Establishment of a method for high performance liquid chromatography (HPLC) characteristic chromatogram of a Huatan prescription preparation and a method for determining the content of components

Abstract

The application relates to a method for establishing a high-performance liquid chromatography characteristic spectrum of a preparation of a Huatan recipe and a method for determining the component content, and belongs to the field of medicine analysis. The method for establishing the high-performance liquid chromatography characteristic spectrum of the preparation of the Huatan recipe comprises the following steps: S1, preparation of a reference solution; S2, preparation of a test solution; S3, determination of chromatographic conditions; S4, collection of chromatograms of the reference solution and multiple batches of the test solution, analysis of the chromatograms of the test solutions, calibration of 13 common peaks, and identification of characteristic peaks of six components; and S5, taking p-hydroxycinnamic acid and Baihuahu methine as double-index reference substances, adopting a double-index linear correction method to effectively predict and locate the other 11 characteristic peaks, which is good in scientific accuracy and durability, and is relatively economical. The method is beneficial to comprehensively and effectively controlling the quality of the preparation of the Huatan recipe.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine analysis, in particular to a method for establishing high performance liquid chromatography (HPLC) characteristic spectrum of Huatan prescription preparation and a method for determining the content of components. BACKGROUND

[0002] The Huatan prescription preparation is composed of five components, i.e., Peucedanum praeruptorum, Platycodon grandiflorum, Bambusa pachinoides, Curcuma zedoaria and Shelly, and has the effects of clearing heat and reducing phlegm, relieving cough and asthma, and can be used for treating lung heat cough, excessive phlegm and asthma. SUMMARY

[0003] The present application provides a method for establishing HPLC characteristic spectrum of Huatan prescription preparation and a method for determining the content of components, which can more comprehensively and effectively control the quality of Huatan prescription preparation.

[0004] The technical scheme adopted by the present application to solve the above technical problem is as follows:

[0005] A method for establishing HPLC characteristic spectrum of Huatan prescription preparation, the Huatan prescription preparation is prepared from 9-11 parts of Peucedanum praeruptorum, 9-11 parts of Platycodon grandiflorum, 7-9 parts of Bambusa pachinoides, 8-10 parts of Curcuma zedoaria and 9-15 parts of Shelly according to weight ratio, and the method comprises the following steps:

[0006] S1 Preparation of reference solution: take appropriate amounts of Platycodon grandiflorum saponin D, Platycodon grandiflorum saponin E, Baihuahu A, Baihuahu B, p-hydroxy cinnamic acid and (+)-Ruscogenin-9'-O-glucoside reference substances, accurately weigh and add methanol to prepare mixed reference solution with different concentrations;

[0007] S2 Preparation of test solution: take appropriate amounts of Huatan prescription preparation or traditional decoction lyophilized powder, dissolve in methanol, weigh, ultrasonic, cool, supplement with methanol, shake, filter through a microporous filter, and take the filtrate;

[0008] S3 HPLC conditions

[0009] Determination: use a C18 column, take acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, gradient elution conditions are as follows: 0-40 min, 5%-30% acetonitrile; 40-45 min, 30%-60% acetonitrile; 45-60 min, 60%-90% acetonitrile; injection volume is 10 μL, column temperature is 30℃, and volume flow rate is 1.0 mL·min -1, detection wavelength 0-15 min 210 nm, 15-22 min 280 nm, 22-24 min 230 nm, 24-43 min 210 nm, 43-60 min 321 nm;

[0010] S4 High performance liquid chromatography determination: according to the chromatographic conditions in step S3, the chromatograms of the mixed control solution and the multiple batches of test solution are collected, the chromatograms are analyzed, 13 common peaks are confirmed, and the characteristic peaks of 6 components are identified;

[0011] S5 Determination of predicted retention time: using multiple chromatographic columns of different brands and types, the chromatograms of the mixed control solution and the test solution are collected according to the chromatographic conditions in step S3, and the standard retention time of the 13 common peaks is obtained; using p-hydroxycinnamic acid and peucedanin as double standard controls, the predicted retention time of the remaining peaks is obtained by double standard linear correction method.

[0012] Preferably, the preparation method of the test solution is specifically: taking Huatanfang formula granules or traditional decoction lyophilized powder, grinding, taking 0.5g, accurately weighing, placing in a conical flask with a plug, accurately adding 25mL of methanol, ultrasonic for 30min, cooling, weighing, supplementing the weight loss with methanol, shaking, filtering with 0.45μm microporous membrane, and then filtering the filtrate to obtain the test solution.

[0013] Preferably, the preparation method of the control solution is specifically: taking appropriate amount of anemarrhena aspera saponin D, anemarrhena aspera saponin E, peucedanin, peucedanin, p-hydroxycinnamic acid, and (+)-maesa-9'-O-glucoside control substances, accurately weighing, and preparing a mixed control solution with a concentration of 100.400, 103.360, 110.491, 119.290, 288.400, and 114.050μg·mL -1 respectively.

[0014] Preferably, in step S5, the specific method for obtaining the predicted retention time by double standard linear correction method is: taking the actual retention time of the double standard control p-hydroxycinnamic acid and peucedanin chromatographic peak as the ordinate, and the standard retention time as the abscissa, to obtain the linear fitting equation; the standard retention time of the remaining 11 characteristic peaks is substituted into the equation to obtain the corresponding predicted retention time; wherein the standard retention time is the average value of the test retention time 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 prescription granules is as follows: 6.0000, 2.5714, 0.9000, 0.5625, and 1.2000 g of peucedanum, platycodon, ginger bamboo, radix curculignis, and sea coral are precisely weighed to prepare the Huatan prescription granules.

[0017] Preferably, the preparation method of the Huatan prescription granules is as follows: 6.0000, 2.5714, 0.9000, 0.5625, and 1.2000 g of peucedanum, platycodon, ginger bamboo, radix curculignis, and sea coral are precisely weighed to prepare the Huatan prescription granules.

[0018] A1 weighs radix curculignis and places it in a sand pot, and then adds 12 times the amount of water to soak for 30 min;

[0019] A2 weighs platycodon, peucedanum, ginger bamboo, and sea coral and places them in another sand pot, and then adds 12 times the amount of water to soak for 30 min;

[0020] A3 decocts the soaking liquid with the medicinal materials in A1, and then boils with a strong fire and changes to a weak fire to decoct for 10 min, and then adds the soaking liquid with the medicinal materials in A2 to the sand pot to decoct together, and then boils with a strong fire and changes to a weak fire to decoct for 20 min, and then passes through a 200-mesh sieve while hot;

[0021] A4 adds 10 times the amount of water to the medicinal residues obtained in A3, boils with a strong fire and changes to a weak fire to decoct for 20 min, and then passes through a 200-mesh sieve while hot, and then the filtrate is combined with the filtrate in A3, and then concentrates at 60 DEG C, and then vacuum freeze-dries to obtain the freeze-dried powder.

[0022] The application also provides a component content determination method of the Huatan prescription preparation, which comprises the following steps: liquid chromatography determination is performed according to the steps S1-S3 of the method established in any one of claims 1-8 to obtain a chromatogram, and the contents of each component of p-hydroxycinnamic acid, platycodoside E, praeruptorin A, praeruptorin B, and (+)-lancolophorol-9'-O-glucoside are calculated based on the external standard method through peak area.

[0023] The application has the advantages that:

[0024] 1.The method of the application establishes a method to use p-hydroxycinnamic acid and Baihuahu A as double standard control, uses two control samples to effectively predict and locate the chromatographic peaks of other 11 components by double standard linear correction method, and is obviously better than the relative retention time method commonly used for chromatographic peak quality at the applicability of the chromatographic column and the accuracy of the prediction, greatly eliminates the problem of low accuracy of characteristic peak quality and applicability of chromatographic column of the relative retention time method, and performs well in scientific accuracy and durability, is more economical, improves the simplicity of the experiment and reduces the cost; and provides a reference and basis for quality control of Huatan prescription preparations.

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

[0026] 3.According to the characteristic spectrum obtained by the method of the application, there are 13 effectively separated characteristic peaks, so the characteristic spectrum obtained can effectively represent the quality of the Huatan prescription preparation, and thus is beneficial to more comprehensive monitoring of the quality of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Different wavelength characteristic spectra of the Huatan prescription granules according to the application.

[0028] Figure 2 Different mobile phase characteristic spectra of the Huatan prescription granules according to the application.

[0029] Figure 3 Different column temperature characteristic spectra of the Huatan prescription granules according to the application.

[0030] Figure 4 Characteristic spectra of the traditional decoction lyophilized powder and the prescription granules of multiple batches of Huatan prescription according to the application.

[0031] Figure 5 Characteristic spectrum of the Huatan prescription preparation according to the application. DETAILED DESCRIPTION

[0032] In order to clearly illustrate the technical features of the present application, the following will describe the present application in detail with specific embodiments and in conjunction with the accompanying drawings.

[0033] 1.Preparation of the Huatan prescription

[0034] 1.1Preparation of the traditional decoction lyophilized powder

[0035] Weigh 27g of Tianzhuhuang (Bamboo Shavings) slices and place them in a clay pot. Add 12 times the amount of water and soak for 30 minutes. Weigh 27g each of Haifushi (Pumice), Jiegeng (Platycodon grandiflorus), Qianhu (Radix Peucedanum praeruptorum), and Jiangzhuru (Radix Bambusae incisum) and place them in another clay pot. Add 12 times the amount of water and soak for 30 minutes. First, decoct Tianzhuhuang. Bring to a boil over high heat, then simmer over low heat for 10 minutes. Add the remaining three herbs along with the soaking liquid to the clay pot and decoct together. Bring to a boil over high heat, then simmer over low heat for 20 minutes. Sieve while hot (200 mesh). Add 10 times the amount of water to the dregs, bring to a boil over high heat, then simmer over low heat for another 20 minutes. Sieve while hot (200 mesh). Combine the filtrates, concentrate at 60℃, and then freeze-dry under vacuum to obtain the freeze-dried powder of the traditional decoction for resolving phlegm (experiment number S1-S15).

[0036] 1.2 Preparation of expectorant formula granules

[0037] Accurately weigh 6.0000 g of the following ingredients: Peucedanum praeruptorum, Platycodon grandiflorus, Zingiber officinale (green bamboo), Bambusa textilis (green bamboo), and Pumice granules, respectively, to prepare the expectorant formula granules (experiment number S16-S21, experimental sample information is shown in Table 1).

[0038] Table 1. Formulation Granule 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] Accurately weigh 11.19, 10.53, 11.27, 11.98, 28.84, and 11.58 mg of platycodon saponin D, platycodon saponin E, angelica dahurica glycoside, angelica dahurica glycoside B, p-hydroxycinnamic acid, and (+)-cantharis phenol-9′-O-glucoside reference standards, respectively, and place them in 10 ml volumetric flasks. Dilute to volume with methanol to prepare reference standard stock solutions with concentrations of 1.0040, 1.0336, 1.1049, 1.1929, 2.8840, and 1.1405 mg / ml. Accurately measure 1 ml of each reference standard stock solution into 10 ml volumetric flasks and dilute to volume with methanol to prepare reference standard stock solutions with concentrations of 100.400, 103.360, 110.491, 119.290, 288.400, and 114.050 μg / mL. -1 A mixed reference solution.

[0043] 3. Preparation of the test solution

[0044] 3.1 Selection of Extraction Method

[0045] About 0.1 g of the Quhuatan preparation was subjected to heating reflux and ultrasonic treatment for 30 min, respectively, and the peak area of each marker peak was determined. The results (see Table 2) showed that the peak areas obtained by ultrasonic extraction and reflux extraction were comparable. Considering that ultrasonic extraction is more convenient, the ultrasonic extraction method was selected.

[0046] Table 2 Results of extraction method investigation (peak area)

[0047]

[0048] 3.2 Selection of extraction time

[0049] The reflux time was set to 15 min, 30 min, and 45 min, respectively, and the peak area of each marker peak was investigated. The results (see Table 3) showed that the peak area obtained by ultrasonic extraction for 15 min was slightly smaller, and the characteristic spectra obtained by ultrasonic extraction for 30 min and 45 min presented basically the same material information. Therefore, the ultrasonic extraction time was selected to be 30 min.

[0050] Table 3 Results of extraction time investigation (peak area)

[0051]

[0052] 3.3 Selection of extraction solvent

[0053] The peak area of each marker peak was investigated when the extraction solvent was 10% methanol, 30% methanol, 50% methanol, 70% methanol, or methanol. The results (see Table 4) showed that the peak area of the marker peak obtained by methanol extraction was larger, and the extraction solvent was finally selected to be methanol.

[0054] Table 4 Results of extraction solvent investigation (peak area)

[0055]

[0056] In summary, an appropriate amount of Quhuatan preparation granules or traditional decoction lyophilized powder was finely ground, accurately weighed about 0.5 g, placed in a conical flask with a stopper, precisely added with 25 mL of methanol, ultrasonically treated for 30 min, cooled, weighed, supplemented with the lost weight with methanol, shaken well, filtered with a 0.45 μm microporous filter, and then the filtrate was obtained.

[0057] 4. Determination of chromatographic conditions

[0058] The C 18Chromatographic column (4.6*250mm, 5μm), with acetonitrile-0.1% phosphoric acid aqueous solution as mobile phase, gradient elution conditions: 0-40min, 5%-30% acetonitrile; 40-45min, 30%-60% acetonitrile; 45-60min, 60%-90% acetonitrile; injection amount 10μL, column temperature 30℃, volume flow rate 1.0mL·min -1 , detection wavelength: 0-15min 210nm, 15-22min 280nm, 22-24min 230nm, 24-43min 210nm, 43-60min 321nm;

[0059] 4.1 Selection of detection wavelength

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

[0061] 4.2 Selection of mobile phase

[0062] Prepare the test sample solution according to the preparation method of the test sample solution in 3, and accurately take 10μL of each of the above 3 test sample solutions, inject into the high performance liquid chromatograph for detection, the mobile phases are methanol-water, acetonitrile-water, acetonitrile-phosphoric acid water, etc., and the detection results are shown in Table 2. Figure 2 According to the principles of the number of chromatographic peaks, chromatographic peak response value, and whether the baseline is stable, etc., finally, the mobile phase conditions (with acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, gradient elution conditions: 0-40min, 5%-30% acetonitrile; 40-45min, 30%-60% acetonitrile; 45-60min, 60%-90% acetonitrile) of the application are selected as the mobile phase of the characteristic spectrum of the Huatan prescription.

[0063] 4.3 Selection of column temperature

[0064] Prepare the test sample solution according to the preparation method of the test sample solution in 3, and accurately take 10μL of each of the above 2 test sample solutions, inject into the high performance liquid chromatograph for detection, the column temperatures are 25℃, 30℃, etc., and the detection results are shown in Table 3. Figure 3The peak separation degree is better at a column temperature of 30 DEG C, and finally, the column temperature condition of 30 DEG C is selected as the detection column temperature of the characteristic spectrum of Huatanfang.

[0065] 5. Establishment of HPLC characteristic spectrum of Huatanfang standard decoction and dispensing granules

[0066] Take 15 batches of traditional Huatanfang decoction and 6 batches of Huatanfang dispensing granules as samples, prepare the sample solution according to the method in item 3, and determine according to the chromatographic conditions in item 4, record the chromatogram and import the data into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version), and the superimposed chromatogram is as follows: Figure 4 Select 13 common peaks with stable peak, good peak shape and separation degree as characteristic peaks, and identify 6 components among them by comparison with the control, which are (+)-Sargentodoxa Wood Resin Phenol-9'-O-glucoside (peak 4), p-hydroxy cinnamic acid (peak 5), Platycodon Saponin E (peak 8), Platycodon Saponin D (peak 10), Peucedanum Praeruptorum A (peak 11) and Peucedanum Praeruptorum B (peak 13). After multi-point correction and Mark peak matching of the characteristic spectrum of 15 batches of traditional Huatanfang decoction, the control characteristic spectrum of traditional Huatanfang decoction is as follows: Figure 5 After comparison, the similarity of 6 batches of Huatanfang dispensing granules and the control characteristic spectrum of traditional decoction is all >0.95, indicating that the similarity between Huatanfang dispensing granules and traditional decoction is good.

[0067] Using 12 different brands and types of chromatographic columns, the standard retention times of peak 1, peak 2, peak 3, peak 4 ((+)-Sargentodoxa Wood Resin Phenol-9'-O-glucoside), peak 5 (p-hydroxy cinnamic acid), peak 6, peak 7, peak 8 (Platycodon Saponin E), peak 9, peak 10 (Platycodon Saponin D), peak 11 (Peucedanum Praeruptorum A), peak 12, peak 13 (Peucedanum Praeruptorum B) 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, 59.7682 min respectively. Among them, the standard retention time is the average value of the retention time of the sample collected on multiple chromatographic columns; the actual retention time of the double standard control is collected on another chromatographic column, and the linear fitting equation is obtained by using the double standard linear correction method, taking the actual retention time of the double standard control p-hydroxy cinnamic acid and Peucedanum Praeruptorum A chromatographic peak as the ordinate and the standard retention time as the abscissa; 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 time.

[0068] 6. Methodology investigation of characteristic spectrum

[0069] 6.1 Reproducibility test

[0070] Take Huatan Fang formula granules according to the preparation method of test solution under item "3" to prepare 6 test solution, according to the chromatographic conditions under item "4" to detect, record chromatogram. With p-hydroxy cinnamic acid peak as reference peak, the RSD of relative retention time of each common peak was ≤0.06%, the RSD of relative peak area was ≤2.88%, indicating that the reproducibility of the method was good.

[0071] Table 5 Reproducibility of each chromatographic peak of Huatan Fang formula granules relative retention time and relative peak area

[0072]

[0073] 6.2 Solution stability test

[0074] Take Huatan Fang formula granules according to the preparation method of test solution under item "3", respectively at 0, 2, 4, 8, 12, 24h according to the chromatographic conditions under item "4" to detect, record chromatogram. With p-hydroxy cinnamic acid peak as reference peak, the RSD of relative retention time of each common peak was ≤0.12%, the RSD of relative peak area was ≤2.47%, indicating that the test solution had good stability within 24h.

[0075] Table 6 Stability of each chromatographic peak of Huatan Fang formula granules relative retention time and relative peak area

[0076]

[0077]

[0078] 6.3 Precision experiment

[0079] Take Huatan Fang formula granules according to the preparation method of test solution under item "3", according to the chromatographic conditions under item "4" to detect, record chromatogram. With p-hydroxy cinnamic acid peak as reference peak, the RSD of relative retention time of each common peak was ≤0.74%, the RSD of relative peak area was ≤1.97%, indicating that the instrument precision was good.

[0080] Table 7 Precision of each chromatographic peak of Huatan Fang formula granules relative retention time and relative peak area

[0081]

[0082]

[0083] 7 Qualitative study by double standard linear method

[0084] 7.1 Calculation of standard retention time and suitability of chromatographic column

[0085] The Qutuofang Granules were prepared according to the method of item 3 to prepare the test solution, and 12 different chromatographic columns were selected according to the chromatographic conditions of item 4. The average value of the actual retention time of the test solution obtained from the 12 chromatographic columns was used as the standard retention time (SRT), and the SRT of 13 components (peak 1 was 8.3148 min, peak 2 was 9.8035 min, peak 3 was 15.5367 min, (+)-lancolol-9'-O-glucoside was 25.8220 min, p-coumaric acid was 27.5484 min, peak 6 was 27.8364 min, peak 7 was 33.5142 min, platycodin E was 36.4188 min, peak 9 was 37.8652 min, platycodin D was 44.5608 min, praeroside A was 55.1509 min, peak 12 was 57.0867 min, and praeroside B was 59.7682 min) was used as the abscissa, and the actual retention time was used as the ordinate. The fitting results of each chromatographic column were obtained, as shown in Table 8. The linear relationship of the retention times of the 13 components on different chromatographic columns was good.

[0086] Table 8 Retention time relationship of different chromatographic columns

[0087]

[0088]

[0089] 7.2 Selection of double-label compounds

[0090] The chromatogram was imported into the DRS Origin 1.0 software, the reference substance was associated, and the retention time window was set to 2 min. The retention time regression deviation, chromatographic peak prediction accuracy (%), and chromatographic column compliance rate (%) were used as the method optimization evaluation parameters. From the 28 groups of double-label peak combinations, the optimal 10 double-label peak combinations were optimized and screened, as shown in Table 9. The chromatographic peak prediction accuracy was 100%, the double-label peak combination peak 5-peak 11 (p-coumaric acid-praeroside A) with the optimal chromatographic column compliance rate and all identified was selected as the double-label peak.

[0091] Table 9 Optimization scheme of double-label linear correction method

[0092]

[0093] 7.3 Prediction of retention time by double-label linear correction method

[0094] Take Col 3 as an example, the double standard linear correction method (LCTRS method) is used for retention time prediction, and the SRT of the double standard compound (p-hydroxy cinnamic acid 27.5484 min, praeruptorin A 55.1509 min) is used as the abscissa, and the actual retention time (p-hydroxy cinnamic acid 26.9861 min, praeruptorin A 54.3846 min) is used as the ordinate. Two points p-hydroxy cinnamic acid (27.5484, 26.9861) and praeruptorin A (55.1509, 54.3846) are obtained, a linear fitting is performed, a linear equation Y = 0.9926X - 0.3586 is obtained, and r = 1. Then the SRT of the remaining 6 components is substituted into the equation to obtain the predicted retention time: peak 1 is 7.8947 min, peak 2 is 9.3724 min, peak 3 is 15.0631 min, (+)-anabasine-9'-O-glucoside is 25.2723 min, peak 6 is 27.2718 min, peak 7 is 32.9076 min, platycodin E is 35.7907 min, peak 9 is 37.2264 min, platycodin D is 43.8725 min, peak 12 is 56.3057 min, and praeruptorin B is 58.9673 min. The actual retention times are 8.5493 min, 9.9609 min, 15.6893 min, 25.6219 min, 27.7320 min, 33.3654 min, 36.1092 min, 37.8105 min, 44.0640 min, 55.8000 min, 59.0580 min, and the absolute deviations are 0.6546 min, 0.5885 min, 0.6262 min, 0.3496 min, 0.4602 min, 0.4578 min, 0.3185 min, 0.5841 min, 0.1915 min, 0.5057 min, and 0.0907 min, respectively.

[0095] 7.4 Comparison of prediction results of double standard linear correction method and relative retention time method

[0096] With p-hydroxy cinnamic acid as the reference, the average relative retention time of 12 components on 12 chromatographic columns is calculated, which is 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. The maximum absolute deviation and the like are used as indexes to compare the prediction results of LCTRS and RRT methods. The results show that the prediction accuracy of LCTRS is higher, more chromatographic columns are suitable, and LCTRS is better than RRT method. See Tables 10-11.

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

[0098]

[0099] Table 11 Absolute deviation of retention time prediction value of different components in two methods of different chromatographic columns

[0100]

[0101]

[0102] 8. Study on determination of index component content

[0103] 8.1 Determination method

[0104] According to steps S1-S3 of the characteristic spectrum establishment method, liquid chromatography was performed to obtain a chromatogram, and the content of each component of p-hydroxycinnamic acid, platycodin E, praeparil A, praeparil B, and (+)-lindlarresinol-9'-O-glucoside was calculated based on the peak area by external standard method.

[0105] 8.2 Methodology investigation

[0106] 8.2.1 Investigation of linear relationship

[0107] An appropriate amount of mixed reference solution under item "2" was precisely measured, and dilution method was used to dilute it by 2, 4, 8, and 16 times, respectively, and detection was performed under the chromatographic conditions in item "4". The standard curve was drawn by linear regression with the mass concentration of the reference as the abscissa and the peak area as the ordinate, and the regression equation and the correlation coefficient were calculated. It was found that each component had a good linear relationship within a certain concentration range, as shown in Table 12.

[0108] Table 12 Regression equation and linear range

[0109]

[0110]

[0111] 8.2.2 Investigation of repeatability

[0112] Six test sample solutions were prepared in parallel according to the method in item "3" using Huatanfang Dispensing Granules, and detection was performed under the chromatographic conditions in item "4". The results showed that the RSD of the content of p-hydroxycinnamic acid, (+)-lindlarresinol-9'-O-glucoside, platycodin E, praeparil A, and praeparil B was ≤2.23%, indicating that the method had good repeatability.

[0113] Table 13 Results of repeatability investigation

[0114]

[0115] 8.2.3 Stability Investigation

[0116] The test solution of Huatanfang Dispensing Granules was prepared according to the method in item 3, and was detected according to the chromatographic conditions in item 4 at 0, 2, 4, 8, 12 and 24 h after preparation, respectively. The results showed that the RSDs of the contents of p-hydroxycinnamic acid, (+)-lindera resin phenol-9'-O-glucoside, platycodon saponin E, imperatorin, and isoimperatorin were all ≤1.87%, indicating that the test solution had good stability within 24 h.

[0117] Table 14 Stability Investigation Results

[0118]

[0119]

[0120] 8.2.4 Precision Investigation

[0121] The control solution was taken in an appropriate amount, and was continuously injected for 6 times according to the chromatographic conditions in item 2.1.3. The RSDs of the peak areas of p-hydroxycinnamic acid, (+)-lindera resin phenol-9'-O-glucoside, platycodon saponin E, imperatorin, and isoimperatorin were all ≤2.01%, indicating that the instrument had good precision.

[0122] Table 15 Precision Investigation Results

[0123]

[0124] 8.2.5 Sample Spiking Recovery Test

[0125] About 0.25 g of the powder of Huatanfang Dispensing Granules was accurately weighed, and the control solution containing the same amount of the corresponding components as in the sample was added. The test solution was prepared according to the method in item 2.1.2, and 6 samples were prepared in parallel. The chromatographic conditions in item 2.1.3 were used for determination, and the sample spiking recoveries of p-hydroxycinnamic acid, platycodon saponin E, imperatorin, isoimperatorin, and (+)-lindera resin phenol-9'-O-glucoside were 102.69%-104.91%, 90.92%-96.39%, 92.60%-95.84%, 85.26%-86.92%, and 87.79%-92.09%, respectively. The RSDs were 0.75%, 1.86%, 1.60%, 1.89%, and 2.02%, respectively, indicating that the method had good accuracy.

[0126] Table 16 Recovery Rate Results

[0127]

[0128] The above detailed description does not limit the scope of the application, and any alternative improvements or changes made by those skilled in the art to the embodiments of the application fall within the scope of the application. The application is not limited by the details described above, but is limited only by the claims.

Claims

1. A method for establishing a high-performance liquid chromatography (HPLC) characteristic spectrum of an expectorant formula, characterized in that, The expectorant preparation, by weight, is formulated into expectorant granules or freeze-dried powder of standard expectorant decoction, consisting of 9-11 parts of Peucedanum praeruptorum, 9-11 parts of Platycodon grandiflorus, 7-9 parts of Zingiber officinale and Bambusa textilis, 8-10 parts of Bambusa textilis scutellaria, and 9-15 parts of pumice. The method for establishing this preparation includes the following steps: Preparation of S1 reference solution: Take appropriate amounts of platycodon saponin D, platycodon saponin E, angelica dahurica glycoside, angelica dahurica glycoside B, p-hydroxycinnamic acid, and (+)-cantharis resin phenol-9′-O-glucoside reference standards, accurately weigh them, and add methanol to prepare mixed reference solutions of different concentrations. Preparation of S2 test solution: Take an appropriate amount of phlegm-reducing formula granules or traditional decoction freeze-dried powder, dissolve in methanol, weigh, sonicate, cool, add methanol to make up the weight, shake well, filter through a microporous membrane, and take the filtrate. S3 High Performance Liquid Chromatography (HPLC) Conditions: A C18 column was used with acetonitrile-0.1% phosphoric acid aqueous solution 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 was 10 μL, column temperature was 30℃, and flow rate was 1.0 mL / min. -1 The detection wavelengths are 210nm for 0-15min, 280nm for 15-22min, 230nm for 22-24min, 210nm for 24-43min, and 321nm for 43-60min. S4 High Performance Liquid Chromatography 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 a total of 13 peaks, and identify the characteristic peaks of 6 components. S5 Determination of predicted retention time: Using multiple chromatographic columns of different brands and types, chromatograms of the mixed reference solution and the test solution were collected according to the chromatographic conditions in step S3 to obtain the standard retention times of 13 common peaks; using p-hydroxycinnamic acid and angelica dahurica as double standard references, the predicted retention times of the remaining peaks were obtained by double standard linear correction method.

2. The method for establishing the high-performance liquid chromatography characteristic spectrum of the expectorant formula 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 expectorant formula or freeze-dried powder of traditional decoction, grind it into a fine powder, take 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of methanol, sonicate for 30min, cool it, weigh it, make up the weight loss with methanol, shake it well, filter it through a 0.45μm microporous membrane, and take the filtrate to obtain the test solution.

3. The method for establishing the high-performance liquid chromatography characteristic spectrum of the expectorant formula according to claim 1, characterized in that, The preparation method of the reference solutions is as follows: Accurately weigh appropriate amounts of platycodon saponin D, platycodon saponin E, imperatorin, imperatorin B, p-hydroxycinnamic acid, and (+)-cantharis resin phenol-9′-O-glucoside reference standards, and dissolve them in methanol to prepare solutions with concentrations of 100.400, 103.360, 110.491, 119.290, 288.400, and 114.050 μg·mL, respectively. -1 A mixed reference solution.

4. The method for establishing the high-performance liquid chromatography characteristic spectrum of the expectorant formula according to claim 1, characterized in that, In step S5, the specific method for obtaining the predicted retention time using the double-standard linear correction method is as follows: the actual retention time of the chromatographic peaks of the double-standard reference standards p-hydroxycinnamic acid and angelica dahurica is used as the ordinate and the standard retention time is used as the abscissa to obtain the linear fitting equation; the standard retention time of the remaining 11 characteristic peaks is substituted into the equation to obtain the corresponding predicted retention time; wherein, the standard retention time is the average value of the retention time of the test sample collected from multiple chromatographic columns.

5. The method for establishing the high-performance liquid chromatography characteristic spectrum of the expectorant formula 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 high-performance liquid chromatography characteristic spectrum of the expectorant formula according to claim 1, characterized in that, The preparation method of the expectorant formula granules is as follows: accurately weigh 6.0000g, 2.5714g, 0.9000g, 0.5625g, and 1.2000g of the formula granules of Peucedanum praeruptorum, Platycodon grandiflorus, Zingiber officinale, Bambusa textilis, Bambusa textilis, and Pumice, respectively, and prepare the expectorant formula granules.

7. The method for establishing the high-performance liquid chromatography characteristic spectrum of the expectorant formula according to claim 1, characterized in that, The chromatographic column has a specification of 4.6×250mm and a diameter of 5μm.

8. The method for establishing the high-performance liquid chromatography characteristic spectrum of the expectorant formula according to claim 1, characterized in that, The preparation method of the lyophilized powder of the standard decoction for expectoration includes the following steps: A1 Weigh out the Tianzhuhuang slices, place them in a clay pot, add 12 times the amount of water and soak for 30 minutes; A2 Weigh out Platycodon grandiflorus, Peucedanum praeruptorum, Zingiber officinale, and pumice and place them in another clay pot. Add 12 times the amount of water and soak for 30 minutes. A3 is used to decoct the soaking liquid containing the medicinal materials in A1. After boiling over high heat, it is simmered over low heat for 10 minutes. Then, the soaking liquid containing the medicinal materials in A2 is added to the clay pot and simmered together. After boiling over high heat, it is simmered over low heat for 20 minutes. Then, it is passed through a 200-mesh sieve while hot. Add 10 times the amount of water to the dregs obtained in A3, bring to a boil over high heat, then simmer over low heat for 20 minutes. While still hot, pass the liquid through a 200-mesh sieve. Combine the filtrate obtained with the filtrate in A3, concentrate at 60°C, and then freeze-dry under vacuum to obtain the final product.

9. A method for determining the content of components in a phlegm-reducing formula, characterized in that, The method includes the following steps: performing liquid chromatography determination according to steps S1-S3 of the method described in any one of claims 1-8 to obtain a chromatogram, and calculating the content of each component, including platycodon saponin E, imperatorin, imperatorin B, p-hydroxycinnamic acid, and (+)-cantharis resin phenol-9′-O-glucoside, by peak area based on the external standard method.

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