Construction method and application of HPLC (High Performance Liquid Chromatography) standard fingerprint spectrum of Gaoguaping capsules

Abstract

The invention discloses a construction method and application of an HPLC (High Performance Liquid Chromatography) standard fingerprint spectrum of Gaoguaping capsules, and belongs to the technical field of quality control of traditional Chinese medicine preparations. According to the construction method of the HPLC standard fingerprint spectrum of the Gaoguaping capsule, multiple active ingredients in the capsule are accurately separated and detected through a high performance liquid chromatography technology in combination with gradient elution and specific chromatographic conditions. The constructed standard fingerprint spectrum contains 23 common peaks and 13 characteristic fingerprint peaks, and can ensure the consistency and stability of different batches of capsules. The fingerprint spectrum can be used for quickly and accurately judging the quality of capsules and improving the detection efficiency. In addition, the fingerprint also reveals the distribution of each medicinal material component in the fingerprint, and a clue is provided for medicinal material traceability and quality control. The method has the advantages of being simple, efficient and good in repeatability and stability, can be used for quality control and product quality identification of the Gaoguaping capsules, and provides effective guarantee for clinical application.

Description

Technical Field

[0001] The present invention relates to, but is not limited to, the technical field of quality control of traditional Chinese medicine preparations, and specifically to a method for constructing a HPLC standard fingerprint of Gaoguanping capsules and an application thereof. Background Art

[0002] Gaoguanping Capsule (Shaanxi Medicine Manufacturing Code Z20120008) is an in-hospital preparation product of Shanhaidan Hospital. It is composed of 19 Chinese medicines, including astragalus, salvia, white peony, Panax notoginseng, red ginseng, ganoderma, pollen, safflower, kudzu root, Achyranthes bidentata, cassia seed, bergamot, and processed Cyperus rotundus. It has the effects of nourishing qi and blood, promoting blood circulation and removing blood stasis, and unblocking meridians and suppressing yang. It is mainly used in the treatment of hypertension and hypertensive heart disease. Among them, astragalus replenishes qi and strengthens the body, and replenishes the deficiency of ancestral qi; salvia promotes blood circulation and removes blood stasis, nourishes blood and calms the mind; red ginseng replenishes qi and strengthens the root, and helps astragalus replenish qi; safflower promotes blood circulation and unblocks meridians, removes blood stasis and relieves pain; Panax notoginseng removes blood stasis and stops bleeding, promotes blood circulation and relieves pain, etc. All the medicines are used together to achieve the effects of nourishing qi and blood, promoting blood circulation and removing blood stasis, unblocking meridians and suppressing yang. The active ingredients in Gaoguanping Capsules include astragaloside I, astragaloside, tanshinone I, cryptotanshinone, tanshinone IIA, puerarin, 3'-methoxypuerarin, hydroxysafflor yellow A, saponins, etc., which are the material basis for the clinical treatment of hypertension and hypertensive heart disease.

[0003] At present, the quality standard of this variety only establishes a thin layer identification method for some of the main ingredients in Gaoguanping Capsules. Although this method can identify some ingredients, it cannot cover all active ingredients, especially those with low content but important contributions to the efficacy. Therefore, the limitations of this method lead to incomplete quality control. The existing quality evaluation methods lack systematicity and cannot evaluate the quality of Gaoguanping Capsules as a whole. The efficacy of Chinese herbal compound preparations often comes from the synergistic effect of multiple ingredients, while existing methods may only focus on the content of a single ingredient or a few ingredients, ignoring the interaction between ingredients and the overall effect.

[0004] As a quality control technology, Chinese medicine fingerprint has the remarkable characteristics of systematicity, integrity and stability. It can not only comprehensively reflect the chemical composition and characteristics of Chinese medicine compound preparations, but also provide scientific basis and strong guarantee for the quality control and clinical application of Chinese medicine compound preparations.

[0005] Gaoguanping Capsules are a traditional Chinese medicine compound preparation, and their efficacy often comes from the synergistic effect of multiple ingredients. However, existing quality evaluation methods may only focus on the content of a single ingredient or a few ingredients, ignoring the interaction between ingredients and the overall effect. Therefore, traditional Chinese medicine fingerprint technology provides a new idea and solution for the quality control of Gaoguanping Capsules. However, there are currently no literature or patent reports on the construction of the HPLC standard fingerprint of Gaoguanping Capsules. Summary of the invention

[0006] In view of the complex ingredients of Gaoguanping capsules, the existing quality evaluation methods of Gaoguanping capsules only focus on the content of a single ingredient or a few ingredients, ignoring the interaction between the ingredients and the overall effect. As a result, the existing quality evaluation methods lack systematic evaluation, insufficient stability evaluation, and cannot fully reflect the material basis of drug efficacy. The present invention aims to provide a method for establishing an HPLC fingerprint of Gaoguanping capsules, and a standard HPLC fingerprint of Gaoguanping capsules established by the method. The standard fingerprint can be used to comprehensively evaluate and control the quality of Gaoguanping capsules, thereby ensuring the stability of product quality and the safety and effectiveness of clinical medication.

[0007] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a method for constructing a HPLC standard fingerprint of Gaoguanping capsules, comprising: S1, using different batches of Gaoguanping capsules as test samples to prepare Gaoguanping capsule solutions; S2, using gallic acid, puerarin, 3'-methoxypuerarin, paeoniflorin, ferulic acid, calycosin glucoside, hesperidin, rutin, quercetin, icariin, tanshinone I, tanshinone IIA and cryptotanshinone as reference substances to prepare reference substance solutions; S3, performing high performance liquid chromatography analysis on the test Gaoguanping capsule solution and the reference solution under the same conditions, and recording the corresponding chromatograms; S4, processing the chromatograms of the test sample and the reference sample obtained in S3 using fingerprint software to generate a HPLC standard fingerprint of Gaoguanping Capsules consisting of common characteristic peaks.

[0008] In S1, the contents of Gaoguanping capsules from different batches were taken, 50%-90% methanol was added for ultrasonic treatment for 30 min-50 min, and the mixture was centrifuged at 10000 r / min for 10 min. The supernatant was taken and filtered through a 0.45 μm microporous filter membrane to obtain the test Gaoguanping capsule solution.

[0009] In S2, the reference solution contained 10.55 µg of gallic acid, 8.97 µg of puerarin, 10.83 µg of 3'-methoxypuerarin, 62.38 µg of paeoniflorin, 12.21 µg of ferulic acid, 9.86 µg of calycosin glucoside, 196.08 µg of hesperidin, 20.72 µg of rutin, 7.99 µg of quercetin, 19.85 µg of icariin, 4.77 µg of tanshinone I, 2.02 µg of tanshinone IIA and 12.66 µg of cryptotanshinone per 1 mL.

[0010] In S3, the HPLC detection wavelength is 230 nm ~ 270 nm, the flow rate is 0.8 mL / min ~ 1.2 mL / min; the column temperature is 25 ℃ ~ 35 ℃, and the injection volume is 5 μL ~ 15 μL.

[0011] Furthermore, the HPLC detection wavelength is 250 nm, the flow rate is 0.8 mL / min; the column temperature is 30°C, and the injection volume is 10 μL.

[0012] In S3, the HPLC assay conditions are: Agilent C 18 Column; mobile phase A is any one of methanol, acetonitrile solution or a mixed solution of the two with a volume percentage of 50% to 100%; mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.05% to 0.2%; gradient elution is adopted, and the gradient elution program is as follows: .

[0013] Furthermore, the mobile phase A is 100% methanol, and the mobile phase B is a 0.1% by volume phosphoric acid aqueous solution.

[0014] The HPLC standard fingerprint of Gaoguanping Capsule contains 23 common peaks and has 13 characteristic fingerprint peaks; the 13 characteristic fingerprint peaks take peak 3 puerarin as the reference peak, peak 1 is gallic acid, peak 3 is puerarin, peak 4 is 3'-methoxypuerarin, peak 5 is paeoniflorin, peak 8 is ferulic acid, peak 9 is calycosin glucoside, peak 10 is rutin, peak 11 is hesperidin, peak 16 is quercetin, peak 19 is icariin, peak 20 is tanshinone I, peak 21 is tanshinone IIA, and peak 22 is cryptotanshinone.

[0015] The relative retention times of the 23 common peaks are: peak 1 0.354, peak 2 0.774, peak 3 1.000, peak 4 1.066, peak 5 1.116, peak 6 1.140, peak 7 1.203, peak 8 1.376, peak 9 1.428, peak 10 1.842, peak 11 1.887, peak 12 1.990, peak 13 2.022, peak 14 2.062, peak 15 2.107, peak 16 2.409, peak 17 2.425, peak 18 2.643, peak 19 2.686, peak 20 3.264, peak 21 3.282, peak 22 3.489, and peak 23 3.717.

[0016] Furthermore, after obtaining the HPLC standard fingerprint of Gaoguanping Capsules, the results of the HPLC standard fingerprint of Gaoguanping Capsules were attributed and analyzed using Astragalus, Salvia miltiorrhiza, Pueraria lobata, Carthamus tinctorius, White Peony Root, Polygonum multiflorum, Citron, Cassia seed, Pollen Typhae and Epimedium as single medicinal material samples.

[0017] The 23 common peaks, peaks 1, 8, 9, 13, 14, 15, 17, 18, and 23 are derived from Astragalus, peaks 12, 13, 15, 20, 21, and 22 are derived from Salvia miltiorrhiza; peaks 2, 3, 4, 6, 7, and 10 are derived from Pueraria; peaks 1, 2, 7, 8, 10, 14, 15, 17, and 23 are derived from Carthamus tinctorius; peaks 1, 2, 5, and 23 are derived from Paeonia lactiflora; peaks 1, 10, 14, 18, and 23 are derived from Polygonum multiflorum; peaks 8, 10, 11, 17, and 23 are derived from Citron; peaks 2, 8, 10, 12, 14, 15, 17, 18, and 23 are derived from Cassia seed; peaks 13, 14, 15, 16, 18, and 23 are derived from Pollen Typhae; and peaks 1, 2, 13, 17, 18, and 19 are derived from Epimedium.

[0018] The present invention provides a HPLC standard fingerprint of Gaoguanping capsules obtained by the above-mentioned construction method.

[0019] The present invention provides a method for detecting the quality of Gaoguanping capsules, and utilizes the Gaoguanping capsules HPLC standard fingerprint spectrum obtained by the above-mentioned construction method to detect the quality of Gaoguanping capsule samples.

[0020] Compared with the prior art, the technical solution of the present invention has achieved the following beneficial technical effects: The method for constructing the HPLC standard fingerprint of Gaoguanping capsule provided by the present invention can accurately separate and detect multiple active ingredients in Gaoguanping capsule by high-performance liquid chromatography technology, combined with gradient elution and specific chromatographic conditions, with high precision, good reproducibility, good separation and high stability. The standard fingerprint constructed by this method can ensure the consistency and stability between different batches of Gaoguanping capsules, and provide a reliable reference standard for the quality control of Gaoguanping capsules. By evaluating the similarity with the standard fingerprint, it is possible to quickly and accurately judge whether the quality of Gaoguanping capsule samples meets the requirements. Compared with traditional quality detection methods, the HPLC standard fingerprint technology has higher detection efficiency, can detect a large number of samples in a short time, and automatically generate a test result report, which greatly shortens the detection cycle, improves work efficiency, provides scientific guidance for the production of medicines, and provides effective protection for clinical applications.

[0021] The HPLC standard fingerprint of Gaoguanping Capsules of the present invention contains 23 common peaks, and 13 characteristic fingerprint peaks are identified among them. These characteristic fingerprint peaks represent the main active ingredients or landmark compounds in the capsules, have high specificity and accuracy, are helpful for rapid identification and analysis of key chemical components in samples, provide strong support for quality control, and can serve as an important reference for quality control of Gaoguanping Capsules.

[0022] Furthermore, the sources of the 23 common peaks in the HPLC standard fingerprint of Gaoguanping Capsules reveal the distribution of various medicinal ingredients in Gaoguanping Capsules in the fingerprint, which helps to deeply understand the chemical composition of Gaoguanping Capsules and provide important clues for medicinal material traceability and quality control; the relative retention time of the 23 common peaks helps to further verify the accuracy and reliability of the fingerprint, providing more detailed data support for the quality control of Gaoguanping Capsules.

[0023] The quality detection method of Gaoguanping capsules of the present invention can quickly and accurately identify products from different batches and different manufacturers. By comparing the presence or absence of common peaks in the obtained fingerprint spectrum, the quality of the preparation can be evaluated more comprehensively, and the quality of the product can be more effectively guaranteed. The method has the advantages of simplicity, high efficiency, good repeatability and stability, and can be used for quality control of Gaoguanping capsules and identification of product quality. By comparing the difference between the actual product and the standard fingerprint spectrum, problems in the production process can be discovered and corrected in time, which helps to ensure the quality consistency between different batches of products, establish a quality database of Gaoguanping capsules, record the fingerprint spectrum information of each batch of capsules, and enhance the traceability of quality control. By studying different components in the fingerprint spectrum, the pharmacological action and efficacy mechanism of Gaoguanping capsules can be deeply understood. In clinical applications, the standard fingerprint spectrum can be used as an important basis for efficacy evaluation and safety monitoring of Gaoguanping capsules. By monitoring the changes of related components in patients, the therapeutic effect of the drug can be evaluated and the treatment plan can be adjusted. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 HPLC fingerprints of 10 batches of Gaoguanping capsules of the present invention; Figure 2 This is the HPLC fingerprint of the Gaoguanping capsule of the present invention, 1-23 are 23 common peaks; Figure 3 is the HPLC fingerprint of the reference solution of the present invention; Figure 4 The HPLC chromatogram of the single medicinal material Astragalus membranaceus of the present invention and the attribution of its compounds; Figure 5 The HPLC chromatogram of the single medicinal material Danshen of the present invention and the attribution of its compounds; Figure 6The HPLC chromatogram of the single medicinal material Puerariae Radix of the present invention and the attribution of its compounds; Figure 7 The HPLC chromatogram of the single medicinal material safflower of the present invention and the attribution of its compounds; Figure 8 The HPLC chromatogram of the single medicinal material white peony root of the present invention and the attribution of its compounds; Fig. 9 The HPLC chromatogram of the single medicinal material Polygonum multiflorum of the present invention and the attribution of its compounds; Fig.10 The HPLC chromatogram of the single medicinal material bergamot of the present invention and the attribution of its compounds; Fig.11 The HPLC chromatogram of the single medicinal material Cassiae Semen of the present invention and the attribution of its compounds; Fig.12 The HPLC chromatogram of the single medicinal material Typha acuminata of the present invention and the attribution of its compounds; Fig.13 The HPLC chromatogram of the single medicinal material Epimedium of the present invention and the attribution of its compounds. DETAILED DESCRIPTION

[0025] The following examples are used to further illustrate the present invention, but these examples are only for better understanding of the invention and are not used to limit the scope or implementation principle of the present invention. The implementation of the present invention is not limited to the following contents. Unless otherwise specified, the test methods used are conventional methods, and the raw materials used are commercially available products.

[0026] 1. Instrument Waters e2695 high performance liquid chromatograph (Waters, USA), equipped with UV detector (Waters2489); electronic analytical balance (XPR2 / A, graduation value 0.001 mg; AG135, graduation value 0.01 mg; PB3002-SDeltaRange, graduation value 0.01 mg; all from Mettler Toledo, Switzerland); ultrasonic cleaner (KQ-250DE, Kunshan Ultrasonic Instrument Co., Ltd.).

[0027] The present invention provides a method for constructing a HPLC standard fingerprint of Gaoguanping capsules, comprising: S1, using different batches of Gaoguanping capsules as test samples to prepare Gaoguanping capsule solutions; S2, using gallic acid, puerarin, 3'-methoxypuerarin, paeoniflorin, ferulic acid, calycosin glucoside, hesperidin, rutin, quercetin, icariin, tanshinone I, tanshinone IIA and cryptotanshinone as reference substances to prepare reference substance solutions; S3, performing high performance liquid chromatography analysis on the test Gaoguanping capsule solution and the reference solution under the same conditions, and recording the corresponding chromatograms; S4, processing the chromatograms of the test sample and the reference sample obtained in S3 using fingerprint software to generate a HPLC standard fingerprint of Gaoguanping Capsules consisting of common characteristic peaks, and performing attribution analysis on the HPLC standard fingerprint results of Gaoguanping Capsules.

[0028] The present invention provides the optimization process of the construction method and detection of the above-mentioned Gaoguanping Capsule HPLC standard fingerprint (1) Comparative study of flow phase In this example, four mobile phases, 0.1% phosphoric acid aqueous solution-methanol, 0.3% phosphoric acid aqueous solution-methanol, 0.1% phosphoric acid aqueous solution-acetonitrile, and 0.1% formic acid aqueous solution-methanol, were investigated for gradient elution. The results showed that when 0.1% phosphoric acid aqueous solution-methanol was used as the mobile phase, under the conditions of gradient elution, the peak separation was good, the peak elution time was moderate, and the peak shape was good.

[0029] (2) Comparative study of detection wavelengths The composition of Gaoguanping Capsules is complex, including organic acids, alkaloids, flavonoids, tanshinones, phenolic acids and other ingredients, and the maximum absorption wavelengths of different ingredients are different. This experiment investigated the effects of different wavelengths such as 230 nm, 250 nm, and 270 nm on sample detection. The results showed that the chromatographic peaks at a wavelength of 250 nm were abundant, the overall peak height was more appropriate, the interference of miscellaneous peaks was small, the peak shape was better, and there were multiple characteristic peaks.

[0030] (2) Flow rate comparison Different flow rates will affect the chromatographic peak elution time, and thus affect the sample separation. This experiment investigated the effect of different flow rates of 0.5, 0.8, 1.0, and 1.5 mL / min on the separation of Gaoguanping capsule samples. The results showed that when the flow rate was 1.0 mL / min, the sample separation time was shorter and the chromatographic peak separation was better.

[0031] (3) Column temperature comparison Column temperature affects both sample separation and running time. This experiment investigated the effects of different column temperatures of 25 ℃, 30 ℃, and 35 ℃ on the separation of Gaoguanping capsule samples. The results showed that when the column temperature was 30 ℃, the sample separation was better and the peak shape was good.

[0032] (4) Comparative study of injection volume The UV response value of the sample chromatographic peak is related to the injection volume. In this experiment, the effects of different injection volumes of 5 μL, 10 μL, and 20 μL on the response values ​​of each component of Gaoguanping Capsule were investigated. The results showed that when the injection volume was 10 μL, the response values ​​of each component were higher than 5 μL, and the chromatographic peak separation effect was better than 20 μL, and the baseline was stable. Therefore, the injection volume was selected as 10 μL.

[0033] (5) Sample extraction conditions Since this sample is a capsule prepared after extraction, ultrasonic extraction is used for pretreatment. 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, methanol, and ethanol were used to extract the sample, and the extraction conditions of ultrasonic 30, 40, and 50 min were investigated. The results showed that the ingredients in the compound can be well reflected in the 70% methanol extract, and ultrasonic 40 min can ensure complete extraction. Therefore, 70% methanol ultrasonic 40 min was selected as the extraction condition.

[0034] In summary, the optimal conditions were determined: Agilent ZORBAX Eclipse plus C 18 Column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol as mobile phase A, 0.1% phosphoric acid water (V / V) solution as mobile phase B; gradient elution, elution conditions are shown in Table 1; detection wavelength: 250 nm; column temperature: 30 ℃; injection volume: 10 μL; flow rate: 0.8 mL / min; Table 1: Gradient elution schedule

[0035] Example 1 This embodiment provides a method for constructing a HPLC standard fingerprint of Gaoguanping Capsules, which specifically comprises the following steps: Samples and reference substances: Gaoguanping capsules (60 capsules per bottle, 0.5 g per capsule) 10 batches (batch numbers are shown in Table 2) were provided by Shanhaidan Hospital. Gallic acid (batch number 110721-202220, purity 90.1%), ferulic acid (batch number 110773-201012, purity 99.6%) and quercetin (batch number 11081-200406, purity 100.0%); 3'-methoxypuerarin (batch number HR1054W7, purity 98.0%) were purchased from Baoji Chenguang Biotechnology Co., Ltd.; puerarin (batch number 110752-202217, purity 96.8%), paeoniflorin (batch number 110736-202145, purity 94.6%), calycosin, glucose Glucoside (batch number 111920-202308, purity 96.9%), rutin (batch number 100080-201409, purity 91.9%), hesperidin (batch number 110721-202220, purity 97.2%), icariin (batch number 110737-202017, purity 98.1%), tanshinone I (batch number 110867-201607), tanshinone IIA (batch number 110766-202323, purity 99.5%), cryptotanshinone (batch number 110852-201807, purity 99.0%).

[0036] Medicinal materials: Astragalus (Gansu), White Peony (Anhui), Salvia miltiorrhiza (Shandong), Pollen Typhae (Anhui), Carthamus tinctorius (Xinjiang Uygur Autonomous Region), Pueraria root (Shaanxi), Cassia seed (Shaanxi), Citron (Guangdong), Polygonum multiflorum (Shaanxi), Epimedium (Jiangsu) were provided by Shanhaidan Hospital. Methanol and acetonitrile were chromatographically pure and purchased from Fisher Company, USA; phosphoric acid was analytically pure and purchased from Tianjin Komiou Chemical Reagent Co., Ltd.; purified water was from Hangzhou Wahaha Group Co., Ltd.; the rest of the reagents were analytically pure.

[0037] Table 2: Batch numbers of Gaoguanping capsule test samples

[0038] 1. Preparation of test solution Take the contents of 20 Gaoguanping capsules, grind and mix them carefully, accurately weigh 1.0 g into a stoppered conical flask, accurately add 25 mL of 70% methanol (V / V) solution, weigh the weight, extract by ultrasonication (power 300 W, frequency 50 kHz) for 40 min, cool, weigh again, make up the lost weight with 70% methanol (V / V) solution, shake well, centrifuge at 10000 r / min for 10 min, filter through a 0.45 μm microporous filter membrane, and take the filtrate as the test solution.

[0039] 2. Preparation of reference solution Gallic acid, puerarin, 3'-methoxypuerarin, paeoniflorin, ferulic acid, calycosin glucoside, hesperidin, rutin, quercetin, icariin, tanshinone I, tanshinone IIA and cryptotanshinone were weighed respectively and dissolved in 70% methanol solution to obtain a reference solution. Wherein, each 1 mL of the reference solution contains 10.55 µg of gallic acid, 8.97 µg of puerarin, 10.83 µg of 3'-methoxypuerarin, 62.38 µg of paeoniflorin, 12.21 µg of ferulic acid, 9.86 µg of calycosin glucoside, 196.08 µg of hesperidin, 20.72 µg of rutin, 7.99 µg of quercetin, 19.85 µg of icariin, 4.77 µg of tanshinone I, 2.02 µg of tanshinone IIA and 12.66 µg of cryptotanshinone.

[0040] 3. Preparation of single herbal sample solution Weigh 1.0 g of single medicinal material samples of Astragalus, Salvia miltiorrhiza, Pueraria lobata, Carthamus tinctorius, White Peony Root, Polygonum multiflorum, Citron, Cassia seed, Pollen Typhae and Epimedium respectively, put them into a stoppered conical flask, accurately add 25 mL of 70% methanol (V / V) solution, weigh the weight, extract by ultrasonic (power 300 W, frequency 50 kHz) for 40 min, cool, weigh again, make up the lost weight with 70% methanol (V / V) solution, shake well, centrifuge at 10000 r / min for 10 min, filter through 0.45 μm microporous filter membrane, take the filtrate as the test solution, and prepare the single medicinal material sample solution.

[0041] 4. Reliability confirmation The test solutions of 10 batches of Gaoguanping Capsules (S1-S10) were injected and tested according to the optimal chromatographic conditions, and the chromatograms were recorded and imported into the Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012 A Version). The chromatograms of 10 batches of Gaoguanping Capsules (S1-S10) test solutions were selected (see Appendix Figure 1 ) as common peaks, generate the reference spectrum of Xiaoyin Capsules by the average calculation method, calculate the relative retention time and relative peak area of ​​each common peak; after data import, multi-point correction and data matching, perform similarity analysis; obtain and export the similarity result table between the chromatograms of different batches of Gaoguanping Capsules test solution and the common peak pattern; confirm the reliability of the results based on the similarity result table and the chromatogram of Gaoguanping Capsules test solution. Peak No. 3 in the chromatogram with good peak shape and separation, no interfering chromatographic peaks in adjacent positions, high response value and stable retention time is used as the reference S peak. After calculation, the RSD values ​​of the relative retention time and relative peak area of ​​the common peaks of all test samples are less than 3.0% and 5.0%, respectively, which meets the requirements.

[0042] 5. Establishment of fingerprint The chromatograms of the test solution and the reference solution of Gaoguanping Capsules were combined with the chromatograms of the single herbal medicines. The chromatograms were matched using the "Chinese Herbal Medicine Fingerprint Similarity Evaluation System (2012A)". The median method was used as the reference spectrum generation method, the time window width was 0.5, and the superimposed spectrum of the test sample and the reference spectrum R were automatically matched after multi-point correction. It contained 23 common peaks and 13 characteristic fingerprint peaks; peak 3 of puerarin was used as the reference peak. The reference peaks are as follows: peak 1 is gallic acid, peak 3 is puerarin, peak 4 is 3'-methoxypuerarin, peak 5 is paeoniflorin, peak 8 is ferulic acid, peak 9 is calycosin glucoside, peak 10 is rutin, peak 11 is hesperidin, peak 16 is quercetin, peak 19 is icariin, peak 20 is tanshinone I, peak 21 is tanshinone IIA, and peak 22 is cryptotanshinone. The fingerprint of Gaoguanping Capsule is obtained, see Appendix. Figure 2 As shown, among them, 1-gallic acid; 3-puerarin; 4-3'-methoxypuerarin; 5-peonidin; 8-ferulic acid; 9-calycosin isoflavone glucoside; 10-rutin; 11-hesperidin; 16-quercetin; 19-icariin; 20-tanshinone I; 21-tanshinone IIA; 22-cryptotanshinone.

[0043] 6. Attribution and identification of common peaks Take the reference solution and the single herbal medicine test solution, analyze them according to the optimal chromatographic conditions, and obtain the fingerprints of the reference solution and the single herbal medicine test solution. Figure 3-13 shown.

[0044] By the attached Figure 3-13The data show that after retention time positioning and chromatographic peak analysis of the reference substance, a total of 13 characteristic peaks were identified, namely gallic acid (peak 1), puerarin (peak 3), 3'-methoxypuerarin (peak 4), paeoniflorin (peak 5), ferulic acid (peak 8), calycosin glucoside (peak 9), rutin (peak 10), hesperidin (peak 11), quercetin (peak 16), icariin (peak 19), tanshinone I (peak 20), tanshinone IIA (peak 21), and cryptotanshinone (peak 22). Among them, peaks 1, 8, 9, 13, 14, 15, 17, 18, and 23 are derived from Astragalus, peaks 12, 13, 15, 20, 21, and 22 are derived from Salvia miltiorrhiza; peaks 2, 3, 4, 6, 7, and 10 are derived from Pueraria; peaks 1, 2, 7, 8, 10, 14, 15, 17, and 23 are derived from Carthamus tinctorius; peaks 1, 2, 5, and 23 are derived from Paeonia lactiflora; peaks 1, 10, 14, 18, and 23 are derived from Polygonum multiflorum; peaks 8, 10, 11, 17, and 23 are derived from Citron; peaks 2, 8, 10, 12, 14, 15, 17, 18, and 23 are derived from Cassia seed; peaks 13, 14, 15, 16, 18, and 23 are derived from Pollen Typhae; and peaks 1, 2, 13, 17, 18, and 19 are derived from Epimedium, indicating that Gaoguanping capsule preparation has a good correlation with single medicinal materials.

[0045] Methodological research on detection methods, investigation of fingerprint methodology (1) Similarity evaluation The data of HPLC fingerprints of 10 batches of Gaoguanping Capsules were imported into the software "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" to analyze the similarity of the 10 batches of samples. The similarities between the samples were all greater than 0.9. The S6 sample spectrum was used as a reference spectrum for similarity calculation. The similarities were all above 0.9, and the similarity was good. The specific results are shown in Table 3.

[0046] It can be seen from the data in Table 3 that the overall chemical spectrum peak information shows that the quality of 10 batches of Gaoguanping capsule samples is relatively stable. The results show that the ingredients of different batches of Gaoguanping capsules are highly similar, the quality is relatively stable and the uniformity is good.

[0047] Table 3: Similarity evaluation of 10 batches of Gaoguanping capsules

[0048] (2) Precision test Accurately weigh the appropriate amount of Gaoguanping Capsules (batch number 2407001), inject the sample 6 times continuously according to the optimal chromatographic conditions, and record the chromatogram. Take peak 3 (puerarin) as the reference peak, and analyze the relative retention time and relative peak area of ​​23 common peaks in the sample. As a result, the RSD values ​​of the relative retention time and relative peak area of ​​23 common peaks were less than 3.0% and 5.0%, respectively, indicating that the instrument has good precision.

[0049] (3) Stability test An appropriate amount of Gaoguanping capsules (batch number 2407001) was accurately weighed, and the samples were injected and measured at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 hours after preparation according to the optimal chromatographic conditions. The relative retention time and relative peak area of ​​23 common peaks in the sample were analyzed with Peak 3 (puerarin) as the reference peak. As a result, the RSD values ​​of the relative retention time and relative peak area of ​​the 23 common peaks were less than 3.0% and 5.0%, respectively, indicating that the Gaoguanping capsule test solution had good stability within 48 hours.

[0050] (4) Repeatability test Take an appropriate amount of Gaoguanping capsules (batch number 2407001), accurately weigh 6 portions, and inject and measure according to the optimal chromatographic conditions. Take peak 3 (puerarin) as the reference peak, and analyze the relative retention time and relative peak area of ​​23 common peaks in the sample. As a result, the RSD values ​​of the relative retention time and relative peak area of ​​23 common peaks were less than 3.0% and 5.0%, respectively, indicating that the method has good repeatability.

[0051] The above embodiments are only preferred implementations of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that for those skilled in the art, local modifications and improvements can be made to the invention in different ways without departing from the principles and concepts of the invention. The protection scope of the present invention shall be based on the claims and shall not be limited by the above specific implementations. All implementations within its scope are subject to the constraints of the present invention and belong to the protection scope of the present invention.

Claims

1. A method for constructing a HPLC standard fingerprint of Gaoguanping Capsules, characterized in that: include: S1, using different batches of Gaoguanping capsules as test samples to prepare Gaoguanping capsule solutions; S2, using gallic acid, puerarin, 3'-methoxypuerarin, paeoniflorin, ferulic acid, calycosin glucoside, hesperidin, rutin, quercetin, icariin, tanshinone I, tanshinone IIA and cryptotanshinone as reference substances to prepare reference substance solutions; S3, performing high performance liquid chromatography analysis on the test Gaoguanping capsule solution and the reference solution under the same conditions, and recording the corresponding chromatograms; S4, processing the chromatograms of the test sample and the reference sample obtained in S3 using fingerprint software to generate a HPLC standard fingerprint of Gaoguanping Capsules consisting of common characteristic peaks.

2. The method for constructing a HPLC standard fingerprint of Gaoguanping Capsules according to claim 1, characterized in that: In S1, the contents of Gaoguanping capsules from different batches were taken, 50%-90% methanol was added for ultrasonic treatment for 30 min-50 min, and the mixture was centrifuged at 10000 r / min for 10 min. The supernatant was taken and filtered through a 0.45 μm microporous filter membrane to obtain the test Gaoguanping capsule solution.

3. The method for constructing a HPLC standard fingerprint of Gaoguanping Capsules according to claim 1, characterized in that: In S3, the HPLC detection wavelength is 230 nm ~270 nm, the flow rate is 0.8 mL / min~1.2 mL / min; the column temperature is 25 ℃~35 ℃; and the injection volume is 5 μL~15 μL.

4. The method for constructing a HPLC standard fingerprint of Gaoguanping Capsules according to claim 1, characterized in that: In S3, the HPLC assay conditions are: Agilent C 18 Column; mobile phase A is any one of methanol, acetonitrile solution or a mixed solution of the two with a volume percentage of 50% to 100%; mobile phase B is a phosphoric acid aqueous solution with a volume percentage of 0.05% to 0.2%; gradient elution is adopted, and the gradient elution program is as follows: 。 5. The method for constructing a HPLC standard fingerprint of Gaoguanping Capsules according to claim 1, characterized in that: The HPLC standard fingerprint of Gaoguanping Capsule contains 23 common peaks and has 13 characteristic fingerprint peaks; the 13 characteristic fingerprint peaks take peak 3 puerarin as the reference peak, peak 1 is gallic acid, peak 3 is puerarin, peak 4 is 3'-methoxypuerarin, peak 5 is paeoniflorin, peak 8 is ferulic acid, peak 9 is calycosin glucoside, peak 10 is rutin, peak 11 is hesperidin, peak 16 is quercetin, peak 19 is icariin, peak 20 is tanshinone I, peak 21 is tanshinone IIA, and peak 22 is cryptotanshinone.

6. The method for constructing a HPLC standard fingerprint of Gaoguanping Capsules according to claim 5, characterized in that: The 23 common peaks, peaks 1, 8, 9, 13, 14, 15, 17, 18, and 23 are derived from Astragalus, peaks 12, 13, 15, 20, 21, and 22 are derived from Salvia miltiorrhiza; peaks 2, 3, 4, 6, 7, and 10 are derived from Pueraria; peaks 1, 2, 7, 8, 10, 14, 15, 17, and 23 are derived from Carthamus tinctorius; peaks 1, 2, 5, and 23 are derived from Paeonia lactiflora; peaks 1, 10, 14, 18, and 23 are derived from Polygonum multiflorum; peaks 8, 10, 11, 17, and 23 are derived from Citron; peaks 2, 8, 10, 12, 14, 15, 17, 18, and 23 are derived from Cassia seed; peaks 13, 14, 15, 16, 18, and 23 are derived from Pollen Typhae; and peaks 1, 2, 13, 17, 18, and 19 are derived from Epimedium.

7. The method for constructing a HPLC standard fingerprint of Gaoguanping Capsules according to claim 5, characterized in that: The relative retention times of the 23 common peaks are: peak 1 0.354, peak 2 0.774, peak 3 1.000, peak 4 1.066, peak 5 1.116, peak 6 1.140, peak 7 1.203, peak 8 1.376, peak 9 1.428, peak 10 1.842, peak 11 1.887, peak 12 1.990, peak 13 2.022, peak 14 2.062, peak 15 2.107, peak 16 2.409, peak 17 2.425, peak 18 2.643, peak 19 2.686, peak 20 3.264, peak 21 3.282, peak 22 3.489, and peak 23 3.

717.

8. The method for constructing a HPLC standard fingerprint of Gaoguanping Capsules according to claim 1, characterized in that: After obtaining the HPLC standard fingerprint of Gaoguanping Capsules, the results of the HPLC standard fingerprint of Gaoguanping Capsules were attributed and analyzed using Astragalus, Salvia miltiorrhiza, Pueraria lobata, Carthamus tinctorius, Paeonia lactiflora, Polygonum multiflorum, Citron, Cassia seed, Pollen Typhae and Epimedium as single medicinal material samples.

9. The HPLC standard fingerprint of Gaoguanping Capsules obtained according to the construction method according to any one of claims 1 to 8.

10. A method for detecting the quality of Gaoguanping capsules, characterized in that: The quality of Gaoguanping capsule samples is detected by using the Gaoguanping capsule HPLC standard fingerprint obtained by the construction method described in any one of claims 1 to 8.

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