A method for constructing an UPLC fingerprint spectrum of cold fever relieving granules and application thereof

Abstract

The application discloses a method for constructing a UPLC fingerprint spectrum of a cold fever relieving granule and application of the method. By using the method, 33 chromatographic peaks can be confirmed, and four raw medicinal materials in the cold fever relieving granule, namely, isatis leaf, radix isatidis, forsythia and bupleurum, all have corresponding attribution peaks. By comparison with reference substances and in combination with ultraviolet absorption spectrum, characteristic peaks of seven chemical components, i.e., gallic acid, (R, S)-adelfungin, protocatechuic acid, isoviolanthin, rutin, forsythia ester glycoside A and forsythia glycoside, are identified, wherein isoviolanthin can be used as a characteristic peak of isatis leaf, and (R, S)-adelfungin can be used as a characteristic peak of radix isatidis, and the two characteristic peaks can distinguish the raw medicinal materials isatis leaf and radix isatidis. The method for constructing the UPLC fingerprint spectrum can obtain a fingerprint spectrum with good separation degree and comprehensive chromatographic peaks, can more objectively and comprehensively evaluate the overall quality of the cold fever relieving granule, and lays a foundation for quality control of the cold fever relieving granule.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine analysis and detection, in particular to a method for constructing UPLC fingerprint of Ganmao Tuire Granules and application thereof. BACKGROUND

[0002] The Ganmao Tuire Granules is composed of Isatis indigotica Fort, Radix Isatidis, Forsythia suspensa and Bistorta, which is decocted, alcohol precipitated and water precipitated to form granules. The Ganmao Tuire Granules has the effects of clearing heat and resolving toxin and dispelling wind and relieving superficies, and is mainly used for upper respiratory tract infection, acute tonsillitis and pharyngitis caused by wind-heat and heat-toxin accumulation, with symptoms of fever and sore throat. Although the Ganmao Tuire Granules is included in the Chinese Pharmacopoeia, its quality standard is still relatively weak, and only the thin-layer identification of indigotin and forsythoside and the content determination of forsythoside are performed.

[0003] A large number of studies have shown that traditional Chinese medicine, especially Chinese patent medicine prepared from multiple Chinese medicines, exerts therapeutic effects by acting on multiple targets in the body through multiple active ingredients. At present, the Ganmao Tuire Granules is controlled in quality by one or several ingredients, which cannot effectively guarantee its therapeutic effect. Moreover, the preparation process of the Ganmao Tuire Granules is relatively complex, and needs to be alcohol precipitated and water precipitated, and only one ingredient (forsythoside) is used to evaluate the production process, which cannot reflect the whole quality transmission of traditional Chinese medicine prepared by modern technology. In addition, the raw materials of the Ganmao Tuire Granules, Isatis indigotica Fort and Radix Isatidis, are derived from different parts of the same medicinal plant, and have similar chemical components. If Isatis indigotica Fort or Radix Isatidis is not added during the production of the Ganmao Tuire Granules, it is difficult to find or control it by the existing quality standard or technology. The existing technology 1 (Quality Control of Ganmao Tuire Granules Decoction, published on June 1, 2004) discloses the HPLC fingerprint of the Ganmao Tuire Granules decoction, and identifies six peaks of isofraxidin, forsythoside, gallic acid, chlorogenic acid, uridine and adenosine. However, among the six peaks, uridine and adenosine are common components of Isatis indigotica Fort and Radix Isatidis, and the remaining five peaks are attributed to Forsythia suspensa and Bistorta, respectively. Therefore, the fingerprint cannot effectively identify Isatis indigotica Fort and Radix Isatidis in the Ganmao Tuire Granules, and thus cannot well qualitatively and quantitatively evaluate the quality of the Ganmao Tuire Granules.

[0004] Therefore, there is an urgent need for a method for controlling the quality of the Ganmao Tuire Granules from multiple ingredients and multiple angles, and detecting whether four ingredients are not completely added during the production process, especially Isatis indigotica Fort and Radix Isatidis derived from different parts of the same medicinal plant. SUMMARY

[0005] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present application provides a method for constructing UPLC fingerprint of Ganmao Tuire Granules and application thereof.

[0006] The first object of the present application is to provide a pretreatment method for detecting cold fever relieving granules by ultra performance liquid chromatography.

[0007] The second object of the present application is to provide an ultra performance liquid chromatography method.

[0008] The third object of the present application is to provide the application of the above-mentioned pretreatment method and / or ultra performance liquid chromatography method in the establishment of the UPLC fingerprint of cold fever relieving granules and / or the quality control of cold fever relieving granules.

[0009] The fourth object of the present application is to provide a method for constructing the UPLC fingerprint of cold fever relieving granules.

[0010] The fifth object of the present application is to provide a quality detection method for cold fever relieving granules.

[0011] Therefore, the present application claims the following contents:

[0012] A pretreatment method for detecting cold fever relieving granules by ultra performance liquid chromatography, taking the sample of the cold fever relieving granules to be detected, and ultrasonically extracting the sample with a methanol aqueous solution with a volume fraction of 25-35% for 40-50 min.

[0013] The cold fever relieving granules are prepared according to the Pharmacopeia of the People's Republic of China: 2020 edition (Part I). Specifically, 435 g of Isatis indigotica, 435 g of Radix Isatidis, 217 g of Forsythia suspensa and 217 g of Bistorta are weighed, and water is added for decoction twice, each time for 1.5 h. The decoction liquid is combined, filtered, and concentrated to a clear extract with a relative density of about 1.08 (90-95°C). After cooling to room temperature, an equal amount of ethanol is added to precipitate, and the supernatant is concentrated to a clear extract with a relative density of 1.20 (60°C). An equal amount of water is added, stirred, and left to stand for 8 h. The supernatant is concentrated to a thick extract with a relative density of 1.38-1.40 (60°C). An appropriate amount of sucrose powder, dextrin and ethanol are added to prepare granules, which are dried to produce 1000 g of cold fever relieving granules (sucrose-containing type). Alternatively, the supernatant is concentrated to a clear extract with a relative density of 1.09-1.11 (60°C), and an appropriate amount of dextrin and flavoring agent are added, mixed uniformly, and spray dried to produce 250 g of cold fever relieving granules (sucrose-free type). The cold fever relieving granules are brownish yellow granules.

[0014] Preferably, the volume fraction of the methanol aqueous solution is 30%.

[0015] Preferably, the sample of the cold fever relieving granules to be detected is a sucrose-free sample, and the mass-volume ratio of the sample of the cold fever relieving granules to be detected to the methanol aqueous solution is 1 g:(12-13) mL.

[0016] The sample to be tested is a sample containing sucrose, and the mass-volume ratio of the sample to be tested to the methanol solution is 1g:(3-4)mL.

[0017] More preferably, the sample to be tested is a sample not containing sucrose, and the mass-volume ratio of the sample to be tested to the methanol solution is 1g:12.5mL.

[0018] The sample to be tested is a sample containing sucrose, and the mass-volume ratio of the sample to be tested to the methanol solution is 1g:3.125mL.

[0019] More preferably, the frequency of the ultrasonic is 35-45kHz, and the power is 490-630W.

[0020] More preferably, the frequency of the ultrasonic is 40kHz, and the power is 560W.

[0021] More preferably, the time of the ultrasonic is 45min.

[0022] An ultra-high performance liquid chromatography method, comprising the following steps:

[0023] Reference solution: (R,S)-adonis solution, isofraxidin solution, forsythoside solution, forsythoside A solution, rutin solution, protocatechuic acid solution and / or gallic acid solution;

[0024] Test sample solution: prepared by using any of the above pretreatment methods;

[0025] Chromatographic conditions: octadecylsilane-bonded silica gel as the filler of the chromatographic column, acetonitrile as the mobile phase A, 0.08-0.12% formic acid aqueous solution as the mobile phase B, in the gradient elution program, the volume percentage of the mobile phase B in the mobile phase system changes as follows:

[0026] 0-15min, the mobile phase B decreases from 100% to 95%;

[0027] 15-25min, the mobile phase decreases from 95% to 90%;

[0028] 25-40min, the mobile phase decreases from 90% to 85%;

[0029] 40-50min, the mobile phase is 85% to 84%;

[0030] 50-60min, the mobile phase decreases from 84% to 80%;

[0031] 60-80min, the mobile phase is 80% to 70%;

[0032] The detection wavelength is 254 nm, and the theoretical plate number calculated according to (R, S)-jadeite should not be less than 5000.

[0033] Preferably, the preparation method of the (R, S)-jadeite solution is that (R, S)-jadeite is uniformly mixed with methanol, and the mass-volume ratio of (R, S)-jadeite to methanol is (15-25) μg: 1 mL;

[0034] The preparation method of the isofraxidilin solution is that isofraxidilin is uniformly mixed with methanol, and the mass-volume ratio of isofraxidilin to methanol is (75-85) μg: 1 mL;

[0035] The preparation method of the forsythoside solution is that forsythoside is uniformly mixed with methanol, and the mass-volume ratio of forsythoside to methanol is (75-85) μg: 1 mL;

[0036] The preparation method of the forsythoside A solution is that forsythoside A is uniformly mixed with methanol, and the mass-volume ratio of forsythoside A to methanol is (195-205) μg: 1 mL;

[0037] The preparation method of the rutin solution is that rutin is uniformly mixed with methanol, and the mass-volume ratio of rutin to methanol is (65-75) μg: 1 mL;

[0038] The preparation method of the protocatechuic acid solution is that protocatechuic acid is uniformly mixed with methanol, and the mass-volume ratio of protocatechuic acid to methanol is (35-45) μg: 1 mL;

[0039] The preparation method of the gallic acid solution is that gallic acid is uniformly mixed with methanol, and the mass-volume ratio of gallic acid to methanol is (35-45) μg: 1 mL.

[0040] More preferably, the mass-volume ratio of (R, S)-jadeite to methanol is 20 μg: 1 mL;

[0041] The mass-volume ratio of isofraxidilin to methanol is 80 μg: 1 mL;

[0042] The mass-volume ratio of forsythoside to methanol is 80 μg: 1 mL;

[0043] The mass-volume ratio of forsythoside A to methanol is 200 μg: 1 mL;

[0044] The mass-volume ratio of rutin to methanol is 70 μg: 1 mL;

[0045] The mass-volume ratio of protocatechuic acid to methanol is 40 μg: 1 mL;

[0046] The mass-volume ratio of the gallic acid to methanol is 40 μg: 1 mL.

[0047] Preferably, the column temperature of the chromatographic column is 20-30℃.

[0048] More preferably, the column temperature of the chromatographic column is 25℃.

[0049] Preferably, the flow rate of the gradient elution is 0.2-0.4 mL / min.

[0050] More preferably, the flow rate of the gradient elution is 0.3 mL / min.

[0051] Preferably, the injection volume of the reference solution and the test solution is 1-3 μL.

[0052] More preferably, the injection volume of the reference solution and the test solution is 2 μL.

[0053] Preferably, the chromatographic column is an ACQUITY HSS T3 C18 chromatographic column with a size of 1.8 μm, 150 mm x 2.1 mm.

[0054] Preferably, the volume fraction of the aqueous formic acid solution is 0.1%.

[0055] The pre-treatment method and / or the ultra-high performance liquid chromatography method described above are used for establishing the UPLC fingerprint of the Ganmao Tuire Granules and / or the quality control of the Ganmao Tuire Granules.

[0056] A method for establishing the UPLC fingerprint of the Ganmao Tuire Granules, wherein different batches of Ganmao Tuire Granules samples are detected by using the ultra-high performance liquid chromatography method described above, the chromatograms are recorded, the chromatograms are introduced into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, the common peaks are screened and confirmed, and the UPLC fingerprint of the Ganmao Tuire Granules is obtained.

[0057] The UPLC fingerprint of the Ganmao Tuire Granules contains 33 fingerprint peaks, wherein the peak No. 9 is the S peak, and the relative retention times of the 33 common peaks are as follows:

[0058] 1st peak 0.38, 2nd peak 0.45, 3rd peak 0.56, 4th peak 0.61, 5th peak 0.68, 6th peak 0.71, 7th peak 0.83, 8th peak 0.88, 9th peak (S peak) 1.00, 10th peak 1.09, 11th peak 1.42, 12th peak 1.83, 13th peak 1.85, 14th peak 1.88, 15th peak 2.13, 16th peak 2.27, 17th peak 2.34, 18th peak 2.67, 19th peak 2.80, 20th peak 2.83, 21st peak 2.87, 22nd peak 2.93, 23rd peak 3.10, 24th peak 3.27, 25th peak 3.32, 26th peak 3.44, 27th peak 3.50, 28th peak 3.54, 29th peak 3.88, 30th peak 4.06, 31st peak 4.52, 32nd peak 4.71, 33rd peak 5.00;

[0059] The 3rd peak is a characteristic chromatographic peak of gallic acid, the 8th peak is a characteristic chromatographic peak of (R, S)-adonirubin, the 9th peak is a characteristic chromatographic peak of protocatechuic acid, the 24th peak is a characteristic chromatographic peak of isorhamnetin, the 25th peak is a characteristic chromatographic peak of rutin, the 28th peak is a characteristic chromatographic peak of forsythoside A, and the 32nd peak is a characteristic chromatographic peak of forsythoside.

[0060] The different batches are not less than 15 batches.

[0061] A quality detection method of a cold fever relieving granule, comprising the following steps:

[0062] S1. Taking a cold fever relieving granule sample to be detected, using any of the above-mentioned ultra-high performance liquid chromatography methods for detection, recording the chromatogram to obtain the UPLC spectrum of the cold fever relieving granule sample to be detected;

[0063] S2. Importing the UPLC fingerprint spectrum of the cold fever relieving granule and the UPLC spectrum of the cold fever relieving granule sample to be detected into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system for comparison, and only when the following two conditions are met can it be determined to be qualified:

[0064] The UPLC spectrum of the cold fever relieving granule sample to be detected presents a fingerprint peak within ±5% of the specified value corresponding to the retention time in the UPLC fingerprint spectrum;

[0065] The similarity of the UPLC spectrum of the cold fever relieving granule sample to be detected and the UPLC fingerprint spectrum is calculated, and the similarity of the UPLC spectrum of the cold fever relieving granule sample to be detected and the UPLC fingerprint spectrum is not less than 0.85;

[0066] The UPLC fingerprint spectrum contains 33 fingerprint peaks, and the 9th peak is an S peak, and the specified values are:

[0067] 1st peak 0.38, 2nd peak 0.45, 3rd peak 0.56, 4th peak 0.61, 5th peak 0.68, 6th peak 0.71, 7th peak 0.83, 8th peak 0.88, 9th peak (S peak) 1.00, 10th peak 1.09, 11th peak 1.42, 12th peak 1.83, 13th peak 1.85, 14th peak 1.88, 15th peak 2.13, 16th peak 2.27, 17th peak 2.34, 18th peak 2.67, 19th peak 2.80, 20th peak 2.83, 21st peak 2.87, 22nd peak 2.93, 23rd peak 3.10, 24th peak 3.27, 25th peak 3.32, 26th peak 3.44, 27th peak 3.50, 28th peak 3.54, 29th peak 3.88, 30th peak 4.06, 31st peak 4.52, 32nd peak 4.71, 33rd peak 5.00.

[0068] The application of any of the above ultra-high performance liquid chromatography methods in detecting the content of isatis leaf, radix isatidis, forsythia and / or anemarrhena in Ganmao Tuire Granules.

[0069] Compared with the prior art, the application has the following beneficial effects:

[0070] The application discloses a Ganmao Tuire Granule UPLC fingerprint spectrum construction method and application. By using the method, 33 chromatographic peaks can be confirmed, and the four raw medicinal materials in the Ganmao Tuire Granule, namely isatis leaf, radix isatidis, forsythia and anemarrhena, all have corresponding attribution peaks. By comparison with reference substances and in combination with ultraviolet absorption spectrum, characteristic peaks of seven chemical components, namely gallic acid, (R, S)-chaulmoogryd, protocatechuic acid, isovitexin, rutin, forsythia ester glycoside A and forsythia glycoside, are identified, wherein isovitexin can be used as a characteristic peak of isatis leaf, and (R, S)-chaulmoogryd can be used as a characteristic peak of radix isatidis, and the two characteristic peaks can distinguish isatis leaf and radix isatidis. The UPLC fingerprint spectrum construction method disclosed by the application can obtain a fingerprint spectrum with good separation degree and comprehensive chromatographic peaks, and can more objectively and comprehensively evaluate the overall quality of the Ganmao Tuire Granule, thereby laying a foundation for quality control of the Ganmao Tuire Granule. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 Test sample chromatogram obtained by using the mobile phase elution gradient in Table 1.

[0072] Figure 2 Influence of different extraction solvents on the test sample chromatogram; S1-S5 are methanol, 70% methanol solution (v / v), 50% methanol solution (v / v), 30% methanol solution (v / v) and water respectively.

[0073] Figure 3Effects of different extraction methods and extraction time on the chromatogram of the test sample; S1-S3 are ultrasonic extraction for 30 min, 45 min and 60 min, respectively, and S4-S6 are heating reflux extraction for 30 min, 45 min and 60 min, respectively.

[0074] Figure 4 Effects of different solid-liquid ratios on the chromatogram of the test sample; S1-S3 are 0.5 g:25 mL, 1.0 g:25 mL, 2.0 g:25 mL and 4.0 g:25 mL, respectively.

[0075] Figure 5 Full wavelength scan of the test sample solution.

[0076] Figure 6 Effects of different mobile phase systems on the chromatogram of the test sample; S1-S3 are methanol-water solution, acetonitrile-water solution, methanol-0.1% formic acid aqueous solution (v / v) and acetonitrile-0.1% formic acid aqueous solution (v / v), respectively.

[0077] Figure 7 Effects of different acid types in the mobile phase system on the chromatogram of the test sample; S1-S3 are acetonitrile-0.1% phosphoric acid aqueous solution (v / v), acetonitrile-0.1% acetic acid aqueous solution (v / v) and acetonitrile-0.1% formic acid aqueous solution (v / v), respectively.

[0078] Figure 8 Effects of different formic acid concentrations in the mobile phase system on the chromatogram of the test sample; S1-S2 are acetonitrile-0.1% formic acid aqueous solution (v / v) and acetonitrile-0.3% formic acid aqueous solution (v / v), respectively.

[0079] Figure 9 Effects of different elution gradients in Table 2 on the chromatogram of the test sample.

[0080] Figure 10 Peak identification results of the chromatogram using gradient 13; S1-S9 are Ganmaotui Re Granules, (R,S)-Emetine, Isoformononetin, Galuteolin, Phillyrin, Phillyrin A, Rutin, Protocatechuic Acid and Gallic Acid, respectively.

[0081] Figure 11 Effects of different elution gradients on the chromatogram of the test sample and reference materials; S1-S5 are the test sample solution (gradient 24), Phillyrin (gradient 24), the test sample solution (gradient 25), Phillyrin (gradient 25) and the test sample solution (gradient 26), respectively.

[0082] Figure 12 Effects of different elution flow rates on the chromatogram of the test sample; S1-S4 are 0.1 mL / min, 0.2 mL / min, 0.3 mL / min and 0.4 mL / min, respectively.

[0083] Figure 13 The effect of different column temperatures on the chromatogram of the test sample; S1-S5 are 20℃, 25℃, 30℃, 35℃, and 40℃, respectively.

[0084] Figure 14 The effect of different injection volumes on the chromatogram of the test sample; S1-S5 are 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL, respectively.

[0085] Figure 15 The fingerprint of the Ganmao TuiRe granules.

[0086] Figure 16 The peak identification results.

[0087] Figure 17 The peak assignment results to Isatidis Radix.

[0088] Figure 18 The peak assignment results to Baphicacanthis Radix.

[0089] Figure 19 The peak assignment results to Forsythiae Fructus.

[0090] Figure 20 The peak assignment results to Bistortae Rhizoma.

[0091] Figure 21 The characteristic peak confirmation results of Isatidis Radix and Baphicacanthis Radix.

[0092] Figure 22 The specificity results.

[0093] Figure 23 The instrument precision results.

[0094] Figure 24 The repeatability results.

[0095] Figure 25 The stability results.

[0096] Figure 26 The column durability results. DETAILED DESCRIPTION

[0097] The present application will be further described below in connection with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods, and apparatus used in the present application are conventional in the art.

[0098] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0099] Information of the reference substances used in the examples of the present application:

[0100] Reference substance Batch number Content Source (R,S)-adonirubin 111753-202007 100％ China National Institute for Food and Drug Control Isoquercitrin A0681 99.7％ Chengdu Man Sit Biological Technology Co., Ltd. Phillygenin 110821-202117 94.9％ China National Institute for Food and Drug Control Forsythoside A 111810-202108 100％ China National Institute for Food and Drug Control Rutin 100080-202012 91.6％ China National Institute for Food and Drug Control Protocatechuic acid 110809-202207 97.5％ China National Institute for Food and Drug Control Gallic acid 110831-201906 91.5％ China National Institute for Food and Drug Control

[0101] The test sample information used in the embodiments of the present application: the cold fever granules are obtained from commercially available samples of major manufacturers or pilot samples. The cold fever granules are prepared according to the Pharmacopeia of the People's Republic of China: 2020 Edition (Part I). Specifically, 435 g of isatis leaf, 435 g of radix isatidis, 217 g of forsythia and 217 g of bupleurum are weighed, and boiled twice for 1.5 h each time, the boiling liquids are combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of about 1.08 (90-95℃). When cooled to room temperature, an equal amount of ethanol is added to precipitate, and after standing, the supernatant is concentrated to a clear paste with a relative density of 1.20 (60℃). An equal amount of water is added, stirred, and allowed to stand for 8 h. The supernatant is concentrated to a thick paste with a relative density of 1.38-1.40 (60℃). An appropriate amount of sucrose powder, dextrin and ethanol is added to prepare granules, which are dried to obtain 1000 g of cold fever granules (containing sucrose type) or 250 g of cold fever granules (sucrose-free type). The cold fever granules are brownish yellow granules.

[0102] Example 1 Influence of different elution gradient conditions on the UPLC fingerprint of cold fever granules

[0103] 1. Experimental method

[0104] 0.5 g of the cold fever granule extract paste (p = 1.23) is precisely weighed and placed in a conical flask with a plug, 25 mL of 50% methanol solution (v / v) is precisely added, the weight is determined, ultrasonic treatment (power 490 W, frequency 40 kHz) is performed for 30 min, the weight is determined again, the lost weight is made up with 50% methanol solution (v / v), shaken well, filtered, and the filtrate is collected to obtain the test sample solution.

[0105] Chromatographic conditions: ACQUITY HSS T3 C18 column (1.8 μm, 100 mm x 2.1 mm, column 1) with octadecylsilane-bonded silica gel as the filler, ACQUITY HSS T3 C18 column (1.8 μm, 150 mm x 2.1 mm, column 2); acetonitrile as mobile phase A, 0.1% formic acid aqueous solution (v / v) as mobile phase B, water as mobile phase C (only for gradient 1), elution according to the gradient in Table 1; detection wavelength 254 nm, comparison of the responses of the main peaks under different gradients, flow rate 0.4 mL / min, column temperature 25℃. HSS T3 C18 column (1.8 μm, 100 mm x 2.1 mm, column 1), ACQUITY HSS T3 C18 column (1.8 μm, 150 mm x 2.1 mm, column 2); acetonitrile as mobile phase A, 0.1% formic acid aqueous solution (v / v) as mobile phase B, water as mobile phase C (only for gradient 1), elution according to the gradient in Table 1; detection wavelength 254 nm, comparison of the responses of the main peaks under different gradients, flow rate 0.4 mL / min, column temperature 25℃.

[0106] Table 1 Elution gradient of mobile phase

[0107]

[0108] (2) Experimental results

[0109] The results are shown in Table 1. The peak shape and resolution of each main chromatographic peak were good, and the baseline was relatively flat under the elution of gradient 13. Therefore, gradient 13 was selected as the gradient for investigating the preparation method of the test solution, i.e., the ACQUITY UPLC HSS T3 C18 column (1.8 μm, 150 mm x 2.1 mm) was used. Figure 1 The HSS T3 C18 column (1.8 μm, 150 mm x 2.1 mm) was used; the mobile phase was acetonitrile-0.1% formic acid aqueous solution (v / v); the gradient elution was as follows: 0 min-15 min-25 min-40 min-50 min-60 min, the volume fraction of acetonitrile was changed to 0%-5%-10%-15%-16%-20%; the flow rate was 0.4 mL / min; the column temperature was 25°C; the detection wavelength was 254 nm; and the injection amount of the test solution was 2 μL.

[0110] Effect of the preparation method of the test solution on the UPLC fingerprint of Ganmao Tuire Granules

[0111] I. Extraction solvent

[0112] 1. Experimental method

[0113] About 2.0 g of Ganmao Tuire Granules was precisely weighed into a conical flask with a plug, and 25 mL of methanol, 70% methanol solution (v / v), 50% methanol solution (v / v), 30% methanol solution (v / v), or water was precisely added, respectively. The weight was determined, and the sample was ultrasonically treated for 30 min (power 560 W, frequency 40 kHz). After cooling, the weight was determined again, and the lost weight was made up with the corresponding extraction solvent. The sample was shaken and filtered, and the filtrate was collected.

[0114] The chromatographic conditions were in accordance with the conditions of gradient 13 in Example 1.

[0115] 2. Experimental results

[0116] The results are shown in Table 1. The peak shape and resolution of each main chromatographic peak were good, and the baseline was relatively flat under the elution of gradient 13. Therefore, gradient 13 was selected as the gradient for investigating the preparation method of the test solution, i.e., the ACQUITY UPLC HSS T3 C18 column (1.8 μm, 150 mm x 2.1 mm) was used. Figure 2 As shown in Table 2, the number of chromatographic peaks was the least when methanol was used for extraction, and there was little difference in the number of chromatographic peaks when 70% methanol solution (v / v), 50% methanol solution (v / v), 30% methanol solution (v / v), or water was used for extraction. The response of the main chromatographic peaks also had little difference. The solvent peak was smaller when 30% methanol solution (v / v) or water was used for extraction, which made the overall peak shape better. Moreover, 30% methanol solution (v / v) was more conducive to the stable storage of the test solution during the analysis process. Therefore, 30% methanol solution (v / v) was selected as the extraction solvent for the fingerprint of Ganmao Tuire Granules.

[0117] II. Extraction method

[0118] 1. Experimental method ​

[0119] Take about 2.0 g of Ganmaotui Re Granules, 6 portions, accurately weigh, place in a conical flask with a stopper, accurately add 25 mL of 30% methanol solution (v / v), weigh, heat reflux extraction for 30 min, 45 min, 60 min, and ultrasonic treatment for 30 min, 45 min, 60 min (power 560 W, frequency 40 kHz) respectively, cool, weigh again, make up the weight loss with 30% methanol solution (v / v), shake well, filter, take the filtrate.

[0120] The chromatographic conditions are carried out according to the conditions of gradient 13 in Example 1.

[0121] 2. Experimental results

[0122] As shown in the results, the sample of ultrasonic extraction for 30 min presents slightly different chromatographic peaks from the other five extraction modes, with fewer component peaks; the number of chromatographic peaks and the peak area of the main chromatographic peaks of the samples under the other five extraction modes have little difference. Therefore, based on the convenience of operation and analysis time, ultrasonic extraction for 45 min is selected as the final extraction mode and time of Ganmaotui Re Granules. Figure 3

[0123] III. Solid-liquid ratio

[0124] 1. Experimental method

[0125] Take 0.5 g, 1.0 g, 2.0 g, 4.0 g of Ganmaotui Re Granules (sucrose-free type) 4 portions, accurately weigh, place in a conical flask with a stopper, accurately add 30% methanol solution (v / v) according to the solid-liquid ratio of 0.5 g:25 mL, 1.0 g:25 mL, 2.0 g:25 mL, 4.0 g:25 mL respectively, weigh, ultrasonic extraction for 45 min, cool, weigh again, make up the weight loss with 30% methanol solution (v / v), shake well, filter, take the filtrate.

[0126] The chromatographic conditions are carried out according to the conditions of gradient 13 in Example 1.

[0127] 2. Experimental results

[0128] As shown in the results, the number of chromatographic peaks in sample 1 (0.5 g:25 mL) is slightly different from the other five samples, with fewer chromatographic peaks, but the differences between the remaining three samples are not large; the chromatographic peak response in sample 2 (1.0 g:25 mL) is relatively small, resulting in a slight baseline shift; the concentration of sample 4 (4.0 g:25 mL) is relatively large, the sample filtration is difficult, and the resolution of some chromatographic peaks decreases. Therefore, the solid-liquid ratio of 2.0 g:25 mL is selected as the final solid-liquid ratio of Ganmaotui Re Granules. Figure 4

[0129] ​​Pre-treatment method of UPLC fingerprint of cold fever relieving granules

[0130] Take cold fever relieving granules sample about 2.0g (sucrose-free type) or 8.0g (sucrose-containing type, converted according to preparation process), accurately weigh, place in a conical flask with plug, accurately add 30% methanol solution (v / v) 25mL, weigh, treat with ultrasonic (40kHz, 560W) for 45min, take out, cool to room temperature, make up the weight loss with 30% methanol solution (v / v), shake well, filter, take the filtrate, and obtain.

[0131] Effect of chromatographic conditions on UPLC fingerprint of cold fever relieving granules

[0132] I. Analysis wavelength

[0133] 1. Experimental method

[0134] Prepare cold fever relieving granules test sample solution according to the pre-treatment method of Example 3.

[0135] Perform full wavelength scanning on the test sample solution, and select the optimal detection wavelength from its 3D graph. The indicators for investigation are: the analysis signal response value is large at this wavelength, the peak information amount is large, the peak distribution is uniform and the baseline is stable, and the separation degree of each peak is good.

[0136] 2. Experimental results

[0137] The results, as shown in Table 1, show that the peak information amount of the sample at 254nm is larger, and the response value is larger, so 254nm is selected as the detection wavelength. Figure 5

[0138] II. Mobile phase system

[0139] 1. Experimental method

[0140] Prepare cold fever relieving granules test sample solution according to the pre-treatment method of Example 3.

[0141] The chromatographic conditions are carried out according to the gradient 13 of Example 1, with the difference being that the mobile phase system is set as: methanol-water solution, acetonitrile-water solution, methanol-0.1% formic acid aqueous solution (v / v), acetonitrile-0.1% formic acid aqueous solution (v / v), respectively. The selection criteria are: the number of peaks in the sample spectrum is large, the peak distribution is uniform, the peak shape and the separation effect of the main component peaks are good, and the baseline is stable.

[0142] 2. Experimental results

[0143] The results, as shown in Table 2, show that the peak information amount of the sample at 254nm is larger, and the response value is larger, so 254nm is selected as the detection wavelength. Figure 6 ​As shown, the test sample obtained using acetonitrile-0.1% formic acid aqueous solution (v / v) as the mobile phase system had more peak information, a more stable baseline, and better resolution and peak shape. Therefore, the mobile phase system was finally determined to be acetonitrile-0.1% formic acid aqueous solution (v / v).

[0144] III. Different Types of Acids

[0145] 1. Experimental Methods

[0146] The cold and fever granule test solution was prepared according to the pretreatment method in Example 3.

[0147] The chromatographic conditions were performed according to gradient 13 of Example 1, except that the mobile phase systems were set as acetonitrile-0.1% phosphoric acid aqueous solution (w / v), acetonitrile-0.1% acetic acid aqueous solution (v / v), and acetonitrile-0.1% formic acid aqueous solution (v / v), respectively. The selection criteria were based on the number of peaks, uniform peak distribution, good peak shape and separation of main component peaks, and stable baseline.

[0148] 2. Experimental Results

[0149] The results are as follows Figure 7 As shown, the baseline drift of the chromatogram in the acetonitrile-0.1% phosphoric acid aqueous solution (w / v) system was severe, and the peak resolution of the chromatogram in the acetonitrile-0.1% acetic acid aqueous solution (v / v) system was poor. Therefore, the acetonitrile-0.1% formic acid aqueous solution (v / v) system was selected as the mobile phase system.

[0150] IV. Formic acid aqueous solution concentration

[0151] 1. Experimental Methods

[0152] The cold and fever granule test solution was prepared according to the pretreatment method in Example 3.

[0153] Chromatographic conditions were performed according to gradient 13 of Example 1, except that the mobile phase systems were set as acetonitrile-0.1% formic acid aqueous solution (v / v) and acetonitrile-0.3% formic acid aqueous solution (v / v), respectively. The selection criteria were based on the number of peaks, uniform peak distribution, good peak shape and separation of main component peaks, and stable baseline.

[0154] 2. Experimental Results

[0155] The results are as follows Figure 8 As shown, increasing the concentration of formic acid aqueous solution enhances the absorption of the solvent peak, which leads to a decrease in the relative response of the effective chromatographic peak. In addition, the resolution of some chromatographic peaks decreases, and the baseline becomes slightly unstable. Therefore, acetonitrile-0.1% formic acid aqueous solution (v / v) was ultimately chosen as the mobile phase system.

[0156] V. Elution gradient

[0157] 1. Experimental method

[0158] The test solution of Ganmao Tuire Granules was prepared according to the pretreatment method of Example 3.

[0159] The chromatographic conditions were performed according to the gradient 13 of Example 1, except that the elution was performed according to the gradient in Table 2, and the responses of each main peak under different gradients were compared.

[0160] Table 2 Elution gradient of mobile phase

[0161]

[0162] 2. Experimental results

[0163] The results are shown in Table 3. Figure 9 As shown in the above table, none of the above gradients can achieve the separation effect of gradient 13, so gradient 13 is still selected as the final elution gradient.

[0164] Six, identification of chromatographic peaks

[0165] 1. Experimental method

[0166] The test solution of Ganmao Tuire Granules was prepared according to the pretreatment method of Example 3.

[0167] Preparation of reference solution:

[0168] An appropriate amount of (R, S)-diosbulbin B reference substance was accurately weighed and dissolved in methanol to prepare a reference solution containing 20 μg of (R, S)-diosbulbin B per 1 mL.

[0169] An appropriate amount of isoflavonoid reference substance was accurately weighed and dissolved in methanol to prepare a reference solution containing 80 μg of isoflavonoid per 1 mL.

[0170] An appropriate amount of forsythoside reference substance was accurately weighed and dissolved in methanol to prepare a reference solution containing 80 μg of forsythoside per 1 mL.

[0171] An appropriate amount of forsythoside A reference substance was accurately weighed and dissolved in methanol to prepare a reference solution containing 200 μg of forsythoside A per 1 mL.

[0172] An appropriate amount of rutin reference substance was accurately weighed and dissolved in methanol to prepare a reference solution containing 70 μg of rutin per 1 mL.

[0173] An appropriate amount of protocatechuic acid reference substance was accurately weighed and dissolved in methanol to prepare a reference solution containing 40 μg of protocatechuic acid per 1 mL.

[0174] An appropriate amount of gallic acid reference substance was accurately weighed and dissolved in methanol to prepare a reference solution containing 40 μg of gallic acid per 1 mL.

[0175] The chromatographic conditions were performed according to gradient 13 of Example 1.

[0176] 2. Experimental Results

[0177] The results are as follows Figure 10 As shown, forsythoside was not eluted under this chromatographic gradient, so the acetonitrile concentration needs to be increased to elute it.

[0178] VII. Elution gradient

[0179] 1. Experimental Methods

[0180] The experimental method in "VI. Chromatographic Peak Identification" is followed, except that elution is performed according to the gradient in Table 3.

[0181] Table 3 Elution gradient of mobile phase

[0182]

[0183]

[0184] 2. Experimental Results

[0185] The results are as follows Figure 11 As shown, forsythoside can be eluted under the above three chromatographic gradients, but gradients 24 and 26 show severe baseline drift in the later stages of retention time. Therefore, gradient 25 was selected as the final elution gradient.

[0186] 8. Elution flow rate

[0187] 1. Experimental Methods

[0188] The cold and fever granule test solution was prepared according to the pretreatment method in Example 3.

[0189] ACQUITY using octadecylsilane-bonded silica gel as a filler An HSS T3 C18 column (1.8 μm, 150 mm × 2.1 mm) was used as the chromatographic column. Acetonitrile was used as the mobile phase A, and 0.1% formic acid aqueous solution (v / v) was used as the mobile phase B. The detection wavelength was 254 nm. The elution gradient of the mobile phase was set according to gradient 25 in "VII. Elution Gradient". The column temperature was 25 °C, and the elution flow rates were set to 0.1 mL / min, 0.2 mL / min, 0.3 mL / min and 0.4 mL / min, respectively.

[0190] 2. Experimental Results

[0191] The results are as follows Figure 12 As shown, compared with other flow rates, when the elution flow rate is 0.3 mL / min, the peak elution time of the sample spectrum is moderate, the peak resolution is high, and the distribution is uniform. Therefore, 0.3 mL / min was selected as the final elution flow rate.

[0192] Nine, column temperature

[0193] 1. Experimental method

[0194] According to the experimental method of "Eight, elution flow rate", set the elution flow rate to 0.3 mL / min, and set the column temperature to 20°C, 25°C, 30°C, 35°C and 40°C, respectively.

[0195] 2. Experimental results

[0196] As shown in the results, with the increase of column temperature, the separation degree of each main chromatographic peak slightly decreases. Combined with the actual environment of the experiment, 25°C is finally selected as the final column temperature. Figure 13

[0197] Ten, sample volume

[0198] 1. Experimental method

[0199] According to the experimental method of "Eight, elution flow rate", set the elution flow rate to 0.3 mL / min, set the column temperature to 25°C, and set the sample volume to 1 μL, 2 μL, 3 μL, 4 μL and 5 μL, respectively.

[0200] 2. Experimental results

[0201] As shown in the results, the sample volume has a certain influence on the separation degree of each main peak. With the increase of sample volume, the response of each main peak significantly increases, but the separation degree of the main component peak significantly decreases, so 2 μL is selected as the final sample volume with moderate response and stable baseline. Figure 14 Example 5 A method for constructing the UPLC fingerprint of a cold fever granule

[0202] 1. Preparation of reference solution

[0203] Take the (R, S)-adimolol reference substance, accurately weigh, add methanol to prepare a reference solution containing (R, S)-adimolol 20 μg per 1 mL, and obtain it.

[0204] Take the isofukinol reference substance, accurately weigh, add methanol to prepare a reference solution containing isofukinol 80 μg per 1 mL, and obtain it.

[0205] Take the forsythoside reference substance, accurately weigh, add methanol to prepare a reference solution containing forsythoside 80 μg per 1 mL, and obtain it.

[0206] Take the forsythoside A reference substance, accurately weigh, add methanol to prepare a reference solution containing forsythoside A 200 μg per 1 mL, and obtain it.

[0207]

[0208] ​​Accurately weigh Rutin reference substance, add methanol to prepare reference solution containing 70 μg Rutin per 1 mL.

[0209] Accurately weigh Protocatechuic acid reference substance, add methanol to prepare reference solution containing 40 μg Protocatechuic acid per 1 mL.

[0210] Accurately weigh Gallic acid reference substance, add methanol to prepare reference solution containing 40 μg Gallic acid per 1 mL.

[0211] 2. Preparation of test solution

[0212] Accurately weigh about 2.0 g (sucrose-free type) or 8.0 g (sucrose-containing type, converted according to the preparation process) of cold and fever relieving granules of different batches, place in a conical flask with a plug, accurately add 25 mL of 30% methanol solution (v / v), weigh, treat with ultrasound (40 kHz, 560 W) for 45 min, remove, cool to room temperature, make up the weight loss with 30% methanol solution (v / v), shake well, filter, and take the filtrate, to obtain the test solution.

[0213] 3. Chromatographic conditions and system suitability

[0214] Take octadecylsilane-bonded silica gel as the filler (2.1 mm x 150 mm, 1.8 μm); take acetonitrile as mobile phase A and 0.1% formic acid aqueous solution (v / v) as mobile phase B, perform gradient elution according to the conditions in Table 4; the detection wavelength is 254 nm; the column temperature is 25°C; and the flow rate is 0.3 mL per minute. The theoretical plate number should not be less than 5000, calculated according to (R, S)-aduchun.

[0215] Table 4 Elution gradient of mobile phase

[0216]

[0217] 4. Assay

[0218] Accurately take 2 μL of the reference solution and the test solution, inject into the ultra-high performance liquid chromatograph, determine, record the chromatogram, introduce the chromatogram into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, screen and confirm the common peaks, and generate the UPLC fingerprint of cold and fever relieving granules through software.

[0219] 5. Identification of chromatographic peaks and selection of reference peaks

[0220] The retention time (8.18 min, 12.99 min, 14.71 min, 48.13 min, 48.86 min, 52.11 min, 69.22 min) and DAD spectrum of the peaks 3, 8, 9, 24, 25, 28 and 32 in the reference substance chromatogram corresponded to those of the peaks 3, 8, 9, 24, 25, 28 and 32 in the sample chromatogram (see Figure 15 and Figure 16 ), so the peaks 3, 8, 9, 24, 25, 28 and 32 were determined to be gallic acid, (R, S)-adelfangchin, protocatechuic acid, isofraxidin, rutin, forsythoside A and forsythoside, respectively. The peak 9 (protocatechuic acid) was well separated and located in the middle, so the peak 9 was determined to be the reference peak (S peak).

[0221] The relative retention time of the characteristic peaks 1-33 was calculated with the peak 9 as the reference peak (S peak), and the relative retention time was within ±5% of the specified value; the specified value was 0.38 for peak 1, 0.45 for peak 2, 0.56 for peak 3, 0.61 for peak 4, 0.68 for peak 5, 0.71 for peak 6, 0.83 for peak 7, 0.88 for peak 8, 1.00 for peak 9 (S peak), 1.09 for peak 10, 1.42 for peak 11, 1.83 for peak 12, 1.85 for peak 13, 1.88 for peak 14, 2.13 for peak 15, 2.27 for peak 16, 2.34 for peak 17, 2.67 for peak 18, 2.80 for peak 19, 2.83 for peak 20, 2.87 for peak 21, 2.93 for peak 22, 3.10 for peak 23, 3.27 for peak 24, 3.32 for peak 25, 3.44 for peak 26, 3.50 for peak 27, 3.54 for peak 28, 3.88 for peak 29, 4.06 for peak 30, 4.52 for peak 31, 4.71 for peak 32 and 5.00 for peak 33.

[0222] Example 6: Drug taste attribution of the chromatographic peaks and characteristic peaks of the raw drug tastes of Isatidis Folium and Radix Isatidis

[0223] I. Experimental method

[0224] The sample solution and the test solutions of the four drug tastes were each injected into the ultra-high performance liquid chromatograph by 2 μL, analyzed according to the chromatographic conditions of Example 5, and the chromatogram was recorded.

[0225] The test solution was prepared according to the method of "2. Preparation of test solution" in Example 5, and the four kinds of medicinal herb test solutions were prepared according to the method of "2. Preparation of test solution" in Example 5, i.e. 2 g of each of Isatis indigotica, Radix Baphlai, Forsythia suspensa and Gomphnaa hirtella were accurately weighed into a conical flask with a stopper, 25 mL of 30% methanol solution (v / v) was accurately added, the weight was determined, and the sample was treated by ultrasonic (40 kHz, 560 W) for 45 min. After cooling to room temperature, the lost weight was made up with 30% methanol solution (v / v), and the mixture was shaken and filtered. The filtrate was collected to obtain the Isatis indigotica test solution, Radix Baphlai test solution, Forsythia suspensa test solution and Gomphnaa hirtella test solution.

[0226] II. Experimental results

[0227] 1. Assignment of chromatographic peaks to medicinal herbs

[0228] The results of the assignment of chromatographic peaks of Ganmao Tuire Granules to Isatis indigotica are shown in Table 1, which shows that chromatographic peaks 1, 2, 3, 4, 5, 6, 7, 8, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 27, 29 in the test solution of Ganmao Tuire Granules belong to the medicinal herb Isatis indigotica. Figure 17 The results of the assignment of chromatographic peaks of Ganmao Tuire Granules to Radix Baphlai are shown in Table 2, which shows that chromatographic peaks 1, 4, 6, 7, 8, 10, 13, 16, 17, 20 in the test solution of Ganmao Tuire Granules belong to the medicinal herb Radix Baphlai.

[0229] Figure 18 The results of the assignment of chromatographic peaks of Ganmao Tuire Granules to Forsythia suspensa are shown in Table 3, which shows that chromatographic peaks 2, 9, 11, 13, 23, 25, 26, 28, 30, 31, 32, 33 in the test solution of Ganmao Tuire Granules belong to the medicinal herb Forsythia suspensa.

[0230] The results of the assignment of chromatographic peaks of Ganmao Tuire Granules to Gomphnaa hirtella are shown in Table 4, which shows that chromatographic peaks 5, 9, 12, 13 in the test solution of Ganmao Tuire Granules belong to the medicinal herb Gomphnaa hirtella. Figure 19

[0231] Figure 20 The results of the assignment of chromatographic peaks of Ganmao Tuire Granules to Gomphnaa hirtella are shown in Table 4, which shows that chromatographic peaks 5, 9, 12, 13 in the test solution of Ganmao Tuire Granules belong to the medicinal herb Gomphnaa hirtella.

[0232] 2. Confirmation of characteristic peaks of raw medicinal herbs Isatis indigotica and Radix Baphlai

[0233] The results of the confirmation of characteristic peaks of raw medicinal herbs Isatis indigotica and Radix Baphlai of Ganmao Tuire Granules are shown in Table 5, which shows that the characteristic peaks of the raw medicinal herbs Isatis indigotica and Radix Baphlai of Ganmao Tuire Granules are consistent with the characteristic peaks of the raw medicinal herbs Isatis indigotica and Radix Baphlai. Figure 21 ​​​As shown, Isatis indigotica and Radix Isatidis contain more identical chromatographic peaks. Among the 7 identified characteristic peaks, (R, S)-adelferin (peak 8) has a high content in Radix Isatidis, but a very low content in Isatis indigotica; isovitexin (peak 24) has a high content in Isatis indigotica, but a very low content or no content in Radix Isatidis. Therefore, (R, S)-adelferin identified in this method can be used as a characteristic peak of Radix Isatidis, and isovitexin can be used as a characteristic peak of Isatis indigotica, and the presence or absence of the peaks can be used to determine whether the cold and fever relieving granules are made of Radix Isatidis or Isatis indigotica.

[0234] Example 7 A quality detection method of a cold and fever relieving granule

[0235] 1. UPLC spectrum of a cold and fever relieving granule to be tested

[0236] According to “2. Preparation of test sample solution” in Example 5, the cold and fever relieving granule to be tested was prepared into a test sample solution.

[0237] According to “3. Chromatographic conditions and system suitability” in Example 5, 2 μL of the test sample solution was precisely taken and injected into an ultra-high performance liquid chromatograph to record a chromatogram, thereby obtaining the UPLC spectrum of the cold and fever relieving granule to be tested.

[0238] 2. UPLC fingerprint spectrum

[0239] Different batches of cold and fever relieving granule samples were taken, and the different batches of cold and fever relieving granule samples were detected according to the method of Example 5. The chromatograms were recorded, and the chromatograms were introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to screen and confirm common peaks. The UPLC fingerprint spectrum of the cold and fever relieving granule was generated by software;

[0240] The UPLC fingerprint spectrum contains 33 fingerprint peaks, and peak No. 9 is the S peak. The specified values are as follows:

[0241] 0.38 for peak No. 1, 0.45 for peak No. 2, 0.56 for peak No. 3, 0.61 for peak No. 4, 0.68 for peak No. 5, 0.71 for peak No. 6, 0.83 for peak No. 7, 0.88 for peak No. 8, 1.00 for peak No. 9 (S peak), 1.09 for peak No. 10, 1.42 for peak No. 11, 1.83 for peak No. 12, 1.85 for peak No. 13, 1.88 for peak No. 14, 2.13 for peak No. 15, 2.27 for peak No. 16, 2.34 for peak No. 17, 2.67 for peak No. 18, 2.80 for peak No. 19, 2.83 for peak No. 20, 2.87 for peak No. 21, 2.93 for peak No. 22, 3.10 for peak No. 23, 3.27 for peak No. 24, 3.32 for peak No. 25, 3.44 for peak No. 26, 3.50 for peak No. 27, 3.54 for peak No. 28, 3.88 for peak No. 29, 4.06 for peak No. 30, 4.52 for peak No. 31, 4.71 for peak No. 32, and 5.00 for peak No. 33.

[0242] 3. Judgment criteria

[0243] The cold and fever relieving granules under test sample is determined to be qualified only when the following two conditions are met:

[0244] The UPLC chromatogram of the cold and fever relieving granules under test sample presents the fingerprint peaks within ±5% of the specified values at the corresponding retention time in the UPLC fingerprint chromatogram;

[0245] The UPLC chromatogram of the cold and fever relieving granules under test sample and the UPLC fingerprint chromatogram are imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (National Pharmacopoeia Committee, 2012 edition) together. After the chromatographic peaks in the UPLC chromatogram of the under test sample are matched with the 33 fingerprint peaks in the UPLC fingerprint chromatogram, the similarity is calculated. When the similarity of the UPLC chromatogram of the under test sample to the UPLC fingerprint chromatogram is greater than 0.85, it is determined to be qualified.

[0246] Methodology investigation of Example 8

[0247] I. Specificity investigation

[0248] 1. Experimental method

[0249] The cold and fever relieving granules were taken and the sample solution and the blank sample solution were prepared according to the method of Example 5. The sample was injected and analyzed according to the chromatographic conditions of Example 5, and the chromatogram was recorded.

[0250] 2. Experimental results

[0251] The results are shown in Table 1 and Table 2. Figure 22 As shown in Table 1 and Table 2, the extraction solvent and elution solvent of the sample have no absorption under the chromatographic conditions and do not interfere with the chromatographic analysis of the sample, indicating that the established method has good specificity.

[0252] II. Instrument precision investigation

[0253] 1. Experimental method

[0254] The cold and fever relieving granules were taken and the sample solution was prepared according to the method of Example 5. The sample was injected continuously for 6 times, and analyzed according to the chromatographic conditions of Example 5, and the chromatogram was recorded. The obtained 6 chromatograms were analyzed by data using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 edition) of the National Pharmacopoeia Committee.

[0255] 2. Experimental results

[0256] The results are shown in Table 5, Table 6, Table 7 and Table 8. Figure 23 As shown in Table 5, Table 6, Table 7 and Table 8, the full spectrum similarity of the 6 chromatograms is 1.000, and the RSD values of the relative retention time and the relative peak area of the 33 main component peaks are all less than 5%. The investigation results show that the instrument precision meets the requirements.

[0257] Table 5 Relative retention time of precision investigation

[0258]

[0259] Table 6 Relative peak area of precision investigation

[0260]

[0261]

[0262] Table 7 Similarity of instrument precision investigation spectrum

[0263]

[0264] Three, repeatability investigation

[0265] 1. Experimental method

[0266] Take 6 portions of Ganmao Tuire Granules, respectively prepare into test solution according to the method of Example 5, analyze according to the chromatographic condition of Example 5, record the chromatogram. Adopt the data analysis of the obtained 6 spectra by the National Pharmacopoeia Committee "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition).

[0267] 2. Experimental results

[0268] The results are shown in Table 8, Table 9, Table 10 and Figure 24 The full spectrum similarity of the 6 spectra is 0.998, and the RSD values of the relative retention time and relative peak area of the 33 main component peaks are all less than 7%. The investigation results show that the repeatability of the method meets the requirements.

[0269] Table 8 Relative retention time of repeatability investigation

[0270]

[0271] Table 9 Relative peak area of repeatability investigation

[0272]

[0273]

[0274] Table 10 Similarity of repeatability investigation spectrum

[0275]

[0276] Four, stability investigation

[0277] 1. Experimental method

[0278] Take the cold fever granules, prepared into test solution according to the method of Example 5, respectively at 0h, 2h, 4h, 8h, 12h and 24h after preparation, analyzed according to the chromatographic conditions of Example 5, and recorded the chromatograms. The obtained 6 chromatograms were analyzed by data analysis using the National Pharmacopoeia Committee "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition).

[0279] 2. Experimental results

[0280] The results are shown in Tables 11, 12, 13 and Figure 25 The full-spectrum similarity of the 6 chromatograms all reached 0.999, and the RSD values of the relative retention time and relative peak area of the 33 main component peaks were all less than 6%. The results of the investigation showed that the sample had good stability within 24h and met the requirements of the methodology.

[0281] Table 11 Relative retention time of stability investigation

[0282]

[0283]

[0284] Table 12 Relative peak area of stability investigation

[0285]

[0286]

[0287] Table 13 Similarity of chromatograms of stability investigation

[0288]

[0289] V. Durability investigation

[0290] 1. Experimental method

[0291] Take the cold fever granules, prepared into test solution according to the method of Example 5, respectively using HSS T3C18 chromatographic column (1.8μm, 150mm×2.1mm), T3C18 chromatographic column (1.6μm, 150mm×2.1mm), Peptide HSS T3C18 chromatographic column (1.8μm, 150mm×2.1mm), C18+ chromatographic column (1.6μm, 150mm×2.1mm) four different types of chromatographic columns, analyzed according to the chromatographic conditions of Example 5, and recorded the chromatograms. The obtained 4 chromatograms were analyzed by data analysis using the National Pharmacopoeia Committee "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition).

[0292] 2. Experimental Results

[0293] The results are as follows Figure 26 As shown, using T3C18 chromatographic column, ACQUITY PeptideHSS T3C18 chromatographic column, ACQUITY cannot be achieved when using a C18+ column. The HSS T3C18 column showed poor or absent resolution of the main chromatographic peaks, making similarity analysis impossible. Further analysis using ACQUITY is necessary. UPLC fingerprint analysis of cold and fever relieving granules was performed using an HSS T3 C18 column.

[0294] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a UPLC fingerprint of a cold and fever-reducing granule, characterized in that, The construction method includes the following steps: Preparation of test solution: Take the sample of cold and fever granules to be tested and extract it with a methanol aqueous solution with a volume fraction of 25-35% by ultrasonic extraction for 40-50 min; Reference solutions: (R, S)-Gouyichun solution, isovitexin solution, forsythoside solution, forsythoside A solution, rutin solution, protocatechuic acid solution and gallic acid solution; Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the column packing material; acetonitrile was used as mobile phase A; and a 0.08–0.12% (v / v) formic acid aqueous solution was used as mobile phase B. In the gradient elution program, the volume percentage change of mobile phase B in the mobile phase system was as follows: From 0 to 15 minutes, the mobile phase B decreased from 100% to 95%. Within 15–25 minutes, the mobile phase B decreased from 95% to 90%. Over 25–40 minutes, the mobile phase B decreased from 90% to 85%. After 40–50 minutes, the mobile phase B decreased from 85% to 84%. In 50–60 minutes, the mobile phase B decreased from 84% to 80%. At 60–80 min, the mobile phase B decreased from 80% to 70%. The detection wavelength is 254 nm, and the theoretical plate number, calculated according to (R, S) - Gao Yichun, should not be less than 5000; Using the above construction method, different batches of cold and fever relieving granules were tested, chromatograms were recorded, and the chromatograms were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to screen and confirm common peaks, thus obtaining the UPLC fingerprint of the cold and fever relieving granules.

2. The construction method according to claim 1, characterized in that, The cold and fever granule sample to be tested was a sugar-free sample, and the mass-to-volume ratio of the cold and fever granule sample to the methanol aqueous solution was 1 g: (12-13) mL. The cold and fever granule sample to be tested is a sucrose-containing sample, and the mass-volume ratio of the cold and fever granule sample to the methanol aqueous solution is 1 g: (3-4) mL.

3. The construction method according to claim 1, characterized in that, The preparation method of the (R,S)-gayocin solution is as follows: (R,S)-gayocin is thoroughly mixed with methanol, and the mass-volume ratio of (R,S)-gayocin to methanol is (15-25) μg: 1 mL. The method for preparing the isovitillin solution is as follows: isovitillin and methanol are thoroughly mixed, and the mass-to-volume ratio of isovitillin to methanol is (75-85) μg: 1 mL; The preparation method of the forsythoside solution is as follows: forsythoside and methanol are thoroughly mixed, and the mass-volume ratio of forsythoside to methanol is (75-85) μg: 1 mL; The preparation method of the forsythoside A solution is as follows: forsythoside A and methanol are thoroughly mixed, and the mass-volume ratio of forsythoside A to methanol is (195-205) μg: 1 mL. The rutin solution is prepared by mixing rutin and methanol thoroughly, wherein the mass-to-volume ratio of rutin to methanol is (65-75) μg: 1 mL. The method for preparing the protocatechuic acid solution is as follows: protocatechuic acid and methanol are thoroughly mixed, and the mass-volume ratio of protocatechuic acid to methanol is (35-45) μg: 1 mL; The gallic acid solution is prepared by mixing gallic acid and methanol thoroughly, wherein the mass-to-volume ratio of gallic acid to methanol is (35-45) μg: 1 mL.

4. The construction method according to claim 1, characterized in that, The column temperature of the chromatographic column is 20–30°C.

5. The construction method according to claim 1, characterized in that, The gradient elution flow rate is 0.2–0.4 mL / min.

6. The construction method according to claim 1, characterized in that, The injection volume of the reference solution and the test solution is 1–3 μL.

7. The application of the construction method according to any one of claims 1 to 6 in establishing the UPLC fingerprint of cold and fever granules and / or in the quality control of cold and fever granules.

8. A quality testing method for cold and fever-reducing granules, characterized in that, Includes the following steps: S1. Take a sample of cold and fever granules to be tested, and use the construction method described in any one of claims 1 to 6 to perform the test, record the chromatogram, and obtain the UPLC chromatogram of the cold and fever granules to be tested; S2. The UPLC fingerprint of the cold and fever-reducing granules described in claim 1 is compared with the UPLC fingerprint of the cold and fever-reducing granules sample to be tested in the traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The sample is deemed qualified only if it meets the following two conditions: The UPLC chromatogram of the cold and fever granule sample to be tested shows fingerprint peaks with corresponding retention times within ±5% of the specified value in the UPLC fingerprint chromatogram; The similarity between the UPLC spectrum and the UPLC fingerprint spectrum of the cold and fever granule sample to be tested was calculated. The similarity between the UPLC spectrum and the UPLC fingerprint spectrum of the cold and fever granule sample to be tested was not less than 0.

85. The UPLC fingerprint spectrum contains 33 fingerprint peaks, with peak number 9 designated as peak S. The specified values ​​are as follows: Peak 1: 0.38; Peak 2: 0.45; Peak 3: 0.56; Peak 4: 0.61; Peak 5: 0.68; Peak 6: 0.71; Peak 7: 0.83; Peak 8: 0.88; Peak 9 (S Peak): 1.00; Peak 10: 1.09; Peak 11: 1.42; Peak 12: 1.83; Peak 13: 1.85; Peak 14: 1.88; Peak 15: 2.13; Peak 16: 2.27; Peak 17: 2.

3. Peak 4, Peak 18 2.67, Peak 19 2.80, Peak 20 2.83, Peak 21 2.87, Peak 22 2.93, Peak 23 3.10, Peak 24 3.27, Peak 25 3.32, Peak 26 3.44, Peak 27 3.50, Peak 28 3.54, Peak 29 3.88, Peak 30 4.06, Peak 31 4.52, Peak 32 4.71, Peak 33 5.00.

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