate test of ammonium glycyrrhizate, respectively.

[0197] Each of the above 9 samples was added to 25 mL of 70% methanol solution, weighed, heated under reflux for 30 minutes, cooled, weighed again, and the lost weight was replenished with solvent. The samples were shaken well, filtered, and the filtrate was collected to obtain the 50%, 100%, and 150% accuracy sample solutions.

[0198] 5. Accurately weigh appropriate amounts of luteolin, cassia seed extract, and ammonium glycyrrhizate reference standards, and add 70% methanol solution to prepare a mixed solution containing 40 μg luteolin, 20 μg cassia seed extract, and 80 μg ammonium glycyrrhizate per mL. This solution is used as the reference solution. Prepare two aliquots using the same method.

[0199] II. HPLC Detection

[0200] Accurately measure 5 μl of each of the accuracy sample and reference solution and inject them into the liquid chromatograph. The liquid chromatography conditions are the same as in Example 2. Measure the peak area and determine the contents of luteolin, cassia seed extract, and ammonium glycyrrhizate in the accuracy sample. Calculate the recovery rate. The results are shown in the table below:

[0201] Table 6. Experimental results of luteolin recovery rate

[0202]

[0203] Table 7. Experimental Results of the Recovery Rate of Cassia Seed Extract

[0204]

[0205]

[0206] Table 8 Experimental Results of Ammonium Glycyrrhizate Recovery Rate

[0207]

[0208] The results above show that when known amounts of cassia seed extract, luteolin, and ammonium glycyrrhizate reference standards were added to the sample, the average recoveries (n=9) of the three components were 95.63%, 100.92%, and 102.29%, respectively, with RSDs of 1.24%, 3.28%, and 2.70%, respectively, indicating that the method has good accuracy.

[0209] Example 8: Determination of the content of luteolin, cassia seed extract, and ammonium glycyrrhizate in a traditional Chinese medicine preparation made from abalone shell.

[0210] I. Reagent Preparation

[0211] Take appropriate amounts of luteolin, cassia aurantium, and ammonium glycyrrhizate reference standards, accurately weigh them, and add 70% methanol solution to prepare a mixed solution containing 40 μg of luteolin, 20 μg of cassia aurantium, and 80 μg of ammonium glycyrrhizate per 1 mL, as the reference solution.

[0212] Take appropriate amounts of three batches of traditional Chinese medicine preparations containing abalone shell, grind them into a fine powder, take 0.5g, accurately weigh it, add 25mL of 70% methanol solution, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, and make up the weight loss with solvent, shake it well, filter it, and take the filtrate as the test solution.

[0213] II. HPLC Detection

[0214] Accurately measure 5 μl each of the reference solution and the test solution, and inject them into the liquid chromatograph. The liquid chromatography conditions are the same as in Example 2. Record the chromatogram, calculate the content of the sample, and the results are as follows:

[0215] Table 9. Results of content determination for three batches of samples

[0216]

[0217] The above results indicate that the detection method provided by this invention is suitable for detecting the content of cassia seed extract, luteolin, and glycyrrhizic acid ammonium in abalone shell traditional Chinese medicine preparations. The method is simple to operate, has good specificity, solution stability, and linearity, and its repeatability and accuracy meet the requirements.

[0218] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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 detecting the active ingredients in traditional Chinese medicine preparations containing abalone shell, characterized in that, include: The raw materials for preparing the traditional Chinese medicine preparation containing abalone shell include: 250 parts by weight of abalone shell, 250 parts by weight of cassia seed, 250 parts by weight of schizonepeta, 250 parts by weight of chrysanthemum and 125 parts by weight of licorice. The preparation method of the traditional Chinese medicine preparation containing abalone shell includes: adding water to abalone shell, cassia seed, schizonepeta, chrysanthemum and licorice and decocting twice, with the amount of water added and the decoction time being 12 times the amount of water for 2 hours and 10 times the amount of water for 1.5 hours respectively, filtering, combining the filtrates, taking the filtrate and concentrating it, adding an appropriate amount of maltodextrin, granulating and drying to obtain the product. The test solution was obtained by extracting the traditional Chinese medicine preparation containing abalone shell with a 50 vol%~70 vol% methanol aqueous solution, and then detected by high performance liquid chromatography. One or more of luteolin, cassia seed extract, or ammonium glycyrrhizate were dissolved in a 50 vol% to 70 vol% methanol aqueous solution to obtain reference solutions. The reference solutions were analyzed by high performance liquid chromatography to obtain chromatograms of the reference substances. The components of traditional Chinese medicine preparations containing abalone shell were qualitatively and quantitatively analyzed based on the chromatograms of the reference substances. Chromatographic parameters include: The chromatographic column was a C18 column; the column temperature was 25℃~40℃; and the mobile phase was gradient elution. Mobile phase A is acetonitrile, and mobile phase B is a 0.05%~2% phosphoric acid solution; The gradient elution procedure includes: From 0 to 6 minutes, the volume fraction of mobile phase A was 16%. Over 6 to 8 minutes, the volume fraction of mobile phase A increased from 16% to 33%. From 8 min to 20 min, the volume fraction of mobile phase A increased from 33% to 36%. Over 20-24 minutes, the volume fraction of mobile phase A increased from 36% to 70%. Over 24-25 minutes, the volume fraction of mobile phase A decreased from 70% to 16%. The volume fraction of mobile phase A is 16% from 25 to 30 minutes. The detector used in high-performance liquid chromatography is a diode array detector; The detection wavelength is 348nm from 0 to 12 minutes. The detection wavelength was 284 nm for 12 min to 14.5 min. The detection wavelength is 252nm from 14.5min to 30min.

2. The detection method according to claim 1, characterized in that, The preparation of the test solution includes: mixing a traditional Chinese medicine preparation containing abalone shell with a solvent, heating under reflux for 15-60 minutes, and filtering to obtain the test solution.

3. The detection method according to claim 1 or 2, characterized in that, The ratio of the mass (g) to the volume (mL) of the traditional Chinese medicine preparation containing abalone shell and the solvent is 0.5g:(25~50)mL.

4. The detection method according to claim 1, characterized in that, The chromatographic column was an Agilent Poroshell ECC column. 18 100mm×4.6mm, 2.7μm; column temperature 30℃.

5. The detection method according to claim 1 or 4, characterized in that, The mobile phase flow rate was 1.4–1.6 mL / min; the injection volume was 1–10 μL.

6. The detection method according to claim 1, characterized in that, The separation degree between luteolin and cassia seed extract was 44.07~49.24, and the separation degree between cassia seed extract and ammonium glycyrrhizate was 9.16~12.

23. The linear range of luteolin is 19.88–795.00 ng, the linear range of cassia aurantium is 11.33–566.5 ng, and the linear range of ammonium glycyrrhizate is 40.03–2001.25 ng.

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