Construction method and detection method of fingerprint spectrum of preparation for nourishing yin and moistening lung
By constructing a fingerprint of the yin-heating lung preparation and using liquid chromatography technology to measure the preparation, the problem of difficulty in quality control of traditional Chinese medicine preparations is solved, a more comprehensive and effective evaluation of the quality of the preparation is achieved, and market competitiveness is improved.
Patent Information
- Application Number
- CN202510363880.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-26
- Publication Date
- 2025-04-25
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Due to its complex and multi-component nature, traditional Chinese medicine preparations are difficult to reflect their quality through a single indicator, resulting in difficulty in quality control and affecting market competitiveness.
By constructing a fingerprint of the yin-heating lung preparation, the preparation is measured using liquid chromatography technology, combining gradient elution procedures and specific chromatographic conditions, the characteristic common peaks are matched to establish a corresponding fingerprint map.
It has achieved a more comprehensive and effective evaluation of the quality of the yin-heating lung preparation, improved the accuracy and comprehensiveness of quality control of traditional Chinese medicine, and enhanced market competitiveness.
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Figure CN119881173A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing and detecting a fingerprint spectrum of a Yin-nourishing and lung-moistening preparation. Background Art
[0002] Lily Solid Gold Preparation is a traditional Chinese medicine preparation made from ten medicinal materials: lily, rehmannia, rehmannia root, ophiopogon, angelica, Scrophularia, Fritillaria cirrhosa, Platycodon, licorice and white peony root. It has the effects of nourishing yin and moistening the lungs, resolving phlegm and relieving cough. It can be used for lung and kidney yin deficiency, dry cough with little phlegm, dry throat and sore throat.
[0003] As a complex system with multiple components, it is difficult to reflect the true quality of Chinese medicine preparations through a single indicator. Fingerprint is a breakthrough in the modernization of Chinese medicine and one of the key technologies. In order to more comprehensively control product quality and improve market competitiveness, it is necessary to establish the fingerprint of Lily Solid Gold preparations to evaluate the quality of Chinese medicine more accurately and comprehensively. Summary of the invention
[0004] Based on this, the first aspect of the present invention provides a method for constructing a fingerprint of a nourishing yin and moistening lung preparation, and its technical solution is as follows:
[0005] A method for constructing a fingerprint spectrum of a Yin-nourishing and lung-moistening preparation comprises the following steps:
[0006] Taking a nourishing yin and moistening lung preparation, adding an extraction solvent for extraction treatment, collecting the extract, and preparing a test solution, wherein the raw materials for preparing the nourishing yin and moistening lung preparation include lily, rehmannia root, prepared rehmannia root, ophiopogon japonicus, angelica sinensis, Scrophularia ningpoensis, Fritillaria cirrhosa, Platycodon grandiflorum, licorice and white peony root;
[0007] Taking the test solution for liquid chromatography determination;
[0008] The chromatographic conditions of the liquid chromatography assay include:
[0009] The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A includes aqueous phosphoric acid solution, and the mobile phase B includes acetonitrile, and the elution is performed by gradient elution;
[0010] The gradient elution includes the following procedures:
[0011] From 0min to 10min, the volume fraction of the mobile phase A decreased from 98% to 87%, and the volume fraction of the mobile phase B increased from 2% to 13%;
[0012] From 10 min to 15 min, the volume fraction of the mobile phase A decreased from 87% to 83%, and the volume fraction of the mobile phase B increased from 13% to 18%;
[0013] From 15min to 21min, the volume fraction of the mobile phase A decreased from 83% to 80%, and the volume fraction of the mobile phase B increased from 18% to 20%;
[0014] From 21min to 26min, the volume fraction of the mobile phase A decreased from 80% to 77%, and the volume fraction of the mobile phase B increased from 20% to 23%;
[0015] From 26 min to 35 min, the volume fraction of the mobile phase A decreased from 77% to 65%, and the volume fraction of the mobile phase B increased from 23% to 35%;
[0016] From 35 min to 45 min, the volume fraction of the mobile phase A is reduced from 65% to 50%, and the volume fraction of the mobile phase B is increased from 35% to 50%;
[0017] From 45 min to 55 min, the volume fraction of the mobile phase A decreased from 50% to 35%, and the volume fraction of the mobile phase B increased from 50% to 65%;
[0018] From 55 min to 60 min, the volume fraction of the mobile phase A decreased from 35% to 30%, and the volume fraction of the mobile phase B increased from 65% to 70%;
[0019] From 60 min to 65 min, the volume fraction of the mobile phase A was maintained at 30%, and the volume fraction of the mobile phase B was maintained at 70%.
[0020] In some embodiments, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%. For example, the mass fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05%, 0.08%, 0.1%, 0.15%, or 0.2%.
[0021] In some embodiments, the stationary phase includes octadecylsilane bonded silica gel. It can be understood that the stationary phase is the filler of the chromatographic column. In the above embodiment, the filler of the chromatographic column includes octadecylsilane bonded silica gel.
[0022] In some embodiments, the chromatographic conditions of the liquid chromatography determination further include: a detection wavelength of 210 nm to 250 nm. For example, the detection wavelength is 210 nm, 220 nm, 230 nm, 240 nm, and 250 nm.
[0023] In some embodiments, the chromatographic conditions of the liquid chromatography determination further include: a column temperature of 22° C. to 28° C. For example, the column temperature is 22° C., 24° C., 26° C., or 28° C.
[0024] In some embodiments, the chromatographic conditions of the liquid chromatography determination further include: a flow rate of 0.5 mL / min to 1.5 mL / min. For example, the flow rate is 0.5 mL / min, 0.8 mL / min, 1.0 mL / min, 1.2 mL / min, 1.5 mL / min.
[0025] In some embodiments, the chromatographic conditions of the liquid chromatography determination further include: the injection volume is 2 μL to 8 μL. For example, the injection volume is 2 μL, 5 μL, or 8 μL.
[0026] In some embodiments, the method for extracting the Yin-nourishing and lung-moistening preparation is ultrasonic extraction. Optionally, the ultrasonic extraction time is 30 min to 60 min. For example, the ultrasonic extraction time is 30 min, 40 min, 50 min, or 60 min.
[0027] In some embodiments, the extraction solvent includes ethanol. Optionally, 20 mL to 30 mL of the extraction solvent is added to every 2 g of the Yangyin Runfei preparation. For example, 20 mL, 25 mL, and 30 mL of the extraction solvent are added to every 2 g of the Yangyin Runfei preparation. Optionally, the extraction solvent is an ethanol-water solution, and the volume fraction of ethanol in the ethanol-water solution is 20% to 80%. For example, the volume fraction of ethanol in the ethanol-water solution is 20%, 30%, 40%, 50%, 60%, 70%, and 80%.
[0028] Wherein, the raw materials for preparing the nourishing yin and moistening lung preparation include lily, rehmannia root, prepared rehmannia root, ophiopogon japonicus, angelica root, Scrophularia ningpoensis, Fritillaria cirrhosa, Platycodon grandiflorum, Glycyrrhiza uralensis and white peony root. Optionally, the nourishing yin and moistening lung preparation is a lily solid gold preparation. Optionally, the lily solid gold preparation includes lily solid gold tablets. Lily solid gold tablets are a dosage form of lily solid gold preparation, which is a second-level protected variety of traditional Chinese medicine. The product is orally administered, easy to use, and has a definite therapeutic effect.
[0029] The second aspect of the present application provides a method for detecting a Yin-nourishing and lung-moistening preparation, and its technical solution is as follows:
[0030] A method for detecting a Yin-nourishing and lung-moistening preparation comprises the following steps:
[0031] Take the to-be-tested nourishing yin and moistening lung preparation, add an extraction solvent for extraction treatment, collect the extract, and prepare a to-be-tested sample solution, wherein the raw materials for preparing the to-be-tested nourishing yin and moistening lung preparation include lily, rehmannia root, prepared rehmannia root, ophiopogon japonicus, angelica sinensis, scrophularia ningpoensis, fritillaria cirrhosa, platycodon grandiflorum, licorice, and white peony root;
[0032] The sample solution to be tested is subjected to liquid chromatography according to the chromatographic conditions as described above, and the obtained chromatogram is matched with the fingerprint spectrum of the Yangyin Runfei preparation as described above to determine whether the Yangyin Runfei preparation to be tested is qualified.
[0033] In some embodiments, the method for extracting the tested Yangyin Runfei preparation is ultrasonic extraction. Optionally, the ultrasonic extraction time is 30 min to 60 min. For example, the ultrasonic extraction time is 30 min, 40 min, 50 min, or 60 min.
[0034] In some embodiments, the extraction solvent includes ethanol. Optionally, 20 mL to 30 mL of the extraction solvent is added to every 2 g of the Yangyin Runfei preparation to be tested. For example, 20 mL, 25 mL, and 30 mL of the extraction solvent are added to every 2 g of the Yangyin Runfei preparation to be tested. Optionally, the extraction solvent is an ethanol-water solution, and the volume fraction of ethanol in the ethanol-water solution is 20% to 80%. For example, the volume fraction of ethanol in the ethanol-water solution is 20%, 30%, 40%, 50%, 60%, 70%, and 80%.
[0035] Wherein, the raw materials for preparing the yin nourishing and lung moistening preparation to be tested include lily, rehmannia root, prepared rehmannia root, ophiopogon japonicus, angelica root, Scrophularia ningpoensis, Fritillaria cirrhosa, Platycodon grandiflorum, Glycyrrhiza uralensis and white peony root. Optionally, the yin nourishing and lung moistening preparation to be tested is a lily solid gold preparation to be tested. Optionally, the lily solid gold preparation to be tested includes a lily solid gold tablet to be tested.
[0036] Compared with the traditional solution, the present invention has the following beneficial effects:
[0037] This application establishes a method for constructing a fingerprint spectrum of a nourishing yin and moistening lung preparation. By preparing a test solution and screening chromatographic conditions, 20 common peaks are matched, and 4 of them are identified, namely, paeoniflorin (peak 6), liquiritin (peak 8), ferulic acid (peak 9) and ammonium glycyrrhizinate (peak 15). The constructed fingerprint spectrum can more comprehensively and effectively understand the quality of the nourishing yin and moistening lung preparation, which is beneficial to the quality control of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.
[0039] Figure 1 The superimposed fingerprints of 11 batches of Lily Solid Gold Tablets and the reference fingerprint R;
[0040] Figure 2 is the chromatogram of the mixed reference solution;
[0041] Figure 3 The chromatogram of Baihe Gujin Tablet and the chromatogram of each reference medicinal material solution;
[0042] Figure 4 The first is the chromatogram of Baihe Gujin Tablet and the second is the chromatogram of each reference medicinal material solution;
[0043] Figure 5 Examine the chromatogram for specificity;
[0044] Figure 6 To investigate the chromatogram for the intermediate precision of liquid phase instruments;
[0045] Figure 7 This is a chromatogram to investigate the intermediate precision of the chromatographic column. DETAILED DESCRIPTION
[0046] The present invention will be further described in detail below in conjunction with specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.
[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.
[0048] 1. Instruments and Materials
[0049] 1.1 Instrument
[0050] High performance liquid chromatography Thermo Fisher U3000 (equipment number: J-20-C-093, J-20-C-008).
[0051] 1.2 Materials
[0052] The batch number information and sources of the 11 batches of Lily Gold Tablets are shown in Table 1.
[0053] The batch information and sources of the paeoniflorin reference substance, liquiritin reference substance, ferulic acid reference substance and ammonium glycyrrhizinate reference substance are shown in Table 1.
[0054] For the batch information and sources of the reference medicinal materials of Licorice, Angelica, White Peony, Rehmannia, Rehmannia, Scrophularia, Ophiopogon, Fritillaria, Lilium, and Platycodon, please refer to Table 1.
[0055] Table 1
[0056]
[0057] 2. Methods and Results
[0058] 2.1 Chromatographic conditions
[0059] The chromatographic column was ODS C18 (4.6×250mm, 5μm), numbered JZ-SY-C002, with 0.1% volume fraction of phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution was performed according to the elution program shown in Table 2. The detection wavelength was 230nm, the column temperature was 25℃, and the flow rate was 1mL / min.
[0060] Table 2
[0061]
[0062] 2.2 Preparation of test solution
[0063] Take 2 g of each of the 11 batches of Lily Gold Tablets, accurately weigh them, place them in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 min. Cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain batches of test solution S1-S11.
[0064] 2.3 Preparation of mixed reference solution and reference medicinal material solution
[0065] Take appropriate amounts of paeoniflorin reference substance, liquiritin reference substance and ammonium glycyrrhizinate reference substance respectively, weigh them accurately, add methanol to make a mixed solution containing 100 μg of paeoniflorin, 100 μg of liquiritin and 100 μg of ammonium glycyrrhizinate per 1 mL to obtain a mixed reference substance solution.
[0066] Take 0.23 g of licorice control medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the licorice control medicinal material solution.
[0067] Take 0.23 g of Angelica sinensis reference medicinal material and accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate to obtain the Angelica sinensis reference medicinal material solution.
[0068] Take 0.23 g of white peony root control medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% ethanol aqueous solution by volume, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% ethanol aqueous solution by volume, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the white peony root control medicinal material solution.
[0069] Take 0.46 g of Rehmannia glutinosa control medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the Rehmannia glutinosa control medicinal material solution.
[0070] Take 0.69 g of Rehmannia glutinosa control medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the Rehmannia glutinosa control medicinal material solution.
[0071] Take 0.18 g of Scrophularia ningpoensis reference medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the Scrophularia ningpoensis reference medicinal material solution.
[0072] Take 0.34 g of Ophiopogon japonicus reference medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the Ophiopogon japonicus reference medicinal material solution.
[0073] Take 0.23 g of Fritillaria cirrhosa control medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the Fritillaria cirrhosa control medicinal material solution.
[0074] Take 0.23 g of lily reference medicinal material, weigh it accurately, put it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate to obtain the lily reference medicinal material solution.
[0075] Take 0.18 g of Platycodon grandiflorum reference medicinal material, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 25 mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45 minutes, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45 μm microporous filter membrane, collect the filtrate, and obtain the Platycodon grandiflorum reference medicinal material solution.
[0076] 2.4 Determination method
[0077] Accurately pipette 5 μL of the test solution under "2.2", the mixed reference solution under "2.3" and each reference medicinal material solution under "2.3", inject into high performance liquid chromatograph (equipment number: J-20-C-093), measure according to the chromatographic conditions under "2.1", record the chromatogram, and obtain the chromatograms of 11 batches of Lily Solid Gold Tablets, the chromatograms of the mixed reference solution and the chromatograms of each reference medicinal material solution.
[0078] 2.5 Establishment of fingerprint and identification of common peaks
[0079] The chromatograms of 11 batches of Baihe Gujin Tablets were imported into the "Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition)", and the chromatogram of the Baihe Gujin Tablets of batch S1 was used as the reference spectrum. The full spectrum peaks were automatically matched to generate superimposed fingerprints, and the average method was used to generate the reference fingerprint R, see Figure 1 The chromatogram of the mixed reference solution is shown in Figure 2 The common peaks were identified by comparing the chromatogram with that of the mixed reference solution.
[0080] The results showed that 20 common peaks were matched in the chromatograms of 11 batches of Lily Solid Gold Tablets. By comparing with the chromatogram of the mixed reference solution, 4 common peaks were identified, namely paeoniflorin (peak 6), liquiritin (peak 8), ferulic acid (peak 9) and ammonium glycyrrhizinate (peak 15).
[0081] 2.6 Similarity analysis of fingerprints
[0082] By comparing the chromatograms, 20 characteristic peaks with good separation and stable peaks in each batch of fingerprints were determined. The 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" was used to generate the reference fingerprint R of the 20 characteristic peaks to perform similarity analysis on the chromatograms of each batch. The similarity evaluation results are shown in Table 3.
[0083] Table 3
[0084]
[0085] The results showed that the similarity of the lowest batch was 0.973, and the others were basically above 0.99. This shows that the similarity of each batch of Lily Solid Gold Tablets is high, and the generated control fingerprint R can be used as an overall similarity evaluation of the material basis of Lily Solid Gold Tablets.
[0086] 2.7 Attribution of each characteristic peak to a single herbal medicine
[0087] Compare the chromatogram of the S5 batch of Lily Solid Gold Tablets with the chromatograms of the control herbal medicine solutions, see Figure 3 and Figure 4 , Figure 3 Among them, A is the chromatogram of the reference medicinal material solution of Licorice; B is the chromatogram of the reference medicinal material solution of Angelica sinensis; C is the chromatogram of the reference medicinal material solution of White Peony; D is the chromatogram of the reference medicinal material solution of Rehmannia glutinosa; E is the chromatogram of the reference medicinal material solution of Rehmannia glutinosa; Figure 4 In the figure, A is the chromatogram of the reference medicinal solution of Scrophularia; B is the chromatogram of the reference medicinal solution of Ophiopogon; C is the chromatogram of the reference medicinal solution of Fritillaria; D is the chromatogram of the reference medicinal solution of Lilium; E is the chromatogram of the reference medicinal solution of Platycodon; F is the chromatogram of Lily Solid Gold Tablets of batch S5.
[0088] The medicinal material identities of the characteristic peaks in the chromatogram of Baihe Gujin Tablets are shown in Table 4.
[0089] Table 4
[0090]
[0091] 3 Methodological validation
[0092] 3.1 Exclusivity Investigation
[0093] Accurately pipette 5 μL of the Lily Solid Gold Tablets test solution of batch S1 under "2.2", the mixed reference solution under "2.3" and the blank solvent, inject them into the HPLC, and measure according to the chromatographic conditions under "2.1" to check whether there is interference with the extraction solvent. The results are shown in Figure 5 , where A is the chromatogram of the test solution of Lily Solid Gold Tablets from batch S1; B is the chromatogram of the mixed reference solution; and C is the chromatogram of the blank solvent.
[0094] The results showed that the extraction solvent had no interference.
[0095] 3.2 Repeatability study
[0096] Take 6 portions of Lily Solid Gold Tablets from batch S1, and prepare 6 portions of Lily Solid Gold Tablets test solution according to the test solution preparation method under "2.2". Accurately pipette 5 μL and inject into the HPLC. Measure according to the chromatographic conditions under "2.1", record the peak area and retention time, use Peak 6 as the reference peak, calculate the relative peak area and RSD value, and see Table 5 for the results. Calculate the relative retention time and RSD value, and see Table 6 for the results.
[0097] Table 5
[0098]
[0099] Table 6
[0100]
[0101] The results showed that the RSD of the relative peak areas of the common peaks were all less than 3.56% (n=6), and the RSD of the relative retention time were all less than 0.09% (n=6), indicating that the method had good repeatability.
[0102] 3.3 Precision investigation
[0103] Accurately pipette 5 μL of the test solution of Lily Solid Gold Tablets in batch S1 under item “2.2” and inject it into the HPLC for 6 consecutive injections. Determine according to the chromatographic conditions under item “2.1”, record the peak area and retention time, take peak No. 6 as the reference peak, calculate the relative peak area and RSD value, and refer to Table 7 for the results. Calculate the relative retention time and RSD value, and refer to Table 8 for the results.
[0104] Table 7
[0105]
[0106] Table 8
[0107]
[0108] The results showed that the RSD of the relative peak areas of the common peaks were all less than 3.94% (n=6), and the RSD of the relative retention time were all less than 0.90% (n=6), indicating that the method had good precision.
[0109] 3.4 Stability investigation
[0110] Prepare batch S1 of Lily Solid Gold Tablets test solution according to the test solution preparation method under "2.2". At 0h, 6h, 12h, 18h, 24h, 30h and 36h after preparation, accurately pipette 5μL and inject into HPLC. Measure according to the chromatographic conditions under "2.1", record the peak area and retention time, take Peak 6 as the reference peak, calculate the relative peak area and RSD value, and see Table 9 for the results. Calculate the relative retention time and RSD value, and see Table 10 for the results.
[0111] Table 9
[0112]
[0113] Table 10
[0114]
[0115] The results showed that the RSD of the relative peak areas of the common peaks were all less than 3.94% (n=7), and the RSD of the relative retention time were all less than 0.12% (n=7), indicating that the method had good stability.
[0116] 3.5 Intermediate precision study
[0117] 3.5.1 Investigation of the intermediate precision of liquid phase instruments
[0118] Accurately pipette 5 μL of the sample solution of Lily Solid Gold Tablets from batch S1 under item "2.2" and inject it into the HPLC (equipment number: J-20-C-008) and HPLC (equipment number: J-20-C-093) respectively. Determine according to the chromatographic conditions under item "2.1" and record the chromatogram. Figure 6 , where A is the chromatogram measured by J-20-C-008 equipment; B is the chromatogram measured by J-20-C-093 equipment.
[0119] Taking the control fingerprint R as reference, the 2012 edition of “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine” was used to perform similarity analysis on the above chromatograms. The similarity evaluation results are shown in Table 11.
[0120] Table 11
[0121]
[0122] The results showed that the similarity of fingerprint spectra measured in HPLCs with different numbers reached 0.985, indicating that the intermediate precision of the HPLC was good.
[0123] 3.5.2 Investigation of intermediate precision of chromatographic columns
[0124] Accurately pipette 5 μL of the sample solution of Lily Solid Gold Tablets in batch S1 under "2.2" and inject it into the HPLC. Determine according to the chromatographic conditions under "2.1". The chromatographic columns are ODS C18 (4.6×250 mm, 5 μm), which are numbered JZ-SY-C002, QC-C18-24-0401 and QC-C18-22-0604. Record the chromatogram. Figure 7 , where A is the chromatogram of JZ-SY-C002; B is the chromatogram of QC-C18-24-0401; and C is the chromatogram of QC-C18-22-0604.
[0125] Taking the control fingerprint R as reference, the 2012 edition of “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine” was used to perform similarity analysis on the above chromatograms. The similarity evaluation results are shown in Table 12.
[0126] Table 12
[0127]
[0128] The results showed that the similarity of the fingerprint spectra measured under different numbered chromatographic columns was about 0.995, indicating that the intermediate precision of the chromatographic columns was good.
[0129] 4. Testing of Lily Solid Gold Sheets
[0130] 4.1 Preparation of sample solution
[0131] Take 2g of the lily solid gold tablets to be tested, accurately weigh it, place it in a 150mL stoppered conical flask, accurately add 25mL of 50% by volume ethanol aqueous solution, seal it, weigh it and then ultrasonically treat it for 45min, cool it to room temperature, weigh it again, make up the lost weight with 50% by volume ethanol aqueous solution, shake it well, filter it with a 0.45μm microporous filter membrane, collect the filtrate to obtain the sample solution to be tested.
[0132] 4.2 Determination method
[0133] Accurately pipette 5 μL of the sample solution to be tested under item "4.1" and inject it into the high performance liquid chromatograph (equipment number: J-20-C-093). Determine according to the chromatographic conditions under item "2.1" and record the chromatogram to obtain the chromatogram of the sample solution to be tested.
[0134] The chromatogram of the sample solution to be tested is compared with the above-mentioned control fingerprint spectrum R. If the similarity is ≥0.9, it can be preliminarily considered that the quality of the Lily Solid Gold Tablets to be tested is qualified.
[0135] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0136] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A method for constructing a fingerprint of a Yin-nourishing and lung-moistening preparation, characterized in that: The following steps are involved: Taking a nourishing yin and moistening lung preparation, adding an extraction solvent for extraction treatment, collecting the extract, and preparing a test solution, wherein the raw materials for preparing the nourishing yin and moistening lung preparation include lily, rehmannia root, prepared rehmannia root, ophiopogon japonicus, angelica sinensis, Scrophularia ningpoensis, Fritillaria cirrhosa, Platycodon grandiflorum, licorice and white peony root; Taking the test solution for liquid chromatography determination; The chromatographic conditions of the liquid chromatography assay include: The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A includes aqueous phosphoric acid solution, and the mobile phase B includes acetonitrile, and the elution is performed by gradient elution; The gradient elution includes the following procedures: From 0min to 10min, the volume fraction of the mobile phase A decreased from 98% to 87%, and the volume fraction of the mobile phase B increased from 2% to 13%; From 10 min to 15 min, the volume fraction of the mobile phase A decreased from 87% to 83%, and the volume fraction of the mobile phase B increased from 13% to 18%; From 15min to 21min, the volume fraction of the mobile phase A decreased from 83% to 80%, and the volume fraction of the mobile phase B increased from 18% to 20%; From 21min to 26min, the volume fraction of the mobile phase A decreased from 80% to 77%, and the volume fraction of the mobile phase B increased from 20% to 23%; From 26 min to 35 min, the volume fraction of the mobile phase A decreased from 77% to 65%, and the volume fraction of the mobile phase B increased from 23% to 35%; From 35 min to 45 min, the volume fraction of the mobile phase A is reduced from 65% to 50%, and the volume fraction of the mobile phase B is increased from 35% to 50%; From 45 min to 55 min, the volume fraction of the mobile phase A decreased from 50% to 35%, and the volume fraction of the mobile phase B increased from 50% to 65%; From 55 min to 60 min, the volume fraction of the mobile phase A decreased from 35% to 30%, and the volume fraction of the mobile phase B increased from 65% to 70%; From 60 min to 65 min, the volume fraction of the mobile phase A was maintained at 30%, and the volume fraction of the mobile phase B was maintained at 70%.
2. The method for constructing the fingerprint of the nourishing yin and moistening lung preparation according to claim 1, characterized in that: Includes at least one of the following features: (1) The volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%; (2) The stationary phase comprises octadecylsilane bonded silica gel.
3. The method for constructing the fingerprint of the nourishing yin and moistening lung preparation according to claim 1, characterized in that: The chromatographic conditions of the liquid chromatography determination also include at least one of the following conditions: (1) The detection wavelength is 210nm~250nm; (2) Column temperature is 22°C~28°C; (3) Flow rate: 0.5 mL / min~1.5 mL / min; (4) The injection volume is 2 μL~8 μL.
4. The method for constructing the fingerprint of the Yangyin Runfei preparation according to any one of claims 1 to 3, characterized in that: Also includes at least one of the following features: (1) The method for extracting the Yin-nourishing and lung-moistening preparation is ultrasonic extraction; (2) The extraction solvent includes ethanol.
5. The method for constructing the fingerprint of the nourishing yin and moistening lung preparation according to claim 4, characterized in that: Also includes at least one of the following features: (1) Add 20 mL to 30 mL of the extraction solvent for every 2 g of the Yangyin Runfei preparation; (2) The extraction solvent is an ethanol aqueous solution, and the volume fraction of ethanol in the ethanol aqueous solution is 20% to 80%.
6. The method for constructing the fingerprint of the nourishing yin and moistening lung preparation according to claim 4, characterized in that: The ultrasonic extraction time is 30min to 60min.
7. A method for detecting a Yin-nourishing and lung-moistening preparation, characterized in that: The following steps are involved: Take the to-be-tested nourishing yin and moistening lung preparation, add an extraction solvent for extraction treatment, collect the extract, and prepare a to-be-tested sample solution, wherein the raw materials for preparing the to-be-tested nourishing yin and moistening lung preparation include lily, rehmannia root, prepared rehmannia root, ophiopogon japonicus, angelica sinensis, scrophularia ningpoensis, fritillaria cirrhosa, platycodon grandiflorum, licorice, and white peony root; The sample solution to be tested is subjected to liquid chromatography according to the chromatographic conditions described in any one of claims 1 to 6, and the obtained chromatogram is matched with the fingerprint of the Yangyin Runfei preparation described in any one of claims 1 to 6 to determine whether the Yangyin Runfei preparation to be tested is qualified.
8. The detection method of the Yin-nourishing and lung-moistening preparation according to claim 7, characterized in that: Also includes at least one of the following features: (1) The method for extracting the Yin-nourishing and lung-moistening preparation to be tested is ultrasonic extraction; (2) The extraction solvent includes ethanol.
9. The detection method of the Yin-nourishing and lung-moistening preparation according to claim 8, characterized in that: Also includes at least one of the following features: (1) Add 20 mL to 30 mL of the extraction solvent for every 2 g of the Yangyin Runfei preparation to be tested; (2) The extraction solvent is an ethanol aqueous solution, and the volume fraction of ethanol in the ethanol aqueous solution is 20% to 80%.
10. The detection method of the Yin-nourishing and lung-moistening preparation according to claim 8, characterized in that: The ultrasonic extraction time is 30min to 60min.