Establishment of fingerprint of Jintongqingbi granules

Abstract

The application discloses a method for establishing HPLC fingerprint spectrum of Shengteng Qingbi granules, which comprises preparation of a test sample solution, determination of HPLC chromatographic conditions and production of an HPLC standard fingerprint spectrum. The application also discloses the HPLC standard fingerprint spectrum of Shengteng Qingbi granules obtained by the method, which has 35 common peaks. The quality detection method is simple in operation, high in stability and good in reproducibility, and the obtained spectrum has many characteristic peaks. Through comparison of the common peaks of the standard fingerprint spectrum, the quality of Shengteng Qingbi granules can be comprehensively evaluated and controlled, which is beneficial to accurate evaluation of the internal quality of the preparation and ensures the safety and effectiveness of clinical medication.

Description

TECHNICAL FIELD

[0001] The application discloses a method for establishing HPLC fingerprint of Jindeng Qingbi granules, and belongs to the field of Chinese medicine preparation analysis. BACKGROUND

[0002] Jindeng Qingbi granules (Guojiazhunzi Z20123065, produced by Lunan Houpu Pharmaceutical Co., Ltd.) are prepared from 11 Chinese medicines, i.e. honeysuckle, caulis sinomenii, belamcanda chinensis, figwort, rehmannia glutinosa, monotropistra, pedunculate galangal, licorice, angelica, radix paeoniae alba and centipede, and mainly have the effects of clearing heat and resolving toxin, promoting blood circulation to remove stasis, dredging collaterals to relieve pain. The granules are used for treating rheumatoid arthritis in active stage, belong to the syndrome of toxic heat accumulation and dampness-heat blocking collaterals, and are suitable for treating joint swelling, pain and pressing pain, morning stiffness, touch-induced fever, or skin redness, body heat, excessive sweating, dry mouth, dry stool and yellow urine, red tongue, yellow and greasy coating, and slippery and rapid pulse.

[0003] The honeysuckle and caulis sinomenii are used as monarch drugs, belamcanda chinensis, figwort, rehmannia glutinosa, monotropistra and pedunculate galangal are used as ministerial drugs, and radix paeoniae alba, centipede and licorice are used as auxiliary drugs; the drugs have the effects of clearing heat and resolving toxin, promoting blood circulation to remove stasis, dredging collaterals to relieve pain. The honeysuckle is rich in volatile oil, flavones and organic acid, and the water decoction, oral liquid and injection of the honeysuckle have the effects of relieving fever, resisting inflammation, regulating immune function and the like. The caulis sinomenii mainly contains sinomenine and magnoflorine, and has the effects of resisting inflammation and pain, resisting rheumatism and immune suppression, and is a commonly used traditional Chinese medicine for treating rheumatoid arthritis. Angelica, radix paeoniae alba, belamcanda chinensis, pedunculate galangal, figwort and monotropistra have the effects of tonifying blood and promoting blood circulation, relieving pain, strengthening muscles and bones; figwort, rehmannia glutinosa, licorice and monotropistra have the effects of nourishing yin and blood, purging fire and resolving toxin; centipede has the effects of promoting blood circulation to remove stasis, attacking toxin to remove stagnation, dredging collaterals to relieve pain, and pharmacological research shows that the centipede can improve microcirculation and has the effects of relieving pain and resisting inflammation. The drugs are used together to have the effects of clearing heat and resolving toxin, promoting blood circulation to remove stasis, removing stasis, dredging collaterals to relieve pain.

[0004] The Jindeng Qingbi granules are traditional Chinese medicine compound preparations, contain multiple traditional Chinese medicines and have complex chemical components, and therefore, comprehensive and effective quality control is an important basis for guaranteeing clinical curative effect. At present, there are few reports on the quality control of the Jindeng Qingbi granules, and the quality control is mainly quantitative analysis of single component of the medicines and qualitative identification of partial medicine components, the effective components are not clear, and there is lack of comprehensive and overall quality control research on the Jindeng Qingbi granules. In order to stabilize and guarantee product quality, it is necessary to comprehensively research the quality detection and control method. The traditional Chinese medicine fingerprint is a quality control technology and method of traditional Chinese medicine, has the characteristics of integrity and fuzziness, and can comprehensively reflect the overall quality of traditional Chinese medicine. At present, there is no related patent publication and literature report on the quality control of the Jindeng Qingbi granules by using the traditional Chinese medicine fingerprint.

[0005] The application discloses a method for establishing an HPLC fingerprint of Jintong Qingbi granules, and an HPLC standard fingerprint of Jintong Qingbi granules prepared by the method. SUMMARY

[0006] In view of the deficiencies of the existing quality detection methods of Jintong Qingbi granules, the application aims to provide a method for establishing a fingerprint of Jintong Qingbi granules.

[0007] The application is a method for establishing an HPLC fingerprint of Jintong Qingbi granules, comprising the following steps:

[0008] 1) Preparation of a test sample solution: grind Jintong Qingbi granule samples, take an appropriate amount, place in a conical flask with a plug, add 50%-90% methanol, weigh, ultrasonic extraction for 20-50 min, cool to room temperature, add 50%-90% methanol to make up for the weight loss, shake well, filter, take the filtrate as the test sample solution.

[0009] 2) Determination of HPLC chromatographic conditions: Waters Xselect CSH C18 column, acetonitrile-methanol as mobile phase A, 0.05%-0.15% formic acid water (V / V) as mobile phase B, gradient elution.

[0010] The gradient elution conditions are as follows:

[0011] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0-13 0.7 5％ 95％ 13-26 0.7-0.9 5％→15％ 95％→85％ 26-45 0.9 15％→20％ 85％→80％ 45-75 0.9-1.0 20％→29％ 80％→71％ 75-85 1.0 29％→40％ 71％→60％ 85-100 1.0 40％→55％ 60％→45％ 100-115 1.0 55％→80％ 45％→20％ 115-117 1.0-0.7 80％→5％ 20％→95％ 117-122 0.7 5％ 95％

[0012] 3) Preparation of the fingerprint: analyze and compare the test sample solution of Jintong Qingbi granules according to the chromatographic conditions in step 2), and obtain the HPLC standard fingerprint of Jintong Qingbi granules composed of common characteristic peaks of samples.

[0013] Preferably, the preparation of the test sample solution in step 1) is as follows: grind Jintong Qingbi granule samples, take an appropriate amount, place in a conical flask with a plug, add an appropriate amount of 70% methanol, weigh, ultrasonic extraction for 30 min, cool to room temperature, add 70% methanol to make up for the weight loss, shake well, filter, take the filtrate as the test sample solution.

[0014] Further preferably, the preparation of the test sample solution in step 1): take the sample of Jintong Qingbi granules, grind, take about 2g, accurately weigh, put into a conical flask with a plug, accurately add 25mL of 70% methanol, weigh, ultrasonic extraction for 30min, cool to room temperature, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and obtain the test sample solution.

[0015] Preferably, the mobile phase A in step 2) is acetonitrile-methanol (1:1), and the mobile phase B is 0.1% formic acid water (V / V).

[0016] Preferably, the specification of the chromatographic column in step 2) is 4.6*150mm, 2.5um.

[0017] In the present application, in the chromatographic conditions in step 2), the detection wavelength is 263nm, the column temperature is 25℃, and the injection volume is 5ul.

[0018] Preferably, take the sample of Jintong Qingbi granules to be detected, prepare the test sample solution according to step 1), perform HPLC chromatographic analysis according to step 2), and prepare the standard fingerprint according to step 3).

[0019] The HPLC standard fingerprint of Jintong Qingbi granules is evaluated for similarity by using the software of “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition”.

[0020] The present application also provides the HPLC standard fingerprint of Jintong Qingbi granules obtained by the above method, and the specific steps are as follows: 15 batches of Jintong Qingbi granule samples are prepared into test sample solutions according to the above method, separated and detected by HPLC, analyzed by using the software of “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition” recommended by the National Pharmacopoeia Committee, and the HPLC standard fingerprint of Jintong Qingbi granules is obtained.

[0021] In the present application, the HPLC standard fingerprint of Jintong Qingbi granules has 35 common peaks, and chlorogenic acid is used as a reference peak, and the relative retention time t of the common peaks is as follows: R 0.048, 0.050, 0.052, 0.068, 0.080, 0.089, 0.095, 0.106, 0.111, 0.115, 0.164, 0.183, 0.221, 0.292, 0.378, 0.739, 0.757, 0.805, 0.840, 0.874, 0.935, 0.965, 1.000, 1.083, 1.203, 1.506, 1.573, 1.635, 1.816, 1.987, 2.026, 2.277, 2.496, 2.928, 3.068.

[0022] By comparing with the reference substance, wherein peak No.15 is sinomenine, peak No.16 is magnoflorine, peak No.17 is neochlorogenic acid, peak No.21 is cryptochlorogenic acid, peak No.23 is chlorogenic acid, peak No.26 is glycyrrhizin, peak No.30 is isochlorogenic acid B, peak No.32 is isochlorogenic acid C, peak No.33 is harpagide, peak No.34 is ligustilide, and peak No.35 is ammonium glycyrrhizinate.

[0023] Compared with the prior art, the technical scheme of the present application has the following beneficial technical effects:

[0024] 1) The method for establishing the HPLC fingerprint of Jintong Qingbi granules overcomes the shortcomings of the prior art, such as single detection index and inability to reflect the internal quality, and has high precision and good reproducibility. The presence or absence of common peaks and the similarity degree can be compared to comprehensively evaluate the quality of Jintong Qingbi granules, thereby effectively ensuring the quality of the finished product.

[0025] 2) According to the prescription composition characteristics of Jintong Qingbi granules, the optimal mobile phase composition, gradient elution program, flow rate, detection wavelength and the like are selected, thereby solving the problems of difficulty in separating the characteristic peaks and large impurity peak interference. The obtained fingerprint has high separation degree and good peak shape, and each characteristic chromatographic peak is well separated from the baseline. The method has good stability, many characteristic peaks, and can comprehensively and accurately evaluate the internal quality of the preparation, and is suitable for controlling the quality of Jintong Qingbi granule products. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure is the HPLC standard fingerprint of Jintong Qingbi granules (1-35 are 35 common peaks);

[0027] Figure 2 The figure is the HPLC mixed control fingerprint of Jintong Qingbi granules;

[0028] The peak No.15 is sinomenine, the peak No.16 is magnoflorine, the peak No.17 is neochlorogenic acid, the peak No.21 is cryptochlorogenic acid, the peak No.23 is chlorogenic acid, the peak No.26 is glycyrrhizin, the peak No.30 is isochlorogenic acid B, the peak No.32 is isochlorogenic acid C, the peak No.33 is harpagide, the peak No.34 is ligustilide, and the peak No.35 is ammonium glycyrrhizinate.

[0029] Figure 3 The figure is the HPLC blank fingerprint of Jintong Qingbi granules.

[0030] Figure 4 The figure is the HPLC fingerprint superimposition of 15 batches of Jintong Qingbi granules. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of this application clearer and more explicit, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the following embodiments are only a part of, and not all of, the embodiments of this application. Therefore, the detailed description of the embodiments below does not limit the scope of protection claimed in this application, and equivalent substitutions or modifications made by those skilled in the art without creative effort should be included within the scope of protection of this invention.

[0032] Example 1: Establishment of HPLC standard fingerprint of Jinteng Qingbi Granules

[0033] 1. Instruments and reagents

[0034] 1.1 Instruments

[0035] WatersArc ultra-high performance liquid chromatograph (Waters Corporation), Waters 2489 UV / Vis detector (Waters Corporation); Dingtai Hengsheng JY-15 ultrasonic cleaner (Dingtai (Hubei) Biochemical Technology Equipment Manufacturing Co., Ltd.).

[0036] 1.2 Test Drugs

[0037] The 15 batches of Jinteng Qingbi Granules were supplied by Lunan Houpu Pharmaceutical Co., Ltd. (batch numbers are shown in Table 1). Acetonitrile and methanol were of chromatographic grade, water was redistilled water, and all other reagents were of analytical grade.

[0038] Table 1. Batch numbers of Jinteng Qingbi Granules (batch number 15)

[0039]

[0040] 2. Methods and Results

[0041] 2.1 Preparation of test solution: Take Jinteng Qingbi granules, grind them into a fine powder, weigh about 2g accurately, place them in a stoppered conical flask, add 25mL of 70% methanol accurately, extract by sonication for 30min, cool to room temperature, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate as the test solution.

[0042] 2.2 Preparation of reference solution: Accurately weigh the mixed reference standards of sinomenine, magnoflorine, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, glycyrrhizin, isochlorogenic acid B, isochlorogenic acid C, harpagoside, ligustilide, and ammonium glycyrrhizate, dissolve them in 70% methanol, and use the solution as the mixed reference standard.

[0043] 2.3 Chromatographic conditions: Column: Waters Xselect CSH C18 column (4.6 × 150 mm, 2.5 μm); Mobile phase: Acetonitrile-methanol (1:1) as mobile phase A, and 0.1% formic acid aqueous solution (V / V) as mobile phase B, with gradient elution performed according to the table below:

[0044] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0-13 0.7 5％ 95％ 13-26 0.7-0.9 5％→15％ 95％→85％ 26-45 0.9 15％→20％ 85％→80％ 45-75 0.9-1.0 20％→29％ 80％→71％ 75-85 1.0 29％→40％ 71％→60％ 85-100 1.0 40％→55％ 60％→45％ 100-115 1.0 55％→80％ 45％→20％ 115-117 1.0-0.7 80％→5％ 20％→95％ 117-122 0.7 5％ 95％

[0045] Detection wavelength: 263nm; column temperature: 25℃; injection volume: 5μL.

[0046] 2.4 Establishment of fingerprint map

[0047] The HPLC fingerprints of 15 batches of Jinteng Qingbi Granules were determined and analyzed, and the HPLC standard fingerprint of Jinteng Qingbi Granules composed of their common characteristic peaks was obtained (see Appendix). Figure 1 , 3 ), where chlorogenic acid is used as the reference peak, and the relative retention time t of its common peak. R The values ​​are as follows: 0.048, 0.050, 0.052, 0.068, 0.080, 0.089, 0.095, 0.106, 0.111, 0.115, 0.164, 0.183, 0.221, 0.292, 0.378, 0.739, 0.757, 0.805, 0.840, 0.874, 0.935, 0.965, 1.000, 1.083, 1.203, 1.506, 1.573, 1.635, 1.816, 1.987, 2.026, 2.277, 2.496, 2.928, 3.068.

[0048] Peak 15 is sinomenine, peak 16 is magnoflorine, peak 17 is neochlorogenic acid, peak 21 is cryptochlorogenic acid, peak 23 is chlorogenic acid, peak 26 is glycyrrhizin, peak 30 is isochlorogenic acid B, peak 32 is isochlorogenic acid C, peak 33 is harpagoside, peak 34 is ligustilide, and peak 35 is ammonium glycyrrhizate.

[0049] The HPLC fingerprint chromatograms of 15 batches of Jinteng Qingbi granules were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software recommended by the Pharmacopoeia Commission. Chromatographic peak matching was performed, and 35 common peaks were identified as characteristic peaks constituting the fingerprint chromatogram of Jinteng Qingbi granules. Chlorogenic acid peak No. 23 was used as the reference peak (S). The relative retention times of the common peaks in the samples are listed in Table 2, and the relative peak areas of the common peaks in the 15 batches of Jinteng Qingbi granules are shown in Table 3. The similarity calculation results between the 15 batches of Jinteng Qingbi granules and the standard fingerprint chromatogram were 0.939, 0.988, 0.994, 0.994, 0.996, 0.990, 0.990, 0.989, 0.987, 0.992, 0.995, 0.984, 0.996, 0.994, and 0.994, respectively.

[0050] Table 2. Relative retention times (t) of common peaks in 15 batches of Jinteng Qingbi Granules R )

[0051]

[0052]

[0053] Table 3. Relative peak area (%) of common peaks in 15 batches of Jinteng Qingbi Granules

[0054]

[0055]

[0056] 2.5 Methodological Examination

[0057] 2.5.1 Precision Test

[0058] Take sample S1 and prepare the test solution according to the method in section 2.2. Inject the sample 6 times consecutively and record its fingerprint spectrum. Using chlorogenic acid peak 23 as the reference peak, calculate the relative retention time RSD of common peaks 1-35. The RSD of the relative peak area is <0.85% and <2.82%, indicating that the instrument precision is good.

[0059] 2.5.2 Stability Test

[0060] Take sample S1 and prepare the test solution according to the method in section 2.2. Inject the sample at 0, 3, 6, 9, 12, 18 and 24 h respectively. Using chlorogenic acid peak No. 23 as the reference peak, the relative retention time RSD of common peaks No. 1-35 is calculated to be <0.66% and the relative peak area RSD is <2.97%, indicating that the test solution is stable within 24 hours.

[0061] 2.5.3 Repeatability Test

[0062] Six portions of the same batch number (S1) were accurately weighed and prepared according to the method in section 2.2. The samples were injected separately. Using the chlorogenic acid peak No. 23 as the reference peak, the relative retention time RSD of the common peaks No. 1-35 was calculated to be <0.64%, and the relative peak area RSD was <2.77%, indicating that the method has good repeatability.

[0063] The above description is merely a preferred embodiment of the present invention, and while it is quite specific and detailed, it should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the principles and concept of the present invention, and these should also fall within the scope of protection of the present invention.

Claims

1. A method for establishing an HPLC fingerprint of Jinteng Qingbi granules, characterized in that, Includes the following steps: 1) Preparation of test solution: Take Jinteng Qingbi granules, grind them into a fine powder, weigh an appropriate amount, place them in a stoppered conical flask, add 50%-90% methanol, weigh the sample, extract with ultrasound for 20-50 min, cool to room temperature, replenish the lost weight with 50%-90% methanol, shake well, filter, and take the filtrate as the test solution. 2) Determination of HPLC chromatographic conditions: Waters Xselect CSH C18 column, with acetonitrile:methanol = 1:1 as mobile phase A and 0.05%-0.15% formic acid in water as mobile phase B, detection wavelength: 263 nm, gradient elution; the gradient elution conditions were as follows: ， 3) Preparation of fingerprint spectrum: The test solution was analyzed and compared according to the chromatographic conditions in step 2) to obtain the HPLC standard fingerprint spectrum of Jinteng Qingbi Granules composed of common characteristic peaks of the samples. The standard fingerprint spectrum has 35 common peaks, of which peak 15 is sinomenine, peak 16 is magnoflorine, peak 17 is neochlorogenic acid, peak 21 is cryptochlorogenic acid, peak 23 is chlorogenic acid, peak 26 is glycyrrhizin, peak 30 is isochlorogenic acid B, peak 32 is isochlorogenic acid C, peak 33 is harpagoside, peak 34 is ligustilide, and peak 35 is ammonium glycyrrhizate.

2. The method as described in claim 1, characterized in that, The test solution mentioned in step 1) is prepared according to the following steps: Take a sample of Jinteng Qingbi Granules, grind it into a fine powder, weigh an appropriate amount, place it in a stoppered conical flask, add an appropriate amount of 70% methanol, weigh it, extract it by sonication for 30 min, cool it to room temperature, replenish the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

3. The method as described in claim 1, characterized in that, Step 2) The mobile phase B is a 0.1% formic acid aqueous solution.

4. The method as described in claim 1, characterized in that, The chromatographic column has a size of 4.6 × 150 mm and a diameter of 2.5 μm.

5. The method as described in claim 1, characterized in that, The chromatographic conditions for step 2) are: column temperature: 25℃; injection volume: 5μL.

6. The method as described in claim 1, characterized in that, The HPLC standard fingerprint of Jinteng Qingbi Granules was evaluated for similarity using the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)".

7. The method according to any one of claims 1-6, characterized in that, Using chlorogenic acid as a reference peak, the relative retention time t of the common peaks in the fingerprint spectrum R The values ​​are as follows: 0.048, 0.050, 0.052, 0.068, 0.080, 0.089, 0.095, 0.106, 0.111, 0.115, 0.164, 0.183, 0.221, 0.292, 0.378, 0.739, 0.757, 0.805, 0.840, 0.874, 0.935, 0.965, 1.000, 1.083, 1.203, 1.506, 1.573, 1.635, 1.816, 1.987, 2.026, 2.277, 2.496, 2.928, 3.068.

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