Construction method of Yuquan capsule characteristic spectrum

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method of a Yuquan capsule characteristic spectrum. Comprising the following steps: 1, preparing a reference substance solution; 2, preparing a test solution; 3, determining conditions; and 4, generating a contrast characteristic spectrum. According to the invention, methodological investigation is carried out on the constructed Yuquan capsule characteristic spectrum method, and the methodological investigation comprises an instrument precision experiment, a method repeatability experiment and a sample stability experiment. In each experiment result, the RSD value of the relative retention time of each peak is less than or equal to 1.0%, and the RSD value of the relative peak area is less than or equal to 10.0%. By means of the method, large types of chemical components in the Yuquan capsules can be reflected, and the overall quality of the Yuquan capsules can be controlled more efficiently and rapidly.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for constructing a characteristic spectrum of Yuquan capsules. Background Art

[0002] Yuquan Capsules are a modified and refined version of Yuquan Powder, described in Ye Tianshi's "Zhongfutang Gongxuan Liangfang" and "Ye Tianshi Shouji Mifang" during the Qing Dynasty. It is a traditional Chinese medicine compound preparation extracted and processed from ten medicinal ingredients: Radix Trichosanthis, Radix Puerariae, Radix Ophiopogonis, Ginseng, Poria, Prunus mume, Radix Astragali, Licorice, Rehmannia glutinosa, and Schisandra chinensis. It is primarily used for symptoms such as Qi and Yin deficiency, thirst and excessive drinking, poor digestion and increased hunger, and diabetes. Due to its outstanding efficacy in treating diabetes, it is favored by clinical physicians. To effectively control the overall quality of this preparation and ensure its clinical efficacy, a characteristic profile is employed to control its overall quality.

[0003] At present, the patent has not yet conducted research on its characteristic spectrum, and the literature has only studied the content and thin-layer chromatography of puerarin, without controlling its overall quality.

[0004] Yuquan Capsules are a multi-ingredient compound preparation, containing ten herbs, including Radix Trichosanthis, Radix Puerariae, and Radix Ophiopogonis. Each herb contains diverse active ingredients, and substances with similar chemical properties (such as flavonoids and saponins) may also be present. High-performance liquid chromatography (HPLC) analysis presents a primary technical challenge in achieving efficient separation of these components while avoiding peak overlap. Detection of minor or low-level components, in particular, requires optimized chromatographic conditions to ensure accurate identification and localization of all key peaks.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a method for constructing a characteristic spectrum of Yuquan Capsules, which can comprehensively, systematically and accurately control and evaluate the drug quality of Yuquan Capsules.

[0007] The second purpose of the present invention is to apply the characteristic spectrum method provided by the present invention to the overall control and evaluation of the drug quality of Yuquan Capsules.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: The present invention provides a method for constructing a characteristic spectrum of Yuquan capsule, comprising the following steps: Preparation of reference solution: accurately weigh the reference substance, add methanol to prepare a solution containing 70 μg per 1 mL, shake well, and use it as the reference solution; the reference substances are: gallic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritigenin, ammonium glycyrrhizate, and schisandrin A; Preparation of the test solution: Take 0.5 g of the contents of Yuquan Capsules, accurately weigh it, add 30 mL of methanol-acetonitrile-0.1% (mass percentage) phosphoric acid aqueous solution, ultrasonically extract, shake well, filter, and collect the filtrate; use a C18 solid phase extraction column, activate it with 5 mL of methanol and 5 mL of purified water in sequence; load the filtrate after ultrasonic extraction onto the activated C18 column at a flow rate of 1 mL / min, and discard the initial filtrate; the subsequent filtrate is rinsed with 5 mL of purified water in sequence to remove polar impurities, and then the target component is eluted with 5 mL of methanol-acetonitrile solution with a volume ratio of 70:30, and the eluate is collected; the eluate is concentrated to near dryness under nitrogen blowing at 40°C, the residue is re-dissolved with 2 mL of 70% methanol solution, filtered through a 0.22 μm microporous filter membrane, and the subsequent filtrate is used as the test solution; Determination: Inject the reference solution and the test solution into the HPLC under the following chromatographic conditions, inject 10 μL of each sample, and record the chromatogram; Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); acetonitrile-methanol-tetrahydrofuran (volume ratio, 70:25:5) as mobile phase A, and 0.1% aqueous phosphoric acid (pH = 2.5) + 5 mM sodium dodecyl sulfate as mobile phase B, elution according to the specified gradient; column temperature, 30°C; flow rate, 1 mL / min; detection wavelength, 270 nm; UV-visible detector, detection wavelength, 270 nm; gradient conditions: From 0 to 45 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 27:73; From 45 to 60 min, the volume ratio of mobile phase A:phase B was changed from 27:73 to 60:40; From 60 to 63 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95; 63-75 min, mobile phase A:B, volume ratio 5:95; Generate a reference characteristic spectrum: Use the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System" developed by the State Pharmacopoeia Committee to select the chromatographic peaks that exist in the chromatograms of different batches of Yuquan Capsules as common peaks, and use the average value calculation method to generate a reference characteristic spectrum of Yuquan Capsules.

[0009] Preferably, the volume ratio of methanol-acetonitrile-0.1% (mass ratio) phosphoric acid aqueous solution in the extraction solvent in step (2) is 50:30:20.

[0010] Preferably, the extraction method in step (2) is: ultrasonic extraction conditions are 250 W, 40 kHz ultrasound for 30 minutes.

[0011] Preferably, the characteristic spectrum generated in step (4) includes 13 chromatographic common peaks, including: peak 1 corresponding to gallic acid, peak 2 corresponding to 5-hydroxymethylfurfural, peak 5 corresponding to puerarin, peak 8 corresponding to liquiritin, peak 11 corresponding to liquiritigenin, peak 12 corresponding to ammonium glycyrrhizate, and peak 13 corresponding to schisandrin.

[0012] Preferably, the characteristic peak is based on the puerarin chromatographic peak as the reference peak, and the relative retention times of the 13 chromatographic peaks are 0.32 (peak 1), 0.42 (peak 2), 0.64 (peak 3), 0.81 (peak 4), 1.05 (peak 6), 1.21 (peak 7), 1.46 (peak 8), 1.56 (peak 9), 2.12 (peak 10), 2.17 (peak 11), 2.42 (peak 12), and 2.64 (peak 13), respectively, and the relative retention times are all within ±10% of the specified value.

[0013] Beneficial effects of the present invention: (1) The present invention established an HPLC characteristic spectrum for Yuquan Capsules, identified 13 characteristic peaks, and more fully demonstrated the chemical characteristics of Yuquan Capsules; (2) The present invention investigated the extraction solvent and extraction method of the sample. Within the investigated range, the chromatographic method showed 13 characteristic peaks, and the relative retention time of each peak had a small change, with RDS < 1.0%; (3) The methodological investigation of the constructed Yuquan Capsule characteristic spectrum method was carried out, including instrument precision experiments, method repeatability experiments, and sample stability experiments. The RSD values ​​of the relative retention time of each peak in each experimental result were all ≤1.0%, and the RSD values ​​of the relative peak area were all ≤10.0%. This shows that the method is good and can reflect the major chemical components in Yuquan Capsule. The overall quality of Yuquan Capsule can be controlled more efficiently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a specificity chromatogram, where S1: negative solution; S2: gallic acid control; S3: 5-hydroxymethylfurfural control; S4: puerarin control; S5: liquiritin control; S6: liquiritigenin control; S7: ammonium glycyrrhizate S8: schisandrin A S9: test solution; Figure 2 It is the chromatogram of precision test; Among them, S1~S6: precision 1~6; Figure 3 is the repeatability test chromatogram; among them, S1~S6: repeatability 1~6; Figure 4 Chromatogram of stability test; S1~S6: stability 1~6; Figure 5The chromatograms of eight batches of Yuquan capsule samples are shown below. Among them, S1: 230202; S2: 230316; S3: 230408; S4: 230511; S5: 230619; S6: 230723; S7: 230801; S8: 231127; Figure 6 This is the reference characteristic spectrum of Yuquan Capsule. DETAILED DESCRIPTION

[0015] To facilitate understanding by those skilled in the art, the present invention is further described below with reference to embodiments and drawings.

[0016] Example 1 1. Instruments and test drugs 1.1 Instruments and Equipment .

[0017] 1.2 Test materials .

[0018] 1.3 Reference Material Information .

[0019] 1.4 Sample Information .

[0020] Example 2 Investigation of the method for constructing the characteristic spectrum of Yuquan Capsules 2.1 Investigation of test sample preparation methods 2.1.1 Investigation of extraction solvents for test samples Chromatographic conditions Chromatographic column: Luna C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-tetrahydrofuran (volume ratio: 70:25:5); mobile phase B: 0.1% aqueous phosphoric acid (pH 2.5) + 5 mM sodium dodecyl sulfate; gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the puerarin peak should be no less than 5000.

[0021] .

[0022] Preparation of reference solution Take gallic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritigenin, ammonium glycyrrhizate, and schisandrin A reference substances, add methanol to make a solution containing 70 μg per 1 mL, shake well, and use it as the reference solution.

[0023] Preparation of test solution 0.5 g of the contents of Yuquan capsule (batch number 230202) was accurately weighed and added with 30 mL of a 50:30 volume ratio of methanol-acetonitrile solution, a 50:30:20 volume ratio of methanol-acetonitrile-0.1% phosphoric acid aqueous solution, and a 30:50:20 volume ratio of methanol-acetonitrile-0.1% phosphoric acid aqueous solution, respectively. Ultrasonic extraction was performed at 250 W and 40 kHz for 30 min, shaken, filtered, and the filtrate was collected. A C18 solid phase extraction column was activated with 5 mL of methanol and 5 mL of purified water in sequence. The filtrate was loaded onto the activated C18 column at a flow rate of 1 mL / min, and the initial filtrate was discarded. The polar impurities were rinsed with 5 mL of purified water in sequence, and the target component was eluted with 5 mL of a 70:30 volume ratio of methanol-acetonitrile solution, and the eluate was collected. The eluate was concentrated to near dryness under nitrogen purge at 40°C, and the residue was reconstituted with 2 mL of 70% methanol solution and filtered through 0.22 μm microporous filter membrane, and take the filtrate as the test solution.

[0024] Assay Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0025] Solvent investigations revealed that peak separation and peak shape were better when the extraction solvent contained 0.1% phosphoric acid and the solvent volume ratio was 50:30:20. However, when phosphoric acid was absent from the extraction solvent, peak shape was poor and peaks were missing, failing to achieve the desired effect. Therefore, a methanol-acetonitrile-0.1% phosphoric acid aqueous solution with a volume ratio of 50:30:20 was selected as the extraction solvent.

[0026] Table 1 Results of relative retention time of solvent investigation .

[0027] 2.1.2 Investigation of test sample extraction methods Preparation of test solution 0.5 g of Yuquan capsules (batch number 230202) were accurately weighed and added to 30 mL of a 50:30:20 (volume ratio) methanol-acetonitrile-0.1% (mass percentage) phosphoric acid aqueous solution. Ultrasonic extraction was performed at 250 W, 40 kHz and then refluxed for 30 min. The extraction was then shaken and filtered, and the filtrate was collected. A C18 solid-phase extraction column was activated with 5 mL of methanol and then 5 mL of purified water. The filtrate was loaded onto the activated C18 column at a flow rate of 1 mL / min, and the initial filtrate was discarded. The column was washed with 5 mL of purified water to remove polar impurities, and the target component was eluted with 5 mL of a 70:30 (volume ratio) methanol-acetonitrile solution. The eluate was collected and concentrated to near dryness at 40°C under nitrogen purge. The residue was reconstituted with 2 mL of 70% methanol solution and filtered through a 0.22 μm microporous filter. The subsequent filtrate was used as the test solution.

[0028] The chromatographic conditions are the same as those in item “2.1.1”; the reference solution is the reference solution in item “2.1.1”.

[0029] Assay Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0030] The results of the extraction method investigation showed that ultrasonic extraction had better peak separation and better peak shape, while heating and reflux extraction had poor peak shape and missing peaks, which did not achieve the desired effect. Therefore, ultrasonic extraction was selected as the extraction method for Yuquan Capsules.

[0031] Table 2 Relative retention time results of extraction methods .

[0032] 2.2 Investigation of chromatographic conditions 2.2.1 Investigation of mobile phase types Chromatographic conditions 1: Luna C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-tetrahydrofuran (volume ratio: 70:25:5); mobile phase B: 0.1% aqueous phosphoric acid (pH 2.5) + 5 mM sodium dodecyl sulfate (SDS); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The theoretical plate number (TPN) calculated based on the puerarin peak should be no less than 5000.

[0033] .

[0034] Chromatographic conditions 2: Luna C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-tetrahydrofuran (volume ratio: 70:25:5); mobile phase B: 0.1% aqueous phosphoric acid (pH 2.5); gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the puerarin peak should be no less than 5000.

[0035] .

[0036] Chromatographic Conditions 3: Luna C18 (4.6x250mm, 5μm); mobile phase A: acetonitrile-methanol (volume ratio: 70:25); mobile phase B: 0.1% aqueous phosphoric acid (pH 2.5) + 5 mM sodium dodecyl sulfate; gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The theoretical plate number calculated based on the puerarin peak should be no less than 5000.

[0037] .

[0038] Preparation of reference solution Take appropriate amounts of gallic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritigenin, ammonium glycyrrhizate, and schisandrin A reference substances, add methanol to make a solution containing 70 μg per 1 mL, shake well, and use as reference solution.

[0039] Preparation of test solution 0.5 g of the contents of Yuquan Capsules (batch number 230202) were accurately weighed and added to 30 mL of methanol-acetonitrile-0.1% phosphoric acid aqueous solution with a volume ratio of 50:30:20. Ultrasonic extraction was performed at 250 W and 40 kHz for 30 min, shaken, filtered, and the filtrate was collected. A C18 solid-phase extraction column was activated with 5 mL of methanol and 5 mL of purified water in sequence. The filtrate was loaded onto the activated C18 column at a flow rate of 1 mL / min, and the initial filtrate was discarded. The polar impurities were rinsed with 5 mL of purified water in sequence, and the target component was eluted with 5 mL of methanol-acetonitrile solution with a volume ratio of 70:30. The eluate was collected and concentrated to near dryness under nitrogen blow at 40°C. The residue was reconstituted with 2 mL of 70% methanol solution, filtered through a 0.22 μm microporous filter membrane, and the subsequent filtrate was used as the test solution.

[0040] Assay Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0041] The results showed that when elution was performed with acetonitrile-methanol-tetrahydrofuran in a volume ratio of 70:25:5 as mobile phase A and 0.1% phosphoric acid aqueous solution at pH = 2.5 + 5 mM sodium dodecyl sulfate as mobile phase B, the test sample chromatogram presented a total of 13 characteristic peaks, with good peak separation and excellent peak shape; when elution was performed with acetonitrile-methanol-tetrahydrofuran in a volume ratio of 70:25:5 as mobile phase A and 0.1% phosphoric acid aqueous solution at pH = 2.5 as mobile phase B or with acetonitrile-methanol in a volume ratio of 70:25 as mobile phase A and 0.1% phosphoric acid aqueous solution at pH = 2.5 + 5 mM sodium dodecyl sulfate as mobile phase B, all peaks were missing, and the corresponding effect was not achieved.

[0042] Table 3 Relative retention time results of mobile phase types .

[0043] 2.2.2 Investigation of mobile phase ratio Chromatographic conditions Chromatographic column: Luna C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-tetrahydrofuran (volume ratio: 70:25:5); mobile phase B: 0.1% aqueous phosphoric acid (pH 2.5) + 5 mM sodium dodecyl sulfate; gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the puerarin peak should be no less than 5000.

[0044] Mobile phase ratio 1: .

[0045] Mobile phase ratio 2: .

[0046] Mobile phase ratio 3: .

[0047] The reference solution is the same as that in item “2.2.1”.

[0048] 0.5 g of the contents of Yuquan Capsules (batch number 230202) were accurately weighed and added to 30 mL of methanol-acetonitrile-0.1% phosphoric acid aqueous solution with a volume ratio of 50:30:20. Ultrasonic extraction was performed at 250 W and 40 kHz for 30 min, shaken, filtered, and the filtrate was collected. A C18 solid-phase extraction column was activated with 5 mL of methanol and 5 mL of purified water in sequence. The filtrate was loaded onto the activated C18 column at a flow rate of 1 mL / min, and the initial filtrate was discarded. The polar impurities were rinsed with 5 mL of purified water in sequence, and the target component was eluted with 5 mL of methanol-acetonitrile solution with a volume ratio of 70:30. The eluate was collected and concentrated to near dryness under nitrogen blow at 40°C. The residue was reconstituted with 2 mL of 70% methanol solution, filtered through a 0.22 μm microporous filter membrane, and the subsequent filtrate was used as the test solution.

[0049] Assay Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0050] The results showed that when eluted with a mobile phase ratio of 1, the test sample chromatogram showed a total of 12 characteristic peaks, with good peak separation and excellent peak shape; when eluted with a mobile phase ratio of 2 or a mobile phase ratio of 3, each peak was missing and the corresponding effect was not achieved.

[0051] Table 4 Relative retention time results of mobile phase ratio study .

[0052] Example 3 Yuquan Capsule Characteristic Spectrum Methodology Verification 3.1 Exclusivity Chromatographic conditions Chromatographic column: Luna C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-tetrahydrofuran (volume ratio: 70:25:5); mobile phase B: 0.1% aqueous phosphoric acid (pH 2.5) + 5 mM sodium dodecyl sulfate; gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the puerarin peak should be no less than 5000.

[0053] .

[0054] Preparation of reference solution Take appropriate amounts of gallic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritigenin, ammonium glycyrrhizate, and schisandrin A reference substances, add methanol to make a solution containing 70 μg per 1 mL, shake well, and use as reference solution.

[0055] Preparation of test solution 0.5 g of the contents of Yuquan Capsules (batch number 230202) were accurately weighed and added to 30 mL of methanol-acetonitrile-0.1% phosphoric acid aqueous solution with a volume ratio of 50:30:20. Ultrasonic extraction was performed at 250 W and 40 kHz for 30 min, shaken, filtered, and the filtrate was collected. A C18 solid-phase extraction column was activated with 5 mL of methanol and 5 mL of purified water in sequence. The filtrate was loaded onto the activated C18 column at a flow rate of 1 mL / min, and the initial filtrate was discarded. The polar impurities were rinsed with 5 mL of purified water in sequence, and the target component was eluted with 5 mL of methanol-acetonitrile solution with a volume ratio of 70:30. The eluate was collected and concentrated to near dryness under nitrogen blow at 40°C. The residue was reconstituted with 2 mL of 70% methanol solution, filtered through a 0.22 μm microporous filter membrane, and the subsequent filtrate was used as the test solution.

[0056] Preparation of negative solution Take an appropriate amount of auxiliary material, take 0.5g, add 30mL of 70% methanol solution, ultrasonically extract (250W, 40Hz) for 30 minutes, shake well, filter, and take the filtrate to obtain the product.

[0057] Assay Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0058] The results showed that the negative solution had no interference and good specificity. Figure 1 .

[0059] 3.2 Precision Take 0.5 g of the contents of Yuquan Capsules (Batch No.: Batch No. 230202), prepare the test solution according to the test solution preparation method under "3.1", inject 6 times according to the chromatographic conditions under "3.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0060] Table 5 Precision relative retention time results .

[0061] Table 6 Precision relative peak area results .

[0062] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, indicating good precision. The chromatogram is attached. Figure 2 .

[0063] 3.3 Repeatability Take 0.5 g of the contents of Yuquan Capsules (batch number: batch number 230202), a total of 6 portions, prepare the test solution according to the test solution preparation method under "3.1", inject the sample according to the chromatographic conditions under "3.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0064] Table 7 Repeatability relative retention time results .

[0065] Table 8 Repeatability relative peak area results .

[0066] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, with good repeatability. The chromatogram is attached. Figure 3 .

[0067] 3.4 Stability Take 0.5 g of the contents of Yuquan Capsules (batch number 230202) and prepare the test solution according to the test solution preparation method under "3.1". Inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic conditions under "3.1". Record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0068] Table 9 Stability relative retention time results .

[0069] Table 10 Stability relative peak area results .

[0070] The results showed that the relative retention time RSD of each chromatographic peak was less than 1.0%, and the relative peak area RSD was less than 10.0%, indicating that the solution was stable. The chromatogram is attached. Figure 4 .

[0071] In summary, the method demonstrated good specificity through specificity testing, with no interference from the negative solution. In precision testing, the relative retention time RSDs for each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 10.0%, indicating good precision. In repeatability testing, the relative retention time RSDs for each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 10.0%, indicating good repeatability. In stability testing, the relative retention time RSDs for each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 10.0%, indicating good solution stability. This method has been methodologically validated, and the results are reliable and accurate.

[0072] Example 4 Construction of Yuquan Capsule Control Characteristic Spectrum Chromatographic conditions Chromatographic column: Luna C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-tetrahydrofuran (volume ratio: 70:25:5); mobile phase B: 0.1% aqueous phosphoric acid (pH 2.5) + 5 mM sodium dodecyl sulfate; gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 270 nm. The number of theoretical plates calculated based on the puerarin peak should be no less than 5000.

[0073] .

[0074] Preparation of reference solution: Take appropriate amount of gallic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritigenin, ammonium glycyrrhizate, and schisandrin A reference substances, add methanol to make a solution containing 70 μg per 1 mL, shake well, and use as reference solution.

[0075] Preparation of test solution 0.5 g of the contents of Yuquan Capsules (batch number 230202) were accurately weighed and added to 30 mL of methanol-acetonitrile-0.1% phosphoric acid aqueous solution with a volume ratio of 50:30:20. Ultrasonic extraction was performed at 250 W and 40 kHz for 30 min, shaken, filtered, and the filtrate was collected. A C18 solid-phase extraction column was activated with 5 mL of methanol and 5 mL of purified water in sequence. The filtrate was loaded onto the activated C18 column at a flow rate of 1 mL / min, and the initial filtrate was discarded. The polar impurities were rinsed with 5 mL of purified water in sequence, and the target component was eluted with 5 mL of methanol-acetonitrile solution with a volume ratio of 70:30. The eluate was collected and concentrated to near dryness under nitrogen blow at 40°C. The residue was reconstituted with 2 mL of 70% methanol solution, filtered through a 0.22 μm microporous filter membrane, and the subsequent filtrate was used as the test solution.

[0076] Assay Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0077] The chromatograms of the test samples of 8 batches of Yuquan Capsules were imported into the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System" and the chromatographic peaks present in the chromatograms of different batches of Yuquan Capsules were selected as common peaks; the control characteristic spectrum was generated using the average calculation method, and the relative retention time and relative peak area of ​​each common peak were calculated. Figure 5 、 6 .

[0078] Table 11 Relative retention time results of eight batches of Yuquan capsule samples .

[0079] Table 12 Relative peak area results of eight batches of Yuquan capsule samples .

[0080] The chromatograms of the eight batches of Yuquan Capsule samples all showed 13 common peaks. The RSDs of the relative retention times of the 13 common peaks were all less than 1.0%, and the RSDs of the relative peak areas were all less than 10.0%.

[0081] The test sample chromatogram shows 13 characteristic peaks, of which Peak 1, Peak 2, Peak 5, Peak 8, Peak 11, Peak 12, and Peak 13 should correspond to gallic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritigenin, ammonium glycyrrhizate, and Schisandrin A, respectively. The peak corresponding to the peak of the puerarin reference substance is the S peak. Calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.32 (Peak 1), 0.42 (Peak 2), 0.64 (Peak 3), 0.81 (Peak 4), 1.05 (Peak 6), 1.21 (Peak 7), 1.46 (Peak 8), 1.56 (Peak 9), 2.12 (Peak 10), 2.17 (Peak 11), 2.42 (Peak 12), and 2.64 (Peak 13).

Claims

1. A method for constructing a characteristic spectrum of Yuquan Capsules, characterized in that: The method can simultaneously detect schisandraic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritigenin, ammonium glycyrrhizate, and schisandrin in Yuquan capsules, and comprises the following steps: (1) Preparation of reference solution: accurately weigh the reference substance, add methanol to prepare a solution containing 70 μg per 1 mL, shake well, and use it as the reference solution; the reference substances are: gallic acid, 5-hydroxymethylfurfural, puerarin, liquiritin, liquiritin, ammonium glycyrrhizate, and schisandrin A; (2) Preparation of the test solution: Take the contents of Yuquan capsules, accurately weigh them, add methanol-acetonitrile-0.1% phosphoric acid aqueous solution, perform ultrasonic extraction, shake well, filter, and collect the filtrate; use a C18 solid phase extraction column, activate it with 5 mL of methanol and 5 mL of purified water in turn; load the filtrate obtained after ultrasonication onto the activated C18 column at a flow rate of 1 mL / min, and discard the initial filtrate; then rinse with 5 mL of purified water in turn to remove polar impurities, and then elute the target component with methanol-acetonitrile solution, and collect the eluate; concentrate the eluate at 40°C with nitrogen blowing to near dryness, re-dissolve the residue with 2 mL of 70% methanol solution, filter it through a 0.22 μm microporous filter membrane, and take the subsequent filtrate as the test solution; (3) Determination: Inject the reference solution and the test solution into a high performance liquid chromatograph under the following chromatographic conditions, inject 10 μL of each sample, and record the chromatogram; Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile-methanol-tetrahydrofuran (volume ratio: 70:25:5) as the mobile phase A; 0.1% aqueous phosphoric acid solution at pH 2.5 + 5 mM sodium dodecyl sulfate as the mobile phase B, eluting according to the specified gradient; column temperature: 30°C; flow rate: 1 mL / min; UV-visible detector: detection wavelength: 270 nm; gradient conditions: From 0 to 45 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 27:73; From 45 to 60 min, the volume ratio of mobile phase A:phase B was changed from 27:73 to 60:40; From 60 to 63 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95; 63-75 min, mobile phase A:B, volume ratio 5:95; (4) Generate a control characteristic spectrum: Select the chromatographic peaks that exist in the chromatograms of different batches of Yuquan Capsules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of Yuquan Capsules.

2. The method for constructing a characteristic spectrum of Yuquan Capsule according to claim 1, characterized in that: The volume ratio of the extraction solvent methanol-acetonitrile-0.1% phosphoric acid aqueous solution in step (2) is 50:30:20, and the amount used is 30 mL.

3. The method for constructing a characteristic spectrum of Yuquan Capsule according to claim 1, characterized in that: The ultrasonic extraction conditions in step (2) were 250 W, 40 kHz ultrasound for 30 minutes.

4. The method for constructing a characteristic spectrum of Yuquan Capsule according to claim 1, characterized in that: The volume of methanol-acetonitrile solution used in step (2) is 5 mL, with a volume ratio of 70:

30.

5. The method for constructing a characteristic spectrum of Yuquan Capsule according to claim 1, characterized in that: The chromatographic column in step (3) is: Luna C18, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

6. The method for constructing a characteristic spectrum of Yuquan Capsule according to claim 1, characterized in that: The characteristic spectrum generated in step (4) includes 13 common chromatographic peaks, specifically: Peak 1 corresponding to gallic acid, Peak 2 corresponding to 5-hydroxymethylfurfural, Peak 5 corresponding to puerarin, Peak 8 corresponding to liquiritin, Peak 11 corresponding to liquiritigenin, Peak 12 corresponding to ammonium glycyrrhizate, and Peak 13 corresponding to schisandrin.

7. The method for constructing a characteristic spectrum of Yuquan Capsule according to claim 1, characterized in that: The characteristic peaks were taken as reference peaks using the puerarin chromatographic peak. The relative retention times of the 13 chromatographic peaks were 0.32 for peak 1, 0.42 for peak 2, 0.64 for peak 3, 0.81 for peak 4, 1.05 for peak 6, 1.21 for peak 7, 1.46 for peak 8, 1.56 for peak 9, 2.12 for peak 10, 2.17 for peak 11, 2.42 for peak 12 and 2.64 for peak 13. The relative retention times were all within ±10% of the specified values.

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