Establishment method of medicine fingerprint spectrum

Abstract

The invention discloses a method for establishing a medicine fingerprint spectrum, which adopts a high performance liquid chromatography method and takes octadecylsilane chemically bonded silica as a filler; acetonitrile is used as a mobile phase A, and a 0.1% phosphoric acid solution is used as a mobile phase B; and carrying out gradient elution. The method provided by the invention develops and determines the chromatographic conditions of the characteristic chromatogram of the bitter sweet granules and the preparation method of the test solution, and provides a method basis for subsequent analysis method verification and process change research. Under the conditions of different flow rates, column temperatures, wavelengths, mobile phases, chromatographic columns and instruments, the determination results are basically consistent, the relative retention time of characteristic peaks fluctuates within the range of + / -10% under the original method, and the similarity evaluation results are not less than 0.990, which shows that the method has good durability.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and in particular relates to a method for establishing a drug fingerprint. Background Art

[0002] Kugan Granules are included in the 2020 Chinese Pharmacopoeia Volume 1. It is composed of 9 herbs: ephedra, mint, cicada shell, honeysuckle, scutellaria, bitter almond, platycodon, Fritillaria thunbergii, and licorice. This prescription has the effects of dispelling wind and heat, promoting lung function and resolving phlegm, and relieving cough and asthma.

[0003] Publicly available literature provides various methods for determining components, such as the HPLC method for determining the content of ephedrine hydrochloride in Kugan granules. The method uses a Hypersil ODSC18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid solution (5:95); detection wavelength: 210 nm; column temperature: room temperature; flow rate: 1.0 ml / min, injection volume: 10 μL. Under these conditions, the retention time of ephedrine hydrochloride is approximately 20 minutes. However, when the inventors repeated this method, they found that different column models produced different results, and not all components in Kugan granules could be detected.

[0004] Therefore, if an analytical method with good repeatability and the ability to detect more components can be provided, it will greatly facilitate the analysis of this drug.

[0005] According to the requirements of the "Technical Guidelines for Research on Pharmaceutical Changes in Marketed Traditional Chinese Medicines", characteristic spectrum evaluation indicators must be added when the process is changed to comprehensively evaluate the consistency of drug quality before and after the change. Therefore, establishing a characteristic spectrum detection method for Kugan Granules is of great value for evaluating product quality before and after process changes. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides a method for establishing a drug fingerprint, which adopts high performance liquid chromatography, octadecylsilane bonded silica gel as a filler; acetonitrile as mobile phase A, and 0.08% to 0.12% phosphoric acid solution as mobile phase B; gradient elution is performed according to the provisions in the following table;

[0007]

[0008] The medicine comprises at least one component selected from scutellaria baicalensis, liquorice, honeysuckle, mint, bitter almond or ephedra.

[0009] Furthermore, the medicine is Kugan granules.

[0010] Furthermore, the bitter and sweet granules contain aspartame.

[0011] The method includes preparing a test solution: taking an appropriate amount of Kugan granules, grinding them into powder, taking about 2.5g of the powder, accurately weighing it, placing it in a stoppered conical flask, accurately adding 50ml of solvent, weighing it, ultrasonically treating it for 30 minutes, cooling it, making up the lost weight with solvent, shaking it, weighing it again, letting it stand for a period of time or centrifuging it to take the supernatant, filtering it, and taking the filtrate to obtain it.

[0012] The reference solution was prepared as follows: an appropriate amount of baicalin reference substance was taken, accurately weighed, and methanol was added to prepare a solution containing 40 to 60 μg per 1 ml.

[0013] Preferably, the solvent is selected from 30% to 100% methanol. Test results show that 30% methanol solution, 50% methanol solution, and 75% methanol solution have no effect on the number of peaks of the test solution, and the peak areas are slightly different. All of the above methanol solutions can be used as extraction solvents.

[0014] Preferably, the detection wavelength is 190 nm to 220 nm, preferably 210 nm.

[0015] Preferably, the chromatographic column is Agilent ZORBAX SB-C18, 4.6*250 mm, 5 μm; or ZORBAX Eclipseplus C18, 4.6*250 mm, 5 μm.

[0016] Preferably, the flow rate is 0.8 to 1.2 ml / min; and the column temperature is 25°C to 40°C, preferably 30°C.

[0017] The characteristic spectrum of the test sample should present 18 characteristic peaks. The peak corresponding to the baicalin reference peak is taken as the S peak. The relative retention time of each characteristic peak is calculated. The relative retention time should be within ±10% of the specified value. The specified value is peak 1: 0.164, peak 2: 0.177, peak 3: 0.354, peak 4: 0.370, peak 5: 0.380, peak 6: 0.414, peak 7: 0.742, peak 8: 0.864, peak 9: 0.905, peak 10: 0.922, peak 11: 0.958, peak 12: 0.966, peak 14: 1.037, peak 15: 1.058, peak 16: 1.079, peak 17: 1.123, peak 18: 1.173.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This method development preliminarily determined the chromatographic conditions and test solution preparation method for a drug characteristic profile. The drug contains at least one component from Scutellaria baicalensis, Licorice, Honeysuckle, Menthol, Bitter Almond or Ephedra, providing a methodological basis for subsequent analytical method validation and process change research for similar drugs.

[0020] 2. This method preliminarily identified 18 characteristic peaks of Kugan Granules, and identified 5 components by comparison with reference substances. At the same time, it was determined that the 18 characteristic peaks were mainly attributed to 6 medicinal herbs: Scutellaria baicalensis, Licorice, Honeysuckle, Menthol, Bitter Apricot Kernel, and Ephedra. Based on the characteristics of each characteristic peak, the peak of baicalin was preliminarily determined to be the S peak, and the relative retention time of each characteristic peak was calculated, thereby achieving the positioning of each characteristic peak.

[0021] 3. This method development studied the effect of aspartame on Kugan Granules (sweet type). The results showed that aspartame had no interference with the characteristic spectrum of Kugan Granules.

[0022] 4. The results of the durability study of the method of the present invention show that the measurement results are basically consistent under different flow rates, column temperatures, wavelengths, mobile phases, chromatographic columns, and instrument conditions. The relative retention times of the characteristic peaks fluctuate within the range of ±10% under the original method, and the similarity evaluation results are not less than 0.990, indicating that the method has good durability.

[0023] 5. A characteristic spectrum study of 11 batches of Kugan Granules (including 3 batches of sweet type) was conducted based on the method of the present invention. The results showed that the RRT of each characteristic peak of the 11 batches of Kugan Granules was basically consistent. The average value of the RRT of each characteristic peak of the 11 batches was temporarily set as the specified value of the characteristic spectrum, providing an evaluation basis for subsequent characteristic spectrum research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 This is the characteristic peak attribution diagram of Kugan Granule in Example 1; wherein, S1: Fritillaria thunbergii, S2: Platycodon grandiflorum, S3: Ephedra sinica, S4: Bitter Apricot kernel, S5: Menthol, S6: Honeysuckle, S7: Licorice, S8: Scutellaria baicalensis, S9: 1323004, S10: Mixed reference solution;

[0026] Figure 2 This is the characteristic spectrum of Kugan Granules (sweet type) in Example 1; wherein, S1 to S3 are Kugan Granules (sweet type) batches 1422015, 1422024, and 1423011, and S4 is aspartame;

[0027] Figure 3 The chromatograms of different methanol solvents used in the test solution preparation and solvent selection experiment in Example 3 are as follows: S1: methanol solvent, S2: 30% methanol solvent, S3: 75% methanol solvent, and S4: 50% methanol solvent.

[0028] Figure 4 This is the 3D graph of the 190nm wavelength in the wavelength selection experiment in Example 3;

[0029] Figure 5 This is the 3D graph of the 210 nm wavelength in the wavelength selection experiment in Example 3;

[0030] Figure 6 This is the 3D graph of the 230nm wavelength in the wavelength selection experiment in Example 3;

[0031] Figure 7 This is the 3D graph of the 250nm wavelength in the wavelength selection experiment in Example 3;

[0032] Figure 8 This is the 3D graph of the 270nm wavelength in the wavelength selection experiment in Example 3;

[0033] Figure 9 This is the 3D graph of the 290nm wavelength in the wavelength selection experiment in Example 3;

[0034] Figure 10 This is the 3D graph of the 310 nm wavelength in the wavelength selection experiment in Example 3;

[0035] Figure 11 The chromatograms are obtained from the experiments with different phosphoric acid solution concentrations, mobile phase flow rates, column temperatures, and wavelengths in Example 3; wherein, S1: original method, S2: 0.08% phosphoric acid solution, S3: 0.12% phosphoric acid solution, S4: flow rate 0.9 ml / min, S5: flow rate 1.1 ml / min, S6: column temperature 28°C, S7: column temperature 32°C, S8: wavelength 208 nm, and S9: wavelength 212 nm.

[0036] Figure 12 The chromatograms of the different chromatographic column experiments in Example 3 are shown; wherein, S1: original method (Aiglent 1200, chromatographic column 3), S2: chromatographic column 1, S3: chromatographic column 2, S4: Aiglent 1260;

[0037] Figure 13 These are the test results for 11 batches of Kugan Granules in Example 4; S1 to S8 are different batches of Kugan Granules (1322027, 1322030, 1323004, 1323006, 1323007, 1323020, 1223024, and 1323026), and S9 to S11 are different batches of sweet-flavored Kugan Granules (1422015, 1422024, and 1423011).

[0038] Figure 14 The relative retention times of the characteristic peaks of 11 batches of Kugan granules in Example 4 are summarized;

[0039] Figure 15 The similarity evaluation was performed for the test results of 11 batches of Kugan granules in Example 4;

[0040] Figure 16 This is the characteristic peak attribution diagram of Kugan Granule in Comparative Example 1; wherein, S1: Fritillaria thunbergii, S2: Ephedra, S3: Bitter Apricot, S4: Platycodon grandiflorum, S5: Honeysuckle, S6: Menthol, S7: Licorice, S8: Scutellaria baicalensis, S9: Kugan Granule (batch 1323004);

[0041] Figure 17 The fingerprints of the reference solution and 10 batches of Kugan granule test solution in Comparative Example 1 are shown; wherein, S1: 1322004, S2: 1322010, S3: 1322013, S4: 1322017, S5: 1322024, S6: 1322030, S7: 1323001, S8: 1323004, S9: 1323006, S10: 1323007, S11: reference solution (the order of peaks is chlorogenic acid, liquiritin, luteolin, and ammonium glycyrrhizate);

[0042] Figure 18 This is the chromatogram under the conditions of gradient elution procedure 1 in Comparative Example 2;

[0043] Figure 19 This is the chromatogram under the conditions of gradient elution program 2 in Comparative Example 2;

[0044] Figure 20 This is the chromatogram under the conditions of gradient elution program 3 in Comparative Example 2. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0046] Among them, cicada slough is an animal medicine, and the current Chinese Pharmacopoeia 2020 edition does not have chemical component identification and content determination detection. Therefore, this method development mainly focuses on the research of 8 medicinal materials other than cicada slough.

[0047] Prepare single-herb control samples for each of the eight herbs above using the Kugan Granule preparation method. Prepare test solutions for each single-herb sample using the test solution preparation method described above. Perform the assay using the chromatographic conditions described above to preliminarily determine the number of characteristic peaks and the identity of the medicinal flavors in the Kugan Granules.

[0048] Table 1 Instruments and chromatographic columns

[0049]

[0050]

[0051] Example 1,

[0052] [Characteristic spectrum] Determined by high performance liquid chromatography (General Chapter 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0053] The chromatographic conditions and system suitability test were conducted using octadecylsilane bonded silica gel as the filler (Agilent ZORBARSB-C18, 4.6 mm × 250 mm, 5 μm; Agilent ZORBAX Eclipse plus C18, 4.6*250 mm, 5 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, with gradient elution as specified in Table 2; detection wavelength of 210 nm; column oven temperature of 30°C; and a flow rate of 1.0 ml / min.

[0054] Table 2

[0055]

[0056] Preparation of reference solution: Take an appropriate amount of baicalin reference substance, weigh accurately, and add methanol to make a solution containing 60 μg per 1 ml.

[0057] Take an appropriate amount of bitter and sweet granules for the test solution, grind them into powder, take about 2.5g of powder, accurately weigh it, put it in a stoppered conical flask, accurately add 50ml of 50% methanol, weigh the weight, ultrasonically treat it for 30 minutes, let it cool, make up the lost weight with 50% methanol, shake it well, weigh the weight again, let it stand for a while or centrifuge it, take the supernatant, filter it, and take the filtrate to obtain it.

[0058] Determination method: Accurately aspirate 5μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and record the chromatographic peak.

[0059] There should be 18 characteristic peaks in the characteristic spectrum of the test sample. The characteristic peak of baicalin is the most obvious, with high content and good peak shape. It is defined as the reference peak (S peak). The relative retention time of each characteristic peak is calculated. The relative retention time should be within ±10% of the specified value. The specified value is 0.164 (peak 1), 0.177 (peak 2), 0.354 (peak 3), 0.370 (peak 4), 0.380 (peak 5), 0.414 (peak 6), 0.742 (peak 7), 0.864 (peak 8), 0.905 (peak 9), 0.922 (peak 10), 0.958 (peak 11), 0.966 (peak 12), 1.037 (peak 14), 1.058 (peak 15), 1.079 (peak 16), 1.123 (peak 17), and 1.173 (peak 18).

[0060] like Figure 1As shown in the figure, the Kugan granules in the method developed this time contain about 18 characteristic peaks. Five known components can be confirmed by comparison with reference substances, among which peaks 1 and 2 are ephedrine hydrochloride and pseudoephedrine hydrochloride, peak 3 is chlorogenic acid, peak 7 is glycyrrhizin, and peak 13 is baicalin.

[0061] The peak attribution of each single herb showed that peaks 1 and 2 were unique to ephedra, peaks 3, 6, and 11 were shared by honeysuckle and mint, peaks 9 and 10 were unique to honeysuckle, peak 12 was unique to mint, peaks 4 and 5 were unique to bitter almonds, peak 7 was unique to liquorice, and peaks 8, 13, 14, 15, 16, 17, and 18 were unique to scutellaria. Ultimately, the characteristic peaks of six herbs in Kugan Granules were determined: ephedra, honeysuckle, mint, bitter almonds, liquorice, and scutellaria. Fritillaria thunbergii and Platycodon grandiflorum had no characteristic peaks.

[0062] This method development studied the effect of aspartame on the characteristic spectrum of Kugan Granules (sweet type). Aspartame, also known as aspartame, is an important sweetener. The results showed that aspartame peaked at 47.106 min and had no interference with the 18 characteristic peaks of Kugan Granules (sweet type). It can be preliminarily determined that this method is also applicable to the characteristic spectrum study of Kugan Granules (sweet type). Figure 2 .

[0063] Example 2, method exploration and comparative test

[0064] 2.1 Preparation of test solution and selection of solvent

[0065] Take an appropriate amount of Kugan granules, grind them finely, and take approximately 2.5g of the powder. Accurately weigh it and place it in a stoppered conical flask. Accurately add 50ml of 50% methanol and weigh it. Ultrasonicate for 30 minutes. Let cool. Make up the lost weight with 50% methanol, shake well, and weigh it again. Let it stand for a while or centrifuge. Take the supernatant, filter it, and take the filtrate. Prepare the test solution in the same way using methanol, 30% methanol solution, and 75% methanol solution as solvents.

[0066] The results are as follows Figure 3 The results showed that different solutions had no effect on the number of peaks of the test solution, and the peak areas were slightly different. The methanol solutions in all the above proportions could be used as extraction solvents.

[0067] 2.2 Wavelength selection

[0068] The DAD detector in Table 1 was used to scan the test solution at a wavelength of 190 nm to 400 nm. The wavelengths of 190 nm, 210 nm, 230 nm, 250 nm, 270 nm, 290 nm, and 310 nm were selected for analysis through the 3D graph. The results are shown in Figure 1. Figures 4-10As shown, the chromatographic peak information is the most at wavelengths of 190 nm and 210 nm, but the baseline is more stable at a wavelength of 210 nm, so 210 nm is selected as the detection wavelength.

[0069] Example 3, method verification test

[0070] 3.1. Exclusivity

[0071] A test solution was prepared according to the method provided in Example 1 and injected into a liquid chromatograph. Full-wavelength (190-400 nm) detection was performed using a diode array detector (DAD) under the chromatographic conditions described in Example 1. Peak purity was calculated using Empower 3 software, with the purity requirement being: purity angle value < purity threshold. The purity calculations revealed that the purity angle value for ephedrine hydrochloride was 1.309 < purity threshold 4.846; the purity angle value for pseudoephedrine hydrochloride was 1.538 < purity threshold 4.362; the purity angle value for chlorogenic acid was 0.455 < purity threshold 2.002; the purity angle value for liquiritin was 1.088 < purity threshold 4.526; and the purity angle value for baicalin was 0.139 < purity threshold 1.030. Peak purity calculations confirmed that the purity of all five components met the criteria, demonstrating the high specificity of this method.

[0072] 3.2 Precision

[0073] The reference solution was prepared according to the method of Example 1, and 5 injections were made continuously. The peak area results showed that the RSD value of each component was less than 2.0%, and the system adaptability was good.

[0074] 3.3 Repeatability

[0075] Six test solutions were prepared according to the method of Example 1. The RSD of the concentration of each component was less than 2.0%, and the repeatability was good.

[0076] 3.4 Linear Relationship Investigation

[0077] The reference substance stock solution was prepared according to the method of Example 1. Five reference substance solutions of different concentrations were prepared by accurately measuring each reference substance stock solution and injected into the liquid chromatograph. The standard curve was drawn with the injection amount (ng) as the abscissa and the peak area as the ordinate, and linear regression was performed. The results showed that the linear equation of baicalin in the range of 1036.2ng to 10362.0ng was y=3781.1x+30280, R 2 =0.9997; the linear equation of ephedrine hydrochloride in the range of 43.28ng~649.20ng is y=2234.3499x-10076.8114, R 2 =0.9999; the linear equation of pseudoephedrine hydrochloride in the range of 40.10ng~601.49ng is y=2117.5497x+6794.6192, R 2=0.9991; the linear equation of chlorogenic acid in the range of 41.25ng~1237.50ng is y=1993.7578x+6252.5309, R 2 =0.9998; the linear equation of liquiritin in the range of 40.22ng~804.30ng is y=3457.2572x-1836.4654,

[0078] R 2 =0.9996, the linear regression equation correlation coefficient R of each component was greater than 0.9990, and the linear relationship between each component was good.

[0079] 3.5 Accuracy

[0080] Using the sample addition recovery method, approximately 1.25g of Kugan granules (6 portions) were accurately weighed and placed in stoppered conical flasks. Precisely measured amounts of reference stock solutions of ephedrine hydrochloride, pseudoephedrine hydrochloride, chlorogenic acid, liquiritin, and baicalin were then loaded onto a liquid chromatograph for determination. The results showed that the recoveries of the components were 94.48% for ephedrine hydrochloride, 105.41% for pseudoephedrine hydrochloride, 99.32% for chlorogenic acid, 93.68% for liquiritin, and 100.90% for baicalin. The contents of ephedrine hydrochloride and pseudoephedrine hydrochloride are typically calculated as two total amounts. The average recovery of the two components was 99.94%, thus meeting the pharmacopoeial standard of 92% to 105% and demonstrating good recovery rates.

[0081] 3.6 Sensitivity

[0082] One of the test solutions in 3.3 Repeatability was taken for sensitivity investigation. When the test solution was diluted 10 times, the signal-to-noise ratio (s / n) of each component was 15.5 for ephedrine hydrochloride, 18.1 for pseudoephedrine hydrochloride, 41.2 for chlorogenic acid, 11.0 for liquiritin, and 1641.3 for baicalin. The corresponding concentrations were 0.07738 mg / ml for ephedrine hydrochloride, 0.095628 mg / ml for pseudoephedrine hydrochloride, 0.3299 mg / ml for chlorogenic acid, 0.06847 mg / ml for baicalin, and 2.8687 mg / ml for baicalin.

[0083] 3.6 Method Durability

[0084] 3.6.1 Effects of different flow rates and column temperatures on characteristic spectra

[0085] The instruments and chromatographic columns shown in Table 1 were used to investigate the effects of different mobile phase flow rates and column temperatures on the characteristic spectra.

[0086] The results are shown in Tables 3, 4 and Figure 11As shown, different flow rates and column temperatures have some impact on the retention time of chromatographic peaks, but there are no significant differences in the number of peaks or peak separation, indicating that this method has good robustness to flow rate and column temperature. The RRTs for different flow rates and column temperatures are all within ±10% of the RRT for Kugan Granules, meeting the requirements.

[0087] Table 3 Summary of relative retention time at different flow rates

[0088]

[0089]

[0090] Table 4 Summary of relative retention time at different column temperatures

[0091]

[0092] 3.6.2 Effects of different wavelengths and phosphoric acid solutions on characteristic spectra

[0093] The instruments and chromatographic columns shown in Table 1 were used to investigate the effects of different wavelengths and phosphoric acid solutions in the mobile phase on the characteristic spectra.

[0094] The results are shown in Tables 5, 6 and Figure 11 As shown, different wavelengths and different phosphoric acid solution ratios have minimal effects on chromatographic peak retention time, peak number, and peak separation, demonstrating the robustness of this method for different wavelengths and phosphoric acid solution ratios. The RRTs for different wavelengths and phosphoric acid solution ratios were all within ±10% of the RRT of Kugan Granules (see Tables 5 and 6), meeting the requirements.

[0095] Table 5 Summary of relative retention times at different wavelengths

[0096]

[0097] Table 6 Summary of relative retention times of different phosphoric acid ratios

[0098]

[0099]

[0100] 3.6.3 Effects of different chromatographic columns and instruments on characteristic spectra

[0101] The instruments and chromatographic columns shown in Table 1 were used to investigate the effects of different wavelengths and phosphoric acid solutions in the mobile phase on the characteristic spectra.

[0102] The results are shown in Tables 7, 8 and Figure 12As shown, different chromatographic columns had no effect on the number of chromatographic peaks, but did have some influence on retention time and separation, though the differences were small. There were no significant differences in the number of chromatographic peaks, retention time, or resolution between different instruments, indicating that this method is robust to different chromatographic columns and instruments. The RRTs for different columns and instruments were all within ±10% of the RRT for Kugan Granules, meeting the requirements.

[0103] Table 7 Summary of relative retention times of different chromatographic columns

[0104]

[0105] Table 8 Summary of relative retention times of different instruments

[0106]

[0107] 3.6.4 Analysis of Similarity Evaluation Results

[0108] The fingerprint similarity evaluation system of Chinese medicine chromatographic fingerprint (version 2012.130723) was used to evaluate the fingerprint similarity between the chromatograms under various conditions of the durability pre-study and the chromatograms of the original method, and the chromatogram of S1 (original method) was used as the reference chromatogram. The results are shown in Tables 9, 10 and Figures 14-15 As shown, compared with the original method, the similarity under different chromatographic conditions is greater than 0.990 (generally required to be no less than 0.900), indicating that the method has good durability under different flow rates, column temperatures, wavelengths, columns, phosphoric acid ratios, and instruments.

[0109] Table 9 Similarity results under different chromatographic conditions

[0110]

[0111]

[0112] S1: original method, S2: 0.08% phosphoric acid solution, S3: 0.12% phosphoric acid solution, S4: flow rate 0.9 ml / min, S5: flow rate 1.1 ml / min,

[0113] S6: column temperature 28°C, S7: column temperature 32°C, S8: wavelength 208nm, S9: wavelength 212nm

[0114] Table 10 Similarity evaluation results of different chromatographic columns and instruments

[0115] S1(R) S2 S3 S4 S1(R) 1 1 1 1 S2 1 1 1 1 S3 1 1 1 1 S4 1 1 1 1 Calculate the corrected peak group 0 groups

[0116] S1: original method (Aiglent 1200, chromatographic column 3), S2: chromatographic column 1, S3: chromatographic column 2, S4: Aiglent 1260 Example 4, Characteristic Spectrum Study of Multiple Batches of Kugan Granules

[0117] After the method was initially determined, the characteristic spectrum of 11 batches of Kugan granules (including 3 batches of sweet type) was studied and the relative retention time of each characteristic peak was calculated. Figure 13 .

[0118] The relative retention times of the characteristic peaks of 11 batches of Kugan granules are summarized as follows: Figure 14 shown.

[0119] The characteristic spectra of 11 batches of Kugan granules all showed 18 characteristic peaks. The chromatographic peak corresponding to baicalin (peak 13) was taken as the S peak. The RRT of characteristic peaks 1 to 18 and the S peak and the average of the RRT of each characteristic peak were calculated. The average of the RRT of 18 characteristic peaks in 11 batches was tentatively set as the specified value of this method, which was 0.164 (peak 1), 0.177 (peak 2), 0.354 (peak 3), 0.370 (peak 4), and 0.490 (peak 5). 4), 0.380 (peak 5), 0.414 (peak 6), 0.742 (peak 7), 0.864 (peak 8), 0.905 (peak 9), 0.922 (peak 10), 0.958 (peak 11), 0.966 (peak 12), 1.037 (peak 14), 1.058 (peak 15), 1.079 (peak 16), 1.123 (peak 17), 1.173 (peak 18). In subsequent studies on the characteristic spectrum of Kugan Granules, the RRT of each characteristic peak should be within ±10% of the specified value.

[0120] The similarity evaluation system of Chinese medicine chromatographic fingerprints (version 2012.130723) was used to evaluate the similarity of the test results of 11 batches of Kugan granules. The results were all greater than 0.900, see Figure 15 .

[0121] Comparative Example 1

[0122] The fingerprint of Kugan granules was determined using the "A method for establishing an HPLC fingerprint of a cold medicine" (application number: CN201610463280.9). The number of peaks, peak identification, and comparison with reference substances were investigated. The specific method is as follows:

[0123] The chromatographic conditions are:

[0124] Instrument: Agilent 1260

[0125] Column: Waters symmetry C18, 150×2.1mm, 3.5μm

[0126] Flow rate: 0.3 ml / min; wavelength: 254 nm; injection volume: 20 μl; column temperature: 25°C.

[0127] The mobile phase elution gradient is:

[0128]

[0129] Preparation of reference solution: Accurately weigh appropriate amounts of ammonium glycyrrhizate, liquiritin, chlorogenic acid, and luteolin, dissolve and dilute to 50 ml with mobile phase solution, and shake well to obtain the reference solution (each 1 ml contains 0.5, 0.4, 0.2, and 0.3 mg / ml of ammonium glycyrrhizate, liquiritin, chlorogenic acid, and luteolin reference substances, respectively).

[0130] Preparation of the test solution: Accurately weigh 4 g of Kugan granules, add 100 mL of methanol, ultrasonicate for 30 min, place in the refrigerator, let it stand for 2 h, and filter through a microporous filter membrane to prepare the test solution.

[0131] Determination method: Accurately aspirate 20 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0132] result:

[0133] 1. Peak number and peak identification

[0134] According to the above-mentioned method for preparing the test solution, Kugan Granules (batch 1323004) and samples of each single herb in the prescription (except cicada slough) were prepared respectively. About 4 g of Kugan Granules and each single herb control sample (except cicada slough) were accurately weighed and placed in a stoppered conical flask. 100 mL of methanol was added respectively. After ultrasonication for 30 minutes, the mixture was placed in a refrigerator and allowed to stand for 2 hours. The mixture was filtered through a 0.45 μm microporous filter membrane and the filtrate was obtained.

[0135] The results are as follows Figure 16 As shown in the figure, compared with the results of each single herb, Kugan granules separated a total of 13 characteristic peaks, which were attributed to four herbs: honeysuckle, mint, liquorice, and scutellaria. There were no characteristic peaks for ephedra, bitter almond, fritillaria thunbergii, and platycodon. The number of characteristic peaks and peak attribution were less than those of the newly developed fingerprint method.

[0136] 2. Comparison with reference substances and testing of multiple batches of Kugan granules

[0137] The fingerprints of the reference solution and 10 batches of Kugan granule test solution were prepared and measured according to the above preparation method, and analyzed and compared.

[0138] like Figure 17 As shown in the figure, the peaks of the fingerprints of 10 batches of Kugan Granules determined by this method are basically consistent. Compared with the reference solution, the fingerprints of 10 batches of Kugan Granules can only identify the characteristic peaks of chlorogenic acid and ammonium glycyrrhizate, and there are no characteristic peaks of liquiritin and luteolin.

[0139] The results showed that this method separated 13 characteristic peaks in Kugan Granules, which were attributed to 4 medicinal materials. The peaks of 10 batches of Kugan Granules were basically the same. However, through comparison with the reference solution, only chlorogenic acid and ammonium glycyrrhizate could be identified in the 10 batches of Kugan Granules, and there were no characteristic peaks of liquiritin and luteolin. The reason for this was analyzed to be that the method was not sensitive enough for the detection of Kugan Granules, resulting in the inability to detect some substances.

[0140] The newly developed method is superior to the determination method in the patent "A method for establishing an HPLC fingerprint of a medicine for treating colds" in terms of the number of peaks, peak identification and comparison with reference substances.

[0141] Comparative Example 2, Effect of Different Elution Gradients

[0142] 2.1, the chromatographic conditions of Example 1 were used, except that the following gradient elution procedure 1 was used:

[0143] 0-25min: acetonitrile 7-8%;

[0144] 25-30 min: acetonitrile 8-13%;

[0145] 30-60 min: acetonitrile 13-20%;

[0146] 60-75 min: acetonitrile 20-26%;

[0147] 75-90min: acetonitrile 26-80%.

[0148] The results are as follows Figure 18 As shown, the chromatographic peaks of chlorogenic acid were poorly separated under this elution gradient.

[0149] 2.2, the chromatographic conditions of Example 1 were used, except that the following gradient elution program 2 was used: 0-10 min: acetonitrile 5-7%;

[0150] 10-30 min: acetonitrile 7-7%;

[0151] 30-35 min: acetonitrile 7-12%;

[0152] 35-45 min: acetonitrile 12-12%;

[0153] 45-60 min: acetonitrile 12-14%;

[0154] 60-75 min: acetonitrile 14-19%;

[0155] 75-90 min: acetonitrile 19-25%;

[0156] 90-110 min: acetonitrile 25-35%;

[0157] 110-120min: acetonitrile 35-80%.

[0158] The results are as follows Figure 19 As shown in the figure, the chromatographic peak separation of chlorogenic acid is improved, but there is still interference from impurities.

[0159] 2.3, the chromatographic conditions of Example 1 were used, except that the following gradient elution procedure 3 was used:

[0160] 0-16 min: acetonitrile 8-9%;

[0161] 16-20 min: acetonitrile 9-14%;

[0162] 20-28 min: acetonitrile 14-17%;

[0163] 28-40 min: acetonitrile 17-19%;

[0164] 40-65 min: acetonitrile 19-30%;

[0165] 65-75 min: 30-60% acetonitrile;

[0166] 75-85min: acetonitrile 60-90%.

[0167] The results are as follows Figure 20 As shown in the figure, the chromatographic peak separation of chlorogenic acid was improved and the characteristic peak of bitter almond was separated, but the characteristic peak of ephedra was poorly separated.

[0168] The above results show that during the early method development process, it was found that the gradient elution procedure was beyond the scope of this application method, and the separation effect of components such as chlorogenic acid, the main component in honeysuckle, and ephedrine hydrochloride in ephedra was poor.

[0169] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for establishing a drug fingerprint, characterized in that: High performance liquid chromatography was used, with octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, and 0.08% to 0.12% phosphoric acid solution as mobile phase B; gradient elution was performed as specified in the table below; 2. The method for establishing a drug fingerprint according to claim 1, characterized in that: The medicine comprises at least one component selected from scutellaria baicalensis, liquorice, honeysuckle, mint, bitter almond or ephedra.

3. The method for establishing a drug fingerprint according to claim 1, characterized in that: The medicine is Kugan granules.

4. The method for establishing a drug fingerprint according to claim 3, wherein: The bitter and sweet granules contain aspartame.

5. The method for establishing a drug fingerprint according to claim 1, wherein: The method comprises the following steps: taking an appropriate amount of Kugan granules, grinding them into powder, taking 2.5 g of the powder, accurately weighing it, placing it in a stoppered conical flask, accurately adding 50 ml of solvent, weighing it, ultrasonically treating it, cooling it, making up the lost weight with solvent, shaking it, weighing it again, letting it stand for a period of time or centrifuging it, taking the supernatant, filtering it, and taking the filtrate to obtain the product; The reference solution was prepared as follows: an appropriate amount of baicalin reference substance was taken, accurately weighed, and methanol was added to prepare a solution containing 40 to 60 μg per 1 ml.

6. The method for establishing a drug fingerprint according to claim 5, characterized in that: The solvent is selected from 30% to 100% methanol.

7. The method for establishing a drug fingerprint according to claim 1, wherein: The detection wavelength is 190 nm to 220 nm, preferably 210 nm.

8. The method for establishing a drug fingerprint according to claim 1, wherein: The chromatographic column is Agilent ZORBAX SB-C18, 4.6*250mm, 5μm or ZORBAX Eclipse plus C18, 4.6*250mm, 5μm.

9. The method for establishing a drug fingerprint according to claim 1, wherein: The flow rate is 0.8-1.2 ml / min; the column temperature is 25°C-40°C, preferably 30°C.

10. The method for establishing a drug fingerprint according to claim 1, wherein: The characteristic spectrum of the test sample should show 18 characteristic peaks. The peak corresponding to the baicalin reference peak is the S peak. The relative retention time of each characteristic peak is calculated. The relative retention time should be within ±10% of the specified value. The specified value is peak 1: 0.164, peak 2: 0.177, peak 3: 0.354, peak 4: 0.370, peak 5: 0.380, peak 6: 0.414, peak 7: 0.742, peak 8: 0.864, peak 9: 0.905, peak 10: 0.922, peak 11: 0.958, peak 12: 0.966, peak 14: 1.037, peak 15: 1.058, Peak 16: 1.079, Peak 17: 1.123, Peak 18: 1.173.

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