Establishment method and application of fingerprint spectrum of Sapocorrhiza angustifolius mixture

Abstract

The invention discloses an establishment method and application of a fingerprint spectrum of a Sanyone-fish-dragon mixture. The establishment method mainly comprises the steps of preparation of a test solution, preparation of a mixed reference substance solution, high performance liquid chromatography detection, establishment of a standard fingerprint spectrum and the like. According to the invention, the standard fingerprint spectrum of the Sapocorrhiza mixture is established, and the method is high in sensitivity, strong in specificity, easy to operate and accurate in result. The standard fingerprint spectrum constructed by the method has 36 characteristic peaks, 8 chemical components such as hesperidin, naringin, ammonium glycyrrhizinate, liquiritin, amygdalin, ferulic acid, hyperoside, quercetin and the like are effectively identified, the problem that the evaluation index of the Sanyone mixture is single is solved, the overall quality condition can be efficiently and comprehensively reflected, and the method has good application prospects. And a basis is provided for quality control and identification of the Sapococalyx mixture.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine analysis and detection, and specifically relates to a method for establishing a fingerprint spectrum of Sanao Yulong mixture and its application. Background Art

[0002] Sanao Yulong Mixture is made from 8 kinds of herbs, including dried tangerine peel, licorice, fried bitter almonds, dried houttuynia, ephedra, earthworms, fried yam and Fritillaria thunbergii. It has the effects of clearing the lungs and relieving exterior symptoms, benefiting the lungs and relieving asthma, and relieving cough and eliminating phlegm. This medicine is an in-hospital preparation of Taihe County Hospital of Traditional Chinese Medicine, with the approval number: Wanyaozhizi Z20210005000. It has a history of clinical use for nearly 30 years, with definite efficacy, and is widely recognized by patients.

[0003] Chinese medicine mixture is an oral liquid preparation made by extracting the decoction pieces with water or other solvents using appropriate methods. It is a complex multi-component system. At present, the evaluation index for Sanao Yulong Mixture is single, which cannot efficiently and comprehensively reflect the overall quality situation. Therefore, it is necessary to establish a complete set of Chinese medicine fingerprints that can better reveal the types and quantities of chemical components it contains, realize the overall characterization and evaluation of the quality of Sanao Yulong Mixture, and have a far-reaching impact on the improvement of the quality of Sanao Yulong Mixture. Summary of the invention

[0004] In view of the shortcomings of the prior art, the primary purpose of the present invention is to provide a method for establishing a fingerprint spectrum of Sanao Ichthyosaur mixture. This method has high sensitivity and strong specificity. The constructed standard fingerprint spectrum has a total of 36 characteristic peaks, which effectively identify 8 chemical components and can achieve overall characterization and evaluation of the quality of Sanao Ichthyosaur mixture.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for establishing a fingerprint spectrum of Sanaoyulong mixture comprises the following steps:

[0007] (a) Preparation of the test solution: accurately pipette the Sanaoyulong mixture, add methanol to dilute to volume, filter, and take the filtrate to obtain the test solution;

[0008] (b) Preparation of mixed reference solution: accurately weigh hesperidin, rutin, ammonium glycyrrhizinate, liquiritin, amygdalin, ferulic acid, hyperoside and quercetin reference substances, dilute to volume with methanol, filter, and take the filtrate to obtain a mixed reference solution;

[0009] (c) High performance liquid chromatography detection: The mixed reference solution and multiple batches of test solution are respectively injected into a high performance liquid chromatograph for determination to obtain a chromatogram, wherein:

[0010] Chromatographic conditions are as follows: chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile (A)-0.1% formic acid (B); gradient elution program: 0-10 min, 10% A→15% A; 10-24 min, 15% A→25% A; 24-28 min, 25% A→26% A; 28-30 min, 26% A→30% A; 30-40 min, 30% A→70% A; 40-45 min, 70% A→10% A; 45-50 min, 10% A; detection wavelength: 283 nm;

[0011] (d) Establishment of standard fingerprint: The obtained chromatogram was imported into the 2012 version of the Chinese medicine fingerprint similarity evaluation software, the S1 batch was set as the reference spectrum, the average method was selected for the control spectrum generation method, the time window width was set to 0.1, and peak matching was performed after multi-point correction to obtain the standard fingerprint spectrum.

[0012] Preferably, in step (a), the mass concentration of the methanol is 100%; and the filtration is performed using a 0.22 μm organic microfiltration membrane.

[0013] Preferably, in step (a), the volume ratio of the Sanaoyulong mixture to methanol is 1:3-5, preferably 1:4.

[0014] Preferably, in step (b), the mass concentration of methanol is 75% to 85%.

[0015] Preferably, in step (c), the chromatographic column is East Laboratory Epic C18, with specifications of 4.6 mm×250 mm and 5 μm.

[0016] Preferably, in step (c), the flow rate is 0.6-1.0 mL / min; the column temperature is 25-35° C.; and the injection volume is 10 μL.

[0017] Preferably, in step (d), the standard fingerprint has a total of 36 characteristic peaks, and 8 characteristic peaks are identified by the reference substance, namely, peak 11 amygdalin, peak 24 licorice glycoside, peak 25 hyperoside, peak 26 ferulic acid, peak 27 naringin, peak 28 hesperidin, peak 35 quercetin, and peak 36 ammonium glycyrrhizinate.

[0018] The present invention also provides the application of the method for establishing the fingerprint spectrum of the Sanao Ichthyosaur mixture in quality inspection. The chromatogram of the Sanao Ichthyosaur mixture sample to be tested is calculated for similarity with the standard fingerprint spectrum, and the quality of the sample to be tested is evaluated according to the similarity calculation result. If the similarity is greater than 0.9, it is qualified, otherwise it is unqualified.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention establishes a standard fingerprint spectrum of Sanao Yulong mixture, which has high sensitivity, strong specificity, is easy to operate and has accurate results. The standard fingerprint spectrum constructed by the method has 36 characteristic peaks, effectively identifying 8 chemical components such as hesperidin, naringin, ammonium glycyrrhizinate, liquiritin, amygdalin, ferulic acid, hyperoside, and quercetin, solving the problem of single evaluation index of Sanao Yulong mixture, and being able to efficiently and comprehensively reflect the overall quality situation, providing a basis for quality control and identification of Sanao Yulong mixture. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the fingerprints of 15 batches of test products and their reference patterns;

[0022] Figure 2 It is the chromatogram of negative samples, the chromatogram of single herb and the chromatogram of mixed reference substances;

[0023] Figure 3 It is the chromatogram of the test sample using different mobile phases;

[0024] Figure 4 It is the chromatogram of the test sample using different detection wavelengths;

[0025] Figure 5 It is the chromatogram of the test sample using different column temperatures;

[0026] Figure 6 It is the chromatogram of the test product using different flow rates;

[0027] Figure 7 It is the chromatogram of the test sample using different dilution solvents;

[0028] Figure 8 These are the chromatograms of the test products using different elution gradient programs. DETAILED DESCRIPTION

[0029] In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. However, the implementation methods of the present invention are not limited to the following embodiments.

[0030] Example 1: Method for establishing fingerprint of Sanaoyulong mixture

[0031] 1. Instruments and reagents

[0032] 1.1 Instrument

[0033] Waters 2489 high performance liquid chromatograph (Waters, USA); YL-060ST ultrasonic cleaner (Shenzhen Yulu Cleaning Equipment Co., Ltd.); AB135-S electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.);

[0034] 1.2 Drug testing

[0035] 15 batches of test samples for Sanaoyulong mixture (batch numbers are 24013001, 24012601, 24012301, 24030601, 24010801, 24012901, 24012801, 24012701, 24031901, 24031201, 24032801, 24032701, 24032901, 24030801, 24030701, numbered S1-S15, all provided by Taihe County Hospital of Traditional Chinese Medicine); dried tangerine peel, licorice, stir-fried bitter almond, dried houttuynia cordata, ephedra, earthworm, stir-fried yam, and thunbergia thunbergii were all provided by Taihe County Hospital of Traditional Chinese Medicine; hesperidin (batch number PS 011588), naringin (batch number PS011583), ammonium glycyrrhizinate (batch number PS011584), amygdalin (batch number PS011718), ferulic acid (batch number PS012244), hyperoside (batch number PS011442), and quercetin (batch number PS012980) reference substances were purchased from Chengdu Pusi Biotechnology Co., Ltd. with a purity of ≥98.0%; liquiritin (batch number 551-15-5) was purchased from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd. with a purity of ≥98.0%; acetonitrile (chromatographic grade) and methanol (chromatographic grade) were purchased from Shanghai Anage Technology Co., Ltd., formic acid (analytical grade), and water was Wahaha purified water.

[0036] 2 Methods and Results

[0037] 2.1 Chromatographic conditions

[0038] The chromatographic column was East Laboratory Epic C18 (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile (A)-0.1% formic acid solution (B), gradient elution (0-10 min, 10% A→15% A; 10-24 min, 15% A→25% A; 24-28 min, 25% A→26% A; 28-30 min, 26% A→30% A; 30-40 min, 30% A→70% A; 40-45 min, 70% A→10% A; 45-50 min, 10% A); the flow rate was 0.8 mL / min; the column temperature was 30°C; the injection volume was 10 μL; and the detection wavelength was 283 nm.

[0039] 2.2 Solution preparation

[0040] 2.2.1 Preparation of test solution

[0041] Accurately pipette 1 mL of Sanao Yulong mixture into a 5 mL volumetric flask, add methanol to the mark, shake evenly, filter with a 0.22 μm organic microfiltration membrane, and take the filtrate to prepare the Sanao Yulong mixture test solution. 2.2.2 Preparation of mixed reference solution

[0042] Accurately weigh hesperidin, rutin, ammonium glycyrrhizinate, liquiritin, amygdalin, ferulic acid, hyperoside and quercetin reference substances, and use 80% methanol as solvent to prepare mixed reference substance solutions with mass concentrations of 300.00, 66.40, 74.80, 42.00, 12.00, 100.00, 12.40 and 15.70 μg / mL, respectively.

[0043] 2.2.3 Preparation of negative sample solution

[0044] Prepare it according to the current process of Sanao Yulong mixture. Weigh 173g of dried tangerine peel, licorice, fried bitter almonds, dried houttuynia cordata, ephedra, earthworm, fried yam and Fritillaria thunbergii according to the prescribed dosage, add 10 times the water and boil three times, 2 hours each time, combine the decoctions, let stand, concentrate, filter, cool, add water to adjust the total amount to 1000mL, stir well, and filter. Prepare samples lacking dried tangerine peel, licorice, ephedra, fried bitter almonds, Fritillaria thunbergii, dried houttuynia cordata, yam and earthworm, and then prepare each negative control test solution according to the method under "2.2.1". 2.2.4 Preparation of test solutions for single-ingredient decoction pieces

[0045] Add 0.5 g of coarse powder each of dried tangerine peel, licorice, fried bitter almonds, dried houttuynia, ephedra, earthworm, fried yam and thunbergia thunbergii into a conical flask, add 50 mL of 80% methanol, and extract by ultrasound (frequency 40 kHz, power 360 W) for 30 min; make up for the weight loss, take the supernatant, filter with a 0.22 μm organic microporous filter membrane, take the filtrate, and prepare the test solution of each single herbal decoction piece.

[0046] 2.3 Methodological investigation

[0047] 2.3.1 Precision

[0048] Take the sample S1, prepare the sample solution according to the method under 2.2.1, and perform the measurement for 6 consecutive times according to the chromatographic conditions under 2.1. Take the peak No. 28 (hesperidin) as the reference peak, and calculate the relative retention time RSD of each common peak <0.19%, and the relative peak area RSD <2.90% (n=6), respectively, indicating that the instrument has good precision and meets the requirements of Chinese herbal medicine fingerprint. The similarity evaluation software of Chinese herbal medicine fingerprint was used for similarity evaluation in 2012, and the result was 1.000, which meets the requirements of Chinese herbal medicine fingerprint.

[0049] 2.3.2 Repeatability

[0050] Take the test sample S1, prepare 6 test solutions in parallel according to the method under 2.2.1, and measure and obtain chromatograms according to the chromatographic conditions under "2.3". Taking peak 28 (hesperidin) as the reference peak, the relative retention time RSD of each common peak was calculated to be less than 0.27%, and the relative peak area RSD was less than 4.17% (n=6), indicating that the method has good repeatability and meets the requirements of Chinese herbal medicine fingerprint. The 2012 version of Chinese herbal medicine fingerprint similarity evaluation software was used for similarity evaluation, and the result was 1.000, which meets the requirements of Chinese herbal medicine fingerprint.

[0051] 2.3.3 Stability

[0052] Take the sample S1, prepare the sample solution according to the method under 2.2.1, and test it according to the chromatographic conditions under "2.3" at room temperature for 0, 2, 4, 8, 12, and 24 hours to obtain the chromatogram. Taking peak 28 (hesperidin) as the reference peak, the relative retention time RSD of each common peak was calculated to be less than 0.26%, and the relative peak area RSD was less than 3.68% (n=6). The results showed that the sample was stable within 24 hours and met the requirements of the fingerprint of traditional Chinese medicine. The similarity evaluation software of traditional Chinese medicine fingerprint was used for similarity evaluation in 2012, and the result was 1.000, which met the requirements of the fingerprint of traditional Chinese medicine.

[0053] 2.4 Fingerprint establishment and similarity evaluation

[0054] Take 15 batches of test samples numbered S1 to S15, prepare test sample solutions according to the method under 2.2.1, and measure them according to the chromatographic conditions under 2.1. 283nm is used as the fingerprint collection wavelength, and the obtained chromatogram is input into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", with S1 as the reference spectrum, and the reference spectrum is generated by the average method, with the time window width set to 0.1. After multiple point corrections, the reference spectrum R and the overlapping spectrum of 15 batches are obtained by peak matching, see Figure 1 The results showed that there were 36 peaks in total, among which peak 28 (hesperidin) had the largest peak area and good separation, so peak 28 was selected as the reference peak; the similarity between each batch and the reference spectrum was ≥0.990, indicating that there was no significant difference between the batches. The results are shown in Table 1.

[0055] Table 1 Similarity evaluation results of 15 batches of test products

[0056] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S1 1 S2 0.999 1 S3 0.999 1 1 S4 0.998 0.999 0.998 1 S5 0.998 0.999 0.999 0.998 1 S6 0.999 0.999 1 0.998 0.999 1 S7 0.999 1 1 0.998 0.999 1 1 S8 0.999 1 1 0.998 0.999 1 1 1 S9 0.998 0.998 0.997 0.999 0.997 0.997 0.997 0.997 1 S10 0.998 0.998 0.998 0.998 0.997 0.997 0.997 0.998 0.998 1 S11 0.998 0.998 0.997 0.998 0.996 0.996 0.996 0.997 0.999 0.998 1 S12 0.999 0.998 0.998 0.999 0.998 0.998 0.998 0.998 0.999 0.998 0.999 1 S13 0.996 0.995 0.994 0.996 0.993 0.994 0.993 0.995 0.997 0.998 0.998 0.996 1 S14 0.998 0.998 0.997 0.998 0.997 0.997 0.997 0.998 0.998 1 0.998 0.998 0.998 1 S15 0.999 0.999 0.999 0.998 0.997 0.999 0.999 0.999 0.998 0.998 0.997 0.998 0.995 0.997 1 R 0.999 1 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.999 0.997 0.999 0.999

[0057] 2.5 Identification and attribution of common peaks

[0058] Take the mixed reference solution under 2.2.2, the negative control test solution under 2.2.3, and the single herbal decoction piece test solution under 2.2.4, and measure them respectively according to the chromatographic conditions under 2.1. Figure 2 As shown, by comparing the reference spectrum R with the mixed reference spectrum, peak 11 was identified as amygdalin, peak 24 as liquiritigenin, peak 25 as hyperoside, peak 26 as ferulic acid, peak 27 as naringin, peak 28 as hesperidin, peak 35 as quercetin, and peak 36 as glycyrrhizic acid. By comparing the control spectrum R with the negative chromatograms of the products lacking dried tangerine peel, lacking earthworm, lacking licorice, lacking bitter almond, lacking ephedra, lacking yam, lacking houttuynia cordata, and lacking Fritillaria thunbergii and the chromatograms of each medicinal piece, it was found that peaks 2 and 11 belonged to bitter almond; peak 29 belonged to Fritillaria thunbergii; peaks 4, 25, 26, and 35 belonged to houttuynia cordata; peaks 23, 24, 30, 31, and 36 belonged to licorice; peaks 5, 6, 9, 15, 16, 21, 27, 28, 32, and 34 belonged to dried tangerine peel; peaks 7, 8, 14, 17-20, and 22 belonged to ephedra; peak 1 belonged to both earthworm and Fritillaria thunbergii; peaks 3, 12, and 13 belonged to both dried tangerine peel and ephedra; peak 10 belonged to both houttuynia cordata and licorice.

[0059] 3 Optimization of chromatographic conditions

[0060] 3.1 Optimization of mobile phase

[0061] The present invention investigates four different mobile phase systems: methanol (A)-water (B), methanol (A)-0.1% formic acid (B), acetonitrile (A)-water (B), and acetonitrile (A)-0.1% formic acid (B). Figure 3 As shown in the figure, since the elution ability of the methanol-water mobile phase system is weaker than that of the acetonitrile-water mobile phase system, the chromatographic peak is relatively late, which increases the analysis time, so the acetonitrile-water mobile phase system with stronger elution ability is selected. By comparing the acetonitrile-water and acetonitrile 0.1% formic acid mobile phase systems, it can be found that after adding 0.1% formic acid to the water phase, the number of chromatographic peaks and the response are increased, so acetonitrile (A)-0.1% formic acid (B) is finally selected as the mobile phase.

[0062] 3.2 Wavelength Optimization

[0063] The present invention investigates 8 different detection wavelengths of 220nm, 240nm, 254nm, 260nm, 283nm, 300nm, 320nm, and 340nm. Figure 4As shown, at the detection wavelengths of 220nm and 240nm, there are terminal absorptions to varying degrees, and the chromatographic peaks are few and the response is low; at the detection wavelengths of 254nm, 260nm, 320nm and 340nm, there are fewer chromatographic peaks, and at the two detection wavelengths of 283nm and 300nm, the chromatograms are relatively similar, but at the wavelength of 283nm, the number of chromatographic peaks is the largest, the response is high, and the separation is good, so the present invention selects 283nm as the detection wavelength.

[0064] 3.3 Optimization of column temperature

[0065] The present invention investigated three different column temperatures of 25°C, 30°C, and 35°C. Figure 5 As shown, when the column temperature is 30°C, the peak shapes of the chromatograms are relatively symmetrical and the separation is good, so 30°C is finally selected as the column temperature of the chromatographic column of the present invention;

[0066] 3.4 Optimization of flow rate

[0067] The present invention investigated three different flow rates of 0.6 mL / min, 0.8 mL / min, and 1.0 mL / min. Figure 6 As shown in the figure, with the increase of flow rate, the sample peak time is also advanced, but when the flow rate is 0.8mL / min, the separation of chromatographic peaks is the best and the solvent is saved, so the flow rate is finally selected as 0.8mL / min.

[0068] 3.5 Optimization of test sample dilution ratio

[0069] The present invention investigates the dilution of the test sample: methanol solvent at four different ratios of 4:1, 1:1, 1:4, and 1:9. Figure 7 As shown in the figure, when the ratio of the test sample to methanol solvent was 4:1 and 1:1, the peak at the retention time of 14.5min was bifurcated and the filtration was difficult. When the ratio of the test sample to methanol solvent was 1:9, there were fewer chromatographic peaks, many chemical components did not respond, and the separation was low. Therefore, the final ratio of the test sample to methanol solvent was 1:4, and the chromatogram peak shape was more symmetrical.

[0070] 3.6 Optimization of gradient elution program

[0071] The present invention investigated 6 different gradient elution procedures, and the method is as follows:

[0072] Method 1: acetonitrile (A)-0.1% formic acid solution (B), 0-15 min, 10% A→30% A; 15-19 min, 30% A→31% A; 19-29 min, 31% A→33% A; 29-35 min, 33% A→40% A; 35-45 min, 40% A→70% A; 45-50 min, 70% A→10% A; 50-55 min, 10% A.

[0073] Method 2: acetonitrile (A)-0.1% formic acid solution (B), 0-8 min, 10% A→15% A; 8-20 min, 15% A→25% A; 20-28 min, 25% A→26% A; 28-32 min, 26% A→30% A; 32-40 min, 30% A→70% A; 40-45 min, 70% A→10% A; 45-50 min, 10% A.

[0074] Method 3: acetonitrile (A)-0.1% formic acid solution (B), 0-10 min, 10% A→15% A; 10-24 min, 15% A→25% A; 24-28 min, 25% A→26% A; 28-30 min, 26% A→30% A; 30-40 min, 30% A→40% A; 40-50 min, 40% A→70% A; 50-55 min, 60% A→10% A; 55-60 min, 10% A.

[0075] Method 4: acetonitrile (A)-0.1% formic acid solution (B), 0-10 min, 10% A→15% A; 10-24 min, 15% A→25% A; 24-28 min, 25% A→26% A; 28-30 min, 26% A→30% A; 30-45 min, 30% A→33% A; 45-50 min, 33% A→40% A; 50-55 min, 40% A→60% A, 55-60 min, 60% A→10% A; 60-65 min, 10% A.

[0076] Method 5: acetonitrile (A)-0.1% formic acid solution (B), 0-10 min, 10% A→15% A; 10-30 min, 15% A→30% A; 30-40 min, 30% A→60% A; 40-45 min, 60% A→10% A; 45-50 min, 10% A.

[0077] Method 6: acetonitrile (A)-0.1% formic acid solution (B), 0-10 min, 10% A→15% A; 10-24 min, 15% A→25% A; 24-28 min, 25% A→26% A; 28-30 min, 26% A→30% A; 30-40 min, 30% A→70% A; 40-45 min, 70% A→10% A; 45-50 min, 10% A.

[0078] The results are as follows Figure 8As shown, method 1 is not suitable as an analysis condition because the initial acetonitrile concentration of the mobile phase is too high, and each component peaks within 19 minutes, the component signal peaks are piled up and the number is small; method 2 has fewer chromatographic peaks, many chemical components have no response, and the separation degree is low; method 3 is improved based on method 2, and it can be seen that the chromatographic peaks increase, but the signal peaks are too concentrated, and deformed peaks appear at retention times of 13.5 minutes and 18.5 minutes; method 4 is further optimized than method 3, but the separation degree is poor at the retention time of 35 minutes; methods 5 and 6 are further optimized than method 4, and the chromatographic peak separation effect of gradient 5 is greatly improved, but the separation of individual peaks is still poor; and the chromatographic peaks in the fingerprint spectrum obtained by gradient 6 all achieve good separation effects, so method 6 is finally selected as the gradient elution procedure of the present invention.

[0079] Example 2: Application of the method for establishing the fingerprint spectrum of Sanaoyulong mixture in quality inspection

[0080] The similarity between the chromatogram of the San'ao Yulong mixture sample to be tested and the standard fingerprint spectrum is calculated, and the quality of the sample to be tested is evaluated based on the similarity calculation result. If the similarity is greater than 0.9, it is qualified, otherwise it is unqualified.

Claims

1. A method for establishing a fingerprint spectrum of Sanao Yulong mixture, characterized in that: The steps include: (a) Preparation of the test solution: accurately pipette the Sanaoyulong mixture, add methanol to dilute to volume, filter, and take the filtrate to obtain the test solution; (b) Preparation of mixed reference solution: accurately weigh hesperidin, rutin, ammonium glycyrrhizinate, liquiritin, amygdalin, ferulic acid, hyperoside and quercetin reference substances, dilute to volume with methanol, filter, and take the filtrate to obtain a mixed reference solution; (c) High performance liquid chromatography detection: The mixed reference solution and multiple batches of test solution are respectively injected into a high performance liquid chromatograph for determination to obtain a chromatogram, wherein: Chromatographic conditions were as follows: chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile (A)-0.1% formic acid (B); gradient elution program: 0-10 min, 10%A→15%A; 10-24 min, 15%A→25%A; 24-28 min, 25%A→26%A; 28-30 min, 26%A→30%A; 30-40 min, 30%A→70%A; 40-45 min, 70%A→10%A; 45-50 min, 10%A; detection wavelength was 283 nm; (d) Establishment of standard fingerprint: The obtained chromatogram was imported into the 2012 version of the Chinese medicine fingerprint similarity evaluation software, the S1 batch was set as the reference spectrum, the average method was selected for the control spectrum generation method, the time window width was set to 0.1, and peak matching was performed after multi-point correction to obtain the standard fingerprint spectrum.

2. The method for establishing the fingerprint spectrum of Sanaoyulong mixture according to claim 1, characterized in that: In step (a), the mass concentration of the methanol is 100%; and the filtration is performed using a 0.22 µm organic microfiltration membrane.

3. The method for establishing the fingerprint spectrum of Sanaoyulong mixture according to claim 1, characterized in that: In step (a), the volume ratio of the Sanaoyulong mixture to methanol is 1:3-5, preferably 1:

4.

4. The method for establishing the fingerprint spectrum of Sanaoyulong mixture according to claim 1, characterized in that: In step (b), the mass concentration of the methanol is 75% to 85%.

5. The method for establishing the fingerprint spectrum of Sanaoyulong mixture according to claim 1, characterized in that: In step (c), the chromatographic column is East Laboratory Epic C18, with specifications of 4.6 mm×250 mm, 5 μm.

6. The method for establishing the fingerprint spectrum of Sanaoyulong mixture according to claim 1, characterized in that: In step (c), the flow rate is 0.6-1.0 mL / min; the column temperature is 25-35 °C; and the injection volume is 10 μL.

7. The method for establishing the fingerprint spectrum of Sanaoyulong mixture according to claim 1, characterized in that: In step (d), the standard fingerprint has a total of 36 characteristic peaks, and 8 characteristic peaks are identified by the reference substance, namely, peak 11 amygdalin, peak 24 licorice glycoside, peak 25 hyperoside, peak 26 ferulic acid, peak 27 naringin, peak 28 hesperidin, peak 35 quercetin, and peak 36 ammonium glycyrrhizinate.

8. Application of the method for establishing the fingerprint spectrum of Sanao Yulong mixture according to any one of claims 1 to 7 in quality inspection.

9. The use according to claim 8, characterized in that: The similarity between the chromatogram of the San'ao Yulong mixture sample to be tested and the standard fingerprint spectrum is calculated, and the quality of the sample to be tested is evaluated based on the similarity calculation result. If the similarity is greater than 0.9, it is qualified, otherwise it is unqualified.

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