A method for establishing HPLC fingerprint of a traditional Chinese medicine composition and the application of the fingerprint.

Abstract

This invention discloses a method for establishing an HPLC fingerprint of a traditional Chinese medicine composition. The method includes the preparation of a reference standard and a test solution, the determination of HPLC chromatographic conditions, and the preparation of an HPLC standard fingerprint. The HPLC standard fingerprint of the traditional Chinese medicine composition obtained by this method is also disclosed. This fingerprint has 20 peaks, with 14 common peaks identified by the reference standard. The method is simple, stable, and reproducible, and the obtained chromatogram has comprehensive characteristic peaks. By comparing the common peaks of the standard fingerprint, the quality of the traditional Chinese medicine composition can be comprehensively evaluated and controlled, which is beneficial for accurately assessing the intrinsic quality of the preparation and ensuring its safety and efficacy.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation analysis. This invention discloses a method for establishing HPLC fingerprint of traditional Chinese medicine composition and the application of the fingerprint. Background Technology

[0002] The traditional Chinese medicine composition consists of eight herbs: American ginseng, Poria cocos, Ziziphus jujuba seed, mulberry, wolfberry, raspberry, Cornus officinalis fruit, and tangerine peel. It has the effects of tonifying the kidneys and replenishing essence, nourishing yin and marrow, calming the mind and soothing the nerves, thereby enhancing memory and anti-aging.

[0003] Traditional Chinese medicine preparations contain multiple herbs and have complex chemical compositions; therefore, effective quality control is crucial for ensuring their clinical efficacy. To guarantee and stabilize product quality, a comprehensive study of quality testing and control methods is necessary.

[0004] This patent provides a method for establishing a fingerprint spectrum of a traditional Chinese medicine composition. The HPLC fingerprint spectrum of the traditional Chinese medicine composition has 20 common peaks, which can effectively reflect the integrity and complexity of the chemical components of the traditional Chinese medicine composition, and comprehensively evaluate and control the quality of the preparation, thereby ensuring its safety and efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a method for establishing fingerprint spectra of traditional Chinese medicine (TCM) compositions. Specifically, this method prepares the TCM composition into a test solution, which is then separated and detected by HPLC to obtain a standard fingerprint spectrum of the TCM composition. This enables comprehensive and multi-faceted detection, improves the quality testing system, and demonstrates good stability and durability. Furthermore, it provides a reliable basis for identifying the authenticity and intrinsic quality of TCM compositions, ensuring their effectiveness.

[0006] The present invention discloses a method for establishing an HPLC fingerprint of a traditional Chinese medicine composition, characterized by comprising the following steps.

[0007] 1) Preparation of reference solutions: Weigh appropriate amounts of 5-hydroxymethylfurfural, monoglycine, chlorogenic acid, cryptochlorogenic acid, loganin, scopolamine, spinosin, rutin, kaempferol-3-O-rutinoside, hesperidin, tiolinin, ginsenoside Rb1, nobiletin and tangeretin reference standards, and dissolve them in methanol to prepare reference solutions.

[0008] 2) Preparation of test solution: Grind the Chinese herbal medicine composition into a fine powder, weigh accurately, add 25-50 ml of 80%-100% methanol, extract by ultrasonication for 10-30 min, cool, filter, and collect the filtrate to obtain the test solution.

[0009] 3) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A and 0.1%~0.3% phosphoric acid solution was used as mobile phase B, with gradient elution; detection wavelength: 203~240nm; flow rate: 0.5~1.0ml / min; column temperature: 20℃~40℃.

[0010] 4) Determination method: Inject the reference solution and the test solution into the chromatographic conditions of step 3) and record the chromatograms. Obtain the HPLC standard fingerprint of the traditional Chinese medicine composition composed of common characteristic peaks of the samples.

[0011] 5) Fingerprint chromatogram preparation: Import the chromatogram obtained in step (4) into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system for data processing to generate a standard fingerprint chromatogram.

[0012] Preferably, the test solution in step 2) is prepared as follows: take the traditional Chinese medicine composition, grind it into a fine powder, take about 2.0g, accurately weigh it, add 25ml of methanol, sonicate for 30min, cool, filter, and take the filtrate to obtain the test solution.

[0013] Preferably, the chromatographic column in step 2) is a Dikma Technologies Kromasil 100Å C18 column.

[0014] Further preferably, the chromatographic column has a size of 4.6 × 250 mm and a diameter of 5 μm.

[0015] Preferably, the mobile phase in step 2) is a 0.1% phosphoric acid solution.

[0016] Step 2) The gradient elution conditions are as follows:

[0017] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0~10 2→5 98→95 10~11 5→10 95→90 11~70 10→20 90→80 70-110 20→38 80→62 110-130 38→50 62→50 130-130.1 50→2 50→98 130.1-150 2 98

[0018] Preferably, the detection wavelength is 240nm for 0~35min and 203nm for 35.1~150min; the flow rate is 1.0ml / min and the column temperature is 30℃.

[0019] Another objective of this invention is to provide a standard HPLC fingerprint of the traditional Chinese medicine composition. Ten batches of traditional Chinese medicine composition samples were prepared into test solutions according to the above method, separated and detected by HPLC, and analyzed using the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission to obtain the standard HPLC fingerprint of the traditional Chinese medicine composition.

[0020] The fingerprint spectrum includes 20 peaks, with hesperidin as the reference peak. The ratio of the retention time of each common peak to the retention time of the reference peak is calculated and should be within ±10% of the specified value. The specified values ​​are: 0.194, 0.308, 0.332, 0.356, 0.427, 0.594, 0.698, 0.714, 0.725, 0.877, 1.210, 1.338, 1.360, 1.495, 1.555, 1.572, 1.671, 1.780, 1.897.

[0021] By comparing with reference standards, 14 peaks were identified as follows: peak 1 is 5-hydroxymethylfurfural, peak 2 is monoglycine, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 5 is loganin, peak 6 is scopolamine lactone, peak 8 is spinosin, peak 9 is rutin, peak 10 is kaempferol-3-O-rutin, peak 11 is hesperidin, peak 14 is linaloside, peak 16 is ginsenoside Rb1, peak 19 is norihesperidin, and peak 20 is tangeretin.

[0022] The fingerprint spectrum detection method established in this invention is used to detect samples. The standard for quality control of traditional Chinese medicine compositions is that the similarity between the sample to be tested and the standard fingerprint spectrum is not less than 0.90.

[0023] This invention is the first to construct an HPLC fingerprint detection method for a traditional Chinese medicine composition. Based on the gradient of the prescription components of the traditional Chinese medicine composition, the optimal HPLC conditions are selected, and the obtained fingerprint spectrum has high resolution, good peak shape, and good separation of each characteristic chromatographic peak. It has good stability and many characteristic peaks, and can comprehensively and accurately evaluate the quality of the traditional Chinese medicine composition. It can be used for the quality control of traditional Chinese medicine composition products.

[0024] The detection method of this invention uses hesperidin as a reference peak and identifies 20 chromatographic peaks that constitute the common peaks of the fingerprint spectrum of the traditional Chinese medicine composition. From the common peaks, 14 characteristic peaks are identified, including 5-hydroxymethylfurfural, monoglucoside, chlorogenic acid, cryptochlorogenic acid, loganin, scopolamine, spinosin, rutin, kaempferol-3-O-rutinoside, hesperidin, tiolinin, ginsenoside Rb1, nobiletin, and citrusin, making the fingerprint spectrum composition richer and more comprehensive.

[0025] The HPLC fingerprint chromatogram of traditional Chinese medicine composition established in this invention overcomes the shortcomings of existing technologies, such as the single detection index and inability to reflect intrinsic quality. The method for establishing HPLC fingerprint chromatograms of traditional Chinese medicine composition provided has high precision and good reproducibility. By comparing the presence or absence of common peaks in the obtained fingerprint chromatograms, the quality of traditional Chinese medicine composition can be comprehensively evaluated, effectively ensuring the quality of the finished product. Attached Figure Description

[0026] Figure 1HPLC mixed control chromatogram of traditional Chinese medicine composition

[0027] Among them, 5-hydroxymethylfurfural (peak 1), mononoside (peak 2), chlorogenic acid (peak 3), cryptochlorogenic acid (peak 4), loganin (peak 5), scopolamine (peak 6), spinosin (peak 7), rutin (peak 8), kaempferol-3-O-rutin (peak 9), hesperidin (peak 10), tiolinin (peak 11), ginsenoside Rb1 (peak 12), norihesperidin (peak 13), and tangeretin (peak 14).

[0028] Figure 2 HPLC standard fingerprint of traditional Chinese medicine composition

[0029] Figure 2 The peaks marked 1-20 are 20 common peaks, including 5-hydroxymethylfurfural (peak 1), mononoside (peak 2), chlorogenic acid (peak 3), cryptochlorogenic acid (peak 4), loganin (peak 5), scopolamine (peak 6), spinosin (peak 8), rutin (peak 9), kaempferol-3-O-rutin (peak 10), hesperidin (peak 11), tiolinin (peak 14), ginsenoside Rb1 (peak 16), norihesperidin (peak 19), and tangeretin (peak 20).

[0030] Figure 3 HPLC fingerprint overlay of 10 batches of traditional Chinese medicine compositions

[0031] Among them are 5-hydroxymethylfurfural (peak 1), mononoside (peak 2), chlorogenic acid (peak 3), cryptochlorogenic acid (peak 4), loganin (peak 5), scopolamine (peak 6), spinosin (peak 8), rutin (peak 9), kaempferol-3-O-rutin (peak 10), hesperidin (peak 11), tiolinin (peak 14), ginsenoside Rb1 (peak 16), norihesperidin (peak 19), and tangeretin (peak 20). Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this application clearer and more explicit, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. It is obvious that the following embodiments are part of, but not all of, the implementation details of this application. Therefore, the detailed description of the embodiments below does not limit the scope of protection claimed in this application, and all equivalent substitutions or modifications made by those skilled in the art without inventive effort should be included within the scope of this invention.

[0033] Example 1: Establishment of HPLC standard fingerprint of traditional Chinese medicine composition

[0034] 1. Instruments and reagents

[0035] 1.1 Instruments

[0036] ACCHROM Organizer S3000 (Huapu S3000) 3410UV detector; AK-040SD ultrasonic cleaner (Shenzhen Yujie Cleaning Equipment Co., Ltd.); Balance ESJ30-5A (Shanghai Precision Instruments Co., Ltd.).

[0037] 1.2 Test Drugs

[0038] Ten batches of traditional Chinese medicine compositions were collected. The sample batch numbers are shown in Table 1. Acetonitrile was of chromatographic grade, water was purified water, and all other reagents were of analytical grade.

[0039] Table 1. Batch numbers of test samples of traditional Chinese medicine compositions

[0040] sample batch number sample batch number S1 20251001 S6 20251202 S2 20251002 S7 20260101 S3 20251003 S8 20260102 S4 20251101 S9 20260201 S5 20251201 S10 20260202

[0041] 2. Methods and Results

[0042] 2.1 Chromatographic conditions: Dikma Technologies Kromasil 100Å C18 column (5μm, 4.6×250mm), column temperature 30℃, flow rate 1.0ml / min, detection wavelength 240nm for 0~35min, 203nm for 35.1~150min, injection volume 10μl; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B. Gradient elution was performed according to the table below.

[0043] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0~10 2→5 98→95 10~11 5→10 95→90 11~70 10→20 90→80 70-110 20→38 80→62 110-130 38→50 62→50 130-130.1 50→2 50→98 130.1-150 2 98

[0044] 2.2 Preparation of reference and test solutions.

[0045] Accurately weigh appropriate amounts of 5-hydroxymethylfurfural, chlorogenic acid, cryptochlorogenic acid, monoglucoside, loganin, scopolamine lactone, spinosin, rutin, kaempferol-3-O-rutinoside, hesperidin, tiolinin, ginsenoside Rb1, nobiletin, and tangeretin reference standards, and dissolve them in methanol to prepare reference solutions.

[0046] Grind the Chinese herbal medicine composition into a fine powder, take about 2.0g, accurately weigh it, add 25ml of methanol, sonicate for 30min, cool, filter, and collect the filtrate to obtain the final product.

[0047] 2.3 Establishment of fingerprint patterns.

[0048] The above-mentioned reference solution and test solution were injected separately into an HPLC chromatographic system to determine the HPLC fingerprint chromatograms of 10 batches of traditional Chinese medicine compositions. The results were analyzed and compared to obtain the HPLC standard fingerprint chromatograms of the traditional Chinese medicine compositions composed of their common characteristic peaks. The chromatogram of the reference solution is shown below. Figure 1As shown, the HPLC standard fingerprint of the traditional Chinese medicine composition is as follows: Figure 2 As shown, with the hesperidin peak (11) as the reference peak, the relative retention time (t) of the common peaks in the standard fingerprint spectrum was calculated. R The specified values ​​are: 0.196, 0.309, 0.333, 0.358, 0.427, 0.597, 0.700, 0.714, 0.725, 0.878, 1.213, 1.338, 1.362, 1.497, 1.556, 1.574, 1.673, 1.784, and 1.902. The calculated relative retention time of the sample should be within ±10% of the specified value.

[0049] Among them, 5-hydroxymethylfurfural (peak 1), mononoside (peak 2), chlorogenic acid (peak 3), cryptochlorogenic acid (peak 4), loganin (peak 5), scopolamine (peak 6), spinosin (peak 8), rutin (peak 9), kaempferol-3-O-rutin (peak 10), hesperidin (peak 11), tiolinin (peak 14), ginsenoside Rb1 (peak 16), norihesperidin (peak 19), and tangeretin (peak 20).

[0050] HPLC fingerprint data of 10 batches of traditional Chinese medicine (TCM) compositions were imported into the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" software recommended by the National Pharmacopoeia Commission for chromatographic peak matching. Twenty common peaks were identified as characteristic peaks of the TCM composition fingerprints. The hesperidin peak (No. 11) was used as the reference peak. The relative retention times of the common peaks in the 10 batches of TCM composition samples are shown in Table 2, and the relative peak areas are shown in Table 3. The similarity calculation results between the 10 batches of TCM compositions and the standard fingerprint chromatograms were 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, respectively. Considering the source of the medicinal materials and the impact of large-scale production, the similarity limit between this product and the reference fingerprint chromatogram was set as follows: according to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicines, the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram should not be lower than 0.90.

[0051] The overlay of HPLC fingerprint chromatograms of 10 batches of traditional Chinese medicine compositions is shown below. Figure 3 As shown.

[0052] Table 2. Relative retention times (t) of common peaks in 10 batches of traditional Chinese medicine compositions R )

[0053] Sample peak number S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 RSD (%) 1 0.196 0.196 0.195 0.195 0.196 0.196 0.196 0.196 0.196 0.196 0.2 2 0.309 0.309 0.308 0.309 0.310 0.310 0.310 0.309 0.310 0.309 0.05 3 0.334 0.333 0.332 0.333 0.334 0.334 0.334 0.333 0.334 0.333 0.06 4 0.358 0.357 0.356 0.357 0.358 0.359 0.358 0.358 0.358 0.358 0.09 5 0.427 0.427 0.426 0.427 0.426 0.428 0.428 0.427 0.427 0.427 0.2 6 0.595 0.595 0.594 0.596 0.596 0.597 0.597 0.596 0.596 0.597 0.16 7 0.697 0.699 0.698 0.697 0.700 0.701 0.701 0.700 0.697 0.700 0.22 8 0.711 0.715 0.714 0.712 0.716 0.717 0.716 0.714 0.711 0.714 0.3 9 0.722 0.725 0.724 0.723 0.726 0.727 0.727 0.725 0.722 0.725 0.27 10 0.876 0.878 0.877 0.877 0.876 0.879 0.877 0.878 0.876 0.878 0.24 11 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.2 12 1.213 1.212 1.209 1.211 1.216 1.216 1.216 1.213 1.215 1.213 0.08 13 1.340 1.335 1.334 1.337 1.342 1.341 1.341 1.337 1.340 1.338 0.04 14 1.363 1.358 1.358 1.360 1.366 1.365 1.364 1.361 1.363 1.362 0.04 15 1.499 1.495 1.495 1.496 1.502 1.501 1.500 1.496 1.498 1.497 0.07 16 1.559 1.555 1.555 1.556 1.561 1.561 1.560 1.555 1.558 1.556 0.09 17 1.576 1.572 1.571 1.573 1.579 1.578 1.577 1.573 1.576 1.574 0.07 18 1.675 1.671 1.670 1.672 1.678 1.677 1.676 1.672 1.675 1.673 0.06 19 1.786 1.782 1.781 1.783 1.789 1.788 1.788 1.783 1.786 1.784 0.06 20 1.905 1.900 1.898 1.900 1.908 1.907 1.907 1.901 1.905 1.902 0.04

[0054] Table 3. Relative peak area (%) of common peaks in 10 batches of traditional Chinese medicine compositions

[0055] Sample peak number S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 RSD (%) 1 0.013 0.012 0.012 0.011 0.012 0.013 0.012 0.012 0.012 0.010 8.22 2 0.289 0.313 0.291 0.313 0.312 0.304 0.288 0.311 0.297 0.286 3.39 3 0.028 0.028 0.027 0.028 0.027 0.027 0.027 0.026 0.026 0.029 2.86 4 0.010 0.011 0.011 0.010 0.011 0.011 0.011 0.011 0.010 0.011 4.4 5 0.139 0.138 0.137 0.133 0.138 0.138 0.139 0.138 0.138 0.139 1.93 6 0.021 0.023 0.023 0.024 0.022 0.021 0.021 0.022 0.019 0.021 5.52 7 0.055 0.057 0.056 0.057 0.058 0.058 0.057 0.059 0.055 0.058 1.96 8 0.026 0.023 0.022 0.023 0.024 0.023 0.024 0.025 0.025 0.025 6.36 9 0.092 0.090 0.089 0.092 0.090 0.091 0.093 0.091 0.092 0.091 1.59 10 0.104 0.102 0.094 0.102 0.100 0.102 0.103 0.100 0.099 0.100 2.61 11 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.07 12 0.038 0.033 0.037 0.036 0.036 0.037 0.036 0.035 0.034 0.041 6.28 13 0.039 0.039 0.038 0.040 0.040 0.040 0.040 0.039 0.037 0.037 1.7 14 0.022 0.023 0.022 0.025 0.025 0.025 0.022 0.023 0.023 0.021 5.12 15 0.022 0.022 0.018 0.022 0.022 0.022 0.017 0.020 0.020 0.021 8.43 16 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.015 0.014 0.014 1.89 17 0.012 0.013 0.012 0.012 0.014 0.013 0.012 0.011 0.011 0.013 5.9 18 0.015 0.016 0.015 0.016 0.016 0.016 0.016 0.014 0.015 0.014 4.59 19 0.137 0.136 0.135 0.137 0.137 0.137 0.138 0.136 0.135 0.135 0.88 20 0.051 0.050 0.049 0.051 0.051 0.051 0.050 0.046 0.049 0.050 2.83

[0056] Table 4. Calculation results of common peak similarity for 10 batches of traditional Chinese medicine compositions.

[0057] sample S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S1 1.000 S2 1.000 S3 1.000 1.000 S4 1.000 1.000 1.000 S5 1.000 1.000 1.000 1.000 S6 1.000 1.000 1.000 1.000 1.000 S7 1.000 1.000 1.000 1.000 1.000 1.000 S8 1.000 1.000 1.000 1.000 1.000 1.000 .000 S9 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 S10 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Comparison of fingerprint patterns 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0058] 2.4 Methodological examination.

[0059] 2.4.1 Precision test.

[0060] Take sample (20260101), prepare test solution according to the method in section 2.2, and inject continuously 6 times. Using the hesperidin peak No. 11 as the reference peak, calculate the RSD% of the relative retention time of the common chromatographic peaks as <0.6% and the RSD% of the relative peak area as <5.0%. At the same time, use the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software to calculate that the similarity of the fingerprint spectrum of each chromatographic peak is greater than 0.99, indicating that the instrument is stable and has good precision.

[0061] Table 5. Relative retention times (tR) of common peaks in precision experiments.

[0062] Serial number Peak number 1 2 3 4 5 6 mean RSD (%) S1 0.197 0.197 0.197 0.197 0.196 0.196 0.197 0.508 S2 0.309 0.309 0.309 0.309 0.309 0.309 0.309 0.000 S3 0.334 0.334 0.334 0.334 0.333 0.333 0.334 0.299 S4 0.359 0.359 0.359 0.359 0.358 0.358 0.359 0.279 S5 0.426 0.426 0.426 0.426 0.426 0.426 0.426 0.000 S6 0.598 0.598 0.598 0.598 0.597 0.596 0.598 0.167 S7 0.701 0.701 0.701 0.701 0.700 0.699 0.701 0.143 S8 0.714 0.714 0.714 0.714 0.714 0.714 0.714 0.000 S9 0.725 0.725 0.725 0.725 0.725 0.725 0.725 0.000 S10 0.878 0.878 0.878 0.878 0.878 0.878 0.878 0.000 S12 1.214 1.214 1.214 1.215 1.213 1.213 1.214 0.082 S13 1.338 1.338 1.338 1.339 1.338 1.338 1.338 0.000 S14 1.362 1.362 1.362 1.363 1.361 1.362 1.362 0.073 S15 1.496 1.496 1.496 1.497 1.495 1.496 1.496 0.067 S16 1.555 1.555 1.555 1.556 1.555 1.555 1.555 0.000 S17 1.573 1.573 1.573 1.574 1.573 1.573 1.573 0.000 S18 1.673 1.673 1.673 1.674 1.672 1.673 1.673 0.060 S19 1.784 1.784 1.784 1.786 1.783 1.784 1.784 0.056 S20 1.903 1.903 1.903 1.904 1.905 1.906 1.904 0.053

[0063] Table 6. Relative peak area (%) of each common peak in the precision experiment.

[0064] Serial number Peak number S1 S2 S3 S4 S5 S6 RSD (%) S1 0.012 0.013 0.013 0.012 0.012 0.012 1.560 S2 0.289 0.286 0.286 0.287 0.287 0.290 1.060 S3 0.027 0.028 0.028 0.028 0.028 0.028 0.810 S4 0.012 0.012 0.012 0.011 0.012 0.012 1.680 S5 0.124 0.137 0.137 0.138 0.136 0.123 4.830 S6 0.017 0.020 0.018 0.018 0.018 0.018 4.890 S7 0.051 0.054 0.052 0.053 0.053 0.051 1.960 S8 0.026 0.025 0.026 0.025 0.027 0.025 2.930 S9 0.090 0.090 0.091 0.091 0.091 0.091 0.760 S10 0.102 0.103 0.103 0.103 0.104 0.102 0.710 S11 1.000 1.000 1.000 1.000 1.000 1.000 0.520 S12 0.044 0.041 0.042 0.043 0.044 0.043 3.450 S13 0.038 0.039 0.036 0.039 0.038 0.039 2.660 S14 0.020 0.023 0.021 0.022 0.022 0.021 3.900 S15 0.022 0.020 0.021 0.021 0.021 0.020 4.490 S16 0.016 0.015 0.016 0.015 0.016 0.016 3.100 S17 0.012 0.013 0.012 0.012 0.012 0.011 4.470 S18 0.016 0.015 0.015 0.015 0.016 0.016 3.280 S19 0.133 0.137 0.137 0.137 0.136 0.135 0.940 S20 0.052 0.053 0.054 0.052 0.053 0.052 1.280

[0065] Table 7. Similarity Results of Precision Experiments

[0066] Serial Number S1 S2 S3 S4 S5 S6 S1 1.000 S2 1.000 S3 1.000 1.000 S4 1.000 1.000 1.000 S5 1.000 1.000 1.000 1.000 S6 1.000 1.000 1.000 1.000 1.000 Comparison of fingerprint patterns 1.000 1.000 1.000 1.000 1.000 1.000

[0067] 2.4.2 Stability test.

[0068] Take sample (20260101), prepare test solution according to the method in section 2.2, and inject at 0, 3, 6, 9, 12, 15, 18, 21, and 24 h respectively. Using hesperidin peak No. 11 as reference peak, calculate the RSD% of relative retention time of common chromatographic peaks as <0.5% and the RSD% of relative peak area as <5.0%. At the same time, use the 2012 version of the Chinese herbal chromatographic fingerprint similarity evaluation system software to calculate that the similarity of fingerprint chromatographic peaks is greater than 0.99, indicating that the test solution is stable within 24 hours.

[0069] Table 8. Relative retention times of common peaks in stability experiments (t) R )

[0070] Serial number Peak number 0h 3h 6h 9h 12h 15h 18h 21h 24h RSD (%) S1 0.195 0.196 0.196 0.196 0.196 0.196 0.196 0.196 0.195 0.000 S2 0.309 0.309 0.309 0.309 0.309 0.309 0.309 0.309 0.309 0.000 S3 0.333 0.334 0.334 0.334 0.334 0.333 0.333 0.333 0.333 0.003 S4 0.358 0.358 0.359 0.359 0.358 0.358 0.358 0.357 0.358 0.003 S5 0.426 0.427 0.427 0.427 0.426 0.426 0.426 0.427 0.426 0.002 S6 0.596 0.597 0.597 0.597 0.597 0.597 0.597 0.596 0.596 0.002 S7 0.698 0.699 0.699 0.700 0.699 0.699 0.699 0.698 0.698 0.001 S8 0.713 0.713 0.713 0.714 0.713 0.713 0.713 0.714 0.713 0.000 S9 0.724 0.724 0.725 0.725 0.725 0.724 0.724 0.724 0.724 0.001 S10 0.878 0.877 0.878 0.878 0.877 0.878 0.877 0.877 0.877 0.001 S11 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 S12 1.213 1.213 1.213 1.213 1.215 1.213 1.214 1.213 1.213 0.001 S13 1.338 1.338 1.338 1.339 1.339 1.338 1.340 1.339 1.339 0.001 S14 1.361 1.361 1.362 1.362 1.363 1.361 1.363 1.362 1.362 0.001 S15 1.495 1.495 1.496 1.496 1.497 1.495 1.498 1.497 1.496 0.001 S16 1.554 1.554 1.555 1.555 1.555 1.554 1.557 1.556 1.556 0.001 S17 1.572 1.572 1.573 1.574 1.574 1.573 1.575 1.574 1.574 0.001 S18 1.672 1.672 1.673 1.673 1.674 1.672 1.674 1.673 1.673 0.001 S19 1.783 1.783 1.784 1.784 1.785 1.783 1.785 1.784 1.784 0.001 S20 1.901 1.902 1.903 1.903 1.904 1.902 1.904 1.903 1.903 0.001

[0071] Table 9. Relative peak area (%) of each common peak in the stability test

[0072] Serial number Peak number 0h 3h 6h 9h 12h 15h 18h 21h 24h RSD (%) S1 0.012 0.012 0.013 0.012 0.013 0.013 0.012 0.012 0.012 1.580 S2 0.289 0.286 0.289 0.288 0.286 0.286 0.287 0.287 0.290 0.860 S3 0.027 0.028 0.028 0.028 0.028 0.028 0.028 0.028 0.028 0.940 S4 0.012 0.012 0.012 0.012 0.012 0.012 0.011 0.012 0.012 1.510 S5 0.124 0.133 0.137 0.133 0.137 0.137 0.138 0.136 0.123 3.940 S6 0.017 0.018 0.019 0.019 0.020 0.018 0.018 0.018 0.018 4.140 S7 0.051 0.052 0.052 0.051 0.054 0.052 0.053 0.053 0.051 1.720 S8 0.026 0.026 0.025 0.024 0.025 0.026 0.025 0.027 0.025 2.850 S9 0.090 0.091 0.091 0.091 0.090 0.091 0.091 0.091 0.091 0.660 S10 0.102 0.103 0.103 0.103 0.103 0.103 0.103 0.104 0.102 0.560 S11 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.410 S12 0.044 0.042 0.044 0.043 0.041 0.042 0.043 0.044 0.043 3.040 S13 0.038 0.038 0.038 0.038 0.039 0.036 0.039 0.038 0.039 2.140 S14 0.020 0.022 0.022 0.022 0.023 0.021 0.022 0.022 0.021 3.230 S15 0.022 0.021 0.022 0.022 0.020 0.021 0.021 0.021 0.020 4.070 S16 0.016 0.015 0.015 0.016 0.015 0.016 0.015 0.016 0.016 3.640 S17 0.012 0.012 0.012 0.012 0.013 0.012 0.012 0.012 0.011 3.740 S18 0.016 0.016 0.016 0.015 0.015 0.015 0.015 0.016 0.016 3.020 S19 0.133 0.138 0.138 0.137 0.137 0.137 0.137 0.136 0.135 1.040 S20 0.052 0.053 0.052 0.052 0.053 0.054 0.052 0.053 0.052 1.290

[0073] Table 10. Similarity results of stability experiments

[0074] Serial Number 0h 3h 6h 9h 12h 15h 18h 21h 24h 0h 1.000 3h 1.000 6h 1.000 1.000 9h 1.000 1.000 1.000 12h 1.000 1.000 1.000 1.000 15h 1.000 1.000 1.000 1.000 1.000 18h 1.000 1.000 1.000 1.000 1.000 1.000 21h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 24h 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Comparison of fingerprint patterns 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0075] 2.4.3 Reproducibility test.

[0076] Six portions of the same batch sample (20260101) were precisely measured and prepared according to the method in section 2.2. The samples were injected separately, with the hesperidin peak (No. 11) as the reference peak. The RSD% of the relative retention time of the common chromatographic peaks was calculated to be less than 0.3%, and the RSD% of the relative peak area was less than 5%. At the same time, the similarity of the fingerprint chromatograms of each chromatographic peak was calculated to be greater than 0.99 using the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software, indicating that the method has good reproducibility.

[0077] Table 11 Relative retention times of common peaks in reproducibility experiments (t) R )

[0078] Serial number Peak number 1 2 3 4 5 6 mean RSD% S1 0.197 0.197 0.197 0.197 0.196 0.196 0.197 0.262 S2 0.309 0.309 0.309 0.309 0.309 0.309 0.309 0.000 S3 0.334 0.334 0.334 0.334 0.333 0.333 0.334 0.155 S4 0.359 0.359 0.359 0.359 0.358 0.358 0.359 0.144 S5 0.426 0.426 0.426 0.426 0.426 0.426 0.426 0.000 S6 0.597 0.598 0.598 0.598 0.597 0.596 0.597 0.137 S7 0.700 0.700 0.701 0.701 0.700 0.699 0.700 0.108 S8 0.713 0.714 0.714 0.714 0.714 0.714 0.714 0.057 S9 0.725 0.725 0.725 0.725 0.725 0.725 0.725 0.000 S10 0.878 0.878 0.878 0.878 0.878 0.878 0.878 0.000 S11 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 S12 1.213 1.214 1.214 1.215 1.213 1.213 1.214 0.067 S13 1.338 1.338 1.338 1.339 1.338 1.338 1.338 0.031 S14 1.361 1.362 1.362 1.363 1.361 1.362 1.362 0.055 S15 1.494 1.495 1.496 1.497 1.495 1.496 1.496 0.070 S16 1.553 1.554 1.555 1.556 1.555 1.555 1.555 0.066 S17 1.572 1.573 1.573 1.574 1.573 1.573 1.573 0.040 S18 1.671 1.673 1.673 1.674 1.672 1.673 1.673 0.062 S19 1.783 1.784 1.784 1.786 1.783 1.784 1.784 0.061 S20 1.902 1.903 1.903 1.905 1.902 1.903 1.903 0.058

[0079] Table 12 Relative peak area (%) of each common peak in reproducibility experiment

[0080] Serial number Peak number S1 S2 S3 S4 S5 S6 RSD (%) S1 0.012 0.012 0.013 0.012 0.012 0.013 4.56 S2 0.300 0.292 0.295 0.289 0.291 0.289 0.78 S3 0.030 0.030 0.029 0.028 0.027 0.028 3.6 S4 0.013 0.013 0.012 0.012 0.013 0.012 3.01 S5 0.140 0.139 0.142 0.141 0.139 0.141 1.84 S6 0.021 0.021 0.021 0.020 0.020 0.020 0.95 S7 0.045 0.045 0.045 0.046 0.045 0.042 2.57 S8 0.013 0.014 0.014 0.014 0.014 0.014 3.76 S9 0.084 0.083 0.082 0.084 0.082 0.086 2.8 S10 0.103 0.102 0.103 0.104 0.101 0.102 1.65 S11 1.000 1.000 1.000 1.000 1.000 1.000 1.57 S12 0.040 0.041 0.042 0.040 0.040 0.041 2.72 S13 0.040 0.039 0.038 0.039 0.039 0.039 1.69 S14 0.022 0.024 0.024 0.023 0.023 0.022 3.59 S15 0.018 0.020 0.018 0.019 0.020 0.018 4.27 S16 0.015 0.015 0.015 0.015 0.015 0.015 2.43 S17 0.013 0.012 0.013 0.013 0.013 0.013 2.57 S18 0.016 0.015 0.016 0.016 0.016 0.016 3.67 S19 0.137 0.137 0.137 0.137 0.136 0.135 1.08 S20 0.053 0.053 0.053 0.052 0.052 0.051 1.02

[0081] Table 13 Results of Similarity Calculation for Reproducibility Tests

[0082] Serial Number S1 S2 S3 S4 S5 S6 S1 1.000 S2 1.000 S3 1.000 1.000 S4 1.000 1.000 1.000 S5 1.000 1.000 1.000 1.000 S6 1.000 1.000 1.000 1.000 1.000 Comparison of fingerprint patterns 1.000 1.000 1.000 1.000 1.000 1.000

[0083] 2.4.4 Durability assessment.

[0084] 2.4.4.1 Column temperature investigation.

[0085] One sample from the same batch (20260101) was precisely measured and the test solution was prepared according to the method in section 2.2. The column temperature was set to 32, 30 and 28℃, and the sample was injected separately. The hesperidin peak No. 11 was used as the reference peak. The RSD% of the relative retention time of the common chromatographic peak was calculated to be <2.0. At the same time, the similarity of the fingerprint spectrum of each chromatographic peak was calculated to be greater than 0.99 using the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software. This indicates that the method can accurately determine the sample within the column temperature range of 28℃~32℃ and has good robustness.

[0086] Table 14. Results of relative retention time during the durability column temperature test (t) R )

[0087] Parameter peak number Column temperature 32℃ Column temperature 30℃ Column temperature 28℃ RSD (%) S1 0.194 0.194 0.198 1.184 S2 0.311 0.31 0.308 0.493 S3 0.332 0.334 0.336 0.599 S4 0.355 0.359 0.362 0.978 S5 0.426 0.424 0.426 0.272 S6 0.586 0.593 0.608 1.886 S7 0.688 0.695 0.713 1.845 S8 0.711 0.709 0.717 0.585 S9 0.717 0.721 0.733 1.15 S10 0.873 0.876 0.882 0.523 S11 1.000 1.000 1.000 1.000 S12 1.219 1.215 1.207 0.503 S13 1.354 1.342 1.322 1.207 S14 1.375 1.366 1.348 1.009 S15 1.522 1.502 1.469 1.787 S16 1.585 1.562 1.525 1.944 S17 1.597 1.58 1.549 1.545 S18 1.698 1.679 1.647 1.539 S19 1.812 1.791 1.756 1.584 S20 1.932 1.912 1.874 1.546

[0088] Table 15. Similarity Results of Durability Column Temperature Test

[0089] parameter Column temperature 28℃ Column temperature 30℃ Column temperature 32℃ Column temperature 28 1 Column temperature 30 0.999 Column temperature 32 1 0.999 Comparison of fingerprint patterns 1 1 1

[0090] 2.4.4.2 Flow velocity investigation.

[0091] One sample from the same batch (20260101) was precisely measured and prepared according to the method in section 2.2. The flow rates were set to 0.9, 1.0, and 1.1 ml / min, and the samples were injected separately. The relative retention time of the common chromatographic peaks was calculated using the hesperidin peak (No. 11) as the reference peak. Except for peak 1, the RSD% of the other peaks was <2%. At the same time, the similarity of the fingerprint chromatograms of each chromatographic peak was calculated to be greater than 0.99 using the 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software. This indicates that the method can accurately determine the sample within the flow rate range of 0.9~1.1 ml / min and has good robustness.

[0092] Table 16 Results of relative retention times of common peaks in durability flow velocity studies (t) R )

[0093] Flow rate (ml / min) Peak number 0.9 1.0 1.1 RSD% S1 0.206 0.194 0.188 4.592 S2 0.312 0.31 0.305 1.294 S3 0.338 0.334 0.328 1.502 S4 0.362 0.359 0.361 0.554 S5 0.43 0.424 0.421 1.176 S6 0.601 0.593 0.588 1.178 S7 0.702 0.695 0.693 0.717 S8 0.713 0.709 0.712 0.281 S9 0.725 0.721 0.721 0.277 S10 0.88 0.876 0.874 0.342 S11 1.000 1.000 1.000 1.000 S12 1.204 1.215 1.222 0.741 S13 1.319 1.342 1.357 1.419 S14 1.341 1.366 1.383 1.541 S15 1.467 1.502 1.526 2.003 S16 1.521 1.562 1.59 2.246 S17 1.55 1.58 1.612 1.961 S18 1.649 1.679 1.698 1.493 S19 1.759 1.791 1.812 1.511 S20 1.876 1.912 1.934 1.521

[0094] Table 17 Results of peak similarity pair analysis using flow velocity.

[0095] parameter Flow rate 0.9 Flow rate 1.0 Flow rate 1.1 Flow rate 0.9 1 Flow rate 1.0 1 Flow rate 1.1 0.999 1 Comparison of fingerprint patterns 1 1 1

[0096] Example 2: Establishment of HPLC standard fingerprint of traditional Chinese medicine composition.

[0097] 1. Instruments and reagents.

[0098] 1.1 Instruments.

[0099] ACCHROM Organizer S3000 (Huapu S3000) 3410UV detector; AK-040SD ultrasonic cleaner (Shenzhen Yujie Cleaning Equipment Co., Ltd.); Balance ESJ30-5A (Shanghai Precision Instruments Co., Ltd.).

[0100] 1.2 Drug test.

[0101] Ten batches of traditional Chinese medicine compositions were collected. The sample batch numbers are shown in Table 1. Acetonitrile was of chromatographic grade, water was purified water, and all other reagents were of analytical grade.

[0102] 2. Methods and Results

[0103] 2.1 Chromatographic conditions: Dikma Technologies Kromasil 100Å C18 column (5μm, 4.6×250mm), column temperature 30℃, flow rate 1.0ml / min, detection wavelength 240nm for 0~35min, 203nm for 35.1~150min, injection volume 10μl; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B.

[0104] Perform gradient elution according to the table below.

[0105] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0~10 2→5 98→95 10~11 5→10 95→90 11~70 10→20 90→80 70-110 20→38 80→62 110-130 38→50 62→50 130-130.1 50→2 50→98 130.1-150 2 98

[0106] 2.2 Preparation of reference solution and test solution.

[0107] Accurately weigh appropriate amounts of 5-hydroxymethylfurfural, chlorogenic acid, cryptochlorogenic acid, monoglucoside, loganin, scopolamine lactone, spinosin, rutin, kaempferol-3-O-rutin, hesperidin, linaloside, ginsenoside Rb1, and norihesperidin as reference standards, and dissolve them in methanol to prepare reference solutions.

[0108] Grind the Chinese herbal medicine composition into a fine powder, take about 2.0g, accurately weigh it, add 25ml of 80% methanol, sonicate for 10min, cool, filter, and collect the filtrate to obtain the final product.

[0109] 2.3 Establishment of fingerprint patterns.

[0110] The above-mentioned reference solution and test solution were injected separately into an HPLC chromatographic system to determine the HPLC fingerprint chromatograms of 10 batches of traditional Chinese medicine compositions. The results were analyzed and compared to obtain the HPLC standard fingerprint chromatograms of the traditional Chinese medicine compositions composed of their common characteristic peaks. The chromatogram of the reference solution is shown below. Figure 1 As shown, the HPLC standard fingerprint of the traditional Chinese medicine composition is as follows: Figure 2 As shown, with the hesperidin peak (11) as the reference peak, the relative retention time (t) of the common peaks in the standard fingerprint spectrum was calculated. R The values ​​were: 0.195, 0.307, 0.331, 0.356, 0.423, 0.595, 0.696, 0.711, 0.722, 0.877, 1.213, 1.336, 1.359, 1.493, 1.551, 1.571, 1.670, 1.781, and 1.899, respectively. The similarity calculation results between the 10 batches of traditional Chinese medicine compositions and the standard fingerprint spectrum were 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, and 1.000, respectively.

[0111] Example 3: Establishment of HPLC standard fingerprint of traditional Chinese medicine composition.

[0112] 1. Instruments and reagents.

[0113] 1.1 Instruments.

[0114] ACCHROM Organizer S3000 (Huapu S3000) 3410UV detector; AK-040SD ultrasonic cleaner (Shenzhen Yujie Cleaning Equipment Co., Ltd.); Balance ESJ30-5A (Shanghai Precision Instruments Co., Ltd.).

[0115] 1.2 Drug test.

[0116] Ten batches of traditional Chinese medicine compositions were collected. The sample batch numbers are shown in Table 1. Acetonitrile was of chromatographic grade, water was purified water, and all other reagents were of analytical grade.

[0117] 2. Methods and Results

[0118] 2.1 Chromatographic conditions: Dikma Technologies Kromasil 100Å C18 column (5μm, 4.6×250mm), column temperature 30℃, flow rate 1.0ml / min, detection wavelength 240nm for 0~35min, 203nm for 35.1~150min, injection volume 10μl; mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B.

[0119] Perform gradient elution according to the table below.

[0120] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0~10 2→5 98→95 10~11 5→10 95→90 11~70 10→20 90→80 70-110 20→38 80→62 110-130 38→50 62→50 130-130.1 50→2 50→98 130.1-150 2 98

[0121] 2.2 Preparation of reference solution and test solution.

[0122] Accurately weigh appropriate amounts of 5-hydroxymethylfurfural, chlorogenic acid, cryptochlorogenic acid, monoglucoside, loganin, scopolamine lactone, spinosin, rutin, kaempferol-3-O-rutin, hesperidin, linaloside, ginsenoside Rb1, and norihesperidin as reference standards, and dissolve them in methanol to prepare reference solutions.

[0123] Grind the Chinese herbal medicine composition into a fine powder, take about 2.0g, accurately weigh it, add 50ml of methanol, sonicate for 20min, cool, filter, and collect the filtrate to obtain the final product.

[0124] 2.3 Establishment of fingerprint patterns.

[0125] The above-mentioned reference solution and test solution were injected separately into an HPLC chromatographic system to determine the HPLC fingerprint chromatograms of 10 batches of traditional Chinese medicine compositions. The results were analyzed and compared to obtain the HPLC standard fingerprint chromatograms of the traditional Chinese medicine compositions composed of their common characteristic peaks. The chromatogram of the reference solution is shown below. Figure 1 As shown, the HPLC standard fingerprint of the traditional Chinese medicine composition is as follows: Figure 2 As shown, with the hesperidin peak (11) as the reference peak, the relative retention time (t) of the common peaks in the standard fingerprint spectrum was calculated. R The values ​​were: 0.195, 0.308, 0.332, 0.357, 0.426, 0.596, 0.698, 0.713, 0.723, 0.877, 1.213, 1.338, 1.361, 1.495, 1.554, 1.573, 1.672, 1.783, and 1.902, respectively. The similarity calculation results between the 10 batches of traditional Chinese medicine compositions and the standard fingerprint spectrum were 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, 1.000, and 1.000, respectively.

Claims

1. A method for establishing an HPLC fingerprint of a traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition comprises the following raw materials: American ginseng, Poria cocos, Ziziphus jujuba seed, mulberry, wolfberry, raspberry, Cornus officinalis fruit, and dried tangerine peel, and includes the following steps: 1) Preparation of reference solutions: Weigh appropriate amounts of 5-hydroxymethylfurfural, monoglycine, chlorogenic acid, cryptochlorogenic acid, loganin, scopolamine, spinosin, rutin, kaempferol-3-O-rutinoside, hesperidin, linaloside, ginsenoside Rb1, nobiletin and tangeretin reference standards, and dissolve them in methanol respectively to prepare reference solutions; 2) Preparation of the test solution: Grind the traditional Chinese medicine composition into a fine powder, weigh accurately, add 25-50 ml of 80%-100% methanol, extract by ultrasonication for 10-30 min, cool, filter, and take the filtrate to obtain the test solution; 3) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile was used as mobile phase A, and 0.1%~0.3% phosphoric acid solution was used as mobile phase B, with gradient elution; detection wavelength: 203~240nm; flow rate: 0.5~1.0ml / min; column temperature: 20℃~40℃; 4) Determination method: Inject and analyze the reference solution and test solution under the chromatographic conditions of step 3), and record the chromatograms. Obtain the HPLC standard fingerprint of the traditional Chinese medicine composition composed of common characteristic peaks of the samples; 5) Fingerprint generation: The chromatogram obtained in step (4) is imported into the Chinese herbal chromatographic fingerprint similarity evaluation system for data processing to generate a standard fingerprint.

2. The method for establishing according to claim 1, characterized in that, Step 2) The test solution is prepared as follows: Grind the traditional Chinese medicine composition into a fine powder, weigh about 2.0g accurately, add 25ml of methanol, sonicate for 30min, cool, shake well, filter, and take the filtrate to obtain the test solution.

3. The method for establishing according to claim 1, characterized in that, Step 3) The chromatographic column is a Dikma Technologies Kromasil 100Å C18 column.

4. The method for establishing according to claim 1, characterized in that, The mobile phase B mentioned in step 3) is 0.1% phosphoric acid.

5. The method for establishing according to claim 1, characterized in that, Step 3) Gradient elution conditions are as follows: t0~10min, 2→5%A; t10~11, 5%→10%A; t11~70, 10%→20%A; t70~110, 20%→38%A; t110~130, 38%→50%A; t130~130.1, 50%→2%A; t130.1~150, 2%A.

6. The method for establishing according to claim 1, characterized in that, In step 3), the detection wavelength is 240nm for 0~35min and 203nm for 35.1~150min, the flow rate is 1.0ml / min, and the column temperature is 30℃.

7. The method according to any one of claims 1-6, characterized in that, The fingerprint spectrum obtained by the fingerprinting method includes 20 peaks. Using hesperidin as the reference peak, the ratio of the retention time of each common peak to the retention time of the reference peak is calculated. This ratio should be within ±10% of a specified value, which is as follows: 0.194，0.308，0.332，0.356，0.427，0.594，0.698，0.714，0.725，0.877，1.210，1.338，1.360，1.495，1.555，1.572，1.671，1.780，1.897。 8. The method according to any one of claims 1-7, characterized in that, The fingerprint spectrum includes 20 peaks, namely: peak 1 is 5-hydroxymethylfurfural, peak 2 is monoglycine, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 5 is loganin, peak 6 is scopolamine, peak 8 is spinosin, peak 9 is rutin, peak 10 is kaempferol-3-O-rutin, peak 11 is hesperidin, peak 14 is linaloside, peak 16 is ginsenoside Rb1, peak 19 is norihesperidin, and peak 20 is tangeretin.

9. The use of the fingerprint spectrum obtained by the method according to any one of claims 1-8 in the quality control of the traditional Chinese medicine composition.

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