Construction method and application of UPLC (Ultra Performance Liquid Chromatography) fingerprint spectrum of cold antipyretic granules

Abstract

The invention discloses a construction method and application of a UPLC (Ultra Performance Liquid Chromatography) fingerprint spectrum of cold antipyretic granules. By utilizing the method disclosed by the invention, 33 chromatographic peaks can be confirmed, and the four raw material medicines including folium isatidis, radix isatidis, fructus forsythiae and bistort rhizome in the cold and fever abatement granules all have corresponding attribution peaks. The characteristic peaks of seven chemical components including gallic acid, (R, S)-epigoitrin, protocatechuic acid, isovitexin, rutin, forsythiaside A and forsythin are identified through comparison of reference substances and combination of ultraviolet absorption spectra, the isovitexin can be used as the characteristic peak of folium isatidis, the (R, S)-epigoitrin can be used as the characteristic peak of radix isatidis, the rutin can be used as the characteristic peak of rutin, and the forsythiaside A can be used as the characteristic peak of rutin. The two characteristic peaks can separate the folium isatidis and the isatis root as raw material medicines. According to the construction method of the UPLC fingerprint spectrum, the fingerprint spectrum with good separation degree and comprehensive chromatographic peaks can be obtained, the overall quality of the cold antipyretic granules can be evaluated more objectively and comprehensively, and a foundation is laid for quality control of the cold antipyretic granules.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis and detection, and in particular to a method for constructing a UPLC fingerprint of cold and fever-reducing granules and an application thereof. Background Art

[0002] The prescription of Ganmao Tuire Granules is composed of Folium Isatidis, Radix Isatidis, Fructus Forsythiae and Radix Polygoni Multiflori, which are decocted with water and made into granules after alcohol precipitation and water precipitation. It has the effects of clearing away heat and detoxifying, dispelling wind and relieving exterior symptoms, and is mainly used for upper respiratory tract infection, acute tonsillitis, pharyngitis, which are caused by exogenous wind-heat, heat-toxic congestion, and symptoms of fever and sore throat. Although Ganmao Tuire Granules are included in the Chinese Pharmacopoeia, their quality standards are still relatively weak, and only thin-layer identification of indigo carmine and forsythin and content determination of forsythin are performed.

[0003] A large number of studies have shown that traditional Chinese medicine, especially Chinese patent medicine prepared from multiple Chinese medicines, relies on multiple active ingredients to act on multiple targets of the body to exert its therapeutic effect. At present, Ganmao Tuife Granules only rely on one or several ingredients to control the quality of Chinese patent medicines, which makes it difficult to effectively guarantee its efficacy. Moreover, the preparation process of Ganmao Tuife Granules is relatively complicated, requiring alcohol precipitation and water precipitation processes. The production process is evaluated only by the content of one ingredient (forsythiaside), which cannot reflect the overall picture of the quality transfer of traditional Chinese medicine after preparation by modern technology. In addition, the raw materials of Ganmao Tuife Granules, Daqingye and Banlangen, come from different parts of the same medicinal plant, and the two have similar chemical compositions. If Daqingye or Banlangen are not added during the production and preparation of Ganmao Tuife Granules, it is difficult to discover or control it through existing quality standards or existing technologies. Prior art 1 (Study on Quality Control of Cold and Fever Relief Granule Soup, Publication Date: June 1, 2004) discloses the HPLC fingerprint of the Cold and Fever Relief Granule Soup, and identifies six peaks of isovitexin, forsythin, gallic acid, chlorogenic acid, uridine and adenosine. However, among these six peaks, uridine and adenosine are components commonly contained in Folium Isatidis and Radix Isatidis, while the remaining five peaks are respectively attributed to Fructus Forsythiae and Radix Polygoni Multiflori. Therefore, the fingerprint cannot effectively identify Folium Isatidis and Radix Isatidis in the Cold and Fever Relief Granules, and thus cannot perform a good qualitative and quantitative evaluation of the quality of the Cold and Fever Relief Granules.

[0004] Therefore, there is an urgent need for a method that can control the quality of cold and fever granules from multiple components and multiple angles and detect whether the four medicinal flavors are incompletely added during the production and preparation process, especially the method that uses different medicinal parts of the same medicinal plant, namely, Isatis indigotica and Isatis root. Summary of the invention

[0005] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present invention provides a method for constructing a UPLC fingerprint of cold and fever-reducing granules and its application.

[0006] The first object of the present invention is to provide a pretreatment method for detecting cold and fever-reducing granules by ultra-high performance liquid chromatography.

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

[0008] The third object of the present invention is to provide the application of the above-mentioned pretreatment method and / or ultra-high performance liquid chromatography method in establishing the UPLC fingerprint of Ganmao Tuire Granules and / or the quality control of Ganmao Tuire Granules.

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

[0010] The fifth object of the present invention is to provide a method for detecting the quality of cold and fever-reducing granules.

[0011] Therefore, the present invention claims the following:

[0012] The invention discloses a pretreatment method for detecting cold and fever granules by ultra-high performance liquid chromatography. The method comprises taking a sample of the cold and fever granules to be detected and ultrasonically extracting the sample for 40 to 50 minutes with a methanol aqueous solution with a volume fraction of 25 to 35%.

[0013] The cold and fever-reducing granules are prepared in accordance with the "Pharmacopoeia of the People's Republic of China: 2020 Edition" (Part 1). Specifically, 435g of isatis indigotica, 435g of isatis root, 217g of forsythia and 217g of polygonum multiflorum are weighed, decocted twice with water, each time for 1.5 hours, the decoctions are combined, filtered, and the filtrate is concentrated to a clear paste 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 mixture is allowed to stand. The supernatant is concentrated to a clear paste with a relative density of 1.20 (60°C), an equal amount of water is added, stirred, and allowed to stand for 8 hours. The supernatant is concentrated into a thick paste with a relative density of 1.38-1.40 (60°C), and appropriate amounts of sucrose powder, dextrin and ethanol are added to make granules, which are dried to make 1000g of cold and fever granules (containing sucrose type); or the supernatant is concentrated into a clear paste with a relative density of 1.09-1.11 (60°C), and appropriate amounts of dextrin and flavoring agents are added, mixed, spray-dried, and made into 250g of cold and fever granules (no sucrose type); the cold and fever granules are brown-yellow granules.

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

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

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

[0017] More preferably, the cold and fever relieving granule sample to be tested is a sucrose-free sample, and the mass volume ratio of the cold and fever relieving granule sample to be tested to the methanol aqueous solution is 1 g:12.5 mL;

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

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

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

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

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

[0023] Reference solution: (R, S)-goitrin solution, isovitexin solution, forsythiaside solution, forsythiaside A solution, rutin solution, protocatechuic acid solution and / or gallic acid solution;

[0024] Test solution: Prepare using any of the above pretreatment methods;

[0025] Chromatographic conditions: Octadecylsilane bonded silica gel is used as the filler of the chromatographic column, acetonitrile is used as the mobile phase A, and a formic acid aqueous solution with a volume fraction of 0.08-0.12% is used 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, mobile phase B decreased from 100% to 95%;

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

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

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

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

[0031] 60-80min, mobile phase decreased from 80% to 70%;

[0032] The detection wavelength is 254 nm, and the theoretical plate number calculated according to (R, S)-galactoside should be no less than 5000.

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

[0034] The preparation method of the isovitexin solution is as follows: fully mixing isovitexin and methanol, wherein the mass volume ratio of isovitexin to methanol is (75-85) μg:1 mL;

[0035] The preparation method of the forsythiaside solution is: fully mixing forsythiaside and methanol, wherein the mass volume ratio of forsythiaside to methanol is (75-85) μg:1 mL;

[0036] The preparation method of the forsythiaside A solution is as follows: fully mixing forsythiaside A and methanol, wherein the mass volume ratio of forsythiaside A to methanol is (195-205) μg:1 mL;

[0037] The preparation method of the rutin solution is: rutin and methanol are fully mixed, 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: fully mixing protocatechuic acid and methanol, wherein 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: fully mixing gallic acid and methanol, wherein 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)-gaitrin to methanol is 20 μg:1 mL;

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

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

[0043] The mass volume ratio of the forsythiaside 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°C.

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

[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 to 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 ACQUITY HSS T3 C18 chromatographic column, specification is 1.8μm, 150mm×2.1mm.

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

[0055] Application of any of the above-mentioned pretreatment methods and / or ultra-high performance liquid chromatography methods in establishing the UPLC fingerprint of Ganmao Tuire Granules and / or quality control of Ganmao Tuire Granules.

[0056] A method for constructing a UPLC fingerprint of Ganmao Tuire Granules, comprising: using any of the above-mentioned ultra-high performance liquid chromatography methods to detect different batches of Ganmao Tuire Granules samples, recording the chromatograms, importing the chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, screening and confirming the common peaks, and obtaining the UPLC fingerprint of Ganmao Tuire Granules;

[0057] The UPLC fingerprint of the cold and fever-relief granules contains 33 fingerprint peaks, with peak No. 9 as the S peak. The relative retention times of the 33 common peaks are:

[0058] 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 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;

[0059] The peak No. 3 is the characteristic chromatographic peak of gallic acid, the peak No. 8 is the characteristic chromatographic peak of (R, S)-goitrin, the peak No. 9 is the characteristic chromatographic peak of protocatechuic acid, the peak No. 24 is the characteristic chromatographic peak of isovitexin, the peak No. 25 is the characteristic chromatographic peak of rutin, the peak No. 28 is the characteristic chromatographic peak of forsythiaside A, and the peak No. 32 is the characteristic chromatographic peak of forsythiaside.

[0060] The different batches are no less than 15 batches.

[0061] A quality detection method for cold and fever-reducing granules, comprising the following steps:

[0062] S1. Take the sample of Ganmao Tuire Granules to be tested, use any of the above-mentioned ultra-high performance liquid chromatography methods for detection, record the chromatogram, and obtain the UPLC spectrum of the Ganmao Tuire Granules sample to be tested;

[0063] S2. The UPLC fingerprint of the Ganmao Tuire Granules and the UPLC fingerprint of the Ganmao Tuire Granules sample to be tested are imported into the Chinese medicine chromatographic fingerprint similarity evaluation system for comparison. The qualified ones can be judged to be qualified if the following two conditions are met:

[0064] The UPLC spectrum of the cold and fever-relief granule sample to be tested presents a fingerprint peak whose retention time in the UPLC fingerprint spectrum is within ±5% of the specified value;

[0065] The similarity between the UPLC spectrum of the tested Ganmao Tuire Granule sample and the UPLC fingerprint spectrum is calculated, and the similarity between the UPLC spectrum of the tested Ganmao Tuire Granule sample and the UPLC fingerprint spectrum is not less than 0.85;

[0066] The UPLC fingerprint spectrum contains 33 fingerprint peaks, with peak No. 9 as the S peak, and the specified values ​​are:

[0067] 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 4, Peak No. 18 is 2.67, Peak No. 19 is 2.80, Peak No. 20 is 2.83, Peak No. 21 is 2.87, Peak No. 22 is 2.93, Peak No. 23 is 3.10, Peak No. 24 is 3.27, Peak No. 25 is 3.32, Peak No. 26 is 3.44, Peak No. 27 is 3.50, Peak No. 28 is 3.54, Peak No. 29 is 3.88, Peak No. 30 is 4.06, Peak No. 31 is 4.52, Peak No. 32 is 4.71, and Peak No. 33 is 5.00.

[0068] Application of any of the above-mentioned ultra-high performance liquid chromatography methods in detecting the content of isatis indigotica, isatis root, forsythia and / or polygonum multiflorum in Ganmao Tuire granules.

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

[0070] The present invention discloses a method for constructing a UPLC fingerprint of cold and fever granules and its application. Using the method of the present invention, 33 chromatographic peaks can be confirmed, and the four raw materials of the cold and fever granules, namely, folium isatidis, root of isatis, forsythia and schizonepeta, all have corresponding attribution peaks. By comparing with reference substances and combining with ultraviolet absorption spectra, the characteristic peaks of seven chemical components, namely, gallic acid, (R, S)-goitrin, protocatechuic acid, isovitexin, rutin, forsythiaside A and forsythiaside, are identified, among which isovitexin can be used as the characteristic peak of folium isatidis, and (R, S)-goitrin can be used as the characteristic peak of root of isatis, and these two characteristic peaks can distinguish folium isatis from root of isatis. The construction method of the UPLC fingerprint disclosed in the present invention can obtain a fingerprint with good separation and comprehensive chromatographic peaks, and can more objectively and comprehensively evaluate the overall quality of the cold and fever granules, laying a foundation for the quality control of the cold and fever granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The chromatogram of the test sample obtained by the mobile phase elution gradient in Table 1.

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

[0073] Figure 3The effects of different extraction methods and extraction times on the chromatograms of the test samples; S1~S3 are ultrasonic extractions for 30min, 45min, and 60min, respectively; S4~S6 are heating reflux for 30min, 45min, and 60min, respectively.

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

[0075] Figure 5 This is the full wavelength scan of the test solution.

[0076] Figure 6 The influence 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 water solution (v / v), and acetonitrile-0.1% formic acid water solution (v / v), respectively.

[0077] Figure 7 is the effect 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 The influence 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] Fig. 9 Table 2 shows the effects of different elution gradients on the chromatograms of the test products.

[0080] Fig.10 The chromatographic peak identification results using gradient 13; S1 to S9 are Ganmao Tuire Granules, (R, S)-Gaoichun, Isovitex, Luteolin, Forsythiaside, Forsythiaside A, Rutin, Protocatechuic Acid, and Gallic Acid, respectively.

[0081] Fig.11 The influence of different elution gradients on the chromatograms of the test sample and the reference sample; S1 to S5 are the test sample solution (gradient 24), forsythiaside (gradient 24), the test sample solution (gradient 25), forsythiaside (gradient 25), and the test sample solution (gradient 26), respectively.

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

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

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

[0085] Fig.15 This is the fingerprint of the cold and fever-reducing granules.

[0086] Fig.16 The chromatographic peak identification results.

[0087] Fig.17 The chromatographic peak was attributed to Folium Isatidis.

[0088] Fig.18 The chromatographic peak was assigned to Radix Isatidis.

[0089] Fig.19 The chromatographic peak was assigned to Forsythia suspensa.

[0090] Fig. 20 This is the result of attributing the chromatographic peak to Polygonum multiflorum.

[0091] Fig.21 The results were confirmed by the characteristic peaks of Folium Isatidis and Radix Isatidis.

[0092] Fig. 22 For exclusive results.

[0093] Fig.23 This is the instrument precision result.

[0094] Fig.24 For reproducible results.

[0095] Fig.25 The stability result.

[0096] Fig.26 The durability results of different chromatographic columns are shown in Figure 2. DETAILED DESCRIPTION

[0097] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0099] Reference substance information used in the examples of the present invention:

[0100] Reference batch number content source (R, S)-Gao Yichun 111753-202007 100％ China Food and Drug Inspection Institute Isovitexin A0681 99.7％ Chengdu Mansite Biotechnology Co., Ltd. Forsythiaside 110821-202117 94.9％ China Food and Drug Inspection Institute Forsythiaside A 111810-202108 100％ China Food and Drug Inspection Institute Rutin 100080-202012 91.6％ China Food and Drug Inspection Institute Protocatechuic acid 110809-202207 97.5％ China Food and Drug Inspection Institute gallic acid 110831-201906 91.5％ China Food and Drug Inspection Institute

[0101] The sample information used in the embodiments of the present invention: The cold and fever granules are derived from major commercial manufacturers or pilot samples. The cold and fever granules are prepared in accordance with the "Pharmacopoeia of the People's Republic of China: 2020 Edition" (Part 1). Specifically, 435g of Isatis indigotica, 435g of Isatis indigotica, 217g of Forsythia and 217g of Polygonum multiflorum are weighed, decocted twice with water, each time for 1.5 hours, the decoctions are combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of about 1.08 (90-95°C), cooled to room temperature, and an equal amount of ethanol is added to precipitate, and the supernatant is concentrated to a clear paste with a relative density of 1.20 (60°C), and an equal amount of water is added, stirred, and allowed to stand for 8 hours. The supernatant is concentrated into a thick paste with a relative density of 1.38-1.40 (60°C), and appropriate amounts of sucrose powder, dextrin and ethanol are added to make granules, which are dried to make 1000g of cold and fever granules (containing sucrose type); or the supernatant is concentrated into a clear paste with a relative density of 1.09-1.11 (60°C), and appropriate amounts of dextrin and flavoring agents are added, mixed, spray-dried, and made into 250g of cold and fever granules (no sucrose type); the cold and fever granules are brown-yellow granules.

[0102] Example 1 Effects of different elution gradient conditions on the UPLC fingerprint of Ganmao Tuire Granules

[0103] 1. Experimental Methods

[0104] Take 0.5 g of the extract of Ganmao Tuifever Granules (ρ=1.23), accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 50% methanol solution (v / v), stopper it, weigh it, ultrasonically treat it (power 490 W, frequency 40 kHz) for 30 min, cool it, weigh it again, make up the lost weight with 50% methanol solution (v / v), shake it well, filter it, and take the filtrate to obtain the test solution.

[0105] Chromatographic conditions: ACQUITY HPLC with octadecylsilane bonded silica as filler HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm, column 1), ACQUITY HSS T3 C18 chromatographic column (1.8 μm, 150 mm×2.1 mm, chromatographic column 2); acetonitrile was used as mobile phase A, 0.1% formic acid aqueous solution (v / v) was used as mobile phase B, and water was used as mobile phase C (only for gradient 1), and elution was performed according to the gradient in Table 1; the detection wavelength was 254 nm, and the responses of the main peaks under different gradients were compared. The flow rate was 0.4 mL per minute, and the column temperature was 25°C.

[0106] Table 1 Mobile phase elution gradient

[0107]

[0108] (2) Experimental results

[0109] The results are as follows Figure 1 As shown in Figure 1, under the elution of gradient 13, the peak shape and separation of each main chromatographic peak are good, and the baseline is relatively stable, so gradient 13 is selected as the gradient for investigating the preparation method of the test solution, that is, ACQUITY HSS T3C18 chromatographic column (1.8 μm, 150 mm×2.1 mm); mobile phase is acetonitrile-0.1% formic acid aqueous solution (v / v); gradient elution: 0 min~15 min~25 min~40 min~50 min~60 min, the volume fraction of acetonitrile changes as follows: 0%~5%~10%~15%~16%~20%; flow rate 0.4 mL / min; column temperature 25°C; detection wavelength 254 nm; the injection volume of the test solution is 2 μL.

[0110] Example 2 Effect of the test sample preparation method on the UPLC fingerprint of cold and fever granules

[0111] 1. Extraction solvent

[0112] 1. Experimental Methods

[0113] Take about 2.0g of Ganmao Tuifever Granules, a total of 5 portions, accurately weighed, placed in a stoppered conical flask, accurately added methanol, 70% methanol solution (v / v), 50% methanol solution (v / v), 30% methanol solution (v / v), and 25mL of water, weighed, ultrasonically treated for 30min (power 560W, frequency 40kHz), cooled, weighed again, made up for the lost weight with the corresponding extraction solvent, shaken well, filtered, and took the filtrate.

[0114] The chromatographic conditions were in accordance with those of Gradient 13 in Example 1.

[0115] 2. Experimental results

[0116] The results are as follows Figure 2 As shown, the chromatographic peaks extracted with methanol are the least, and the number of chromatographic peaks extracted with 70% methanol solution (v / v), 50% methanol solution (v / v), 30% methanol solution (v / v) and water is not much different, and the response difference of the main chromatographic peaks is also relatively small. The solvent peaks extracted with 30% methanol solution (v / v) and water are smaller, making the overall peak shape better, and 30% methanol solution (v / v) is more conducive to the stable preservation 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] 2. Extraction method

[0118] 1. Experimental Methods

[0119] Take about 2.0g of cold and fever granules, a total of 6 portions, accurately weighed, placed in a stoppered conical flask, accurately added 25mL of 30% methanol solution (v / v), weighed, heated to reflux extraction for 30min, 45min, 60min and ultrasonic treatment for 30min, 45min, 60min (power 560W, frequency 40kHz), respectively, cooled, weighed again, made up for the lost weight with 30% methanol solution (v / v), shaken well, filtered, and took the filtrate.

[0120] The chromatographic conditions were in accordance with those of Gradient 13 in Example 1.

[0121] 2. Experimental results

[0122] The results are as follows Figure 3 As shown in the figure, the chromatographic peaks presented by the sample extracted by ultrasonic extraction for 30 minutes are slightly different from those of the samples in the other five extraction modes, with fewer component peaks; the number of chromatographic peaks presented by the samples in the other five extraction modes and the peak areas of the main chromatographic peaks are not much different. Therefore, based on the convenience of operation and analysis time, ultrasonic extraction for 45 minutes was selected as the final extraction method and time for Ganmao Tuire Granules.

[0123] 3. Extraction material-liquid ratio

[0124] 1. Experimental Methods

[0125] Take 4 portions of cold and fever granules (sucrose-free type) of 0.5g, 1.0g, 2.0g, and 4.0g respectively, weigh accurately, put them in stoppered conical flasks, and accurately add 30% methanol solution (v / v) according to the solid-liquid ratio of 0.5g:25mL, 1.0g:25mL, 2.0g:25mL, and 4.0g:25mL, respectively, weigh the weight, extract with ultrasound for 45min, cool, weigh again, make up the lost weight with 30% methanol solution (v / v), shake well, filter, and take the filtrate.

[0126] The chromatographic conditions were in accordance with those of Gradient 13 in Example 1.

[0127] 2. Experimental results

[0128] The results are as follows Figure 4 As shown, the number of chromatographic peaks in sample 1 (0.5g: 25mL) is slightly different from the other five samples, with fewer chromatographic peaks, but the difference between the other three samples is not large; the chromatographic peak response in sample 2 (1.0g: 25mL) is relatively small, resulting in a slight drift in the baseline; sample 4 (4.0g: 25mL) has a high concentration, making sample filtration more difficult, and the separation of some chromatographic peaks is reduced. Therefore, a solid-liquid ratio of 2.0g: 25mL was selected as the final solid-liquid ratio of the cold and fever granules.

[0129] Example 3 A pre-treatment method for UPLC fingerprint of cold and fever granules

[0130] Take about 2.0 g (sucrose-free type) or 8.0 g (sucrose-containing type, converted according to the preparation process) of the Ganmao Tuire Granule sample, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 30% methanol solution (v / v), weigh it, treat it with ultrasound (40 kHz, 560 W) for 45 min, take it out, cool it to room temperature, make up the lost weight with 30% methanol solution (v / v), shake it well, filter it, and take the filtrate to obtain it.

[0131] Example 4 Effect of Chromatographic Conditions on UPLC Fingerprint of Ganmao Tuire Granules

[0132] 1. Analysis wavelength

[0133] 1. Experimental Methods

[0134] The cold and fever-reducing granule test solution was prepared according to the pretreatment method of Example 3.

[0135] The test solution is scanned at full wavelength, and the best detection wavelength is selected from its 3D graph. The evaluation indicators are: large analytical signal response value at this wavelength, high peak information, uniform peak distribution and stable baseline, and good peak separation.

[0136] 2. Experimental results

[0137] The results are as follows Figure 5 As shown, the sample peak at 254nm has more complete information and a larger response value, so 254nm is selected as the detection wavelength.

[0138] 2. Mobile Phase System

[0139] 1. Experimental Methods

[0140] The cold and fever-reducing granule test solution was prepared according to the pretreatment method of Example 3.

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

[0142] 2. Experimental results

[0143] The results are as follows Figure 6As shown, the mobile phase system of acetonitrile-0.1% formic acid aqueous solution (v / v) obtained the test sample has more peak information, a more stable baseline, and better separation and peak shape, so the mobile phase system was finally determined to be acetonitrile-0.1% formic acid aqueous solution (v / v).

[0144] 3. Different types of acids

[0145] 1. Experimental Methods

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

[0147] The chromatographic conditions were carried out according to the gradient 13 of Example 1, except that the mobile phase systems were 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). The selection criteria were a large number of peaks in the sample spectrum, uniform peak distribution, good peak shape and main component peak separation, and a 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 is serious, and the separation of the chromatogram peaks in the acetonitrile-0.1% acetic acid aqueous solution (v / v) system is poor. Therefore, acetonitrile-0.1% formic acid aqueous solution (v / v) was selected as the mobile phase system.

[0150] 4. Concentration of formic acid aqueous solution

[0151] 1. Experimental Methods

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

[0153] The chromatographic conditions were carried out according to the gradient 13 of Example 1, except that the mobile phase system was set to acetonitrile-0.1% formic acid aqueous solution (v / v) and acetonitrile-0.3% formic acid aqueous solution (v / v). The selection criteria were a large number of peaks in the sample spectrum, uniform peak distribution, good peak shape and main component peak separation effect, and a stable baseline.

[0154] 2. Experimental results

[0155] The results are as follows Figure 8 As shown, when the concentration of formic acid aqueous solution increases, the absorption of the solvent peak is enhanced, resulting in the relative response of the effective chromatographic peak being depressed. In addition, the separation of some chromatographic peaks is reduced and the baseline is slightly unstable. Therefore, acetonitrile-0.1% formic acid aqueous solution (v / v) is still selected as the mobile phase system.

[0156] 5. Elution gradient

[0157] 1. Experimental Methods

[0158] The cold and fever-reducing granule test solution was prepared according to the pretreatment method of Example 3.

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

[0160] Table 2 Mobile phase elution gradient

[0161]

[0162] 2. Experimental results

[0163] The results are as follows Fig. 9 As shown, the above gradients cannot achieve the separation effect of gradient 13, so gradient 13 is still selected as the final elution gradient.

[0164] 6. Chromatographic peak identification

[0165] 1. Experimental Methods

[0166] The cold and fever-reducing granule test solution was prepared according to the pretreatment method of Example 3.

[0167] Preparation of reference solution:

[0168] Take an appropriate amount of (R, S)-gavezodone reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 20 μg of (R, S)-gavezodone per 1 mL.

[0169] Take an appropriate amount of isovitexin reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 80 μg of isovitexin per 1 mL.

[0170] Take an appropriate amount of forsythiaside reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 80 μg forsythiaside per 1 mL.

[0171] Take an appropriate amount of Forsythiaside A reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 200 μg of Forsythiaside A per 1 mL.

[0172] Take an appropriate amount of rutin reference substance, weigh it accurately, and add methanol to make a reference substance solution containing 70 μg of rutin per 1 mL.

[0173] Take an appropriate amount of protocatechuic acid reference substance, weigh it accurately, and add methanol to make a reference substance solution containing 40 μg of protocatechuic acid per 1 mL.

[0174] Take an appropriate amount of gallic acid reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 40 μg of gallic acid per 1 mL.

[0175] The chromatographic conditions were carried out according to the gradient 13 of Example 1.

[0176] 2. Experimental results

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

[0178] VII. Elution Gradient

[0179] 1. Experimental Methods

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

[0181] Table 3 Mobile phase elution gradient

[0182]

[0183]

[0184] 2. Experimental results

[0185] The results are as follows Fig.11 As shown in the figure, under the above three chromatographic gradients, forsythin can be eluted, but the baseline drift of gradients 24 and 26 is serious at the end of the retention time, so gradient 25 is selected as the final elution gradient.

[0186] 8. Elution flow rate

[0187] 1. Experimental Methods

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

[0189] ACQUITY with octadecylsilane bonded silica as filler HSS T3 C18 (1.8 μm, 150 mm × 2.1 mm) was used as the chromatographic column, acetonitrile was used as mobile phase A, 0.1% formic acid aqueous solution (v / v) was used as mobile phase B, the detection wavelength was 254 nm, the mobile phase elution gradient was according to the 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 Fig.12 As shown, compared with other flow rates, when the elution flow rate is 0.3 mL / min, the peak time of the spectrum of the test sample is moderate, the peak separation is high and the distribution is uniform, so 0.3 mL / min is selected as the final elution flow rate.

[0192] 9. Column temperature

[0193] 1. Experimental Methods

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

[0195] 2. Experimental results

[0196] The results are as follows Fig.13 As shown in the figure, as the column temperature increases, the separation of the main chromatographic peaks decreases slightly. Combined with the actual experimental environment, 25℃ was finally selected as the final chromatographic column temperature.

[0197] 10. Injection volume

[0198] 1. Experimental Methods

[0199] According to the experimental method of "VIII. Elution flow rate", the elution flow rate was set to 0.3 mL / min, the column temperature was set to 25°C, and the injection volumes were set to 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL, respectively.

[0200] 2. Experimental results

[0201] The results are as follows Fig.14 As shown in the figure, the injection volume has a certain influence on the separation of each major peak. With the increase of injection volume, the response of each major peak increases significantly, but the separation of the main component peak decreases significantly. Therefore, 2 μL with moderate response and stable baseline is selected as the final injection volume.

[0202] Example 5 A method for constructing a UPLC fingerprint of cold and fever relieving granules

[0203] 1. Preparation of reference solution

[0204] Take an appropriate amount of (R, S)-gavezodone reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 20 μg of (R, S)-gavezodone per 1 mL.

[0205] Take an appropriate amount of isovitexin reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 80 μg of isovitexin per 1 mL.

[0206] Take an appropriate amount of forsythiaside reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 80 μg forsythiaside per 1 mL.

[0207] Take an appropriate amount of Forsythiaside A reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 200 μg of Forsythiaside A per 1 mL.

[0208] Take an appropriate amount of rutin reference substance, weigh it accurately, and add methanol to make a reference substance solution containing 70 μg of rutin per 1 mL.

[0209] Take an appropriate amount of protocatechuic acid reference substance, weigh it accurately, and add methanol to make a reference substance solution containing 40 μg of protocatechuic acid per 1 mL.

[0210] Take an appropriate amount of gallic acid reference substance, weigh it accurately, and add methanol to prepare a reference substance solution containing 40 μg of gallic acid per 1 mL.

[0211] 2. Preparation of test solution

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

[0213] 3. Chromatographic conditions and system suitability

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

[0215] Table 4 Mobile phase elution gradient

[0216]

[0217] 4. Assay

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

[0219] 5. Chromatographic peak identification and reference peak selection

[0220] The retention times (8.18min, 12.99min, 14.71min, 48.13min, 48.86min, 52.11min, 69.22min) and DAD spectra of the peaks of the gallic acid reference substance, (R, S)-galactoside reference substance, protocatechuic acid reference substance, isovitexin reference substance, rutin reference substance, forsythiaside A reference substance and forsythiaside reference substance corresponded one to one with peaks 3, 8, 9, 24, 25, 28 and 32 in the spectrum of the test substance (see Fig.15 and Fig.16 ), so peaks 3, 8, 9, 24, 25, 28 and 32 were determined to be gallic acid, (R, S)-galactoatechin, protocatechuic acid, isovitexin, rutin, forsythiaside A and forsythiaside, respectively. Among them, peak 9 (protocatechuic acid) had a good separation effect and was located in the middle, so peak 9 was determined to be the reference peak (S peak).

[0221] Taking peak 9 as the reference peak (S peak), the relative retention time of characteristic peaks 1 to 33 was calculated, and the relative retention time was within ±5% of the specified value; the specified value was 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 No. 15 is 2.13, Peak No. 16 is 2.27, Peak No. 17 is 2.34, Peak No. 18 is 2.67, Peak No. 19 is 2.80, Peak No. 20 is 2.83, Peak No. 21 is 2.87, Peak No. 22 is 2.93, Peak No. 23 is 3.10, Peak No. 24 is 3.27, Peak No. 25 is 3.32, Peak No. 26 is 3.44, Peak No. 27 is 3.50, Peak No. 28 is 3.54, Peak No. 29 is 3.88, Peak No. 30 is 4.06, Peak No. 31 is 4.52, Peak No. 32 is 4.71, and Peak No. 33 is 5.00.

[0222] Example 6: Spectral peaks of medicinal properties and characteristic peaks of raw medicinal properties of Folium Isatidis and Radix Isatidis

[0223] 1. Experimental Methods

[0224] 2 μL of the test solution and the four drug taste test solutions were injected into the ultra-high performance liquid chromatograph, analyzed according to the chromatographic conditions of Example 5, and the chromatograms were recorded.

[0225] The test solution was prepared according to the method of "2. Preparation of test solution" in Example 5, and the preparation methods of the four medicinal taste test solutions refer to "2. Preparation of test solution" in Example 5, that is, 2 g each of Isatis indigotica, Isatis indigotica, Forsythia suspensa and Polygonum multiflorum were taken, accurately weighed, placed in a stoppered conical flask, and 25 mL of 30% methanol solution (v / v) was accurately added, weighed, treated with ultrasound (40 kHZ, 560 W) for 45 min, taken out, cooled to room temperature, supplemented with 30% methanol solution (v / v) to make up the lost weight, shaken well, filtered, and the filtrate was taken to obtain Isatis indigotica test solution, Isatis indigotica test solution, Forsythia suspensa test solution and Polygonum multiflorum test solution.

[0226] 2. Experimental Results

[0227] 1. Medicinal taste of chromatographic peaks

[0228] The results of attributing the chromatographic peaks of Ganmao Tuire Granules to Isatis indigotica are as follows: Fig.17 As shown, chromatographic peaks 1, 2, 3, 4, 5, 6, 7, 8, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 27 and 29 in the test solution of Ganmao Tuire Granules belong to the raw material Folium Isatidis.

[0229] The results of attributing the chromatographic peak of Ganmao Tuire Granules to Isatis Root are as follows: Fig.18 As shown, chromatographic peaks 1, 4, 6, 7, 8, 10, 13, 16, 17, and 20 in the Ganmao Tuire Granules test solution belong to the medicinal Radix Isatidis.

[0230] The results of attributing the chromatographic peak of Ganmao Tuire Granules to Forsythia suspensa are as follows: Fig.19 As shown, the chromatographic peaks 2, 9, 11, 13, 23, 25, 26, 28, 30, 31, 32 and 33 in the test solution of Ganmao Tuire Granules belong to the medicinal herb Forsythia suspensa.

[0231] The results of attributing the chromatographic peaks of Ganmao Tuire Granules to Polygonum multiflorum are as follows: Fig. 20 As shown, the chromatographic peaks 5, 9, 12 and 13 in the test solution of Ganmao Tuire Granules belong to the medicinal herb Polygonum multiflorum.

[0232] 2. Confirmation of characteristic peaks of raw materials Daqingye and Isatis root

[0233] The characteristic peaks of the raw materials of Ganmao Tuire Granules, Daqingye and Banlangen, were confirmed as follows: Fig.21As shown, the medicinal flavors of Isatis indigotica and Radix Isatidis contain many identical chromatographic peaks. However, among the 7 characteristic peaks identified, the content of (R, S)-goitrin (peak 8) in Radix Isatidis is high, while the content in Isatis indigotica is very low; the content of isovitexin (peak 24) in Radix Isatidis is high, while the content in Radix Isatidis is very low or absent. Therefore, the (R, S)-goitrin identified in this method can be used as the characteristic peak of Radix Isatidis, and isovitexin can be used as the characteristic peak of Radix Isatidis. The retention or absence of the peaks can be used to determine whether the cold and fever-relief granules are fed with Radix Isatidis or Folium Isatidis.

[0234] Example 7 A quality detection method for cold and fever relieving granules

[0235] 1. UPLC spectrum of the Ganmao Tuire granules sample to be tested

[0236] According to "2. Preparation of test solution" in Example 5, the cold and fever-relief granules to be tested were prepared into a test solution.

[0237] According to "3. Chromatographic conditions and system suitability" in Example 5, 2 μL of the test solution was accurately pipetted and injected into the ultra-high performance liquid chromatograph, and the chromatogram was recorded to obtain the UPLC spectrum of the Ganmao Tuire Granules to be tested.

[0238] 2.UPLC fingerprint

[0239] Taking different batches of Ganmao Tuire Granule samples, testing the different batches of Ganmao Tuire Granule samples according to the method of Example 5, recording the chromatograms, importing the chromatograms into the Chinese medicine chromatographic fingerprint similarity evaluation system, screening and confirming the common peaks, and generating the UPLC fingerprint of Ganmao Tuire Granules through the software;

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

[0241] 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 4, Peak No. 18 is 2.67, Peak No. 19 is 2.80, Peak No. 20 is 2.83, Peak No. 21 is 2.87, Peak No. 22 is 2.93, Peak No. 23 is 3.10, Peak No. 24 is 3.27, Peak No. 25 is 3.32, Peak No. 26 is 3.44, Peak No. 27 is 3.50, Peak No. 28 is 3.54, Peak No. 29 is 3.88, Peak No. 30 is 4.06, Peak No. 31 is 4.52, Peak No. 32 is 4.71, and Peak No. 33 is 5.00.

[0242] 3. Judgment criteria

[0243] The cold and fever-reducing granules sample to be tested can be judged as qualified only if the following two conditions are met:

[0244] The UPLC spectrum of the Ganmao Tuire Granules sample to be tested presents a fingerprint peak whose corresponding retention time in the UPLC fingerprint spectrum is within ±5% of the specified value;

[0245] The UPLC spectrum and UPLC fingerprint of the Ganmao Tuire granules sample to be tested were imported into the Chinese medicine chromatographic fingerprint similarity evaluation software (China Pharmacopoeia Committee, 2012 edition). After the chromatographic peaks in the UPLC spectrum of the sample to be tested were matched with the 33 fingerprint peaks in the UPLC fingerprint spectrum, the similarity was calculated. When the similarity between the UPLC spectrum of the sample to be tested and the UPLC fingerprint spectrum was greater than 0.85, it was judged to be qualified.

[0246] Example 8 Methodological Investigation

[0247] 1. Exclusiveness Investigation

[0248] 1. Experimental Methods

[0249] Take Ganmao Tuire Granules, prepare a test solution and a blank test solution according to the method of Example 5, inject the sample, analyze according to the chromatographic conditions of Example 5, and record the chromatogram.

[0250] 2. Experimental results

[0251] The results are as follows Fig. 22 As shown, the extraction solvent and elution solvent of the test sample have basically no absorption under the chromatographic conditions and have no interference with the chromatographic analysis of the test sample, indicating that the established method has good specificity.

[0252] 2. Instrument Precision Inspection

[0253] 1. Experimental Methods

[0254] Take Ganmao Tuire Granules, prepare a test solution according to the method of Example 5, inject 6 times continuously, analyze according to the chromatographic conditions of Example 5, and record the chromatogram. The 6 obtained chromatograms were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) of the National Pharmacopoeia Committee.

[0255] 2. Experimental results

[0256] The results are shown in Tables 5, 6, 7 and Fig.23 As shown in the figure, the similarity of the whole spectrum of the six spectra reached 1.000, and the RSD values ​​of the relative retention time and relative peak area of ​​the 33 main component peaks were all less than 5%. The inspection results showed that the precision of the instrument met the requirements.

[0257] Table 5 Relative retention time of precision investigation

[0258]

[0259] Table 6 Relative peak areas of precision investigation

[0260]

[0261]

[0262] Table 7 Similarity of spectra in the instrument precision study

[0263]

[0264] 3. Repeatability Study

[0265] 1. Experimental Methods

[0266] Take 6 portions of Ganmao Tuire Granules, prepare test solutions according to the method of Example 5, analyze according to the chromatographic conditions of Example 5, and record the chromatograms. The 6 obtained chromatograms were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) of the National Pharmacopoeia Committee.

[0267] 2. Experimental results

[0268] The results are shown in Tables 8, 9, 10 and Fig.24 As shown in the figure, the similarity of the whole spectrum of the six spectra reached 0.998, and the RSD values ​​of the relative retention time and relative peak area of ​​the 33 main component peaks were all less than 7%. The results showed that the repeatability of the method met the requirements.

[0269] Table 8 Relative retention time of repeatability study

[0270]

[0271] Table 9 Relative peak areas of repeatability study

[0272]

[0273]

[0274] Table 10 Similarity of reproducibility test spectra

[0275]

[0276] 4. Stability inspection

[0277] 1. Experimental Methods

[0278] Take Ganmao Tuire Granules and prepare a test solution according to the method of Example 5. Inject the sample at 0h, 2h, 4h, 8h, 12h and 24h after preparation, analyze according to the chromatographic conditions of Example 5, and record the chromatogram. The 6 obtained spectra were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) of the National Pharmacopoeia Committee.

[0279] 2. Experimental results

[0280] The results are shown in Tables 11, 12, 13 and Fig.25 As shown in the figure, the similarity of the full spectrum of the six spectra 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 showed that the sample had good stability within 24 hours and met the methodological requirements.

[0281] Table 11 Relative retention time of stability study

[0282]

[0283]

[0284] Table 12 Relative peak areas of stability studies

[0285]

[0286]

[0287] Table 13 Similarity of stability test spectra

[0288]

[0289] 5. Durability inspection

[0290] 1. Experimental Methods

[0291] Take the cold and fever-relief granules and prepare the test solution according to the method of Example 5. HSS T3C18 chromatographic column (1.8μm, 150mm×2.1mm), T3C18 chromatographic column (1.6μm, 150mm×2.1mm), Peptide HSS T3C18 column (1.8μm, 150mm×2.1mm), Four different types of chromatographic columns (1.6 μm, 150 mm×2.1 mm) were used for analysis according to the chromatographic conditions of Example 5, and the chromatograms were recorded. The Chinese Pharmacopoeia Committee's "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) was used to analyze the data of the four obtained spectra.

[0292] 2. Experimental results

[0293] The results are as follows Fig.26 As shown, using T3C18 Column, ACQUITY PeptideHSS T3C18 chromatographic column, ACQUITY cannot be achieved on C18+ columns The analytical results of the HSS T3C18 column showed that the main chromatographic peaks were poorly separated or disappeared, and similarity analysis could not be performed. ACQUITY HSS T3 C18 chromatographic column was used to perform UPLC fingerprint analysis of Ganmao Tuire Granules.

[0294] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A pretreatment method for detecting cold and fever-relief granules by ultra-high performance liquid chromatography, characterized in that: Take the cold and fever-relief granule sample to be tested, and use the methanol aqueous solution with a volume fraction of 25-35% for ultrasonic extraction for 40-50 minutes.

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

3. An ultra-high performance liquid chromatography method, characterized in that: The following steps are involved: Reference solution: (R, S)-goitrin solution, isovitexin solution, forsythiaside solution, forsythiaside A solution, rutin solution, protocatechuic acid solution and / or gallic acid solution; Test solution: prepared using the pretreatment method described in claim 1 or 2; Chromatographic conditions: Octadecylsilane bonded silica gel is used as the filler of the chromatographic column, acetonitrile is used as the mobile phase A, and a formic acid aqueous solution with a volume fraction of 0.08-0.12% is used 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: 0-15min, mobile phase B decreased from 100% to 95%; 15-25min, the mobile phase is reduced from 95% to 90%; 25-40 min, mobile phase decreased from 90% to 85%; 40-50min, the mobile phase was reduced from 85% to 84%; 50-60 min, the mobile phase decreased from 84% to 80%; 60-80min, mobile phase decreased from 80% to 70%; The detection wavelength is 254 nm, and the theoretical plate number calculated according to (R, S)-galactoside should be no less than 5000.

4. The ultra-high performance liquid chromatography method according to claim 3, characterized in that: The preparation method of the (R, S)-gaoichun solution is as follows: (R, S)-gaoichun and methanol are fully mixed, and the mass volume ratio of the (R, S)-gaoichun to methanol is (15-25) μg:1 mL; The preparation method of the isovitexin solution is as follows: fully mixing isovitexin and methanol, wherein the mass volume ratio of isovitexin to methanol is (75-85) μg:1 mL; The preparation method of the forsythiaside solution is: fully mixing forsythiaside and methanol, wherein the mass volume ratio of forsythiaside to methanol is (75-85) μg:1 mL; The preparation method of the forsythiaside A solution is as follows: fully mixing forsythiaside A and methanol, wherein the mass volume ratio of forsythiaside A to methanol is (195-205) μg:1 mL; The preparation method of the rutin solution is: rutin and methanol are fully mixed, and the mass volume ratio of rutin to methanol is (65-75) μg: 1 mL; The preparation method of the protocatechuic acid solution is: fully mixing protocatechuic acid and methanol, wherein the mass volume ratio of protocatechuic acid to methanol is (35-45) μg:1 mL; The preparation method of the gallic acid solution is: fully mixing gallic acid and methanol, wherein the mass volume ratio of gallic acid to methanol is (35-45) μg:1 mL.

5. The ultra-high performance liquid chromatography method according to claim 3, characterized in that: The column temperature of the chromatographic column is 20-30°C.

6. The ultra-high performance liquid chromatography method according to claim 3, characterized in that: The flow rate of the gradient elution is 0.2-0.4 mL / min.

7. The ultra-high performance liquid chromatography method according to claim 3, characterized in that: The injection volume of the reference solution and the test solution is 1 to 3 μL.

8. Use of the pretreatment method according to any one of claims 1 to 2 and / or the ultra-high performance liquid chromatography method according to any one of claims 3 to 7 in establishing the UPLC fingerprint of Ganmao Tuire Granules and / or in quality control of Ganmao Tuire Granules.

9. A method for constructing a UPLC fingerprint of cold and fever relieving granules, characterized in that: The ultra-high performance liquid chromatography method described in any one of claims 3 to 7 is used to detect different batches of Ganmao Tuire granules samples, and the chromatograms are recorded. The chromatograms are introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the common peaks are screened and confirmed to obtain the UPLC fingerprint of Ganmao Tuire granules.

10. A quality detection method for cold and fever relieving granules, characterized in that: The following steps are involved: S1. Take the sample of Ganmao Tuire Granules to be tested, detect it using the ultra-high performance liquid chromatography method according to any one of claims 3 to 7, record the chromatogram, and obtain the UPLC spectrum of the Ganmao Tuire Granules sample to be tested; S2. The UPLC fingerprint of the Ganmao Tuire Granules according to claim 9 and the UPLC fingerprint of the Ganmao Tuire Granules sample to be tested are introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system for comparison. The qualified ones can be judged to be qualified if the following two conditions are met: The UPLC spectrum of the cold and fever-relief granule sample to be tested presents a fingerprint peak whose retention time in the UPLC fingerprint spectrum is within ±5% of the specified value; The similarity between the UPLC spectrum of the tested Ganmao Tuire Granule sample and the UPLC fingerprint spectrum is calculated, and the similarity between the UPLC spectrum of the tested Ganmao Tuire Granule sample and the UPLC fingerprint spectrum is not less than 0.85; The UPLC fingerprint spectrum contains 33 fingerprint peaks, with peak No. 9 as the S peak, and the specified values ​​are: 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 4, Peak No. 18 is 2.67, Peak No. 19 is 2.80, Peak No. 20 is 2.83, Peak No. 21 is 2.87, Peak No. 22 is 2.93, Peak No. 23 is 3.10, Peak No. 24 is 3.27, Peak No. 25 is 3.32, Peak No. 26 is 3.44, Peak No. 27 is 3.50, Peak No. 28 is 3.54, Peak No. 29 is 3.88, Peak No. 30 is 4.06, Peak No. 31 is 4.52, Peak No. 32 is 4.71, and Peak No. 33 is 5.00.

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