A method for constructing a characteristic spectrum of Tongfengding capsule

Abstract

The present invention belongs to the technical field of traditional Chinese medicine analysis, and in particular relates to a method for constructing a characteristic spectrum of Tongfengding Capsules. The present invention establishes HPLC characteristic spectrums for Tongfengding Capsules at wavelengths of 270nm and 320nm, respectively. At a wavelength of 270nm, 8 characteristic peaks are identified, and gallic acid, maleinic acid, and gentiopicroside are controlled; at a wavelength of 320nm, 11 characteristic peaks are identified, and gentiopicroside, paeoniflorin, verbascoside, and tetrahydropalmatine are controlled, thereby fully and comprehensively displaying the chemical composition characteristics of Tongfengding Capsules. In addition, the present invention investigates the extraction solvent and extraction method of the sample, and the type and concentration of the acid added in the mobile phase. At wavelengths of 270nm and 320nm, the relative retention time changes of each peak are small, and the RDS is less than 2.0%.

Description

Technical Field

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

[0002] Tongfengding Capsules, now included in the 2020 edition of the Chinese Pharmacopoeia (Volume 1), are a traditional Chinese medicine compound preparation made from eight herbs, including Gentiana macrophylla, Corydalis yanhusuo, and Cyathula officinalis. They are known for their efficacy in clearing heat and dampness, promoting blood circulation and dredging the meridians, and treating gout. Existing quality control standards and literature primarily focus on quantitative control of only one or two ingredients, making it difficult to fully reflect the quality of the preparation. Traditional Chinese medicine characteristic profile technology offers a holistic and fuzzy approach to quality control, enabling comprehensive testing of the effectiveness and stability of traditional Chinese medicines.

[0003] Tongfengding Capsules are composed of eight medicinal herbs with complex chemical compositions and widely varying properties. Effective separation and detection of these multiple components must be achieved under the same chromatographic conditions, placing high demands on mobile phase gradient design, chromatographic column selection, and detection wavelength optimization.

[0004] The present invention constructs a characteristic spectrum method for Tongfengding Capsules, in order to provide an experimental basis for comprehensively evaluating the intrinsic product quality of Tongfengding Capsules. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing a characteristic spectrum of Tongfengding Capsules, which can comprehensively, systematically and accurately control and evaluate the drug quality of Tongfengding Capsules.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0007] The present invention provides a method for constructing a characteristic spectrum of Tongfengding capsule, comprising the following steps:

[0008] (1) Preparation of reference solution: Take an appropriate amount of reference substance, weigh accurately, add methanol to make a solution containing 90 μg per 1 mL, shake well, and use it as the reference solution; the reference substances are: gallic acid, gentiopicroside, gentioside, verbascoside, paeoniflorin, and tetrahydropalmatine;

[0009] (2) Preparation of the test solution: Take 0.5 g of the contents of Tongfengding capsules, accurately weigh it, add 30 mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, where 0.1% phosphoric acid is the mass fraction, and extract for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter through a 0.45 μm microporous filter membrane to obtain a crude extract; select a C18 solid phase extraction column, first activate the column with 5 mL of methanol, and then balance the column with 5 mL of water to neutrality; slowly load the crude extract onto the C18 column with a flow rate of 1 mL / min; rinse with 5 mL of water to remove highly polar impurities; elute the target component with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and collect the eluate; concentrate the eluate at 40°C with nitrogen blowdown to near dryness; make the volume up to 2 mL with methanol, vortex mix, and pass through a 0.22 μm filter membrane to obtain the purified test solution;

[0010] (3) Determination: The reference solution and the test solution were injected into a high performance liquid chromatograph under the following chromatographic conditions, 10 μl of each sample was injected, and the chromatogram was recorded; the chromatographic conditions were: octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 was used as the mobile phase A, 10 mM potassium dihydrogen phosphate buffer with a pH of 2.8 + 0.05% trifluoroacetic acid was used as the mobile phase B, of which 0.05% trifluoroacetic acid was used as the volume fraction, and elution was performed according to the specified gradient; the column temperature was 30°C; the flow rate was 1 mL per minute; the detection wavelength was 270 nm or 320 nm;

[0011] The elution gradient conditions were:

[0012] From 0 to 30 min, the volume ratio of mobile phase A:phase B was changed from 5:95 to 20:80;

[0013] From 30 to 50 min, the volume ratio of mobile phase A:phase B changed from 20:80 to 33:67;

[0014] From 50 to 65 min, the volume ratio of mobile phase A:phase B was changed from 33:67 to 60:40;

[0015] From 65 to 66 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95;

[0016] 66-76 min, mobile phase A:B, volume ratio 5:95;

[0017] (4) Generate a control characteristic spectrum: Use the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” developed by the Chinese Pharmacopoeia Committee to select the chromatographic peaks that exist in the chromatograms of different batches of Tongfengding Capsules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of Tongfengding Capsules.

[0018] Preferably, the volume ratio of the methanol-water-0.1% phosphoric acid solution used in the extraction in step (2) is 70:28:2, and the amount used is 30 mL.

[0019] Preferably, in step (2), the volume ratio of methanol-water-phosphoric acid solution used for elution is 70:30:0.1, and the amount added is 5 mL.

[0020] Preferably, the centrifugation condition is 4000 rpm for 10 minutes.

[0021] Preferably, the control characteristic spectrum generated in step (4) at a wavelength of 270 nm includes 8 common chromatographic peaks, specifically: Peak 1 corresponding to gallic acid, Peak 2 corresponding to maleinic acid, and Peak 5 corresponding to gentiopicroside.

[0022] Preferably, the control characteristic spectrum generated in step (4) at a wavelength of 320 nm includes 11 common chromatographic peaks, specifically: Peak 2 corresponding to gentiopicroside, Peak 5 corresponding to paeoniflorin, Peak 7 corresponding to verbascoside, and Peak 10 corresponding to tetrahydropalmatine.

[0023] Preferably, the characteristic peak at a wavelength of 270 nm is taken as the reference peak with the chromatographic peak of gentiopicroside, and the relative retention times of the eight chromatographic peaks are 0.31 for peak 1, 0.67 for peak 2, 0.84 for peak 3, 0.89 for peak 4, 1.14 for peak 6, 1.56 for peak 7, and 2.10 for peak 8.

[0024] Preferably, the characteristic peak at a wavelength of 320 nm is the chromatographic peak of gentiopicroside as the reference peak, and the relative retention times of the 11 chromatographic peaks are: peak 1 is 0.89, peak 3 is 1.14, peak 4 is 1.19, peak 5 is 1.21, peak 6 is 1.47, peak 7 is 1.51, peak 8 is 1.56, peak 9 is 1.68, peak 10 is 1.76, peak 11 is 2.10

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention establishes HPLC characteristic spectra of Tongfengding Capsules at wavelengths of 270nm and 320nm, respectively. At a wavelength of 270nm, 8 characteristic peaks are identified, and gallic acid, mascinyl acid, and gentiopicroside are controlled; at a wavelength of 320nm, 11 characteristic peaks are identified, and gentiopicroside, paeoniflorin, verbascoside, and tetrahydropalmatine are controlled, which fully and comprehensively demonstrates the chemical composition characteristics of Tongfengding Capsules. In addition, the present invention investigates the extraction solvent and extraction method of the sample, and the type and concentration of the acid added in the mobile phase. At wavelengths of 270nm and 320nm, the relative retention time of each peak changes slightly, and the RDS is less than 2.0%;

[0027] (2) The method for characterizing Tongfengding Capsules was validated by the present invention, including specificity, precision, repeatability, and stability tests. The RSD values ​​of the relative retention times of the peaks in each experimental result were all ≤1.0%, and the RSD values ​​of the relative peak areas were all ≤10.0%. This indicates that the characterizing method is good and can reflect most of the chemical components in Tongfengding Capsules. It can more efficiently and quickly control the overall quality of Tongfengding Capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The extraction method of the test sample was investigated by 270 nm chromatogram; among them, S1: ultrasonic extraction; S2: reflux extraction;

[0029] Figure 2 The extraction method of the test sample was investigated by 320 nm chromatogram; among them, S1: ultrasonic extraction; S2: reflux extraction;

[0030] Figure 3 Specific HPLC chromatogram at 270 nm; S1: negative; S2: gentiopicroside control; S3: gallic acid control; S4: mascinyl acid control; S5: test sample: 2303103;

[0031] Figure 4 Specific HPLC chromatogram at 320 nm; where S1: negative; S2: gentiopicroside control; S3: paeoniflorin control; S4: tetrahydropalmatine control; S5: verbascoside control; S6: test sample: 2303103;

[0032] Figure 5 Precision HPLC chromatogram at 270 nm; S1-S6: precision 1-precision 6;

[0033] Figure 6 Precision HPLC chromatogram at 320 nm; S1-S6: precision 1-precision 6;

[0034] Figure 7 Repeatability HPLC chromatogram at 270 nm; where S1-S6: repeatability 1-repeatability 6;

[0035] Figure 8 Repeatability HPLC chromatogram at 320 nm; where S1-S6: repeatability 1-repeatability 6;

[0036] Figure 9 HPLC chromatogram at 270 nm; wherein, S1: 0 h; S2: 2 h; S3: 4 h; S4: 8 h; S5: 12 h; S6: 24 h;

[0037] Figure 10 320nm chromatogram of stability HPLC; wherein, S1: 0h; S2: 2h; S3: 4h; S4: 8h; S5: 12h; S6: 24h;

[0038] Figure 11 HPLC chromatograms at 270 nm of eight batches of Tongfengding capsule test products; among them, S1: 2303103; S2: 2303102; S3: 2304101; S4: 2304103; S5: 2403101; S6: 2403102; S7: 2404101; S8: 2405102;

[0039] Figure 12 HPLC 320nm chromatograms of eight batches of Tongfengding capsule test products; among them, S1: 2303103; S2: 2303102; S3: 2304101; S4: 2304103; S5: 2403101; S6: 2403102; S7: 2404101; S8: 2405102;

[0040] Figure 13 270nm reference characteristic spectrum of Tongfengding Capsule

[0041] Figure 14 320nm reference characteristic spectrum of Tongfengding Capsule DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the following examples and accompanying drawings. The instruments and equipment used in the examples are as follows:

[0043] 1. Instruments and reagents

[0044] 1.1 Instruments and Equipment

[0045] .

[0046] 1.2 Test materials

[0047] .

[0048] 1.3 Reference Material Information

[0049] .

[0050] 1.4 Sample Information

[0051] .

[0052] Example 1 Investigation of the method for constructing the characteristic spectrum of Tongfengding capsule

[0053] 1.1 Investigation of test sample preparation methods

[0054] 1.1.1 Investigation of the extraction solvent of the test sample

[0055] Chromatographic conditions

[0056] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-formic acid (volume ratio: 70:30:0.1); mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH 2.8) + 0.05% trifluoroacetic acid; gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 270 nm and 320 nm. The number of theoretical plates, calculated based on the gentiopicroside peak, should be no less than 5000.

[0057] .

[0058] Preparation of reference solution

[0059] Take appropriate amount of gallic acid, maleinic acid, gentiopicroside, verbascoside, paeoniflorin, and tetrahydropalmatine reference substances, accurately weigh them, add methanol to make a solution containing 90 μg per 1 mL, shake well, and use them as reference solution.

[0060] Preparation of test solution

[0061] Take 0.5g of the contents of Tongfengding Capsules (batch number: 2303103), accurately weigh them, and add 30mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, methanol-water-0.1% phosphoric acid solution with a volume ratio of 30:70:2, and methanol-water solution with a volume ratio of 70:28, respectively. Extract for 30 minutes; centrifuge at 4000rpm for 10 minutes, take the supernatant, filter through a 0.45μm microporous filter membrane to obtain a crude extract; use a C18 solid phase extraction column First, activate the column with 5 mL of methanol, and then balance the column with 5 mL of water to neutrality; slowly load the crude extract onto the C18 column with a flow rate of 1 mL / min; rinse with 5 mL of water to remove highly polar impurities; elute the target component with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and collect the eluate; concentrate the eluate at 40°C with nitrogen blow-through to near dryness; make up to 2 mL with methanol, vortex mix, and pass through a 0.22 μm filter membrane to obtain the purified test solution.

[0062] Assay

[0063] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0064] Solvent investigation revealed that peak separation and peak shape were better in the 270 nm and 320 nm chromatograms when the extraction solvent contained 0.1% phosphoric acid and the solvent volume ratio was 70:28:2. 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-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2 was selected as the extraction solvent.

[0065] Table 1 Relative retention time results of solvent investigation (270nm)

[0066] .

[0067] Table 2 Relative retention time results of solvent investigation (320nm)

[0068] .

[0069] 1.1.2 Investigation of the extraction method of the test sample

[0070] Preparation of test solution

[0071] Take 0.5g of Tongfengding Capsules (batch number: 2303103), accurately weigh it, add 30mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, and extract by ultrasonication (250W / 40KHz) or heating reflux extraction (60℃) for 30 minutes; centrifuge at 4000rpm for 10 minutes, take the supernatant, filter through 0.45μm microporous membrane to obtain crude extract; use C18 solid phase extraction column, first use 5mL methanol to extract the crude extract. The column was eluted with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and the eluate was collected; the eluate was concentrated to near dryness by nitrogen blow-through at 40°C; the volume was made up to 2 mL with methanol, vortexed and filtered through a 0.22 μm filter membrane to obtain the purified test solution.

[0072] The chromatographic conditions are the same as those in item “1.1.1”; the reference solution is the reference solution in item “1.1.1”.

[0073] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject them into liquid chromatograph, and record the chromatogram.

[0074] The RSD of the relative retention time of the extraction method was less than 2.0%, indicating that the extraction method of ultrasonic extraction (250W / 40KHz) or heating reflux extraction (60°C) had little effect on the relative retention time of each peak in the 270nm and 320nm chromatograms. The results are shown in the attached Figure 1 , Attachment Figure 2.

[0075] Table 3 Relative retention time results of extraction methods (270nm)

[0076] .

[0077] Table 4 Relative retention time results of extraction methods (320nm)

[0078] .

[0079] 1.2 Investigation of chromatographic conditions

[0080] 1.2.1 Investigation of mobile phase types

[0081] Chromatographic conditions 1: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-formic acid (volume ratio: 70:30:0.1); mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH 2.8) + 0.05% trifluoroacetic acid; gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 270 nm and 320 nm. The number of theoretical plates calculated based on the gentiopicroside peak should be no less than 5000.

[0082] .

[0083] Chromatographic conditions 2: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol (70:30, by volume); mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH 2.8) + 0.05% trifluoroacetic acid; gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 270 nm and 320 nm. The number of theoretical plates calculated based on the gentiopicroside peak should be no less than 5000.

[0084] .

[0085] Chromatographic Conditions 3: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-formic acid (volume ratio: 70:30:0.1), mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH 2.8), gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 270 nm and 320 nm. The number of theoretical plates calculated based on the gentiopicroside peak should be no less than 5000.

[0086] .

[0087] Preparation of reference solution

[0088] Take appropriate amount of gallic acid, maleinic acid, gentiopicroside, verbascoside, paeoniflorin, and tetrahydropalmatine reference substances, accurately weigh them, add methanol to make a solution containing 90 μg per 1 mL, shake well, and use them as reference solution.

[0089] Preparation of test solution

[0090] Take 0.5 g of the contents of Tongfengding Capsules (batch number: 2303103), accurately weigh it, add 30 mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, and extract for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter through a 0.45 μm microporous filter membrane to obtain a crude extract; select a C18 solid phase extraction column, first activate the column with 5 mL of methanol, and then balance the column with 5 mL of water to neutrality; slowly load the crude extract onto the C18 column with a flow rate of 1 mL / min; rinse with 5 mL of water to remove highly polar impurities; elute the target component with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and collect the eluate; concentrate the eluate to near dryness with nitrogen blow at 40°C; make up to 2 mL with methanol, vortex mix, and pass through a 0.22 μm filter membrane to obtain the purified test solution.

[0091] Assay

[0092] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0093] The results showed that when elution was performed with acetonitrile-methanol-formic acid solution in a volume ratio of 70:30:0.1 as mobile phase A and 10 mM potassium dihydrogen phosphate buffer at pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, the chromatogram of the test sample presented 8 characteristic peaks at a wavelength of 270 nm and 11 characteristic peaks at a wavelength of 320 nm, with good peak separation and excellent peak shape; when elution was performed with acetonitrile-methanol solution in a volume ratio of 70:30 as mobile phase A and 10 mM potassium dihydrogen phosphate buffer at pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B or with acetonitrile-methanol-formic acid solution in a volume ratio of 70:30:0.1 as mobile phase A and 10 mM potassium dihydrogen phosphate buffer at pH = 2.8 as mobile phase B, all peaks were missing and the corresponding effect was not achieved.

[0094] Table 5 Relative retention time results of mobile phase types (270nm)

[0095] .

[0096] Table 6 Relative retention time results of mobile phase types (320nm)

[0097] .

[0098] 1.2.2 Investigation of mobile phase ratio

[0099] Chromatographic conditions

[0100] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-formic acid (volume ratio: 70:30:0.1); mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH 2.8) + 0.05% trifluoroacetic acid; gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 270 nm and 320 nm. The number of theoretical plates, calculated based on the gentiopicroside peak, should be no less than 5000.

[0101] Mobile phase ratio 1:

[0102] .

[0103] Mobile phase ratio 2:

[0104] .

[0105] Mobile phase ratio 3:

[0106] .

[0107] The reference solution is the same as that in item “1.2.1”.

[0108] Take 0.5 g of the contents of Tongfengding Capsules (batch number: 2303103), accurately weigh it, add 30 mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, and extract for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter through a 0.45 μm microporous filter membrane to obtain a crude extract; select a C18 solid phase extraction column, first activate the column with 5 mL of methanol, and then balance the column with 5 mL of water to neutrality; slowly load the crude extract onto the C18 column with a flow rate of 1 mL / min; rinse with 5 mL of water to remove highly polar impurities; elute the target component with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and collect the eluate; concentrate the eluate to near dryness with nitrogen blow at 40°C; make up to 2 mL with methanol, vortex mix, and pass through a 0.22 μm filter membrane to obtain the purified test solution.

[0109] Assay

[0110] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0111] The results showed that when eluted with a mobile phase ratio of 1, the chromatogram of the test sample presented 8 characteristic peaks at a wavelength of 270nm and 11 characteristic peaks at a wavelength of 320nm, with good peak separation and excellent peak shape; when eluted with a mobile phase ratio of 2 or a mobile phase ratio of 3, all peaks were missing and the corresponding effect was not achieved.

[0112] Table 7 Relative retention time results of mobile phase acid concentration (270nm)

[0113] .

[0114] Table 8 Relative retention time results of mobile phase acid concentration (320nm)

[0115] .

[0116] Example 2 Methodological Verification of Tongfengding Capsules Characteristic Spectrum

[0117] 2.1 Exclusivity

[0118] Chromatographic conditions

[0119] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-formic acid (volume ratio: 70:30:0.1); mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH 2.8) + 0.05% trifluoroacetic acid; gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 270 nm and 320 nm. The number of theoretical plates, calculated based on the gentiopicroside peak, should be no less than 5000.

[0120] .

[0121] Preparation of reference solution

[0122] Take appropriate amount of gallic acid, maleinic acid, gentiopicroside, verbascoside, paeoniflorin, and tetrahydropalmatine reference substances, accurately weigh them, add methanol to make a solution containing 90 μg per 1 mL, shake well, and use them as reference solution.

[0123] Preparation of test solution

[0124] Take 0.5 g of the contents of Tongfengding Capsules (batch number: 2303103), accurately weigh it, add 30 mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, and extract for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter through a 0.45 μm microporous filter membrane to obtain a crude extract; select a C18 solid phase extraction column, first activate the column with 5 mL of methanol, and then balance the column with 5 mL of water to neutrality; slowly load the crude extract onto the C18 column with a flow rate of 1 mL / min; rinse with 5 mL of water to remove highly polar impurities; elute the target component with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and collect the eluate; concentrate the eluate to near dryness with nitrogen blow at 40°C; make up to 2 mL with methanol, vortex mix, and pass through a 0.22 μm filter membrane to obtain the purified test solution.

[0125] Preparation of negative solution

[0126] Take an appropriate amount of auxiliary materials, grind them finely, take 0.5g, weigh accurately, add 30mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, and extract for 30 minutes; centrifuge at 4000rpm for 10 minutes, take the supernatant, filter through a 0.45μm microporous filter membrane to obtain a crude extract; select a C18 solid phase extraction column, first activate the column with 5mL of methanol, and then balance the column with 5mL of water to neutrality; slowly load the crude extract onto the C18 column with a flow rate of 1mL / min; rinse with 5mL of water to remove highly polar impurities; elute the target component with 5mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and collect the eluate; concentrate the eluate to near dryness with nitrogen blow at 40℃; make the volume up to 2mL with methanol, vortex mix, and pass through a 0.22μm filter membrane to obtain.

[0127] Assay

[0128] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0129] The results showed that the negative solution had no interference at 270nm and 320nm wavelengths, and the specificity was good. Figure 3 , Attachment Figure 4 .

[0130] 2.2 Precision

[0131] Take 0.5 g of the contents of Tongfengding Capsules (batch number: 2303103) and prepare the test solution according to the test solution preparation method under "2.1". Inject 6 times according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0132] Table 9 Precision relative retention time results (270nm)

[0133] .

[0134] Table 10 Precision relative retention time results (320nm)

[0135] .

[0136] Table 11 Precision relative peak area results (270nm)

[0137] .

[0138] Table 12 Precision relative peak area results (320nm)

[0139] .

[0140] The results showed that at wavelengths of 270 nm and 320 nm, the relative retention time RSDs of each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 10.0%, indicating good precision. The chromatograms are attached. Figure 5 , Attachment Figure 6 .

[0141] 2.3 Repeatability

[0142] Take 0.5 g of the contents of Tongfengding Capsules (batch number: 2303103), a total of 6 portions, and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample according to the chromatographic conditions under "2.1", record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0143] Table 13 Repeatability relative retention time results (270nm)

[0144] .

[0145] Table 14 Repeatability relative retention time results (320nm)

[0146] .

[0147] Table 15 Repeatability relative peak area results (270nm)

[0148] .

[0149] Table 16 Repeatability relative peak area results (320nm)

[0150] .

[0151] The results showed that at wavelengths of 270nm and 320nm, the relative retention time RSDs of each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 10.0%, indicating good repeatability. The chromatograms are attached. Figure 7 , Attachment Figure 8 .

[0152] 2.4 Stability

[0153] Take 0.5 g of the contents of Tongfengding Capsules (batch number: 2303103) and prepare the test solution according to the test solution preparation method under "2.1". Inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic conditions under "2.1". Record the chromatogram, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0154] Table 17 Stability relative retention time results (270nm)

[0155] .

[0156] Table 18 Stability relative retention time results (320nm)

[0157] .

[0158] Table 19 Stability relative peak area results (270nm)

[0159] .

[0160] Table 20 Stability relative peak area results (320nm)

[0161] .

[0162] The results showed that at wavelengths of 270 nm and 320 nm, the relative retention time RSDs of each chromatographic peak were less than 1.0%, and the relative peak area RSDs were less than 10.0%, indicating that the solution had good stability. The chromatograms are attached. Figure 9 , Attachment Figure 10 .

[0163] In summary, the characteristic spectrum has good specificity and there is no interference from negative samples, indicating that the characteristic detection method has strong specificity. In the precision test, the relative retention time RSDs are all less than 1.0%, and the relative peak area RSDs are all less than 10.0%, indicating good instrument precision. In the repeatability test, the relative retention time RSDs of each chromatographic peak are all less than 1.0%, and the relative peak area RSDs are all less than 10.0%, indicating good repeatability. In the stability test, the relative retention time RSDs of each chromatographic peak are all less than 1.0%, and the relative peak area RSDs are all less than 10.0%, indicating that the test solution is stable within 24 hours. This method has been well validated.

[0164] Example 3 Construction of the control characteristic spectrum of Tongfengding Capsules

[0165] Chromatographic conditions

[0166] Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile-methanol-formic acid (volume ratio: 70:30:0.1); mobile phase B: 10 mM potassium dihydrogen phosphate buffer (pH 2.8) + 0.05% trifluoroacetic acid; gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 270 nm and 320 nm. The number of theoretical plates, calculated based on the gentiopicroside peak, should be no less than 5000.

[0167] .

[0168] Preparation of reference solution: Take appropriate amount of gallic acid, maleinic acid, gentiopicroside, verbascoside, paeoniflorin, and tetrahydropalmatine reference substances, weigh accurately, add methanol to prepare a solution containing 90 μg per 1 mL, shake well, and use as reference solution.

[0169] Preparation of the test solution: 0.5 g of the contents of Tongfengding Capsules (batch number: 2303103) were accurately weighed and added to 30 mL of a methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2. The mixture was extracted for 30 minutes. The mixture was centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected and filtered through a 0.45 μm microporous filter to obtain a crude extract. A C18 solid phase extraction column was selected, which was first activated with 5 mL of methanol and then equilibrated with 5 mL of water to neutrality. The crude extract was slowly loaded onto the C18 column at a flow rate of 1 mL / min. The column was rinsed with 5 mL of water to remove highly polar impurities. The target component was eluted with 5 mL of a methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and the eluate was collected. The eluate was concentrated to near dryness by nitrogen blow-through at 40°C. The volume was made up to 2 mL with methanol, vortexed to mix, and filtered through a 0.22 μm filter to obtain the purified test solution.

[0170] Assay

[0171] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0172] The chromatograms of the test samples of 8 batches of Tongfengding Capsules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and the chromatographic peaks present in the chromatograms of different batches of Tongfengding 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. The results are shown in the attached Figure 11 , Attachment Figure 12 , Attachment Figure 13 , Attachment Figure 14 .

[0173] Table 21 Relative retention time results of eight batches of Tongfengding Capsules (270nm)

[0174] .

[0175] Table 22 Relative retention time results of eight batches of Tongfengding capsules (320nm)

[0176] .

[0177] Table 23 Relative peak area results (270nm) of eight batches of Tongfengding capsules

[0178] .

[0179] Table 24 Relative peak area results (320nm) of eight batches of Tongfengding capsules

[0180] .

[0181] The 270nm wavelength chromatograms of the eight batches of Tongfengding Capsules samples all showed 8 common peaks, the RSDs of the relative retention times of the 8 common peaks were all less than 1.0%, and the RSDs of the relative peak areas were all less than 8.0%; the 320nm wavelength chromatograms of the eight batches of Tongfengding Capsules samples all showed 11 common peaks, the RSDs of the relative retention times of the 11 common peaks were all less than 1.0%, and the RSDs of the relative peak areas were all less than 8.0%.

[0182] At a wavelength of 270 nm, the sample chromatogram exhibits eight characteristic peaks, of which Peaks 1, 2, and 5 correspond to the peaks of gallic acid, magnosinic acid, and gentiopicroside reference substance, respectively. The peak corresponding to the peak of the gentiopicroside 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: 0.31 (Peak 1), 0.67 (Peak 2), 0.84 (Peak 3), 0.89 (Peak 4), 1.14 (Peak 6), 1.56 (Peak 7), and 2.10 (Peak 8).

[0183] At a wavelength of 320 nm, the chromatogram of the test sample showed 11 characteristic peaks, of which Peaks 2, 5, 7, and 10 corresponded to the peaks of the gentiopicroside, paeoniflorin, verbascoside, and tetrahydropalmatine reference substance, respectively. The peak corresponding to the peak of the gentiopicroside reference substance was the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.89 (peak 1), 1.14 (peak 3), 1.19 (peak 4), 1.21 (peak 5), 1.47 (peak 6), 1.51 (peak 7), 1.56 (peak 8), 1.68 (peak 9), 1.76 (peak 10), and 2.10 (peak 11).

Claims

1. A method for constructing a characteristic spectrum of Tongfengding Capsule, characterized in that: The following steps are involved: (1) Preparation of reference solution: accurately weigh the reference substance, add methanol to prepare a solution containing 90 μg per 1 mL, shake well, and use it as the reference solution; the reference substances are: gallic acid, gentiopicroside, gentioside, verbascoside, paeoniflorin, and tetrahydropalmatine; (2) Preparation of the test solution: Take the contents of Tongfengding capsules, weigh accurately, add methanol-water-0.1% phosphoric acid solution, and extract for 30 minutes; after centrifugation, take the supernatant, filter through a 0.45μm microporous filter membrane to obtain a crude extract; select a C18 solid phase extraction column, first activate the column with 5mL of methanol, and then balance the column with 5mL of water to neutrality; slowly load the crude extract onto the C18 column, and control the flow rate at 1mL / min; rinse with 5mL of water to remove highly polar impurities; elute the target component with methanol-water-phosphoric acid solution, and collect the eluate; concentrate the eluate at 40℃ with nitrogen blowing to near dryness; dilute to 2mL with methanol, vortex mix, and pass through a 0.22μm filter membrane to obtain the purified 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-formic acid solution (volume ratio: 70:30:0.1) as mobile phase A, 10 mM potassium dihydrogen phosphate buffer (pH = 2.8) + 0.05% trifluoroacetic acid as mobile phase B, elution according to the specified gradient; column temperature: 30°C; flow rate: 1 mL / min; detection wavelength: 270 nm or 320 nm. The elution gradient conditions were: From 0 to 30 min, the volume ratio of mobile phase A:phase B was changed from 5:95 to 20:80; From 30 to 50 min, the volume ratio of mobile phase A:phase B changed from 20:80 to 33:67; From 50 to 65 min, the volume ratio of mobile phase A:phase B was changed from 33:67 to 60:40; From 65 to 66 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95; 66-76 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 Tongfengding Capsules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of Tongfengding Capsules.

2. The method for constructing a characteristic spectrum of Tongfengding Capsule according to claim 1, characterized in that: The volume ratio of methanol-water-0.1% phosphoric acid solution used for extraction in step (2) is 70:28:2, and the amount added is 30 mL.

3. The method for constructing a characteristic spectrum of Tongfengding Capsule according to claim 1, characterized in that: In step (2), the volume ratio of methanol-water-phosphoric acid solution used for elution is 70:30:0.1, and the amount added is 5 mL.

4. The method for constructing a characteristic spectrum of Tongfengding Capsule according to claim 1, characterized in that: In step (2), the centrifugation condition is 4000 rpm for 10 minutes.

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

6. The method for constructing a characteristic spectrum of Tongfengding Capsule according to claim 1, characterized in that: The control characteristic spectrum generated in step (4) at a wavelength of 270 nm includes 8 common chromatographic peaks, including: Peak 1 corresponding to gallic acid, Peak 2 corresponding to maleinic acid, and Peak 5 corresponding to gentiopicroside.

7. The method for constructing a characteristic spectrum of Tongfengding Capsule according to claim 1, characterized in that: The control characteristic spectrum generated in step (4) at a wavelength of 320 nm includes 11 common chromatographic peaks, including: Peak 2 corresponding to gentiopicroside, Peak 5 corresponding to paeoniflorin, Peak 7 corresponding to verbascoside, and Peak 10 corresponding to tetrahydropalmatine.

8. The method for constructing a characteristic spectrum of Tongfengding Capsule according to claim 1, characterized in that: In step (4), the characteristic peak at a wavelength of 270 nm is referenced to the chromatographic peak of gentiopicroside. The relative retention times of the eight chromatographic peaks are 0.31 for peak 1, 0.67 for peak 2, 0.84 for peak 3, 0.89 for peak 4, 1.14 for peak 6, 1.56 for peak 7, and 2.10 for peak 8.

9. The method for constructing a characteristic spectrum of Tongfengding Capsule according to claim 1, characterized in that: The characteristic peak at a wavelength of 320 nm was taken as the reference peak using the chromatographic peak of gentiopicroside. The relative retention times of the 11 chromatographic peaks were 0.89 for peak 1, 1.14 for peak 3, 1.19 for peak 4, 1.21 for peak 5, 1.47 for peak 6, 1.51 for peak 7, 1.56 for peak 8, 1.68 for peak 9, 1.76 for peak 10, and 2.10 for peak 11.

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