A characteristic spectrum construction method, quality control method and preparation method of a Simiao Yang'an Decoction reference sample

Abstract

The application discloses a characteristic chromatogram construction method, a quality control method and a preparation method of Simiao Yongan Decoction reference sample. Raw medicinal materials of the Simiao Yongan Decoction include honeysuckle, radix scrophulariae, angelica and licorice, and the characteristic chromatogram construction method comprises the following steps: (1) preparing a test sample solution and a control sample solution; (2) detecting by using a high performance liquid phase method, and the chromatographic conditions comprise the following steps: taking octadecylsilane bonded silica gel as a filler; taking acetonitrile as a mobile phase A and 0.2% formic acid as a mobile phase B, and performing gradient elution according to the following table; the detection wavelength is 254 nm; and the theoretical plate number is not less than 5000 according to glycyrrhizic acid. The chromatographic peak information of the characteristic chromatogram constructed by the application is rich, the resolution is good, the specificity, stability and repeatability are good, the quality control method has high resolution, good stability, short detection time and high efficiency.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically, it relates to a method for constructing a characteristic spectrum of the Simiao Yong'an Decoction reference sample, a quality control method, and a preparation method. Background Technology

[0002] Simiao Yong'an Decoction is a classic formula recorded in ancient books, originating from "New Compilation of Effective Prescriptions" written by Bao Xiang'ao in the Qing Dynasty. It is a heat-clearing agent with the effects of clearing heat and detoxifying, promoting blood circulation and relieving pain. The formula consists of 3 liang each of honeysuckle and Scrophularia ningpoensis, 2 liang of Angelica sinensis, and 1 liang of licorice root, decocted in water and taken orally. It is mainly used to treat gangrene caused by intense heat toxicity, characterized by dark red, slightly swollen, burning, ulcerated, foul-smelling limbs with severe pain, or fever, thirst, red tongue, and rapid pulse. Compared to modern Chinese medicine preparations, traditional preparations generally suffer from disadvantages such as outdated technology, unstable quality, and insufficient production efficiency.

[0003] The reference sample is the substance obtained after decoction, in its basic form as a concentrated and dried product. The reference sample loses the morphological fingerprint of the original medicinal slices, and qualitative identification of only the indicator components is insufficient to reflect its quality. As a standard reference for assessing whether the reference sample is essentially consistent with the clinical decoction, its quality should be strengthened through specific identification and overall quality control of multiple components. Therefore, the quality control of the reference sample should emphasize overall quality control based on characteristic chromatograms and the accurate quantitative determination of its main effective components, which is beneficial for better controlling the quality of Simiao Yong'an Decoction.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a method for constructing characteristic chromatograms, a quality control method, and a preparation method for a Simiao Yong'an Decoction reference sample. The preparation method of this invention exhibits high extraction and transfer rates of the effective components, rich chromatographic peak information in the characteristic chromatogram, excellent separation, and good specificity, stability, and repeatability. The quality control method demonstrates high separation, good stability, low detection limit, and high detection efficiency.

[0006] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0007] The first objective of this invention is to provide a method for constructing a characteristic spectrum of a Simiao Yong'an Decoction reference sample. The raw materials of Simiao Yong'an Decoction include honeysuckle, scrophularia, angelica, and licorice, comprising:

[0008] (1) Prepare the test solution and the reference solution;

[0009] (2) Detection was performed by high performance liquid chromatography. The chromatographic conditions included: using octadecylsilane-bonded silica gel as the packing material; using acetonitrile as mobile phase A and 0.2% formic acid as mobile phase B, and performing gradient elution as specified in the table below; the detection wavelength was 254 nm; and the theoretical plate number, calculated based on glycyrrhizic acid, was not less than 5000.

[0010] In a further embodiment, the elution gradient is:

[0011]

[0012] Preferably, the flow rate is 1.0 ml per minute and the column temperature is 25°C.

[0013] Preferably, the chromatographic column used is: InertSμstain C18 4.6×250mm, 5μm.

[0014] In a further embodiment, in step (1), the preparation method of the reference solution includes: taking an appropriate amount of ammonium glycyrrhizate reference standard, accurately weighing it, adding 70% methanol to prepare a reference solution with a concentration of 0.1 mg / ml;

[0015] Preferably, the weight of glycyrrhizic acid = the weight of ammonium glycyrrhizate / 1.0207.

[0016] In a further embodiment, step (1) includes the following method for preparing the test solution:

[0017] Take 0.5g of Simiao Yong'an Decoction sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, sonicate for 15 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the product.

[0018] Preferably, the ultrasonic treatment power is 500W and the frequency is 40kHz.

[0019] The second objective of this invention is to provide an application of the method for constructing the characteristic spectrum of the Simiao Yong'an Decoction reference sample as described above in the quality detection of Simiao Yong'an Decoction.

[0020] Preferably, the constructed characteristic spectrum includes 9 characteristic peaks, with the S peak corresponding to the glycyrrhizic acid reference peak. The relative retention times of the other 7 characteristic peaks are: peak 1 0.20, peak 2 0.21, peak 3 0.62, peak 4 0.66, peak 5 0.74, peak 6 0.92, peak 7 0.99, peak 8 0.99, and peak 9 (S peak) 1.00.

[0021] The third objective of this invention is to provide a quality control method for a reference sample of Simiao Yong'an Decoction. The raw materials of Simiao Yong'an Decoction include honeysuckle, scrophularia, angelica, and licorice. The quality control method includes:

[0022] (1) Prepare reference solutions of chlorogenic acid, harpagoside and ferulic acid; prepare test solution;

[0023] (2) High-performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions included: octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B; harpagoside detection wavelength of 278 nm, and chlorogenic acid and ferulic acid detection wavelength of 324 nm; gradient elution was performed. The theoretical plate number, calculated based on chlorogenic acid, was not less than 10,000.

[0024] A further embodiment of the gradient elution includes:

[0025]

[0026] Preferably, the column temperature is 30°C and the flow rate is 0.5 ml per minute.

[0027] Preferably, the chromatographic column used is ThermoFisher Hypersil, 2.1×100mm, 1.9μm.

[0028] In a further step, the preparation method of the reference solution in step (1) includes:

[0029] Take appropriate amounts of chlorogenic acid reference standard, harpagoside reference standard and ferulic acid reference standard, weigh them accurately, place them in a brown volumetric flask, and add 70% methanol to prepare a reference solution containing 0.16 mg of chlorogenic acid, 30 μg of harpagoside and 10 μg of ferulic acid per 1 ml.

[0030] In a further embodiment, step (1) includes the following method for preparing the test solution:

[0031] Take 0.5g of Simiao Yong'an Decoction sample powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, sonicate for 15 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the product.

[0032] Preferably, the ultrasonic treatment power is 500W and the frequency is 40kHz;

[0033] Preferably, based on the dried product, the Simiao Yong'an Decoction sample should contain 0.025% to 0.047% ferulic acid, 0.77% to 1.43% chlorogenic acid, and 0.064% to 0.120% harpagoside by mass.

[0034] The fourth objective of this invention is to provide a preparation process for Simiao Yong'an Decoction, wherein the raw materials of Simiao Yong'an Decoction include honeysuckle, scrophularia, angelica, and licorice, comprising:

[0035] Mix all the raw medicinal materials, decoct twice, soak in water for 30 minutes, boil over high heat until boiling, then simmer over low heat for 20 minutes, filter, add water for the second decoction, boil over high heat until boiling, then simmer over low heat for 15 minutes, filter, combine the filtrates, concentrate to extract, freeze dry, and you will get the product.

[0036] Preferably, the high heat is 2000W and the low heat is 600W;

[0037] Preferably, the filtration process passes through a 150-mesh sieve;

[0038] Preferably, add 10-12 times the amount of water the first time, and 8-9 times the amount of water the second time;

[0039] The preferred ingredients of Simiao Yong'an Decoction include: honeysuckle 111.90g, scrophularia 111.90g, angelica 74.60g, licorice 37.30g, 4000ml of water added for the first time, and 3000ml of water added for the second time.

[0040] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0041] 1. To enhance the specific identification and multi-component, overall quality control of Simiao Yong'an Decoction, a characteristic chromatographic method was adopted for overall quality evaluation. The established characteristic chromatographic method was validated, and the obtained characteristic chromatograms showed stable baselines, moderate response values ​​for each common peak, good separation, and good stability, repeatability, and robustness, which is beneficial for controlling the quality stability of Simiao Yong'an Decoction.

[0042] 2. To better control the quality of the Simiao Yong'an Decoction reference sample, chlorogenic acid, harpagoside, and ferulic acid were used as the main active ingredients for content determination. A method was established and validated. The determined method met the requirements for repeatability, stability, accuracy, and range. Chlorogenic acid, harpagoside, and ferulic acid can be used as the content determination indicator components for the Simiao Yong'an Decoction reference sample. - This method has a short detection time and high efficiency.

[0043] 3. The preparation method of the Si Miao Yong An Tang reference sample of the present invention is simple, the transfer rate of each effective component is high, and the efficacy is better.

[0044] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0045] The accompanying drawings, as part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0046] Figure 1 This is a superimposed graph of different concentration temperatures in Experiment Example 1 of the present invention, wherein: S1. Extract, S2. 45℃ concentrate, S3. 60℃ concentrate, S4. 75℃ concentrate;

[0047] Figure 2 This is a superimposed graph of different concentration temperatures in Experiment 1 of the present invention, where S1 is the extract, S2 is the concentrate at 45°C, S3 is the concentrate at 60°C, and S4 is the concentrate at 75°C.

[0048] Figure 3 This is a superimposed graph of different drying methods in Experiment Example 1 of the present invention, wherein: S1. extract, S2. freeze-dried concentrate, S3. vacuum-dried concentrate, S4. freeze-dried powder, S5. vacuum-dried powder;

[0049] Figure 4 This is the result graph of chromatographic condition 1 in test example 2 of the present invention, with 210nm, 278nm and 327nm from top to bottom;

[0050] Figure 5 This is the result graph of chromatographic condition 2 in test example 2 of the present invention, with 210nm, 278nm and 324nm from top to bottom;

[0051] Figure 6 This is a graph showing the results of chromatographic condition 3 in Experimental Example 2 of this invention;

[0052] Figure 7 It is a chromatogram after the chromatographic conditions have been determined;

[0053] Figures 8-12 The chromatograms under section 2.1.2 of Experimental Example 2, obtained using wavelengths of 240nm, 254nm, 270nm, 300nm, and 320nm respectively;

[0054] Figure 13 The chromatograms for different extraction methods in Experiment Example 2 are shown from top to bottom as ultrasonic extraction and reflux extraction.

[0055] Figure 14-19 The chromatograms are those obtained by sequentially using water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol as solvents in Experiment Example 2.

[0056] Figure 20These are chromatograms of different extraction times in Experiment Example 2, from top to bottom representing extraction times of 15 minutes, 30 minutes, and 60 minutes.

[0057] Figure 21 This is a selected chromatogram of reference substances, where S1 is the Simiao Yong'an Decoction reference sample, S2 is the mixed reference standard, 1. chlorogenic acid, 2. ferulic acid, 3. glycyrrhizin, 4. luteolin, 5. ligustilide, 6. 3,5-di-O-caffeoylquinic acid, 7. 4,5-di-O-caffeoylquinic acid, 8. cinnamic acid, 9. harpagoside, and 10. ammonium glycyrrhizate.

[0058] Figure 22 The diagram shows the specificity of test example 2, with 70% methanol and the test sample arranged from top to bottom.

[0059] Figure 23 This is a diagram illustrating the overall assessment of Experimental Example 2;

[0060] Figure 24 and Figure 25 Chromatograms of different column temperatures in Experiment Example 2;

[0061] Figure 26 Chromatograms with different flow rates were used for Experimental Example 2: S1, flow rate 0.9 ml / min; S2, flow rate 1.0 ml / min; S3, flow rate 1.1 ml / min.

[0062] Figure 27 The chromatograms for Experimental Example 2 were obtained using different chromatographic columns: S1. WondaSil C18, S2. Agilent ZORBAX SB-C18, and S3. InertSustain C18.

[0063] Figure 28 For the specificity study of honeysuckle, S1. reference sample, S2. honeysuckle-deficient negative sample, S3. honeysuckle-positive sample;

[0064] Figure 29 For the specificity study of licorice, S1. reference sample, S2. licorice-deficient negative sample, S3. licorice-positive sample;

[0065] Figure 30 For the specificity study of Angelica sinensis, S1 is the reference sample, S2 is the negative sample lacking Angelica sinensis, and S3 is the positive sample of Angelica sinensis.

[0066] Figure 31 For the specificity study of Scrophularia ningpoensis, S1 is the reference sample, S2 is the negative sample lacking Scrophularia ningpoensis, and S3 is the positive sample of Scrophularia ningpoensis.

[0067] Figure 32The characteristic spectral peaks of Simiao Yong'an Decoction were located as follows: 1. chlorogenic acid, 2. ferulic acid, 3. glycyrrhizin, 4. luteolin, 5. ligustilide, 6. 3,5-di-O-caffeoylquinic acid, 7. 4,5-di-O-caffeoylquinic acid, 8. cinnamic acid, 9. harpagoside, and 10. ammonium glycyrrhizate.

[0068] Figure 33 This is a chromatogram of 17 batches of Simiao Yong'an Decoction reference samples.

[0069] Figure 34 The chromatogram of Method 1 in Example 3 shows that the detection wavelengths from top to bottom are 210 nm, 278 nm and 327 nm, respectively.

[0070] Figure 35 The chromatogram of Method 2 in Experiment Example 3 shows the detection wavelengths from top to bottom as 210 nm, 278 nm and 324 nm; 1 harpagoside, 2 ferulic acid, 3 3,5-di-O-caffeoylquinic acid, 4 harpagoside;

[0071] Figure 36 This is the chromatogram of Method 3 in Experiment Example 3. The detection wavelengths from top to bottom are 210 nm, 278 nm and 324 nm, respectively; 1 harpagoside, 2 ferulic acid, 3 3,5-di-O-caffeoylquinic acid, 4 harpagoside;

[0072] Figure 37 The chromatogram of Method 4 in Example 3 shows the detection wavelengths from top to bottom as 210 nm, 278 nm and 327 nm; 1 harpagoside, 2 ferulic acid, 3 3,5-di-O-caffeoylquinic acid, 4 harpagoside;

[0073] Figure 38 The chromatogram is the final condition of Experiment Example 3, with detection wavelengths from top to bottom of 210 nm, 278 nm and 324 nm; 1 harpagoside, 2 ferulic acid, 3 3,5-di-O-caffeoylquinic acid, 4 harpagoside;

[0074] Figure 39 The results of harpagoside (278nm) specificity in Experiment 3 are shown in the following order from top to bottom: mixed standard reference, Simiao Yong'an Decoction reference sample, and negative sample lacking Scrophularia ningpoensis.

[0075] Figure 40 The specificity results of 3,5-di-O-caffeoylquinic acid (324nm) in Experiment Example 3 are as follows: from top to bottom, mixed standard reference, Simiao Yong'an Decoction reference sample, and negative sample lacking honeysuckle.

[0076] Figure 41 The specificity results of ferulic acid (324nm) in Experiment Example 3 are shown in the following order from top to bottom: mixed standard reference, Simiao Yong'an Decoction reference sample, and negative sample lacking Angelica sinensis.

[0077] Figure 42 The results show the specificity of chlorogenic acid (324nm) in Experiment Example 3. From top to bottom, the results are: mixed standard reference, Simiao Yong'an Decoction reference sample, and negative control lacking honeysuckle.

[0078] Figure 43 The chromatogram of the mixed standard reference in Experiment Example 3 is shown, which includes 1. chlorogenic acid, 2. ferulic acid, and 3. harpagoside.

[0079] Figure 44 The chromatograms for different flow rates detected in Experimental Example 3 are as follows: 1. Chlorogenic acid, 2. Ferulic acid, 3. Harpagoside, S1. 0.48 ml / ml, S2. 0.50 ml / ml, S3. 0.52 ml / ml;

[0080] Figure 45 The chromatograms for detection using different phosphoric acid solution concentrations in Experiment Example 3 are as follows: 1. Chlorogenic acid, 2. Ferulic acid, 3. Harpagoside, S1. 0.08% phosphoric acid solution, S2. 0.10% phosphoric acid solution, S3. 0.12% phosphoric acid solution.

[0081] Figure 46 The results of the column temperature investigation in Experiment Example 3 (278nm) are as follows: from top to bottom, the column temperatures are 25℃, 30℃, and 35℃.

[0082] Figure 47 The results of the column temperature investigation in Experiment Example 3 (324nm) are as follows: from top to bottom, the column temperatures are 25℃, 30℃, and 35℃.

[0083] Figure 48 The results of the column robustness test in Experiment 3 (278 nm) are shown below, from top to bottom: Hypersil GOLD C18 and Shim-pack Scepter HD-C18-80.

[0084] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0085] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0086] Example 1: Preparation process of Si Miao Yong An Tang reference sample

[0087] This product is a dried powder made from honeysuckle, scrophularia, angelica, and licorice (licorice root).

[0088] Prescription: Honeysuckle 111.90g, Scrophularia ningpoensis 111.90g, Angelica sinensis 74.60g, Licorice root 37.30g

[0089] Preparation: Add 4000ml of water to the above four ingredients and soak for 30 minutes. Bring to a boil over high heat (2000W), then simmer over low heat (600W) for 20 minutes. Filter through a 150-mesh sieve. For the second decoction, add 3000ml of water, bring to a boil over high heat (2000W), then simmer over low heat (600W) for 15 minutes. Filter through a 150-mesh sieve. Combine the two filtrates, mix them, and concentrate under reduced pressure to approximately 500g of clear extract. Freeze-dry to obtain the final product. (Yield range: 40.9%–50.0%)

[0090] It should be noted that the powder described in the following examples or test cases is the Simiao Yong'an Decoction reference sample prepared using the method of Example 1.

[0091] Example 2: Construction of the characteristic spectrum of the Simiao Yong'an Decoction reference sample

[0092] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.2% formic acid was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the detection wavelength was 254 nm; the flow rate was 1.0 mL / min, and the column temperature was 25 °C. The theoretical plate number, calculated based on glycyrrhizic acid, was not less than 5000. Column: InertSμstain C18 (4.6 × 250 mm, 5 μm).

[0093] Table 1

[0094]

[0095] Preparation of reference solution: Take an appropriate amount of ammonium glycyrrhizate reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 0.1 mg per ml, which is the result of (weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207).

[0096] Preparation of the test solution: Accurately weigh approximately 0.5 g of the powder (passed through a No. 2 sieve) and place it in a stoppered conical flask. Accurately add 25 ml of 70% methanol, weigh the solution, and sonicate (500 W, 40 kHz) for 15 minutes. Cool the flask, weigh the solution again, and replenish the lost weight with 70% methanol. Shake well, filter the solution, and collect the filtrate. Assay: Accurately inject 20 μl each of the reference solution and the test solution into the liquid chromatograph.

[0097] The characteristic chromatogram of the test sample should show 9 characteristic peaks. The peak corresponding to the glycyrrhizic acid reference is the S peak. Calculate the retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.20 (peak 1), 0.21 (peak 2), 0.62 (peak 3), 0.66 (peak 4), 0.74 (peak 5), 0.92 (peak 6), 0.99 (peak 7), 0.99 (peak 8), 1.00 [peak 9 (S)]. The characteristic chromatogram is shown below. Figure 1 As shown.

[0098] Example 3: Quality Control Method of Simiao Yong'an Decoction

[0099] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase (ThermoFisher Hypersil 2.1 × 100 mm, 1.9 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B; harpagoside was detected at 278 nm, and chlorogenic acid and ferulic acid were detected at 324 nm; the flow rate was 0.5 mL / min, and the column temperature was 30.0 °C, with gradient elution performed according to the table below. The theoretical plate number, calculated based on chlorogenic acid, should not be less than 10,000. Specifically, the chromatographic column was a ThermoFisher Hypersil GOLD C18.

[0100] Table 2

[0101]

[0102] Preparation of the test solution: Take about 0.5 g of the powder (passed through a No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 15 minutes, cool it, weigh it again, make up the weight loss with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0103] Preparation of reference solution: Take appropriate amounts of chlorogenic acid reference standard, harpagoside reference standard and ferulic acid reference standard, accurately weigh them, place them in a brown volumetric flask, add 70% methanol to prepare a solution containing 0.16 mg of chlorogenic acid, 30 μg of harpagoside and 10 μg of ferulic acid per 1 ml.

[0104] The assay involves precisely pipetting 1 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the concentration.

[0105] This product, calculated on a dried basis, should contain 0.025%–0.047% ferulic acid (C10H10O4), 0.77%–1.43% chlorogenic acid (C16H8O9), and 0.064%–0.120% harpagoside (C24H30O11).

[0106] Study on the preparation process of Experimental Example 1

[0107] 1. Raw materials: Angelica sinensis slices: batch numbers YP18B-19091601-2, YP18B-19091102, YP18B-20201302; Honeysuckle: batch numbers YC104-21032403, YC104-21032402, YC104-21031201; Scrophularia ningpoensis slices: batch numbers YP75B-21031204, YP75B-21031202, YP75B-21031210; Licorice slices: batch numbers YP07B-19111202, YP07B-19111208, YP07B-21022501.

[0108] 2. Reagents: Chlorogenic acid: purity 96.1%, specification 20mg, batch number 110753-202018, National Institutes for Food and Drug Control (NIFDC); Harpagoside: purity 96.0%, specification 20mg, batch number 111730-201508, NIFDC; Ferulic acid: purity 99.4%, specification 50mg, batch number 111773-201915, NIFDC; Methanol: AR, Shanghai Titan Technology Co., Ltd.; Acetonitrile: HPLC grade, Merck & Co., Ltd.; Purified water: 596ml, Hangzhou Wahaha Group Co., Ltd.; Phosphoric acid: HPLC grade, Honeywell Trading (Shanghai) Co., Ltd.

[0109] 3. Process Study of Simiao Yong'an Decoction Standard Sample

[0110] All experiments were conducted using single-factor experiments. The main evaluation indicators for the entire experiment were the yield rate, transfer rate of ferulic acid, chlorogenic acid and harpagoside content, and fingerprint spectrum.

[0111] 3.1 Determination of the number of decoctions, dosage, and decoction time

[0112] The book *New Compilation of Prescriptions* (Qing Dynasty, Bao Xiang'ao) states: "Honeysuckle and Scrophularia, three liang each; Angelica sinensis, two liang; licorice, one liang. Take with warm water." The dosage of Qing Dynasty prescriptions disclosed in the *Key Information Table of Ancient Classic Prescriptions* published by the State Administration of Traditional Chinese Medicine can be calculated as 37.30g for one liang. Furthermore, the original prescription for Simiao Yong'an Decoction does not record specific decoction methods. According to the *Technical Guidelines for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Catalogue of Ancient Classic Prescriptions (Trial Implementation)* issued by the Center for Drug Evaluation of the National Medical Products Administration, decoction and administration should be carried out in accordance with the *Management Standards for Traditional Chinese Medicine Decoction Rooms in Medical Institutions*. The preparation method for Simiao Yong'an Decoction can be tentatively determined as follows: Take 111.9g each of honeysuckle and Scrophularia ningpoensis, 37.3g of licorice, and 74.6g of Angelica sinensis, add water, soak for 30 minutes, then decoct twice. The first decoction should be 20 minutes, and the second decoction should be 15 minutes. Filter the decoction to obtain Simiao Yong'an Decoction.

[0113] 3.2 Determination of the water absorption of medicinal slices

[0114] Weigh out 2 portions of the medicinal slices, which is 1 / 10 of the prescription amount, and place them in a decoction pot. Add water until it covers the slices by 5cm. Soak for 30 minutes, then bring to a boil over high heat. After boiling, continue to simmer over low heat for 1 hour. Filter the resulting liquid, pour out the dregs, weigh them, and calculate the amount of water absorbed by the slices.

[0115] Table 3 Results of the investigation on the water absorption of medicinal slices

[0116]

[0117] The results showed that after being fully decocted, the water absorption rate of the medicinal slices in the Simiao Yong'an Decoction prescription was about three times the total weight of the medicinal slices. Therefore, when decocting multiple times, the amount of water added in subsequent decoctions should be reduced by three times compared to the first decoction.

[0118] 3.3 Determining the water addition ratio

[0119] Take 1 / 10 of the prescription amount of medicinal slices, totaling 3 groups (2 portions per group), and place them in a decoction pot. For the first decoction, add 10, 12, and 15 times the amount of water respectively, soak for 30 minutes, then bring to a boil over high heat (2000W), followed by simmering over low heat (600W) for 20 minutes. Filter the decoction. For the second decoction, add 7, 9, and 12 times the amount of water respectively, bring to a boil over high heat, and continue simmering over low heat for 15 minutes. Filter the decoction, mix well, and determine the content of ferulic acid, harpagoside, and chlorogenic acid, as well as the total solids content. Calculate the extract yield and transfer rate. The experimental results are shown in Table 4.

[0120] Table 4 Results of Water Addition Dosage

[0121]

[0122] The results showed that when the initial water addition was 10 times the normal amount, all indicators were significantly lower than those at 12 times and 15 times the normal amount, and the liquid level was less than 2 cm above the medicinal slices at this time. When the water addition reached 15 times the normal amount, all indicators increased, but compared with the 12 times the normal amount, only the extract yield and harpagoside transfer rate increased significantly. Considering that Scrophularia ningpoensis and harpagoside have good water solubility, and that the future preparation will be scaled up by 10 times, which will prolong the heating time and increase the transfer rate of each indicator component, adjusting the water addition ratio to 12 / 10 times is more appropriate. This can reduce the amount of medicinal liquid used while achieving the highest possible extraction efficiency.

[0123] 3.4 Selection of different frying power

[0124] Take 8 portions of the herbal slices (1 / 10 of the prescription amount), place them in a decoction pot, add 400ml of water, soak for 30 minutes, then decoct on an electric ceramic stove. Bring to a boil over high heat (2000W), then simmer over low heat for 20 minutes. Filter. For the second decoction, add 300ml of water, bring to a boil over high heat, then simmer over low heat for 15 minutes. Combine the filtrates. The simmering heat was set at 200W, 400W, 600W, and 800W. The content and total solids of the decoction were measured, and the yield and transfer rate were compared. The results are shown in Table 5.

[0125] Table 5 Results of different boiling powers

[0126]

[0127] The results above show that when using 200W to 800W for decoction, the ratio of the minimum to the maximum value of different indicators is around 90%. The yield of extract is significantly reduced at 800W. The trend of the transfer rate of indicator components with the decoction power is uncertain, and the fluctuation range is within ±10% of the relative value, with no significant impact. In terms of the amount of decoction, the amount of decoction is more and more stable at 400W and below. Above 400W, the amount of decoction gradually decreases with the increase of decoction power, and the decrease is more significant at 600W. Considering that the decoction will be scaled up in the future when preparing according to the prescription, the amount of decoction will increase by a factor of two, and the extraction rate will also increase accordingly. Based on the comprehensive evaluation, 600W is selected for decoction.

[0128] 3.5 Filter mesh size selection

[0129] Six portions of the extract from the above experiment were taken, with half the volume of the decoction obtained from each portion. After mixing evenly, the mixtures were filtered through 100-mesh, 150-mesh, and 200-mesh nylon sieves, respectively. The total solid content and the content of index components were measured. The appropriate filtration mesh size was determined based on the filtration speed, the condition of the dregs, and the yield of the extract. The experimental results are shown in Tables 6 and 7.

[0130] Table 6 Filter mesh count results

[0131]

[0132]

[0133] Table 7. Results of Filter Mesh Count Study

[0134]

[0135] As shown in the table, filtration with the above-mentioned mesh sizes can achieve the filtration purpose, and the yield of paste is not much different. When filtration is performed with 100 mesh, the transfer rate of index components in the filtrate is lower than that of 150 mesh and 200 mesh. However, the transfer rate of index components in the filtrate after filtration with 150 mesh is not much different from that after filtration with 200 mesh. Considering the filtration speed and the phenomenon, filtration with 150 mesh is recommended.

[0136] 3.6 Concentration Temperature Study

[0137] After preparing three portions of Simiao Yong'an Decoction according to the method determined above, the medicinal liquid was mixed evenly and divided into three equal portions. These portions were then concentrated under reduced pressure at temperatures of 45℃, 60℃, and 75℃, and vacuum degrees of -0.09 to -0.10 MPa, respectively, to a final concentration of 35g to 70g. The content, total solids, and characteristic chromatograms of the unconcentrated and concentrated medicinal liquids were determined, and the extract yield and transfer rate were calculated. The results are shown in Figures 8 and 9. Figure 2 .

[0138] Table 8 Concentration Temperature Results

[0139]

[0140] The results show that when the Simiao Yong'an Decoction was concentrated at the above-mentioned temperatures, the yield of the concentrated liquid and the transfer rates of harpagoside, chlorogenic acid, and ferulic acid were slightly reduced compared to the original liquid. The transfer rates decreased significantly at a concentration temperature of 75℃. Furthermore, the similarity of the concentrated liquid was greater than 0.9, therefore, the concentration temperature was determined to be no higher than 60℃.

[0141] 3.7 Research on Drying Methods

[0142] Two reference samples were prepared according to the method determined above, mixed thoroughly, and the two portions of the drug solution were concentrated under reduced pressure, serving as test sample 1 and test sample 2, respectively. The test samples were then subjected to freeze-drying (pre-freezing temperature: -20 to -45℃, drying cold trap temperature: -45 to -65℃, vacuum degree: <100 Pa) and reduced pressure drying (temperature: 60℃, vacuum degree: -0.09 to -0.10 MPa), respectively. The drying process was observed, the solid was weighed, the content and fingerprint spectrum were measured, and the dry powder rate and transfer rate were calculated. The experimental results are shown in Table 9.

[0143] Table 9 Results of the investigation on drying methods

[0144]

[0145] Note: Solid moisture content was not deducted when calculating the dry powder yield.

[0146] The results in the table above show that, since the moisture content of the dry powder was not measured, the yield of the dry powder was higher than that of the concentrated liquid. Regarding the transfer rate of the index components, both the concentrated liquid and the dry powder had lower transfer rates than the extract. When vacuum drying was used, the transfer rate of ferulic acid in the dry powder decreased significantly, while the chlorogenic acid content increased slightly. From the characteristic chromatograms, except for the vacuum-dried powder, the similarity of the other samples to the extract was higher than 0.9. In conclusion, freeze drying better preserves the overall characteristics of the medicinal liquid; therefore, freeze drying was chosen as the preparation method for the Simiao Yong'an Decoction reference sample.

[0147] 3.8 Process Validation

[0148] Based on the preparation process parameters of the Simiao Yong'an Decoction reference sample obtained from the above research, medicinal slices from the same batch and different batches of raw materials were used to form the prescription. Reference samples were prepared according to the prescription amount, and the extracts and dry powders were tested respectively. The results are as follows:

[0149] Table 10 Process Validation Formulation Ingredient List

[0150]

[0151] Table 11 Results of the original process validation for the reference samples

[0152]

[0153] Note: During the original formula verification, batches 1, 2, and 3 of raw materials were completely identical, while batches 4 and 5 of raw materials were completely different.

[0154] The results above show that when the decoction and dry powder were prepared according to the original prescription, the fluctuations in various quality attributes were small and the process was stable.

[0155] In summary, the final standard sample preparation process for Simiao Yong'an Decoction is as follows: Take 111.90g of honeysuckle, 111.90g of scrophularia, 74.60g of angelica, and 37.30g of licorice, place them in a 6L decoction pot, add 4000ml of water, soak for 30 minutes, then cover and decoct. Bring to a boil over high heat (2000W), then simmer over low heat (600W) for 20 minutes. Filter through a 150-mesh sieve. Add 3000ml of water to the dregs, bring to a boil over high heat (2000W), then simmer over low heat (600W) for another 15 minutes. Concentrate the filtrate to approximately 500g of clear extract, then freeze-dry to obtain the final product.

[0156] Experimental Example 2

[0157] 1.1 Raw materials: Simiao Yong'an Decoction reference sample, batch number: WZJZ14-21043001P; Simiao Yong'an Decoction negative sample (missing Scrophularia ningpoensis), batch number: WZJZ14-21060701P; Simiao Yong'an Decoction negative sample (missing Angelica sinensis), batch number: WZJZ14-21060702P; Simiao Yong'an Decoction negative sample (missing Lonicera japonica), batch number: WZJZ14-21060703P; Simiao Yong'an Decoction negative sample (missing Glycyrrhiza uralensis), batch number: WZJZ14-21060704P; Simiao Yong'an Decoction positive sample (Lonicera japonica), batch number: WZJZ14-21092904P; Positive samples of Simiao Yong'an Decoction (Scrophularia ningpoensis slices), batch number: WZJZ14-21092902P; Positive samples of Simiao Yong'an Decoction (Angelica sinensis slices), batch number: WZJZ14-21092903P; Positive samples of Simiao Yong'an Decoction (Glycyrrhiza uralensis slices), batch number: WZJZ14-21092905P; Positive samples of Simiao Yong'an Decoction (Scrophularia ningpoensis), batch number: WZJZ14-21121302P; Positive samples of Simiao Yong'an Decoction (Glycyrrhiza uralensis), batch number: WZJZ14-21122303P; Positive samples of Simiao Yong'an Decoction (Angelica sinensis), batch number: WZJZ14-21122301P

[0158] 1.2 Reagents: Methanol: HPLC grade, Shanghai Anpu Scientific Instruments Co., Ltd.; Acetonitrile: HPLC grade, Shanghai Anpu Scientific Instruments Co., Ltd.; Formic acid: HPLC grade, Shanghai Titan Technology Co., Ltd.; Purified water: Hangzhou Wahaha Group Co., Ltd.; Methanol: AR grade, Shanghai Titan Technology Co., Ltd.; Harpagoside reference standard: batch number 111729-201506, China National Institutes for Food and Drug Control; Harpagoside reference standard: batch number 111730-201709, China National Institutes for Food and Drug Control; 3,5-O-dicaffeine Acylquinic acid reference standard: batch number 111782-201807, National Institutes for Food and Drug Control (NIFDC); 4,5-O-dicaffeoylquinic acid reference standard: batch number 111894-202103, NIFDC; Chlorogenic acid reference standard: batch number 110753-202018, NIFDC; Luteolin reference standard: batch number 111720-201609, NIFDC; Ferulic acid reference standard: batch number 110773-201915, NIFDC; Ligusticum lactone reference standard: batch number 111737-201910, NIFDC. Ammonium glycyrrhizate reference standard: batch number 110731-202021, National Institutes for Food and Drug Control; Glycyrrhizin reference standard: batch number 111610-201908, National Institutes for Food and Drug Control; Glycyrrhizin reference standard: batch number MUST-17022104, Chengdu Mansite Biotechnology Co., Ltd.; Cinnamic acid reference standard: batch number 110786-201604, National Institutes for Food and Drug Control.

[0159] 2. Experimental Procedure and Results

[0160] 2.1 Chromatographic conditions

[0161] Preparation method of test sample: Take 0.5g of the powder of this product (passed through No. 2 sieve), place it in a stoppered conical flask, accurately add 25ml of 50% methanol, weigh it, sonicate (500W, 40kHz) for 30 minutes, cool, weigh it again, make up the weight loss with 50% methanol, shake well, filter, and take the filtrate to obtain the test sample.

[0162] 2.1.1 Investigation of mobile phase ratio

[0163] Condition 1: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, detection wavelengths were 210 nm, 278 nm, and 327 nm, flow rate was 1.0 ml per minute, column temperature was 30.0 ℃, injection volume was 20 μl, and elution was performed according to the gradient in the table below.

[0164] Table 12

[0165]

[0166] Condition 2: Use octadecylsilane-bonded silica gel as the packing material; use acetonitrile as mobile phase A and 0.4% phosphoric acid as mobile phase B, and elute according to the gradient specified in the table below; the detection wavelengths are 210 nm, 278 nm, and 324 nm, the column temperature is 30 °C, and the injection volume is 20 μl.

[0167] Table 13

[0168]

[0169] Condition 3: Use octadecylsilane-bonded silica gel as the packing material; use acetonitrile as mobile phase A and 0.1% phosphoric acid as mobile phase B, and elute according to the gradient specified in the table below; the detection wavelength is 254 nm, the column temperature is 30 °C, and the injection volume is 20 μl.

[0170] Table 14

[0171]

[0172]

[0173] The results of conditions 1-3 are as follows: Figure 4-6 As shown: From the chromatograms of the three preliminary experimental results above, the chromatogram obtained under condition 1 has poor peak shape and concentrated peaks; the chromatogram under condition 2 has clear peaks, but fewer in number; the chromatogram under condition 3 has more peaks, richer chromatographic information, and better resolution of each peak. Therefore, based on condition 3, a method for determining the characteristic chromatogram of Simiao Yong'an Decoction was developed. The chlorogenic acid peak, 3,5-di-O-caffeoylquinic acid peak, and 4,5-di-O-caffeoylquinic acid peak were separated. Formic acid, which has a lower acidity, was used as the buffer salt solution; that is, 0.1% phosphoric acid solution was replaced with 0.2% formic acid solution. After adjusting the elution ratio, the final chromatographic conditions were determined, as shown in the table below. The chromatograms are as follows. Figure 7 As shown in the figure, the separation is good.

[0174] Table 15

[0175]

[0176] 2.1.2 Selection of detection wavelength

[0177] The detection wavelengths reported in the literature are often for the determination of a specific component and may not be entirely suitable for the needs of fingerprint spectroscopy. In order to obtain multi-level information, a diode array detector is used, and an appropriate amount of the test solution is taken and scanned at a wavelength of 190-400 nm.

[0178] Chromatograms within the 240nm–320nm range were analyzed, with comparative analyses performed on spectra at wavelengths of 240nm, 254nm, 270nm, 300nm, and 320nm. The chromatographic results are as follows: Figure 8-12 As shown.

[0179] As can be seen from the chromatograms of the above wavelengths, the sample absorption is mainly in the wavelength range of 240nm to 270nm. Observation of the chromatograms at different wavelengths revealed that the baseline of the chromatogram at 240nm wavelength showed severe drift when eluted with a high proportion of organic phase; the baseline of the chromatogram after 254nm was stable and the information peaks were abundant. It was observed that as the absorption wavelength increased, the absorption of the chromatographic peaks in the high proportion of organic phase elution gradually weakened. In comparison, the baseline was stable at 254nm wavelength, with a large amount of information and good chromatographic peak resolution. Therefore, 254nm was finally selected as the determination wavelength.

[0180] 2.2 Investigation on the preparation of the test solution

[0181] 2.2.1 Selection of the preparation method for the test sample solution

[0182] Preparation of the test solution: Accurately weigh 0.5 g of the powder (passed through a No. 2 sieve), place it in a stoppered conical flask, and accurately add 25 mL of 50% methanol. Sonicate the solution (500 W power, 40 kHz frequency). Z Heat and reflux for 30 minutes each, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0183] The chromatogram is shown in Figure 13. The experimental results show that, overall, the two extraction methods have no effect on the chromatographic peak information, with only a slight difference in retention time. For ease of operation, the ultrasonic method is more suitable, and the extraction method is determined to be ultrasonic treatment.

[0184] 2.2.2 Selection of solvent for preparing test sample solution

[0185] Take 6 portions of the powder (passed through a No. 2 sieve), each 0.5 g, accurately weigh them, and place them in a stoppered conical flask. Accurately add 25 mL each of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol. Sonicate the mixture (500 W power, 40 kHz frequency) for 30 minutes, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake well, filter it, and collect the filtrate to obtain the final product.

[0186] Figure 14-19The chromatograms are shown in order using water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol as solvents. Experimental results show that when the methanol concentration is below 70%, the chromatographic peak information in the obtained chromatograms is basically consistent. However, when methanol or ethanol is used as the extraction solvent, the chromatographic peak information decreases, and peak adhesion occurs. Especially when ethanol is used as the extraction solvent, the chromatographic peaks are almost unresponsive. To maintain consistency with the content determination, 70% methanol is used as the extraction solvent.

[0187] 2.2.3 Examination of extraction time

[0188] Take three portions of the powder (passed through a No. 2 sieve), each 0.5g, accurately weigh them, place them in a stoppered conical flask, accurately add 25mL of 70% methanol, and sonicate them (power 500W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively. After cooling, weigh them again, make up the weight loss with 70% methanol, shake well, filter, and collect the filtrate to obtain the product.

[0189] The results are as follows Figure 20 As shown, from top to bottom, extraction times are 15 minutes, 30 minutes, and 60 minutes. The results indicate that extraction time has no effect on the retention time or response of any chromatogram; therefore, 15 minutes of sonication is sufficient.

[0190] 2.3 Selection of reference objects and identification of relevant components

[0191] The test solution was prepared according to the preparation method determined in Example 2. Chlorogenic acid, glycyrrhizin, cinnamic acid, glycyrrhizic acid, ferulic acid, harpagoside, luteolin, ligustilide, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid were used as reference solutions. 10 μl of each solution was accurately pipetted into the liquid chromatograph and determined.

[0192] The results are as follows Figure 21 As shown, S1. Simiao Yong'an Decoction reference sample, S2. Mixed reference standard, 1. Chlorogenic acid, 2. Ferulic acid, 3. Glycyrrhizin, 4. Luteolin, 5. Ligusticolide, 6. 3,5-di-O-caffeoylquinic acid, 7. 4,5-di-O-caffeoylquinic acid, 8. Cinnamic acid, 9. Harpagoside, 10. Ammonium glycyrrhizate. The results showed that all peaks of the test sample were clearly identifiable in the chromatograms compared to the above-mentioned reference substances. Furthermore, the glycyrrhizic acid peak response in the chromatogram of the test sample solution was stable, and the baseline was separated; therefore, glycyrrhizic acid was ultimately selected as the reference substance.

[0193] 2.5 Methodological Validation

[0194] 2.5.1 Specificity Examination

[0195] The specificity was examined, and the results were as follows: Figure 22As shown, from top to bottom, the samples are 70% methanol and the test sample. The experimental results indicate that the blank solvent has no interference, demonstrating good specificity.

[0196] 2.5.2 Holistic Examination

[0197] Extend the elution time by 30 minutes and continue eluting with a high organic phase ratio, observing whether the chromatogram shows complete elution. Overall results are as follows: Figure 23 As shown. The results indicate that the sample was completely eluted after 120 minutes, and the final chromatographic elution conditions were determined as follows:

[0198] Table 16 Chromatographic Elution Conditions

[0199]

[0200] 2.5.3 Instrument Precision Test

[0201] Take approximately 0.5 g of the powder (passed through a No. 2 sieve), accurately weigh it, and proceed according to the preparation and determination method of the test solution in Example 2. Inject the sample 6 times consecutively, calculate the relative retention time, and the results are as follows.

[0202] Table 17 Calculation Results of Instrument Precision-Relative Retention Time

[0203]

[0204] The results show that the relative retention time (RSD) of each characteristic peak is less than 1.0%, indicating that the instrument precision of this method is good.

[0205] 2.5.4 Repeatability Test

[0206] Take approximately 0.5 g of the powder (passed through a No. 2 sieve) (6 portions in total), weigh accurately, and operate according to the preparation and determination method of the test solution in Example 2. Calculate the relative retention time, and the results are as follows.

[0207] Table 18 Results of Repeatability-Relative Retention Time Calculation

[0208]

[0209] The results show that the relative retention time (RSD) of each characteristic peak is less than 1%, indicating that the method has good repeatability.

[0210] 2.5.5 Solvent stability test

[0211] Take approximately 0.5 g of the powder (passed through a No. 2 sieve), accurately weigh it, and operate according to the preparation and determination method of the test solution in Example 2. Inject the sample and determine the relative retention time after 0, 2, 16, 18, 20, 22, 24, and 38 hours after preparation. The results are as follows.

[0212] Table 19 Solvent stability - Relative retention time calculation results

[0213]

[0214] The results showed that the relative retention time (RSD) of each characteristic peak was less than 2.0%, indicating that the sample had good stability within 38 hours.

[0215] 2.5.6 Durability Test

[0216] 2.5.6.1 Selection of Column Temperature

[0217] The column temperatures were set at 25℃, 28℃, 30℃, 32℃, and 35℃ respectively for investigation. The chromatograms are shown below. Figure 24 As shown, S1 is a column temperature of 25℃, S2 is a column temperature of 28℃, S3 is a column temperature of 30℃, S4 is a column temperature of 32℃, and S5 is a column temperature of 35℃.

[0218] Experimental results show that column temperature has a significant impact on the characteristic chromatogram of the Simiao Yong'an Decoction reference sample. As the column temperature increases, the elution time of the chromatographic peaks advances, and the separation effect changes. Based on the principle of completeness of chromatographic peak information, a column temperature of 25℃ was selected after comprehensive evaluation. Experiments were then conducted with column temperatures set to 20℃, 22℃, and 25℃, and the results are as follows. Figure 25 As shown, S1 has a column temperature of 20℃, S2 has a column temperature of 22℃, and S3 has a column temperature of 25℃. The calculated results of the relative retention times of each characteristic peak are shown in Table 20.

[0219] Table 20 Calculation results of relative retention time during column temperature investigation.

[0220]

[0221]

[0222] As the column temperature increases, the elution time of the main chromatographic peaks advances, while the elution time of the S peak is delayed. The relative retention times of all characteristic peaks remain within ±10% of the mean. Therefore, this method exhibits good column temperature robustness.

[0223] 2.5.6.2 Selection of Flow Rate

[0224] The flow rate was set at 0.9 ml, 1.0 ml, and 1.1 ml per minute for the study. The results are as follows: Figure 26 As shown, S1. Flow rate 0.9 ml / min, S2. Flow rate 1.0 ml / min, S3. Flow rate 1.1 ml / min.

[0225] Experimental results show that flow rate has a significant impact on the characteristic chromatogram of Simiao Yong'an Decoction reference sample. As the flow rate increases, the retention time of each chromatographic peak gradually advances. Peaks 4 and 5 are not completely separated from nearby impurity peaks. The overall evaluation shows that a flow rate of 1.0 ml per minute is optimal. The provisional flow rate is 1.0 ml per minute. However, the flow rate durability of this method is poor.

[0226] 2.5.6.3 Selection of Formic Acid Solutions of Different Concentrations

[0227] The results were investigated using acetonitrile-0.15% formic acid solution, acetonitrile-0.2% formic acid solution, and acetonitrile-0.25% formic acid solution, respectively. The results are shown in the table below.

[0228] Table 21 Results of Calculation of Relative Retention Time Based on Acid Concentration

[0229]

[0230] The results showed that the number of major chromatographic peaks remained constant under different acid concentrations, and the separation was good. The relative retention times of each characteristic peak were all within the range of mean ± 10%. Therefore, this method is robust to different acid concentrations.

[0231] 2.5.6.4 Selection of Chromatographic Column

[0232] The HPLC chromatograms of the Simiao Yong'an Decoction reference sample were investigated using InertSustain C18 (4.6×250 mm, 5 μm) columns, Agilent ZORBAX SB-C18 (4.6×250 mm, 5 μm) columns, and WondaSil C18 (4.6×250 mm, 5 μm) columns, respectively. The chromatographic results are shown below. Figure 27 As shown, where: S1. WondaSil C18 S2. Agilent ZORBAX SB-C18 S3. InertSustain C18.

[0233] Table 22 Calculation results of relative retention times for different chromatographic columns

[0234]

[0235] Experimental results showed that different brands of chromatographic columns had varying separation effects on the HPLC chromatographic peaks of the Simiao Yong'an Decoction reference sample. Comparison of the three chromatographic columns revealed that, observing the peaks around 60-80 min, the Agilent ZORBAX SB-C18 column contained less peak information than the other two columns, making peaks 3-5 difficult to identify in the chromatogram. The InertSustain C18 and WondaSil C18 columns showed similar peak information and could both be used for the determination of the characteristic chromatograms of Simiao Yong'an Decoction.

[0236] 2.5.7 Intermediate Precision Experiment

[0237] Based on the above experimental results, six test samples were prepared in parallel by different personnel on different dates, and characteristic spectra were determined using high-performance liquid chromatography (HPLC) instruments of different brands. The retention times of each characteristic peak were calculated as follows:

[0238] Table 23 Characteristic Map Study - Intermediate Precision

[0239]

[0240] Table 24 Results of Intermediate Precision-Relative Retention Time Calculation

[0241]

[0242]

[0243] The results showed that the relative retention times of each characteristic peak in the spectra obtained from different devices did not vary much, the RSD was less than 5%, and the intermediate precision was good.

[0244] 3. Identification of characteristic peaks

[0245] 3.1 Chromatographic Peak Assignment

[0246] Following the preparation process for the Simiao Yong'an Decoction reference sample, negative samples lacking honeysuckle, positive and negative samples lacking scrophularia, negative samples lacking angelica, negative samples lacking licorice, positive samples of honeysuckle, positive samples of scrophularia, positive samples of licorice, and positive samples of angelica were prepared respectively. The preparation and determination methods were performed according to the test sample preparation section of Example 2. The results are as follows: Figure 28-31 As shown, Figure 28 For the specificity study of honeysuckle, S1. reference sample, S2. honeysuckle-deficient negative sample, S3. honeysuckle-positive sample; Figure 29 For the specificity study of licorice, S1. reference sample, S2. licorice-deficient negative sample, S3. licorice-positive sample; Figure 30 For the specificity study of Angelica sinensis, S1 is the reference sample, S2 is the negative sample lacking Angelica sinensis, and S3 is the positive sample of Angelica sinensis. Figure 31 For the specificity study of Scrophularia ningpoensis, S1 is the reference sample, S2 is the negative sample lacking Scrophularia ningpoensis, and S3 is the positive sample of Scrophularia ningpoensis.

[0247] The results showed that unique characteristic peaks could be found in the feature maps of the four medicinal materials.

[0248] 3.2 Characteristic Peak Calibration Results

[0249] The reference standards for each indicator component detected in the Chinese Pharmacopoeia were prepared into a reference solution of appropriate concentration using 70% methanol. Following the method in Example 2, the chemical components of the characteristic peaks were identified. The characteristic peak locations of Simiao Yong'an Decoction are shown in the chromatogram. Figure 32 As shown, the reference standards from left to right are 1 to 10, namely 1. chlorogenic acid, 2. ferulic acid, 3. glycyrrhizin, 4. luteolin, 5. ligustilide, 6. 3,5-di-O-caffeoylquinic acid, 7. 4,5-di-O-caffeoylquinic acid, 8. cinnamic acid, 9. harpagoside, and 10. ammonium glycyrrhizate.

[0250] The results showed that the above 10 compounds could be identified using reference standards in the characteristic spectrum of the Simiao Yong'an Decoction reference sample.

[0251] 4. Establishment of Feature Maps

[0252] 4.1 Identification of common peaks in characteristic spectra

[0253] The method of Example 2 was used to determine the reference peaks of 17 batches of Simiao Yong'an Decoction. The obtained chromatograms were analyzed, and nine characteristic peaks with good stability and suitable response values ​​were selected for calibration. Peak 9 showed moderate response value and good separation, therefore it was chosen as the reference peak. The results are as follows: Figure 33 As shown, where: S1.WZJZ14-21102101, S2.WZJZ14-21102102, S3.WZJZ14-21102103, S4.WZJZ14-21102104, S5.WZJZ14-21102105, S6.WZJZ14-21102801, S7.WZJZ14-21102802, S8.WZJZ14-21102803, S9.WZJZ14-2 1102804, S10.WZJZ14-21102805, S11.WZJZ14-21102806S12.WZJZ14-21110801, S13.WZJZ14-2111 0802S14.WZJZ14-21110803, S15.WZJZ14-21110804, S16.WZJZ14-21110805S17.WZJZ14-21110806.

[0254] 4.2 Requirements for relative retention time and relative peak area

[0255] The relative retention times and relative peak areas of nine characteristic peaks in the characteristic spectra of 17 batches of Simiao Yong'an Decoction reference samples were calculated, and the results are as follows.

[0256] Table 25 Calculation results of relative retention times for 17 batches of reference samples

[0257]

[0258] Table 26 Calculation results of relative retained peak areas of 17 batches of reference samples

[0259]

[0260]

[0261] Based on the above results, the RSD of the relative retention time of each characteristic peak does not exceed 2.0%, and can be included in the standard of characteristic spectrum; except for the S peak, the RSD of the relative retention peak area of ​​each characteristic peak to the S peak is large, all exceeding 2%, and should not be used as the standard for inclusion in the characteristic spectrum of Simiao Yong'an Decoction.

[0262] 4.3 Theoretical Plate Requirement

[0263] The theoretical plate number of glycyrrhizic acid peaks in the characteristic spectra of 17 batches of reference samples was analyzed and statistically analyzed. The results are shown in the table below.

[0264] Table 27

[0265]

[0266] The results showed that in the characteristic chromatograms of the 17 batches of reference samples, the theoretical plate number of the glycyrrhizic acid peak in each batch was not less than 3,000,000, while the theoretical plate number required for system suitability was determined to be not less than 5,000 based on the chlorogenic acid peak.

[0267] 4.4 Establishment of the reference map

[0268] A reference chromatogram was established using the HPLC chromatograms of 17 batches of Simiao Yong'an Decoction reference samples from the software system for evaluating the similarity of chromatographic characteristic chromatograms of traditional Chinese medicines (version 2012.130723) issued by the Chinese Pharmacopoeia Commission. The established chromatograms are shown below. Figure 1 As shown.

[0269] The chromatogram of the test sample should show 9 characteristic peaks. The peak corresponding to the reference is the S peak. Calculate the retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.20 (peak 1), 0.21 (peak 2), 0.62 (peak 3), 0.66 (peak 4), 0.74 (peak 5), 0.92 (peak 6), 0.99 (peak 7), 0.99 (peak 8), 1.00 [peak 9 (S)].

[0270] In summary, the characteristic spectral method established in this study exhibits good specificity, instrument precision, repeatability, stability, and robustness, and can be used as an important means to control the quality of Simiao Yong'an Decoction reference samples.

[0271] Experimental Example 3

[0272] The raw materials and reagents used are the same as in Experimental Example 2.

[0273] 2. Chromatographic Conditions Study

[0274] Preparation of the test sample: Take about 0.5g of the powder (passed through a No. 2 sieve), place it in a stoppered conical flask, weigh it accurately, add 25ml of 50% methanol accurately, weigh the total amount, sonicate (power 500W, frequency 40kHz) for 30 minutes, cool, weigh it again, make up the weight loss with 50% methanol, shake well, filter, and take the filtrate to obtain the test sample.

[0275] 2.1 Investigation of Mobile Phase Proportion

[0276] (1) Method 1:

[0277] An InertSustain C18 (4.6×250mm, 5μm) column was used; acetonitrile was used as mobile phase A, and 0.4% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the specifications in the table; the flow rate was 1.0 mL per minute; the detection wavelengths were 210 nm for harpagoside, 278 nm for harpagoside, and 327 nm for ferulic acid and 3,5-di-O-caffeoylquinic acid; the flow rate was 1.0 mL per minute; the column temperature was 38.0℃; and the injection volume was 20 μl.

[0278] Table 28 Investigation of Mobile Phase Ratio - Method 1

[0279]

[0280]

[0281] Mobile phase ratio determination - Chromatogram of Method 1 is shown below Figure 34 As shown, the detection wavelengths from top to bottom are 210 nm, 278 nm, and 327 nm; 1. harpagoside, 2. ferulic acid, 3. 3,5-di-O-caffeoylquinic acid, 4. harpagoside. The chromatograms show that the major peaks are largely separated. Harpagoside can be identified in the 210 nm spectrum, as can harpagoside in the 278 nm spectrum, and ferulic acid and 3,5-di-O-caffeoylquinic acid can be identified in the 327 nm spectrum.

[0282] (2) Method Two

[0283] An InertSustain C18 (4.6×250mm, 5μm) column was used; acetonitrile was used as mobile phase A, and 0.4% phosphoric acid was used as mobile phase B, with elution performed according to the gradient specified in the table below; the detection wavelengths for harpagoside and ferulic acid were 210nm and 278nm, respectively, and for 324nm, respectively; the column temperature was 30℃, and the injection volume was 20μl.

[0284] Table 29 Investigation of Mobile Phase Ratio - Method Two

[0285]

[0286] The chromatogram of method two for investigating the proportion of mobile phase is shown below. Figure 35 As shown, the detection wavelengths from top to bottom are 210 nm, 278 nm, and 324 nm; 1. harpagoside, 2. ferulic acid, 3. 3,5-di-O-caffeoylquinic acid, 4. harpagoside. It can be seen from the figure that the major chromatographic peaks have been largely separated. Harpagoside can be identified in the 210 nm spectrum, harpagoside in the 278 nm spectrum, and ferulic acid and 3,5-di-O-caffeoylquinic acid can be identified in the 324 nm spectrum.

[0287] Both chromatographic conditions above can be used for content determination. However, the pH value of 0.4% phosphoric acid is too low. Therefore, only the mobile phase B is changed to 0.1% phosphoric acid, which becomes Method 3 and Method 4. The above two conditions are then used to re-examine the content.

[0288] (3) Method 3 and Method 4

[0289] An InertSustain C18 (4.6×250mm, 5μm) column was used; acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the specifications in Method 1 and Method 2, and the results are as follows.

[0290] The chromatogram of method three for mobile phase ratio determination is shown below. Figure 36 As shown, the detection wavelengths from top to bottom are 210nm, 278nm and 324nm; 1 harpagoside, 2 ferulic acid, 3 3,5-di-O-caffeoylquinic acid, 4 harpagoside;

[0291] The chromatogram of method four for mobile phase ratio determination is shown below. Figure 37 As shown, the detection wavelengths from top to bottom are 210nm, 278nm and 327nm; 1 harpagoside, 2 ferulic acid, 3 3,5-di-O-caffeoylquinic acid, 4 harpagoside.

[0292] The results showed that after changing the mobile phase to 0.1% phosphoric acid, the chromatographic peaks of each indicator component were symmetrical, and the resolution met the detection requirements (R > 1.5), but method three ( Figure 36 The matching degree of the harpagoside in the peak was only 919 (less than 950), which can be considered as insufficient purity of this peak.

[0293] Method 4 Figure 37All peaks met the resolution and purity requirements. Under these conditions, the method was converted to an ultra-high efficiency method using a Hypersil GOLD C18 (2.1×100mm, 1.9μm). After optimization, it was found that after the purity of the other chromatographic peaks reached a high value (matching degree > 990), the purity of harbazoside was low. Therefore, the harbazoside index was discarded, and the other three components were detected instead.

[0294] (4) Final Method

[0295] A Hypersil GOLD C18 (2.1×100 mm, 1.9 μm) column was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B; the detection wavelengths for harpagoside and ferulic acid were 210 nm and 278 nm, respectively, while the detection wavelengths for ferulic acid and 3,5-di-O-caffeoylquinic acid were 324 nm. The column temperature was 30 °C, and the injection volume was 1 μl.

[0296] Table 30

[0297]

[0298] Investigation of mobile phase ratio - final conditions as follows Figure 38 As shown, the detection wavelengths from top to bottom are 210nm, 278nm and 324nm; 1 harpagoside, 2 ferulic acid, 3 3,5-di-O-caffeoylquinic acid, 4 harpagoside.

[0299] Under these mobile phase conditions, good separation of each peak can be achieved in a relatively short time, and the chromatographic peaks of each index component are symmetrical and have high purity.

[0300] The original method used a 5 μm particle size column for elution, while the optimized method uses a 1.9 μm particle size column for elution. The elution time is reduced to 28% of the original method, which improves the detection efficiency. The buffer salt solution was changed from 0.4% phosphoric acid to 0.1% phosphoric acid, which better protects the equipment and column. The mobile phase elution rate was reduced from 1.0 ml per minute to 0.5 ml per minute, which saves at least 80% of the mobile phase solution, making the detection method more economical.

[0301] 2.2 Selection of detection wavelength and check of peak purity of index components

[0302] Based on the tentatively established chromatographic conditions described above, the test solution was scanned using a diode array detector at wavelengths of 190–400 nm, with a step size of 2 nm. Simultaneously, the purity of the chromatographic peaks of the indicator components in the test solution chromatogram was checked. The results are shown in the table below.

[0303] Table 31 Purity test of each indicator component in the test sample

[0304]

[0305]

[0306] Table 32 Peak information of each indicator component in the test sample

[0307]

[0308] The test results showed that in the chromatogram of the reference sample, the peak matching index of each peak at the same retention time of the reference standard peak exceeded 980, indicating good peak purity. The spectral absorption curves showed that ferulic acid had a maximum absorption at approximately 325 nm, close to the 324 nm specified in the method; 3,5-di-O-caffeoylquinic acid had a maximum absorption at 327 nm, also close to 324 nm; and harpagoside had a maximum absorption at approximately 281 nm, close to the 278 nm specified in the method. Therefore, the 278 nm and 324 nm wavelengths selected under the chromatographic conditions are suitable as the detection wavelengths for the determination of the content of the Simiao Yong'an Decoction reference sample.

[0309] 2.3 Results of Chromatographic Condition Investigation

[0310] Based on the above experiments, the chromatographic conditions for determining the content of Simiao Yong'an Decoction in the reference sample are tentatively set as follows:

[0311] A chromatographic column (2.1 × 100 mm, 1.9 μm) was used; acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B; harpagoside was detected at a wavelength of 278 nm, ferulic acid and 3,5-di-O-caffeoylquinic acid were detected at a wavelength of 324 nm; the column temperature was 30 °C.

[0312] Table 33 Mobile phase elution conditions

[0313]

[0314] Preparation of reference solutions: Weigh appropriate amounts of ferulic acid, 3,5-di-O-caffeoylquinic acid, and harpagoside reference standards accurately, place them in a brown volumetric flask, and add 50% methanol to prepare a mixed solution containing 10 μg, 30 μg, and 30 μg per ml, respectively.

[0315] The assay involves precisely pipetting 1 μl of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0316] 3. Specificity Research

[0317] Following the preparation process of the Simiao Yong'an Decoction reference sample in Example 1, negative samples of Simiao Yong'an Decoction lacking Scrophularia ningpoensis (batch number: WZJZ14-21060701P), negative samples of Simiao Yong'an Decoction lacking Angelica sinensis (batch number: WZJZ14-21060702P), and negative samples of Simiao Yong'an Decoction lacking Lonicera japonica (batch number: WZJZ14-21060703P) were prepared. The above samples and the Simiao Yong'an Decoction reference sample (batch number: WZJZ14-21043001P) were taken, and their contents were determined according to "2.3 Chromatographic Conditions Exploration Results". The results are as follows: Figures 39-41 As shown. Figure 39 For the specificity results of harpagoside (278nm), from top to bottom, the mixed standard reference, the Simiao Yong'an Decoction reference sample, and the negative sample lacking Scrophularia ningpoensis; Figure 40 The specificity results for 3,5-di-O-caffeoylquinic acid (324nm) are as follows: from top to bottom, mixed standard reference, Simiao Yong'an Decoction reference sample, and negative sample lacking honeysuckle. Figure 41 For the specificity results of ferulic acid (324nm), from top to bottom are the mixed standard reference, the Simiao Yong'an Decoction reference sample, and the negative sample lacking Angelica sinensis.

[0318] The results above show that the blank solvent has no effect on the chromatographic peaks of the index components. Ferulic acid and harpagoside have strong specificity. 3,5-Di-O-caffeoylquinic acid shows a small peak at the same position in the chromatogram of the honeysuckle-deficient negative sample, with a peak area approximately 8% larger than that of the reference sample, which may affect the determination results. Furthermore, the peak matching index of this peak in the negative sample is 872, indicating that this peak is impure. Therefore, 3,5-Di-O-caffeoylquinic acid was removed from the list of index components, and chlorogenic acid was added as the index content. The specificity verification results of chlorogenic acid (324nm) are as follows: Figure 42 As shown, from top to bottom are the mixed standard reference, the Simiao Yong'an Decoction reference sample, and the negative control lacking honeysuckle.

[0319] The results showed that a small peak, approximately 1% of the area, appeared at the corresponding position in the chromatogram of the chlorogenic acid reference standard in the negative sample lacking honeysuckle. The purity of this peak in the negative sample was 966, indicating acceptable purity. Therefore, the specificity of this method for chlorogenic acid was considered good. The absorption spectrum showed that the maximum absorption of chlorogenic acid was at 326 nm, very close to the 324 nm specified in the method; therefore, 324 nm was used as the detection wavelength for chlorogenic acid. The chromatogram of the mixed standard reference standard is shown below. Figure 43 As shown, 1. chlorogenic acid, 2. ferulic acid, and 3. harpagoside.

[0320] The final chromatographic conditions for content determination are as follows:

[0321] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel (ThermoFisher Hypersil 2.1 × 100 mm, 1.9 μm) as the stationary phase; acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B; harpagoside was detected at 278 nm, and chlorogenic acid and ferulic acid were detected at 324 nm; the flow rate was 0.5 mL / min, and the column temperature was 30.0 °C, with gradient elution performed according to the table below. The theoretical plate number, calculated based on chlorogenic acid, should not be less than 10,000.

[0322] Table 34

[0323]

[0324] 4. Study on the preparation method of the test solution

[0325] 4.1 Examination of Extraction Methods

[0326] Take three groups of powdered product (passed through a No. 2 sieve), two portions of 0.5g each, accurately weighed, and place them in separate stoppered conical flasks. Accurately add 25ml of 70% methanol to each flask. Perform reflux heating, ultrasonic treatment (500W power, 40kHz frequency), and shaking for 30 minutes respectively. Cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate to obtain the test solution. Determine the sample according to the method in Example 3, compare the results, and select a suitable extraction method.

[0327] Table 35. Results of the Examination of Extraction Methods

[0328]

[0329] The test results showed that the content values ​​of the three extraction methods were relatively close. Among them, the extraction rate of ferulic acid and harpagoside was higher when using ultrasonic extraction, and the operation was convenient. Therefore, ultrasonic extraction was finally selected.

[0330] 4.2 Investigation of Extraction Solvents

[0331] Take six groups of powdered product, two portions of each group, each 0.5g, accurately weighed, and place them in separate stoppered conical flasks. Accurately add 25ml each of water, 30% methanol, 50% methanol, 70% methanol, methanol, and ethanol to each flask. Sonicate (500W power, 40kHz frequency) for 30 minutes, cool, and weigh again. Make up the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate to obtain the test solution. Determine the sample according to Example 3, compare the test results, and select a suitable extraction solvent.

[0332] Table 36 Results of Solvent Overload Test

[0333]

[0334] The test results showed that different solvents had different extraction rates for harpagoside, chlorogenic acid and ferulic acid. When the solvent was 70% methanol, the extraction rates of all three components were relatively high. Therefore, 70% methanol was chosen as the extraction solvent.

[0335] 4.3 Extraction volume assessment

[0336] Take three groups of powdered product, two portions of each group, each 0.5g, accurately weighed, and place them in separate stoppered conical flasks. Accurately add 25ml of 70% methanol to each flask, and sonicate (500W power, 40kHz frequency) for 15, 30, and 60 minutes respectively. After cooling, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate to obtain the test solution. Determine the sample according to the method in Example 3, compare the results, and select an appropriate extraction volume.

[0337] Table 37 Results of Extraction Solvent Testing

[0338]

[0339] The test results showed that the extraction volume affected the extraction of harpagoside, chlorogenic acid, and ferulic acid. When using 10 ml of solvent for extraction, the content of the three components decreased significantly. The content was higher when using 25 ml and 50 ml of solvent. The measured values ​​at 25 ml were more than 95% higher than those at 50 ml. After comprehensive consideration, the extraction solvent volume was selected as 25 ml, at which point the extraction volume was 50 times.

[0340] 4.4 Examination of extraction time

[0341] Take three groups of powdered product, two portions of each group, each 0.5g, accurately weighed, and place them in separate stoppered conical flasks. Accurately add 25ml of 70% methanol to each flask, and sonicate (500W power, 40kHz frequency) for 15, 30, and 60 minutes respectively. After cooling, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate to obtain the test solution. Determine according to Example 3, compare the test results, and select an appropriate extraction time.

[0342] Table 38 Results of Extraction Time Examination

[0343]

[0344] The test results showed that when using ultrasound to extract the sample powder, the extraction time had little effect on the determination results of the contents of the three components. Therefore, the extraction time was selected as 15 minutes.

[0345] 4.5 Examination of the number of extractions

[0346] Take three groups of this product powder, two portions of each group, each portion about 0.5g, weigh accurately, and place them in a stoppered conical flask.

[0347] Add 25 ml of 70% methanol to Group 1, seal tightly, weigh, sonicate (500W power, 40kHz frequency) for 15 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain an extract.

[0348] The second group was extracted by repeating the above steps. The insoluble matter with filter paper was placed in a stoppered conical flask, 25 ml of 70% methanol was accurately added, the flask was sealed tightly, the weight was measured, and the flask was extracted by sonication for 15 minutes. After cooling, the flask was weighed again, and the weight was replenished with 70% methanol to make up the weight loss. The flask was shaken well, filtered, and the second extract was obtained. The filtrates were combined.

[0349] The third group repeated the above steps to obtain the first and second extracts. The insoluble matter from the second extraction, along with filter paper, was placed in a stoppered conical flask, and 25 ml of 70% methanol was precisely added. The flask was sealed tightly, weighed, and ultrasonically extracted for 15 minutes. After cooling, the flask was weighed again, and the lost weight was replenished with 70% methanol. The flask was shaken well and filtered to obtain the third extract. The filtrates were combined. The results were compared according to the method in Example 3 to determine the appropriate number of extractions.

[0350] Table 39 Results of content detection based on extraction frequency

[0351]

[0352] The test results showed that harpagoside and ferulic acid were basically completely extracted with one extraction, and the extraction rate of chlorogenic acid reached 95.7% compared with three extractions. Considering the reproducibility and operability of the method, one extraction was selected.

[0353] 5. Methodological Validation Studies

[0354] Referring to the relevant content of the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China, the robustness, precision, linearity, and accuracy of the methods established in the above studies were verified.

[0355] 5.1 Instrument Precision

[0356] Take one sample of Simiao Yong'an Decoction (WZJZ14-21043001P) and test it according to the method of "Example 3". Inject the sample 6 times consecutively, record the peak area of ​​each reference standard, and calculate the deviation. See Table 40.

[0357] Table 40 Calculation results of instrument precision deviation

[0358]

[0359] The results showed that the RSD of each chromatographic peak did not exceed 2.0%, indicating good instrument precision.

[0360] 5.2 Linearity Examination

[0361] Accurately weigh appropriate amounts of harpagoside reference standard, ferulic acid reference standard, and chlorogenic acid, and place them in a brown volumetric flask. Add 70% methanol to prepare a mixed solution containing 30 μg, 10 μg, and 0.16 mg per ml, with actual concentrations of 30.5664 μg / ml, 9.749152 μg / ml, and 177.13152 μg / ml, respectively. Inject 0.2, 0.5, 1.0, 2.0, 3.0, and 4.0 μl of each solution, respectively, and detect according to the method in "Example 3". Plot the reference standard mass (ng) on ​​the x-axis and the peak area on the y-axis, and perform linear regression calculation.

[0362] The linear regression equation for harpagoside is: Y = 0.0618X + 0.0308

[0363] Correlation coefficient: R = 0.9999

[0364] The results showed that harpagoside exhibited good linearity in the range of 6.1133 ng to 122.2656 ng.

[0365] Table 41 Results of linearity assay for harpagoside

[0366]

[0367] Linear regression equation for chlorogenic acid: Y = 0.0826X + 0.2302 Correlation coefficient: R = 1.0000

[0368] The results showed that chlorogenic acid exhibited good linearity in the range of 35.4263 ng to 708.5261 ng.

[0369] Table 42 Results of linearity testing for chlorogenic acid

[0370]

[0371] Linear regression equation for ferulic acid: Y = 0.1437X + 0.0136;

[0372] Correlation coefficient: R = 0.9999

[0373] The results showed that ferulic acid exhibited good linearity in the range of 1.9483 ng to 38.9966 ng.

[0374] Table 43 Results of linearity test for ferulic acid

[0375]

[0376] The results showed that harpagoside exhibited good linearity in the range of 6.11328 ng to 122.2656 ng; chlorogenic acid exhibited good linearity in the range of 35.426304 ng to 708.52608 ng; and ferulic acid exhibited good linearity in the range of 1.948304 ng to 38.996608 ng.

[0377] 5.3 Precision Test

[0378] 5.3.1 Repeatability Test

[0379] Take 6 samples of the Simiao Yong'an Decoction reference sample (passed through a No. 2 sieve) and determine the content according to the method in "Example 3".

[0380] Table 44 Results of Repeatability Testing

[0381]

[0382] The results showed that RSD% < 2.0%, and the repeatability met the requirements.

[0383] 5.3.2 Intermediate Precision Examination

[0384] The content was determined by different analysts on different dates using the Simiao Yong'an Decoction reference sample (batch number: WZJZ14-21102806) according to the method in "Example 3".

[0385] Table 45 Results of Intermediate Precision Testing

[0386]

[0387] The results showed that RSD% < 5.0%, and the intermediate precision met the requirements.

[0388] 5.4 Accuracy Examination

[0389] Using the equal-volume addition method, nine portions of the Simiao Yong'an Decoction reference sample (batch number: WZJZ14-21043001P), each approximately 0.3g, were accurately weighed and placed into stoppered conical flasks.

[0390] Accurately weigh 11.99 mg of harpagoside reference standard (batch number: 111730-201508, purity 96.0%), place it in a 100 ml volumetric flask, dilute to the mark with 70% methanol, and shake well to obtain the harpagoside reference standard stock solution. Accurately pipette 1.5 ml, 3.0 ml, and 4.5 ml (three portions each) into the above-mentioned conical flasks.

[0391] Accurately weigh 37.48 mg of chlorogenic acid reference standard (batch number: 110753-202018, purity 96.1%), place it in a 100 ml volumetric flask, dilute to the mark with 70% methanol, and shake well to obtain the chlorogenic acid reference standard stock solution. Accurately pipette 2.5 ml, 5.0 ml, and 7.5 ml (3 portions each) into the above-mentioned conical flasks.

[0392] Accurately weigh 9.98 mg of ferulic acid reference standard (batch number: 110773-201915, purity 99.4%), place it in a 100 ml volumetric flask, dilute to the mark with 70% methanol, and shake well to obtain the ferulic acid reference standard stock solution. Accurately pipette 0.5 ml, 1 ml, and 1.5 ml (three portions each) into the above conical flasks.

[0393] Add 20.5 ml, 16 ml, and 11.5 ml of 70% methanol (three portions each) precisely, seal tightly, and weigh. Sonicate each portion (500 W, 40 kHz) for 15 minutes, cool, weigh again, add 70% methanol to make up the lost weight, shake well, filter, and collect the filtrate to obtain the test solution. Determine the content according to the chromatographic conditions of "Example 3". Calculate the recovery rate. The detection results are shown in the table below.

[0394] Table 46 Accuracy Test Results - Hapagoside

[0395]

[0396]

[0397] Table 47 Accuracy Test Results - Chlorogenic Acid

[0398]

[0399] Table 48 Accuracy Test Results - Ferulic Acid

[0400]

[0401] The content of harpagoside, chlorogenic acid, and ferulic acid in the Si Miao Yong An Tang reference sample (batch number: WZJZ14-21043001P) was 0.123%.

[0402] The results showed that the average recoveries of harpagoside, chlorogenic acid, and ferulic acid were 99.07%, 95.75%, and 94.84%, respectively, with RSDs all less than 3.0%, indicating that the method had good accuracy.

[0403] 5.5 Durability

[0404] Two samples of Simiao Yong'an Decoction (passed through a No. 2 sieve) were taken and their content was determined according to the method in "Example 3". The robustness of the column temperature, flow rate, phosphoric acid solution concentration, chromatographic column, and solvent stability in the method was investigated, and the results are as follows.

[0405] Figure 44 Chromatograms were obtained using different flow rates: 1. Chlorogenic acid, 2. Ferulic acid, 3. Harpagoside, S1. 0.48 ml / ml, S2. 0.50 ml / ml, S3. 0.52 ml / ml.

[0406] Figure 45 Chromatograms were obtained using different phosphoric acid solution concentrations: 1. Chlorogenic acid, 2. Ferulic acid, 3. Harpagoside, S1. 0.08% phosphoric acid solution, S2. 0.10% phosphoric acid solution, S3. 0.12% phosphoric acid solution.

[0407] Table 49 Results of the durability study of harpagoside

[0408]

[0409] Table 50 Results of Chlorogenic Acid Durability Study

[0410]

[0411] Table 51 Results of Ferulic Acid Durability Study

[0412]

[0413]

[0414] The results show that the peak resolution of harpagoside reference standard improves with increasing flow rate. At a flow rate of 0.48 ml / min, the resolution between the harpagoside chromatographic peak and the leading edge is less than 1.5, making quantitative determination impossible. This method has poor durability with varying flow rates and requires a flow rate of at least 0.5 ml / min. Changes in acid concentration have little impact on the elution time and resolution of each chromatographic peak, indicating good durability with varying acid concentrations.

[0415] Figure 46 These are the column temperature results (278nm), from top to bottom: 25℃, 30℃, 35℃; Figure 47 The results of the column temperature investigation (324nm) are shown from top to bottom: 25℃, 30℃, and 35℃.

[0416] When investigating column temperature, the method was set at 25℃, 30℃, and 35℃. Results showed that at 25℃, the harpagoside peak overlapped with surrounding peaks. After calculating its content, it was found that the content at 25℃ was 0.199%, significantly higher than the normal range of 0.12%–0.13%. At 35℃, a small peak appeared after the ferulic acid reference standard peak, and separation was poor. Analysis of the sample at 35℃ revealed impurities in the ferulic acid peak, affecting the integration. Therefore, this method recommends using a column temperature of 30℃.

[0417] When studying column types, columns of different brands with the same packing material, particle size, column length, and diameter were used for testing and comparison. The results are as follows: Figure 48 As shown. Figure 48 The results of the column robustness test (278nm) are shown below, from top to bottom: Hypersil GOLD C18 and Shim-pack Scepter HD-C18-80.

[0418] The results showed that when using the Shimadzu Shim-pack Scepter HD-C18-80 column, small peaks appeared at the tails of the chromatographic peaks of chlorogenic acid and harpagoside, resulting in a resolution of less than 1.5, which would affect the content calculation. Therefore, the Thermo Fisher Hypersil GOLD C18 column is more suitable.

[0419] Table 52 Results of Solvent Stability Study

[0420]

[0421] The results showed that the RSD of the peak area of ​​the test sample did not exceed 3.0%, indicating that the test sample solution had good stability within 18 hours.

[0422] 5.6 Theoretical Plate Requirement

[0423] Seventeen batches of Simiao Yong'an Decoction reference samples were tested using the above method. Systematic adaptability analysis was performed on the chromatographic peak of chlorogenic acid, and its theoretical plate number distribution was statistically analyzed.

[0424] Table 53

[0425]

[0426] Based on the above statistical results, the theoretical plate number of the chlorogenic acid peak is consistently above 50,000. Referring to the national drug standard YBZ-PFKL-2021073 "Honeysuckle Formula Granules" [Content Determination], the theoretical plate number calculated based on the chlorogenic acid peak should not be less than 10,000. Therefore, the theoretical plate number in the system suitability requirement is determined to be no less than 10,000 calculated based on the chlorogenic acid peak.

[0427] 6. Determination of content in reference samples

[0428] Following the procedure described in Example 3 for the preparation and determination of the test solution, the contents of chlorogenic acid, ferulic acid, and harpagoside in 17 batches of reference samples were determined. The results are shown in Table 54.

[0429] Table 541 Results of content determination in reference samples

[0430]

[0431]

[0432] The results showed that the content of harpagoside in 17 batches of Simiao Yong'an Decoction reference samples ranged from 0.047% to 0.149%, with an average of 0.092%. Considering various influencing factors in actual production, and allowing for fluctuations within ±30% of the average, it was tentatively determined that this product, calculated on a dried basis, contains harpagoside (C... 24 H 30 O 11 The value should be between 0.064% and 0.120%.

[0433] The chlorogenic acid content of 17 batches of reference samples ranged from 0.94% to 1.26%, with an average of 1.10%. Considering various influencing factors in actual production, the content was adjusted to fluctuate within ±30% of the average. Therefore, based on the dried product, the content of chlorogenic acid (C0.05) is tentatively determined. 16 The concentration of H8O9 should be between 0.77% and 1.43%.

[0434] The ferulic acid content of 17 batches of reference samples ranged from 0.028% to 0.061%, with an average of 0.036%. Considering various influencing factors in actual production, the content was adjusted to fluctuate within ±30% of the average. Therefore, based on the dried product, the content of ferulic acid (C...) is tentatively determined. 10 H 10 O4) should be 0.025% to 0.047%.

[0435] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a Simiao Yong'an Decoction reference sample, wherein the raw materials of Simiao Yong'an Decoction include honeysuckle, scrophularia, angelica, and licorice, characterized in that... include: (1) Prepare the test solution and reference solution of the reference sample; The preparation method of the test solution includes: taking 0.5g of Simiao Yong'an Decoction sample powder, accurately weighing it, placing it in a stoppered conical flask, accurately adding 25ml of 70% methanol, weighing it, sonicating it for 15 minutes, cooling it, weighing it again, replenishing the lost weight with 70% methanol, shaking it well, filtering it, and taking the filtrate to obtain the test solution. (2) The detection was performed by high performance liquid chromatography. The chromatographic conditions included: using octadecylsilane-bonded silica gel as the packing material; using acetonitrile as mobile phase A and 0.2% formic acid as mobile phase B, and performing gradient elution according to the specifications in the table below; the detection wavelength was 254 nm; and the theoretical plate number, calculated based on glycyrrhizic acid, was not less than 5000. The elution gradient is as follows: The reference standards include: chlorogenic acid, ferulic acid, glycyrrhizin, luteolin, ligustilide, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, cinnamic acid, harpagoside, and ammonium glycyrrhizate.

2. The construction method according to claim 1, characterized in that, The flow rate was 1.0 ml per minute, and the column temperature was 25°C.

3. The construction method according to claim 1, characterized in that, The chromatographic column used was: InertSμstain C184.6×250mm, 5μm.

4. The construction method according to any one of claims 1-3, characterized in that, In step (1), the ultrasonic treatment power is 500W and the frequency is 40kHz.

5. The application of a method for constructing a characteristic spectrum of a Simiao Yong'an Decoction reference sample as described in any one of claims 1-4 in the quality testing of Simiao Yong'an Decoction.

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