A high performance liquid chromatography fingerprint analysis method for Fufangteng capsules and its application

Abstract

The present invention discloses a high performance liquid chromatography fingerprint analysis method for compound Fufangteng capsule and its application, which belongs to the field of chemical analysis technology. A mixed solution of protocatechuic acid, calycosin glucoside, thorn formononetin, calycosin, and formononetin is used as a standard solution, and a methanol extract of compound Fufangteng capsule is used as a test solution for high performance liquid chromatography analysis to establish a compound Fufangteng capsule HPLC fingerprint; wherein, the high performance liquid chromatography analysis uses acetonitrile 0.2% phosphoric acid solution as the mobile phase, the elution mode is gradient elution, and the detection wavelength is 254nm. The method provided by the present invention has good repeatability and high durability, and can be used for compound Fufangteng capsule quality control.

Description

Technical Field

[0001] The present 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 existing technology, the fingerprint detection of Fufang Fufang Teng Capsules mainly adopts methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS), and qualitative and quantitative analysis of its main active ingredients (such as flavonoids, phenols, saponins, etc.) is performed to characterize its chemical composition and quality consistency. As a comprehensive quality control method, fingerprints can reflect the overall chemical characteristics of traditional Chinese medicine compound preparations, and therefore play an important role in the quality evaluation of traditional Chinese medicine. However, there are still some problems in the existing technology: First, the ingredients of Fufang Fufang Teng Capsules are complex and the matrix interference is large, resulting in poor separation of some components and difficulty in fully reflecting their chemical characteristics; second, the existing fingerprints are mostly based on a single detection method and lack 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 still imperfect, 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 insufficiently studied. Existing research has focused on qualitative and quantitative analysis of chemical components, but lacks in-depth exploration 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 existing technology, the establishment of the common pattern of the HPLC fingerprint of compound Fufang Teng mixture by Qin Jieping et al. and its application in preparation quality control, as well as the research on the HPLC fingerprint determination method of compound Fufang Teng mixture by Qin Jieping et al., disclose the use of HPLC method to detect the fingerprint of compound Fufang Teng 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 technicians 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 Fufangteng Capsules and its application.

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

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

[0008] 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 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 isoflavone glucoside was 0.0202 mg·mL -1 The concentration of formononetin was 0.0088 mg·mL -1 The concentration of calycosin isoflavones was 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 their shells and ground into powder, and then methanol is added to the obtained compound fufangteng capsule powder for ultrasonic extraction. After the extraction is completed, the sample 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 present 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, and uses an acetonitrile-0.2% phosphoric acid solution as a mobile phase, while limiting the specific conditions of gradient elution to analyze and detect the fingerprint of compound Fufangteng capsules. The HPLC fingerprint of the compound Fufangteng capsules is established, 20 common peaks are determined, 5 common peaks are identified, and the similarities of 15 batches of samples are all greater than 0.90. The method provided by the present invention has good repeatability and can be used for quality control of compound Fufangteng capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0023] Figure 1 This is an overlay of HPLC images of 15 batches of Fufangteng capsule samples;

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

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

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts 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] Isoflavone glucoside was purchased from the China Food and Drug Identification Institute (Batch No. 111920-202308);

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

[0032] 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] 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 capsules (0.37g capsules) of Fufangteng capsule samples from each batch. -1 ), remove the capsule shell, grind finely, accurately weigh 1.0 g of powder, place it in a 50 mL conical flask, accurately add 25 mL of methanol, weigh it, extract it by ultrasonic for 30 min, let it cool, weigh it again, make up the lost amount with methanol, shake it well, filter it, and pass the filtrate through a 0.45 μm 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 reference substances, respectively, and place them in a 10 mL volumetric flask. Accurately weigh 17.94 mg and 19.12 mg of calycosin glucoside and formononetin standard reference substances, respectively, and place them in a 20 mL volumetric flask. Methanol was added to dissolve and make up the volume to obtain the stock solution of each single reference substance.

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

[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] The above 15 batches of test sample solutions were measured under the above chromatographic conditions and the chromatograms were recorded. The chromatogram data of the 15 batches of samples were imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine (2012A Edition)", and S1 was set as the reference spectrum. After multi-point correction and Mark peak matching, the fingerprints and common patterns (R) of the 15 batches of Fufangteng Capsules were constructed. The fingerprints had 20 common peaks. By comparing with standard solutions, five components were identified: protocatechuic acid ((Peak 7)), calycosin glucoside (Peak 14), formononetin (Peak 17), calycosin (Peak 18), and formononetin (Peak 20); 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 batches of 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 batches of samples (2)

[0065]

[0066]

[0067] (5) Similarity evaluation

[0068] The similarity of the HPLC chromatograms of 15 batches of Fufangteng Capsules was calculated using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)." The results showed that the similarity of the chromatograms of each batch of Fufangteng Capsules sample was greater than 0.9 compared to the control fingerprint. The results are shown in Tables 5 and 6, indicating that the chromatograms of the Fufangteng Capsules sample batches were highly similar.

[0069] Table 5 Similarity of HPLC chromatograms of 15 batches of Fufangteng capsules (1)

[0070]

[0071]

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

[0073]

[0074] Technical effect:

[0075] 1. Precision test

[0076] Accurately weigh 10 Fufangteng capsules (S1) (0.37g / 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 according to the chromatographic conditions of step (3) 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, and 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 precision is good. 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 Common peak precision experiment - relative peak area (n = 6)

[0080]

[0081]

[0082] 2. Stability test

[0083] Accurately weigh 10 Fufangteng capsules (S1) (0.37g / 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 the sample at 0h, 2h, 4h, 8h, 12h, and 24h according to the chromatographic conditions of step (3) 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, and the RSD value range of the relative retention time of each common peak is 0.09% to 0.60%, and the RSD value range of the relative peak area 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) (0.37g·capsule -1), remove the capsule shell, grind it into powder, accurately weigh 1.0g of powder, a total of 6 parts, 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 of each common peak ranges from 0.11% to 0.60%, and the relative peak area RSD value ranges from 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 reproducibility 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 merely preferred embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules, characterized in that: The HPLC fingerprint of Fufangteng Capsule was established by using the mixed solution of protocatechuic acid, calycosin glucoside, formononetin, calycosin and formononetin as the standard solution and the methanol extract of Fufangteng Capsule as the test solution. The high performance liquid chromatography analysis used acetonitrile-0.2% phosphoric acid solution as the mobile phase, the elution mode was gradient elution, and the detection wavelength was 254 nm; 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; Wherein, A is acetonitrile and B is phosphoric acid solution; The chromatographic column used in the HPLC analysis was Acclaim TM 120 C18 column.

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

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

4. The high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules according to claim 3, characterized in that: The ratio of the added amount of the compound Fufangteng capsule powder to methanol is 1g:25mL.

5. The high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules according to claim 3, characterized in that: Before the filtration, the method 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.

6. The high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules according to claim 3, characterized in that: The filtration is through a 0.45 μm microporous filter membrane.

7. The high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules according to claim 1, characterized in that: The conditions for the HPLC analysis were: injection volume 10 μL, flow rate 1.0 mL·min -1 , column temperature 30℃.

8. Use of the high performance liquid chromatography fingerprint analysis method for Fufangteng Capsules according to any one of claims 1 to 7 in the quality control of Fufangteng Capsules.

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