Multi-component material basis-based Ganhai Weikang capsule characteristic chromatogram as well as construction method and application of Ganhai Weikang capsule characteristic chromatogram

Abstract

The invention discloses a Ganhai Weikang capsule specific chromatogram based on a multi-component material basis as well as a construction method and application thereof, and belongs to the technical field of quality control and evaluation of Chinese patent medicines. A high performance liquid chromatography-high resolution mass spectrometry method is adopted for detection, and glycyrrhizic acid, liquiritin, liquiritigenin, apiose liquiritin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, narirutin, p-coumaric acid and kaempferide are taken as reference substances; the specific chromatogram is successfully constructed by detecting different batches of Ganhai Weikang capsules. The method for detecting the specific chromatogram of the Ganhai Weikang capsule, established by the invention, is simple and convenient to operate, high in precision and rapid in analysis, can realize efficient and synchronous detection of 12 characteristic components, and has good stability, accuracy and reproducibility. According to the method, synchronous and rapid analysis of multiple components is realized, the detection efficiency is remarkably improved, and reliable scientific basis and technical support are provided for quality control and clinical curative effect guarantee of the Ganhai Weikang capsules.

Description

Technical Field

[0001] This invention belongs to the field of quality control and evaluation technology of traditional Chinese medicine, specifically involving a characteristic spectrum of Ganhaiweikang capsules based on multi-component material basis, its construction method and application. Background Technology

[0002] Ganhai Weikang Capsules (National Drug Approval Number: Z20025708) are composed of eight medicinal herbs: licorice, cuttlebone, sea buckthorn, immature bitter orange, atractylodes macrocephala, phellodendron bark, corydalis rhizome, and total glycosides of gynostemma pentaphyllum. In this formula, licorice replenishes qi and generates fluids, and has anti-inflammatory and stomach-protecting effects; cuttlebone neutralizes acid and relieves pain; atractylodes macrocephala strengthens the spleen and dries dampness; immature bitter orange breaks up qi stagnation, eliminates stagnation, resolves phlegm, and disperses masses; sea buckthorn strengthens the spleen and aids digestion; phellodendron bark clears heat and dries dampness; corydalis rhizome invigorates blood and relieves pain; and total glycosides of gynostemma pentaphyllum nourish the heart and strengthen the spleen, replenish qi and blood. The entire formula simultaneously strengthens the spleen and replenishes qi, promotes qi circulation and eliminates stagnation, and combines astringent and analgesic effects with clearing heat and drying dampness, achieving the combined effects of strengthening the spleen and stomach, and astringing and relieving pain. Clinically, Ganhai Weikang Capsules can be used for gastric and duodenal ulcers, chronic gastritis, and reflux esophagitis caused by spleen deficiency and qi stagnation.

[0003] Currently, research on the quality testing of Ganhai Weikang capsules is still in its infancy. Existing literature only reports thin-layer chromatography identification of individual medicinal materials in the prescription and the determination of ammonium glycyrrhizate content. However, as a compound preparation composed of multiple traditional Chinese medicines, Ganhai Weikang capsules have a complex and diverse chemical composition, with synergistic and interactive effects between the medicinal ingredients. Relying solely on the detection of a single component or individual medicinal material is insufficient to comprehensively reflect its overall quality characteristics and cannot effectively support stability evaluation and production consistency control. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a characteristic spectrum of Ganhaiweikang capsules based on multi-component material basis, its construction method and application, so as to solve the technical problem that the existing detection methods of Ganhaiweikang capsules can only reflect individual components and are difficult to comprehensively evaluate the overall quality of the compound.

[0005] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of this invention discloses a method for constructing a characteristic spectrum of Ganhaiweikang capsules based on a multi-component material basis, comprising the following steps: S1. Take different batches of Ganhaiweikang capsules as test samples and prepare test sample solutions; S2. Prepare single reference solutions using glycyrrhizic acid, glycyrrhizin, glycyrrhizin, celery glycyrrhizin, naringin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin naringin, p-coumaric acid and kaempferol as reference standards; S3. Perform chromatographic analysis on the test solution obtained in S1 and the single reference solution obtained in S2 to obtain the corresponding chromatograms; S4. Perform similarity analysis on the chromatograms of the test solution obtained in S3; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram. Import the total ion chromatogram into Xcalibur software and perform data analysis based on the peak elution of the chromatogram to obtain the mass spectrometry results of each chemical component. S6. Based on the chromatograms of the test solution and the single reference solution obtained in S3, and combined with the total ion chromatogram and the mass spectrometry results of the chemical components obtained in S5, determine the chemical components of each peak in the characteristic spectrum and obtain the characteristic spectrum of Ganhaiweikang capsules.

[0006] In a second aspect, the present invention discloses the characteristic chromatogram of Ganhaiweikang capsules obtained by the above-described construction method.

[0007] A third aspect of the present invention discloses the application of the above-described construction method in the quality control or quality evaluation of Ganhaiweikang capsules or similar drugs.

[0008] In a fourth aspect, the present invention discloses a method for detecting the quality of Ganhaiweikang capsules. The method utilizes the aforementioned characteristic chromatogram of Ganhaiweikang capsules to detect the quality of Ganhaiweikang capsules or its equivalent drugs, using one or more of the following as quality markers: glycyrrhizic acid, glycyrrhizin, glycyrrhizin, celery glycyrrhizin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin-naringenin, p-coumaric acid, and kaempferol.

[0009] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for constructing a characteristic spectrum of Gan Hai Wei Kang capsules based on a multi-component material basis. Using glycyrrhizic acid, glycyrrhizin, glycyrrhizin, apigenin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin, p-coumaric acid, and kaempferol as reference standards, high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HDMS) are coupled to simultaneously detect multiple components in Gan Hai Wei Kang capsules, achieving a holistic characterization of chemical components and overcoming the limitations of traditional single-component detection. This method comprehensively reflects the main active ingredient characteristics of each medicinal material in Gan Hai Wei Kang capsules and reveals the synergistic effects of the compound formulation. Through characteristic peak comparison and similarity analysis, different batches of samples can be effectively distinguished and abnormal samples identified, ensuring the consistency of product origin and formulation. This method is scientifically sound, with rapid and simple pretreatment. Methodological verification, including precision, repeatability, and stability tests, demonstrates good reproducibility and stability. It effectively overcomes the limitations of traditional detection methods, fully meets the practical needs of quality standard setting and industrial quality control, and accurately, clearly, and objectively evaluates the quality of Ganhaiweikang capsules. It provides a quantitative and traceable chemical basis for establishing quality standards for Ganhaiweikang capsules, playing a vital role and value in controlling the quality of Ganhaiweikang capsules and ensuring clinical efficacy. This method also contributes to promoting the modernization and standardization of quality control for traditional Chinese medicine compound preparations.

[0010] Furthermore, using methanol solution as the extraction solvent ensures a stable chromatographic peak baseline, a large number of components, and good extraction effect; the preparation of the test solution by ultrasonic followed by rotary evaporation concentration ensures a large peak area in the chromatogram and a large number of detected components.

[0011] Furthermore, in the chromatographic analysis, a column temperature of 30℃ was selected to ensure good separation of each component in the chromatographic peak and good peak shape; a detection wavelength of 280 nm was selected to ensure the most comprehensive information contained in the chromatogram and a stable baseline; a flow rate of 0.6 mL / min was selected to ensure high separation of each target component and symmetrical peak shape; acetonitrile-0.1% phosphoric acid was used as the mobile phase to ensure good separation of each component in Ganhaiweikang capsules; and a specific elution program was selected to ensure good separation of each component in Ganhaiweikang capsules, high peak height, stable baseline, and complete chromatographic information. Attached Figure Description

[0012] Figure 1 The characteristic chromatogram of the Ganhai Weikang capsule of the present invention; Figure 2 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test sample solution according to the present invention; Figure 3 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test sample solution according to the present invention; Figure 4This is a chromatogram obtained by optimizing column temperature under chromatographic conditions according to the present invention; Figure 5 The chromatogram and full-wavelength scan (190~800nm) obtained by optimizing the detection wavelength under chromatographic conditions in this invention are shown. Figure 6 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention; Figure 7 This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention; Figure 8 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention; Figure 9 The chromatogram (A) and mass spectrum (B) of the hesperidin standard of the present invention are shown below. Figure 10 The UV spectrum (A) and 3D image (B) of the hesperidin standard of the present invention are shown. Figure 11 The chromatogram (A) and mass spectrum (B) of the eugenol standard of the present invention are shown below. Figure 12 The UV spectrum (A) and 3D image (B) of the eugenol standard of the present invention are shown. Figure 13 The chromatogram (A) and mass spectrum (B) of the p-coumaric acid standard of the present invention are shown below. Figure 14 The UV spectrum (A) and 3D image (B) of the p-coumaric acid standard of the present invention are shown below. Figure 15 The chromatogram (A) and mass spectrum (B) of the glycyrrhizin standard of the present invention are shown below. Figure 16 The UV spectrum (A) and 3D image (B) of the glycyrrhizin standard of the present invention are shown. Figure 17 The chromatogram (A) and mass spectrum (B) of the glycyrrhizin standard of the present invention are shown below. Figure 18 The UV spectrum (A) and 3D image (B) of the glycyrrhizin standard of the present invention are shown. Figure 19 The chromatogram (A) and mass spectrum (B) of the glycyrrhizic acid standard of the present invention are shown below. Figure 20 The UV spectrum (A) and 3D image (B) of the glycyrrhizic acid standard of the present invention are shown. Figure 21 The chromatogram (A) and mass spectrum (B) of the chlorogenic acid standard of the present invention are shown below. Figure 22 The UV spectrum (A) and 3D image (B) of the chlorogenic acid standard of the present invention are shown. Figure 23The chromatogram (A) and mass spectrum (B) of the apigenin glycyrrhizin standard of the present invention are shown below. Figure 24 The UV spectrum (A) and 3D image (B) of the apigenin glycyrrhizin standard of the present invention are shown. Figure 25 The chromatogram (A) and mass spectrum (B) of the kaempferol standard of the present invention are shown below. Figure 26 The UV spectrum (A) and 3D image (B) of the kaempferol standard of the present invention are shown. Figure 27 The chromatogram (A) and mass spectrum (B) of the naringenin standard of the present invention are shown below. Figure 28 The UV spectrum (A) and 3D image (B) of the naringenin standard of the present invention are shown. Figure 29 The chromatogram (A) and mass spectrum (B) of the protocatechuic acid standard of the present invention are shown below. Figure 30 The UV spectrum (A) and 3D image (B) of the protocatechuic acid standard of the present invention are shown. Figure 31 The chromatogram (A) and mass spectrum (B) of the rutin standard of the present invention are shown. Figure 32 The UV spectrum (A) and 3D image (B) of the rutin standard of the present invention are shown. Figure 33 The characteristic chromatograms of 15 batches of Ganhaiweikang capsules tested according to the present invention are shown. Detailed Implementation

[0013] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0014] This invention provides a method for constructing a characteristic spectrum of Ganhai Weikang capsules based on a multi-component material basis, comprising the following steps: S1. Weigh 1.0 g of the contents of different batches of Ganhaiweikang capsules, add 20 mL of methanol solution, extract by ultrasonication (ultrasonic frequency: 40 kHz, power: 720 W) for 30 min, filter, concentrate by rotary evaporation to 5 mL, filter through a 0.45 µm microporous membrane to obtain the Ganhaiweikang capsule test solution.

[0015] S2. Accurately weigh glycyrrhizic acid, glycyrrhizin, glycyrrhizin, apigenin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin, p-coumaric acid, and kaempferol, and dissolve them in methanol solution to prepare a single reference solution containing 0.42 mg glycyrrhizic acid, 0.32 mg glycyrrhizin, 0.38 mg glycyrrhizin, 0.41 mg apigenin, 0.43 mg naringenin, 0.36 mg chlorogenic acid, 0.49 mg syringic acid, 0.49 mg protocatechuic acid, 0.31 mg hesperidin, 0.39 mg rutin, 0.44 mg p-coumaric acid, and 0.41 mg kaempferol per 1 mL.

[0016] S3. Inject the test solution and the single reference solution into the high performance liquid chromatograph, perform chromatographic analysis under the same conditions, and record the corresponding chromatograms. The liquid chromatography conditions were as follows: column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; detector: diode array detector, detection wavelength: 280 nm; flow rate: 0.6 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution.

[0017] S4. Import the chromatograms of different batches of Ganhaiweikang capsule test solutions obtained in S3 into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; select the chromatographic peaks present in all chromatograms of different batches of Ganhaiweikang capsule test solutions as common peaks, generate the reference chromatogram of Ganhaiweikang capsules using the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak; perform similarity analysis after data import, multi-point correction and data matching; obtain and export the similarity result table between the chromatograms of different batches of Ganhaiweikang capsule test solutions and the common peak patterns; confirm the reliability of the results based on the similarity result table and the chromatograms of Ganhaiweikang capsule test solutions. S5. Perform high-resolution mass spectrometry analysis on the test solution. The high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, mass-to-charge ratio scan range m / z is 100-1500, and the total ion chromatogram is obtained. Import the total ion chromatogram into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peak elution of the chromatogram to obtain the mass spectrometry results of each chemical component.

[0018] S6. Based on the chromatograms of the Ganhaiweikang capsule test solution and the single reference solution obtained in S3, and by comparing them with the total ion chromatogram and the mass spectrometry results of the chemical components obtained in S5, the following peaks were identified in the chromatograms: Peak 5 is protocatechuic acid, retention time 12.617 min; Peak 7 is chlorogenic acid, retention time 15.310 min; Peak 14 is syringic acid, retention time 19.267 min; Peak 18 is apigenin, retention time 22.890 min; Peak 19 is glycyrrhizin, retention time 23.753 min; Peak 20 is coumaric acid, retention time 24.123 min; Peak 21 is naringin, retention time 25.027 min; Peak 23 is hesperidin, retention time 26.730 min; Peak 27 is glycyrrhizin, retention time 35.590 min. Peak 30 is naringenin, with a retention time of 40.437 min; peak 31 is glycyrrhizic acid, with a retention time of 42.003 min; peak 36 is kaempferol, with a retention time of 49.417 min. The characteristic chromatogram of Ganhaiweikang capsules was obtained.

[0019] This invention also provides an optimization process for the above-mentioned feature map detection, including the following steps: 1. Optimization of test solution preparation: This invention investigates different extraction methods, including ultrasonic extraction, reflux extraction, maceration extraction, and ultrasonic followed by rotary evaporation concentration extraction, and the results are as follows: Figure 2 As shown, analysis revealed that the chromatogram obtained by ultrasonic rotary evaporation concentration extraction had a large peak area and a large number of detected components. Therefore, ultrasonic rotary evaporation concentration extraction method was adopted. This invention compared the extraction effects of five extraction solvents: ethanol solution, methanol solution, 50% methanol solution, 80% methanol solution, and aqueous solution. The results are as follows: Figure 3 As shown in the figure, analysis revealed that when methanol, 50% methanol, and 80% methanol were used as extraction solvents, the number of components detected in the chromatograms was relatively large, indicating good extraction efficiency. However, when methanol solution was used as the extraction solvent, the resulting chromatographic peak baseline was more stable; therefore, methanol solution was chosen for extraction. 2. Optimization of chromatographic conditions: This invention screened column temperatures of 25℃, 30℃, and 35℃, and the results are as follows: Figure 4 As shown, the separation effect of each component in the chromatographic peak is better and the peak shape is better when the column temperature is maintained at 30℃. Therefore, the column temperature of 30℃ was finally selected. This invention employs a diode array detector to investigate the detection wavelength. The sample is scanned across the entire wavelength range of 190–800 nm, and chromatograms at 237 nm, 265 nm, 275 nm, and 280 nm are extracted. The results are as follows: Figure 5As shown, the chromatogram contains the most comprehensive information and has a stable baseline when the detection wavelength is 280 nm. Therefore, this method is selected as the detection wavelength condition. This invention screened different flow rates of 0.6 mL / min, 0.8 mL / min, and 1.0 mL / min, and the results are as follows: Figure 6 As shown, a flow rate of 0.6 mL / min resulted in high separation of the target components and symmetrical peak shapes. Considering both separation performance and system stability, a flow rate of 0.6 mL / min was ultimately selected. This invention compares the elution effects of several different elution systems—methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, methanol-0.05% phosphoric acid, acetonitrile-0.05% phosphoric acid, methanol-0.1% formic acid, acetonitrile-0.1% formic acid, methanol-water, and acetonitrile-water—under different elution gradients. The results are as follows: Figure 7 As shown, compared to the methanol system, the acetonitrile system exhibits a greater number of detectable characteristic peaks and larger peak areas for each major active ingredient in its chromatogram. The acetonitrile-water elution system has relatively low separation efficiency, and while the acetonitrile-0.1% formic acid elution system possesses some separation capability, its peak shape and elution characteristics are not as ideal as those of the acetonitrile-0.1% phosphoric acid system. Using acetonitrile-0.1% phosphoric acid as the mobile phase resulted in better separation of the components in Gan Hai Wei Kang capsules; therefore, acetonitrile-0.1% phosphoric acid was ultimately selected as the mobile phase.

[0020] After determining the optimal mobile phase composition, this invention screened the optimal gradient elution program through numerous experiments. Some of the elution programs are as follows: Table 1 Elution Procedure 1

[0021] Table 2 Elution Procedure 2

[0022] Table 3 Elution Procedure 3

[0023] Table 4 Elution Procedure 4

[0024] Table 5 Elution Procedure 5

[0025] Table 6 Elution Procedure 6

[0026] Test results as follows Figure 8 As shown, elution program 6 has good resolution, high peak height, stable baseline, and complete chromatographic information. Therefore, elution program 6 is selected as the optimal elution program.

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] The instruments used in the following examples are shown in Table 7. All batches of Gan Hai Wei Kang capsules were purchased from Shaanxi Dongke Pharmaceutical Co., Ltd., and the reagents used are shown in Table 8. The reference standards used were: glycyrrhizin (batch number: 111610-202209, purity 95.2%), glycyrrhizin (batch number: 112126-202401, purity 95.6%), chlorogenic acid (batch number: 110753-20520, purity 97.9%), protocatechuic acid (batch number: 110809-202207, purity 97.5%), and hesperidin (batch number: 110721-202220, purity 97.2%), all purchased from the China National Institutes for Food and Drug Control. Naringenin (batch number: AFCB2758, purity 98%) was purchased from Chengdu Efa Biotechnology Co., Ltd. Glycyrrhizic acid (batch number: 20070203, purity 98.27%) and kaempferol (batch number: 21102501, purity 99.97%) were purchased from Chengdu Pufeide Biotechnology Co., Ltd. Apigenin (batch number: PS011457, purity 98%), syringic acid (batch number: PS010564, purity 98.0%), and p-coumaric acid (batch number: PS020451, purity 98%) were all purchased from Chengdu Pusi Biotechnology Co., Ltd. Rutin (batch number: AB1564, purity 98.0%) was purchased from Chengdu Aiboke Biotechnology Co., Ltd. Experimental methods in the following examples, unless otherwise specified, were generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art.

[0029] Table 7 Instruments

[0030] Table 8 Reagents

[0031] Example 1: Method for constructing the characteristic spectrum of Ganhai Weikang capsules based on multi-component material basis S1. Preparation of Ganhaiweikang Capsule Test Solution: Weigh 1.0 g of the contents of 15 batches of Ganhaiweikang Capsules and place them in a stoppered conical flask. Add 20 mL of methanol solution and extract by ultrasonication for 30 min at an ultrasonic frequency of 40 kHz and a power of 720 W. Filter, concentrate by rotary evaporation to 5 mL, and filter through a 0.45 µm microporous membrane to obtain the Ganhaiweikang Capsule Test Solution.

[0032] S2. Preparation of reference solutions: Accurately weigh glycyrrhizic acid, glycyrrhizin, glycyrrhizin, apigenin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin, p-coumaric acid, and kaempferol reference standards, respectively, and dissolve them in methanol solution to prepare single reference solutions containing 0.42 mg glycyrrhizic acid, 0.32 mg glycyrrhizin, 0.38 mg glycyrrhizin, apigenin, 0.41 mg naringenin, 0.43 mg chlorogenic acid, 0.36 mg syringic acid, 0.49 mg protocatechuic acid, 0.49 mg hesperidin, 0.31 mg rutin, 0.39 mg p-coumaric acid, and 0.44 mg kaempferol per 1 mL.

[0033] S3. The Ganhaiweikang capsule test solution obtained in S1 and the single reference solution obtained in S2 were injected into a high-performance liquid chromatograph (HPLC) for chromatographic analysis. Detection was performed under the same conditions: Column: Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm) column; Detector: Diode array detector, detection wavelength: 280 nm; Flow rate: 0.6 mL / min; Injection volume: 10 μL; Column temperature: 30℃; Mobile phase: Acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution, elution program as shown in Table 6. The corresponding chromatograms were recorded. Figure 1 ; Figure 9 , 11 In numbers 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31 (A), Figure 10 , 12 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32). S4. Import the chromatograms of the 15 batches of Ganhaiweikang capsule test solutions obtained in S3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; select the chromatographic peaks present in all chromatograms of the 15 batches of Ganhaiweikang capsule test solutions as common peaks, generate the reference chromatogram of Ganhaiweikang capsules using the average value calculation method, and calculate the relative retention time and relative peak area of ​​each common peak; perform similarity analysis after data import, multi-point correction and data matching; obtain and export the similarity result table between the chromatograms of the 15 batches of Ganhaiweikang capsule test solutions and the common peak patterns; confirm the reliability of the results based on the similarity result table and the chromatograms of the Ganhaiweikang capsule test solutions.

[0034] S5. Perform high-resolution mass spectrometry analysis on the test solution. The high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, mass-to-charge ratio scan range m / z 100-1500, to obtain the total ion chromatogram; import the total ion chromatogram into Xcalibur software, enter the Qual Browser interface, and perform data analysis based on the peak elution of the chromatogram of the test solution to obtain the mass spectrometry results of each chemical component. Figure 9 , 11 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 (B)).

[0035] S6. Based on the chromatograms of the Ganhaiweikang capsule test solution and the single reference solution obtained in S3, and by comparing them with the total ion chromatogram and the mass spectrometry results of the chemical components obtained in S5, the following peaks were identified in the chromatograms: Peak 5 is protocatechuic acid, retention time 12.617 min; Peak 7 is chlorogenic acid, retention time 15.310 min; Peak 14 is syringic acid, retention time 19.267 min; Peak 18 is apigenin, retention time 22.890 min; Peak 19 is glycyrrhizin, retention time 23.753 min; Peak 20 is coumaric acid, retention time 24.123 min; Peak 21 is naringin, retention time 25.027 min; Peak 23 is hesperidin, retention time 26.730 min; Peak 27 is glycyrrhizin, retention time 35.590 min. Peak 30 is naringenin, retention time 40.437 min; peak 31 is glycyrrhizic acid, retention time 42.003 min; peak 36 is kaempferol, retention time 49.417 min. The characteristic chromatogram of Ganhaiweikang capsules was obtained. Figure 33Subsequently, the characteristic chromatogram results of Ganhai Weikang capsules were analyzed for attribution. Glycyrrhizic acid, glycyrrhizin, glycyrrhizin, naringenin, and apigenin were derived from licorice; chlorogenic acid, kaempferol, and protocatechuic acid were derived from total glycosides of Gynostemma pentaphyllum; syringic acid, protocatechuic acid, and p-coumaric acid were derived from Hippophae rhamnoides; and rutin and hesperidin were derived from Citrus aurantium.

[0036] Meanwhile, the automatically generated reference chromatogram R was used to generate a common chromatographic peak pattern. Analysis and calculation showed that the common chromatographic peaks of 15 batches of Ganhaiweikang capsules were relatively similar, indicating that the characteristic chromatogram of Ganhaiweikang capsules established by this method can effectively detect the quality of Ganhaiweikang capsules and 15 batches of Ganhaiweikang capsules (Table 10).

[0037] Table 9. Similarity between each batch of samples and the common chromatographic peak pattern

[0038] Example 2: Methodological Study of Feature Map Detection Method S1, Precision Study The test solution prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Six parallel injections were performed with an injection volume of 10 μL. The peak area and retention time were analyzed and the RSD value was calculated. The results are shown in Table 10. It can be seen that the RSD of relative retention time and relative peak area are both less than 3%, indicating that the parallel injection precision of the device is good.

[0039] Table 10 Peak area and retention time in precision studies

[0040] S2, Stability Study The test solution prepared by the method in Example 1 was analyzed according to the detection method in Example 1. Injections were performed at different times (0, 2, 6, 12, 18, and 24 h) with an injection volume of 10 μL. The peak area and retention time of the samples were analyzed, and the RSD value was calculated. The results are shown in Table 11. It can be seen that the RSD of both the relative retention time and the relative peak area is less than 3%, indicating that the chromatographic peak of the Ganhaiweikang capsule test solution hardly changes within 24 h, demonstrating good stability.

[0041] Table 11 Peak area and retention time in stability study

[0042] S3, Repeatability Studies Six batches of sample solutions were prepared according to the test solution method in Example 1. Under the chromatographic conditions of Example 1, the injection volume was 10 μL. The peak area and retention time were analyzed and the RSD value was calculated. The results are shown in Table 12. It can be seen that the RSD of relative retention time and relative peak area are both less than 3%, indicating that the sample chromatographic peak reproducibility is good and the repeatability of the method is good.

[0043] Table 12 Peak area and retention time in repeatability studies

[0044] The above experimental results show that the characteristic spectrum construction method of Ganhaiweikang capsules provided by the present invention has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Ganhaiweikang capsules, providing quality assurance for clinical efficacy.

[0045] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a characteristic spectrum of Gan Hai Wei Kang capsules based on a multi-component material basis, characterized in that, Includes the following steps: S1. Take different batches of Ganhaiweikang capsules as test samples and prepare test sample solutions; S2. Prepare single reference solutions using glycyrrhizic acid, glycyrrhizin, glycyrrhizin, celery glycyrrhizin, naringin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin naringin, p-coumaric acid and kaempferol as reference standards; S3. Perform chromatographic analysis on the test solution obtained in S1 and the single reference solution obtained in S2 to obtain the corresponding chromatograms; S4. Perform similarity analysis on the chromatograms of the test solution obtained in S3; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram. Import the total ion chromatogram into Xcalibur software and perform data analysis based on the peak elution of the chromatogram to obtain the mass spectrometry results of each chemical component. S6. Based on the chromatograms of the test solution and the single reference solution obtained in S3, and combined with the total ion chromatogram and the mass spectrometry results of the chemical components obtained in S5, determine the chemical components of each peak in the characteristic spectrum and obtain the characteristic spectrum of Ganhaiweikang capsules.

2. The method for constructing the characteristic spectrum of Ganhai Weikang capsules based on a multi-component material basis according to claim 1, characterized in that, In S1, the preparation method of the test solution of Ganhaiweikang capsules is as follows: weigh 1.0 g of the contents of different batches of Ganhaiweikang capsules, add 20 mL of methanol solution, extract by ultrasonication for 30 min, filter, concentrate by rotary evaporation, and filter through a 0.45 µm microporous membrane to obtain the test solution of Ganhaiweikang capsules.

3. The method for constructing the characteristic spectrum of Ganhai Weikang capsules based on a multi-component material basis according to claim 1, characterized in that, In S2, the preparation method of the single reference solution is as follows: accurately weigh glycyrrhizic acid, glycyrrhizin, glycyrrhizin, apigenin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin, p-coumaric acid, and kaempferol, dissolve them in methanol solution to prepare a single reference solution containing 0.42 mg of glycyrrhizic acid, 0.32 mg of glycyrrhizin, 0.38 mg of glycyrrhizin, 0.41 mg of apigenin, 0.43 mg of naringenin, 0.36 mg of chlorogenic acid, 0.49 mg of syringic acid, 0.49 mg of protocatechuic acid, 0.31 mg of hesperidin, 0.39 mg of rutin, 0.44 mg of p-coumaric acid, and 0.41 mg of kaempferol per 1 mL.

4. The method for constructing a characteristic spectrum of Ganhai Weikang capsules based on a multi-component material basis according to claim 1, characterized in that, In S3, the chromatographic conditions were as follows: Column: Shim-pack VP-ODS, 250 mm × 4.6 mm, 5 μm; Detector: Diode array detector, detection wavelength 280 nm; Flow rate: 0.6 mL / min; Injection volume: 10 μL; Column temperature: 30℃; Gradient elution program is shown in the table below: 。 5. The method for constructing a characteristic spectrum of Ganhai Weikang capsules based on a multi-component material basis according to claim 1, characterized in that, In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode is full scan mode, and mass-to-charge ratio scanning range m / z is 100-1500.

6. The method for constructing a characteristic spectrum of Ganhai Weikang capsules based on a multi-component material basis according to claim 1, characterized in that, Based on the chromatograms of the test solution and the single reference solution obtained in S3, and by comparing them with the total ion chromatogram and the mass spectrometry results of the chemical components obtained in S5, the following peaks were identified in the chromatograms: Peak 5 is protocatechuic acid, retention time 12.617 min; Peak 7 is chlorogenic acid, retention time 15.310 min; Peak 14 is syringic acid, retention time 19.267 min; Peak 18 is apigenin, retention time 22.890 min; Peak 19 is glycyrrhizin, retention time 23.753 min; Peak 20 is coumaric acid, retention time 24.123 min; Peak 21 is naringin, retention time 25.027 min; Peak 23 is hesperidin, retention time 26.730 min; and Peak 27 is glycyrrhizin, retention time 35.590 min. Peak 30 is naringenin, with a retention time of 40.437 min; peak 31 is glycyrrhizic acid, with a retention time of 42.003 min; peak 36 is kaempferol, with a retention time of 49.417 min. The characteristic chromatogram of Ganhaiweikang capsules was obtained.

7. The method for constructing a characteristic spectrum of Ganhai Weikang capsules based on a multi-component material basis according to claim 1, characterized in that, After obtaining the characteristic spectrum of Ganhaiweikang capsules, an attribution analysis was performed on the characteristic spectrum results of Ganhaiweikang capsules. Glycyrrhizic acid, glycyrrhizin, glycyrrhizin, naringenin, and apigenin were derived from licorice; chlorogenic acid, kaempferol, and protocatechuic acid were derived from total glycosides of Gynostemma pentaphyllum; syringic acid, protocatechuic acid, and p-coumaric acid were derived from Hippophae rhamnoides; and rutin and hesperidin were derived from Citrus aurantium.

8. The characteristic chromatogram of Ganhaiweikang capsules obtained by the construction method according to any one of claims 1 to 7.

9. A method for detecting the quality of Gan Hai Wei Kang capsules, characterized in that, Using the characteristic chromatogram of Ganhaiweikang capsules as described in claim 8, the quality of Ganhaiweikang capsules or its equivalent drugs is detected, with one or more of glycyrrhizic acid, glycyrrhizin, glycyrrhizin, celery glycyrrhizin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin, p-coumaric acid and kaempferol as quality markers.

10. The method for detecting the quality of Gan Hai Wei Kang capsules according to claim 9, characterized in that, The quality basis of Gan Hai Wei Kang capsules or its equivalent drugs includes the following detected ingredients: glycyrrhizic acid, glycyrrhizin, glycyrrhizin, celery glycyrrhizin, naringenin, chlorogenic acid, syringic acid, protocatechuic acid, hesperidin, rutin, p-coumaric acid and kaempferol.

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