High performance liquid chromatography fingerprint spectrum analysis method for compound wintercreeper capsule and application of high performance liquid chromatography fingerprint spectrum analysis method

Abstract

The invention discloses a compound wintercreeper capsule high performance liquid chromatography fingerprint spectrum analysis method and application thereof, and belongs to the technical field of chemical analysis. A mixed solution of protocatechuic acid, calycosin glucoside, formononin, calycosin and formononetin is used as a standard solution; performing high performance liquid chromatography analysis by taking a methanol extracting solution of the compound wintercreeper capsule as a test solution, and establishing an HPLC fingerprint spectrum of the compound wintercreeper capsule; wherein in the high performance liquid chromatography analysis, an acetonitrile-0. 2% phosphoric acid solution is used as a mobile phase, the elution mode is gradient elution, and the detection wavelength is 254 nm. The method provided by the invention is good in repeatability and high in durability, and can be used for controlling the quality of the compound wintercreeper capsule.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a high performance liquid chromatography fingerprint analysis method for compound fufangteng capsules and an application thereof. Background Art

[0002] In the prior art, the fingerprint detection of Fufangteng Capsule mainly adopts high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) and other methods, and qualitative and quantitative analysis is performed on its main active ingredients (such as flavonoids, phenols, saponins, etc.) to characterize its chemical composition and quality consistency. As a comprehensive quality control method, fingerprint can reflect the overall chemical characteristics of Chinese medicine compound preparations, so it plays an important role in the quality evaluation of Chinese medicine. However, there are still some problems in the prior art: first, the ingredients of Fufangteng Capsule are complex and the matrix interference is large, resulting in poor separation of some components and difficulty in fully reflecting its chemical characteristics; secondly, the existing fingerprints are mostly based on a single detection method, lacking systematic research on the combination of multiple technologies, which easily leads to the omission of certain trace or thermally unstable components; in addition, the standardization and consistency evaluation system of fingerprints is not yet perfect, and the data comparability between different laboratories or batches is poor, which affects the accuracy and reliability of quality control. More importantly, the correlation between fingerprints and drug efficacy is insufficient. Existing studies focus on the qualitative and quantitative analysis of chemical components, but lack in-depth discussion of the relationship between chemical components and drug efficacy. These technical bottlenecks limit the further application of Fufangteng Capsule fingerprints in quality control and drug efficacy evaluation.

[0003] In the prior art, the establishment of the common pattern of the HPLC fingerprint of compound Fuyuancao mixture by Qin Jieping et al. and its application in preparation quality control, as well as the research on the determination method of the HPLC fingerprint of compound Fuyuancao mixture by Qin Jieping et al. disclosed the use of HPLC method to detect the fingerprint of compound Fuyuancao mixture, but the above methods have the problem of not comparing the characteristic components.

[0004] Therefore, how to provide a fingerprint detection method for Fufangteng Capsules is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a high performance liquid chromatography fingerprint analysis method for compound Fufangteng capsules and its application.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A high performance liquid chromatography fingerprint analysis method for Fufangteng capsules, using a mixed solution of protocatechuic acid, calycosin glucoside, formononetin, calycosin and formononetin as a standard solution, and using a methanol extract of Fufangteng capsules as a test solution for high performance liquid chromatography analysis, to establish a HPLC fingerprint of Fufangteng capsules;

[0008] The HPLC analysis uses acetonitrile-0.2% phosphoric acid solution as the mobile phase, the elution method is gradient elution, and the detection wavelength is 254 nm. The 0.2% phosphoric acid solution is a phosphoric acid solution with a mass fraction of 0.2%.

[0009] Preferably, the gradient elution is:

[0010] 0-20min, 5%A, 95%B; 20-25min, 5%→12%A, 95%→88%B; 25-36min, 12%A, 88%B; 36-40min, 12 %→17%A, 88%→83%B; 40-56min, 17%A, 83%B; 56-60min, 17%→21%A, 83%→79%B; 60-78min, 21% A, 79% B; 78-82min, 21% → 29% A, 79% → 71% B; 82-90min, 29% A, 71% B; 90-93min, 29% → 36% A, 71% →64%B; 93-110min, 36%A, 64%B; 110-115min, 36%→5%A, 64%→95%B; 115-120min, 5%A, 95%B;

[0011] Among them, A is acetonitrile and B is phosphoric acid solution.

[0012] Preferably, the concentration of protocatechuic acid in the standard solution is 0.0180 mg·mL -1 The concentration of calycosin glucoside was 0.0202 mg·mL -1 The concentration of formononetin was 0.0088 mg·mL -1 The concentration of calycosin isoflavone is 0.0183 mg·mL -1 , the concentration of formononetin was 0.0096 mg·mL -1 .

[0013] Preferably, the method for preparing the test solution comprises the following steps:

[0014] The compound fufangteng capsules are removed from the capsule shell and then ground into powder, and then methanol is added to the obtained compound fufangteng capsule powder for ultrasonic extraction. After the extraction is completed, the test solution is obtained by filtering.

[0015] Preferably, the ratio of the added amount of the compound Fufangteng capsule powder to methanol is 1g:25mL.

[0016] Preferably, before filtering, the process further comprises: cooling the product obtained after ultrasonic extraction, weighing the amount of methanol lost during the extraction process, and then adding methanol to make up the amount and shaking well.

[0017] Preferably, the filtration is through a 0.45 μm microporous filter membrane.

[0018] Preferably, the conditions for the HPLC analysis are: the chromatographic column is Acclaim TM 120C18 column, injection volume 10 μL, flow rate 1.0 mL min -1 , column temperature 30℃.

[0019] Application of a high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules in the quality control of Fufangteng Capsules.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The invention uses a mixed solution of protocatechuic acid, calycosin glucoside, formononetin, calycosin and formononetin as a standard solution, adopts an Acclaim 120-C18 chromatographic column, uses acetonitrile-0.2% phosphoric acid solution as a mobile phase, and limits the specific conditions of gradient elution to analyze and detect the fingerprint of compound Fufangteng capsule, establishes an HPLC fingerprint of the compound Fufangteng capsule, establishes 20 common peaks, identifies 5 common peaks, and the similarities of 15 batches of samples are all greater than 0.90. The method provided by the invention has good repeatability and can be used for quality control of the compound Fufangteng capsule. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0023] Figure 1 This is the HPLC overlay of 15 batches of Fufangteng Capsule samples;

[0024] Figure 2 The HPLC graph of the mixed reference substance (A), the HPLC reference fingerprint of Fufangteng Capsule (B) and the blank control (C);

[0025] The labels are: 7-protocatechuic acid, 14-calycosin glucoside, 17-formononetin, 18-calycosin glucoside, and 20-formononetin. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] Unless otherwise specified, the raw materials in the examples of the present invention were purchased from commercial sources;

[0029] Some of the raw materials were sourced as follows: protocatechuic acid was purchased from the China Food and Drug Identification Institute (batch number: 110809-202207);

[0030] Calycosin isoflavone glucoside was purchased from China Food and Drug Identification Institute (Batch No.: 111920-202308);

[0031] Formononetin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Batch No.: JB247303);

[0032] Calycosin isoflavones were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Batch No.: S29IB227691);

[0033] Formononetin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Batch No.: N08IB231271);

[0034] Acetonitrile and methanol were chromatographically pure (Fisher);

[0035] Phosphoric acid was chromatographically pure and purchased from Xilong Scientific Co., Ltd.;

[0036] Purified water was purchased from Hangzhou Wahaha Group Co., Ltd.;

[0037] Fufang Fufangteng Capsules were provided by Guangxi University of Chinese Medicine Bai Nian Le Pharmaceutical Co., Ltd. (Batch numbers: 20240601, 20240602, 20240603, 20240604, 20270101, 20240702, 20240703, 20240704, 20240705, 20240801, 20240802, 20240803, 20240901, 20240902, 20240903, numbered S1 to S15 respectively)

[0038] Some of the instruments used in the embodiments of the present invention are:

[0039] 1260 high performance liquid chromatograph (Agilent, USA);

[0040] SG250HE ultrasonic cleaner (Shanghai Guante Ultrasonic Instrument Co., Ltd.);

[0041] ME104E 1 / 10,000 electronic balance (Mettler-Toledo Instrument Co., Ltd.);

[0042] MS205U 1 / 100,000 electronic analytical balance (Mettler-Toledo Instrument Co., Ltd.).

[0043] Example 1

[0044] A high performance liquid chromatography fingerprint analysis method for Fufangteng capsules comprises the following steps:

[0045] (1) Preparation of test solution

[0046] Take 15 batches of Fufangteng capsule samples, and take 10 Fufangteng capsule samples (0.37g capsules) from each batch. -1 ), remove the capsule shell, grind it into powder, accurately weigh 1.0g of powder, put it in a 50mL conical flask, accurately add 25mL of methanol, weigh it, extract it by ultrasonic for 30min, cool it, weigh it again, make up the lost amount with methanol, shake it well, filter it, and pass the filtrate through a 0.45μm microporous filter membrane to obtain 15 batches of test solution.

[0047] (2) Preparation of standard solution

[0048] Accurately weigh 23.96 mg, 17.57 mg, and 19.12 mg of protocatechuic acid, formononetin, and calycosin isoflavone standard reference substances, respectively, and place them in a 10 mL volumetric flask. Accurately weigh 17.94 mg and 19.12 mg of calycosin isoflavone glucoside and formononetin standard reference substances, respectively, and place them in a 20 mL volumetric flask. Add methanol to dissolve and make up to volume to obtain each single reference substance stock solution.

[0049] Accurately add protocatechuic acid reference stock solution, calycosin glucoside reference stock solution, formononetin reference stock solution, calycosin reference stock solution, and formononetin reference stock solution into the same 20 mL volumetric flask, add methanol to the scale to make the mass concentrations 0.0180 and 0.0202 mg mL -1 , 0.0088mg·mL -1 , 0.0183mg·mL -1 , 0.0096mg·mL -1 The mixed reference solution is used as the standard solution for later use.

[0050] (3) Chromatographic conditions

[0051] The chromatographic column is Acclaim TM 120 C18 column, (250*4.6mm, 5um), mobile phase: acetonitrile (A)-0.2% phosphoric acid solution (B); wherein, 0.2% phosphoric acid solution is a phosphoric acid solution with a mass fraction of 0.2%.

[0052] Gradient elution:

[0053] 0-20min, 5%A, 95%B; 20-25min, 5%→12%A, 95%→88%B; 25-36min, 12%A, 88%B; 36-40min, 12 %→17%A, 88%→83%B; 40-56min, 17%A, 83%B; 56-60min, 17%→21%A, 83%→79%B; 60-78min, 21% A, 79% B; 78-82min, 21% → 29% A, 79% → 71% B; 82-90min, 29% A, 71% B; 90-93min, 29% → 36% A, 71% →64%B; 93-110min, 36%A, 64%B, 110-115min, 36%→5%A, 64%→95%B, 115-120min, 5%A, 95%B;

[0054] The detection wavelength was 254 nm, the injection volume was 10 μL, and the flow rate was 1.0 mL min -1 , column temperature 30℃.

[0055] (4) Establishment of HPLC fingerprint

[0056] Take the above 15 batches of test sample solutions, measure them under the above chromatographic conditions, and record the chromatograms. Import the 15 batches of sample chromatogram data into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012A Edition)", set S1 as the reference spectrum, and construct the fingerprints and common patterns (R) of 15 batches of Fufangteng Capsules after multi-point calibration and Mark peak matching. The fingerprints have 20 common peaks. Through standard solution comparison, five components, including protocatechuic acid ((Peak 7)), calycosin glucoside (Peak 14), formononetin (Peak 17), calycosin (Peak 18), and formononetin (Peak 20), were identified; see Figure 1 and Tables 1-4.

[0057] Table 115 Retention time of Fufangteng capsule samples (1)

[0058] Serial number S1 S2 S3 S4 S5 S6 S7 S8 Peak 1 0.2487 0.2473 0.2478 0.2484 0.2481 0.2482 0.2477 0.2477 Peak 2 0.2630 0.2615 0.2618 0.2628 0.2622 0.2622 0.2616 0.2618 Peak 3 0.4788 0.4758 0.4770 0.4786 0.4776 0.4775 0.4764 0.4764 Peak 4 0.5400 0.5376 0.5386 0.5403 0.5396 0.5395 0.5379 0.5380 Peak 5 0.6642 0.6615 0.6623 0.6640 0.6631 0.6631 0.6614 0.6614 Peak 6 0.7532 0.7513 0.7516 0.7529 0.7523 0.7522 0.7512 0.7512 Peak 7 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 Peak 8 1.1839 1.1810 1.1812 1.1841 1.1828 1.1819 1.1814 1.1814 Peak 9 1.7185 1.7122 1.7135 1.7191 1.7157 1.7164 1.7127 1.7127 Peak 10 1.9944 1.9853 1.9892 1.9952 1.9911 1.9913 1.9870 1.9870 Peak 11 2.1669 2.1583 2.1634 2.1706 2.1663 2.1653 2.1614 2.1614 Peak 12 2.9288 2.9178 2.9260 2.9345 2.9277 2.9571 2.9206 2.9206 Peak 13 2.9594 2.9486 2.9574 2.9664 2.9592 2.9571 2.9516 2.9519 Peak 14 3.0119 2.9991 3.0076 3.0167 3.0096 3.0079 3.0020 3.0020 Peak 15 3.4427 3.4378 3.4536 3.4616 3.4526 3.4505 3.4413 3.4413 Peak 16 4.4132 4.4019 4.4183 4.4251 4.4148 4.4143 4.4089 4.4038 Peak 17 4.5974 4.5830 4.6001 4.6086 4.5973 4.5958 4.5842 4.5842 Peak 18 5.2163 5.1952 5.2061 5.2194 5.2074 5.2090 5.1980 5.1980 Peak 19 5.2717 5.2503 5.2605 5.2741 5.2617 5.2640 5.2529 5.2529 Peak 20 6.2940 6.2689 6.2787 6.2962 6.2820 6.2845 6.2726 6.2726

[0059] Table 215 Retention time of Fufangteng capsule samples (2)

[0060]

[0061]

[0062] Table 3 Peak area of ​​15 batch samples (1)

[0063] Serial number S1 S2 S3 S4 S5 S6 S7 S8 Peak 1 0.7871 0.5429 0.6000 0.3874 0.3977 0.4384 0.4439 0.4028 Peak 2 0.6433 0.5231 0.5483 0.4749 0.4667 0.4769 0.4749 0.4375 Peak 3 0.5722 0.3386 0.3450 0.2996 0.3378 0.3508 0.3460 0.4112 Peak 4 0.0968 0.1742 0.1827 0.1714 0.1740 0.1626 0.1740 0.1760 Peak 5 0.4774 0.6852 0.7080 1.0064 0.9920 0.8340 0.9740 0.8420 Peak 6 1.7081 1.3147 1.3811 1.1083 1.1174 1.1610 1.1120 1.2218 Peak 7 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 Peak 8 0.9381 0.9367 0.9464 0.6479 0.6325 0.7742 0.9048 0.7294 Peak 9 0.7984 0.7746 0.7862 0.7436 0.7400 0.7754 0.5493 0.8173 Peak 10 0.5474 0.5229 0.5375 0.5400 0.5525 0.5267 0.4454 0.5299 Peak 11 0.3724 0.3123 0.3208 0.4186 0.4232 0.3648 0.2772 0.3295 Peak 12 0.4712 0.2967 0.2824 0.2247 0.2396 0.2383 0.1806 0.2392 Peak 13 0.2096 0.2016 0.2071 0.1185 0.1243 0.1496 0.1052 0.1034 Peak 14 1.0221 1.6634 1.7719 1.5700 1.6382 1.6081 1.4344 1.7012 Peak 15 1.7918 1.4249 1.5007 1.2973 1.4662 1.4237 1.1808 1.3398 Peak 16 0.2003 0.2280 0.2470 0.2439 0.2568 0.2336 0.2714 0.2121 Peak 17 0.5143 0.9157 0.9713 0.9580 1.0207 0.9519 0.9760 1.0030 Peak 18 2.2333 1.6439 1.6958 1.5820 1.6713 1.6338 1.8141 1.6772 Peak 19 0.8015 1.0171 1.0810 1.0949 1.1786 1.0794 1.2100 1.1083 Peak 20 0.8510 0.7526 0.7588 0.8010 0.8620 0.7959 0.8063 0.9310

[0064] Table 4 Peak area of ​​15 batch samples (2)

[0065]

[0066]

[0067] (5) Similarity evaluation

[0068] The similarity of HPLC spectra of 15 batches of Fufangteng capsules was calculated using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". The results showed that compared with the control fingerprint, the similarity of the spectra of each batch of Fufangteng capsule samples was greater than 0.9. The results are shown in Tables 5 and 6. The results show that the spectra of different batches of Fufangteng capsule samples are similar.

[0069] Table 5 Similarity of HPLC spectra of batch 15 Fufangteng capsules (1)

[0070]

[0071]

[0072] Table 6 Similarity of HPLC spectra of batch 15 Fufangteng capsules (2)

[0073]

[0074] Technical effect:

[0075] 1. Precision test

[0076] Accurately weigh 10 Fufangteng capsules (S1) (0.37 g / capsule) -1 ), remove the capsule shell, grind it into powder, accurately weigh 1.0g of powder, prepare the test solution according to step (1) of Example 1, and continuously inject 6 times under the chromatographic conditions of step (3) of Example 1, and use protocatechuic acid (peak 7) as the reference peak to calculate the relative retention time and relative peak area of ​​each common peak. The RSD value range of the relative retention time of each common peak is 0.09% to 0.28%, and the RSD value range of the relative peak area of ​​each common peak is 0.20% to 0.85%, indicating that the instrument has good precision. The relative retention time of the common peak precision experiment is shown in Table 7, and the relative peak area of ​​the common peak precision experiment is shown in Table 8.

[0077] Table 7 Common peak precision experiment-relative retention time (n=6)

[0078] Number of peaks 1 2 3 4 5 6 RSD% 1 0.2482 0.2481 0.2482 0.2467 0.2482 0.2475 0.25 2 0.2626 0.2626 0.2627 0.2609 0.2628 0.2620 0.28 3 0.4775 0.4775 0.4773 0.4754 0.4771 0.4764 0.17 4 0.5407 0.5408 0.5406 0.5396 0.5400 0.5405 0.09 5 0.6628 0.6627 0.6624 0.6596 0.6624 0.6623 0.18 6 0.7518 0.7519 0.7514 0.7494 0.7510 0.7515 0.12 7 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 8 1.1795 1.1794 1.1794 1.1749 1.1792 1.1772 0.16 9 1.7173 1.7169 1.7169 1.7078 1.7177 1.7124 0.23 10 1.9924 1.9916 1.9914 1.9793 1.9912 1.9868 0.25 11 2.1653 2.1638 2.1629 2.1506 2.1626 2.1593 0.25 12 2.9308 2.9287 2.9274 2.9117 2.9280 2.9197 0.25 13 2.9623 2.9601 2.9590 2.9429 2.9592 2.9522 0.24 14 3.0101 3.0082 3.0071 2.9906 3.0074 2.9988 0.25 15 3.4545 3.4516 3.4489 3.4291 3.4484 3.4422 0.27 16 4.4162 4.4122 4.4105 4.3859 4.4121 4.3998 0.26 17 4.5977 4.5934 4.5911 4.5655 4.5920 4.5802 0.26 18 5.2124 5.2087 5.2081 5.1767 5.2074 5.1938 0.26 19 5.2685 5.2655 5.2646 5.2323 5.2636 5.2492 0.27 20 6.2875 6.2839 6.2819 6.2447 6.2811 6.2647 0.26

[0079] Table 8 Total peak precision experiment - relative peak area (n = 6)

[0080]

[0081]

[0082] 2. Stability test

[0083] Accurately weigh 10 Fufangteng capsules (S1) (0.37 g / capsule) -1 ), remove the capsule shell, grind it into powder, accurately weigh 1.0g of powder, prepare the test solution according to step (1) of Example 1, and inject and measure according to the chromatographic conditions of step (3) of Example 1 at 0h, 2h, 4h, 8h, 12h, and 24h, respectively, and calculate the relative retention time and relative peak area of ​​each common peak with protocatechuic acid (peak 7) as the reference peak. The relative retention time RSD value range of each common peak is 0.09% to 0.60%, and the relative peak area RSD value range of each common peak is 0.56% to 1.66%, indicating that the test solution has good stability within 24h. The relative retention time of the common peak stability experiment is shown in Table 9, and the relative peak area of ​​the common peak stability experiment is shown in Table 10.

[0084] Table 9 Common peak stability experiment-relative retention time (n=6)

[0085]

[0086]

[0087] Table 10 Common peak stability test - relative peak area (n = 6)

[0088]

[0089]

[0090] 3. Repeatability test

[0091] Accurately weigh the same batch of Fufangteng Capsules (S1) (0.37 g / capsule) -1), remove the capsule shell, grind it into powder, accurately weigh 1.0g of powder, a total of 6 portions, prepare the test solution according to step (1) of Example 1, and inject and measure according to the chromatographic conditions of step (1) of Example 1. Take protocatechuic acid (peak 7) as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak, the relative retention time RSD value range of each common peak is 0.11% to 0.60%, and the relative peak area RSD value range of each common peak is 0.20% to 0.85%, indicating that the method has good repeatability. Common peak repeatability experiment-relative retention time is shown in Table 11, and common peak repeatability experiment-relative peak area is shown in Table 12.

[0092] Table 11 Common peak repeatability experiment-relative retention time (n=6)

[0093]

[0094]

[0095] Table 12 Common peak repeatability experiment-relative peak area (n=6)

[0096]

[0097]

[0098] It can be seen from the above experimental data that the method established in the present invention has good repeatability and can be used for quality control of Fufang Fufangteng Capsules.

[0099] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules, characterized in that: The mixed solution of protocatechuic acid, calycosin glucoside, formononetin, calycosin and formononetin was used as the standard solution, and the methanol extract of Fufangteng Capsule was used as the test solution to carry out HPLC analysis, and the HPLC fingerprint of Fufangteng Capsule was established; Wherein, the high performance liquid chromatography analysis uses acetonitrile-0.2% phosphoric acid solution as the mobile phase, the elution method is gradient elution, and the detection wavelength is 254nm.

2. The high performance liquid chromatography fingerprint analysis method of Fufangteng Capsule according to claim 1 is characterized in that: The gradient elution is: 0-20min, 5%A, 95%B; 20-25min, 5%→12%A, 95%→88%B; 25-36min, 12%A, 88%B; 36-40min, 12 %→17%A, 88%→83%B; 40-56min, 17%A, 83%B; 56-60min, 17%→21%A, 83%→79%B; 60-78min, 21% A, 79% B; 78-82min, 21% → 29% A, 79% → 71% B; 82-90min, 29% A, 71% B; 90-93min, 29% → 36% A, 71% →64%B; 93-110min, 36%A, 64%B; 110-115min, 36%→5%A, 64%→95%B; 115-120min, 5%A, 95%B; Among them, A is acetonitrile and B is phosphoric acid solution.

3. The high performance liquid chromatography fingerprint analysis method of Fufangteng Capsule according to claim 1 is characterized in that: In the standard solution, the concentration of protocatechuic acid is 0.0180 mg·mL -1 The concentration of calycosin glucoside was 0.0202 mg·mL -1 The concentration of formononetin was 0.0088 mg·mL -1 The concentration of calycosin isoflavone is 0.0183 mg·mL -1 , the concentration of formononetin was 0.0096 mg·mL -1 .

4. The high performance liquid chromatography fingerprint analysis method of Fufangteng Capsule according to claim 1 is characterized in that: The preparation method of the test solution comprises the following steps: The compound fufangteng capsules are removed from the capsule shell and then ground into powder, and then methanol is added to the obtained compound fufangteng capsule powder for ultrasonic extraction. After the extraction is completed, the test solution is obtained by filtering.

5. The high performance liquid chromatography fingerprint analysis method of Fufangteng Capsules according to claim 4 is characterized in that: The ratio of the added amount of the compound fufangteng capsule powder to methanol is 1g:25mL.

6. The high performance liquid chromatography fingerprint analysis method of Fufangteng Capsules according to claim 4 is characterized in that: Before filtering, the method further includes: cooling the product obtained after ultrasonic extraction, weighing the amount of methanol lost during the extraction process, and then adding methanol to make up the amount and shaking well.

7. The high performance liquid chromatography fingerprint analysis method of Fufangteng Capsules according to claim 4 is characterized in that: The filtration is through a 0.45 μm microporous filter membrane.

8. The high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules according to claim 1, characterized in that: The conditions of the HPLC analysis are as follows: the chromatographic column is Acclaim TM 120C18 column, injection volume 10 μL, flow rate 1.0 mL min -1 , column temperature 30℃.

9. Application of the high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules as described in any one of claims 1 to 8 in the quality control of Fufangteng Capsules.

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