A method for constructing a characteristic map of synovitis capsule and its application

Abstract

The present invention relates to the field of traditional Chinese medicine analysis technology, and more particularly to a method for constructing a characteristic spectrum for synovitis capsules and its application. The method comprises the following steps: 1. preparing a reference solution; 2. preparing a test solution; 3. determining the assay conditions; and 4. importing the chromatogram into a traditional Chinese medicine spectrum to generate a control characteristic spectrum. This characteristic method identifies 13 characteristic peaks, demonstrates high repeatability and precision, and comprehensively and comprehensively displays the chemical composition characteristics of synovitis capsules, enabling comprehensive control of the capsule's pharmaceutical quality.

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 application thereof. Background Art

[0002] Synovitis capsule is a traditional Chinese medicine compound preparation extracted and processed from 13 medicinal materials including Prunella Vulgaris, Ligustrum lucidum fruit, Holly leaf, Astragalus membranaceus, Stephania tetrandra, Coix seed, Smilax glabra, Luffa loofah, Zedoariae rhizome, Salvia miltiorrhiza, Angelica sinensis, Cyathula capitata, and Siegesbeckia sibiricum. It has the effects of clearing away heat and dampness, promoting blood circulation and unblocking meridians; it is used for acute and chronic synovitis.

[0003] Traditional Chinese medicine (TCM) characteristic spectrum technology is both holistic and ambiguous in TCM quality control. It emphasizes the relative proportions, order of arrangement, and interrelationships of multiple components (shared characteristic peaks), providing comprehensive information. It can comprehensively assess the effectiveness and stability of TCM quality. Currently, patents do not include research on characteristic spectrum technology, and literature only studies the content and thin-layer chromatography of tetrandrine, without addressing overall quality control.

[0004] Furthermore, the complexity and interference of the ingredients in the synovitis capsule compound are significant: the 13-ingredient compound contains hundreds of chemical components with widely varying polarity and solubility, which can easily lead to chromatographic peak overlap or baseline interference. Interactions between the components (such as hydrogen bonding and hydrophobic interactions) can alter the extraction efficiency or stability of the target component. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a method for constructing a characteristic map 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 the Synovial Inflammation Capsule.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0008] The present invention provides a method for constructing a characteristic map of a synovitis capsule, comprising the following steps:

[0009] (1) Preparation of reference solution: Take the reference substance, accurately weigh it, 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;

[0010] (2) Preparation of test solution: Take the test sample of synovitis capsule, accurately weigh 1.0g, put it into a round-bottom flask, add 50ml of 50% methanol solution, extract it by ultrasonic extraction at 250W and 40Hz for 40 minutes, centrifuge it 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 it with pure water for 3 times the column volume, then elute it 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 it up to 10ml with 50% methanol solution, filter it through a 0.45μm microporous filter membrane, and take the filtrate.

[0011] (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;

[0012] (4) Chromatographic conditions were as follows: 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 / min; UV-visible detector detection wavelength 300-340 nm;

[0013] The gradient elution program is as follows:

[0014] From 0 to 25 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 21:79;

[0015] From 25 to 50 min, the volume ratio of mobile phase A:phase B changed from 21:79 to 27:63;

[0016] From 50 to 60 min, the volume ratio of mobile phase A:phase B was changed from 27:63 to 60:40;

[0017] From 60 to 63 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95;

[0018] 63-73 min, mobile phase A:B, volume ratio 5:95;

[0019] (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.

[0020] Preferably, in step (3), the volume ratio of trifluoroacetic acid:tetrahydrofuran:water is 5:80:1000.

[0021] Preferably, the chromatographic column in step (3) is: Topsil-C18 4.6x250mm, 5μm.

[0022] Preferably, the detection wavelength in step (3) is 320 nm.

[0023] Preferably, the characteristic peak uses the caffeic acid chromatographic peak as the 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.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention established an HPLC characteristic spectrum for the synovitis capsule, identifying 13 characteristic peaks, which fully and comprehensively demonstrated the chemical composition characteristics of the synovitis capsule. The present invention investigated the extraction solvent and extraction method of the sample. Within the scope of the investigation, the preparation method of the test solution, chromatographic conditions and elution gradient were optimized. The characteristic spectrum measured by the optimized method showed 13 characteristic peaks, and the peak shape and separation of each characteristic peak were good.

[0026] (2) The method for characterizing the synovitis capsule 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 ≤2.0%, and the RSD values ​​of the relative peak areas were all ≤3.0%. This indicates that the characterizing method is good and can reflect the major chemical components in the synovitis capsule. This method can more efficiently and quickly control the overall quality of the synovitis capsule. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings.

[0028] Figure 1 The chromatograms of the test sample extraction solvents were examined; among them, S1: 40% methanol; S2: 50% methanol; S3: 80% methanol;

[0029] Figure 2 The chromatograms of the test sample extraction methods were examined; among them, S1: ultrasonic extraction; S2: reflux extraction;

[0030] Figure 3 Wavelength inspection chromatogram; among them, S1: 300nm; S2: 320nm; S3: 340nm;

[0031] Figure 4Specific HPLC chromatogram; where 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 sample: 23101911;

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

[0033] Figure 6 Repeatability HPLC chromatogram; where S1-S6: repeatability 1-repeatability 6;

[0034] Figure 7 Stability HPLC chromatogram; wherein, S1: 0h; S2: 2h; S3: 4h; S4: 8h; S5: 12h; S6: 24h;

[0035] 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;

[0036] Figure 9 Comparative characteristic spectrum of Synovitis Capsule. DETAILED DESCRIPTION

[0037] 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:

[0038] 1. Instruments and reagents

[0039] 1.1 Instruments and Equipment

[0040] .

[0041] 1.2 Test materials

[0042] .

[0043] 1.3 Reference Material Information

[0044] .

[0045] 1.4 Sample Information

[0046] .

[0047] Example 1 Investigation of the method for constructing the characteristic map of synovitis capsule

[0048] 1.1 Investigation of test sample preparation methods

[0049] 1.1.1 Investigation of the extraction solvent of the test sample

[0050] Chromatographic conditions

[0051] Chromatographic column: Welch Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: 0.2% phosphoric acid solution, gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number calculated based on the caffeic acid peak should be no less than 5000.

[0052] .

[0053] Preparation of reference solution

[0054] Take appropriate amount of salvianolic acid B, caffeic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid reference substances, accurately weigh them, add methanol to make solutions containing 45 μg per 1 ml, shake well, and use them as reference solutions.

[0055] Preparation of test solution

[0056] Take an appropriate amount of the contents of Huamoyan Capsule (batch number: 23101911), accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 40% methanol, 50% methanol, and 80% methanol solution respectively, extract under 250W, 40 Hz ultrasonication for 40 minutes, centrifuge at 3000 rpm for 10 minutes, and take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate under reduced pressure at 40°C to about 20 ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3-1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / 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 under reduced pressure at 40°C to near dryness, dilute to 10 ml with 50% methanol solution, filter through a 0.45 μm microporous membrane, and take the filtrate.

[0057] Assay

[0058] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0059] The RSD of the relative retention time of the solvent investigation results were all 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 .

[0060] Table 1 Results of relative retention time of solvent investigation

[0061] .

[0062] 1.1.2 Investigation of the extraction method of the test sample

[0063] Preparation of test solution

[0064] Take an appropriate amount of the contents of the Synovial Inflammation Capsule (batch number: 23101911), accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 50% methanol solution, and perform ultrasonic extraction (250W, 40 Hz) or heating reflux (80℃ / 60min) for 40 minutes, centrifuge at 3000 rpm for 10 minutes, and 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 boil at 4 Concentrate under reduced pressure at 0°C to approximately 20 ml; apply the concentrate to a D101 macroporous adsorption resin column with a particle size of 0.3-1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / min. First, rinse with pure water for 3 times the column volume, then elute with 5 times the column volume of 50% ethanol solution, and collect the eluate; concentrate the eluate under reduced pressure at 40°C to near dryness, make up to 10 ml with 50% methanol solution, filter through a 0.45 μm microporous filter membrane, and collect the filtrate to obtain the product;

[0065] 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”.

[0066] Assay

[0067] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0068] The RSD of the relative retention time of the extraction method 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 .

[0069] Table 2 Results of relative retention time of extraction method

[0070] .

[0071] 1.2 Investigation of chromatographic conditions

[0072] 1.2.1 Wavelength Investigation

[0073] Chromatographic conditions

[0074] Chromatographic column: Welch Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: trifluoroacetic acid: tetrahydrofuran: water (5:80:1000), gradient elution as specified in the table below; column temperature: 30°C; detection wavelengths: 300 nm, 320 nm, and 340 nm. The number of theoretical plates calculated based on the caffeic acid peak should be no less than 5000.

[0075] .

[0076] The reference solution is the same as that in item “1.1.1”.

[0077] Preparation of test solution: Take an appropriate amount of the contents of the synovialitis capsule, accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 50% methanol solution, extract it under 250W and 40 Hz ultrasound for 40 minutes, centrifuge it at 3000 rpm for 10 minutes, and take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat the ultrasound extraction for 20 minutes, combine the two extracts, and concentrate them under reduced pressure at 40°C to about 20 ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / min, first rinse it with pure water for 3 times the column volume, then elute it with 50% ethanol solution for 5 times the column volume, and collect the eluate; the eluate is concentrated under reduced pressure at 40°C to near dryness, dilute to 10 ml with 50% methanol solution, filter it through a 0.45 μm microporous membrane, and take the filtrate.

[0078] Assay

[0079] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0080] The results showed that 13 characteristic peaks could be detected at wavelengths between 300 nm and 340 nm, and the RSDs of the relative retention times were all less than 1.0%. Therefore, wavelengths between 300 nm and 340 nm can meet the requirements for characteristic spectrum detection.

[0081] Table 3 Results of relative retention time of wavelength investigation

[0082] .

[0083] 1.2.2 Investigation of mobile phase types

[0084] Chromatographic conditions 1: Welch Topsil-C18 column (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: trifluoroacetic acid: tetrahydrofuran: water (5:80:1000), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number (theoretical plate number) calculated based on caffeic acid should be no less than 5000.

[0085] .

[0086] Chromatographic Conditions 2: Welch Topsil-C18 column (4.6 x 250 mm, 5 μm); acetonitrile as mobile phase A, tetrahydrofuran:water (100:1000) as mobile phase B, gradient elution as specified in the table below; column temperature 30°C; detection wavelength 320 nm. The theoretical plate number calculated based on caffeic acid should be no less than 5000.

[0087] .

[0088] Chromatographic Conditions 3: Welch Topsil-C18 column (4.6 x 250 mm, 5 μm); 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 320 nm. The theoretical plate number calculated based on caffeic acid should be no less than 5000.

[0089] .

[0090] The reference solution is the same as that in item “1.1.1”.

[0091] Preparation of test solution

[0092] Take an appropriate amount of the contents of the synovialitis capsule, accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 50% methanol solution, extract under 250W and 40 Hz ultrasound for 40 minutes, centrifuge at 3000 rpm for 10 minutes, and take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate under reduced pressure at 40°C to about 20 ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / 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 under reduced pressure at 40°C to near dryness, dilute to 10 ml with 50% methanol solution, pass through a 0.45 μm microporous filter membrane, and take the filtrate.

[0093] Assay

[0094] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0095] 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 presented a total of 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 when 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 peaks were missing, and the corresponding effect was not achieved.

[0096] Table 4 Relative retention time results of mobile phase acid types

[0097] .

[0098] 1.2.3 Investigation of mobile phase ratio

[0099] Chromatographic conditions

[0100] Chromatographic column: Welch Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: trifluoroacetic acid: tetrahydrofuran: water (5:80:1000), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the caffeic acid 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 “1.1.1”.

[0108] Preparation of test solution

[0109] Take an appropriate amount of the contents of the synovialitis capsule, accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 50% methanol solution, extract under 250W and 40 Hz ultrasound for 40 minutes, centrifuge at 3000 rpm for 10 minutes, and take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate under reduced pressure at 40°C to about 20 ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / 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 under reduced pressure at 40°C to near dryness, dilute to 10 ml with 50% methanol solution, pass through a 0.45 μm microporous filter membrane, and take the filtrate.

[0110] Assay

[0111] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0112] The results showed that when eluted with a mobile phase ratio of 1, the test sample chromatogram showed a total of 13 characteristic peaks, with good peak separation and excellent peak shape; when eluted with a mobile phase ratio of 2 or a mobile phase ratio of 3, there were poor peak shapes and peak missing, and the corresponding effect was not achieved.

[0113] Table 5 Results of relative retention time of mobile phase ratio

[0114] .

[0115] Example 2 Methodological Verification of Characteristic Spectrum of Synovitis Capsules

[0116] 2.1 Exclusivity

[0117] Chromatographic conditions

[0118] Chromatographic column: Welch Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: trifluoroacetic acid: tetrahydrofuran: water (5:80:1000), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the caffeic acid peak, should be no less than 5000.

[0119] .

[0120] Preparation of reference solution

[0121] Take appropriate amount of salvianolic acid B, caffeic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid reference substances, accurately weigh them, add methanol to make solutions containing 45 μg per 1 ml, shake well, and use them as reference solutions.

[0122] Preparation of test solution

[0123] Take an appropriate amount of the contents of the synovialitis capsule, accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 50% methanol solution, extract under 250W and 40 Hz ultrasound for 40 minutes, centrifuge at 3000 rpm for 10 minutes, and take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate under reduced pressure at 40°C to about 20 ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / 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 under reduced pressure at 40°C to near dryness, dilute to 10 ml with 50% methanol solution, pass through a 0.45 μm microporous filter membrane, and take the filtrate.

[0124] Preparation of negative solution

[0125] Take an appropriate amount of excipients, accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 50% methanol solution, extract under 250W and 40 Hz ultrasound for 40 minutes, centrifuge at 3000 rpm for 10 minutes, and take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate under reduced pressure at 40°C to about 20 ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / 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 under reduced pressure at 40°C to near dryness, dilute to 10 ml with 50% methanol solution, pass through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the product.

[0126] Assay

[0127] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0128] The results showed that the negative solution had no interference and good specificity. Figure 4 .

[0129] 2.2 Precision

[0130] Take 1.0 g of the contents of Synovial Inflammation Capsules (Batch No.: 23101911) 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.

[0131] Table 6 Precision relative retention time results

[0132] .

[0133] Table 7 Precision relative peak area results

[0134] .

[0135] 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 .

[0136] 2.3 Repeatability

[0137] Take 1.0 g of the contents of Synovial Inflammation Capsules (Batch No.: 23101911), and make 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, measure the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the RSD.

[0138] Table 8 Repeatability relative retention time results

[0139] .

[0140] Table 9 Repeatability relative peak area results

[0141] .

[0142] 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 .

[0143] 2.4 Stability

[0144] Take 1.0 g of the contents of Synovial Inflammation Capsules (Batch No.: 23101911) 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.

[0145] Table 10 Stability relative retention time results

[0146] .

[0147] Table 11 Stability relative peak area results

[0148] .

[0149] 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 was stable. The chromatogram is attached. Figure 7 .

[0150] In summary, the characteristic spectrum has good specificity and no interference from negative samples, indicating that the characteristic detection method has strong specificity. In the precision test, the relative retention time RSDs were all less than 1.0%, and the relative peak area RSDs were all less than 1.0%, indicating good instrument precision. In the repeatability test, the relative retention time RSDs of each chromatographic peak were all less than 1.0%, and the relative peak area RSDs were all less than 3.0%, indicating good repeatability. In the stability test, the relative retention time RSDs of each chromatographic peak were all less than 1.0%, and the relative peak area RSDs were all less than 2.0%, indicating that the test solution was stable within 24 hours. This method has been well validated.

[0151] Example 3 Construction of the control characteristic spectrum of Huamoyan Capsule

[0152] Chromatographic conditions

[0153] Chromatographic column: Welch Topsil-C18 (4.6 x 250 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: trifluoroacetic acid: tetrahydrofuran: water (5:80:1000), gradient elution as specified in the table below; column temperature: 30°C; detection wavelength: 320 nm. The theoretical plate number, calculated based on the caffeic acid peak, should be no less than 5000.

[0154] .

[0155] Preparation of reference solution

[0156] Take appropriate amount of salvianolic acid B, caffeic acid, chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid reference substances, accurately weigh them, add methanol to make solutions containing 45 μg per 1 ml, shake well, and use them as reference solutions.

[0157] Preparation of test solution

[0158] Take an appropriate amount of the contents of the synovialitis capsule, accurately weigh 1.0 g, place it in a round-bottom flask, add 50 ml of 50% methanol solution, extract under 250W and 40 Hz ultrasound for 40 minutes, centrifuge at 3000 rpm for 10 minutes, and take the supernatant; add 30 ml of 50% methanol solution to the residue again, repeat the ultrasonic extraction for 20 minutes, combine the two extracts, and concentrate under reduced pressure at 40°C to about 20 ml; load the concentrate onto a D101 macroporous adsorption resin column with a particle size of 0.3~1.25 mm and a column volume of 10 ml at a flow rate of 1.0 ml / 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 under reduced pressure at 40°C to near dryness, dilute to 10 ml with 50% methanol solution, pass through a 0.45 μm microporous filter membrane, and take the filtrate.

[0159] Assay

[0160] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0161] The chromatograms of the test samples of 8 batches of Huamoyan Capsules were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and the chromatographic peaks present in the chromatograms of different batches of Huamoyan Capsules were selected as common peaks; the average value calculation method was used to generate the control characteristic spectrum, 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.

[0162] Table 12 Relative retention time results of eight batches of Huamoyan Capsule samples

[0163] .

[0164] Table 13 Relative peak area results of eight batches of Huamoyan Capsule samples

[0165] .

[0166] The chromatograms of the eight batches of Huamoyan Capsule samples all showed 13 common peaks. The RSDs of the relative retention times of the 13 common peaks were all less than 1.0%, and the RSDs of the relative peak areas were all less than 8.0%.

[0167] The chromatogram of the test sample showed a total of 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 caffeic acid 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: 0.35 (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), and 2.95 (Peak 13).

Claims

1. A method for constructing a characteristic map of a synovitis capsule, characterized in that: The following steps are involved: (1) Preparation of reference solution: Take the reference substance, accurately weigh it, 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 synovitis capsule, accurately weigh 1.0g, put it into a round-bottom flask, add 50ml of 50% methanol solution, extract it by ultrasonic extraction at 250W and 40Hz for 40 minutes, centrifuge it 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 it with pure water for 3 times the column volume, then elute it 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 it up to 10ml with 50% methanol solution, filter it 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 sample, and record the chromatogram; Chromatographic conditions were as follows: octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, trifluoroacetic acid:tetrahydrofuran:water as the mobile phase B, where the volume ratio of trifluoroacetic acid:tetrahydrofuran:water was 5:80:1000, and elution was performed according to the prescribed gradient; the column temperature was 30°C; the flow rate was 1 ml / min; and the detection wavelength of the UV-visible detector was 300-340 nm. The gradient elution program is as follows: From 0 to 25 min, the volume ratio of mobile phase A:phase B changed from 5:95 to 21:79; From 25 to 50 min, the volume ratio of mobile phase A:phase B changed from 21:79 to 27:63; From 50 to 60 min, the volume ratio of mobile phase A:phase B was changed from 27:63 to 60:40; From 60 to 63 min, the volume ratio of mobile phase A:phase B was changed from 60:40 to 5:95; 63-73 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 The chromatographic column in step (3) is: Topsil-C18 4.6x250mm, 5μm.

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

4. The method according to claim 1, wherein The characteristic peaks were referenced with the caffeic acid chromatographic peak. There were 13 common chromatographic peaks, among which peak 2 was neochlorogenic acid with a relative retention time of 0.65; peak 5 was chlorogenic acid with a relative retention time of 0.87; peak 6 was cryptochlorogenic acid with a relative retention time of 0.92; peak 7 was caffeic acid; and peak 11 was salvianolic acid B with a relative retention time of 2.

24.

5. Use of the characteristic spectrum of synovitis capsule obtained by the method according to any one of claims 1 to 4 in the quality rating of synovitis capsule.

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