Establishment method of high performance liquid characteristic chromatogram of phlegm reducing preparation and component content determination method

Abstract

The invention relates to an establishment method of a high performance liquid characteristic chromatogram of a phlegm reducing preparation and a component content determination method, and belongs to the field of medicine analys.The establishment method of the high performance liquid characteristic chromatogram of the phlegm reducing preparation comprises the following steps that S1, a reference substance solution is prepared; s2, preparing a test solution; s3, determining chromatographic conditions; s4, collecting chromatograms of the reference substance solution and multiple batches of test substance solutions, analyzing the chromatograms of the test substance solutions, calibrating 13 common peaks, and identifying characteristic peaks of 6 components; s5, p-hydroxycinnamic acid and praeruptorin A are taken as double-standard reference substances, other 11 characteristic peaks are effectively predicted and positioned through two reference substances by adopting a double-standard linear correction method, the method is good in scientific accuracy and durability and economical, and the method is beneficial to comprehensively and effectively controlling the quality of the phlegm reducing preparation.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a method for establishing a high-performance liquid phase characteristic spectrum of a phlegm-removing prescription and a method for determining the content of its components. Background Art

[0002] The Huatan prescription, composed of five ingredients: Peucedanum chinense, Platycodon grandiflorum, Rhizoma Zingiberis, Rhizoma Cibotii, and Pumice stone, clears heat and resolves phlegm, relieves cough and asthma, and is used to treat lung-heat cough, as well as wheezing caused by excessive phlegm. However, no relevant characteristic profile inspection methods or standards have been established for this Huatan prescription, making it impossible to fully reflect its quality and ensure comprehensive and accurate quality control. Summary of the Invention

[0003] In order to make up for the deficiencies of the prior art, the present invention provides a method for establishing a high-performance liquid chromatography characteristic spectrum of a phlegm-removing prescription preparation and a method for determining the content of components, which is beneficial to improving the quality of a more comprehensive and effective phlegm-removing prescription preparation.

[0004] The technical solution adopted by the present invention to solve the above technical problems is:

[0005] A method for establishing a high-performance liquid chromatography characteristic spectrum of a phlegm-removing prescription preparation, wherein the phlegm-removing prescription preparation is prepared from 9-11 parts of peucedanum chinense, 9-11 parts of platycodon grandiflorum, 7-9 parts of rhizoma zingiberis, 8-10 parts of rhizoma damicornis, and 9-15 parts of pumice stone, in parts by weight, to form phlegm-removing prescription granules or phlegm-removing prescription standard decoction freeze-dried powder, the method comprising the following steps:

[0006] Preparation of S1 reference solution: Accurately weigh appropriate amounts of platycoside D, platycoside E, praeruptoside A, praeruptoside B, p-hydroxycinnamic acid, and (+)-candelilla resin phenol-9′-O-glucoside reference substances, and add methanol to prepare mixed reference solutions of different concentrations.

[0007] Preparation of S2 test solution: Take an appropriate amount of Huatan formula granules or traditional decoction freeze-dried powder, dissolve in methanol, weigh, sonicate, cool, add methanol to the weight, shake well, filter through a microporous membrane, and obtain the filtrate;

[0008] S3 HPLC conditions

[0009] Determination of the concentration of acetonitrile: A C18 column was used with acetonitrile-0.1% aqueous phosphoric acid as the mobile phase. The gradient elution conditions were: 0-40 min, 5%-30% acetonitrile; 40-45 min, 30%-60% acetonitrile; 45-60 min, 60%-90% acetonitrile; injection volume 10 μL, column temperature 30°C, volume flow rate 1.0 mL min -1, detection wavelength 0-15min is 210nm, 15-22min is 280nm, 22-24min is 230nm, 24-43min is 210nm, 43-60min is 321nm;

[0010] S4 HPLC determination: Collect chromatograms of the mixed reference solution and multiple batches of test solution according to the chromatographic conditions in step S3, analyze the chromatograms, confirm that there are 13 common peaks, and identify the characteristic peaks of 6 components;

[0011] S5 Determination of predicted retention time: Using multiple chromatographic columns of different brands and types, collect chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step S3 to obtain the standard retention times of 13 common peaks; using p-hydroxycinnamic acid and praeruptorin A as double-labeled reference substances, use the double-labeled linear calibration method to obtain the predicted retention times of the remaining peaks.

[0012] Preferably, the preparation method of the test solution is as follows: take an appropriate amount of granules of the Huatan prescription or freeze-dried powder of the traditional decoction, grind it into powder, take 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of methanol, ultrasonicate for 30 minutes, cool it, weigh it, make up the lost weight with methanol, shake it well, filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain it.

[0013] Preferably, the preparation method of the reference solution is as follows: respectively take appropriate amounts of platycoside D, platycoside E, praeruptoside A, praeruptoside B, p-hydroxycinnamic acid, and (+)-candelilla resin phenol-9′-O-glucoside reference substances, accurately weigh them, and add methanol to prepare the concentrations to be 100.400, 103.360, 110.491, 119.290, 288.400, and 114.050 μg·mL, respectively. -1 mixed reference solution.

[0014] Preferably, in step S5, the specific method for obtaining the predicted retention time using the double-label linear correction method is: using the actual retention time of the chromatographic peaks of the double-label reference substances p-hydroxycinnamic acid and praeruptorin A as the ordinate and the standard retention time as the abscissa to obtain a linear fitting equation; substituting the standard retention times of the remaining 11 characteristic peaks into the equation to obtain the corresponding predicted retention times; wherein the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns.

[0015] Preferably, in step S5, the standard retention times of the 13 peaks are 8.3148 min, 9.8035 min, 15.5367 min, 25.8220 min, 27.5484 min, 27.8364 min, 33.5142 min, 36.4188 min, 37.8652 min, 44.5608 min, 55.1509 min, 57.0867 min, and 59.7682 min, respectively.

[0016] Preferably, the preparation method of the Huatan formula granules is as follows: accurately weigh 6.0000, 2.5714, 0.9000, 0.5625, and 1.2000 g of peucedanum, platycodon grandiflorum, ginger bamboo shavings, rhizoma dandruff, and pumice stone formula granules to prepare the Huatan formula granules.

[0017] Preferably, the preparation method of the freeze-dried powder of the standard decoction of Huatan Fang comprises the following steps:

[0018] A1 Weigh out Tianzhuhuang slices, place in a casserole, add 12 times the amount of water and soak for 30 minutes;

[0019] A2 Weigh Platycodon grandiflorum, Peucedanum chinense, Rhizoma Zingiberis, and Pumice stone and place them in another casserole. Add 12 times the amount of water and soak for 30 minutes.

[0020] A3 decocts the soaking liquid with medicinal materials in A1, brings it to a boil over high heat, then simmers it for 10 minutes. Then, add the soaking liquid with medicinal materials in A2 into the casserole and simmer together, brings it to a boil over high heat, then simmers it for 20 minutes, and sieve it through a 200-mesh sieve while it is still hot.

[0021] A4: Add 10 times the amount of water to the residue obtained in A3, boil over high heat, then simmer for 20 minutes. Pass the residue through a 200-mesh sieve while hot. Combine the filtrate with the filtrate from A3, concentrate at 60°C, and freeze-dry in a vacuum.

[0022] The present application also provides a method for determining the content of ingredients in a phlegm-resolving prescription, comprising the following steps: performing liquid chromatography determination according to steps S1-S3 of the method established according to any one of claims 1-8 to obtain a chromatogram, and calculating the content of each component, including p-hydroxycinnamic acid, platycodon saponin E, peucedanum praeruptoside A, peucedanum praeruptoside B, and (+)-candelilla resin phenol-9′-O-glucoside, by peak area based on an external standard method.

[0023] The present invention adopts the above structure and has the advantages that:

[0024] 1. The present application establishes a method, using p-hydroxycinnamic acid and praeruptorin A as double-label reference substances, and adopts a double-label linear correction method to effectively predict and locate the chromatographic peaks of the other 11 components with two reference substances. Based on multi-point linear regression correction, the method is significantly superior to the relative retention time method currently commonly used for chromatographic peak qualitative analysis in terms of chromatographic column applicability and prediction accuracy, greatly eliminating the problems of insufficient characteristic peak qualitative accuracy and chromatographic column applicability of the relative retention time method, performing well in scientific accuracy and durability, and being relatively economical, improving the simplicity of the experiment and reducing costs; and providing a reference and basis for the quality control of phlegm-resolving prescription preparations.

[0025] 2. This method has the advantages of simplicity, stability, high precision and good reproducibility.

[0026] 3. The characteristic spectrum obtained by the method established in this application has 13 effectively separated characteristic peaks. Therefore, the obtained characteristic spectrum can more effectively characterize the quality of the phlegm-resolving prescription preparation, thereby facilitating a more comprehensive monitoring of the quality of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are characteristic spectra of the granules of the phlegm-removing prescription at different wavelengths.

[0028] Figure 2 The graphs are characteristic spectra of different mobile phases for the granules of the Huatan prescription of the present invention.

[0029] Figure 3 The graphs are characteristic graphs of different column temperatures for the granules of the Huatan prescription of the present invention.

[0030] Figure 4 The characteristic spectra of multiple batches of freeze-dried powder and formula granules of the traditional decoction of Huatan Decoction of the present invention.

[0031] Figure 5 It is the characteristic spectrum of the phlegm-resolving prescription of the present invention. DETAILED DESCRIPTION

[0032] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings.

[0033] 1. Preparation of phlegm-resolving prescription

[0034] 1.1 Preparation of freeze-dried powder of traditional decoction

[0035] Weigh 27g of Tianzhuhuang slices and place them in a casserole. Add 12 times the amount of water and soak for 30 minutes. Weigh 27g each of Haifushi, Platycodon grandiflorum, Peucedanum chinense, and Zingiberis radix scutellariae indica and place them in another casserole. Add 12 times the amount of water and soak for 30 minutes. First, decoct Tianzhuhuang slices, bringing them to a boil over high heat, then simmering for 10 minutes. Add the remaining three herbs along with the soaking liquid to the casserole and decoct together. Bring them to a boil over high heat, then simmer for 20 minutes. Sieve them while hot (200 mesh). Add 10 times the amount of water to the medicinal residues, bring them to a boil over high heat, then simmer for another 20 minutes. Sieve them while hot (200 mesh). Combine the filtrates, concentrate at 60°C, and freeze-dry under vacuum to obtain freeze-dried powders of the traditional Huatan Fang decoction (experiment numbers S1-S15).

[0036] 1.2 Preparation of Huatan Decoction Granules

[0037] Accurately weigh 6.0000, 2.5714, 0.9000, 0.5625, and 1.2000 g of Peucedanum chinense, Platycodon grandiflorum, Rhizoma Zingiberis (green stalk bamboo), Rhizoma Cibotii (green peel bamboo), and Pumice stone granules to prepare the Huatan formula granules (experimental numbers S16-S21, experimental sample information see Table 1).

[0038] Table 1 Formula particle information and combination

[0039]

[0040] Note: A—Shandong Hongjitang Pharmaceutical Group Co., Ltd.; B—Anhui Xiehecheng Pharmaceutical Co., Ltd.; C—Jiangyin Tianjiang Pharmaceutical Co., Ltd.; D—Beijing Kangrentang Pharmaceutical Co., Ltd.

[0041] 2. Preparation of Mixed Reference Solution

[0042] Platycoside D, platycoside E, peucedanin A, peucedanin B, p-hydroxycinnamic acid, and (+)-candelilla resin phenol-9′-O-glucoside reference substances 11.19, 10.53, 11.27, 11.98, 28.84, and 11.58 mg were taken, accurately weighed, and placed in a 10 ml volumetric flask. Methanol was added to the volume to prepare the reference substance mother solutions with concentrations of 1.0040, 1.0336, 1.1049, 1.1929, 2.8840, and 1.1405 mg / ml, respectively. 1 ml of each reference substance mother solution was accurately measured and placed in a 10 ml volumetric flask. Methanol was added to the volume to prepare the reference substance mother solutions with concentrations of 100.400, 103.360, 110.491, 119.290, 288.400, and 114.050 μg·mL, respectively. -1 mixed reference solution.

[0043] 3. Preparation of Test Solution

[0044] 3.1 Selection of extraction method

[0045] About 0.1 g of the Huatan prescription was taken and subjected to heating reflux and ultrasonic treatment for 30 minutes respectively. The peak area of ​​each identified peak was measured. The results showed (see Table 2) that the peak areas obtained by ultrasonic extraction and reflux extraction were equivalent. Considering that ultrasonic extraction is more convenient, ultrasonic extraction was selected.

[0046] Table 2 Extraction method investigation results (peak area)

[0047]

[0048] 3.2 Selection of extraction time

[0049] The reflux times were set to 15 min, 30 min, and 45 min, respectively, and the peak areas of the identified peaks were examined. The results showed (see Table 3) that the peak area of ​​the chromatogram obtained by ultrasound at 15 min was slightly smaller, and the material information presented by the characteristic spectra obtained at 30 min and 45 min was basically the same, so the ultrasound time was selected as 30 min.

[0050] Table 3 Extraction time investigation results (peak area)

[0051]

[0052] 3.3 Selection of extraction solvent

[0053] The peak areas of the identified peaks when the extraction solvents were 10%, 30%, 50%, 70% and methanol, respectively, were investigated. The results showed (see Table 4) that the peak area of ​​the identified peaks extracted with methanol was larger, and methanol was finally selected as the extraction solvent.

[0054] Table 4 Extraction solvent investigation results (peak area)

[0055]

[0056] In summary, take an appropriate amount of granules of the phlegm-resolving prescription or freeze-dried powder of the traditional decoction, grind it into powder, take about 0.5 g, weigh it accurately, put it in a stoppered conical flask, add 25 mL of methanol accurately, ultrasonicate for 30 minutes, let it cool, weigh it, make up the lost weight with methanol, shake it well, filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain the product.

[0057] 4. Determination of Chromatographic Conditions

[0058] Using C 18The chromatographic column was 4.6 × 250 mm, 5 μm. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution. The gradient elution conditions were: 0-40 min, 5%-30% acetonitrile; 40-45 min, 30%-60% acetonitrile; 45-60 min, 60%-90% acetonitrile; the injection volume was 10 μL, the column temperature was 30 °C, and the volume flow rate was 1.0 mL min. -1 , detection wavelengths are: 210 nm for 0-15 min, 280 nm for 15-22 min, 230 nm for 22-24 min, 210 nm for 24-43 min, and 321 nm for 43-60 min;

[0059] 4.1 Selection of detection wavelength

[0060] Prepare the test solution according to the preparation method of the test solution in 3, accurately weigh 10 μL of each of the above 5 test solutions, and inject them into the high performance liquid chromatography for detection. The detection wavelengths are 210 nm, 230 nm, 254 nm, 280 nm, 321 nm, etc. The test results are shown in Figure 1 Based on the number of chromatographic peaks, chromatographic peak response values, and whether the baseline is stable, the wavelength conditions of the present invention (210 nm for 0-15 min, 280 nm for 15-22 min, 230 nm for 22-24 min, 210 nm for 24-43 min, and 321 nm for 43-60 min) were finally selected as the detection wavelength of the characteristic spectrum of the Huatan prescription.

[0061] 4.2 Selection of mobile phase

[0062] Prepare the test solution according to the preparation method of the 3 test solutions. Accurately weigh 10 μL of each of the 3 test solutions and inject them into the high performance liquid chromatography for detection. The mobile phases are methanol-water, acetonitrile-water, acetonitrile-phosphoric acid water, etc. The test results are shown in Figure 2 Based on the number of chromatographic peaks, chromatographic peak response values, and baseline stability, the mobile phase conditions of the present invention (acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, gradient elution conditions: 0-40 min, 5%-30% acetonitrile; 40-45 min, 30%-60% acetonitrile; 45-60 min, 60%-90% acetonitrile) were finally selected as the mobile phase for the characteristic spectrum of the Huatan recipe.

[0063] 4.3 Selection of column temperature

[0064] Prepare the test solution according to the preparation method of the test solution in 3, accurately weigh 10 μL of each of the above two test solutions, and inject them into the high performance liquid chromatography for detection. The column temperature is 25℃, 30℃, etc., respectively. The test results are shown in Figure 3The chromatographic peak separation was better when the column temperature was 30°C, and the column temperature condition of 30°C of the present invention was finally selected as the detection column temperature for the characteristic spectrum of Huatan Recipe.

[0065] 5. Establishment of HPLC characteristic patterns of standard decoction and granules of Huatan Recipe

[0066] Take 15 batches of traditional decoction of Huatan Fang and 6 batches of granule samples of Huatan Fang, prepare the test solution according to the method under "3", measure according to the chromatographic conditions under "4", record the chromatogram and import the data into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version), and the superimposed chromatograms are shown in Figure 4 . 13 common peaks with good peak stability, peak shape and separation were selected as characteristic peaks. After comparison with the reference substance, 6 components were identified, namely (+)-candelilla resin phenol-9′-O-glucoside (peak 4), p-hydroxycinnamic acid (peak 5), platycodon saponin E (peak 8), platycodon saponin D (peak 10), peucedanum praeruptoside A (peak 11), and peucedanum praeruptoside B (peak 13). After multi-point correction and Mark peak matching of the characteristic spectra of 15 batches of traditional decoctions of Huatan Fang, the control characteristic spectra of traditional decoctions of Huatan Fang were generated. Figure 5 After comparison, the similarity of the characteristic spectra of the six batches of Huatan Fang granules and the traditional decoction control was >0.95, indicating that the similarity between the Huatan Fang granules and the traditional decoction was good.

[0067] Using 12 chromatographic columns of different brands and types, the standard retention times of peak 1, peak 2, peak 3, peak 4 ((+)-candelilla resin phenol-9′-O-glucoside), peak 5 (p-hydroxycinnamic acid), peak 6, peak 7, peak 8 (platycoside E), peak 9, peak 10 (platycoside D), peak 11 (peuerculin A), peak 12, peak 13 (peuerculin B) were 8.3148 min, 9.8035 min, 15.5367 min, 25.8220 min, 27.5484 min, 27.8364 min, 33.5142 min, 36.4188 min, 37.8652 min, 44.5608 min, 55.1509 min, 57.0867 min, and 59.7682 min, respectively. Among them, the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns; the actual retention time of the double-labeled reference substance is collected on another chromatographic column, and the double-labeled linear correction method is adopted, with the actual retention time of the chromatographic peaks of the double-labeled reference substances p-hydroxycinnamic acid and praeruptorin A as the ordinate and the standard retention time as the abscissa to obtain the linear fitting equation; the standard retention times of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 10, peak 12, and peak 13 are substituted into the equation to obtain the corresponding predicted retention times.

[0068] 6. Methodological Investigation of Feature Maps

[0069] 6.1 Repeatability test

[0070] Prepare six parallel test solutions of the Huatan Formula granules according to the test solution preparation method under "3." Test the solutions according to the chromatographic conditions under "4," and record the chromatograms. Using the p-hydroxycinnamic acid peak as the reference peak, the relative retention time RSDs of the common peaks were ≤0.06%, and the relative peak area RSDs were ≤2.88%, demonstrating good reproducibility of the method.

[0071] Table 5 Relative retention time and peak area of ​​each chromatographic peak in the repeatability of Huatan Fang granules

[0072]

[0073] 6.2 Solution stability test

[0074] Prepare the test solution using the Huatan Recipe granules according to item "3". Perform the test according to the chromatographic conditions under item "4" at 0, 2, 4, 8, 12, and 24 hours after preparation, and record the chromatogram. Using the p-hydroxycinnamic acid peak as the reference peak, the RSDs of the relative retention times of the common peaks were ≤0.12%, and the RSDs of the relative peak areas were ≤2.47%, indicating that the test solution had good stability within 24 hours.

[0075] Table 6 Relative retention time and relative peak area of ​​each chromatographic peak in the stability of Huatan prescription granules

[0076]

[0077]

[0078] 6.3 Precision experiment

[0079] Prepare the test solution of Huatan Recipe granules according to the chromatographic conditions in item "3". Inject the sample six times continuously according to the chromatographic conditions in item "4" and record the chromatogram. Using the p-hydroxycinnamic acid peak as the reference peak, the RSDs of the relative retention times of the common peaks were ≤0.74%, and the RSDs of the relative peak areas were ≤1.97%, indicating good instrument precision.

[0080] Table 7 Relative retention time and relative peak area of ​​each chromatographic peak of Huatan Fang granules precision

[0081]

[0082]

[0083] 7 Qualitative Study of Double-Standardized Linear Method

[0084] 7.1 Calculation of Standard Retention Time and Column Suitability

[0085] Take the Huatan formula granules and prepare the test solution according to item "3". Select Col 1 to Col 12 different chromatographic columns and detect them according to the chromatographic conditions under item "4". The average actual retention time of the test solution obtained by 12 chromatographic columns is used as the standard retention time (SRT). The SRT of 13 components (peak 1 is 8.3148min, peak 2 is 9.8035min, peak 3 is 15.5367min, (+)-candelilla resin phenol-9'-O-glucoside is 25.8220min, p-hydroxycinnamic acid is 27.5484min, peak 6 is 27.836 4min, peak 7 is 33.5142min, platycoside E is 36.4188min, peak 9 is 37.8652min, platycoside D is 44.5608min, pecuculline A is 55.1509min, peak 12 is 57.0867min, and pecuculline B is 59.7682min) as the abscissa, and the actual retention time is the ordinate, and the fitting results of each chromatographic column are obtained, as shown in Table 8. The retention times of the 13 components on different chromatographic columns have a good linear relationship.

[0086] Table 8 Relationship between retention times of different chromatographic columns

[0087]

[0088]

[0089] 7.2 Selection of double-labeled compounds

[0090] The spectra were imported into DRS Origin 1.0 software, correlated with reference standards, and set a retention time window of 2 minutes. Retention time regression deviation, chromatographic peak prediction accuracy (%), and column consistency (%) were used as method optimization evaluation parameters. Ten optimal double-label peak combinations were selected from 28 double-label peak combinations, as shown in Table 9. The peak prediction accuracy was 100% for all combinations, and peaks 5 to 11 (p-hydroxycinnamic acid-peucedanum praeruptolide A), which had the best column consistency and had been identified, were selected as the double-label peaks.

[0091] Table 9 Optimization scheme of double-standard linear calibration method

[0092]

[0093] 7.3 Prediction of retention time using the double-label linear calibration method

[0094] Taking Col 3 as an example, the double-label linear calibration method (LCTRS method) was used to predict the retention time. The SRT of the double-label compound (p-hydroxycinnamic acid 27.5484min, praeruptorin A 55.1509min) was used as the abscissa, and the actual retention time (p-hydroxycinnamic acid 26.9861min, praeruptorin A 54.3846min) was used as the ordinate. Two points of p-hydroxycinnamic acid (27.5484, 26.9861) and praeruptorin A (55.1509, 54.3846) were obtained. Linear fitting was performed to obtain the linear equation Y = 0.9926X-0.3586, r = 1 Then, the SRTs of the remaining six components were substituted into the equation to obtain the predicted retention times: peak 1 was 7.8947 min, peak 2 was 9.3724 min, peak 3 was 15.0631 min, (+)-candelilla resin phenol-9′-O-glucoside was 25.2723 min, peak 6 was 27.2718 min, peak 7 was 32.9076 min, platycoside E was 35.7907 min, peak 9 was 37.2264 min, platycoside D was 43.8725 min, peak 12 was 56.3057 min, and peucedanum butylene glycol was 58.9673 min. The actual retention times were 8.5493min, 9.9609min, 15.6893min, 25.6219min, 27.7320min, 33.3654min, 36.1092min, 37.8105min, 44.0640min, 55.8000min, and 59.0580min, and the absolute deviations were 0.6546min, 0.5885min, 0.6262min, 0.3496min, 0.4602min, 0.4578min, 0.3185min, 0.5841min, 0.1915min, 0.5057min, and 0.0907min, respectively.

[0095] 7.4 Comparison of prediction results between the double-label linear calibration method and the relative retention time method

[0096] Using p-hydroxycinnamic acid as a reference, the mean relative retention times of 12 components on 12 columns were calculated to be 0.3005, 0.3526, 0.5585, 0.9379, 1.0000, 1.0134, 1.2268, 1.3229, 1.3752, 1.6186, 2.0030, 2.0738, and 2.1712, respectively. The prediction results of LCTRS and RRT were compared using metrics such as maximum absolute deviation. The results showed that LCTRS had higher prediction accuracy and was applicable to more columns, outperforming the RRT method. (See Tables 10-11.)

[0097] Table 10 Comparison of prediction results between LCTRS method and RRT method

[0098]

[0099] Table 11 Absolute deviations of the predicted retention times of different components using two methods on different chromatographic columns

[0100]

[0101]

[0102] 8. Research on the determination of index component content

[0103] 8.1 Assay method

[0104] Liquid chromatography was performed according to steps S1-S3 of the characteristic spectrum establishment method to obtain a chromatogram, and the contents of p-hydroxycinnamic acid, platycoside E, peucedanum praeruptoside A, peucedanum praeruptoside B, and (+)-candelilla resin phenol-9′-O-glucoside were calculated by peak area based on the external standard method.

[0105] 8.2 Methodological Review

[0106] 8.2.1 Linear Relationship Investigation

[0107] Accurately measure an appropriate amount of the mixed reference solution under "2" and dilute it 2-, 4-, 8-, and 16-fold using the multiple dilution method. Perform the assay according to the chromatographic conditions under "4." Perform a linear regression using reference concentration as the abscissa and peak area as the ordinate, plot a standard curve, and calculate the regression equation and correlation coefficient. Results indicate good linear relationships for each component within a specific concentration range (see Table 12).

[0108] Table 12 Regression equation and linear range

[0109]

[0110]

[0111] 8.2.2 Repeatability Study

[0112] Six test sample solutions were prepared in parallel using the Huatan formula granules according to the method under "3". The solutions were tested according to the chromatographic conditions under "4". The results showed that the RSDs of the contents of p-hydroxycinnamic acid, (+)-candelilla resin phenol-9′-O-glucoside, platycoside E, peucedanum arvense A, and peucedanum arvense B were all ≤2.23%, indicating that the method had good repeatability.

[0113] Table 13 Repeatability test results

[0114]

[0115] 8.2.3 Stability Study

[0116] The test solution was prepared from the Huatan Decoction granules according to the method under "3". The test was carried out according to the chromatographic conditions under "4" at 0, 2, 4, 8, 12, and 24 hours after preparation. The results showed that the RSDs of the contents of p-hydroxycinnamic acid, (+)-candelilla resin phenol-9′-O-glucoside, platycoside E, peucedanum arvense A, and peucedanum arvense B were all ≤1.87%, indicating that the test solution had good stability within 24 hours.

[0117] Table 14 Stability test results

[0118]

[0119]

[0120] 8.2.4 Precision investigation

[0121] Take an appropriate amount of the reference solution and inject it continuously for 6 times according to the chromatographic conditions under "2.1.3". The RSDs of the peak areas of p-hydroxycinnamic acid, (+)-candelilla resin phenol-9'-O-glucoside, platycodon saponin E, peucedanum praeruptoside A, and peucedanum praeruptoside B were all ≤2.01%, indicating that the instrument precision was good.

[0122] Table 15 Precision inspection results

[0123]

[0124] 8.2.5 Sample recovery test

[0125] Take about 0.25g of the granule powder of Huatan Recipe, weigh it accurately, add the reference solution in the same amount as the corresponding component in the sample, prepare the test solution according to the method under "2.1.2", prepare 6 copies in parallel, and determine according to the chromatographic conditions under "2.1.3". The calculated recoveries of p-hydroxycinnamic acid, platycoside E, peucedanum acuminoids A, peucedanum acuminoids B, and (+)-candelilla resin phenol-9′-O-glucoside are 102.69%-104.91%, 90.92%-96.39%, 92.60%-95.84%, 85.26%-86.92%, and 87.79%-92.09%, respectively. The RSDs are 0.75%, 1.86%, 1.60%, 1.89%, and 2.02%, respectively, indicating that the method has good accuracy.

[0126] Table 16 Recovery results

[0127]

[0128] The above specific embodiments are not intended to limit the scope of protection of the present invention. For those skilled in the art, any substitution, improvement, or modification of the embodiments of the present invention falls within the scope of protection of the present invention. Any details not described in the present invention are generally known to those skilled in the art.

Claims

1. A method for establishing a high performance liquid chromatography characteristic spectrum of a phlegm-resolving prescription, characterized in that: The phlegm-removing prescription preparation is prepared from 9-11 parts of Peucedanum chinense, 9-11 parts of Platycodon grandiflorum, 7-9 parts of Rhizoma Zingiberis, 8-10 parts of Radix Glehniae and 9-15 parts of Pumice stone in parts by weight to form phlegm-removing prescription granules or phlegm-removing prescription standard decoction freeze-dried powder. The preparation method comprises the following steps: Preparation of S1 reference solution: Accurately weigh appropriate amounts of platycoside D, platycoside E, praeruptoside A, praeruptoside B, p-hydroxycinnamic acid, and (+)-candelilla resin phenol-9′-O-glucoside reference substances, and add methanol to prepare mixed reference solutions of different concentrations. Preparation of S2 test solution: Take an appropriate amount of Huatan formula granules or traditional decoction freeze-dried powder, dissolve in methanol, weigh, sonicate, cool, add methanol to the weight, shake well, filter through a microporous membrane, and obtain the filtrate; Determination of S3 HPLC conditions: A C18 column was used with acetonitrile-0.1% aqueous phosphoric acid as the mobile phase. The gradient elution conditions were: 0-40 min, 5%-30% acetonitrile; 40-45 min, 30%-60% acetonitrile; 45-60 min, 60%-90% acetonitrile; injection volume 10 μL, column temperature 30°C, volume flow rate 1.0 mL min -1 , detection wavelength 0-15min is 210nm, 15-22min is 280nm, 22-24min is 230nm, 24-43min is 210nm, 43-60min is 321nm; S4 HPLC determination: Collect chromatograms of the mixed reference solution and multiple batches of test solution according to the chromatographic conditions in step S3, analyze the chromatograms, confirm that there are 13 common peaks, and identify the characteristic peaks of 6 components; S5 Determination of predicted retention time: Using multiple chromatographic columns of different brands and types, collect chromatograms of the mixed reference solution and the test solution according to the chromatographic conditions in step S3 to obtain the standard retention times of 13 common peaks; using p-hydroxycinnamic acid and praeruptorin A as double-labeled reference substances, use the double-labeled linear calibration method to obtain the predicted retention times of the remaining peaks.

2. The method for establishing the HPLC characteristic spectrum of the phlegm-removing prescription according to claim 1, characterized in that: The specific method for preparing the test solution is as follows: take an appropriate amount of granules of the Huatan prescription or freeze-dried powder of the traditional decoction, grind it into powder, take 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of methanol, ultrasonicate for 30 minutes, cool it, weigh it, make up the lost weight with methanol, shake it evenly, filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain it.

3. The method for establishing the HPLC characteristic spectrum of the phlegm-removing prescription according to claim 1, characterized in that: The preparation method of the reference solution is as follows: take appropriate amounts of platycoside D, platycoside E, praeruptoside A, praeruptoside B, p-hydroxycinnamic acid, and (+)-candelilla resin phenol-9′-O-glucoside reference substances, accurately weigh them, and add methanol to prepare the concentrations to 100.400, 103.360, 110.491, 119.290, 288.400, and 114.050 μg·mL, respectively. -1 mixed reference solution.

4. The method for establishing the HPLC characteristic spectrum of the phlegm-removing prescription according to claim 1, characterized in that: In step S5, the specific method for obtaining the predicted retention time using the double-label linear correction method is: using the actual retention time of the chromatographic peaks of the double-label reference substances p-hydroxycinnamic acid and white flower peucedanum as the ordinate and the standard retention time as the abscissa to obtain a linear fitting equation; substituting the standard retention times of the remaining 11 characteristic peaks into the equation to obtain the corresponding predicted retention times; wherein the standard retention time is the average retention time of the test sample collected on multiple chromatographic columns.

5. The method for establishing the HPLC characteristic spectrum of the phlegm-removing prescription according to claim 1, characterized in that: In step S5, the standard retention times of the 13 peaks are 8.3148 min, 9.8035 min, 15.5367 min, 25.8220 min, 27.5484 min, 27.8364 min, 33.5142 min, 36.4188 min, 37.8652 min, 44.5608 min, 55.1509 min, 57.0867 min, and 59.7682 min, respectively.

6. The method for establishing the HPLC characteristic spectrum of the phlegm-removing prescription according to claim 1, characterized in that: The preparation method of the Huatan formula granules is as follows: accurately weigh 6.0000, 2.5714, 0.9000, 0.5625, and 1.2000 g of the formula granules of Peucedanum chinense, Platycodon grandiflorum, Rhizoma Zingiberis, Radix Glycyrrhizae, and Pumice stone to prepare the Huatan formula granules.

7. The method for establishing the HPLC characteristic spectrum of the phlegm-removing prescription according to claim 1, characterized in that: The specifications of the chromatographic column are 4.6×250 mm, 5 μm.

8. The method for establishing the HPLC characteristic spectrum of the phlegm-removing prescription according to claim 1, characterized in that: The preparation method of the freeze-dried powder of the standard decoction of Huatan Fang comprises the following steps: A1 Weigh out Tianzhuhuang slices, place in a casserole, add 12 times the amount of water and soak for 30 minutes; A2 Weigh Platycodon grandiflorum, Peucedanum chinense, Rhizoma Zingiberis, and Pumice stone and place them in another casserole. Add 12 times the amount of water and soak for 30 minutes. A3 decocts the soaking liquid with medicinal materials in A1, brings it to a boil over high heat, then simmers it for 10 minutes. Then, add the soaking liquid with medicinal materials in A2 into the casserole and simmer together, brings it to a boil over high heat, then simmers it for 20 minutes, and sieve it through a 200-mesh sieve while it is still hot. A4: Add 10 times the amount of water to the residue obtained in A3, boil over high heat, then simmer for 20 minutes. Pass the residue through a 200-mesh sieve while hot. Combine the filtrate with the filtrate from A3, concentrate at 60°C, and freeze-dry in a vacuum.

9. A method for determining the content of ingredients in a phlegm-removing prescription, characterized in that: The method comprises the following steps: performing liquid chromatography determination according to steps S1-S3 of the method established according to any one of claims 1-8 to obtain a chromatogram, and calculating the content of platycodon saponin E, praeruptoside A, praeruptoside B, p-hydroxycinnamic acid, and (+)-candelilla resin phenol-9′-O-glucoside by peak area based on an external standard method.

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