Construction method of specific chromatogram of gout-treating capsule

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method of a specific chromatogram of a gout-treating capsule. According to the method disclosed by the invention, HPLC characteristic chromatograms are respectively established under the wavelengths of 270nm and 320nm for the gout-determining capsule, eight characteristic peaks are defined under the wavelength of 270nm, and gallic acid, mannuronic acid and gentiopicroside are controlled; under the wavelength of 320nm, 11 characteristic peaks are defined, gentiopicroside, paeoniflorin, verbascoside and tetrahydropalmatine are controlled, and the chemical component characteristics of the gout treating capsule are relatively fully and integrally displayed. The extraction solvent and the extraction mode of the sample and the type and the concentration of the acid added in the mobile phase are investigated, the relative retention time change of each peak is small under the wavelength of 270 nm and 320 nm, and the RDS is less than 2.0%.

Description

Technical Field

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

[0002] Tongfengding capsules are currently included in the first part of the Chinese Pharmacopoeia (2020 Edition). It is a traditional Chinese medicine compound preparation extracted and processed from 8 kinds of medicinal materials such as Gentianae Macrophyllae Radix, Corydalis Rhizoma, Cyathulae Radix, etc., and has the effects of clearing heat and removing dampness, promoting blood circulation and dredging collaterals, and treating gout. Most of the existing quality control standards and literature only quantitatively control 1 to 2 of its components, and it is difficult to comprehensively reflect the quality of the preparation. The technology of traditional Chinese medicine characteristic chromatogram has integrity and ambiguity in the quality control of traditional Chinese medicine, and can comprehensively detect the effectiveness and stability of the quality of traditional Chinese medicine.

[0003] Tongfengding capsules are composed of 8 kinds of medicinal materials, with complex chemical components and large property differences. It is necessary to achieve effective separation and detection of multiple components under the same chromatographic conditions, which puts extremely high requirements on the design of mobile phase gradient, the selection of chromatographic column and the optimization of detection wavelength.

[0004] The present invention constructs a method for the characteristic chromatogram of Tongfengding capsules, in order to provide an experimental basis for comprehensively evaluating the internal 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 chromatogram 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 object of the present invention, the following technical solutions are specifically adopted: The present invention provides a method for constructing a characteristic chromatogram of Tongfengding capsules, including the following steps: (1) Preparation of reference substance solution: Take an appropriate amount of reference substance, accurately weigh it, and separately add methanol to make a solution containing 90 μg per 1 mL, shake well, and use it as the reference substance solution; The reference substances are: gallic acid, loganic acid, gentiopicroside, acteoside, paeoniflorin, tetrahydropalmatine; (2) Preparation of the test solution: Weigh 0.5 g of the content of Tongfengding capsules precisely, add 30 mL of a methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2 (where 0.1% phosphoric acid is the mass fraction), extract for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter it 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 until neutral; slowly load the crude extract onto the C18 column, controlling the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of a methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40°C; make up the volume to 2 mL with methanol, vortex and mix evenly, and filter through a 0.22 μm filter membrane to obtain the purified test solution; (3) Determination: The reference solution and the test solution are injected into a high-performance liquid chromatograph under the following chromatographic conditions, each injecting 10 μl, and record the chromatogram; the chromatographic conditions are: using octadecylsilane-bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); using an acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A, and a 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B (where 0.05% trifluoroacetic acid is the volume fraction), and perform elution according to the specified gradient; the column temperature is 30°C; the flow rate is 1 mL per minute; the detection wavelength is 270 nm or 320 nm; The elution gradient conditions are: 0 - 30 min, mobile phase A: B, volume ratio changes from 5:95 to 20:80; 30 - 50 min, mobile phase A: B, volume ratio changes from 20:80 to 33:67; 50 - 65 min, mobile phase A: B, volume ratio changes from 33:67 to 60:40; 65 - 66 min, mobile phase A: B, volume ratio changes from 60:40 to 5:95; 66 - 76 min, mobile phase A: B, volume ratio is 5:95; (4) Generating a reference characteristic chromatogram: Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" formulated by the National Pharmacopoeia Commission, select the chromatographic peaks that exist in the chromatograms of different batches of Tongfengding capsules as common peaks, and generate the reference characteristic chromatogram of Tongfengding capsules by the average value calculation method.

[0007] Preferably, in step (2), the volume ratio of the methanol-water-0.1% phosphoric acid solution for extraction is 70:28:2, and the dosage is 30 mL.

[0008] Preferably, in step (2), the volume ratio of the methanol-water-phosphoric acid solution for elution is 70:30:0.1, and the addition amount is 5 mL.

[0009] Preferably, the centrifugation conditions are centrifugation at 4000 rpm for 10 minutes.

[0010] Preferably, the control characteristic chromatogram generated at a wavelength of 270 nm in step (4) includes 8 common chromatographic peaks, specifically: peak 1 corresponding to gallic acid, peak 2 corresponding to loganic acid, and peak 5 corresponding to gentiopicroside.

[0011] Preferably, the control characteristic chromatogram generated at a wavelength of 320 nm in step (4) includes 11 common chromatographic peaks, specifically: peak 2 corresponding to gentiopicroside, peak 5 corresponding to paeoniflorin, peak 7 corresponding to acteoside, and peak 10 corresponding to tetrahydropalmatine.

[0012] Preferably, at a wavelength of 270 nm, with the gentiopicroside chromatographic peak as the reference peak, the relative retention times of the 8 chromatographic peaks are: peak 1 is 0.31, peak 2 is 0.67, peak 3 is 0.84, peak 4 is 0.89, peak 6 is 1.14, peak 7 is 1.56, and peak 8 is 2.10.

[0013] Preferably, at a wavelength of 320 nm, with the gentiopicroside chromatographic peak as the reference peak, 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, and peak 11 is 2.10. Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention has established HPLC characteristic chromatograms for Tongfengding capsules at wavelengths of 270 nm and 320 nm respectively. At a wavelength of 270 nm, 8 characteristic peaks are identified, and gallic acid, loganic acid, and gentiopicroside are controlled; at a wavelength of 320 nm, 11 characteristic peaks are identified, and gentiopicroside, paeoniflorin, acteoside, and tetrahydropalmatine are controlled, which more fully and comprehensively demonstrates the chemical composition characteristics of Tongfengding capsules; moreover, the present invention has investigated the extraction solvent and extraction method of the sample, as well as the type and concentration of acid added to the mobile phase. At wavelengths of 270 nm and 320 nm, the relative retention times of each peak vary little, and the RSDs are all less than 2.0%. (2) The present invention has carried out methodological verification on the characteristic fingerprint method of Tongfengding capsules constructed, including specificity, precision, repeatability, and stability experiments. In the results of each experiment, the RSD values of the relative retention times of each peak are all ≤ 1.0%, and the RSD values of the relative peak areas are all ≤ 10.0%. This indicates that the characteristic fingerprint method is good, and this method can reflect most of the chemical components in Tongfengding capsules. It can control the overall quality of Tongfengding capsules more efficiently and quickly. Description of the Drawings

[0014] Figure 1 Chromatogram at 270 nm for investigating the extraction method of the test sample; among them, S1: ultrasonic extraction; S2: reflux extraction; Figure 2 Chromatogram at 320 nm for investigating the extraction method of the test sample; among them, S1: ultrasonic extraction; S2: reflux extraction; Figure 3 Chromatogram at 270 nm of specificity HPLC; S1: negative; S2: gentiopicroside control; S3: gallic acid control; S4: loganin acid control; S5: test sample: 2303103; Figure 4 Chromatogram at 320 nm of specificity HPLC; among them, S1: negative; S2: gentiopicroside control; S3: paeoniflorin control; S4: tetrahydropalmatine control; S5: acteoside control; S6: test sample: 2303103; Figure 5 Chromatogram at 270 nm of precision HPLC; among them, S1 - S6: precision 1 - precision 6; Figure 6 Chromatogram at 320 nm of precision HPLC; among them, S1 - S6: precision 1 - precision 6; Figure 7 Chromatogram at 270 nm of repeatability HPLC; among them, S1 - S6: repeatability 1 - repeatability 6; Figure 8 Chromatogram at 320 nm of repeatability HPLC; among them, S1 - S6: repeatability 1 - repeatability 6; Figure 9 Chromatogram at 270 nm of stability HPLC; among them, S1: 0 h; S2: 2 h; S3: 4 h; S4: 8 h; S5: 12 h; S6: 24 h; Figure 10 Chromatogram at 320 nm of stability HPLC; among them, S1: 0 h; S2: 2 h; S3: 4 h; S4: 8 h; S5: 12 h; S6: 24 h; Figure 11HPLC chromatograms of eight batches of Tongfengding capsules for test at 270 nm; among them, S1: 2303103; S2: 2303102; S3: 2304101; S4: 2304103; S5: 2403101; S6: 2403102; S7: 2404101; S8: 2405102; Figure 12 HPLC chromatograms of eight batches of Tongfengding capsules for test at 320 nm; among them, S1: 2303103; S2: 2303102; S3: 2304101; S4: 2304103; S5: 2403101; S6: 2403102; S7: 2404101; S8: 2405102; Figure 13 Reference characteristic chromatogram of Tongfengding capsules at 270 nm Figure 14 Reference characteristic chromatogram of Tongfengding capsules at 320 nm Detailed implementation manners

[0015] The present invention will be further described below in conjunction with the embodiments and the drawings. The instruments and equipment used in the embodiments are as follows: I. Instruments and test drugs 1.1 Instruments and equipment .

[0016] 1.2 Test materials .

[0017] 1.3 Reference substance information .

[0018] 1.4 Sample information .

[0019] Investigation on the construction method of the characteristic chromatogram of Tongfengding capsules in Example 1 1.1 Investigation on the preparation method of the test sample 1.1.1 Investigation on the extraction solvent of the test sample Chromatographic conditions Chromatographic column: Alltima-C18 (4.6x250 mm, 5 μm); acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A, 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the column temperature was 30 °C; the detection wavelengths were 270 nm and 320 nm. The number of theoretical plates calculated based on the gentianopicroside peak should be not less than 5000.

[0020] .

[0021] Preparation of reference substance solution Appropriately weigh a proper amount of reference substances of gallic acid, loganic acid, gentiopicroside, acteoside, paeoniflorin, and tetrahydropalmatine, and accurately weigh them. Respectively dissolve them in methanol to prepare solutions containing 90 μg per 1 mL, shake well, and use them as reference substance solutions.

[0022] Preparation of test solution Take 0.5 g of the content of Tongfengding Capsules (batch number: 2303103), accurately weigh it, and respectively add 30 mL 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, and extract for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter it 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, and control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, collect the eluate; concentrate the eluate to nearly dry at 40 °C by nitrogen blowing; dilute to 2 mL with methanol, vortex and mix well, and filter through a 0.22 μm filter membrane to obtain the purified test solution.

[0023] Determination method Precisely pipette 10 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0024] The results of solvent investigation show that in the chromatograms at 270 nm and 320 nm, when the extraction solvent contains 0.1% phosphoric acid and the volume ratio of the extraction solvent is 70:28:2, the peak resolution is better and the peak shape is better; when there is no phosphoric acid in the extraction solvent, there are situations of poor peak shape and peak missing, and the corresponding effects cannot be achieved. Therefore, select the methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2 as the extraction solvent.

[0025] Table 1 Results of relative retention time of solvent investigation (270 nm) 。

[0026] Table 2 Results of relative retention time of solvent investigation (320 nm) 。

[0027] 1.1.2 Investigation of test sample extraction method Preparation of test solution Take 0.5 g of the content 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 by ultrasonic extraction (250 W / 40 KHz) or heat reflux extraction (60 °C) for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter it 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, control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C; dissolve it to 2 mL with methanol, vortex and mix well, and filter through a 0.22 μm filter membrane to obtain the purified test solution.

[0028] The chromatographic conditions are the same as those under "1.1.1"; the reference substance solution is taken as the reference substance solution under "1.1.1".

[0029] Determination method: Accurately pipette 10 μL of the reference substance solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0030] The RSDs of the relative retention times in the results of the extraction method investigation are all less than 2.0%, indicating that when the extraction method is ultrasonic extraction (250 W / 40 KHz) or heat reflux extraction (60 °C), the influence on the relative retention times of each peak in the chromatograms at 270 nm and 320 nm is small. The results are shown in Appendix Figure 1 and Appendix Figure 2 .

[0031] Table 3 Results of the relative retention times in the extraction method investigation (270 nm) .

[0032] Table 4 Results of the relative retention times in the extraction method investigation (320 nm) .

[0033] 1.2 Chromatographic condition investigation 1.2.1 Investigation of the type of mobile phase Chromatographic condition 1: Alltima-C18 (4.6 x 250 mm, 5 μm); use acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A, and 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, and perform gradient elution according to the regulations in the following table; the column temperature is 30 °C; the detection wavelengths are 270 nm and 320 nm. The number of theoretical plates calculated based on the gentiopicroside peak should be not less than 5000.

[0034] 。

[0035] Chromatographic conditions 2: Alltima-C18 (4.6x250mm, 5μm); acetonitrile-methanol solution with a volume ratio of 70:30 as mobile phase A, 10 mM potassium dihydrogen phosphate buffer with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelengths were 270 nm and 320 nm. The number of theoretical plates calculated based on the gentiopicroside peak should be not less than 5000.

[0036] 。

[0037] Chromatographic conditions 3: Alltima-C18 (4.6x250mm, 5μm); acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A, 10 mM potassium dihydrogen phosphate buffer with pH = 2.8 as mobile phase B, gradient elution was carried out according to the regulations in the following table; column temperature was 30°C; detection wavelengths were 270 nm and 320 nm. The number of theoretical plates calculated based on the gentiopicroside peak should be not less than 5000.

[0038] 。

[0039] Preparation of reference substance solution Appropriately weigh a suitable amount of reference substances of gallic acid, loganic acid, gentiopicroside, acteoside, paeoniflorin, and tetrahydropalmatine, and accurately weigh them. Respectively dissolve them in methanol to prepare solutions containing 90 μg per 1 mL, shake well, and use them as reference substance solutions.

[0040] Preparation of test solution Take 0.5 g of the content 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, extract for 30 minutes; centrifuge at 4000 rpm for 10 minutes, take the supernatant, filter it 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 neutral; slowly load the crude extract onto the C18 column, control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, collect the eluate; concentrate the eluate to nearly dry at 40°C under nitrogen blowing; dissolve it in methanol to a volume of 2 mL, vortex and mix well, filter through a 0.22 μm filter membrane to obtain the purified test solution.

[0041] Determination method Precisely pipette 10 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0042] The results showed that when eluting with acetonitrile - methanol - formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A and 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, the test sample chromatogram presented 8 characteristic peaks at a wavelength of 270 nm and 11 characteristic peaks at a wavelength of 320 nm, and the peak resolution was good and the peak shape was excellent; when eluting with acetonitrile - methanol solution with a volume ratio of 70:30 as mobile phase A and 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B or acetonitrile - methanol - formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A and 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 as mobile phase B, each peak was missing and the corresponding effects could not be achieved.

[0043] Table 5 Results of relative retention time for investigation of mobile phase types (270 nm) 。

[0044] Table 6 Results of relative retention time for investigation of mobile phase types (320 nm) 。

[0045] 1.2.2 Investigation of mobile phase ratio Chromatographic conditions Chromatographic column: Alltima - C18 (4.6x250 mm, 5 μm); acetonitrile - methanol - formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A, 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, and gradient elution was carried out according to the regulations in the following table; column temperature was 30 °C; detection wavelengths were 270 nm and 320 nm. The theoretical plate number calculated by the gentiopicroside peak should be not less than 5000.

[0046] Mobile phase ratio 1: 。

[0047] Mobile phase ratio 2: 。

[0048] Mobile phase ratio 3: 。

[0049] The reference substance solution was taken from the reference substance solution under item "1.2.1".

[0050] Take 0.5 g of the content of Tongfengding Capsules (batch number: 2303103), accurately weigh it, add 30 mL of a 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 it 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, and control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of a methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, and collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C; dilute to 2 mL with methanol, vortex and mix evenly, and filter through a 0.22 μm filter membrane to obtain the purified test solution.

[0051] Assay method Precisely pipette 10 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0052] The results showed that when eluting with mobile phase ratio 1, the test solution chromatogram showed 8 characteristic peaks at a wavelength of 270 nm and 11 characteristic peaks at a wavelength of 320 nm, and the peak separation was good and the peak shape was excellent; when eluting with mobile phase ratio 2 or mobile phase ratio 3, each peak was missing and the corresponding effects were not achieved.

[0053] Table 7 Results of relative retention time for investigating the acid concentration of the mobile phase (270 nm) .

[0054] Table 8 Results of relative retention time for investigating the acid concentration of the mobile phase (320 nm) .

[0055] Example 2 Methodological verification of the characteristic chromatogram of Tongfengding Capsules 2.1 Specificity Chromatographic conditions Chromatographic column: Alltima-C18 (4.6 x 250 mm, 5 μm); use an acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A, and a 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, and perform gradient elution according to the regulations in the following table; the column temperature is 30 °C; the detection wavelengths are 270 nm and 320 nm. The number of theoretical plates calculated based on the gentiopicroside peak should be not less than 5000.

[0056] .

[0057] Preparation of the reference solution Weigh appropriate amounts of gallic acid, loganic acid, gentiopicroside, acteoside, paeoniflorin, and tetrahydropalmatine reference substances accurately, and dissolve them separately in methanol to prepare solutions containing 90 μg per 1 mL. Shake well to obtain the reference substance solutions.

[0058] Preparation of Test Solution Take 0.5 g of the content of Tongfengding Capsules (batch number: 2303103), weigh accurately, add 30 mL of a 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 membrane to obtain the crude extract; select a C18 solid - phase extraction column, first activate the column with 5 mL of methanol, then balance the column with 5 mL of water until neutral; slowly load the crude extract onto the C18 column, control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of a methanol - water - phosphoric acid solution with a volume ratio of 70:30:0.1, collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C; dissolve and dilute to 2 mL with methanol, vortex and mix well, filter through a 0.22 μm membrane to obtain the purified test solution.

[0059] Preparation of Negative Solution Take appropriate amounts of excipients, grind them finely, take 0.5 g, weigh accurately, add 30 mL of a 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 membrane to obtain the crude extract; select a C18 solid - phase extraction column, first activate the column with 5 mL of methanol, then balance the column with 5 mL of water until neutral; slowly load the crude extract onto the C18 column, control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of a methanol - water - phosphoric acid solution with a volume ratio of 70:30:0.1, collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C; dissolve and dilute to 2 mL with methanol, vortex and mix well, filter through a 0.22 μm membrane to obtain the negative solution.

[0060] Determination Method Precisely pipette 10 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0061] The results show that at wavelengths of 270 nm and 320 nm, the negative solution has no interference and the specificity is good. The results are shown in Appendix Figure 3 and Appendix Figure 4 .

[0062] 2.2 Precision Take the content of Tongfengding Capsules (batch number: 2303103), take 0.5 g, prepare the test solution according to the preparation method of the test solution under item "2.1", inject 6 needles according to the chromatographic conditions under item "2.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0063] Table 9 Results of relative retention time for precision (270 nm) 。

[0064] Table 10 Results of relative retention time for precision (320 nm) 。

[0065] Table 11 Results of relative peak area for precision (270 nm) 。

[0066] Table 12 Results of relative peak area for precision (320 nm) 。

[0067] The results show that at wavelengths of 270 nm and 320 nm, the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 10.0%, indicating good precision. The chromatograms are shown in Appendix Figure 5 、Appendix Figure 6 。

[0068] 2.3 Repeatability Take the content of Tongfengding Capsules (batch number: 2303103), take 0.5 g, a total of 6 portions, prepare the test solution according to the preparation method of the test solution under item "2.1", inject according to the chromatographic conditions under item "2.1", record the chromatogram, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0069] Table 13 Results of relative retention time for repeatability (270 nm) 。

[0070] Table 14 Results of relative retention time for repeatability (320 nm) 。

[0071] Table 15 Results of relative peak area for repeatability (270 nm) 。

[0072] Table 16 Results of relative peak area for repeatability (320 nm) 。

[0073] The results showed that at wavelengths of 270 nm and 320 nm, the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 10.0%, indicating good repeatability. The chromatograms are shown in Attachment Figure 7 and Attachment Figure 8 .

[0074] 2.4 Stability Take the content of Tongfengding Capsules (batch number: 2303103), weigh 0.5 g, prepare the test solution according to the preparation method of the test solution under item "2.1", and inject samples at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively according to the chromatographic conditions under item "2.1", record the chromatograms, determine the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD.

[0075] Table 17 Results of relative retention time for stability (270 nm) .

[0076] Table 18 Results of relative retention time for stability (320 nm) .

[0077] Table 19 Results of relative peak area for stability (270 nm) .

[0078] Table 20 Results of relative peak area for stability (320 nm) .

[0079] The results showed that at wavelengths of 270 nm and 320 nm, the RSD of the relative retention time of each chromatographic peak was less than 1.0%, and the RSD of the relative peak area was less than 10.0%, indicating good solution stability. The chromatograms are shown in Attachment Figure 9 and Attachment Figure 10 .

[0080] In summary, the specificity of this characteristic chromatogram is good, and the negative sample has no interference, indicating that the characteristic detection method has strong specificity; in the precision test, the RSD of the relative retention time is less than 1.0%, and the RSD of the relative peak area is less than 10.0%, indicating good instrument precision; in the repeatability test, the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 10.0%, indicating good repeatability; in the stability test, the RSD of the relative retention time of each chromatographic peak is less than 1.0%, and the RSD of the relative peak area is less than 10.0%, so the test solution is stable within 24 h. This method has been well verified by methodology.

[0081] Example 3 Construction of the reference characteristic chromatogram of Tongfengding Capsules Chromatographic conditions Chromatographic column: Alltima-C18 (4.6x250mm, 5μm); Acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 was used as mobile phase A, and 10 mM potassium dihydrogen phosphate buffer with pH = 2.8 + 0.05% trifluoroacetic acid was used as mobile phase B. Gradient elution was carried out according to the regulations in the following table; the column temperature was 30°C; the detection wavelengths were 270 nm and 320 nm. The number of theoretical plates calculated by the swertiamarin peak should be not less than 5000.

[0082] 。

[0083] Preparation of reference solution Weigh an appropriate amount of gallic acid, loganin acid, swertiamarin, acteoside, paeoniflorin, and tetrahydropalmatine reference substances accurately, and dissolve them in methanol respectively to make solutions containing 90 μg per 1 mL. Shake well to obtain the reference solution.

[0084] Preparation of test solution Take 0.5 g of the content of Tongfengding Capsules (batch number: 2303103), weigh accurately, add 30 mL of methanol-water-0.1% phosphoric acid solution with a volume ratio of 70:28:2, 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 the 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, and control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with 5 mL of methanol-water-phosphoric acid solution with a volume ratio of 70:30:0.1, collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40°C; dilute to 2 mL with methanol, vortex and mix well, filter through a 0.22 μm filter membrane to obtain the purified test solution.

[0085] Determination method Precisely pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0086] Import the test chromatograms of 8 batches of Tongfengding Capsules into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints", and select the chromatographic peaks that exist in the chromatograms of different batches of Tongfengding Capsules as the common peaks; generate the reference characteristic fingerprint by the average value calculation method, and calculate the relative retention time and relative peak area of each common peak. The results are shown in Appendix Figure 11 、Appendix Figure 12 、Appendix Figure 13 、Appendix Figure 14 。

[0087] Table 21 Results of relative retention time of eight batches of Tongfengding Capsules (270 nm) 。

[0088] Table 22 Results of Relative Retention Time of Eight Batches of Tongfengding Capsules (320 nm) 。

[0089] Table 23 Results of Relative Peak Area of Eight Batches of Tongfengding Capsules (270 nm) 。

[0090] Table 24 Results of Relative Peak Area of Eight Batches of Tongfengding Capsules (320 nm) 。

[0091] In the chromatograms of eight batches of Tongfengding capsule samples at a wavelength of 270 nm, 8 common peaks were presented. 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%. In the chromatograms of eight batches of Tongfengding capsule samples at a wavelength of 320 nm, 11 common peaks were presented. 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%.

[0092] At a wavelength of 270 nm, 8 characteristic peaks were presented in the chromatogram of the test sample. Among them, Peak 1, Peak 2, and Peak 5 corresponded to the reference peaks of gallic acid, loganic acid, and gentiopicroside respectively. The peak corresponding to the reference peak of gentiopicroside was the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and their relative retention times should all be within the range of ±10% of the specified values. 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), 2.10 (Peak 8).

[0093] At a wavelength of 320 nm, 11 characteristic peaks were presented in the chromatogram of the test sample. Among them, Peak 2, Peak 5, Peak 7, and Peak 10 corresponded to the reference peaks of gentiopicroside, paeoniflorin, acteoside, and tetrahydropalmatine respectively. The peak corresponding to the reference peak of gentiopicroside was the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and their relative retention times should all be within the range of ±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), 2.10 (Peak 11).

Claims

1. A method for constructing a characteristic chromatogram of Tongfengding capsules, characterized in that It includes the following steps: (1) Preparation of the reference solution: Take the reference substances, weigh accurately, and dissolve them in methanol respectively to prepare solutions containing 90 μg per 1 mL, shake well, and use them as the reference solutions; the reference substances are: gallic acid, loganic acid, gentiopicroside, acteoside, paeoniflorin, tetrahydropalmatine; (2) Preparation of the test solution: Take the content of Tongfengding capsules, weigh accurately, add methanol-water-0.1% phosphoric acid solution, extract for 30 minutes; after centrifugation, take the supernatant, filter it through a 0.45 μm microporous filter membrane to obtain the 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 neutral; slowly load the crude extract onto the C18 column, and control the flow rate at 1 mL / min; wash with 5 mL of water to remove strongly polar impurities; elute the target components with methanol-water-phosphoric acid solution, collect the eluate; concentrate the eluate to nearly dry by nitrogen blowing at 40 °C; dissolve it in methanol to a volume of 2 mL, vortex and mix well, and filter through a 0.22 μm 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 each, and record the chromatogram; The chromatographic conditions are: using octadecylsilane chemically bonded silica as the filler; using an acetonitrile-methanol-formic acid solution with a volume ratio of 70:30:0.1 as mobile phase A, and using a 10 mM potassium dihydrogen phosphate buffer solution with pH = 2.8 + 0.05% trifluoroacetic acid as mobile phase B, and perform elution according to the specified gradient; the column temperature is 30 °C; the flow rate is 1 mL per minute; the detection wavelength is 270 nm or 320 nm; The elution gradient conditions are: 0 - 30 min, mobile phase A: mobile phase B, volume ratio changes from 5:95 to 20:80; 30 - 50 min, mobile phase A: mobile phase B, volume ratio changes from 20:80 to 33:67; 50 - 65 min, mobile phase A: mobile phase B, volume ratio changes from 33:67 to 60:40; 65 - 66 min, mobile phase A: mobile phase B, volume ratio changes from 60:40 to 5:95; 66 - 76 min, mobile phase A: mobile phase B, volume ratio is 5:95; (4) Generate the reference characteristic chromatogram: Select the chromatographic peaks that exist in the chromatograms of different batches of Tongfengding capsules as the common peaks, and generate the reference characteristic chromatogram of Tongfengding capsules by the average value calculation method.

2. The construction method of the characteristic chromatogram of a gout-relieving capsule according to claim 1, characterized in that In step (2), the volume ratio of the methanol-water-0.1% phosphoric acid solution used for extraction is 70:28:2, and the addition amount is 30 mL.

3. The method for constructing the characteristic spectrum of a gout-relieving capsule according to claim 1, wherein In step (2), the volume ratio of the methanol-water-phosphoric acid solution used for elution is 70:30:0.1, and the addition amount is 5 mL.

4. The construction method of the characteristic fingerprint of a gout-relieving capsule according to claim 1, wherein, In step (2), the centrifugation conditions are centrifugation at 4000 rpm for 10 minutes.

5. The method for constructing the characteristic chromatogram of a gout-relieving capsule according to claim 1, wherein 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 the characteristic chromatogram of a gout-relieving capsule according to claim 1, wherein, The reference characteristic chromatogram generated at a wavelength of 270 nm in step (4) includes 8 common chromatographic peaks, specifically: peak 1 corresponding to gallic acid, peak 2 corresponding to loganic acid, and peak 5 corresponding to gentiopicroside.

7. The method for constructing the characteristic chromatogram of a gout-relieving capsule according to claim 1, characterized in that, The control characteristic chromatogram 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 acteoside, and peak 10 corresponding to tetrahydropalmatine.

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

10.

9. The method for constructing the characteristic chromatogram of Tongfengding capsules according to claim 1, characterized in that, The characteristic peaks at a wavelength of 320 nm are referenced to the gentiopicroside chromatographic peak. The relative retention times of the 11 chromatographic peaks are as follows: 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, and peak 11 is 2.10.

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