A method for determining the fingerprint of a traditional Chinese medicine composition

Abstract

The application claims a fingerprint spectrum determination method of a traditional Chinese medicine composition, the traditional Chinese medicine composition is composed of the following raw medicinal materials: Ephedra, gypsum, forsythia, radix scutellariae, mulberry bark, bitter apricot kernel, radix peucedani, pinellia, dried tangerine or orange peel, fritillaria, burdock seed, honeysuckle, rhubarb, platycodon, licorice, the fingerprint spectrum determination method calibrates 22 main chromatographic peaks, identifies 14 of them, which are neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoforsythoside A, forsythoside A, hesperidin, baicalin, arctiin, aloe emodin, ammonium glycyrrhizinate, chrysophanol, emodin, rhein, emodin methyl ether.

Description

Technical Field

[0001] This invention relates to a method for determining the fingerprint spectrum of a traditional Chinese medicine composition. It belongs to the field of traditional Chinese medicine detection technology. Background Technology

[0002] Traditional Chinese medicine (TCM) fingerprinting refers to the chromatograms or spectra obtained by appropriate analysis of certain TCM materials or preparations after proper processing. It is a comprehensive and quantifiable identification method based on systematic research into the chemical components of TCM, primarily used to evaluate the authenticity, quality, and stability of TCM materials and semi-finished TCM preparations. Its prominent characteristics are "holistic" and "fuzzy" properties.

[0003] Traditional Chinese medicine (TCM) and its preparations are complex multi-component systems. Therefore, evaluating their quality requires appropriate detection methods that provide rich identification information. However, current methods such as microscopic identification, physicochemical identification, and content determination are insufficient to address this issue. Establishing a fingerprint spectrum for TCM can comprehensively reflect the types and quantities of chemical components contained in TCM and its preparations, thereby providing a holistic description and evaluation of drug quality. This aligns perfectly with the holistic theory of TCM. Furthermore, further research into spectroscopic efficacy can truly integrate the quality of TCM with its efficacy, helping to elucidate the mechanisms of action of TCM. In conclusion, the research and establishment of TCM fingerprint spectra are of great significance for improving the quality of TCM and promoting its modernization.

[0004] Currently, fingerprinting technology for traditional Chinese medicine (TCM) involves numerous methods, including chromatographic methods such as thin-layer chromatography (TLCS), high-performance liquid chromatography (HPLC), gas chromatography (GC), and high-performance capillary electrophoresis (HPCE), as well as spectroscopic methods such as ultraviolet (UV) spectroscopy, infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance (NMR), and X-ray diffraction. Chromatographic methods are the mainstream, especially HPLC, TLCS, and GC, which have become three recognized routine analytical techniques. HPLC is favored for its high separation efficiency, high selectivity, high detection sensitivity, fast analysis speed, and wide application range; most TCM components can be analyzed and detected on HPLC, and there is considerable experience in its application. Therefore, HPLC has become the preferred method for TCM fingerprinting technology. With the application of coupled techniques such as HPLC-MS and GC-MS, TCM fingerprinting technology is becoming increasingly sophisticated.

[0005] Patent application number 200810089447.5, entitled "A Drug for Treating Bronchitis Containing Ephedra and Its Preparation Method," discloses a traditional Chinese medicine composition containing ephedra, forsythia, bitter almond, and pinellia, and its preparation method. This application, as a quality control method for this traditional Chinese medicine composition, examines its fingerprint spectrum determination. This determination method has not been previously disclosed. Using this fingerprint spectrum to control the quality of this marketed product effectively ensures the safety of medication for the public. Summary of the Invention

[0006] This invention provides a method for determining the fingerprint spectrum of a traditional Chinese medicine composition.

[0007] The fingerprint spectrum determination method is as follows: Preparation of test solution: Take 0.5-1.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10-50mL of 40-100% methanol, weigh it, sonicate for 20-40 minutes, cool it, weigh it again, replenish the lost weight with 40-100% methanol, shake well, filter it, and take the filtrate to obtain the test solution; Preparation of reference solutions: Weigh out the reference standards of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, rhein, and rhein methyl ether, add methanol, and prepare a mixed reference solution. Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column is a C18 column; the detection wavelength is 245-254 nm, and the flow rate is 0.1-0.5 ml / min. Determination method: Accurately pipette 1-10 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

[0008] The fingerprint spectroscopy determination method of the present invention detects a traditional Chinese medicine composition preparation made from the following raw materials in parts by weight: Ephedra 52; Gypsum 324; Forsythia 194; Scutellaria baicalensis 78; Mulberry bark 194; Bitter almond 130; Peucedanum praeruptorum 78; Pinellia ternata 130; Tangerine peel 78; Fritillaria cirrhosa 78; Arctium lappa 130; Honeysuckle 130; Rhubarb 39; Platycodon grandiflorus 76; Licorice 65.

[0009] The fingerprint spectroscopy determination method of the present invention preferably uses traditional Chinese medicine composition preparations made from the following raw materials in parts by weight: Ephedra 86; Gypsum 194; Forsythia 324; Scutellaria baicalensis 130; Mulberry bark 324; Bitter almond 78; Peucedanum praeruptorum 130; Pinellia ternata 78; Tangerine peel 130; Fritillaria cirrhosa 130; Arctium lappa 78; Honeysuckle 78; Rheum palmatum 65; Platycodon grandiflorus 46; Licorice 39.

[0010] The fingerprint spectroscopy determination method of the present invention can preferably be used to detect traditional Chinese medicine composition preparations made from the following raw materials in parts by weight: Ephedra 69g; Gypsum 25g; Forsythia 25g; Scutellaria baicalensis 104g; Mulberry bark 25g; Bitter almond 104g; Peucedanum praeruptorum 104g; Pinellia ternata 104g; Tangerine peel 104g; Fritillaria cirrhosa 104g; Arctium lappa 104g; Honeysuckle 104g; Rheum palmatum 52g; Platycodon grandiflorus 61g; Licorice 52g.

[0011] The fingerprint spectroscopy determination method of the present invention can preferably be used to detect traditional Chinese medicine composition preparations made from the following raw materials in parts by weight: Ephedra 55; Gypsum 254; Forsythia 318; Scutellaria baicalensis 107; Mulberry bark 203; Bitter almond 107; Peucedanum praeruptorum 82; Pinellia ternata 105; Tangerine peel 84; Fritillaria cirrhosa 125; Arctium lappa 122; Honeysuckle 113; Rheum palmatum 42; Platycodon grandiflorus 60; Licorice 50.

[0012] The fingerprint spectroscopy determination method of the present invention includes the preparation method of the active ingredient of the traditional Chinese medicine composition to be detected as follows: A. Weigh out the fritillaria bulbs according to the formula ratio, grind them into fine powder, and set aside. B. Weigh out ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb according to the formula ratio, and extract twice with 40-70% ethanol, each time for 1-4 hours. Add 8-10 times the amount for the first extraction and 6-9 times the amount for the second extraction. Combine the extracts, filter, recover the ethanol from the filtrate under reduced pressure, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Set aside for later use. C. Weigh out gypsum, mulberry bark, angelica root, tangerine peel, honeysuckle, platycodon root, and licorice root according to the formula ratio. Add water and decoct twice, each time for 1-4 hours. Add 9-11 times the amount for the first decoction and 7-9 times the amount for the second decoction. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Combine this extract with the clear extract obtained in step B and set aside. The fine powder obtained in step A and the combined clear extract obtained in step C together constitute the active ingredient of the pharmaceutical composition.

[0013] The fingerprint spectrum determination method of the present invention can be used to detect traditional Chinese medicine compositions that can be prepared into capsules, tablets, granules, or oral liquids. The preparation method for tablets is as follows: A. Weigh out the fritillaria bulbs according to the formula ratio, grind them into fine powder, and set aside. B. Weigh out ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb according to the formula ratio, add 50% ethanol and reflux twice for 3 hours each time. Add 10 times the amount for the first time and 6 times the amount for the second time. Combine the extracts, filter, recover the ethanol from the filtrate under reduced pressure, and concentrate to a clear extract with a relative density of 1.15 measured at 60℃. Set aside for later use. C. Weigh out gypsum, mulberry bark, angelica root, tangerine peel, honeysuckle, platycodon root, and licorice root according to the formula ratio. Add water and decoct twice, 2 hours each time. Add 10 times the amount for the first decoction and 7 times the amount for the second decoction. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.15 measured at 60°C. Combine this extract with the clear extract obtained in step B and set aside. D. Spray dry the combined clear paste obtained in step C, and collect the spray powder for later use; E. The spray powder obtained in step D and the fine powder obtained in step A are mixed with ethanol as a binder to form a soft mass. After sieving, granulating, drying and granulating, sodium carboxymethyl starch, microcrystalline cellulose and magnesium stearate are added, mixed well and compressed into tablets to obtain the final product.

[0014] The fingerprint spectrum determination method of the present invention preferably includes the following steps: Preparation of test solution: Take 1-1.2 g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.07-0.09 mg / ml neochlorogenic acid, 0.07-0.09 mg / ml chlorogenic acid, 0.07-0.09 mg / ml cryptochlorogenic acid, 0.07-0.09 mg / ml isoflavone A, 0.1-0.3 mg / ml forsythoside A, 0.1-0.3 mg / ml hesperidin, 0.1-0.3 mg / ml baicalin, 0.1-0.3 mg / ml arctiin, and 0.04-0.06 mg / ml forsythoside A, forsythoside A, hesperidin, baicalin, arctiin, and forsythoside A, respectively. The solution is prepared by mixing a standard solution of glycyrrhizic acid (mg / ml), aloe-emodin (0.01-0.02 mg / ml), rhein (0.01-0.03 mg / ml), emodin (0.01-0.03 mg / ml), chrysophanol (0.01-0.03 mg / ml), and emodin methyl ether (0.005-0.01 mg / ml). Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column was a C18 column; the detection wavelength was 254 nm, and the flow rate was 0.3 ml / min. Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

[0015] The fingerprint spectrum determination method of the present invention, more preferably comprising the following steps: Preparation of test solution: Take 1 g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.07-0.09 mg / ml neochlorogenic acid, 0.07-0.09 mg / ml chlorogenic acid, 0.07-0.09 mg / ml cryptochlorogenic acid, 0.07-0.09 mg / ml isoflavone A, 0.1-0.3 mg / ml forsythoside A, 0.1-0.3 mg / ml hesperidin, 0.1-0.3 mg / ml baicalin, 0.1-0.3 mg / ml arctiin, and 0.04-0.05 mg / ml forsythoside A, forsythoside A, hesperidin, baicalin, arctiin, and forsythoside A, respectively. The solution is prepared by mixing a standard solution of glycyrrhizic acid (mg / ml), aloe-emodin (0.01-0.02 mg / ml), rhein (0.01-0.03 mg / ml), emodin (0.01-0.02 mg / ml), chrysophanol (0.01-0.02 mg / ml), and emodin methyl ether (0.005-0.009 mg / ml). Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column was a C18 column; the column length was 100 mm, the inner diameter was 2.1 mm, and the particle size was 1.8 µm; the detection wavelength was 254 nm, the flow rate was 0.3 ml / min; the column temperature was 30 ℃; and the injection volume was 1 μL. Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

[0016] The feasibility of the fingerprint spectral determination method for the traditional Chinese medicine composition of the present invention was evaluated from multiple aspects using the sample prepared in Example 1. The evaluation method is as follows: 1. Instruments and Materials 1.1 Instruments Waters ACQUITY UPLC H-CLASS ultra-high performance liquid chromatograph (PDA detector, Empower 3.0 chromatography workstation), circulating multi-purpose vacuum pump (Zhengzhou Changcheng Science & Industry Trade Co., Ltd.), KQ250DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), and Swiss METTLER TOLEDOAL electronic analytical balance.

[0017] 1.2 Materials 1.2.1 Reference Standard Information 1.2.2 Sample Information The traditional Chinese medicine composition of this invention (Shijiazhuang Yiling Pharmaceutical Co., Ltd.) 1.2.3 Reagents Acetonitrile (chromatographic grade, Merck, batch number JB091230), methanol (chromatographic grade, Merck, batch number l1061607948), phosphoric acid (analytical grade, Tianjin Kemeo Chemical Reagent Co., Ltd., batch number 20190518). 2. Selection of chromatographic conditions 2.1 Selection of detection wavelength The traditional Chinese medicine composition sample was subjected to full-wavelength determination. Chromatograms were extracted at different wavelengths (220nm, 254nm, 280nm, 300nm, and 330nm). After careful comparison, the most chromatographic peaks were extracted at a wavelength of 254nm, which can more comprehensively reflect the chromatographic information of the sample. Therefore, 254nm was selected as the detection wavelength.

[0018] 2.2 Selection of Mobile Phase System This study investigated the chromatographic peaks in methanol-water, methanol-0.1% formic acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, acetonitrile-water, and acetonitrile-0.1% formic acid aqueous solution. The results showed that the peak shapes were asymmetrical, and some peaks could not achieve baseline separation. The acetonitrile-0.1% phosphoric acid aqueous solution was investigated, and under this condition, the peak shapes were better and baseline separation was achieved. The results showed that the acetonitrile-0.1% phosphoric acid system had the best peak resolution; therefore, the mobile phase system was: acetonitrile-acid-water (0.1% phosphoric acid aqueous solution), flow rate: 0.3 mL / min. The gradient elution table is shown in Table 2. The chromatographic column was determined to be ACQUITY UPLC HSS T3 C18 1.8 μm, 2.1*100 mm; the following parameters were set as initial conditions: wavelength 254 nm; column temperature: 30℃; injection volume: 1 μL. .

[0019] 2.3 Selection of Flow Rate The effects of different flow rates (0.2 ml / min, 0.3 ml / min, and 0.4 ml / min) on the chromatograms were investigated. At a flow rate of 0.2 ml / min, compounds with longer elution times and larger peak areas exhibited poor peak symmetry. At a flow rate of 0.4 ml / min, most peaks failed to reach baseline separation. Considering all factors, the chromatograms at a flow rate of 0.3 ml / min showed better peak shapes and most peaks achieved near-baseline separation. Therefore, a flow rate of 0.3 ml / min was selected.

[0020] 2.4 Column Temperature Selection The chromatographic column was investigated at different column temperatures. At a column temperature of 25℃, the chromatographic peak shape was tailed. At 30℃, the chromatographic peak was excellent. At a column temperature of 35℃, the chromatographic peak with a larger peak area had poor symmetry. After comprehensive comparison, the column temperature of 30℃ was selected.

[0021] 2.5 Selection of Chromatographic Column Different chromatographic columns were investigated: ACQUITY UPLC HSS T3 C18 1.8μm, 2.1*100mm; CORTECS UPLC C18 1.6μm, 2.1*100mm; and ACQUITY UPLC BEH C18 1.7μm, 2.1*100mm. Based on a comprehensive comparison of the number and shape of chromatographic peaks, ACQUITY UPLC HSS T3 C18 1.8μm, 2.1*100mm was selected.

[0022] 2.6 Fingerprint chromatograms of reference standard and test sample Under the established chromatographic conditions, the reference solution and the test solution were injected separately. Fourteen major chromatographic peaks were identified in the chromatogram of the test solution. Based on the retention times and spectral characteristics of the peaks, the following peaks were identified in the fingerprint chromatogram: Peak 1: Neochlorogenic acid; Peak 2: Chlorogenic acid; Peak 3: Cryptochlorogenic acid; Peak 5: Isoflavone A; Peak 7: Forsythoside A; Peak 10: Hesperidin; Peak 12: Baicalin; Peak 13: Arctiin; Peak 17: Aloe-emodin; Peak 18: Ammonium glycyrrhizate; Peak 19: Rhein; Peak 20: Emodin; Peak 21: Rhein; Peak 22: Emodin methyl ether. The chromatograms and spectra of the reference and test solutions are attached. Figure 1-2 .

[0023] 3. Preparation of reference standards and samples 3.1 Preparation of reference solution Preparation of neochlorogenic acid reference solution: Accurately weigh 21.95 mg of neochlorogenic acid reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0024] Preparation of chlorogenic acid reference solution: Accurately weigh 20.23 mg of chlorogenic acid reference standard, place it in a 5 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0025] Preparation of cryptochlorogenic acid reference solution: Accurately weigh 19.07 mg of cryptochlorogenic acid reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0026] Preparation of isoflavone A reference solution: Accurately weigh 10.19 mg of isoflavone A reference standard, place it in a 5 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0027] Preparation of forsythoside A reference solution: Accurately weigh 11.89 mg of forsythoside A reference standard, place it in a 5 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0028] Preparation of baicalin reference solution: Accurately weigh 15.42 mg of baicalin reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0029] Preparation of hesperidin reference solution: Accurately weigh 13.63 mg of hesperidin reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0030] Preparation of arctiin reference solution: Accurately weigh 12.80 mg of arctiin reference standard, place it in a 5 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0031] Preparation of ammonium glycyrrhizate reference solution: Accurately weigh 11.36 mg of ammonium glycyrrhizate reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0032] Preparation of stock solutions of aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether reference standards: Preparation of aloe-emodin reference solution: Accurately weigh 8.29 mg of aloe-emodin reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0033] Preparation of rhein reference solution: Accurately weigh 10.58 mg of rhein reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0034] Preparation of emodin reference solution: Accurately weigh 12.84 mg of reference standard, place it in a 25 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0035] Preparation of rhein reference solution: Accurately weigh 10.56 mg of rhein reference standard, place it in a 10 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0036] Preparation of emodin methyl ether reference solution: Accurately weigh about 9.58 mg of emodin methyl ether reference standard, place it in a 25 mL volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and set aside.

[0037] Preparation of a mixed reference solution of aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether: Accurately measure 1 mL of aloe-emodin reference solution, 1 mL of rhein reference solution, 1 mL of emodin reference solution, 1 mL of chrysophanol reference solution, and 1 mL of emodin methyl ether reference solution into a 50 mL volumetric flask, and dilute to the mark with methanol to prepare a mixed reference solution of aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether.

[0038] Accurately measure 1 mL of neochlorogenic acid reference solution, 0.5 mL of chlorogenic acid reference solution, 1 mL of cryptochlorogenic acid reference solution, 1 mL of isoflavone A reference solution, 2 mL of forsythoside A reference solution, 2 mL of baicalin reference solution, 3 mL of hesperidin reference solution, 2 mL of arctiin reference solution, 1 mL of glycyrrhizic acid reference solution, and 10 mL of a mixed reference solution of aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether into a 25 mL volumetric flask. Dilute to the mark with methanol to prepare a mixed reference solution for later use.

[0039] 3.2 Preparation of the test solution Take an appropriate amount of the Chinese herbal composition, grind it into a fine powder, take 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake it well, centrifuge it (centrifuge at 1400rpm for 10min), take the supernatant, filter it through 0.22µm, and take the filtrate to obtain the final product.

[0040] 4. Preparation of test solution and reference solution 4.1 Investigation of extraction solvent The extraction effects of 50% methanol, 80% methanol, and 100% methanol were compared respectively, as shown in the appendix. Figure 3 Appendix Figure 4 Appendix Figure 5 The results showed that the fingerprint spectrum obtained by 100% methanol extraction had the most chromatographic peaks, so methanol was chosen as the extraction solvent.

[0041] 4.2 Examination of Extraction Methods The ultrasonic and reflux extraction methods were compared separately (see appendix). Figure 6 Appendix Figure 7 The number and intensity of the chromatographic peaks in the fingerprint chromatograms obtained by ultrasonic extraction and reflux extraction were not significantly different. Since ultrasonic extraction is simple, it was chosen.

[0042] 4.3 Examination of ultrasound time Comparisons were made of ultrasound at 20 minutes, 30 minutes, and 40 minutes. The results showed that the fingerprints obtained after 30 minutes and 40 minutes of ultrasound had a large amount of chromatographic peak information and no significant difference. Therefore, the final ultrasound time was determined to be 30 minutes. See the appendix for details. Figure 8 Appendix Figure 9 Appendix Figure 10 .

[0043] 4.4 Preparation of reference solution Accurately weigh an appropriate amount of baicalin reference standard, and dissolve it in methanol to prepare a solution containing 60 μg per ml. The chromatographic information of the reference standard is attached. Figure 11 .

[0044] 4.5 Determination method: Accurately pipette 1µL of the test solution and inject it into the ultra-high performance liquid chromatograph. Measure and record the chromatogram to obtain the result.

[0045] Chromatographic information of the test sample is attached. Figure 12 .

[0046] 5. Methodological Examination 5.1 Precision Test: The sample of the traditional Chinese medicine composition was prepared according to the "Preparation Method of Test Solution" in 3.2. The sample was injected six times consecutively under the chromatographic conditions. The RSD of the relative retention time and relative peak area of ​​each major chromatographic peak was less than 2%, indicating good injection precision. The results are shown in Tables 3 and 4. .

[0047] 5.2 Repeatability Test: Six portions of the contents of the traditional Chinese medicine composition were prepared according to the "Preparation Method of Test Solution" in 3.2, and injected into a liquid chromatograph for detection. The RSD of the retention time of each major chromatographic peak was less than 2%, and the RSD of the peak area was less than 3%, indicating that the method has good repeatability. The results are shown in Tables 5 and 6. .

[0048] 5.3 Stability Test: The test solution was injected at 0h, 2h, 4h, 8h, 12h, and 24h. The RSD of the retention time of each major chromatographic peak was less than 2%, and the RSD of the peak area was less than 3%, indicating that the test solution was stable within 24h. The results are shown in Tables 7 and 8. .

[0049] 5.4 Fingerprint pattern establishment and similarity analysis Seven batches of the described traditional Chinese medicine composition samples were weighed, and test solutions were prepared according to the requirements in section 3.2. The samples were then injected and analyzed under the above chromatographic conditions to obtain fingerprint chromatograms of the seven batches of the described traditional Chinese medicine composition samples. Specific chromatographic information is provided in the appendix. Figure 13 The fingerprint spectra of seven batches of samples were analyzed using the 2012.0 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System software developed by the National Pharmacopoeia Commission. Multi-point correction and automatic matching of chromatographic peaks in each fingerprint spectrum were performed using the median method. Similarity was calculated and a control fingerprint spectrum was generated. The similarity results are shown in Table 9, and the control fingerprint spectrum is attached. Figure 14 There were a total of 22 chromatographic peaks, among which peak number 12, baicalin, was used as the reference peak due to its stable elution and large peak area. .

[0050] 5.5 For the content determination of the seven batches of traditional Chinese medicine composition samples, the test solution from 5.4 and the mixed standard solution from 3.1 were injected into the liquid chromatograph for detection. The results are shown in Table 10. .

[0051] The traditional Chinese medicine composition described in this invention is a marketed traditional Chinese medicine product. Currently, the quality control of this product relies on the individual identification of some medicinal materials. However, as this is a complex traditional Chinese medicine product, limited identification is insufficient to reflect its quality. To better ensure product quality and protect public medication safety, we studied the fingerprint spectrum of this traditional Chinese medicine composition. Fingerprint spectrum studies were conducted on seven different batches of the drug. The fingerprint spectrum analysis identified 22 representative common peaks, and 14 of these peaks were identified. The separation of each peak was good. Using fingerprint spectrum for quality control can comprehensively reflect the overall quality of the traditional Chinese medicine product, effectively avoiding the drawbacks of simply adding only the individual components that need to be tested during the market inspection process.

[0052] The fingerprint spectrum detection method described in this invention is a complete technical solution, including several technical features. For each of these features, the inventors have conducted numerous experimental method studies. Although each technical feature may seem simple, the overall technical solution composed of countless features represents the optimal solution for quality control of the formulation. In particular, the fingerprint spectrum similarity among seven batches tested using this method is greater than 95%, indicating that the product is stable and controllable.

[0053] This method is simple to operate, highly feasible, and quick, solving the problem of quality control in large compound traditional Chinese medicine preparations. The experimental results show that this method has good precision, stability, and repeatability, providing a new approach to improve the quality control of this traditional Chinese medicine composition. Attached Figure Description

[0054] Figure 1 Reference chromatogram Figure 2 fingerprint spectrum of the test sample Figure 3 Extraction efficiency of 50% methanol Figure 4 Extraction efficiency of 80% methanol Figure 5 Extraction efficiency of 100% methanol Figure 6 Ultrasonic fingerprint extraction Figure 7 Reflux extraction fingerprint spectrum Figure 8 Ultrasonic extraction of fingerprints after 20 minutes Figure 9 Ultrasonic extraction of fingerprints for 30 minutes Figure 10 Fingerprint analysis after 40 minutes of ultrasonic extraction Figure 11 chromatogram of reference solution Figure 12 Chromatogram of the test solution Figure 13 Chromatographic fingerprints of the seven batches of the traditional Chinese medicine compositions Figure 14 The traditional Chinese medicine composition was compared with the fingerprint spectrum. Detailed Implementation

[0055] Example 1 Weigh out the following ingredients in the specified proportions: Ephedra 52g; Gypsum 324g; Forsythia 194g; Scutellaria baicalensis 78g; Mulberry bark 194g; Bitter almond 130g; Peucedanum praeruptorum 78g; Pinellia ternata 130g; Tangerine peel 78g; Fritillaria cirrhosa 78g; Arctium lappa 130g; Honeysuckle 130g; Rhubarb 39g; Platycodon grandiflorus 76g; Licorice 65g.

[0056] Extracted using the following process: A. Weigh out the fritillaria bulbs according to the formula ratio, grind them into fine powder, and set aside. B. Weigh out ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb according to the formula ratio, add 50% ethanol and reflux twice for 3 hours each time. Add 10 times the amount for the first time and 6 times the amount for the second time. Combine the extracts, filter, recover the ethanol from the filtrate under reduced pressure, and concentrate to a clear extract with a relative density of 1.15 measured at 60℃. Set aside for later use. C. Weigh out gypsum, mulberry bark, angelica root, tangerine peel, honeysuckle, platycodon root, and licorice root according to the formula ratio. Add water and decoct twice, 2 hours each time. Add 10 times the amount for the first decoction and 7 times the amount for the second decoction. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.15 measured at 60°C. Combine this extract with the clear extract obtained in step B and set aside. D. Spray dry the combined clear paste obtained in step C, and collect the spray powder for later use; E. Prepare capsules using conventional methods.

[0057] Fingerprint spectroscopy determination method: Preparation of test solution: Take 1g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, rhein, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.088 mg / ml neochlorogenic acid, 0.078 mg / ml chlorogenic acid, 0.076 mg / ml cryptochlorogenic acid, 0.080 mg / ml isoflavone A, 0.177 mg / ml forsythoside A, 0.157 mg / ml hesperidin, 0.115 mg / ml baicalin, 0.202 mg / ml arctiin, 0.045 mg / ml glycyrrhizic acid ammonium, 0.016 mg / ml aloe-emodin, 0.021 mg / ml rhein, and 0.010 mg / ml rhein methyl ether. The standard solution is obtained by mixing 0.015 mg / ml emodin, 0.008 mg / ml chrysophanol, and 0.008 mg / ml emodin methyl ether. Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column was determined to be ACQUITY UPLC HSS T3 C18 1.8μm, 2.1*100mm; the following parameters were set as initial conditions: flow rate: 0.3mL / min; wavelength: 254nm; column temperature: 30 ℃; injection volume: 1μL.

[0058] Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

[0059] Example 2 Weigh out the following proportions: Ephedra 86g; Gypsum 194g; Forsythia 324g; Scutellaria baicalensis 130g; Mulberry bark 324g; Bitter almond 78g; Peucedanum praeruptorum 130g; Pinellia ternata 78g; Tangerine peel 130g; Fritillaria cirrhosa 130g; Arctium lappa 78g; Honeysuckle 78g; Rheum palmatum 65g; Platycodon grandiflorus 46g; Licorice root 39g. Extracted using the following process: A. Weigh out the fritillaria bulbs according to the formula ratio, grind them into fine powder, and set aside. B. Weigh out ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb according to the formula ratio, add 50% ethanol and reflux twice, 1 hour each time. Add 8 times the amount for the first time and 9 times the amount for the second time. Combine the extracts, filter, recover the ethanol from the filtrate under reduced pressure, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Set aside for later use. C. Weigh out gypsum, mulberry bark, angelica root, tangerine peel, honeysuckle, platycodon root, and licorice according to the formula ratio, add water and decoct twice, 2 hours each time. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Combine with the clear extract obtained in step B and set aside. D. The combined extract obtained in step C is spray-dried, the spray powder is collected, and tablets are made using conventional methods.

[0060] Fingerprint spectroscopy determination method: Preparation of test solution: Take 1 g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, rhein, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.079 mg / ml neochlorogenic acid, 0.088 mg / ml chlorogenic acid, 0.085 mg / ml cryptochlorogenic acid, 0.081 mg / ml isoflavone A, 0.179 mg / ml forsythoside A, 0.148 mg / ml hesperidin, 0.109 mg / ml baicalin, 0.226 mg / ml arctiin, 0.049 mg / ml glycyrrhizic acid ammonium, 0.018 mg / ml aloe-emodin, 0.025 mg / ml rhein, and 0.015 mg / ml rhein methyl ether. The standard solution is obtained by mixing 0.018 mg / ml emodin, 0.009 mg / ml chrysophanol, and 0.009 mg / ml emodin methyl ether. Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column was determined to be ACQUITY UPLC HSS T3 C18, 1.8 μm, 2.1*100 mm; the following parameters were set as initial conditions: flow rate: 0.3 mL / min; wavelength: 254 nm; column temperature: 30 ℃; injection volume: 1 μL. Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

[0061] Example 3 The raw material formula is as follows: Ephedra 69g; Gypsum 259g; Forsythia 259g; Scutellaria baicalensis 104g; Mulberry bark 259g; Bitter almond 104g; Peucedanum praeruptorum 104g; Pinellia ternata 104g; Tangerine peel 104g; Fritillaria cirrhosa 104g; Arctium lappa 104g; Honeysuckle 104g; Rheum palmatum 52g; Platycodon grandiflorus 61g; Licorice 52g.

[0062] Extracted using the following process: A. Weigh out the fritillaria bulbs according to the formula ratio, grind them into fine powder, and set aside. B. Weigh out ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb according to the formula ratio, add 70% ethanol and reflux twice, 2 hours each time. Add 10 times the amount for the first time and 9 times the amount for the second time. Combine the extracts, filter, recover the ethanol from the filtrate under reduced pressure, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Set aside for later use. C. Weigh out gypsum, mulberry bark, angelica root, tangerine peel, honeysuckle, platycodon root, and licorice root according to the formula ratio. Add water and decoct twice, 3 hours each time. Add 10 times the amount for the first decoction and 10 times the amount for the second decoction. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Combine this extract with the clear extract obtained in step B and set aside. D. The combined extract obtained in step C is spray-dried, the spray powder is collected, and it is made into granules using conventional methods.

[0063] Fingerprint spectroscopy determination method: Preparation of test solution: Take 0.5 g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 50% methanol, weigh it, sonicate for 20 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, rhein, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.080 mg / ml neochlorogenic acid, 0.082 mg / ml chlorogenic acid, 0.085 mg / ml cryptochlorogenic acid, 0.085 mg / ml isoflavone A, 0.180 mg / ml forsythoside A, 0.166 mg / ml hesperidin, 0.116 mg / ml baicalin, 0.232 mg / ml arctiin, 0.046 mg / ml glycyrrhizic acid ammonium, 0.019 mg / ml aloe-emodin, 0.020 mg / ml rhein, and 0.011 mg / ml rhein methyl ether. The standard solution is obtained by mixing 0.016 mg / ml emodin, 0.009 mg / ml chrysophanol, and 0.009 mg / ml emodin methyl ether. Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column was an ACQUITY UPLC BEH C18 1.7μm, 2.1*100mm column; the detection wavelength was 245nm, and the flow rate was 0.1ml / min. Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

[0064] Example 4 The active pharmaceutical ingredient (API) formula is as follows: Weigh out the following proportions: Ephedra 55g; Gypsum 254g; Forsythia 318g; Scutellaria baicalensis 107g; Mulberry bark 203g; Bitter almond 107g; Peucedanum praeruptorum 82g; Pinellia ternata 105g; Tangerine peel 84g; Fritillaria cirrhosa 125g; Arctium lappa 122g; Honeysuckle 113g; Rheum palmatum 42g; Platycodon grandiflorus 60g; Licorice 50g.

[0065] Extracted using the following process: A. Weigh out the fritillaria bulbs according to the formula ratio, grind them into fine powder, and set aside. B. Weigh out ephedra, forsythia, bitter almond, pinellia, burdock seed, and rhubarb according to the formula ratio, add 40-70% ethanol and reflux twice for 3 hours each time. Add 8 times the amount for the first time and 7 times the amount for the second time. Combine the extracts, filter, recover the ethanol from the filtrate under reduced pressure, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Set aside for later use. C. Weigh out gypsum, mulberry bark, angelica root, tangerine peel, honeysuckle, platycodon root, and licorice root according to the formula ratio. Add water and decoct twice, 2 hours each time. Add 10 times the amount for the first decoction and 8 times the amount for the second decoction. Combine the decoctions, filter, and concentrate to a clear extract with a relative density of 1.04-1.16 measured at 60℃. Combine this extract with the clear extract obtained in step B and set aside. D. The combined extract obtained in step C is spray-dried, the spray powder is collected, and it is made into pills using conventional methods.

[0066] Fingerprint spectroscopy determination method: Preparation of test solution: Take 1.5g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 80% methanol, weigh it, sonicate for 40 minutes, cool it, weigh it again, make up the lost weight with 80% methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, rhein, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.080 mg / ml neochlorogenic acid, 0.082 mg / ml chlorogenic acid, 0.085 mg / ml cryptochlorogenic acid, 0.085 mg / ml isoflavone A, 0.180 mg / ml forsythoside A, 0.166 mg / ml hesperidin, 0.116 mg / ml baicalin, 0.232 mg / ml arctiin, 0.046 mg / ml glycyrrhizic acid ammonium, 0.019 mg / ml aloe-emodin, 0.020 mg / ml rhein, and 0.011 mg / ml rhein methyl ether. The standard solution is obtained by mixing 0.016 mg / ml emodin, 0.009 mg / ml chrysophanol, and 0.009 mg / ml emodin methyl ether. Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column was a CORTECS UPLC C18 1.6μm, 2.1*100mm column; the detection wavelength was 245nm, and the flow rate was 0.1ml / min. Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

Claims

1. A method for determining the fingerprint spectrum of a traditional Chinese medicine composition, wherein the composition is made from the following raw materials in parts by weight: Ephedra 52-86; Gypsum 194-324; Forsythia 194-324; Scutellaria baicalensis 78-130; Morus alba root bark 194-324; Bitter almond 78-130; Peucedanum praeruptorum 78-130; Pinellia ternata 78-130; Tangerine peel 78-130; Fritillaria cirrhosa 78-130; Arctium lappa 78-130; Honeysuckle 78-130; Rheum palmatum 39-65; Platycodon grandiflorus 46-76; Licorice 39-65, characterized in that... The method for determining the fingerprint spectrum of this traditional Chinese medicine composition is as follows: Preparation of test solution: Take 0.5-1.5g of the traditional Chinese medicine composition, accurately weigh it, place it in a stoppered conical flask, accurately add 10-50mL of 40-100% methanol, weigh it, sonicate for 20-40 minutes, cool it, weigh it again, replenish the lost weight with 40-100% methanol, shake well, filter it, and take the filtrate to obtain the test solution; Preparation of reference solutions: Weigh out the reference standards of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, rhein, and rhein methyl ether, add methanol, and prepare a mixed reference solution. Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column is C10. 18 Chromatographic column; detection wavelength 254 nm, flow rate 0.1-0.5 ml / min; Determination method: Accurately pipette 1-10 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

2. The fingerprint spectrum determination method as described in claim 1, characterized in that... The method for determining the fingerprint spectrum is as follows: Preparation of test solution: Take 1-1.2g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.07-0.09 mg / ml neochlorogenic acid, 0.07-0.09 mg / ml chlorogenic acid, 0.07-0.09 mg / ml cryptochlorogenic acid, 0.07-0.09 mg / ml isoflavone A, 0.1-0.3 mg / ml forsythoside A, 0.1-0.3 mg / ml hesperidin, 0.1-0.3 mg / ml baicalin, 0.1-0.3 mg / ml arctiin, and 0.04-0.06 mg / ml forsythoside A, forsythoside A, hesperidin, baicalin, arctiin, and forsythoside A, respectively. The solution is prepared by mixing a standard solution of glycyrrhizic acid (mg / ml), aloe-emodin (0.01-0.02 mg / ml), rhein (0.01-0.03 mg / ml), emodin (0.01-0.03 mg / ml), chrysophanol (0.01-0.03 mg / ml), and emodin methyl ether (0.005-0.01 mg / ml). Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column is C10. 18 Chromatographic column; detection wavelength 254 nm, flow rate 0.3 ml / min; column temperature 30℃; Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

3. The fingerprint spectrum determination method as described in claim 2, characterized in that... The method for determining the fingerprint spectrum is as follows: Preparation of test solution: Take 1g of the Chinese herbal composition, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the test solution. Preparation of reference solutions: Weigh out the following reference standards: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoflavone A, forsythoside A, hesperidin, baicalin, arctiin, aloe-emodin, glycyrrhizic acid ammonium, rhein, emodin, chrysophanol, and rhein methyl ether. Add methanol to prepare reference solutions containing 0.07-0.09 mg / ml neochlorogenic acid, 0.07-0.09 mg / ml chlorogenic acid, 0.07-0.09 mg / ml cryptochlorogenic acid, 0.07-0.09 mg / ml isoflavone A, 0.1-0.3 mg / ml forsythoside A, 0.1-0.3 mg / ml hesperidin, 0.1-0.3 mg / ml baicalin, 0.1-0.3 mg / ml arctiin, and 0.04-0.05 mg / ml forsythoside A, forsythoside A, hesperidin, baicalin, arctiin, and forsythoside A, respectively. The solution is prepared by mixing a standard solution of glycyrrhizic acid (mg / ml), aloe-emodin (0.01-0.02 mg / ml), rhein (0.01-0.03 mg / ml), emodin (0.01-0.02 mg / ml), chrysophanol (0.01-0.02 mg / ml), and emodin methyl ether (0.005-0.009 mg / ml). Preparation of reference solution: Take an appropriate amount of baicalin reference standard, accurately weigh it, and add methanol to prepare a solution containing 60 µg per ml. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with gradient elution. The elution ratios are shown in the table below: The chromatographic column is C10. 18 Chromatographic column; column length 100 mm, inner diameter 2.1 mm, particle size 1.8 µm; detection wavelength 254 nm, flow rate 0.3 ml / min; column temperature 30 °C; Determination method: Accurately pipette 1 µL of the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatogram to obtain the result.

4. The method for determining fingerprint spectra as described in any one of claims 1-3, characterized in that... The chromatographic column used was an ACQUITY UPLC HSS T3 C. 18 1.8μm, 2.1*100mm.

5. The method for determining fingerprint spectra as described in any one of claims 1-3, characterized in that... It also includes the following steps: a. Obtaining a reference fingerprint: Import the chromatogram of the test sample that has passed the current standard evaluation into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" to synthesize a reference fingerprint; b. Similarity evaluation: Import the fingerprint chromatogram of the sample to be evaluated into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" and calculate the similarity. The similarity between the fingerprint chromatogram of the sample and the control fingerprint chromatogram shall not be less than 0.9.

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