Construction method and application of specific chromatogram of gout eliminating preparation

Abstract

The invention discloses a construction method and application of a specific chromatogram of a gout eliminating preparation, and the construction method comprises the following steps: (1) taking a reference substance, and adding a dissolving solvent to prepare a reference substance solution; (2) extracting the gout clearing and eliminating preparation by using an extraction solvent to prepare a test solution; and (3) respectively carrying out high performance liquid chromatography detection on the test solution and the reference substance solution to respectively obtain a fingerprint spectrum and a reference spectrum of the gout clearing and eliminating preparation, carrying out common peak identification on chromatographic peaks of the fingerprint spectrum of the gout clearing and eliminating preparation according to the reference spectrum, and constructing the characteristic spectrum of the gout clearing and eliminating preparation. According to the specific chromatogram construction method provided by the invention, 22 characteristic peaks are obtained, 6 identified characteristic peaks are obtained, the method is good in stability and repeatability, small in difference among different groups and batches, good in uniformity and high in stability among the same batches, and the quality of the gout clearing and eliminating preparation product can be more comprehensively controlled; the specificity identification and the overall quality control of the gout clearing and eliminating preparation are enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a construction method and application of a characteristic spectrum of a Tongfengqingxiao preparation. BACKGROUND

[0002] Tongfengqingxiao formula is a classic prescription for treating gout. The prescription is a protocol formula formed on the basis of the ancient prescription "Simiao Powder" according to clinical application, which is composed of fifteen kinds of medicines such as Baizhu, Futo Cangzhu, Futo Yiyiren, Yan Guanhuabai, Doukou, Chuan Niuxi, Tufuling, Jinqiancao, Cheqiancao, Xuchangqing, Pugongying, Mianbidi, Zhonglou, Shancigu, and Qingfengteng, and is used for treating acute gouty arthritis due to dampness-heat accumulation. The symptoms include muscle or joint redness, pain, refusal to press, local burning, ease with cooling, heaviness, difficulty walking, fever, thirst, restlessness, yellow urine, red tongue, yellow and greasy fur, and slippery pulse. The formula has been used in hospital clinics for decades. According to the feedback from patients and pharmacological experiment data, after two courses of treatment, the symptoms of gouty arthritis are significantly improved, and the inflammatory factors and uric acid levels in the body are also significantly reduced. Pharmacological studies show that the formula mainly treats gouty arthritis by increasing uric acid metabolism and enhancing the body's immunity. The Guzhongqingxiao granules prepared and registered by the applicant's team are also based on the basic formula of Tongfengqingxiao, and are medical institution preparations (Gan medicine preparation Z20250013000) developed in accordance with the "Medical Institution Preparation Registration Management Method".

[0003] Regarding the Tongfengqingxiao preparation, the current quality control mainly relies on thin layer chromatography identification and determination of single content of Qingteng alkaloids, which is difficult to meet the overall quality evaluation needs of the synergistic effect of multiple components of the compound preparation. In view of the complex characteristics of traditional Chinese medicine compound components, the traditional Chinese medicine fingerprint technology can establish a quality evaluation model containing multiple characteristic peaks by systematically analyzing the chemical substance group, which can not only reflect the unique chemical characteristic pattern of the preparation, but also realize batch stability monitoring through similarity analysis.

[0004] In order to more comprehensively and effectively control the quality of Tongfengqingxiao preparation for clinical use, and ensure its safety and effectiveness, it is necessary to establish a fingerprint spectrum to more comprehensively control the overall quality of Tongfengqingxiao preparation. SUMMARY

[0005] The present application aims to overcome the defects of the prior gout Qingxiao preparation that only controls the quality with single component, lacks characteristic spectrum and is difficult to comprehensively control the preparation quality standard, thereby providing a construction method and application of the characteristic spectrum of gout Qingxiao preparation, which is good in stability and repeatability, small in difference between different batches, good in uniformity between the same batches and high in stability, can more comprehensively control the product quality of gout Qingxiao preparation and strengthen the specific identification and overall quality control of gout Qingxiao preparation. In addition, the present application optimizes the extraction mode, extraction solvent, extraction time and high performance liquid chromatography detection wavelength of the test solution preparation of gout Qingxiao preparation, elutes with a specific gradient program, and obtains the fingerprint spectrum of gout Qingxiao preparation with relatively good chromatographic peak separation effect and peak type.

[0006] The technical scheme of the present application is as follows: The construction method of the characteristic spectrum of gout Qingxiao preparation comprises the following steps: (1) taking a control sample, adding a dissolution solvent to prepare a control sample solution; (2) using an extraction solvent to extract gout Qingxiao preparation to prepare a test solution; (3) respectively performing high performance liquid chromatography detection on the test solution and the control sample solution to obtain the fingerprint spectrum and the control spectrum of gout Qingxiao preparation, identifying the common peaks of the chromatographic peaks of the fingerprint spectrum of gout Qingxiao preparation according to the control spectrum, and constructing the characteristic spectrum of gout Qingxiao preparation.

[0007] The conditions of high performance liquid chromatography detection include: the detection wavelength is 280 nm, acetonitrile is used as the mobile phase A, and phosphoric acid aqueous solution or formic acid aqueous solution is used as the mobile phase B for gradient elution.

[0008] The gradient elution conditions are as follows: 0-5 min, the volume percentage of the mobile phase A and the mobile phase B changes by 5%-5% and 95%-95%, respectively; 5-10 min, the volume percentage of the mobile phase A and the mobile phase B changes by 5%-7% and 95%-93%, respectively; 10-25 min, the volume percentage of the mobile phase A and the mobile phase B changes by 7%-9% and 93%-91%, respectively; 25-35 min, the volume percentage of the mobile phase A and the mobile phase B changes by 9%-12% and 91%-88%, respectively; 35-50 min, the volume percentage of the mobile phase A and the mobile phase B changes by 12%-15% and 88%-85%, respectively; 50-55 min, the volume percentage of the mobile phase A and the mobile phase B changes by 15%-18% and 85%-82%, respectively; 55-70min, the volume percentage of mobile phase A and mobile phase B changes to 18%-20% and 82%-80%, respectively; 70-80min, the volume percentage of mobile phase A and mobile phase B changes to 20%-25% and 80%-75%, respectively; 80-85min, the volume percentage of mobile phase A and mobile phase B changes to 25%-30% and 75%-70%, respectively; 85-90min, the volume percentage of mobile phase A and mobile phase B changes to 30%-30% and 70%-70%, respectively.

[0009] Preferably, the gout clearing and eliminating preparation is gout clearing and eliminating granules or gout clearing and eliminating decoction.

[0010] Preferably, in step (1), the reference substance includes any one or several of sinomenine, caffeic acid, magnoflorine, astilbin, chicoric acid, and berberine hydrochloride. The concentration of the reference substance solution is 0.1-0.5 mg / ml, and the dissolving solvent is methanol. More preferably, the concentration of the reference substance solution is: sinomenine 0.312 mg / ml, caffeic acid 0.31 mg / ml, magnoflorine 0.292 mg / ml, astilbin 0.38 mg / ml, chicoric acid 0.1993 mg / ml, and berberine hydrochloride 0.318 mg / ml.

[0011] Preferably, in step (2), the extraction method is ultrasonic extraction or reflux extraction, the extraction solvent is water, methanol or a methanol solution, the volume concentration of the methanol solution is 30-60%, and the extraction time is 15-60 min. More preferably, the extraction method is ultrasonic extraction, the ultrasonic power is 200-400 W, the ultrasonic frequency is 30-50 kHz, the extraction solvent is a methanol aqueous solution with a volume concentration of 60%, and the extraction time is 15 min.

[0012] Preferably, in step (3), the conditions for high performance liquid chromatography detection further include: a chromatographic column with octadecylsilane bonded silica gel as the filler, a chromatographic column size of 4.6 x 250 mm, a particle size of 5 μm, a column temperature of 30-38 ℃, a flow rate of 0.8-1.2 ml / min, and an injection volume of 10-30 μL. More preferably, the column temperature is 35 ℃, the flow rate is 1 ml / min, and the injection volume is 20 μL.

[0013] Preferably, the volume concentration of the phosphoric acid aqueous solution or the formic acid aqueous solution is 0.1-0.5%. More preferably, the mobile phase B is a 0.1% volume concentration phosphoric acid aqueous solution.

[0014] Preferably, the construction method further comprises: taking single medicinal material of the Gout Clearing and Eliminating Preparation according to the prescription to carry out water extraction and dry distillation, then using the extraction solvent to carry out extraction (the extraction method is the same as that in step (2) of the Gout Clearing and Eliminating Preparation), preparing the test solution of the single medicine, carrying out high performance liquid chromatography detection, and determining the peak attribution of the fingerprint of the Gout Clearing and Eliminating Preparation.

[0015] Preferably, the fingerprint of the Gout Clearing and Eliminating Preparation comprises 22 common peaks, and the identified 6 characteristic peaks comprise: peak No. 6 is sinomine, peak No. 7 is caffeic acid, peak No. 8 is magnoflorine, peak No. 15 is astilbin, peak No. 16 is chicoric acid, and peak No. 22 is berberine hydrochloride.

[0016] Among them, peak No. 6 and peak No. 8 belong to Sinomenium acutum, peak No. 7 belongs to Cyathula officinalis, Taraxacum mongolicum and Plantago asiatica, peak No. 15 belongs to Smilax glabra, peak No. 16 belongs to Taraxacum mongolicum, and peak No. 22 belongs to Cyathula officinalis and Phellodendri chinensis.

[0017] Taking peak No. 8 magnoflorine as a reference peak, the relative retention time and relative peak area of the 6 characteristic peaks are as follows: The relative retention time of peak No. 6 is 0.5988-0.6019, and the relative peak area is 0.3578-0.4502; The relative retention time of peak No. 7 is 0.7875-0.7905, and the relative peak area is 0.2504-0.3045; The relative retention time of peak No. 8 is 1.0000, and the relative peak area is 1.0000; The relative retention time of peak No. 15 is 1.5610-1.5730, and the relative peak area is 0.4877-0.6120; The relative retention time of peak No. 16 is 1.5953-1.6014, and the relative peak area is 0.3634-0.4664; The relative retention time of peak No. 22 is 2.0608-2.0699, and the relative peak area is 0.2754-0.3275.

[0018] The application further provides the characteristic spectrum of the Gout Clearing and Eliminating Preparation obtained by the construction method.

[0019] The application further provides application of the construction method of the characteristic spectrum of the Gout Clearing and Eliminating Preparation in quality control of the Gout Clearing and Eliminating Preparation.

[0020] The application has the following beneficial effects: 1. The method for constructing the characteristic map of the gout clearing and eliminating preparation provided by the application has 22 characteristic peaks, 6 identified characteristic peaks, good stability and repeatability, small difference between different groups, good uniformity between batches, high stability, can more comprehensively control the product quality of the gout clearing and eliminating preparation, and strengthens the specific identification and overall quality control of the gout clearing and eliminating preparation.

[0021] 2. The method for constructing the characteristic map of the gout clearing and eliminating preparation provided by the application has 22 characteristic peaks, 6 identified characteristic peaks, good stability and repeatability, small difference between different groups, good uniformity between batches, high stability, can more comprehensively control the product quality of the gout clearing and eliminating preparation, and strengthens the specific identification and overall quality control of the gout clearing and eliminating preparation.

[0022] 3. The method for constructing the characteristic map of the gout clearing and eliminating preparation provided by the application has 22 characteristic peaks, 6 identified characteristic peaks, good stability and repeatability, small difference between different groups, good uniformity between batches, high stability, can more comprehensively control the product quality of the gout clearing and eliminating preparation, and strengthens the specific identification and overall quality control of the gout clearing and eliminating preparation. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 For the peak attribution of the fingerprint map of the gout clearing and eliminating granules of the application (Note: S1: Radix et Rhizoma Aucklandiae, S2: Radix et Rhizoma Anemarrhenae, S3: Herba Lysimachiae, S4: Rhizoma Atractylodis Macrocephalae, S5: Rhizoma Atractylodis Lanceae, S6: Rhizoma Alpiniae Oxophyllae, S7: Rhizoma Anemarrhenae, S8: Rhizoma Anemarrhenae, S9: Herba Taraxaci, S10: Cortex Phellodendri, S11: Herba Plantaginis, S12: Fructus Amomi Rotundus, S13: Caulis Sargentodoxae, S14: Semen Coicis, S15: Radix Pseudodiscoreae, S16: Gout clearing and eliminating granules); Figure 2 For the confirmation of the characteristic map of the gout clearing and eliminating granules of the application (Note: S1: Gout clearing and eliminating granules, S2: Reference substance); Figure 3 For the fingerprint map of the gout clearing and eliminating granules under different detection wavelength conditions (Note: S1: 220 nm, S2: 254 nm, S3: 280 nm, S4: 310 nm, S5: 340 nm, S6: 370 nm); Figure 4 For the fingerprint map of the gout clearing and eliminating granules under different mobile phase conditions (Note: S1: Acetonitrile-water, S2: Acetonitrile-0.1% formic acid aqueous solution, S3: Acetonitrile-0.05% phosphoric acid aqueous solution, S4: Acetonitrile-0.1% phosphoric acid aqueous solution); Figure 5 For the similarity comparison of the fingerprint maps of 15 batches of gout clearing and eliminating granules. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0025] Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available products, which can be purchased through commercial channels.

[0026] The gout clearing and eliminating formula of the present application is: Bai Zhi, Fu Chao Cang Zhi, Fu Chao Yi Ren, Yan Guan Huang Bai, Dou Qu, Chuan Niu Xi, Tu Fu Ling, Jin Qian Cao, Che Qian Cao, Xu Chang Qing, Pu Gong Ying, Mian Di Cai, Chong Lou, Shan Ci Gu, Qing Feng Teng.

[0027] Example 1: Establishment of a detection method for gout clearing and eliminating granules 1. Instruments and preparations Instruments: Shimadzu LC-20AT high-performance liquid chromatograph, AL-104 analytical balance (Mettler Toledo Shanghai Co., Ltd.), ultrasonic cleaning instrument KQ-250DB (Kunshan Ultrasonic Instrument Co., Ltd.).

[0028] Preparation of gout clearing and eliminating granules: 15 kinds of medicinal materials, such as Bai Zhi, Fu Chao Cang Zhi, Fu Chao Yi Ren, Yan Guan Huang Bai, Dou Qu, Chuan Niu Xi, Tu Fu Ling, Jin Qian Cao, Che Qian Cao, Xu Chang Qing, Pu Gong Ying, Mian Di Cai, Chong Lou, Shan Ci Gu, and Qing Feng Teng, were weighed according to the prescription dosage, and then water was added for decoction twice. The first time, 10 times the volume of water was added for decoction for 1 hour, and the second time, 8 times the volume of water was added for decoction for 1 hour. The decoction was combined, filtered, concentrated to a clear extract with a relative density of 1.15-1.25 (55-60℃), dried, pulverized, and then mixed with an appropriate amount of dextrin and sucralose. After mixing, granules were prepared and dried to obtain gout clearing and eliminating granules.

[0029] 2. Establishment of the method (1) Preparation of the test solution The proposed test preparation method is as follows: 2g of gout clearing and eliminating granules were finely ground and placed in a conical flask with a stopper. 50ml of 60% (volume concentration) methanol aqueous solution was precisely added, the weight was determined, and ultrasonic treatment was performed for 30 minutes. After cooling, the weight was determined again, the lost weight was made up with 60% methanol aqueous solution, and the mixture was shaken and filtered. The filtrate was collected, filtered through a microporous filter membrane, and then obtained.

[0030] (2) Preparation of the control solution The preparation method of the control solution is as follows: taking sinomenine, caffeic acid, magnoflorine, astilbin, chicoric acid and berberine hydrochloride respectively, dissolving them in methanol to prepare the control solution of sinomenine 0.312 mg / ml, caffeic acid 0.31 mg / ml, magnoflorine 0.292 mg / ml, astilbin 0.38 mg / ml, chicoric acid 0.1993 mg / ml and berberine hydrochloride 0.318 mg / ml respectively.

[0031] (3) High performance liquid chromatography detection conditions Considering the baseline, peak number, separation degree of each chromatographic peak and other factors, the chromatographic conditions are determined as follows: using Shimadzu LC-20AT type high performance liquid chromatograph, Diamonsil C 18 chromatographic column (4.6x250mm, 5μm), gradient elution with acetonitrile (phase A)-0.1% phosphoric acid aqueous solution (phase B) as the mobile phase.

[0032] The gradient elution conditions are as follows: 0-5min, B phase 95-95vol%, A phase 5-5vol%; 5-10min, B phase 95-93vol%, A phase 5-7vol%; 10-25min, B phase 93-91vol%, A phase 7-9vol%; 25-35min, B phase 91-88vol%, A phase 9-12vol%; 35-50min, B phase 88-85vol%, A phase 12-15vol%; 50-55min, B phase 85-82vol%, A phase 15-18vol%; 55-70min, B phase 82-80vol%, A phase 18-20vol%; 70-80min, B phase 80-75vol%, A phase 20-25vol%; 80-85min, B phase 75-70vol%, A phase 25-30vol%; 85-90min, B phase 70-70vol%, A phase 30-30vol%.

[0033] Column temperature 35℃, flow rate 1ml / min, wavelength 280nm.

[0034] Example 2 Fingerprint spectrum construction of Tongfengqing granules 1. Peak assignment The prescription proportion was weighed respectively by Atractylodes macrocephala Koidz, Fried with bran Atractylodes lancea, Fried with bran Coix lacryja, salt with Huang Bai, Amomum villosum, Cyathula officinalis Klotz, Smilax glabra, Lysimachia christinae Hance, Plantago asiatica L., Xuanlongqing, Taraxacum mongolicum, Alisma orientale, Paris polyphylla, Caulis Sargentodoxae, and the water was added 25 ml, respectively, and decocted for 30 minutes, and then filtered and evaporated. The test solution of each single medicine was prepared by the method of preparing test solution of example 1 in sequence. According to the determined chromatographic conditions, the sample was injected and detected, and the results were shown in Table 1. Figure 1 and Table 1.

[0035] 22 peak assignment results: peak 1 is 12 common peaks (Atractylodes macrocephala Koidz, Fried with bran Atractylodes lancea, Fried with bran Coix lacryja, Cyathula officinalis Klotz, Smilax glabra, Lysimachia christinae Hance, Taraxacum mongolicum, Alisma orientale, Paris polyphylla, Caulis Sargentodoxae), peak 2 is 9 common peaks (Atractylodes macrocephala Koidz, Fried with bran Atractylodes lancea, Fried with bran Coix lacryja, Cyathula officinalis Klotz, Smilax glabra, Lysimachia christinae Hance, Paris polyphylla, Caulis Sargentodoxae), peak 3 is 10 common peaks (Fried with bran Coix lacryja, Amomum villosum, Smilax glabra, Lysimachia christinae Hance, Taraxacum mongolicum, Alisma orientale, Paris polyphylla, Caulis Sargentodoxae), peak 4 is 3 common peaks (Fried with bran Coix lacryja, Paris polyphylla, Atractylodes macrocephala Koidz), peak 6, peak 8 and peak 12 belong to Caulis Sargentodoxae, peak 7 is 3 common peaks (Cyathula officinalis Klotz, Taraxacum mongolicum, Plantago asiatica L.), peak 10 belongs to Fried with bran Coix lacryja, peak 11 belongs to Lysimachia christinae Hance, peak 13 and peak 17 belong to Plantago asiatica L., peak 9, peak 14, peak 15, peak 18, peak 19, peak 20 and peak 21 belong to Smilax glabra, peak 5 and peak 16 belong to Taraxacum mongolicum, and peak 22 is the common peak of Cyathula officinalis Klotz and salt with Huang Bai.

[0036] Table 1 Peak assignment in fingerprint

[0037] 2. Chromatographic peak identification According to the above determined chromatographic method, the test solution, the control solution (sinomenine, caffeic acid, magnoflorine, astilbin, chicoric acid, berberine hydrochloride) were detected respectively, and the results were shown in Table 2. Figure 2 .

[0038] By comparing the peak time, the spectrum of the control and the test solution, it was determined that peak 6 was sinomenine, peak 7 was caffeic acid, peak 8 was magnoflorine, peak 15 was astilbin, peak 16 was chicoric acid, and peak 22 was berberine hydrochloride.

[0039] Example 3 Parameter optimization and method confirmation in fingerprint construction 1. Parameter optimization (1) Selection of detection wavelength Take the test sample solution prepared in Example 1, compare the chromatograms at wavelengths of 220 nm, 254 nm, 280 nm, 310 nm, 340 nm, and 370 nm, and the results are shown in Table 1. Figure 3 The results show that, by full scanning at wavelengths of 190-400 nm using a diode array detector (DAD), and based on factors such as the number of chromatographic peaks, peak response value and its proportion, peak resolution and tailing factor, baseline stability, and the like, the wavelength of 280 nm is selected for determination of the fingerprint spectrum of Tongfengqing Granules.

[0040] (2) Selection of mobile phase type The mobile phase types investigated were acetonitrile-water, acetonitrile-0.1% formic acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, and acetonitrile-0.05% phosphoric acid aqueous solution, and the results are shown in Table 2. Figure 4 The results show that, when acetonitrile-0.1% phosphoric acid aqueous solution is used as the mobile phase, the separation effect and peak shape of each chromatographic peak of the fingerprint spectrum are relatively good compared with other mobile phases, and therefore acetonitrile-0.1% phosphoric acid aqueous solution with a lower acid concentration is selected as the mobile phase.

[0041] (3) Investigation of extraction method Take Tongfengqing Granules, 2 g in total, accurately weighed, and placed in a conical flask with a stopper. Add 60% methanol aqueous solution 50 ml, and treat by ultrasonic wave (power 300 W, frequency 40 kHz) and heating reflux (80°C) for 30 minutes, respectively. Take out and cool, re-weigh, make up the weight loss with 60% methanol aqueous solution, shake well, filter, and take the filtrate, to obtain the test sample. Determine the fingerprint spectrum according to the determined chromatographic conditions, and the results are shown in Table 2. The results show that the extraction effect of the 6 characteristic peaks is similar in the fingerprint spectrum of the test sample treated by ultrasonic wave and heating reflux, and considering the convenience of operation, the extraction method of Tongfengqing Granules is selected as ultrasonic extraction.

[0042] Table 2 Investigation results of characteristic peak area of different extraction methods

[0043] (4) Investigation of extraction solvent Take Tongfengqing Granules, 2 g in total, accurately weighed, and placed in a conical flask with a stopper. Add water, 30% methanol aqueous solution, 60% methanol aqueous solution, and methanol 50 ml, respectively, and treat by ultrasonic wave (power 300 W, frequency 40 kHz) for 30 minutes. Take out and cool, re-weigh, make up the weight loss with each extraction solvent, shake well, filter, and take the filtrate, to obtain the test sample. Determine the fingerprint spectrum according to the determined chromatographic conditions, and the results are shown in Table 3. The results show that the overall peak shape of the sample treated by 60% methanol aqueous solution is better, and the characteristic peak area is slightly larger than that of the sample treated by the remaining solvents. Therefore, the extraction solvent is selected as 60% methanol aqueous solution.

[0044] Table 3 Investigation results of characteristic peak area of different extraction solvents

[0045] (5) Extraction time Take the Tongfeng Qingxiao granules and grind them finely, take 2g, accurately weigh and place in a conical flask with a plug, accurately add 60% methanol aqueous solution 50ml, ultrasonic treatment (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, take out and cool, re-weigh, make up the weight loss with 60% methanol aqueous solution, shake well, filter, take the filtrate, and obtain. Determine the fingerprint by the determined chromatographic conditions, and the results are shown in Table 4. The results show that the peak areas of the characteristic peaks in the test sample are close, and the ultrasonic extraction time is 15 minutes. The ultrasonic extraction time is 15 minutes.

[0046] Table 4 Investigation results of characteristic peak area of different extraction times

[0047] 2, The fingerprint detection and analysis method of the present application is established After investigation and research from many aspects, the analysis method of Tongfeng Qingxiao granules is determined as follows: Chromatographic conditions: Diamonsil C18 chromatographic column (4.6x250mm, 5um), flow rate 1.0mL / min, column temperature 30℃, wavelength 280nm, gradient elution with acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) as the mobile phase.

[0048] Preparation of test sample solution: take the Tongfeng Qingxiao granules and grind them finely, take 2g, accurately weigh and place in a conical flask with a plug, accurately add 60% methanol aqueous solution 50ml, tightly plug, weigh, ultrasonic treatment for 15 minutes (300W, 40kHz), take out and cool, re-weigh, make up the weight loss with 60% methanol aqueous solution, shake well, filter, take the filtrate, and obtain.

[0049] Determination method: accurately take 20ul of the test sample solution, inject into the liquid chromatograph, determine, record the chromatogram, and obtain.

[0050] Example 4 Verification of the Tongfeng Qingxiao granule detection method in the construction of the fingerprint 1. Reproducibility test Prepare 6 test sample solutions according to the determined analysis method, and detect them in turn to investigate the reproducibility. Take peak 8 (magnolia alkaloid) as the reference peak, calculate the relative retention time and relative peak area of other characteristic peaks. The results are shown in Table 5 and Table 6. The relative retention time RSD of the 6 characteristic peaks is not more than 1.0%, and the relative peak area RSD is not more than 5.0%, indicating that the method has good reproducibility.

[0051] Table 5 Fingerprint repeatability relative retention time results

[0052] Table 6 Fingerprint repeatability relative peak area results

[0053] 2. Stability test Prepare 1 sample solution according to the established analysis method, and determine the fingerprint at 0 h, 8 h, 16 h, 24 h, 32 h, and 48 h, respectively, to investigate the stability. Take peak No. 8 (Magnoflorine) as the reference peak to calculate the relative retention time and relative peak area of other characteristic peaks. The results are shown in Tables 7 and 8. The RSD of the relative retention time of the 6 characteristic peaks is not more than 1.0%, and the RSD of the relative peak area is not more than 5.0%, indicating that the solution is stable within 50 hours.

[0054] Table 7 Fingerprint stability relative retention time results

[0055] Table 8 Fingerprint stability relative peak area results

[0056] 3. Precision test Prepare 1 sample solution according to the established analysis method, and continuously sample 6 times to investigate the precision. Take peak No. 8 (Magnoflorine) as the reference peak to calculate the relative retention time and relative peak area of other characteristic peaks. The results are shown in Tables 9 and 10. The RSD of the relative retention time of the 6 characteristic peaks is not more than 1.0%, and the RSD of the relative peak area is not more than 5.0%, indicating that the instrument precision is good.

[0057] Table 9 Fingerprint precision relative retention time results

[0058] Table 10 Fingerprint precision relative peak area results

[0059] 4. Similarity evaluation The results are shown in Tables 11-13 and Figure 5The RSDs of the relative retention times of the common peaks in the chromatograms of the fifteen batches of the test samples of the Tongfengqing Xiaoke granules were all less than 2%, the RSDs of the relative peak areas were all less than 5%, and the similarity of the chromatograms was all greater than 0.98, indicating that the differences between the batches were small, the preparation process was reliable and stable, and the method was accurate.

[0060] Table 11 Relative retention time results of the characteristic peaks of the granule fingerprint chromatograms of the fifteen batches

[0061] Table 12 Relative peak area results of the characteristic peaks of the granule fingerprint chromatograms of the fifteen batches

[0062] Table 13 Similarity comparison results of the granule fingerprint chromatograms of the fifteen batches

[0063] Finally, it should be noted that the above description is only for the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing the characteristic chromatogram of Tongfengxiao preparation, characterized in that: The construction method comprises the following steps: (1) taking a control sample, adding a dissolving solvent to prepare a control sample solution; (2) using an extraction solvent to extract the gout clearing and eliminating preparation to prepare a test sample solution; (3) respectively detecting the test sample solution and the control sample solution by high performance liquid chromatography to obtain a fingerprint spectrum of the gout clearing and eliminating preparation and a control spectrum, identifying common peaks of the fingerprint spectrum of the gout clearing and eliminating preparation according to the control spectrum to construct a characteristic spectrum of the gout clearing and eliminating preparation; The conditions of high performance liquid chromatography detection include: a detection wavelength of 280 nm, using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution or a formic acid aqueous solution as a mobile phase B for gradient elution, and the gradient elution conditions are as follows: 0-5 min, the volume percentage of the mobile phase A and the mobile phase B changes by 5%-5% and 95%-95% respectively; 5-10 min, the volume percentage of the mobile phase A and the mobile phase B changes by 5%-7% and 95%-93% respectively; 10-25 min, the volume percentage of the mobile phase A and the mobile phase B changes by 7%-9% and 93%-91% respectively; 25-35 min, the volume percentage of the mobile phase A and the mobile phase B changes by 9%-12% and 91%-88% respectively; 35-50 min, the volume percentage of the mobile phase A and the mobile phase B changes by 12%-15% and 88%-85% respectively; 50-55 min, the volume percentage of the mobile phase A and the mobile phase B changes by 15%-18% and 85%-82% respectively; 55-70 min, the volume percentage of the mobile phase A and the mobile phase B changes by 18%-20% and 82%-80% respectively; 70-80 min, the volume percentage of the mobile phase A and the mobile phase B changes by 20%-25% and 80%-75% respectively; 80-85 min, the volume percentage of the mobile phase A and the mobile phase B changes by 25%-30% and 75%-70% respectively; 85-90 min, the volume percentage of the mobile phase A and the mobile phase B changes by 30%-30% and 70%-70% respectively.

2. The method for constructing the characteristic map of the gout Qingxiao preparation according to claim 1, characterized in that: The gout clearing and eliminating preparation is gout clearing and eliminating granules or gout clearing and eliminating decoction.

3. The method for constructing the characteristic map of Tongfengqingxia preparation according to claim 1, characterized in that: In step (1), the control sample comprises any one or several of sinomenine, caffeic acid, magnoflorine, astilbin, chicoric acid and berberine hydrochloride, and the concentration of the control sample solution is 0.1-0.5 mg / ml.

4. The method for constructing the characteristic map of Tongfengqingxia preparation according to claim 1, characterized in that: In step (2), the extraction method is ultrasonic extraction or reflux extraction, the extraction solvent is water, methanol or a methanol solution, the volume concentration of the methanol solution is 30-60%, and the extraction time is 15-60 min.

5. The method for constructing the characteristic map of Tongfengqingxia preparation according to claim 1, characterized in that: In step (3), the conditions of high performance liquid chromatography detection further include: a chromatographic column with octadecylsilane bonded silica gel as a filler, a column temperature of 30-38 ℃, a flow rate of 0.8-1.2 ml / min, and a sample injection amount of 10-30 μL; The volume concentration of the phosphoric acid aqueous solution or the formic acid aqueous solution is 0.1-0.5%.

6. The method for constructing the characteristic map of Tongfengqingxia preparation according to claim 1, characterized in that: The construction method further comprises: taking single medicinal material of the Tongfeng Qingxiao preparation according to a prescription to perform water extraction and dry distillation, then using an extraction solvent to perform extraction, preparing a test sample solution of the single medicine, performing high performance liquid chromatography detection, and determining peak attribution of the fingerprint of the Tongfeng Qingxiao preparation.

7. The method for constructing the characteristic map of the Tongfengqingxia preparation according to claim 4, characterized in that: The extraction mode is ultrasonic extraction, the ultrasonic power is 200-400 W, the ultrasonic frequency is 30-50 kHz, the extraction solvent is a methanol aqueous solution with a volume concentration of 60%, and the extraction time is 15 min.

8. The method for constructing the characteristic map of the gout Qingxiao preparation according to claim 5, characterized in that: In step (3), the conditions of the high performance liquid chromatography detection further comprise: a column temperature of 35 DEG C, a flow rate of 1 ml / min, and a sample injection amount of 20 mu L; and the mobile phase B is a phosphoric acid aqueous solution with a volume concentration of 0.1%.

9. The method for constructing the characteristic map of the Tongfengqing preparation according to any one of claims 1-8, characterized in that: The fingerprint of the Tongfeng Qingxiao preparation comprises 22 common peaks, and the identified characteristic peaks comprise: peak No. 6 is sinomine, peak No. 7 is caffeic acid, peak No. 8 is magnoflorine, peak No. 15 is astilbin, peak No. 16 is chicoric acid, and peak No. 22 is berberine hydrochloride.

10. An application of a construction method of the characteristic spectrum of the Tongfeng Qingxiao preparation according to claim 1 in quality control of the Tongfeng Qingxiao preparation.

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