Qingjin Huatan Granules Characteristic Spectrum Detection Method and Characteristic Spectrum

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a characteristic spectrum detection method of Qingjin Huatan granules and its characteristic spectrum. The characteristic spectrum detection method of Qingjin Huatan granules comprises the following steps: taking a platycodon saponin D reference substance to prepare a reference solution; taking Qingjin Huatan granules to prepare a test solution; respectively injecting the reference solution and the test solution into a liquid chromatograph according to the following chromatographic conditions, and measuring, respectively obtaining a reference solution chromatogram and a test solution chromatogram, and comparing the retention time to obtain a characteristic spectrum of Qingjin Huatan granules; mainly using HPLC-ELSD method for detection. The characteristic spectrum detection method of Qingjin Huatan granules provided by the present invention realizes the quality control of Platycodon grandiflorum, Anemarrhena asphodeloides and Fritillaria thunbergii at the same time, and has the characteristics of strong specificity, good repeatability and good stability, and the present invention also provides its characteristic spectrum.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a characteristic spectrum detection method of Qingjin Huatan granules and a characteristic spectrum thereof. Background Art

[0002] Qingjin Huatan Decoction comes from "Medical General Principles" by Ye Wenling in the Ming Dynasty. It has the effects of clearing away heat and detoxifying, clearing the liver and removing fire, relieving cough and reducing phlegm, relieving cough and relieving asthma, clearing the throat and relieving sore throat. It is composed of 11 medicinal ingredients including Scutellaria baicalensis, Gardenia jasminoides, Platycodon grandiflorum, Ophiopogon japonicus, Morus alba bark, Fritillaria thunbergii, Anemarrhena asphodeloides, Trichosanthes kirilowii, Citrus aurantium, Poria cocos, and Licorice. This prescription is the 56th prescription in the "Catalogue of Ancient Classic Prescriptions (First Batch)" released in April 2018.

[0003] The characteristic spectrum of traditional Chinese medicine is currently a widely accepted model for evaluating the quality of traditional Chinese medicine. Its application reflects the trend of comprehensive quality management of traditional Chinese medicine and conforms to the characteristics of overall characterization analysis of traditional Chinese medicine quality control. Therefore, the development of characteristic spectrum methods is particularly important for the quality control of Qingjin Huatan Decoction and its compound preparations.

[0004] Qingjin Huatan Decoction is composed of 11 medicinal materials with complex ingredients. The characteristic spectrum methods in the existing technology mostly use DAD detectors, which cannot achieve quality control of Platycodon grandiflorum, Fritillaria thunbergii, and Rhizoma Anemarrhenae. For example, in the literature "Study on the Material Benchmark Fingerprint and Multi-index Content Determination of the Classic Prescription Qingjin Huatan Decoction" published by authors Li Yingman, Zhang Linlin, Zhu Qiang, etc., it is disclosed that the material benchmark fingerprint of Qingjin Huatan Decoction was established by using a high-performance liquid chromatograph equipped with a DAD detector, and 23 common peaks were identified, among which peaks 1, 2, 3, 4, 6, 7, 8 (gardenia glycosides), 14, 15, 16, and 17 belong to Gardenia jasminoides; peak 5 is shared by Morus alba and Licorice; peak 9 (mangiferin) belongs to Rhizoma Anemarrhenae; peak 10 belongs to Licorice; peaks 11, 18 (baicalin), 19, 20, 21, 22, and 23 (wogonin) belong to Scutellaria baicalensis; peaks 12 and 13 (hesperidin) belong to Citrus aurantium. This method controlled six medicinal materials, namely Gardenia, Scutellaria, Licorice, Morus alba bark, Citrus aurantium, and Anemarrhena asphodeloides, but failed to characterize the quality of Platycodon, Fritillaria thunbergii, Trichosanthes fruit, Ophiopogon japonicus, and Poria, and only one characteristic peak of mangiferin was detected in Anemarrhena asphodeloides. The representative component of Anemarrhena asphodeloides, which is also the indicator component for content determination in the Chinese Pharmacopoeia, Anemarrhena saponin BⅡ, was not controlled.

[0005] The article "HPLC-ELSD determination of fritillary acid A and fritillary acid B in Jinbei Tankeqing granules (Li Yueying, Yang Guoliang, etc.)" published in Volume 11, Issue 6, 2010, 446, uses high performance liquid chromatography HPLC-ELSD to determine fritillary acid A and fritillary acid B, and uses acetonitrile-water-diethylamine mobile phase for detection. The mobile phase of this method contains 0.03% diethylamine, and the high concentration of diethylamine is easy to damage the chromatographic column and detector. In addition, isocratic elution is used to focus on the detection of fritillary acid A and fritillary acid B, and only the taste of Fritillaria thunbergii can be controlled. In addition, the preparation of the test solution of this method requires heating and reflux in a water bath at 80°C for 2 hours, which is complicated and time-consuming, and is not conducive to daily inspection. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art, provide a characteristic spectrum detection method for Qingjin Huatan granules, realize the quality control of Platycodon grandiflorum, Anemarrhena asphodeloides and Fritillaria thunbergii at the same time, and have the characteristics of strong specificity, good repeatability and good stability. The present invention also provides its characteristic spectrum.

[0007] The Qingjin Huatan granule characteristic spectrum detection method of the present invention comprises the following steps:

[0008] (1) Prepare a reference solution with platycodon saponin D reference substance;

[0009] (2) Prepare the test solution with Qingjin Huatan granules;

[0010] (3) Inject the reference solution, the test solution, and the blank excipient solution into a liquid chromatograph according to the following chromatographic conditions for measurement, obtain the chromatograms of the reference solution and the test solution, and compare the retention times to obtain the characteristic spectrum of Qingjin Huatan Granules;

[0011] The chromatographic conditions are as follows: the chromatographic column uses octadecylsilane bonded silica gel as a filler, uses an evaporative light scattering detector, uses acetonitrile as mobile phase A, uses 0.008%-0.012% triethylamine solution as mobile phase B, performs gradient elution, and has a flow rate of 0.9 mL / min-1.0 mL / min; the column temperature is 33° C.-37° C. The % of the triethylamine solution is the volume percentage.

[0012] The reference solution of step (1) uses an ethanol solution or a methanol solution to dissolve the Platycodon saponin D reference substance. Specifically, take the Platycodon saponin D reference substance, accurately weigh it, and add 60% ethanol to prepare a reference solution containing 150 μg per 1 mL.

[0013] Preparation of the test solution in step (2): Take Qingjin Huatan Granules, first soak them in ammonia water, then add chloroform-methanol or dichloromethane-methanol mixed solution, ultrasonically treat, filter, evaporate the solid to dryness, and use ethanol solution or methanol solution to make up the volume. Specifically, take Qingjin Huatan Granules, grind them, take 0.5g, accurately weigh, place in a stoppered conical flask, add 5mL of ammonia water to soak for 1h, accurately add chloroform-methanol or dichloromethane-methanol (volume ratio 4:1) ) mixed solution 20mL, seal, mix, ultrasonic or reflux treatment for 45min-75min, cool, filter, and rinse the conical flask and filter paper with a mixed solution of chloroform-methanol or dichloromethane-methanol (volume ratio 4:1), transfer all the solution to an evaporating dish, evaporate to dryness, add ethanol solution or methanol solution to dissolve the residue and transfer to a 5mL volumetric flask, dilute to the scale with ethanol solution or methanol solution, shake well, filter, and take the filtrate to obtain.

[0014] The gradient elution conditions of step (3) are:

[0015] From 0 to 20 min, the volume fraction of mobile phase A changes from 20% to 25%, and the volume fraction of mobile phase B changes from 80% to 75%;

[0016] 20-25min, the volume fraction of mobile phase A changes to 25%-25%, and the volume fraction of mobile phase B changes to 75%-75%;

[0017] From 25 to 43 min, the volume fraction of mobile phase A changed from 25% to 40%, and the volume fraction of mobile phase B changed from 75% to 60%;

[0018] From 43 to 55 min, the volume fraction of mobile phase A changed from 40% to 70%, and the volume fraction of mobile phase B changed from 60% to 30%;

[0019] From 55 to 75 min, the volume fraction of mobile phase A changed to 70%-70%, and the volume fraction of mobile phase B changed to 30%-30%.

[0020] The chromatographic conditions of step (3) also include: injection volume of 30 μL; system suitability requirement: theoretical plate number calculated based on the platycodon saponin D peak is not less than 3000.

[0021] The blank auxiliary material solution in step (3) is obtained by dissolving maltodextrin in ethanol solution or methanol solution. Specifically, 0.15 g of maltodextrin is dissolved in 60% ethanol and diluted to the scale, and then shaken to obtain the blank auxiliary material solution.

[0022] The ethanol solution or methanol solution in step (1), step (2) and step (3) is preferably 60% ethanol, 30% methanol, 60% vt methanol, 80% methanol, and more preferably 60% ethanol.

[0023] The chromatographic column used in step (3) was Thermo Acclaim™ 120 C18.

[0024] The evaporative light scattering detector model of step (3) is SEDEX 85 evaporative light detector.

[0025] A characteristic spectrum obtained by the characteristic spectrum detection method of Qingjin Huatan Granules: the test solution presents 6 characteristic peaks, with Peak 2 Platycodon saponin D peak as a reference, wherein Peak 1, Peak 4 and Peak 5 are derived from Anemarrhena asphodeloides medicinal material, Peak 2 and Peak 3 are derived from Platycodon grandiflorum medicinal material, and Peak 6 is derived from Fritillaria thunbergii medicinal material.

[0026] The 6 characteristic peaks were positioned and compared with the reference substances. Peak 1 was timosaponin BⅡ, peak 2 was platycodon saponin D, peak 5 was timosaponin AⅢ, and peak 6 was fritillarin A.

[0027] The characteristic spectrum detection method of Qingjin Huatan granules described in the present invention was verified by the following aspects:

[0028] 1. System suitability investigation: Take the reference solution, adopt the same chromatographic conditions as in the method, inject the sample 6 times continuously, record the chromatogram, and use the retention time and peak area RSD of platycodon saponin D peak as the investigation indicators.

[0029] 2. Specificity investigation: Take blank excipient solution, reference solution and test sample solution, use the same chromatographic conditions as in the method, record the chromatogram, and examine whether the test sample solution has chromatographic peaks at the reference peaks, and whether the blank excipient has interference at each characteristic peak.

[0030] 3. Repeatability investigation: Take the test sample solution and prepare 6 parallel copies. Use the same chromatographic conditions as in the method, inject each solution once, record the chromatogram, take the No. 2 Platycodon saponin D peak as the reference, calculate the relative retention time of peaks 1 and 3-6, and use the RSD of the relative retention time of each peak as the investigation indicator.

[0031] 4. Intermediate precision investigation: Replace the analyst, prepare 6 samples in parallel according to the preparation method of the test solution, replace another liquid chromatograph, adopt the same chromatographic conditions as in the method, inject each solution once, record the chromatogram, take the No. 2 Platycodon saponin D peak as the reference, calculate the relative retention time of peaks 1 and 3-6, and use the RSD of the relative retention time of each peak as the investigation indicator.

[0032] 5. Stability study of the test solution: Take the test solution and place it at room temperature for 0h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h. Use the same chromatographic conditions as in the method, record the chromatogram, take Peak 2, Platycodon saponin D, as the reference, calculate the relative retention time of Peaks 1 and 3-6, and use the RSD of the relative retention time of each peak as the study index.

[0033] 6. Durability investigation: Take the test sample solution, take different column temperatures, different flow rates, different triethylamine concentrations, and different chromatographic columns, use the same chromatographic conditions as in the method, record the chromatogram, use peak No. 2, Platycodon saponin D, as a reference, calculate the relative retention times of peaks 1 and 3-6, and use the RSD of the relative retention time of each peak as the investigation indicator.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The characteristic spectrum detection method of Qingjin Huatan Granules of the present invention adopts the HPLC-ELSD method to establish the characteristic spectrum detection method of Qingjin Huatan Granules, and a total of 6 characteristic peaks are calibrated, and 4 characteristic components, namely, platycodon saponin D, timosaponin BⅡ, timosaponin AⅢ, and fritillarin A, are identified.

[0036] (2) The method of the present invention has good specificity, high precision, good reproducibility, good durability, and good solution stability, and can more comprehensively and effectively control the quality of Qingjin Huatan Decoction and its compound preparations, thereby ensuring efficacy and medication safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the characteristic spectrum of the Qingjin Huatan Granules of Example 1.

[0038] Figure 2 This is the characteristic spectrum of Qingjin Huatan Granules obtained by two extraction methods, namely ultrasonic treatment and reflux extraction, in Example 2.

[0039] Figure 3 The characteristic spectra of the Qingjin Huatan granules in Example 3 with ultrasonic treatment times of 75 min, 1 h, and 45 min respectively.

[0040] Figure 4 The characteristic spectra of Qingjin Huatan Granules in Example 4 are respectively chloroform-methanol mixed solution and dichloromethane-methanol mixed solution.

[0041] Figure 5 The characteristic spectra of Qingjin Huatan Granules in Example 5 where the extraction solvents are 80% methanol, 60% methanol, 30% methanol, 80% ethanol, 60% ethanol, and 30% ethanol, respectively.

[0042] Figure 6 This is a comparison chart of system suitability in method validation.

[0043] Figure 7 This is a comparison chart of specificity in method validation.

[0044] Figure 8 This is a comparison chart of repeatability in method validation.

[0045] Fig. 9 This is a comparison chart of intermediate precision in method validation.

[0046] Fig.10 This is a stability comparison chart of the test solution in method validation.

[0047] Fig.11 This is a comparison chart of column temperature durability in method validation.

[0048] Fig.12 This is a comparison chart of flow rate durability in method validation.

[0049] Fig.13 This is a comparison chart of the durability of triethylamine concentration in mobile phase B during method validation.

[0050] Fig.14 This is a comparison chart of the durability of different chromatographic columns used in method validation. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific embodiments.

[0052] The following instrument models are used:

[0053] Liquid chromatograph: Thermo Ultimate 3000 high performance liquid chromatograph, Thermo Fisher Scientific Inc.;

[0054] Waters Arc high performance liquid chromatograph, Waters Company;

[0055] Evaporative light detector: SEDEX 85 evaporative light detector, SEDERE, France;

[0056] Weighing instrument: BCE951-10CN electronic balance, Beijing Sartorius Scientific Instrument Co., Ltd.;

[0057] AUW220D electronic balance, Shimadzu Corporation, Japan.

[0058] The following sources of medicines are used:

[0059] Qingjin Huatan Granules: Shandong Qidu Pharmaceutical Co., Ltd., batch number: 2404002;

[0060] Platycoside D: China Food and Drug Inspection Institute, batch number: 111851-202209, content: 97.3%;

[0061] Anemarrhena saponin BⅡ: China Food and Drug Administration, batch number: 111839-202308, content: 93.2%;

[0062] Anemarrhena saponin AⅢ: Chengdu Pusi Biotechnology Co., Ltd., batch number: PS011471, purity: 98.67%;

[0063] Fritillaria thunbergii: China Food and Drug Inspection Institute, batch number: 110750-201913, content: 98.4%;

[0064] Methanol: Chengdu Kelong Chemical Reagent Co., Ltd., batch number: 2024041901;

[0065] Acetonitrile: Chengdu Kelong Chemical Reagent Co., Ltd., batch number: 2024022902;

[0066] Ethanol: Chengdu Kelong Chemical Reagent Co., Ltd., batch number: 2024011501;

[0067] Ammonia water: Chengdu Kelong Chemical Reagent Co., Ltd., batch number: 2023103001;

[0068] Chloroform: Chengdu Jinshan Chemical Reagent Co., Ltd., batch number: 20230918;

[0069] Triethylamine: DIKMA, batch number: 8184474;

[0070] Maltodextrin: Xi’an Tianzheng Pharmaceutical Excipients Co., Ltd., batch number: 2023050151.

[0071] The % in the present invention refers to volume percentage.

[0072] Example 1

[0073] The Qingjin Huatan granule characteristic spectrum detection method comprises the following steps:

[0074] (1) Preparation of reference solution: Take Platycodon saponin D reference substance, weigh it accurately, and add 60% ethanol to make a reference solution containing 150 μg per 1 mL.

[0075] (2) Preparation of test solution: Take Qingjin Huatan Granules and soak them with ammonia water, then add chloroform-methanol mixed solution, ultrasonically treat, filter, evaporate the solid to dryness, and make up to volume with 60% ethanol. Specifically: Take Qingjin Huatan Granules, grind them into powder, take 0.5 g, accurately weigh, place in a stoppered conical flask, soak with 5 mL of ammonia water for 1 h, accurately add 20 mL of chloroform-methanol (volume ratio 4:1) mixed solution, stopper, mix well, ultrasonically treat for 1 h, cool, filter, and rinse the conical flask and filter paper with chloroform-methanol (volume ratio 4:1) mixed solution. Transfer all the solution to an evaporating dish, evaporate to dryness, add 60% ethanol to dissolve the residue and transfer to a 5 mL volumetric flask, dilute to the mark with 60% ethanol, shake well, filter, and take the filtrate.

[0076] (3) Prepare blank auxiliary material solution: Take 0.15 g of maltodextrin, add 60% ethanol to dissolve and dilute to scale, shake well.

[0077] (4) The reference solution, the test solution, and the blank excipient solution were respectively injected into a Thermo Ultimate 3000 high performance liquid chromatograph according to the following chromatographic conditions: a Thermo Acclaim™ 120 C18 chromatographic column, a SEDEX 85 evaporative light detector, acetonitrile as mobile phase A, 0.01% triethylamine solution as mobile phase B, gradient elution, the gradient elution conditions were as shown in Table 1, the flow rate was 1.0 mL / min; the column temperature was 35°C, and the injection volume was 30 μL; the system suitability requirement was: the number of theoretical plates calculated based on the platycodon saponin D peak was not less than 3000.

[0078] Table 1 Gradient elution

[0079]

[0080] The determination is performed to obtain the chromatogram of the reference solution and the chromatogram of the test solution respectively;

[0081] The retention time of the chromatogram of the test solution is compared with the retention time of the chromatogram of the reference solution to obtain the characteristic spectrum of the Qingjin Huatan granules, such as Figure 1 As shown, there are 6 characteristic peaks in the chromatogram of the test solution, among which the peak corresponding to the Platycodon saponin D reference peak is the S peak. The relative retention time (characteristic peak / S peak) of each characteristic peak and the S peak (peak 2) is calculated, which are 0.68 (peak 1), 1.03 (peak 3), 1.18 (peak 4), 1.65 (peak 5), and 1.92 (peak 6), respectively. The relative retention times are all within ±10%, which meets the specified values.

[0082] Example 2

[0083] The detection steps in this embodiment are the same as those in embodiment 1, except that "ultrasonic treatment for 1 h" in the preparation of the test solution in step (2) of embodiment 1 is replaced by "reflux extraction for 1 h", and the other steps are the same. The characteristic spectra of the two extraction methods are as follows: Figure 2 (From top to bottom, the extraction methods are ultrasound and reflux respectively), as shown in Figure 2 It can be seen that the extraction effects of heating reflux and ultrasonic extraction are basically the same. In order to facilitate experimental operation, ultrasonic extraction is the preferred extraction method.

[0084] Example 3

[0085] The detection steps in this embodiment are the same as those in embodiment 1, except that "ultrasonic treatment for 1 h" in the preparation of the test solution in step (2) of embodiment 1 is replaced by "ultrasonic treatment for 45 min" and "ultrasonic treatment for 75 min", respectively. The other steps are the same, and characteristic spectra of three ultrasonic time are obtained, such as Figure 3 As shown by Figure 3 (From top to bottom, they are 75 min, 1 h, and 45 min, respectively) It can be seen that there is no obvious difference in the peak shape and separation effect of each characteristic peak at different ultrasonic extraction times. Considering the durability of the method, the preferred ultrasonic time is 1 h.

[0086] Example 4

[0087] The detection steps in this embodiment are the same as those in embodiment 1, except that the "mixed solution of chloroform-methanol (volume ratio 4:1)" in the preparation of the test solution in step (2) of embodiment 1 is replaced by "mixed solution of dichloromethane-methanol (volume ratio 4:1)", and the other steps are the same. The characteristic spectra of the two extraction solvents are obtained, as shown in FIG. Figure 4 As shown by Figure 4 (From top to bottom, chloroform-methanol mixed solution is used as the extraction solvent, and dichloromethane-methanol mixed solution is used as the extraction solvent) It can be seen that when the chloroform-methanol (volume ratio 4:1) mixed solution is used as the extraction solvent, the response values ​​of each characteristic peak are relatively high, and the chloroform-methanol (volume ratio 4:1) mixed solution is preferably used as the extraction solvent.

[0088] Example 5

[0089] The detection steps in this embodiment are the same as those in embodiment 1, except that the "60% ethanol" in the preparation of the test solution in step (2) of embodiment 1 is replaced by "30% ethanol", "80% ethanol", "30% methanol", "60% methanol" and "80% methanol", respectively. The other steps are the same. The characteristic spectra of the six extraction solvents are obtained, as shown in FIG. Figure 5 As shown, Figure 5From top to bottom, they are 80% methanol, 60% methanol, 30% methanol, 80% ethanol, 60% ethanol, and 30% ethanol. The results of different extraction solvents are shown in Table 2. Figure 5 It can be seen that when 30% ethanol and 80% ethanol are used as extraction solvents, the peak shape is poor and there are few peaks. When 30% methanol, 60% methanol, 80% methanol and 60% ethanol are used as extraction solvents, the response values ​​of each characteristic peak are higher. Among them, when chloroform + 60% ethanol is used as the extraction solvent, the content of Fritillaria cirrhosae and the ratio of the Fritillaria cirrhosae peak area to the maximum peak area are greater than those of the other extraction solvents. 60% ethanol is preferably used as the extraction solvent.

[0090] Table 2 Results of investigation on different extraction solvents

[0091]

[0092] Methodological validation

[0093] 2.1 System Applicability

[0094] Take the reference solution of Example 1, repeat the injection 6 times according to the chromatographic conditions of Example 1, record the chromatogram, calculate the RSD of the retention time and peak area of ​​platycodon saponin D, and record its theoretical plate number. Figure 6 (From bottom to top are the chromatograms of parallel samples corresponding to system suitability solutions 1-6). The results are shown in Table 3. The RSD of the peak area of ​​platycoside D was 1.6%, less than 4.0%, and the RSD of the retention time was 0.02%, less than 1.0%. The theoretical plate number of the platycoside D peak was greater than 3000, and the system applicability was good.

[0095] Table 3 System suitability investigation results

[0096]

[0097] 2.2 Exclusivity

[0098] Take the blank auxiliary material solution, reference solution and test solution of Example 1, inject them into the liquid chromatograph according to the chromatographic conditions of Example 1, and record the chromatogram. The comparison chart of blank auxiliary material solution, reference solution and test solution is as follows: Figure 7 As shown, Figure 7 From top to bottom in the figure are the chromatograms of the test solution, reference solution, and blank auxiliary material solution. The results show that there are chromatographic peaks in the test solution at the corresponding positions of the reference peaks, and there are no chromatographic peaks in the blank auxiliary material at the peaks of each characteristic peak. The blank auxiliary material has no interference, and the method has good specificity.

[0099] 2.3 Repeatability

[0100] According to the preparation method of the test solution in Example 1, 6 test solutions were prepared in parallel. According to the chromatographic conditions of Example 1, each solution was injected once, and the chromatogram was recorded. The platycodon saponin D peak was taken as the S peak, and the RSD of the relative retention time (RRT) of each characteristic peak and the S peak was calculated. The repeatability comparison diagram is shown in FIG. Figure 8 (From bottom to top are the chromatograms corresponding to repeatable solutions 1-6), and the results are shown in Table 4. The RSD of the relative retention time of each characteristic peak is between 0.0% and 1.0%, all less than 5.0%, and the repeatability is good.

[0101] Table 4 Repeatability test results

[0102]

[0103] 2.4 Intermediate precision

[0104] Different analysts used different liquid chromatographs to prepare 6 test solutions in parallel according to the preparation method of the test solution in Example 1. According to the chromatographic conditions of Example 1, each solution was injected once, and the chromatogram was recorded. The platycodon saponin D peak was taken as the S peak, and the RSD of the relative retention time of each characteristic peak and the S peak was calculated. The comparison of the intermediate precision of the 6 injections is shown in the figure below. Fig. 9 The results are shown in Table 5. In Table 5, the data of Personnel A refer to the test results of "2.3 Repeatability", and Personnel B refers to the intermediate precision test results. The results show that the RSD values ​​of the relative retention time of each characteristic peak and the S peak in the 6 results determined by different analysts at different times on different instruments are within the range of 0.05%-1.0%, and the RSD values ​​of the relative retention time of each characteristic peak and the S peak in the 12 results determined by different analysts are within the range of 0.3%-1.0%. The relative retention time of the 6 results determined by different personnel and the 12 results determined by 2 people are all less than 5.0%, indicating that the intermediate precision of this method is good.

[0105] Table 5 Intermediate precision and precision inspection results

[0106]

[0107] 2.5 Stability of test solution

[0108] Take the test solution of Example 1, and measure it according to the chromatographic conditions of Example 1 at 0h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h respectively, record the relative retention time of each characteristic peak and the S peak, and investigate the stability of the solution. The comparison chart is as follows Fig.10As shown (from bottom to top are 0h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h chromatograms), the results are shown in Table 6. The RSD of the relative retention time of each characteristic peak is between 0.04% and 0.2%, all less than 4.0%, and the test solution has good stability.

[0109] Table 6 Results of stability study of test solution

[0110]

[0111] 2.6 Durability

[0112] The reference solution and the test solution of Example 1 were taken to examine the column temperature, flow rate, triethylamine concentration in mobile phase B in the chromatographic conditions of Example 1 within a certain range and the durability between different chromatographic columns. The parameters of the durable chromatographic condition variation are shown in Table 7, and the experimental results are shown in Tables 8 to 11. The corresponding chromatograms are shown in Tables 1 to 2. Figure 11-14 shown. Fig.11 This is a comparison chart of column temperature durability (from top to bottom: 35°C, 33°C, 37°C). Fig.12 This is a flow rate durability comparison chart (from top to bottom: 1.0ml / min, 1.1ml / min, 0.9ml / min), Fig.13 This is a comparison chart of the durability of triethylamine concentration in mobile phase B (from top to bottom: 0.01% triethylamine, 0.008% triethylamine, 0.012% triethylamine). Fig.14 This is a comparison chart of the durability of different chromatographic columns (from top to bottom: chromatographic column 1, chromatographic column 3, chromatographic column 2).

[0113] Table 7 Durability chromatographic condition variation parameters

[0114]

[0115] Table 8 Column temperature durability test results

[0116]

[0117] Table 9 Flow rate durability test results

[0118]

[0119] Table 10 Durability test results of triethylamine concentration in mobile phase B

[0120]

[0121] Table 11 Column durability test results

[0122]

[0123] Depend on Figure 11-Figure 14 It can be seen that under different column temperatures, flow rates, and triethylamine concentrations in mobile phase B, the RSD of the relative retention time of each characteristic peak and the S peak is less than 5.0%, indicating that the durability of this method is good. Peak 5 was not detected in the chromatogram of the sample in column 3, and column 3 (Waters XSelect HSS T3 C18, 4.6mm×250mm, 5μm (SN: 01933317912440)) is not suitable for this method. Between columns of the same model but different batches, the RAD of the relative retention time of each characteristic peak and the S peak is less than 5.0%, indicating that the durability of the chromatographic column in this method is good.

Claims

1. A method for detecting characteristic patterns of Qingjin Huatan granules, characterized in that: The following steps are involved: (1) Dissolve the platycodon saponin D reference substance in ethanol solution or methanol solution to prepare a reference solution; (2) Preparation of test solution: Take Qingjin Huatan granules and soak them in ammonia water, then add a mixed solution of chloroform-methanol or dichloromethane-methanol, treat with ultrasound or reflux for 45 min-75 min, filter, evaporate the liquid, and make up to volume with ethanol solution or methanol solution; (3) Injecting the reference solution, the test solution, and the blank auxiliary material solution into a liquid chromatograph according to the following chromatographic conditions for measurement, obtaining a chromatogram of the reference solution and a chromatogram of the test solution, respectively, and comparing the retention times to obtain a characteristic spectrum of the Qingjin Huatan Granules; the blank auxiliary material solution is obtained by dissolving maltodextrin in an ethanol solution or a methanol solution; The test solution presents 6 characteristic peaks, with peak No. 2 platycodon saponin D peak as a reference, wherein peak No. 1, peak No. 4 and peak No. 5 are derived from the medicinal material of Anemarrhena, peak No. 2 and peak No. 3 are derived from the medicinal material of Platycodon, and peak No. 6 is derived from the medicinal material of Fritillaria thunbergii; The six characteristic peaks were compared with timosaponin BⅡ, platycodon saponin D, timosaponin AⅢ, and fritillarin A reference substances, and it was found that peak 1 was timosaponin BⅡ, peak 2 was platycodon saponin D, peak 5 was timosaponin AⅢ, and peak 6 was fritillarin A. The chromatographic conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel, which is Thermo Acclaim™ 120 C18; an evaporative light scattering detector is used, acetonitrile is used as mobile phase A, 0.008%-0.012% triethylamine solution is used as mobile phase B, gradient elution is performed, and the flow rate is 0.9mL / min-1.0mL / min; the column temperature is 33°C-37°C, and the gradient elution conditions are: From 0 to 20 min, the volume fraction of mobile phase A changes from 20% to 25%, and the volume fraction of mobile phase B changes from 80% to 75%; 20-25min, the volume fraction of mobile phase A changes to 25%-25%, and the volume fraction of mobile phase B changes to 75%-75%; From 25 to 43 min, the volume fraction of mobile phase A changed from 25% to 40%, and the volume fraction of mobile phase B changed from 75% to 60%; From 43 to 55 min, the volume fraction of mobile phase A changed from 40% to 70%, and the volume fraction of mobile phase B changed from 60% to 30%; From 55 to 75 min, the volume fraction of mobile phase A changed to 70%-70%, and the volume fraction of mobile phase B changed to 30%-30%.

2. The Qingjin Huatan granule characteristic spectrum detection method according to claim 1, characterized in that: The chromatographic conditions of step (3) also include: the theoretical plate number required for system suitability is not less than 3000 calculated based on the platycodon saponin D peak.

3. The Qingjin Huatan granule characteristic spectrum detection method according to claim 1, characterized in that: The evaporative light scattering detector model of step (3) is SEDEX 85 evaporative light detector.

Images