A method for establishing and applying high-performance liquid chromatography fingerprints of traditional Chinese medicine compound preparations.

Abstract

This invention proposes a method for establishing a high-performance liquid chromatography (HPLC) fingerprint spectrum of a traditional Chinese medicine compound preparation and its application. The method includes: (1) taking an appropriate amount of the traditional Chinese medicine compound preparation, adding a solvent, and sonicating it to obtain a test solution; (2) performing HPLC detection on the test solution obtained in step (1). The detection conditions are: using a C18 column; using acetonitrile as mobile phase A and 0.15% phosphoric acid aqueous solution as mobile phase B for gradient elution; flow rate 0.8-1.2 mL / min, column temperature 28℃-35℃, injection volume 8-12 μL, and detection wavelength 290-300 nm. The HPLC fingerprint spectrum determined by the method described in this application can rapidly and effectively identify six components: chlorogenic acid, cryptochlorogenic acid, polygalactoside, strychnoside, emodin-8-O-β-D-glucoside, and emodin, and has the advantages of stability and high precision.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology. Specifically, it relates to a method for establishing and applying high-performance liquid chromatography (HPLC) fingerprint spectra of traditional Chinese medicine compound preparations. More specifically, it relates to a method for determining and applying HPLC fingerprint spectra of Niaoqingshu granules. Background Technology

[0002] Urinary Clearing and Soothing Granules are a compound preparation composed of six herbs: Akebia trifoliata, Chrysanthemum indicum, Polygonum cuspidatum, Eleutherococcus senticosus, Plantago asiatica, and Paris polyphylla. It has the effects of clearing heat and dampness, promoting urination and relieving strangury. It is used for strangury caused by damp-heat accumulation, characterized by difficulty urinating, painful urination, and chronic prostatitis with the above symptoms.

[0003] The fingerprint spectrum of traditional Chinese medicine is of great significance for effectively controlling the quality of Chinese medicinal materials or prepared Chinese medicines, as it reflects the chemical composition of Chinese medicine to a certain extent.

[0004] The current standard for Niaoqingshu granules is included in the National Drug Standard of the State Food and Drug Administration (WS-10988(ZD-0988)-2002-2011Z). This standard includes quality control indicators such as thin-layer chromatography identification of Polygonum cuspidatum, Chrysanthemum indicum, and Plantago asiatica, and determination of emodin content in Polygonum cuspidatum. Zeng Yongchang et al. studied the determination of polygalactosidin content in Niaoqingshu granules using RP-HPLC. However, these methods are all for the identification or content determination of individual components and are insufficient to comprehensively characterize the quality of Niaoqingshu granules. Therefore, establishing a standard fingerprint spectrum for quality control of Niaoqingshu granules is of great significance. Summary of the Invention

[0005] This invention provides a method for establishing a fingerprint spectrum of urinary tract clearing granules, which is used to control and evaluate the quality of urinary tract clearing granules.

[0006] This invention provides a method for establishing a fingerprint spectrum of urinary ...

[0007] In some embodiments, the solvent of the present invention is a 70-80% by volume methanol solution. In other embodiments, the solvent of the present invention is a 75% by volume methanol solution.

[0008] In some embodiments, the mass-to-volume ratio of the urinary tract cleansing granules to the solvent is 2g:(20-30)mL. In other embodiments, the mass-to-volume ratio of the urinary tract cleansing granules to the solvent is 2g:25mL.

[0009] In some embodiments, the dissolution is performed by ultrasonic treatment for 10-60 minutes. In other embodiments, the ultrasonic treatment time is 10, 30, or 60 minutes. In still other embodiments, the ultrasonic treatment conditions are 500W power, 40kHz frequency, and ultrasonic time of 10 minutes.

[0010] In some embodiments, the ultrasonic treatment products are filtered. In other embodiments, the ultrasonic treatment is performed by filtering the ultrasonic treatment products using a filter membrane.

[0011] In some embodiments, the C18 chromatographic column of the present invention has a size of 250 mm × 4.6 mm and a diameter of 5 μm. In other embodiments, the C18 chromatographic column of the present invention is: 1) Waters Xbridge C18, size 250 mm × 4.6 mm and a diameter of 5 μm; 2) Agilent Eclipse Plus C18, size 250 mm × 4.6 mm and a diameter of 5 μm; 3) Agilent ZORBAXSB-CI8, size 250 mm × 4.6 mm and a diameter of 5 μm; or 4) ACE Excel 5Super C18, size 250 mm × 4.6 mm and a diameter of 5 μm.

[0012] In some embodiments, mobile phase B is a 0.10% or 0.15% aqueous solution of phosphoric acid.

[0013] In some implementations, the gradient described in this invention is:

[0014] Time (min) Phase A (%) Phase B (%) 0～20 5→10 95→90 20～28 10→15 90→85 28～55 15→17 85→83 55～80 17→40 83→60 80～85 40→90 60→10 85～95 90 10

[0015] In some embodiments, the flow rate described in this invention is 1.0 mL / min.

[0016] In some embodiments, the column temperature described in this invention is 30°C.

[0017] In some embodiments, the injection volume of the present invention is 10 μL.

[0018] In some embodiments, the detection wavelength described in this invention is 295 nm.

[0019] The fingerprint chromatogram of this invention identifies six chromatographic peaks: chlorogenic acid, cryptochlorogenic acid, polygalactoside, scutellarin, emodin-8-O-β-D-glucoside, and emodin. Using peak 3 (polygalactoside) as a reference peak, the relative retention times of chlorogenic acid, cryptochlorogenic acid, polygalactoside, scutellarin, emodin-8-O-β-D-glucoside, and emodin are, respectively: 0.53±10%, 0.61±10%, 1, 1.92±10%, 1.99±10%, and 2.39±10%.

[0020] In some implementation schemes, the relative retention times of chlorogenic acid, cryptochlorogenic acid, polygalactoside, scutellarin, emodin-8-O-β-D-glucoside and emodin are 0.53, 0.61, 1.00, 1.92, 1.99 and 2.39, respectively.

[0021] The fingerprint spectrum detection method for Urinary Clearing and Relief Granules established in this invention was used to detect the sample. The standard for quality control of Urinary Clearing and Relief Granules is that the similarity between the sample to be tested and the fingerprint spectrum of Urinary Clearing and Relief Granules is not less than 0.90.

[0022] This invention presents the first high-performance liquid chromatography (HPLC) fingerprinting quality control method for Urinary Relief Granules. Based on the structural characteristics and physicochemical properties of the active ingredients in Urinary Relief Granules, the analytical conditions, including sample processing methods, mobile phase, detection wavelength, elution program, column temperature, and flow rate, were screened and optimized, and the methodology was validated systematically. The resulting fingerprint chromatogram of Urinary Relief Granules exhibits high resolution, good peak shape, and excellent baseline separation for all characteristic chromatographic peaks. It demonstrates good stability and numerous characteristic peaks, enabling a comprehensive and accurate evaluation of the quality of Urinary Relief Granules and is suitable for controlling the quality of this product.

[0023] The high-performance liquid chromatography fingerprint spectrum of Urea Clearing Granules established in this invention overcomes the shortcomings of existing technologies that rely on single detection indicators and cannot reflect intrinsic quality. It identifies common peaks in the fingerprint spectra of multiple Urea Clearing Granules and identifies six chemical components from these common peaks: chlorogenic acid, cryptochlorogenic acid, polygalactoside, monaxanthin, emodin-8-O-β-D-glucoside, and emodin. These components are attributed to the four medicinal herbs in the prescription, enabling a comprehensive evaluation of the quality of Urea Clearing Granules and effectively ensuring the quality of the finished product. Attached Figure Description

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0025] Figure 1 It is the comparative fingerprint spectrum according to the embodiments of the present invention;

[0026] Figure 2This is a superimposed image of chromatograms obtained from 22 batches of urine-clearing and soothing granules test samples according to an embodiment of the present invention, using the fingerprint chromatogram establishment method of urine-clearing and soothing granules designed and screened in this application.

[0027] Figure 3 This is a result diagram of the common peaks of fingerprint spectra obtained by using the fingerprint spectra establishment method of 22 batches of urine-clearing and soothing granules according to the present invention and the fingerprint spectra establishment method of urine-clearing and soothing granules designed and screened in this application.

[0028] Figure 4A This is a result diagram of the detection of common peaks belonging to wild chrysanthemum using the fingerprint spectrum establishment method of urinary cleansing granules designed and screened according to an embodiment of the present invention.

[0029] Figure 4B This is a result diagram of detecting common peaks belonging to Polygonum cuspidatum using the fingerprint spectrum establishment method of the urine cleansing and soothing granules designed and screened according to an embodiment of the present invention.

[0030] Figure 4C This is a diagram showing the results of detecting common peaks belonging to Plantago asiatica using the fingerprint spectrum establishment method for Urinary Clearing and Soothing Granules designed and screened according to an embodiment of the present invention.

[0031] Figure 4D This is a result diagram of the detection of common peaks belonging to Elephantopus scabra using the fingerprint spectrum establishment method of Niaoqingshu granules designed and screened according to an embodiment of the present invention.

[0032] Figure 4E This is a result diagram of the detection of common peaks belonging to Paris polyphylla according to the fingerprint spectrum establishment method of the urine-clearing and soothing granules designed and screened according to the present invention.

[0033] Figure 4F This is a result diagram of the detection of common peaks belonging to Aristolochia debilis using the fingerprint spectrum establishment method of Niaoqingshu granules designed and screened according to an embodiment of the present invention.

[0034] Figure 5 This is a result diagram of the identification of common peaks of chlorogenic acid, cryptochlorogenic acid, buddleja glycoside, euphorbia citrinin, emodin-8-O-β-D-glucoside, and emodin using the fingerprint spectrum establishment method of the urine cleansing granules designed and screened according to an embodiment of the present invention.

[0035] Figure 6A This is a chromatogram obtained by the method for establishing fingerprint chromatograms of urinary cleansing granules using methanol-water as the mobile phase according to an embodiment of the present invention.

[0036] Figure 6BThe chromatogram is obtained by the fingerprinting method of urinary cleansing granules with acetonitrile-water as the mobile phase according to an embodiment of the present invention.

[0037] Figure 6C The chromatogram is obtained by the fingerprinting method of urinary cleansing granules using acetonitrile-phosphoric acid aqueous solution as the mobile phase according to an embodiment of the present invention.

[0038] Figure 6D This is a chromatogram obtained by the fingerprinting method of Niaoqingshu granules using acetonitrile-formic acid aqueous solution as the mobile phase according to an embodiment of the present invention.

[0039] Figure 7A The chromatogram is obtained by the method for establishing the fingerprint spectrum of Niaoqingshu granules using the mobile phase gradient shown in gradient 1 according to an embodiment of the present invention.

[0040] Figure 7B The chromatogram is obtained by the method for establishing the fingerprint spectrum of Niaoqingshu granules using the mobile phase gradient shown in gradient 2 according to an embodiment of the present invention.

[0041] Figure 7C The chromatogram is obtained by the method for establishing the fingerprint spectrum of Niaoqingshu granules using the mobile phase gradient shown in gradient 3 according to an embodiment of the present invention.

[0042] Figure 7D This is a chromatogram obtained by the method for establishing the fingerprint spectrum of Niaoqingshu granules using the mobile phase gradient shown in gradient 4 according to an embodiment of the present invention.

[0043] Figure 7E This is a chromatogram obtained by the method for establishing the fingerprint spectrum of Niaoqingshu granules using the mobile phase gradient shown in gradient 5 according to an embodiment of the present invention.

[0044] Figure 8 The superimposed chromatogram is obtained by the fingerprint spectrum establishment method of Niaoqingshu granules using wavelengths of 210nm, 230nm, 254nm, 280nm, 295nm, and 330nm according to an embodiment of the present invention.

[0045] Figure 9 This is a superimposed chromatogram obtained by the fingerprint chromatogram establishment method of urine-clearing and soothing granules using chromatographic columns 1-4 according to an embodiment of the present invention;

[0046] Figure 10 The superimposed chromatogram is obtained by the fingerprint chromatogram establishment method of urinary cleansing granules with column temperatures of 25°C, 28°C, 30°C and 35°C according to an embodiment of the present invention.

[0047] Figure 11The superimposed chromatograms are obtained by the fingerprinting method for Urine Cleansing and Relief Granules using flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min according to embodiments of the present invention.

[0048] Figure 12A This is a chromatogram obtained by personnel 1 using the fingerprinting method of the Waters high performance liquid chromatograph for urine clearing and soothing granules according to an embodiment of the present invention;

[0049] Figure 12B This is a chromatogram obtained by personnel 2 using the fingerprint chromatogram establishment method of Agilent high performance liquid chromatography according to an embodiment of the present invention;

[0050] Figure 13 This is an experimental result diagram of the repeatability test of the method for establishing the fingerprint spectrum of urine-clearing granules according to an embodiment of the present invention;

[0051] Figure 14 This is an experimental result diagram of the instrument precision detection of the method for establishing the fingerprint spectrum of urine-clearing granules according to an embodiment of the present invention;

[0052] Figure 15 This is an experimental result diagram of the solution stability test of the method for establishing the fingerprint spectrum of Niaoqingshu granules according to an embodiment of the present invention. Detailed Implementation

[0053] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0054] The endpoints and any values ​​of the ranges described in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0055] In this invention, the preparation of the Niaoqingshu granules is in accordance with the standard (trial) WS-10988(ZD-0988)-2002-2011Z of the State Drug Administration of the People's Republic of China, and its prescription is shown in Table 1. The specific preparation method is as follows: Take the above six medicinal materials, add water to decoct Akebia, Polygonum cuspidatum, and Paris polyphylla for 1 hour, then add Chrysanthemum indicum, Eleutherococcus senticosus, and Plantago asiatica and continue to decoct for 30 minutes. Filter and reserve the filtrate. Add water to the dregs and decoct twice, the first time for 1 hour and the second time for 30 minutes. Filter the decoction, combine the two decoctions, and then combine them with the above filtrate. Concentrate under reduced pressure to a thick paste with a relative density of 1.32-1.38 (25℃). Add sucrose and lactose, mix well, and use an appropriate amount of ethanol to make a soft mass. Make granules, dry and granulate to obtain the final product.

[0056] Table 1: Prescription Table for Urine-Clearing and Soothing Granules

[0057] name Weight (g) Shanmutong 200 wild chrysanthemum 240 Polygonum cuspidatum 280 Elephantopus 280 Plantain 280 Paris polyphylla 120 sucrose 520 lactose 270

[0058] The instruments, reagents, and materials involved in this invention are as follows:

[0059] instrument

[0060] Electronic microbalance (Mettler-Toledo XP6); Electronic analytical balance (Mettler-Toledo XS105); Constant temperature water bath (Shanghai Yiheng Scientific Instruments Co., Ltd. HWS24); Pure water system (Shanghai Hetai Instruments Co., Ltd. Masrer-QUT); Ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd. KQ5200DE); Waters e2695 / 2698 high performance liquid chromatograph; Agilent 1260 Infinity high performance liquid chromatograph (with DAD detector); Waters Xbridge C18 (4.6mm×250mm, 5μm) column, ACE Excel 5Super C18 (4.6mm×250mm, 5μm) column, Agilent ZORBAXSB-CI8 (4.6mm×250mm, 5μm) column, Agilent Eclipse Plus C18 (4.6mm×250mm, 5μm) column.

[0061] reagents

[0062] Acetonitrile and phosphoric acid were of chromatographic grade; methanol was of analytical grade.

[0063] Reference

[0064] Rhein reference standard (batch number: 110756-201913, purity 96.0%), Polygonin reference standard (batch number: 111575-201603, purity 87.3%), Buddleja glycoside reference standard (batch number: 111528-201710, purity 96.6%), and chlorogenic acid reference standard (batch number: 110753-202018, purity 96.1%) were purchased from the National Institutes for Food and Drug Control; Rhein-8-O-β-D-glucoside reference standard (batch number: 21-08-2302, purity 97.36%) and cryptochlorogenic acid reference standard (batch number: 21-08-2304, purity 95.81%) were purchased from SINCO.

[0065] medicinal materials

[0066] Aristolochia debilis (batch number: 201201, purchased from Yunnan Taihua Fenggong Pharmaceutical Co., Ltd.), Chrysanthemum indicum (batch number: JZT20210315, purchased from Jiuzhoutong Group Anguo Chinese Medicinal Materials Co., Ltd.), Polygonum cuspidatum (batch number: 21050702, purchased from Anhui Xiehecheng Pharmaceutical Co., Ltd.), Eleutherococcus senticosus (batch number: 20210419001, purchased from Guangdong Pharmaceutical Co., Ltd.), Plantago asiatica (batch number: 21051117, purchased from Anhui Xiehecheng Pharmaceutical Co., Ltd.), and Paris polyphylla (batch number: 20210510001, purchased from Guangdong Pharmaceutical Co., Ltd.).

[0067] Test sample

[0068] Information on the Niaoqingshu granules samples is shown in Table 2.

[0069] Table 2: Sample Information Table for Urine-Clearing and Soothing Granules

[0070] serial number batch number enterprise 1 05319003 Yipinhong Biomedical Co., Ltd. 2 05319005 Yipinhong Biomedical Co., Ltd. 3 05319006 Yipinhong Biomedical Co., Ltd. 4 05320003 Yipinhong Biomedical Co., Ltd. 5 05320015 Yipinhong Biomedical Co., Ltd. 6 05320016 Yipinhong Biomedical Co., Ltd. 7 05320019 Yipinhong Biomedical Co., Ltd. 8 05321001 Yipinhong Biomedical Co., Ltd. 9 05321002 Yipinhong Biomedical Co., Ltd. 10 05321003 Yipinhong Biomedical Co., Ltd. 11 05321004 Yipinhong Biomedical Co., Ltd. 12 05321005 Yipinhong Biomedical Co., Ltd. 13 05321006 Yipinhong Biomedical Co., Ltd. 14 05321007 Yipinhong Biomedical Co., Ltd. 15 05321008 Yipinhong Biomedical Co., Ltd. 16 05321009 Yipinhong Biomedical Co., Ltd. 17 05321012 Yipinhong Biomedical Co., Ltd. 18 05321013 Yipinhong Biomedical Co., Ltd. 19 05321016 Yipinhong Biomedical Co., Ltd. 20 05321021 Yipinhong Biomedical Co., Ltd. 21 05322001 Yipinhong Biomedical Co., Ltd. 22 05322002 Yipinhong Biomedical Co., Ltd.

[0071] negative sample

[0072] Negative samples were prepared according to the standard prescription ratio and standard process method of this product. The negative samples were: * *Aristolochia debilis* (batch number: 21062901), * *Chrysanthemum indicum* (batch number: 21062901), * Polygonum cuspidatum* (batch number: 21062901), * *Elephantopus scabra* (batch number: 21062901), * *Plantago asiatica* (batch number: 21062901), and * *Paris polyphylla* (batch number: 21062901).

[0073] Example 1: Establishment of high-performance liquid chromatography fingerprint of Urinary Clearing and Relief Granules

[0074] 1.1 Preparation of the reference solution

[0075] Take appropriate amounts of strychnoside and polygalactoside reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 25 μg of strychnoside and 40 μg of polygalactoside per 1 mL.

[0076] 1.2 Preparation of the test solution

[0077] Take an appropriate amount of this product, mix well, grind finely, take 2.0g, accurately weigh, place in a stoppered conical flask, accurately add 25mL of 75% methanol, seal tightly, weigh, sonicate for 10 minutes, cool, make up the weight loss with 75% methanol, shake well, filter, and take the filtrate to obtain the product.

[0078] 1.3 Chromatographic conditions and determination

[0079] Using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), with acetonitrile as mobile phase A and 0.15% phosphoric acid aqueous solution as mobile phase B, gradient elution was performed according to the conditions in Table 3; the detection wavelength was 295 nm; the flow rate was 1.0 mL / min; and the column temperature was 30 °C. The theoretical plate number, calculated based on the polygalactosidin peak, should not be less than 3000; the injection volume was 10 μL.

[0080] Table 3: Mobile Phase Gradient Table

[0081] Time / min Mobile phase A (%) Mobile phase B (%) 0～20 5→10 95→90 20～28 10→15 90→85 28～55 15→17 85→83 55～80 17→40 83→60 80～85 40→90 60→10 85～95 90 10

[0082] 1.4 Selection of Reference Peak

[0083] In the chromatograms of different batches of samples, peaks 3 (polygonin) and 4 (scutellarin) exhibit strong specificity, clear chemical composition, moderate retention time, and good separation effect. Therefore, both peaks 3 (polygonin) and 4 (scutellarin) can be used as reference peaks. At a wavelength of 295 nm, the peak area of ​​peak 4 (scutellarin) is smaller than that of the other common peaks; the peak area of ​​peak 3 (polygonin) is larger than that of the other common peaks, and its peak area is moderate. Therefore, peak 3 (polygonin) is determined to be the reference peak (S peak).

[0084] 1.5 Generation of reference maps

[0085] The chromatograms of 22 batches of samples were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software developed by the National Pharmacopoeia Commission for processing. Common peaks were identified, and a high-performance liquid chromatography fingerprint of Niaoqingshu granules was established. A control fingerprint was generated using the median method. The control fingerprint is shown below. Figure 1 As shown.

[0086] 1.6 Setting Similarity Limits

[0087] The similarity between the fingerprint chromatograms of the 22 batches of samples and the control ranged from 0.996 to 1.000, as shown in Table 4. The average similarity of the 22 batches was 0.998. Considering the source of the medicinal materials and the impact of large-scale production, the similarity limit between the fingerprint chromatogram of this product and the control was set as follows: according to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the control should not be lower than 0.90.

[0088]

[0089] Example 2: Confirmation and Attribution of Common Peaks in Urine-Clearing and Soothing Granules

[0090] 2.1 Confirmation of common peaks

[0091] For the 22 batches of Niaoqingshu granules samples mentioned above, the test solutions were prepared according to section "1.2", and the chromatograms were determined under the chromatographic conditions described in section "1.3". The chromatograms were recorded. After statistical integration of the chromatograms of all samples (integration parameters: minimum peak area 50, peak width 0.02, slope 30%), the resulting chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software developed by the National Pharmacopoeia Commission in TXT format for processing. Using chromatogram S1 as the reference chromatogram, a time window width of 0.1 minutes was set, and a control fingerprint chromatogram was generated using the median method. Mark matching of the chromatographic peaks of the 22 batches of Niaoqingshu granules samples was performed using a multi-point correction method to obtain a superimposed chromatogram of the fingerprint chromatograms of the 22 batches of samples. Six common peaks were identified, such as... Figure 2 , 3 As shown.

[0092] 2.2 Assignment of common peaks

[0093] 2.2.1 Classification of medicinal materials

[0094] (1) Preparation of herbal extract

[0095] Take slices of Akebia quinata, Chrysanthemum indicum, Polygonum cuspidatum, Eleutherococcus senticosus, Plantago asiatica, and Paris polyphylla, respectively, and pulverize them. Take about 15g of each powder, add 150mL of water, boil for 90 minutes, and filter. Add 120mL of water, boil for 60 minutes, and filter again. Combine the two filtrates to obtain the extract of Akebia quinata, Chrysanthemum indicum, Polygonum cuspidatum, Eleutherococcus senticosus, Plantago asiatica, and Paris polyphylla. Filter the extract to obtain the final product.

[0096] (2) Preparation of the test solution of medicinal materials

[0097] Take 30 mL of the extracts of Akebia trifoliata, Chrysanthemum indicum, Polygonum cuspidatum, Eleutherococcus senticosus, Plantago asiatica, and Paris polyphylla, respectively, evaporate to about 2 mL, add 25 mL of 75% methanol to the residue, weigh it, extract by sonication for 30 minutes, cool, weigh it again, make up the lost weight with 75% methanol, shake well, filter, and take the filtrate as the test solutions of Akebia trifoliata, Chrysanthemum indicum, Polygonum cuspidatum, Eleutherococcus senticosus, Plantago asiatica, and Paris polyphylla, respectively.

[0098] (3) Preparation of negative sample solution

[0099] Take appropriate amounts of negative samples of Aristolochia debilis, Chrysanthemum indicum, Plantago asiatica, Eleutherococcus senticosus, Polygonum cuspidatum, and Paris polyphylla from the urine-clearing and soothing granules, and prepare them according to the method in section "1.2" to obtain the solutions of each negative sample.

[0100] Take the extracts, test solutions, negative sample solutions, and test solutions from section "1.2" of the above-mentioned medicinal materials, and perform chromatographic analysis according to the chromatographic conditions in section "1.3," recording the chromatogram at 295 nm. Assign the common peaks in the fingerprint chromatogram to the medicinal materials based on their retention times.

[0101] Experimental results are as follows Figures 4A-4F As shown, the results indicate that among the six common peaks, peak 1 belongs to Elephantopus scabra and Chrysanthemum indicum; peak 2 belongs to Elephantopus scabra, Chrysanthemum indicum, and Plantago asiatica; peaks 3, 5, and 6 belong to Polygonum cuspidatum; peak 4 belongs to Chrysanthemum indicum; and no common peak belonging to Aristolochia debilis and Paris polyphylla could be identified.

[0102] 2.2.2 Classification of Chemical Components

[0103] Accurately weigh appropriate amounts of each reference standard and prepare solutions containing 80 μg of emodin and 90 μg of strychnos nucifera glycoside per mL using methanol; prepare solutions containing 40 μg of polygalactoside per mL using 50% methanol; prepare solutions containing 40 μg of chlorogenic acid and 42 μg of cryptochlorogenic acid per mL using 60% methanol; and prepare solutions containing 80 μg of emodin-8-O-β-D-glucoside per mL using 75% methanol. Inject the above reference standard solutions and the test solutions from section "1.2" sequentially according to the chromatographic conditions in section "1.3" and record the chromatograms at 295 nm. Assign chemical components to the common peaks in the fingerprint chromatograms based on the retention time information of the chromatographic peaks.

[0104] Test results as follows Figure 5 As shown, among the six common peaks, peak 1 is chlorogenic acid, peak 2 is cryptochlorogenic acid, peak 3 is polygalactoside, peak 4 is scutellarin, peak 5 is emodin-8-O-β-D-glucoside, and peak 6 is emodin.

[0105] In summary, the six common peaks were classified as medicinal materials and their chemical components were identified. The specific results are shown in Table 5.

[0106] Table 5: List of medicinal materials and chemical components attribution for common peaks in fingerprint chromatograms

[0107]

[0108]

[0109] Example 3: Study on the high-performance liquid chromatography detection method for Urine Clearing and Soothing Granules

[0110] Based on Example 1, the following experiment was conducted:

[0111] 3.1 Examination of Detection Methods

[0112] 3.1.1 Investigation of the mobile phase system

[0113] Using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), experiments were conducted using different mobile phase systems (methanol-water, acetonitrile-water, acetonitrile-phosphoric acid solution, acetonitrile-formic acid solution). The samples were injected and analyzed according to the gradient elution program in Table 6, using a DAD detector at a wavelength of 254 nm, a flow rate of 1.0 mL / min, and a column temperature of 30 °C. The test solution from section "1.2" was used for analysis, with an injection volume of 10 μL. Chromatograms were recorded.

[0114] Table 6:

[0115] Time / min A% (Methanol / Acetonitrile) B% (water / 0.1% phosphoric acid solution / 0.1% formic acid solution) 0～80 5→30 95→70 80～120 30→90 70→10 120～125 90→5 10→95 125～140 5 95

[0116] Experimental results are as follows Figures 6A-6D As shown, the chromatograms obtained using acetonitrile-phosphoric acid aqueous solution as the mobile phase contain more chromatographic peak information, and the peak shapes and separation effects of each chromatographic peak are better. Therefore, the acetonitrile-phosphoric acid aqueous solution system was selected as the mobile phase system.

[0117] 3.1.2 Investigation of the mobile phase gradient

[0118] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size). Gradients 1-4 used acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B; gradient 5 used acetonitrile as mobile phase A and 0.15% phosphoric acid aqueous solution as mobile phase B. A DAD detector was used at a wavelength of 254 nm; the flow rate was 1.0 mL / min; and the column temperature was 30 °C. The test solution from section "1.2" was used for detection, with an injection volume of 10 μL, and eluted according to the different gradients shown in Table 7.

[0119] Table 7:

[0120]

[0121]

[0122] Experimental results are as follows Figures 7A-7E As shown, when using gradient 5 for detection, the corresponding chromatographic peaks can be eluted within the gradient program time with good separation and a relatively stable baseline. Therefore, gradient 5 was selected as the elution gradient.

[0123] 3.1.3 Examination of Detection Wavelength

[0124] Take the test solution from section “1.2”, and scan the sample in the wavelength range of 200-400nm using the chromatographic conditions from section “1.3”, with a column temperature of 30℃, a flow rate of 1.0mL / min and a DAD diode array detector.

[0125] The experimental results were obtained by selecting wavelengths of 210nm, 230nm, 254nm, 280nm, 295nm, and 330nm. Figure 8 As shown, the chromatographic peak at 295nm is rich in information, has good peak resolution, uniform peak area distribution, and stable baseline. Therefore, 295nm was chosen as the detection wavelength.

[0126] 3.1.4 Examination of Extraction Conditions

[0127] The preparation method of the test solution is determined as follows: Take an appropriate amount of this product, mix it well, grind it into a fine powder, take 2.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 75% methanol, stopper it tightly, weigh it, sonicate it (power 500w, frequency 40KHz) for 10 minutes, cool it, make up the weight loss with 75% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0128] 3.1.4.1 Examination of Extraction Methods

[0129] Take an appropriate amount of this product, mix well, grind finely, and accurately weigh 2.0g into 6 portions. Place each portion into a stoppered conical flask, accurately add 25mL of 75% methanol to each portion, and weigh them. After one portion has settled, take the supernatant and filter it directly. Treat the remaining 5 portions with sonication (500W power, 40kHz frequency) or heat under reflux for different times, cool, and weigh them again. Make up the weight loss with 75% methanol, shake well, filter, and take the filtrate. The test solution prepared by different extraction methods and extraction times shall be determined according to the chromatographic conditions under section "2.1".

[0130] The experimental results are shown in Table 8. It was found that the peak areas of the samples after reflux and ultrasonic treatment for 30 minutes were similar, while those after static extraction were less. The peak areas of the samples after ultrasonic treatment (power 500w, frequency 40KHz) for different times were similar. Ultrasonic treatment for 10 minutes can completely extract the samples and is simple to operate. Therefore, ultrasonic treatment for 10 minutes was selected as the extraction method.

[0131] Table 8: Evaluation of Extraction Methods (based on peak area per unit mass)

[0132]

[0133]

[0134] 3.1.4.2 Investigation of Extraction Solvents

[0135] Accurately weigh approximately 2g of the test sample powder, dividing it into 6 portions. Add 25mL of water, methanol solution of different concentrations, or 75% ethanol to each portion precisely, weigh them, and sonicate them (500W, 40kHz) for 30 minutes. Cool them, weigh them again, and replenish the lost weight with the corresponding extraction solution. Shake well, filter, and collect the filtrate. Determine the weight of each test sample solution prepared with different extraction solvents according to the chromatographic conditions described in section "2.1".

[0136] The experimental results are shown in Table 9. It was found that the peak area per unit mass was large when using 75% methanol for extraction. Therefore, 75% methanol was selected as the extraction solvent.

[0137] Table 9: Evaluation of extraction solvents (based on peak area per unit mass)

[0138] Peak water 25% methanol 50% methanol 75% methanol methanol 75% ethanol 1 1767 1787 1755 1699 712 1819 2 1591 1620 1600 1558 791 943 3 9514 13058 19723 20701 14802 21224 4 1514 1742 2301 2416 1976 2427 5 2732 2823 5118 5391 4446 5346 6 533 133 2483 2826 2435 2946

[0139] 3.1.4.3 Investigation into the amount of extraction solvent used

[0140] Accurately weigh approximately 2g of the test sample powder into three portions. Add 10mL, 25mL, and 50mL of 75% methanol to each portion, respectively, and accurately weigh them. Sonicate the mixture (500W power, 40kHz frequency) for 30 minutes. Make up the weight loss with 75% methanol, shake well, filter, and collect the filtrate. Then, perform high-performance liquid chromatography (HPLC) detection according to the chromatographic conditions shown in section "1.3".

[0141] The experimental results are shown in Table 10. It was found that when the extraction solvent volume was 10 mL, the solvent volume was too small, resulting in a large amount of insoluble matter in the sample, and peak 3 could not be effectively extracted. When the solvent volume was 25 mL and 50 mL, the sample extraction was relatively complete, but when the extraction solvent volume was 50 mL, the peak areas of peaks 1 and 2 were small; when the extraction solvent volume was 25 mL, the peak areas of each chromatographic peak were moderate. Therefore, selecting an extraction solvent volume of 25 mL was economical, environmentally friendly, and effective.

[0142] Table 10: Investigation of extraction solvent usage (based on peak area per unit mass of total volume)

[0143] Peak 10mL 25mL 50mL 1 46057 42469 44332 2 38304 38957 40486 3 23980 517529 547872 4 62878 60400 62143 5 129341 134778 139981 6 67990 70641 73655

[0144] 3.1.5 Examination of the chromatographic column

[0145] This experiment tested four different models of chromatographic columns from three brands: Column 1: Waters Xbridge C18 (4.6mm×250mm, 5μm), Column 2: Agilent Eclipse Plus C18 (4.6mm×250mm, 5μm), Column 3: Agilent ZORBAX SB-CI8 (4.6mm×250mm, 5μm), and Column 4: ACE Excel 5Super C18 (4.6mm×250mm, 5μm).

[0146] The experimental results are shown in Table 11 and Figure 9 As shown, the results indicate that all chromatographic columns exhibited good column efficiency, symmetrical peak shapes, and good separation. The chromatographic fingerprints were processed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" software from the National Pharmacopoeia Commission, with the reference fingerprint chromatogram as the reference reference. The similarity between the chromatograms of all four columns and the reference chromatogram was greater than 0.99.

[0147] Table 11: Similarity between fingerprint chromatograms of different columns and reference chromatograms

[0148]

[0149]

[0150] 3.1.6 Investigation of column temperature

[0151] In this experiment, the test solution under section 1.2 was used to detect the chromatographic peak separation effect at different column temperatures (25℃, 28℃, 30℃, 35℃). The other chromatographic conditions were the same as those under section 1.3.

[0152] Experimental results are as follows Figure 10 As shown, the separation of chromatographic peaks was poor when the column temperature was 25℃, while the separation of each chromatographic peak was good under other column temperature conditions.

[0153] 3.1.7 Examination of Flow Velocity

[0154] The effects of different flow rates (0.8 mL / min, 1.0 mL / min, and 1.2 mL / min) on the chromatographic peak separation were investigated. The test solution under section “1.2” was used, and the chromatographic conditions were the same as those under “1.3” except for the flow rate.

[0155] Experimental results are as follows Figure 11 As shown, the separation effect of each chromatographic peak was good under various flow rate conditions, and the separation effect of the chromatographic peak was better when the flow rate was 1.0 mL / min.

[0156] 3.2 Intermediate Precision Test

[0157] Take the test solution shown in section "1.2", and use the same chromatographic conditions as described in "1.3". Investigate the effect of two analysts using two different brands of high performance liquid chromatographs: Instrument A: Agilent 1260 high performance liquid chromatograph, Instrument B: Waters high performance liquid chromatograph on the detection results of the fingerprint chromatogram of the test sample.

[0158] Experimental results are as follows Figure 12A and 12B As shown, the results indicate that the detection results of the two instruments are not significantly different, and the fingerprint peaks in the spectrum are symmetrical, indicating good separation effect.

[0159] 3.3 Repeatability Test

[0160] Take approximately 2.0 g of the same batch of sample (batch number 05321007), accurately weigh it, and divide it into 6 portions. Prepare test solutions according to the method in section "1.2", and determine the chromatograms according to the chromatographic conditions in section "1.3". Select 6 common chromatographic peaks, and use peak 3 (polygonin) as the reference peak S, calculate the relative retention times and relative peak areas of common peaks 1-6.

[0161] The specific experimental results are shown in Tables 12-14 and 14-15. Figure 13 As shown, the results showed that the relative retention time (RSD) of each common chromatographic peak was ≤0.3%, and the relative peak area (RSD) of the common peak was ≤2.1%. The obtained chromatograms were imported into the software of the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) developed by the National Pharmacopoeia Commission in AIA format for processing. The similarity between the chromatograms of the 6 samples and the control chromatogram was 1.000, indicating that the method has good repeatability.

[0162] Table 12 Relative retention times of common peaks in repeatability tests

[0163]

[0164] Table 13 Relative peak areas of common peaks in repeatability tests

[0165]

[0166]

[0167] Table 14. Results of Repeatability Test Similarity Evaluation

[0168] Repeatability similarity Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 Comparison of fingerprint patterns Repeatability 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Repeatability 2 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Repeatability 3 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Repeatability 4 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Repeatability 5 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Repeatability 6 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Comparison of fingerprint patterns 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0169] 3.4 Precision Test

[0170] Take one portion (approximately 2.0 g) of this product (batch number 05321007), accurately weigh it, prepare the test solution according to the method under section "1.2", and determine the chromatographic conditions under section "1.3". Inject the sample six times consecutively and record the chromatogram. Select six common chromatographic peaks, using peak 3 (polygonin) as the reference peak S, and calculate the relative retention time and relative peak area of ​​common peaks 1-6.

[0171] The experimental results are shown in Tables 15-17 and 16. Figure 14 As shown in the figure. The results showed that the relative retention time (RSD) of each common chromatographic peak was ≤0.9%, and the relative peak area (RSD) of the common peak was ≤1.7%. The obtained chromatograms were imported into the software of the Chinese Pharmacopoeia Commission's Chromatographic Fingerprint Similarity Evaluation System (2012 version) in AIA format for processing. The similarity between the chromatograms of the 6 injections and the control chromatograms was 1.000, indicating good precision.

[0172] Table 15: Relative retention times of common peaks in precision tests

[0173]

[0174] Table 16: Relative Peak Areas of Common Peaks in Precision Tests

[0175]

[0176] Table 17: Similarity Evaluation Results of Precision Tests

[0177]

[0178]

[0179] 3.5 Stability Test

[0180] Take this product (batch number 05321007), prepare the test solution according to the method in section "1.2", and inject it into the liquid chromatogram for analysis at 0, 6, 10, 24, 40, and 48 hours according to the chromatographic conditions in section "1.3", and record the chromatograms. Select 6 common peaks, and use peak 3 (polygonin) as the reference peak S, calculate the relative retention time and relative peak area of ​​common peaks 1-6.

[0181] The experimental results are shown in Tables 18-20 and Figure 15As shown, the results showed that the relative retention time (RSD) of each common chromatographic peak was ≤0.2%, and the relative peak area (RSD) of the common peak was ≤1.2%. The obtained chromatograms were imported into the software of the Chinese Pharmacopoeia Commission's Chromatographic Fingerprint Similarity Evaluation System (2012 version) in AIA format for processing. The similarity between the chromatograms of the 6 injections and the control chromatograms was 1.000, indicating that the test solution was stable within 48 hours.

[0182] Table 18: Relative retention times of common peaks in solution stability tests

[0183]

[0184] Table 19: Relative peak areas of common peaks in solution stability tests

[0185]

[0186] Table 20: Similarity Evaluation Results of Solution Stability Tests

[0187] Stability similarity 0h 6h 10h 24h 40h 48h Comparison of fingerprint patterns 0h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 6h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 10h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 24h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 40h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 48h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Comparison of fingerprint patterns 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0188] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0189] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for establishing a high-performance liquid chromatography fingerprint of urine-clearing granules, characterized in that, Includes the following steps: (1) Take an appropriate amount of urine-clearing granules, add solvent to dissolve them, and obtain the test solution; The mass-to-volume ratio of the urine-clearing and solubilizing granules to the solvent is 2g:25mL; The solvent is a 75% (v / v) methanol solution; The dissolution was performed by ultrasonic treatment for 10 minutes; (2) The test solution obtained in step (1) is subjected to high-performance liquid chromatography (HPLC) to obtain the fingerprint spectrum of the urine-clearing granules, wherein the HPLC detection conditions are as follows: A C18 column was used, with dimensions of 250 mm × 4.6 mm and a diameter of 5 μm. The column temperature is 30℃; The detection wavelength is 295nm; Gradient elution was performed using acetonitrile as mobile phase A and 0.15% phosphoric acid aqueous solution as mobile phase B at a flow rate of 1.0 mL / min and an injection volume of 10 μL. The gradient elution conditions are as follows: 。 2. The method according to claim 1, characterized in that, The fingerprint chromatogram of the urine-clearing granules contains six characteristic peaks, corresponding to chlorogenic acid, cryptochlorogenic acid, polygalactoside, monaxioline, emodin-8-O-β-D-glucoside, and emodin, respectively. With the polygalactoside reference peak as the reference peak, the relative retention times of each chromatographic peak are: 0.53±10%, 0.61±10%, 1.00%, 1.92±10%, 1.99±10%, and 2.39±10%.

3. The method according to claim 2, characterized in that, The relative retention times of chlorogenic acid, cryptochlorogenic acid, polygalactoside, monaxioline, emodin-8-O-β-D-glucoside and emodin in the fingerprint spectrum of the urine-clearing granules were 0.53, 0.61, 1.00, 1.92, 1.99 and 2.39, respectively.

4. The use of the fingerprint spectrum obtained by the method according to any one of claims 1-3 in the quality control of Niaoqingshu granules.

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