Establishment method of HPLC fingerprint of Shenyankangfu tablets and its standard fingerprint

Abstract

The present invention provides a method for establishing a HPLC fingerprint spectrum of Shenyan Kangfu Tablets and a standard fingerprint spectrum thereof, comprising the following steps: preparation of a test sample solution; preparation of a reference substance solution; establishment of a fingerprint spectrum; confirmation of a standard fingerprint spectrum, respectively establishing fingerprint spectrums for multiple batches of Shenyan Kangfu Tablets, importing the fingerprint spectrums of multiple batches of Shenyan Kangfu Tablets into the 2012 version software of the "Chinese Medicine Chromatographic Fingerprint Evaluation System" of the National Pharmacopoeia Committee, generating a standard fingerprint spectrum, wherein the standard fingerprint spectrum has 20 common peaks. The method for establishing a HPLC fingerprint spectrum of Shenyan Kangfu Tablets and the standard fingerprint spectrum thereof described in the present invention can comprehensively reflect the characteristics of Shenyan Kangfu Tablets, have good specificity, provide a reference basis for ensuring safe, stable and quality-controlled medication, can realize quality monitoring of Shenyan Kangfu Tablets preparations, effectively control the quality of Shenyan Kangfu Tablets, ensure the efficacy of the drug, and promote the inheritance and development of traditional Chinese medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and in particular relates to a method for establishing a HPLC fingerprint spectrum of a Shenyan Kangfu Tablet and a standard fingerprint spectrum thereof. Background Art

[0002] The key product of Tianjin Tongrentang Group Co., Ltd., Nephritis Recovery Tablets, is derived from the essence of the famous Chinese medicine expert Zhao Enjian's many years of clinical experience in treating chronic nephritis, and is included in the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0003] The medicine is composed of 13 Chinese medicinal herbs including American ginseng, ginseng, rehmannia root, salt ulmoides, yam, oldenlandia diffusa, black bean, wild yam, motherwort, salvia miltiorrhiza, oriental water plantain, imperata root, and platycodon. It has the effects of invigorating qi and nourishing yin, tonifying the kidney and strengthening the spleen, and clearing away residual toxins. It is used for edema caused by deficiency of both qi and yin, insufficiency of the spleen and kidney, and internal retention of water and dampness, with symptoms such as fatigue, sore waist and weak legs, edema of the face and limbs, dizziness and tinnitus; and patients with chronic nephritis, proteinuria, and hematuria with the above symptoms.

[0004] Traditional Chinese medicine preparations contain many chemical components. These chemical components produce an overall therapeutic effect and are the main indicators of important evaluation. A single component cannot truly reflect the actual level of quality of traditional Chinese medicine preparations. The 2020 edition of the Chinese Pharmacopoeia only identifies tanshinone ⅡA in Danshen, stachydrine hydrochloride in Leonurus japonicus, Rb1, Re, Rg, and pseudo-F11 thin layer in Platycodon grandiflorum and American ginseng, and the content of tanshinone ⅡA in Danshen. However, due to the large number of drug components in Nephritis Recovery Tablets, it is difficult to fully and accurately reflect the efficacy and overall quality of the modified preparation by only testing these five medicinal materials. Summary of the invention

[0005] In view of this, the present invention aims to propose a method for establishing the HPLC fingerprint of Nephritis Recovery Tablets and its standard fingerprint, so as to comprehensively reflect the characteristics of Nephritis Recovery Tablets, have good specificity, help control the quality of Nephritis Recovery Tablets, and ensure clinical efficacy.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] The method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets comprises the following steps:

[0008] S1. Preparation of test sample solution: remove the coating of Shenyankangfu tablets, crush, add methanol, reflux in a water bath, filter, and obtain the test sample solution;

[0009] S2. Preparation of reference solution: Weigh reference substances of tanshinone ⅡA, ginsenoside Rb1, leonurine hydrochloride, salvianolic acid B, astilbin, daidzein, pinoresinol diglucoside, p-coumaric acid, and rosmarinic acid, respectively, and add methanol to prepare reference solution;

[0010] S3, establishment of fingerprint: respectively aspirate the test sample solution and the reference solution and inject them into the liquid chromatograph for determination, and obtain the fingerprint of the test sample;

[0011] S4. Confirmation of standard fingerprint: According to the above method, fingerprints of multiple batches of Nephritis Recovery Tablets are established respectively, and the fingerprints of multiple batches of Nephritis Recovery Tablets are imported into the 2012 version of the software "Chinese Medicine Chromatographic Fingerprint Evaluation System" of the State Pharmacopoeia Committee to generate a standard fingerprint. The standard fingerprint has 20 common peaks.

[0012] Furthermore, the concentrations of tanshinone ⅡA, ginsenoside Rb1, leonurine hydrochloride, salvianolic acid B, astilbin, daidzin, pinoresinol diglucoside, p-coumaric acid and rosmarinic acid in the reference solution are 2-30 μg / ml; preferably, the concentration of tanshinone ⅡA in the reference solution is 8 μg / ml, the concentration of ginsenoside Rb1 is 20 μg / ml, the concentration of leonurine hydrochloride is 5 μg / ml, the concentration of salvianolic acid B is 5 μg / ml, the concentration of astilbin is 5 μg / ml, the concentration of daidzin is 10 μg / ml, the concentration of pinoresinol diglucoside is 20 μg / ml, the concentration of p-coumaric acid is 5 μg / ml and the concentration of rosmarinic acid is 10 μg / ml.

[0013] Furthermore, the determination conditions in step S3 are: chromatographic column: Agilent Technologies ZORBAX SB-C18; 4.6×250 mm, 3.5 μm, flow rate: 0.6 ml / min, mobile phase: A: 0.1-0.2% phosphoric acid solution, B: acetonitrile, column temperature: 20°C-35°C, injection volume 6 μl, analysis time: 188 min, scanning wavelength: 203-360 nm.

[0014] Further, from 0 to 10 min, the volume ratio of A:B is 98:2; from 10 to 18 min, the volume ratio of A:B is 98:2-87:13; from 18 to 58 min, the volume ratio of A:B is 87:13-86:14; from 58 to 68 min, the volume ratio of A:B is 86:14-83:17; from 68 to 88 min, the volume ratio of A:B is 83:17-80:20; from 88 to 123 min, the volume ratio of A:B is 80:20-79:21; from 123 to 155 min, the volume ratio of A:B is 79:21-49:51; from 155 to 165 min, the volume ratio of A:B is 49:51-19:81; from 165 to 188 min, the volume ratio of A:B is 19:81-9:91.

[0015] Furthermore, from 0 to 37.5 min, the scanning wavelength is 360 nm; from 37.5 to 52.5 min, the scanning wavelength is changed to 203 nm; from 52.5 to 71 min, the scanning wavelength is changed to 330 nm; from 71 to 83 min, the scanning wavelength is changed to 270 nm; from 83 to 110 min, the scanning wavelength is changed to 210 nm; from 110 to 143 min, the scanning wavelength is changed to 275 nm; from 143 to 146.5 min, the scanning wavelength is changed to 203 nm; from 146.5 to 183 min, the scanning wavelength is changed to 275 nm; from 183 to 188 min, the scanning wavelength is changed to 203 nm.

[0016] Furthermore, the column temperature from 0 to 55 min was 20°C, and the column temperature from 55 to 188 min was 35°C.

[0017] Furthermore, the method further comprises the following steps:

[0018] According to the prescription of Nephritis Recovery Tablets, single medicinal material solutions and single or multiple medicinal material negative control prescription sample solutions are prepared, the single medicinal material solutions, single or multiple medicinal material negative control prescription sample solutions and the reference substance solution in step S2 are injected into a liquid chromatograph for measurement, fingerprints of the single medicinal material solutions and the single or multiple medicinal material negative control prescription sample solutions are established, and compared with 20 common peaks to determine the ownership of the fingerprint peaks.

[0019] Furthermore, fingerprint peaks 1, 3, 7, 9, 10, 11, and 12 are from Smilax glabra, fingerprint peak 2 is from Eucommia ulmoides, fingerprint peaks 4 and 8 are from black beans, fingerprint peak 5 is from Leonurus japonicus, fingerprint peak 6 is from Hedyotis diffusa and Imperata cylindrica, fingerprint peaks 13, 14, 15, 17, 18, 19, and 20 are from Salvia miltiorrhiza, and fingerprint peak 16 is from Panax ginseng and American ginseng.

[0020] According to the standard fingerprint established by any of the above-mentioned establishment methods, the relative retention times of the common peaks are: peak 1 0.329±0.020, peak 2 0.357±0.020, peak 3 0.361±0.020, peak 4 0.395±0.020, peak 5 0.409±0.020, peak 6 0.460±0.020, peak 7 0.615±0.020, peak 8 0.637±0.010, peak 9 0.643±0.010, peak 10 0.709±0.010, peak 11 0.727±0.010, peak 12 0.750±0.010, peak 13 0. 817±0.010, peak 14 0.873±0.010, peak 15 1.000, peak 16 1.216±0.010, peak 17 1.401±0.020, peak 18 1.439±0.020, peak 19 1.476±0.020, peak 20 1.540±0.020, wherein peak 15 is peak S; preferably, peak 2 is pinoresinol diglucoside, peak 4 is daidzein, peak 5 is leonurine hydrochloride, peak 6 is p-coumaric acid, peak 9 is astilbin, peak 14 is rosmarinic acid, peak 15 is salvianolic acid B, peak 17 is ginsenoside Rb1, and peak 19 is tanshinone ⅡA.

[0021] The method for establishing the HPLC fingerprint of the Nephritis Recovery Tablets as described above or the application of the standard fingerprint as described above in the field of Nephritis Recovery Tablets detection.

[0022] Furthermore, the application method is: obtain the fingerprint of the sample of the Nephritis Recovery Tablet according to steps S1-S3, use the standard fingerprint as a control, use the 2012 version of the "Chinese Medicine Chromatographic Fingerprint Evaluation System" software of the National Pharmacopoeia Committee to calculate the similarity of the fingerprints, and use the similarity of not less than 0.90 as the standard to judge whether the sample is qualified.

[0023] Compared with the prior art, the method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets and its standard fingerprint have the following advantages:

[0024] (1) The method for establishing the HPLC fingerprint of the Nephritis Recovery Tablets and its standard fingerprint described in the present invention can comprehensively reflect the characteristics of the Nephritis Recovery Tablets, have good specificity, provide a reference basis for ensuring the safety, stability and quality control of the drug, and can realize the quality monitoring of the Nephritis Recovery Tablets preparation, effectively control the quality of the Nephritis Recovery Tablets, ensure the efficacy of the drug, and promote the inheritance and development of traditional Chinese medicine.

[0025] (2) The method for establishing the HPLC fingerprint of Nephritis Rehabilitation Tablets and its standard fingerprint described in the present invention can detect and evaluate Nephritis Rehabilitation Tablets products that have been marketed in China, so as to control the quality of Nephritis Rehabilitation Tablets commercially available preparations, distinguish the sources of Nephritis Rehabilitation Tablets commercially available preparations, and ensure the clinical efficacy of Nephritis Rehabilitation Tablets commercially available preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0027] Figure 1 The fingerprints of the test sample solutions of 10 batches of Shenyan Kangfu Tablets and the standard fingerprints of Shenyan Kangfu Tablets described in Example 2 of the present invention are shown in FIG. 2, wherein R(20) is the standard fingerprint of Shenyan Kangfu Tablets, and S1(20)-S10(20) are the fingerprints of the test sample solutions of 10 batches of Shenyan Kangfu Tablets respectively;

[0028] Figure 2 This is a schematic diagram of the fingerprint of the mixed reference solution described in Example 2 of the present invention;

[0029] Figure 3 This is a schematic diagram of the standard fingerprint spectrum of the Nephritis Recovery Tablets described in Example 2 of the present invention;

[0030] Figure 4 This is a schematic diagram of the fingerprint spectrum of the negative control prescription sample solution of the medicinal material described in Example 3 of the present invention;

[0031] Figure 5 Schematic diagram of the comparison of the effects of different mobile phase solvents on HPLC chromatograms described in Example 4 of the present invention;

[0032] Figure 6 It is a comparative schematic diagram of the effects of different concentrations of phosphoric acid on HPLC chromatograms described in Example 4 of the present invention;

[0033] Figure 7 Schematic diagram comparing the effects of different column temperatures on HPLC chromatograms as described in Example 5 of the present invention. DETAILED DESCRIPTION

[0034] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0035] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0036] The raw materials and instruments used in each embodiment are as follows:

[0037] Reagents: acetonitrile (Fisher, USA, HPLC grade); phosphoric acid (Fisher, USA, HPLC Grade, 85%); methanol (Fisher, USA, HPLC grade); purified water (Hangzhou Wahaha Group Co., Ltd.);

[0038] Reference substances: Tanshinone ⅡA (batch number: 110766-202022), salvianolic acid B (batch number: 111562-201917), leonurine hydrochloride (batch number: 111823-201704), astilbin (batch number: 111798-201805), soybean glycoside (batch number: 111738-201603), dulcitol diglucoside (batch number: 111537-201706), ginseng Rb1 (batch number: 110704-202129), rosmarinic acid (batch number: 111871-201706) The above reference substances are from: China Food and Drug Inspection Institute; p-coumaric acid (batch number: 240072-201912 source: Shanghai Hongyong Biotechnology Co., Ltd.);

[0039] Medicinal materials: American ginseng (batch number: 1103Z2101001, origin: Jilin), ginseng (batch number: 1234Z2103002, origin: Jilin), Rehmannia root (batch number: 1095Z2102001, origin: Henan), Eucommia ulmoides (batch number: 1073Z2101002, origin: Sichuan), Chinese yam (batch number: 1083Z2101001, origin: Henan), Hedyotis diffusa (batch number: 1215Z2101001, origin: Henan), black bean (batch number: 1141Z2101001, origin: Hebei ), Smilax glabra (batch number: 1223z2101001, origin: Guangxi), Leonurus japonicus (batch number: 1158Z2101001, origin: Hebei), Salvia miltiorrhiza (batch number: 1088Z2102001, origin: Shandong), Alisma orientalis (batch number: 1075Z2105001, origin: Sichuan), Imperata cylindrica (batch number: 1097Z2101001, origin: Hebei), Platycodon grandiflorum (batch number: 1050Z2102001, origin: Guangxi), commercially available medicinal materials, qualified by the quality department of Tianjin Tongrentang Group Co., Ltd.;

[0040] Test samples: A total of 10 batches of Nephritis Recovery Tablets were provided by Tianjin Tongrentang Group Qualified Co., Ltd. The batch numbers are shown in Table 1.

[0041] Table 1 Batch number of Nephritis Recovery Tablets

[0042] serial number batch number serial number batch number S1 CP17055 S6 CP17060 S2 CP17056 S7 CP17061 S3 CP17057 S8 CP17062 S4 CP17058 S9 CP17063 S5 CP17059 S10 CP17064

[0043] instrument

[0044] Agilent 1260 HPLC (workstation: OpenLab CDS2.1, and OpenLabC.01.07, G1311C quaternary pump, G1329B autosampler, G1316A column oven, G1315D DAD detector), AL204 and XS205 (METTLER TOLEDO Instrument Shanghai Co., Ltd.) analytical electronic balance

[0045] Example 1

[0046] In this embodiment, the fingerprint of the test sample solution and the reference solution are obtained by measuring the test sample solution and the reference solution respectively. The two fingerprints are compared, and the corresponding characteristic peaks of the fingerprint of the test sample solution are identified by retention time according to the known characteristic peaks in the fingerprint of the reference solution, so as to locate the index components in the fingerprint of the test sample solution and obtain the fingerprint of the Shenyan Kangfu Tablet, which specifically includes the following steps:

[0047] Preparation of test sample solution: remove the coating of Shenyankangfu tablets, crush them, take 1.0 g, add 20 mL of methanol, reflux in a water bath for 2 hours, take out and cool to room temperature, filter, take the filtrate, and filter it through a 0.22 μm microporous membrane to obtain the solution;

[0048] Preparation of mixed reference solution: accurately weigh appropriate amount of tanshinone ⅡA, ginsenoside Rb1, leonurine hydrochloride, salvianolic acid B, astilbin, daidzin, pinoresinol diglucoside, p-coumaric acid, and rosmarinic acid reference substances, and add methanol to prepare a mixed reference solution, wherein the concentration of tanshinone ⅡA is 8μg / ml, the concentration of ginsenoside Rb1 is 20μg / ml, the concentration of leonurine hydrochloride is 5μg / ml, the concentration of salvianolic acid B is 5μg / ml, the concentration of astilbin is 5μg / ml, the concentration of daidzin is 10μg / ml, the concentration of pinoresinol diglucoside is 20μg / ml, the concentration of p-coumaric acid is 5μg / ml, and the concentration of rosmarinic acid is 10μg / ml;

[0049] Using the same high performance liquid chromatography method, the test sample solution and the mixed reference solution were accurately aspirated and injected into the high performance liquid chromatography instrument for determination, and the liquid fingerprint chromatograms of the test sample solution and the mixed reference solution were obtained respectively. The chromatographic conditions of the high performance liquid chromatography method were as follows: chromatographic column: Agilent Technologies ZORBAX SB-C18; 4.6×250mm, 3.5μm;

[0050] Flow rate: 0.6ml / min;

[0051] Mobile phase: A: 0.1-0.2% phosphoric acid solution, B: acetonitrile,

[0052] 0-10min, A:B volume ratio 98:2;

[0053] 10-18min, A:B volume ratio 98:2-87:13;

[0054] 18-58min, A:B volume ratio 87:13-86:14;

[0055] 58-68min, A:B volume ratio 86:14-83:17;

[0056] 68-88min, A:B volume ratio 83:17-80:20;

[0057] 88-123min, A:B volume ratio 80:20-79:21;

[0058] 123-155min, A:B volume ratio 79:21-49:51;

[0059] 155-165min, A:B volume ratio 49:51-19:81;

[0060] 165-188min, A:B volume ratio 19:81-9:91,

[0061] Column temperature: 0-55min Column temperature: 20℃;

[0062] 55-188min Column temperature: 35°C,

[0063] Injection volume 6μl,

[0064] Analysis time: 188min.

[0065] The Nephritis Recovery Tablets were scanned at a full ultraviolet wavelength of 200-400nm, and the wavelength of each index component was determined based on the pharmacopoeia wavelength required by the index component and the maximum absorption of the index component in the full wavelength scanning results. Among them, the pharmacopoeia detection wavelength of pinulose alcohol diglucoside is 277nm; the pharmacopoeia detection wavelength of soybean glycoside is 248nm; and the pharmacopoeia detection wavelength of leonurine hydrochloride is 277nm. Because the effective retention time of these three components is close, and the characteristic peak absorption area response value is small, resulting in a large difference in peak response with other components, it was found that all three components have a large absorption at 203nm, so it is used as a common detection wavelength. The detection wavelength of p-coumaric acid is 315nm with maximum absorption, but considering the elimination of interference from the absorption peaks of other components, 330nm is selected as the detection wavelength. The pharmacopoeia detection wavelength of astilbin is 291nm, but due to the large characteristic peak absorption area of ​​the single index component of astilbin in the Nephritis Recovery Tablets, the peak response difference with other components is large, which is not conducive to the fingerprint analysis of Nephritis Recovery Tablets, so the wavelength of 270nm is selected as the detection wavelength of astilbin. The detection wavelength of rosmarinic acid is 330nm with maximum absorption. Considering that the characteristic peak absorption area response value near the absorption peak of rosmarinic acid is small, in order to avoid the large difference in peak response with other components, 210nm is selected as the detection wavelength. The pharmacopoeia detection wavelength of salvianolic acid B is 286nm, but due to the large characteristic peak absorption area of ​​the single index component of salvianolic acid B in the Nephritis Recovery Tablets, the peak response difference with other components is large, which is not conducive to the fingerprint analysis of Nephritis Recovery Tablets, so the wavelength of 275nm is selected as the detection wavelength of salvianolic acid B. The pharmacopoeia detection wavelength of ginsenoside Rb1 is 203nm, so 203nm is selected as the detection wavelength. The pharmacopoeia detection wavelength of Tanshinone ⅡA is 275nm, and 275nm is selected as the detection wavelength. Among them, p-coumaric acid was found in both Hedyotis diffusa and Imperata root in the specificity investigation, and ginsenoside Rb1 was found in both ginseng and American ginseng in the specificity investigation. The liquid chromatography behaviors of the components in the Nephritis Recovery Tablets are quite different, and it is impossible to fix the detection wavelength while ensuring that each effective component has a large response value. Considering the detection conditions of each component comprehensively, this embodiment uses the program wavelength as the detection wavelength to ensure that both the small peaks and large peaks of each effective component and other characteristic peaks have suitable detection effects.

[0066] Scanning wavelength: 0-37.5min, scanning wavelength is 360nm;

[0067] 37.5-52.5min, scanning wavelength changed to 203nm;

[0068] 52.5-71min, scanning wavelength changed to 330nm;

[0069] 71-83min, scanning wavelength was changed to 270nm;

[0070] 83-110min, scanning wavelength changed to 210nm;

[0071] 110-143min, scanning wavelength changed to 275nm;

[0072] 143-146.5min, scanning wavelength changed to 203nm;

[0073] 146.5-183min, scanning wavelength changed to 275nm;

[0074] 183-188min, the scanning wavelength was changed to 203nm.

[0075] The fingerprint of the test sample solution is compared with the fingerprint of the mixed reference solution, and the corresponding characteristic peaks in the fingerprint of the test sample solution are identified according to the relative retention time of the known characteristic peaks in the fingerprint of the reference solution, so as to attribute and locate the index components in the fingerprint of the test sample solution and obtain the fingerprint of the Nephritis Recovery Tablets.

[0076] Example 2

[0077] In this example, the method for establishing the fingerprint of the Shenyan Convalescent Tablets established in Example 1 was used to detect 10 batches of Shenyan Convalescent Tablets to obtain the fingerprint of the test sample solution and the fingerprint of the mixed reference solution. The batch numbers of the 10 batches of Shenyan Convalescent Tablets are shown in Table 1. The fingerprints of the test sample solutions of different batches were obtained, and the fingerprints of the mixed reference solution were as follows: Figure 2 As shown, the fingerprint of the test sample solution was imported into the 2012 version of the software "Chinese Medicine Chromatographic Spectrum Similarity Evaluation System" issued by the National Pharmacopoeia Committee, and the S6 sample in Table 1 was used as a reference spectrum. After multi-point correction, automatic matching was adopted and the average was used to generate the standard fingerprint of Shenyan Kangfu Tablets. The superimposed spectrum of 10 batches is shown in Figure 1 As shown, the standard fingerprint of Shenyankangfu Tablets is obtained as Figure 3 The standard fingerprint of the co-calibrated Shenyan Kangfu Tablets includes 20 common peaks, accounting for more than 86% of the total peak area, among which the peak No. 15 has a relatively stable and moderate peak time, so the peak No. 15 is selected as the positioning peak S peak.

[0078] The fingerprints of 10 batches of Shenyan Kangfu Tablets were calculated, and the ratio of the retention time of each chromatographic peak to the retention time of the S peak in the same spectrum was used to obtain the relative retention time. See Table 2. As can be seen from Table 2, the relative retention time RSD < 1%. The fingerprints of the 10 batches of test sample solutions were compared with the standard fingerprints for similarity. The results are shown in Table 3. As can be seen from Table 3, the similarity of the 10 batches of samples is ≥ 0.90, indicating that the components of the 10 batches of Shenyan Kangfu Tablets samples are relatively stable, indicating that the fingerprint is representative.

[0079] Table 2 Average relative retention time of each chromatographic peak of 10 batches of test sample solutions

[0080]

[0081]

[0082] Table 3 Comparison of similarity between the fingerprints of 10 batches of test sample solutions and the standard fingerprints

[0083]

[0084] The standard fingerprint of the above-mentioned Nephritis Recovery Tablets was compared with the fingerprint of the reference solution. According to the known characteristic peaks in the fingerprint of the reference solution, the corresponding characteristic peaks in the standard fingerprint of the Nephritis Recovery Tablets were identified by relative retention time, and the peak No. 2 was located as pinoresinol diglucoside, peak No. 4: daidzein, peak No. 5: leonurine hydrochloride, peak No. 6: p-coumaric acid, peak No. 9: astilbin, peak No. 13: rosmarinic acid, peak No. 15: tanshinone B, peak No. 16: ginseng Rb1, and peak No. 19: tanshinone ⅡA.

[0085] Example 3

[0086] The process of the screening method of the fingerprint spectrum of multiple medicinal materials of Shenyan Kangfu Tablets is as follows:

[0087] Preparation of single herbal medicine solution: Weigh 1.0 g of single herbal medicine powder respectively, put it into a 50 mL conical flask, add 20 mL of methanol, reflux in a water bath for 2 hours, take it out and cool it to room temperature, filter it, and pass the filtrate through a 0.22 μm pore size microporous filter membrane to obtain the single herbal medicine test solution;

[0088] Negative control prescription sample solution of medicinal materials: According to the prescription ratio, prepare a negative control prescription sample that does not contain any one or more medicinal materials according to the preparation method of Shenyan Kangfu Tablets. Take the negative control prescription sample of medicinal materials, grind it into powder, weigh 1.0g of the powder, put it in a 50mL conical flask, add 20mL of methanol, reflux in a water bath for 2 hours, take it out and cool it to room temperature, filter it, and take the filtrate to pass through a 0.22μm pore size microporous filter membrane to obtain the negative control prescription sample solution of medicinal materials;

[0089] Fingerprints of single medicinal material solutions and medicinal material negative samples are obtained by high performance liquid chromatography under the same chromatographic conditions as those in the detection method of the fingerprint of Shenyan Kangfu Tablets in Example 1, thereby obtaining fingerprints of single medicinal material solutions and medicinal material negative control prescription samples;

[0090] The fingerprint of the mixed reference solution is obtained by adopting the same steps as the detection method of the fingerprint of the Shenyankangfu Tablets in Example 1 to obtain the fingerprint of the reference solution;

[0091] Determination of the fingerprint of Shenyan Kangfu Tablets: The fingerprint of Shenyan Kangfu Tablets is obtained by using the same detection method as that of the fingerprint of Shenyan Kangfu Tablets in Example 1;

[0092] Generation of standard fingerprint: The fingerprint standard fingerprint of Shenyan Kangfu Tablets is obtained by adopting the same steps as the detection method of the fingerprint of Shenyan Kangfu Tablets in Example 2;

[0093] Quality inspection: Compare the fingerprint of the reference solution, the fingerprint of the single medicinal material solution, and the fingerprint of the negative control prescription sample solution with the standard fingerprint of Shenyan Kangfu Tablets, identify the corresponding characteristic peaks of the reference solution and the single medicinal material sample solution in the standard spectrum of Shenyan Kangfu Tablets, and use the negative control prescription sample solution for comparison to exclude non-characteristic peaks, so as to attribute and locate the characteristic peaks in the fingerprint of the test solution;

[0094] Technical means for attributing and locating characteristic peaks: Based on the above-mentioned fingerprint quality detection of Nephritis Rehabilitation Tablets, the characteristic peaks in the standard fingerprint of the test solution are attributed and located through the characteristic peak relative retention time and characteristic peak DAD-UV (diode array ultraviolet absorption spectrum, range: 200-400nm) spectral identification method.

[0095] The results are as follows Figure 4 As shown, among them, 1 is the fingerprint of the test sample of Shenyan Kangfu Tablets; 2 is the fingerprint of the negative control prescription sample of Eucommia ulmoides; 3 is the fingerprint of the negative control prescription sample of black bean; 4 is the fingerprint of the negative control prescription sample of Salvia miltiorrhiza; 5 is the fingerprint of the negative control prescription sample of Ginseng; 6 is the fingerprint of the negative control prescription sample of American ginseng; 7 is the fingerprint of the double negative control prescription sample of Ginseng-American ginseng; 8 is the fingerprint of the control prescription sample of Smilax glabra; 9 is the fingerprint of the negative control prescription sample of Hedyotis diffusa; 10 is the fingerprint of the negative control prescription sample of Imperata cylindrica; 11 is the fingerprint of the double negative control prescription sample of Hedyotis diffusa-Imperata cylindrica; 12 is the fingerprint of the negative control prescription sample of Platycodon grandiflorum; 1 3 is the fingerprint of the negative control prescription sample of yam; 14 is the fingerprint of the negative control prescription sample of Leonurus japonicus; 15 is the fingerprint of the negative control prescription sample of Alisma orientalis; 16 is the fingerprint of the negative control prescription sample of Rehmannia glutinosa. The fingerprint of the test sample of Shenyan Kangfu Tablets was compared with the fingerprints of each medicinal material solution and each medicinal material negative control prescription sample solution, and it was determined that peaks 1, 3, 7, 9, 10, 11, and 12 were derived from Smilax glabra; peak 2 was derived from Eucommia ulmoides; peak 4 was derived from black beans; peaks 5 and 8 were derived from Leonurus japonicus; peak 6 was derived from Hedyotis diffusa and Imperata cylindrica; peaks 13, 14, 15, 17, 18, 19, and 20 were derived from Salvia miltiorrhiza; peak 16 was derived from Panax ginseng and American ginseng.

[0096] Example 4

[0097] The selection of mobile phase solvent for the detection method of the fingerprint spectrum of the Nephritis Recovery Tablets in the present invention was investigated.

[0098] 1. Investigation of solvent system

[0099] Under the same conditions, the chromatograms of the high performance liquid chromatography (HPLC) of the test sample solution of Shenyan Kangfu Tablets in the mobile phase system of phosphoric acid aqueous solution-acetonitrile and phosphoric acid aqueous solution-methanol were compared and analyzed. The baseline was stable, the drift was small, and the peak shape effect was good during the gradient elution of acetonitrile. Therefore, phosphoric acid aqueous solution-acetonitrile was selected as the mobile phase. Figure 5 shown.

[0100] 2. Investigation of different concentrations of phosphoric acid

[0101] According to the detection method of the fingerprint spectrum of the Shenyankangfu Tablets in the above Example 1, the test sample solution of the Shenyankangfu Tablets was tested under the conditions of 0.1% phosphoric acid-acetonitrile, 0.15% phosphoric acid-acetonitrile, and 0.2% phosphoric acid-acetonitrile mobile phase. The HPLC chromatograms were compared and analyzed. The separation degree and peak shape effect of 0.15% phosphoric acid-acetonitrile were better. Figure 6 shown.

[0102] Example 5

[0103] The column temperature selection of the detection method of the fingerprint spectrum of the Nephritis Recovery Tablets in the present invention was investigated.

[0104] According to the detection method of the fingerprint spectrum of Shenyankangfu Tablets in Example 1, the column temperature was set to 20 degrees, 30 degrees, 35 degrees and the column temperature gradient (0-55 minutes, column temperature: 20°C; 55-188 minutes, column temperature: 35°C). Figure 7 As shown, in the first 80 minutes, the column temperature of 20 degrees has better effects on separation and peak number than those of 30 degrees and 35 degrees. After 80 minutes, the column temperature of 35 degrees is better than those of 20 degrees and 30 degrees in terms of baseline, peak time and separation. Taking comprehensive consideration, the column temperature gradient (0-55 minutes, column temperature: 20°C; 55-188 minutes, column temperature: 35°C) is determined to be optimal.

[0105] Example 6

[0106] The method for detecting the fingerprint of the Shenyankangfu Tablets in the present invention was verified by methodology, and the detection method and performance index results are as follows:

[0107] 1. Precision: Take a portion of Shenyan Kangfu Tablets No. S4 (Batch No.: CP17058) and test it according to the detection method of the fingerprint spectrum of Shenyan Kangfu Tablets in Example 1 above, and inject the sample 6 times continuously. Take Peak 15 as the reference peak S for calculation, and examine the consistency of the retention time of each main chromatographic peak in the fingerprint spectrum. The results are shown in Table 4. The relative retention time of 20 common peaks is RSD%≤2.0%. The results show that the method has good precision.

[0108] Table 4 Precision results

[0109]

[0110]

[0111] 2. Repeatability: Six samples of Shenyan Kangfu Tablets numbered S4 (batch number: CP17058) were taken and tested according to the detection method of the fingerprint spectrum of Shenyan Kangfu Tablets in Example 1. Peak 15 was used as the reference peak S for calculation. The retention time of each main chromatographic peak in the fingerprint spectrum was examined. The results are shown in Table 5. There are 20 common peak relative retention times, RSD%≤2.0%. The results show that the method of the present invention has good repeatability and high accuracy.

[0112] Table 5 Repeatability results

[0113]

[0114]

[0115] 3. Stability: Take one sample of Shenyan Kangfu Tablets numbered S4 (batch number: CP17058) and test it according to the detection method of the fingerprint spectrum of Shenyan Kangfu Tablets in Example 1. After preparing the test solution of Shenyan Kangfu Tablets, place it for 0h, 5h, 10h, 15h, 20h, and 25h for detection. Peak 15 is used as the reference peak S for calculation, and the retention time of each main chromatographic peak in the fingerprint spectrum is investigated. The results are shown in Table 6. The relative retention time of 20 common peaks is RSD%≤2.0%. The results show that Shenyan Kangfu Tablets have good stability when tested within 25 hours.

[0116] Table 6 Stability results

[0117]

[0118]

Claims

1. The method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets is characterized in that: The following steps are involved: S1. Preparation of test sample solution: remove the coating of Shenyankangfu tablets, crush, add methanol, reflux in a water bath, filter, and obtain a test sample solution; S2. Preparation of reference solution: Weigh reference substances of tanshinone ⅡA, ginsenoside Rb1, leonurine hydrochloride, salvianolic acid B, astilbin, daidzein, pinoresinol diglucoside, p-coumaric acid and rosmarinic acid respectively, add methanol to prepare reference solution; S3, establishment of fingerprint: respectively aspirate the test sample solution and the reference solution and inject them into the liquid chromatograph for determination, and obtain the fingerprint of the test sample; S4. Confirmation of the standard fingerprint: According to the above method, fingerprints of multiple batches of Shenyan Kangfu Tablets are established respectively, and the fingerprints of multiple batches of Shenyan Kangfu Tablets are introduced into the traditional Chinese medicine chromatographic fingerprint evaluation system to generate a standard fingerprint, and the standard fingerprint has 20 common peaks; The 20 common peaks included pinoresinol diglucoside, daidzein, leonurine hydrochloride, p-coumaric acid, astilbin, rosmarinic acid, salvianolic acid B, ginsenoside Rb1, and tanshinone ⅡA; The determination conditions in step S3 are: chromatographic column: Agilent Technologies ZORBAX SB-C18; 4.6×250 mm, 3.5 μm, flow rate: 0.6 ml / min, mobile phase: A: 0.1-0.2% phosphoric acid solution, B: acetonitrile, column temperature: 20° C.-35° C., injection volume 6 μl, analysis time: 188 min; 0-10min, A:B volume ratio 98:2; 10-18min, A:B volume ratio 98:2-87:13; 18-58min, A:B volume ratio 87:13-86:14; 58-68min, A:B volume ratio 86:14-83:17; 68-88min, A:B volume ratio 83:17-80:20; 88-123min, A:B volume ratio 80:20-79:21; 123-155min, A:B volume ratio 79:21-49:51; 155-165min, A:B volume ratio 49:51-19:81; 165-188min, A:B volume ratio : B volume ratio 19:81-9:91; 0-37.5min, scanning wavelength is 360nm; 37.5-52.5min, scanning wavelength is changed to 203nm; 52.5-71min, scanning wavelength is changed to 330nm; 71-83min, scanning wavelength is changed to 270nm; 83-110min, scanning wavelength is changed to 210nm; 110-143min, scanning wavelength is changed to 275nm; 143-146.5min, scanning wavelength is changed to 203nm; 146.5-183min, scanning wavelength is changed to 275nm; 183-188min, scanning wavelength is changed to 203nm.

2. The method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets according to claim 1, characterized in that: The concentrations of tanshinone ⅡA, ginsenoside Rb1, leonurine hydrochloride, salvianolic acid B, astilbin, daidzein, pinoresinol diglucoside, p-coumaric acid and rosmarinic acid in the reference solution are 2-30 μg / ml.

3. The method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets according to claim 2, characterized in that: The concentration of tanshinone ⅡA in the reference solution was 8μg / ml, ginsenoside Rb1 was 20μg / ml, leonurine hydrochloride was 5μg / ml, salvianolic acid B was 5μg / ml, astilbin was 5μg / ml, daidzin was 10μg / ml, pinoresinol diglucoside was 20μg / ml, p-coumaric acid was 5μg / ml, and rosmarinic acid was 10μg / ml.

4. The method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets according to claim 1, characterized in that: 0-55min column temperature: 20℃, 55-188min column temperature: 35℃.

5. The method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets according to claim 1, characterized in that: The following steps are also included: According to the prescription of Nephritis Recovery Tablets, single medicinal material solutions and single or multiple medicinal material negative control prescription sample solutions are prepared, the single medicinal material solutions, single or multiple medicinal material negative control prescription sample solutions and the reference substance solution in step S2 are injected into a liquid chromatograph for measurement, fingerprints of the single medicinal material solutions and the single or multiple medicinal material negative control prescription sample solutions are established, and compared with 20 common peaks to determine the ownership of the fingerprint peaks.

6. The method for establishing the HPLC fingerprint of Shenyankangfu Tablets according to claim 5, characterized in that: Fingerprint peaks 1, 3, 7, 9, 10, 11, and 12 come from Smilax glabra, fingerprint peak 2 comes from Eucommia ulmoides, fingerprint peaks 4 and 8 come from black beans, fingerprint peak 5 comes from Leonurus japonicus, fingerprint peak 6 comes from Hedyotis diffusa and Imperata cylindrica, fingerprint peaks 13, 14, 15, 17, 18, 19, and 20 come from Salvia miltiorrhiza, and fingerprint peak 16 comes from Panax ginseng and American ginseng.

7. The method for establishing the HPLC fingerprint of the Shenyankangfu Tablets according to any one of claims 1 to 6, characterized in that: The relative retention times of the common peaks in the established standard fingerprint are: peak 1 0.329±0.020, peak 2 0.357±0.020, peak 3 0.361±0.020, peak 4 0.395±0.020, peak 5 0.409±0.020, peak 6 0.460±0.020, peak 7 0.615±0.020, peak 8 0.637±0.010, peak 9 0.643±0.010, peak 10 0.709±0.

020. 010, peak 11 0.727±0.010, peak 12 0.750±0.010, peak 13 0.817±0.010, peak 14 0.873±0.010, peak 15 1.000, peak 16 1.216±0.010, peak 17 1.401±0.020, peak 18 1.439±0.020, peak 19 1.476±0.020, peak 20 1.540±0.020, among which peak 15 is S peak.

8. The method for establishing the HPLC fingerprint of Shenyan Kangfu Tablets according to claim 7, characterized in that: Peak 2 is pinoresinol diglucoside, peak 4 is daidzein, peak 5 is leonurine hydrochloride, peak 6 is p-coumaric acid, peak 9 is astilbin, peak 14 is rosmarinic acid, peak 15 is salvianolic acid B, peak 17 is ginsenoside Rb1, and peak 19 is tanshinone ⅡA.

9. Application of the method for establishing HPLC fingerprint of Shenyan Kangfu Tablets as described in any one of claims 1-8: obtain the fingerprint of the Shenyan Kangfu Tablets sample according to steps S1-S3, use the standard fingerprint as a control, use the Chinese Pharmacopoeia Committee Chinese Medicine Chromatographic Fingerprint Evaluation System to calculate the similarity of the fingerprints, and judge whether the sample is qualified based on the similarity not less than 0.90.

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