Construction method and application of specific chromatogram of synovitis capsule

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method and application of a specific chromatogram of a synovitis capsule. Comprising the following steps: 1, preparing a reference substance solution; 2, preparing a test solution; 3, determining conditions; and 4, importing the chromatogram into a traditional Chinese medicine spectrum to generate a control characteristic spectrum. According to the characteristic method, 13 characteristic peaks are defined, the method is good in repeatability and high in precision, the chemical component characteristics of the synovitis capsule are fully and integrally displayed, and the medicine quality of the synovitis capsule is integrally controlled.

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 a synovitis capsule and an application thereof. Background Art

[0002] Synovitis capsules are a Chinese herbal compound preparation made from 13 medicinal materials, including Prunella Vulgaris, Ligustrum lucidum, Hollyhock leaves, Astragalus, Stephania tetrandra, Coix seeds, Smilax glabra, Luffa, Zedoaria, Salvia miltiorrhiza, Angelica sinensis, Cyathula officinalis, and Siegesbeckia sibiricum. It has the effects of clearing away heat and dampness, promoting blood circulation and dredging collaterals; it is used for acute and chronic synovitis.

[0003] The characteristic spectrum technology of traditional Chinese medicine is holistic and fuzzy in the quality control of traditional Chinese medicine. It emphasizes the relative proportion, arrangement order and mutual involvement of multiple components (shared characteristic peaks), and the information reflected is comprehensive. It can comprehensively detect the effectiveness and stability of the quality of traditional Chinese medicine. At present, there is no research on its characteristic spectrum in the patent, and the literature only studies the content and thin layer chromatography of powdered tetrandrine, and does not control its overall quality.

[0004] In addition, the complexity and interference of the compound ingredients of Synovial Inflammation Capsules: The compound preparation composed of 13 herbs contains hundreds of chemical components with large differences in polarity and solubility, which can easily lead to chromatographic peak overlap or baseline interference. Interactions between components (such as hydrogen bonds and hydrophobic interactions) may change the extraction efficiency or stability of the target components. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for constructing a characteristic spectrum of synovitis capsules, which can comprehensively, systematically and accurately control and evaluate the drug quality of synovitis capsules.

[0006] 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 synovitis capsules.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The present invention provides a method for constructing a characteristic spectrum of a synovitis capsule, comprising the following steps: (1) Preparation of reference solution: Take the reference substance, weigh it accurately, add methanol to make a solution containing 45 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, and salvianolic acid B; (2) Preparation of test solution: Take the test sample of synovial inflammation capsule, accurately weigh 1.0g, put it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W and 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant as the extract for later use; add 30ml of 50% methanol solution to the residue obtained after centrifugation again, repeat the ultrasonic extraction for 20 minutes, combine the extracts obtained after two centrifugations, and concentrate the extract to 20ml under reduced pressure at 40℃; then load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; then concentrate the eluate to near dryness under reduced pressure at 40℃, make up to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate; (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 solution, and record the chromatogram; (4) Chromatographic conditions: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, trifluoroacetic acid:tetrahydrofuran:water solution as mobile phase B, elution according to the specified gradient; column temperature 30°C; flow rate 1 ml per minute; UV-visible detector detection wavelength 300-340 nm; The gradient elution program is as follows: 0-25min, mobile phase A:B, volume ratio changed from 5:95 to 21:79; 25-50min, the volume ratio of mobile phase A:B changed from 21:79 to 27:63; 50-60min, the volume ratio of mobile phase A:phase B changed from 27:63 to 60:40; From 60 to 63 min, the volume ratio of mobile phase A to phase B changed from 60:40 to 5:95; 63-73 min, mobile phase A:B, volume ratio 5:95; (5) Generate a control characteristic spectrum: Select the chromatographic peaks that exist in the chromatograms of different batches of synovitis capsules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of synovitis capsules.

[0008] Preferably, in step (3), the volume ratio of trifluoroacetic acid:tetrahydrofuran:water is 5:80:1000.

[0009] Preferably, the chromatographic column in step (3) is: Topsil-C18 4.6x250mm, 5μm.

[0010] Preferably, the detection wavelength in step (3) is 320 nm.

[0011] Preferably, the characteristic peak uses the caffeic acid chromatographic peak as a reference peak, and there are 13 common chromatographic peaks, among which Peak No. 2 is neochlorogenic acid, with a relative retention time of 0.65; Peak No. 5 is chlorogenic acid, with a relative retention time of 0.87; Peak No. 6 is cryptochlorogenic acid, with a relative retention time of 0.92; Peak No. 7 is caffeic acid; Peak No. 11 is salvianolic acid B, with a relative retention time of 2.24.

[0012] Compared with the prior art, the present invention has the following advantages: (1) The present invention established an HPLC characteristic spectrum for the Synovial Inflammation Capsule, identified 13 characteristic peaks, and fully and comprehensively demonstrated the chemical composition characteristics of the Synovial Inflammation Capsule. The present invention investigated the extraction solvent and extraction method of the sample, and within the scope of the investigation, optimized the preparation method of the test solution, the chromatographic conditions and the elution gradient. The characteristic spectrum measured by the optimized method showed 13 characteristic peaks, and the peak shape and separation degree of each characteristic peak were good.

[0013] (2) The method of characteristic spectrum of synovitis capsule constructed in the present invention was verified by methodology, including specificity, precision, repeatability and stability experiments. The RSD value of the relative retention time of each peak in each experimental result was ≤2.0%, and the RSD value of the relative peak area was ≤3.0%. This shows that the characteristic spectrum method is good and can reflect the major chemical components in synovitis capsule. The overall quality of synovitis capsule can be controlled more efficiently and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described below in conjunction with the accompanying drawings.

[0015] Figure 1 The chromatogram of the test sample extraction solvent was examined; among them, S1: 40% methanol; S2: 50% methanol; S3: 80% methanol; Figure 2 The chromatograms of the test sample extraction methods were investigated; among them, S1: ultrasonic extraction; S2: reflux extraction; Figure 3 Wavelength inspection chromatogram; among them, S1: 300nm; S2: 320nm; S3: 340nm; Figure 4 Specific HPLC chromatogram; S1: negative; S2: salvianolic acid B control; S3: caffeic acid control; S4: chlorogenic acid control; S5: neochlorogenic acid control; S6: cryptochlorogenic acid control; S7: test product: 23101911; Figure 5 Precision HPLC chromatogram; where S1-S6: precision 1-precision 6; Figure 6Repeatability HPLC chromatogram; where S1-S6: repeatability 1-repeatability 6; Figure 7 Stability HPLC chromatogram; wherein, S1: 0h; S2: 2h; S3: 4h; S4: 8h; S5: 12h; S6: 24h; Figure 8 HPLC chromatograms of eight batches of synovial capsule test products; among them, S1: 23101911; S2: 23110603; S3: 23112506; S4: 24011511; S5: 24030206; S6: 24031102; S7: 24041908; S8: 24040912; Fig. 9 Comparative characteristic profile of Synovitis Capsules. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with the embodiments and drawings. The instruments and equipment used in the embodiments are as follows: 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 1 Investigation of the method for constructing the characteristic spectrum of synovitis capsule 1.1 Investigation of test sample preparation methods 1.1.1 Investigation of extraction solvents for test samples Chromatographic conditions Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320nm. The theoretical plate number calculated based on the caffeic acid peak should be no less than 5000.

[0021] .

[0022] Preparation of reference solution Take appropriate amount of salvianolic acid B, caffeic acid, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid reference substances, weigh accurately, add methanol to make solutions containing 45 μg per 1 ml, shake well, and use as reference solutions.

[0023] Preparation of test solution Take an appropriate amount of the contents of the Synovial Inflammation Capsule (batch number: 23101911), accurately weigh 1.0g, place in a round-bottom flask, add 40% methanol, 50% methanol, and 80% methanol solution 50ml respectively, ultrasonic extraction at 250W and 40Hz for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant; add 30ml of 50% methanol solution to the residue again, repeat ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate to about 20ml under reduced pressure at 40℃; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated to near dryness under reduced pressure at 40℃, fixed to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate to obtain; Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0024] The RSD of the relative retention time of the solvent investigation results was less than 1.0%, indicating that when the extraction solvent was 40%~80% methanol, it had little effect on the relative retention time of each peak in the chromatogram. Finally, 50% methanol was selected as the extraction solvent. The results are shown in the attached Figure 1 .

[0025] Table 1 Relative retention time results of solvent investigation .

[0026] 1.1.2 Investigation of the extraction method of the test sample Preparation of test solution Take an appropriate amount of the contents of the Synovial Inflammation Capsule (batch number: 23101911), accurately weigh 1.0 g, place in a round-bottom flask, add 50 ml of 50% methanol solution, ultrasonically extract (250 W, 40 Hz) or heat reflux (80 ° C / 60 min) for 40 minutes, centrifuge at 3000 rpm for 10 minutes, take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat ultrasonic extraction for 20 minutes, combine the two extracts, and heat at 4 0℃ vacuum concentration to about 20ml; the concentrate was loaded onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinsed with pure water for 3 times the column volume, then eluted with 50% ethanol solution for 5 times the column volume, and the eluate was collected; the eluate was concentrated to near dryness at 40℃ under vacuum, fixed to 10ml with 50% methanol solution, filtered through a 0.45μm microporous filter membrane, and the filtrate was obtained; The chromatographic conditions are the same as those in item “1.1.1”; the reference solution is the same as that in item “1.1.1”.

[0027] Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0028] The RSD of the relative retention time of the extraction method results was less than 1.0%, indicating that the extraction method of ultrasonic extraction or heating reflux extraction had little effect on the relative retention time of each peak in the chromatogram. The results are shown in the attached Figure 2 .

[0029] Table 2 Results of relative retention time of extraction method .

[0030] 1.2 Investigation of chromatographic conditions 1.2.1 Wavelength Investigation Chromatographic conditions Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, trifluoroacetic acid: tetrahydrofuran: water (5:80:1000) solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelengths selected at 300nm, 320nm, and 340nm, respectively. The theoretical plate number calculated based on the caffeic acid peak should be no less than 5000.

[0031] .

[0032] The reference solution is the same as that in item “1.1.1”.

[0033] Preparation of the test solution Take an appropriate amount of the contents of the synovial capsule, accurately weigh 1.0g, place it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W and 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant; add 30ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate at 40℃ under reduced pressure to about 20ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated at 40℃ under reduced pressure to near dryness, fixed to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate.

[0034] Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0035] The results show that 13 characteristic peaks can be detected at wavelengths between 300nm and 340nm, and the RSD of the relative retention time is less than 1.0%. Therefore, the wavelength between 300nm and 340nm can meet the requirements for characteristic spectrum detection.

[0036] Table 3 Results of relative retention time in wavelength investigation .

[0037] 1.2.2 Investigation of mobile phase types Chromatographic conditions 1: Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, trifluoroacetic acid: tetrahydrofuran: water (5:80:1000) solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320nm. The theoretical plate number calculated based on caffeic acid should be no less than 5000.

[0038] .

[0039] Chromatographic conditions 2: Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, tetrahydrofuran: water (100:1000) solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320nm. The theoretical plate number calculated based on caffeic acid should be no less than 5000.

[0040] .

[0041] Chromatographic conditions 3: Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320nm. The theoretical plate number calculated based on caffeic acid should be no less than 5000.

[0042] .

[0043] The reference solution is the same as that in item “1.1.1”.

[0044] Preparation of test solution Take an appropriate amount of the contents of the Synovial Inflammation Capsule, accurately weigh 1.0g, place it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W, 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant; add 30ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate at 40℃ under reduced pressure to about 20ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated at 40℃ under reduced pressure to near dryness, fixed to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate.

[0045] Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0046] The results showed that when acetonitrile was used as mobile phase A and trifluoroacetic acid:tetrahydrofuran:water (5:80:1000) solution was used as mobile phase B for elution, the chromatogram of the test sample showed 13 characteristic peaks, with good peak separation and excellent peak shape; when acetonitrile was used as mobile phase A and tetrahydrofuran:water (100:1000) solution was used as mobile phase B or acetonitrile was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B for elution, there were poor peak shapes and peak missing, and the corresponding effects could not be achieved.

[0047] Table 4 Relative retention time results of mobile phase acid types .

[0048] 1.2.3 Investigation of mobile phase ratio Chromatographic conditions Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, trifluoroacetic acid: tetrahydrofuran: water (5:80:1000) solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320nm. The theoretical plate number calculated based on the caffeic acid peak should be no less than 5000.

[0049] Mobile phase ratio 1: .

[0050] Mobile phase ratio 2: .

[0051] Mobile phase ratio 3: .

[0052] The reference solution is the same as that in item “1.1.1”.

[0053] Preparation of test solution Take an appropriate amount of the contents of the Synovial Inflammation Capsule, accurately weigh 1.0g, place it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W, 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant; add 30ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate at 40℃ under reduced pressure to about 20ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated at 40℃ under reduced pressure to near dryness, fixed to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate.

[0054] Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0055] The results showed that when eluted with the mobile phase ratio of 1, the chromatogram of the test sample showed 13 characteristic peaks, with good peak separation and excellent peak shape; when eluted with the mobile phase ratio of 2 or the mobile phase ratio of 3, there were poor peak shapes and peak missing, and the corresponding effect could not be achieved.

[0056] Table 5 Relative retention time results of mobile phase ratio investigation .

[0057] Example 2 Methodological verification of the characteristic spectrum of synovitis capsule 2.1 Exclusivity Chromatographic conditions Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, trifluoroacetic acid: tetrahydrofuran: water (5:80:1000) solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320nm. The theoretical plate number calculated based on the caffeic acid peak should be no less than 5000.

[0058] .

[0059] Preparation of reference solution Take appropriate amount of salvianolic acid B, caffeic acid, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid reference substances, weigh accurately, add methanol to make solutions containing 45 μg per 1 ml, shake well, and use as reference solutions.

[0060] Preparation of test solution Take an appropriate amount of the contents of the Synovial Inflammation Capsule, accurately weigh 1.0g, place it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W, 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant; add 30ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate at 40℃ under reduced pressure to about 20ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated at 40℃ under reduced pressure to near dryness, fixed to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate.

[0061] Preparation of negative solution Take an appropriate amount of auxiliary materials, accurately weigh 1.0g, put it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W and 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant; add 30ml of 50% methanol solution to the residue again, repeat ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate at 40℃ under reduced pressure to about 20ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated at 40℃ under reduced pressure to near dryness, fixed to 10ml with 50% methanol solution, pass through a 0.45μm microporous filter membrane, and take the filtrate to obtain.

[0062] Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0063] The results showed that the negative solution had no interference and good specificity. Figure 4 .

[0064] 2.2 Precision Take 1.0 g of the contents of Synovial Inflammation Capsules (batch number: 23101911), 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, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0065] Table 6 Precision relative retention time results .

[0066] Table 7 Precision relative peak area results .

[0067] 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 1.0%, indicating good precision. The chromatogram is attached. Figure 5 .

[0068] 2.3 Repeatability Take 1.0 g of the contents of Synovial Inflammation Capsules (batch number: 23101911), for a total of 6 portions, 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, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0069] Table 8 Repeatability relative retention time results .

[0070] Table 9 Repeatability relative peak area results .

[0071] 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 3.0%, with good repeatability. The chromatogram is attached. Figure 6 .

[0072] 2.4 Stability Take 1.0 g of the contents of Synovial Inflammation Capsules (batch number: 23101911), 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, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0073] Table 10 Stability relative retention time results .

[0074] Table 11 Stability relative peak area results .

[0075] 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 2.0%, indicating that the solution had good stability. The chromatogram is attached. Figure 7 .

[0076] In summary, the specificity of the characteristic spectrum is good, and the negative sample has no interference, indicating that the characteristic detection method has strong specificity; the RSD of the relative retention time in the precision test is less than 1.0%, and the RSD of the relative peak area is less than 1.0%, indicating that the instrument precision is good; 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 3.0%, indicating good repeatability; 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 2.0%, so the test solution is stable within 24 hours. This method has been well validated by methodology.

[0077] Example 3 Construction of the comparative characteristic spectrum of Synovial Inflammation Capsules Chromatographic conditions Chromatographic column: Welch Topsil-C18 (4.6x250mm, 5μm); acetonitrile as mobile phase A, trifluoroacetic acid: tetrahydrofuran: water (5:80:1000) solution as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320nm. The theoretical plate number calculated based on the caffeic acid peak should be no less than 5000.

[0078] .

[0079] Preparation of reference solution Take appropriate amount of salvianolic acid B, caffeic acid, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid reference substances, weigh accurately, add methanol to make solutions containing 45 μg per 1 ml, shake well, and use as reference solutions.

[0080] Preparation of test solution Take an appropriate amount of the contents of the Synovial Inflammation Capsule, accurately weigh 1.0g, place it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W, 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant; add 30ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate at 40℃ under reduced pressure to about 20ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated at 40℃ under reduced pressure to near dryness, fixed to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate.

[0081] Determination method Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and record the chromatogram.

[0082] The chromatograms of the test samples of 8 batches of synovitis capsules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the chromatographic peaks present in the chromatograms of different batches of synovitis capsules were selected as common peaks; the control characteristic spectrum was generated by 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 8 , 9.

[0083] Table 12 Relative retention time results of eight batches of synovial capsule samples .

[0084] Table 13 Relative peak area results of eight batches of synovial inflammation capsule samples .

[0085] The chromatograms of the eight batches of Huamoyan Capsule samples all showed 13 common peaks, and the RSDs of the relative retention times and relative peak areas of the 13 common peaks were all less than 1.0%, and less than 8.0%.

[0086] The chromatogram of the test sample showed 13 characteristic peaks, of which peaks 2, 5, 6, 7 and 11 corresponded to the peaks of the reference substance of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid and salvianolic acid B respectively; the peak corresponding to the peak of the reference substance of caffeic acid was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.35 (peak 1), 0.65 (peak 2), 0.77 (peak 3), 0.80 (peak 4), 0.87 (peak 5), 0.92 (peak 6), 1.15 (peak 8), 1.93 (peak 9), 2.00 (peak 10), 2.24 (peak 11), 2.50 (peak 12), 2.95 (peak 13).

Claims

1. A method for constructing a characteristic spectrum of a synovitis capsule, characterized in that: The following steps are involved: (1) Preparation of reference solution: Take the reference substance, weigh it accurately, add methanol to make a solution containing 45 μg per 1 ml, shake well, and use it as the reference solution; the reference substances are: chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, and salvianolic acid B; (2) Preparation of test solution: Take the test sample of synovial inflammation capsule, accurately weigh 1.0g, put it in a round-bottom flask, add 50ml of 50% methanol solution, extract at 250W and 40Hz ultrasonically for 40 minutes, centrifuge at 3000rpm for 10 minutes, and take the supernatant as the extract for later use; add 30ml of 50% methanol solution to the residue obtained after centrifugation again, repeat the ultrasonic extraction for 20 minutes, combine the extracts obtained after two centrifugations, and concentrate the extract to 20ml under reduced pressure at 40℃; then load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25mm and a column volume of 10ml at a flow rate of 1.0ml / min, first rinse with pure water for 3 times the column volume, then elute with 50% ethanol solution for 5 times the column volume, and collect the eluate; then concentrate the eluate to near dryness under reduced pressure at 40℃, make up to 10ml with 50% methanol solution, filter through a 0.45μm microporous filter membrane, and take the filtrate; (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 solution, and record the chromatogram; The chromatographic conditions were as follows: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, trifluoroacetic acid:tetrahydrofuran: aqueous solution as mobile phase B, elution according to the specified gradient; column temperature 30°C; flow rate 1 ml per minute; UV-visible detector detection wavelength 300-340 nm; The gradient elution program is as follows: 0-25min, mobile phase A:B, volume ratio changed from 5:95 to 21:79; 25-50min, the volume ratio of mobile phase A:B changed from 21:79 to 27:63; 50-60min, the volume ratio of mobile phase A:phase B changed from 27:63 to 60:40; From 60 to 63 min, the volume ratio of mobile phase A to phase B changed from 60:40 to 5:95; 63-73 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 synovitis capsules as common peaks, and use the average value calculation method to generate a control characteristic spectrum of synovitis capsules.

2. The method according to claim 1, characterized in that In step (3), the volume ratio of trifluoroacetic acid:tetrahydrofuran:water is 5:80:1000.

3. The method according to claim 1, characterized in that The chromatographic column in step (3) is: Topsil-C18 4.6x250mm, 5μm.

4. The method according to claim 1, characterized in that: The detection wavelength in step (3) is 320 nm.

5. The method according to claim 1, characterized in that The characteristic peak takes the caffeic acid chromatographic peak as the reference peak, and there are 13 common peaks in the chromatograms, among which peak No. 2 is neochlorogenic acid with a relative retention time of 0.65; peak No. 5 is chlorogenic acid with a relative retention time of 0.87; peak No. 6 is cryptochlorogenic acid with a relative retention time of 0.92; peak No. 7 is caffeic acid; peak No. 11 is salvianolic acid B with a relative retention time of 2.

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6. Application of the characteristic spectrum of Synovial Inflammation Capsules obtained by any one of the methods of claims 1 to 5 in the quality rating of Synovial Inflammation Capsules.

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