Detection method for simultaneously determining contents of various components in lip and tooth stomach clearing pill

Abstract

The invention discloses a detection method for simultaneously determining the content of multiple components in a lip and tooth stomach clearing pill, and belongs to the technical field of quality control of traditional Chinese medicine compound preparations. According to the detection method, the six components including gentiopicroside, geniposide, berberine hydrochloride, hesperidin, baicalin and chrysophanol can be simultaneously determined through an HPLC wavelength switching method; the method gives consideration to the index components of monarch drugs, ministerial drugs and adjuvant drugs in the lip and tooth stomach clearing pills, has the advantages of simplicity and convenience in operation, high specificity, high accuracy and good repeatability and tolerance, and has positive significance in quality control and comprehensive evaluation of the lip and tooth stomach clearing pills.

Description

Technical Field

[0001] This invention relates to the field of quality control technology for traditional Chinese medicine compound preparations, specifically to a detection method for simultaneously determining the content of multiple components in Chunchi Qingwei Pills. Background Technology

[0002] The Lip and Tooth Clearing Stomach Pill is composed of 14 herbs, including rhubarb, gardenia, scutellaria, gentian, phellodendron, and tangerine peel. It has the effect of clearing stomach heat and is used to treat symptoms such as swollen and painful gums, dry mouth and chapped lips, and sore throat caused by stomach heat. In the formula, rhubarb, phellodendron, scutellaria, and gardenia are the principal herbs, clearing heat, purging heat, detoxifying, and promoting urination. Gentian, anemarrhena, cimicifuga, rehmannia, and gypsum are the assistant herbs, clearing heat, cooling blood, moistening dryness, and promoting bowel movements. Tangerine peel, saposhnikovia, borneol, and menthol are the adjuvant herbs, dispelling wind, regulating qi, clearing heat, and opening the orifices. Angelica dahurica guides the medicine through the meridians and harmonizes the other herbs, serving as the guiding herb. The entire formula works together to clear stomach heat.

[0003] Traditional Chinese medicine compound preparations are formulated under the guidance of traditional Chinese medicine theory, following the principle of "principal, assistant, adjuvant, and guide". When selecting quality control indicators, the principal drug should be the first choice, while the assistant, adjuvant, and guide drugs should also be considered. Furthermore, adopting a quality control method that simultaneously quantifies multiple components is more scientific and reasonable, and can more comprehensively reflect the intrinsic quality of traditional Chinese medicine compound preparations.

[0004] The Lip and Tooth Clearing Stomach Pill is included in Part I of the 2025 edition of the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Chinese Pharmacopoeia"). The standard uses HPLC to determine the content of total anthraquinones in rhubarb and baicalin in scutellaria, but the quality control indicators are singular. Although some studies have established quantitative methods for one or more components in the prescription, such as gardenia (detecting geniposide), rhubarb (detecting emodin and chrysophanol), phellodendron (detecting berberine hydrochloride), and scutellaria (detecting baicalin), they have not yet covered the large quantities of gentian and tangerine peel in the prescription, and the quality control indicators are not comprehensive. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a detection method for simultaneously determining the content of multiple components in Chunchi Qingwei Pills, aiming to provide methodological support for the quality control and standard improvement of Chunchi Qingwei Pills.

[0006] The specific technical solution of the present invention is as follows: A method for simultaneously determining the content of multiple components in Chunchi Qingwei Pills includes the following steps: The sample of Chunchi Qingwei Pills was extracted with methanol to obtain a test solution; the test solution was then analyzed by high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Column: C 18 The chromatographic column has dimensions of 250 mm × 4.6 mm and a diameter of 5 μm. Using methanol (A)-0.05~0.15% phosphoric acid (B) as the mobile phase, gradient elution was performed as follows: 0–25 min, 20% A; 25–35 min, 20% A→40% A; 35–45 min, 40% A; 45–55 min, 40% A→50% A; 55–70 min, 50% A→80% A; 70–75 min, 80% A→85% A; 75–85 min, 85% A; 85–86 min, 85% A→20% A; 86–90 min, 20% A. Flow rate: 0.9–1.1 mL / min; column temperature: 25–35 ℃; injection volume: 10 μL; Detection wavelengths: 0–35 min, 240 nm for geniposide and gentiopicrin; 35–75 min, 280 nm for berberine hydrochloride, hesperidin and baicalin; 75–90 min, 254 nm for rhein.

[0007] Preferably, in the above detection method, the mass-to-volume ratio of the Chunchi Qingwei Pill sample to methanol is 1 g: 100 mL.

[0008] Preferably, in the above detection method, the sample of Chunchi Qingwei Pill is extracted by reflux, and the reflux extraction time is 2-3 hours. In some embodiments of the present invention, the preparation method of the test solution is as follows: the Chunchi Qingwei Pill sample is finely ground, accurately weighed and added to methanol, weighed, heated under reflux, cooled, weighed again, the weight loss is made up with methanol, and filtered to obtain the final product.

[0009] Preferably, in the above detection method, the solution is filtered through a 0.45 μm filter membrane before being injected for detection.

[0010] Preferably, the above detection method further includes the following steps: weighing the standards gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein, and preparing mixed reference solutions of different concentrations using methanol as the solvent; performing detection under the same chromatographic conditions as the test solution; obtaining regression equations for each component based on the peak areas of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein at different concentrations; and calculating the content of each component in the test solution based on the regression equations.

[0011] In the above detection method, the chromatographic column can be selected from CAPCELL Peak-C. 18 Pntulips® NT-C 18 Waters sunfire-C 18 wait.

[0012] Preferably, in the above detection method, the flow rate is 1.0 mL / min and the column temperature is 30 ℃.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, by analyzing the proportions of each herb in Chunchi Qingwei Pill and their constituent index components, first determined gentiopicrin (gentian), geniposide (gardenia), berberine hydrochloride (Phellodendron), hesperidin (tangerine peel), baicalin (Scutellaria), and rhein (rhubarb) as quantitative index components to construct a comprehensive evaluation scheme for Chunchi Qingwei Pill. Then, for these six components, simultaneous determination was achieved using HPLC wavelength switching, providing an experimental basis for the quality control and standard improvement of Chunchi Qingwei Pill. Furthermore, the method of this invention has the advantages of simple operation, high specificity, high accuracy, good repeatability, and good tolerability. Attached Figure Description

[0014] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0015] Figure 1 The figures are HPLC chromatograms of the Chunchi Qingwei Pills in Example 2, where Figure A is the mixed reference standard, Figure B is the Chunchi Qingwei Pills test sample, Figure C is the negative sample lacking Gentiana scabra, Figure D is the negative sample lacking Gardenia jasminoides, Figure E is the negative sample lacking Phellodendron chinense, Figure F is the negative sample lacking Citrus reticulata, Figure G is the negative sample lacking Scutellaria baicalensis, and Figure H is the negative sample lacking Rheum palmatum. Figure 2 The HPLC chromatograms of the Lip and Tooth Cleansing Stomach Pills in Example 3 were obtained using different instruments. Figure 3 The HPLC chromatograms of the Lip and Tooth Cleansing Stomach Pills in Example 3 under different chromatographic columns are shown below. Figure 4 The HPLC chromatograms of the Lip and Tooth Cleansing Stomach Pills in Example 3 at different temperatures are shown below. Figure 5 The HPLC chromatograms of the Lip and Tooth Cleansing Stomach Pills in Example 3 at different flow rates are shown below. Figure 6 The HPLC chromatograms of the Lip and Tooth Cleansing Stomach Pills in Example 3 at different phosphoric acid concentrations are shown below. Figure 7 The HPLC chromatogram of Chunchi Qingwei Pills in Comparative Example 1 under acetonitrile-water mobile phase; Explanation of the symbols in the attached figure: Peak 1 is gentiopicrin, Peak 2 is geniposide, Peak 3 is berberine hydrochloride, Peak 4 is hesperidin, Peak 5 is baicalin, and Peak 6 is rhein. Detailed Implementation

[0016] The technical solution of the present invention will be described in detail below in conjunction with the drawings and embodiments.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0018] For traditional Chinese medicine compound preparations, in order to more comprehensively reflect the quality of traditional Chinese medicine compound preparations, a reasonable quality control method should take into account the monarch, minister, assistant, and envoy herbs. Chunchi Qingwei Pills are composed of 14 traditional Chinese medicines, and the existing quality control method for Chunchi Qingwei Pills is single, and only a small number of components can be quantitatively detected simultaneously, so it is difficult to comprehensively evaluate Chunchi Qingwei Pills.

[0019] In order to solve the technical problem that the existing quality control method for Chunchi Qingwei Pills is relatively one-sided, the present invention provides a detection method for simultaneously determining the contents of multiple components in Chunchi Qingwei Pills. This method supports the simultaneous quantitative determination of Gentiana scabra, Gardenia jasminoides, Phellodendron amurense, Citrus reticulata, Scutellaria baicalensis, and Rheum palmatum, which have relatively high contents in Chunchi Qingwei Pills, so as to achieve the purpose of comprehensively evaluating the quality of Chunchi Qingwei Pills.

[0020] The prescription of Chunchi Qingwei Pills contains many medicinal flavors, and each traditional Chinese medicine contains multiple active ingredients. In the previous experiments of the inventors, gentiopicroside (Gentiana scabra), geniposide (Gardenia jasminoides), berberine hydrochloride (Phellodendron amurense), hesperidin (Citrus reticulata), baicalin (Scutellaria baicalensis), mangiferin (Anemarrhena asphodeloides), isoferulic acid (Cimicifuga foetida), imperatorin (Angelica dahurica), [prim-O-glucosylcimifugin, 5- O -O-methylvisammioside (Saposhnikovia divaricata)], [chrysophanol, rhein, emodin, aloe-emodin, physcion (Rheum palmatum)] and other ingredients were selected as the test components. It was found that for the 4 herbs of Anemarrhena asphodeloides, Cimicifuga foetida, Saposhnikovia divaricata, and Angelica dahurica, due to their relatively small proportion in the prescription or the relatively low content of the target components, after being prepared into test samples, the response values of their components were relatively low; and for the other 4 components in Rheum palmatum, there were also problems of relatively low response values and poor repeatability. Therefore, the present invention finally determines gentiopicroside, geniposide, berberine hydrochloride, hesperidin, baicalin, and chrysophanol as the quantitative components to establish a quality control method for Chunchi Qingwei Pills.

[0021] According to the Chinese Pharmacopoeia (2025 edition), different Chinese medicinal herbs are suitable for extraction using different solvents. For example, Phellodendron bark uses acetonitrile-0.1% phosphoric acid (50:50) as the extraction solvent, Scutellaria baicalensis uses 70% ethanol, and Rheum palmatum uses methanol. This invention uses methanol as the extraction solvent and investigated different extraction methods (ultrasound, reflux) and solvent volumes (50, 100 mL). The results showed that adding 100 mL of methanol per 1 g of sample and reflux extraction yielded the best results. The reflux extraction times of 0.5, 1, 1.5, 2, 2.5, and 3 h were also investigated. The results showed that baicalin was basically completely extracted after 2 h of reflux extraction, while the other five components were basically completely extracted after 1 h of reflux extraction.

[0022] The six components, including gentiopicroside, have different structures and therefore different maximum absorbance values. To achieve simultaneous determination of all six components, this invention employs a diode array detector to acquire the spectra of the six components (200–400 nm). The results show that gentiopicroside has absorption peaks at 243 and 274 nm, geniposide at 240 nm, berberine hydrochloride at 228, 265, and 347 nm, hesperidin at 283 nm, baicalin at 276 and 315 nm, and rhein at 223 and 254 nm. Based on the peak times of each component and to improve detection sensitivity, this invention ultimately determines that gentiopicroside and geniposide are detected at 240 nm, berberine hydrochloride, hesperidin, and baicalin at 280 nm, and rhein at 254 nm.

[0023] This invention also investigated two mobile phase systems: acetonitrile-water and methanol-water. It was found that geniposide and gentiopicrin showed poor separation in the acetonitrile-water system but better separation in the methanol-water system. Therefore, methanol was chosen as the organic phase of the mobile phase system. To improve the peak shapes of the six components, different concentrations of formic acid (0.05%, 0.1%, 0.15%) and phosphoric acid (0.05%, 0.1%, 0.15%) were added to the aqueous phase. It was found that adding phosphoric acid had the best effect, resulting in consistent peak shapes for all six components at the three concentrations mentioned above.

[0024] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0025] The 15 batches of Chunchi Qingwei Pills samples used in the following examples (batch numbers: 20250105, 20250103, 20250202, 20250301, 20250101, 20241107, 20241211, 20240701, 20241105, 20240704, 20241103, 20241002, 20241202, 20240301, 20240605) were purchased from Changchun Renmin Pharmaceutical Co., Ltd. (4.5g / bag, shelf life 36 months).

[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0027] Example 1 This example provides an HPLC method for simultaneously determining the content of multiple components in Chunchi Qingwei Pills, including the following steps: (1) Preparation of each test solution.

[0028] Preparation of the test solution: Take the sample of Chunchi Qingwei Pill, grind it into a fine powder, accurately weigh 1 g, place it in a stoppered conical flask, accurately add 100 mL of methanol, weigh it, heat under reflux for 2 h, cool it, weigh it again, make up the weight loss with methanol, filter it, and the test solution is obtained.

[0029] Preparation of mixed reference solution: Accurately weigh 9.68 mg of gentiopicroside, 9.37 mg of geniposide, 10.61 mg of berberine hydrochloride, 9.82 mg of hesperidin, 10.26 mg of baicalin, and 11.64 mg of rhein, and place them into 25, 25, 25, 25, 25, and 50 mL volumetric flasks, respectively. Dissolve in methanol and dilute to the mark. Then, pipette 3, 4, 2, 3, 6, and 1.5 mL of each solution into the same 20 mL volumetric flask, and dilute to the mark with methanol, respectively. The resulting solutions contain 57.92 μg / mL of gentiopicroside, 74.40 μg / mL of geniposide, 40.74 μg / mL of berberine hydrochloride, 56.15 μg / mL of hesperidin, 119.67 μg / mL of baicalin, and 17.32 μg / mL of rhein. Prepare a mixed reference standard stock solution with a concentration of μg / mL; pipette 0.5, 1, 2, 3, 4, and 5 mL of the mixed reference standard stock solution into 5 mL volumetric flasks, add methanol to the mark, and obtain mixed reference standard solutions with gradient concentrations.

[0030] Negative sample solutions: Based on the prescription ratio and preparation process of Chunchi Qingwei Pills, negative samples lacking rhubarb (i.e., without emodin), scutellaria (i.e., without baicalin), tangerine peel (i.e., without hesperidin), phellodendron bark (i.e., without berberine hydrochloride), gardenia (i.e., without geniposide), and gentian (i.e., without gentiopicrin) were prepared. Then, following the preparation method of the test sample solution, six negative sample solutions were prepared.

[0031] (2) HPLC detection.

[0032] Take appropriate amounts of each solution obtained in step (1) as needed, filter them through a 0.45 μm filter membrane, inject them for analysis, and record each chromatogram. Specifically, the chromatographic conditions are as follows: Column: CAPCELL Peak-C 18 (250 mm × 4.6 mm, 5 μm); Mobile phase: methanol (A)-0.1% phosphoric acid (B), gradient elution (0~25 min, 20%A; 25~35 min, 20%A→40%A; 35~45 min, 40%A; 45~55 min, 40%A→50%A; 55~70 min, 50%A→80%A; 70~75 min, 80%A→85%A; 75~85 min, 85%A; 85~86 min, 85%A→20%A; 86~90 min, 20%A); Flow rate: 1.0 mL / min; column temperature: 30 ℃; Detection wavelengths: 240 nm (0–35 min for geniposide and gentiopicrin), 280 nm (35–75 min for berberine hydrochloride, hesperidin, and baicalin), 254 nm (75–90 min for rhein). Injection volume: 10 μL.

[0033] Example 2 Following the detection method in Example 1, this example examines the method as follows: (1) Specificity test.

[0034] Take the test solution, the mixed reference stock solution, and the six negative sample solutions, filter them through a 0.45 μm filter membrane, inject them for analysis, and record the chromatograms.

[0035] The results showed that the retention times of the six components in the chromatogram of the test sample solution were the same as those in the chromatogram of the mixed reference standard, and the resolution with adjacent peaks was greater than 1.5. No gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, or rhein peaks were observed at the corresponding retention times in the negative chromatograms of gentian, gardenia, phellodendron, tangerine peel, scutellaria, and rhubarb, indicating no interference from negative samples (see [link to negative sample chromatogram]). Figure 1 The above results indicate that the method of the present invention has good specificity.

[0036] (2) Examination of linear relationships.

[0037] Six mixed standard solutions of varying concentrations were filtered through a 0.45 μm filter membrane and injected for analysis. The peak areas of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein were recorded at different concentrations. Linear regression was performed with the peak areas of the six components at different concentrations as the ordinate (Y) and the corresponding mass concentrations (μg / mL) as the abscissa (X). The regression equations and linear ranges for each component are shown in Table 1.

[0038] Table 1. Regression equations, correlation coefficients, and linear ranges of the six components in Chunchi Qingwei Pills.

[0039] (3) Instrument precision test.

[0040] The third point of the gradient concentration was filtered through a microporous membrane (0.45 μm), and the sample was continuously injected and measured 6 times.

[0041] The results showed that the peak area RSDs of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein were 0.32%, 0.33%, 0.16%, 0.34%, 0.17%, and 0.21%, respectively, indicating that the instrument had good precision.

[0042] (4) Repeatability test.

[0043] Take the sample with batch number 20250105, accurately weigh 6 portions, each about 1 g, prepare test solutions for each portion, and inject them for testing.

[0044] The results showed that the average contents of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein were 3.449, 4.432, 2.280, 2.547, 8.415, and 0.6913 mg / g, respectively, with RSDs of 1.20%, 1.27%, 0.96%, 1.19%, 1.12%, and 1.28%, respectively, indicating that the method of the present invention has good reproducibility.

[0045] (5) Stability test.

[0046] The test solution from batch number 20250105 was analyzed at 0, 3, 9, 15, 21, and 30 h. The peak area RSDs of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein were 1.36%, 1.20%, 1.00%, 1.29%, 1.27%, and 0.99%, respectively. All components of the test solution were stable within 30 h.

[0047] (6) Intermediate precision test.

[0048] Sample number 20240605 was taken, ground finely, and two researchers prepared the test solution separately. The solution was then analyzed using three different brands of instruments under the chromatographic conditions shown in Example 1 (see Example 1). Figure 2 ).

[0049] The results are as follows Figure 2 As shown, the resolutions of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein were all greater than 1.5 under different instruments, with RSDs of 1.90%, 1.00%, 2.47%, 1.00%, 0.68%, and 0.98%, respectively (see Table 2). These results indicate that the method of this invention has good intermediate precision.

[0050] Table 2. Results of intermediate precision tests on six components of Chunchi Qingwei Pills

[0051] (7) Recovery test.

[0052] Take a sample (batch number 20250105) with known contents of gentiopicroside, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein, accurately weigh 0.5 g, and divide into 6 portions. Add 100% of each portion to a separately prepared single reference solution (6 mL of 0.2924 mg / mL gentiopicroside, 6.5 mL of 0.3456 mg / mL geniposide, 3 mL of 0.3629 mg / mL berberine hydrochloride, 5 mL of 0.2628 mg / mL hesperidin, 15 mL of 0.2902 mg / mL baicalin, and 1.5 mL of 0.2385 mg / mL rhein), and add methanol to make up to 100 mL. Prepare test solutions separately, inject the samples, determine the peak area of ​​each component, and calculate the recovery rate by substituting into the regression equation.

[0053] The results showed that the average recovery rates of the six components were 98.69%–102.42%, and the RSDs were 1.06%–2.29% (see Table 3), indicating that the method of the present invention has good recovery rates and meets the analytical requirements.

[0054] Table 3. Results of the recovery rate test of 6 different substances in Chunchi Qingwei Pills (n=6)

[0055] Example 3 Unlike Example 1, the chromatographic conditions were changed in this example, as follows: Take sample number 20240605, grind it finely, prepare the test solution, and chromatographically analyze it on different columns [CAPCELL Peak-C]. 18 Pntulips® NT-C 18 Waters sunfire-C 18 Samples were injected and measured under different conditions (250 mm × 4.6 mm, 5 μm), different temperatures (25, 30, 35℃), different phosphoric acid concentrations (0.05%, 0.1%, 0.15%), and different flow rates (0.9, 1.0, 1.1 mL / min).

[0056] The results are as follows Figure 3-6 As shown in Table 4, under all experimental conditions, the resolution of each component was greater than 1.5, and the RSD of their contents was less than 3.0%. These results indicate that the method has good robustness.

[0057] Table 4. Durability Study Results of Six Ingredients in Lip and Tooth Cleansing Stomach Pills

[0058] Example 4 Following the method shown in Example 1, 15 batches of Chunchi Qingwei Pills were taken in this example. Two test solutions were prepared for each batch. The peak areas of the six components were measured by injection, and the contents of each component were calculated by substituting them into the regression equation. The test results are shown in Table 5.

[0059] Table 5. Results of determination of the content of 6 components in 15 batches of Chunchi Qingwei Pills (mg / g, n=2)

[0060] Table 5 shows that the contents of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein in different batches of Chunchi Qingwei Pills ranged from 2.343 to 3.736, 4.160 to 5.612, 2.161 to 2.996, 2.478 to 4.138, 6.847 to 8.804, and 0.6037 to 0.8557, respectively. The content of each component fluctuated little, and the quality was relatively stable.

[0061] Comparative Example 1 Unlike Example 1, this example used acetonitrile-water (10:90), acetonitrile-water (12:88), acetonitrile-water (15:85), and acetonitrile-water (18:82) as mobile phases for isocratic elution. The results showed that the separation degree of both gentiopicrin and geniposide was less than 1.5 (…). Figure 7 ).

[0062] In summary, this invention has successfully established an HPLC method for the simultaneous determination of the content of six indicator components in Chunchi Qingwei Pills. This method is simple to operate, highly specific, accurate, repeatable, and well-tolerated, and can be used for the quality control and evaluation of Chunchi Qingwei Pills.

[0063] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for simultaneously determining the content of multiple components in Chunchi Qingwei Pills, characterized in that, Includes the following steps: The sample of Chunchi Qingwei Pills was extracted with methanol to obtain a test solution; the test solution was then analyzed by high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Column: C 18 Chromatographic column, 250 mm × 4.6 mm, 5 μm; Using methanol (A)-0.05~0.15% phosphoric acid (B) as the mobile phase, gradient elution was performed as follows: 0–25 min, 20% A; 25–35 min, 20% A→40% A; 35–45 min, 40% A; 45–55 min, 40% A→50% A; 55–70 min, 50% A→80% A; 70–75 min, 80% A→85% A; 75–85 min, 85% A; 85–86 min, 85% A→20% A; 86–90 min, 20% A. Flow rate: 0.9–1.1 mL / min; column temperature: 25–35 ℃; injection volume: 10 μL; Detection wavelengths: 0–35 min, 240 nm for geniposide and gentiopicrin; 35–75 min, 280 nm for berberine hydrochloride, hesperidin and baicalin; 75–90 min, 254 nm for rhein.

2. The detection method according to claim 1, characterized in that, The mass-to-volume ratio of the Lip and Tooth Cleansing Stomach Pill sample to methanol was 1 g: 100 mL.

3. The detection method according to claim 1 or 2, characterized in that, The sample of the Chunchi Qingwei Pill was extracted by methanol reflux for 2-3 hours.

4. The detection method according to claim 3, characterized in that, The preparation method of the test solution is as follows: grind the sample of Chunchi Qingwei Pill into fine powder, accurately weigh it and add it to methanol, weigh it, heat it under reflux, cool it, weigh it again, make up the weight loss with methanol, and filter it to obtain the solution.

5. The detection method according to claim 1, characterized in that, The test solution was filtered through a 0.45 μm filter membrane before being injected for testing.

6. The detection method according to claim 1, characterized in that, It also includes the following steps: Weigh out the standards gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein, and prepare mixed reference solutions of different concentrations using methanol as the solvent. Detect these solutions under the same chromatographic conditions as the test solution. Based on the peak areas of gentiopicrin, geniposide, berberine hydrochloride, hesperidin, baicalin, and rhein at different concentrations, obtain regression equations for each component. Then, calculate the content of each component in the test solution based on the regression equations.

7. The detection method according to claim 1, characterized in that, The chromatographic column was selected from CAPCELL Peak-C. 18 Pntulips® NT-C 18 Waters sunfire-C 18 .

8. The detection method according to claim 1, characterized in that, The flow rate is 1.0 mL / min.

9. The detection method according to claim 1, characterized in that, The column temperature is 30 ℃.

10. The application of the detection method for simultaneously determining the content of multiple components in Chunchi Qingwei Pills as described in claim 1 in the quality control of Chunchi Qingwei Pills.

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