Method of establishing characteristic spectrum of tangerine peel sputum cough preparation and application

Abstract

The application discloses a characteristic spectrum method for Juhong Tanke preparation and application, wherein (1) the preparation of the test sample solution: the Juhong Tanke preparation is weighed, dissolved in a 50% methanol solution, filtered, and the filtrate is obtained; (2) determination: the test sample solution is taken and injected into a high performance liquid chromatograph for determination, so as to obtain the characteristic spectrum of the Juhong Tanke preparation. The application first establishes a detection method which can simultaneously identify four main medicinal materials in the Juhong Tanke preparation. The characteristic spectrum contains seven characteristic peaks, corresponding to amygdalin, naringin, wild lacquer tree glycoside, orange peel lactone hydrate, isoscutellarein, schisandrol A and a component derived from licorice. Four characteristic components belong to the Juhong Tanke preparation, one characteristic component belongs to the bitter almond, one characteristic component belongs to the Schisandra chinensis, and one characteristic component belongs to the licorice. Through the characterization of the multi-medicinal material components, the system comprehensive evaluation of the overall quality of the Juhong Tanke preparation is realized.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and specifically relates to the construction and application of a characteristic fingerprint method for a tangerine peel cough preparation. Background Art

[0002] The tangerine peel cough preparation is composed of eight traditional Chinese medicines, namely tangerine peel, stemona tuber (honey-fried), poria cocos, pinellia ternata (processed), whitefronted goosefoot, liquorice root, bitter apricot kernel and schizandra berry, and has the effects of regulating qi and resolving phlegm, moistening the lungs and relieving cough. Clinically, it can be used for cough with excessive phlegm, asthma caused by cold, bronchitis and pharyngolaryngitis. However, at present, there are many manufacturers of commercially available tangerine peel cough preparations, and the dosage forms include mixture (oral liquid), decoction extract and granules, etc. However, the quality standards implemented by different dosage forms are different and the overall level is low, which seriously restricts the clinical application of this prescription preparation. Among them, the tangerine peel cough granules implement the drug standard of the National Medical Products Administration (WS3-B-2819-97-2-2020), the tangerine peel cough decoction extract implements the eighteenth volume of the "Drug Standard for Traditional Chinese Medicine Formulas of the Ministry of Health" (WS3-B-3522-98), and the tangerine peel cough liquid is included in the "Chinese Pharmacopoeia" (Part I) of 2025 edition.

[0003] Among the above standards, the current standards for tangerine peel cough granules and tangerine peel cough decoction extracts have not established fingerprint or characteristic fingerprints, lack systematic quality control indicators for the main active substances, and it is difficult to effectively monitor the production process and the quality of finished products, and it is also difficult to meet the actual needs of modern and refined evaluation of the quality standards of traditional Chinese medicines.

[0004] In addition, although the quality standard for tangerine peel cough liquid in the "Chinese Pharmacopoeia" of 2025 edition has included the item of [Characteristic Fingerprint], and unified the preparation method and chromatographic conditions of the test samples under the items of [Characteristic Fingerprint] and [Content Determination], there are still many deficiencies: (1) The gradient elution program adopted is relatively complex and the result reproducibility is poor; (2) The tangerine peel cough liquid is composed of eight medicines, but only the monarch drug tangerine peel is specified under the items of [Characteristic Fingerprint] and [Content Determination], and only whitefronted goosefoot among the remaining medicinal materials is identified by thin-layer chromatography. There is no effective quality control for the bitter apricot kernel, which has certain toxicity in the formula, and the overall quality evaluation coverage is seriously insufficient; (3) The selected detection wavelength of 320 nm in the items of [Characteristic Fingerprint] and [Content Determination] is not the maximum absorption wavelength of the active ingredients naringin and rhoifolin, and the error of content determination at this wavelength is relatively large, affecting the accuracy of the results; (4) The brand and model of the chromatographic column are limited in the [Characteristic Fingerprint], and the method applicability is poor, which seriously restricts the practical application value of this method. Summary of the Invention

[0005] The purpose of this invention is to provide a detection method for tangerine peel cough preparations. This method can simultaneously identify multiple main medicinal materials in the preparation and achieve online synchronous analysis of characteristic spectrum construction and key component content determination. The method has good repeatability, accuracy, applicability, and durability, and also has the advantages of low solvent toxicity and environmental friendliness, enabling full-process monitoring and quality evaluation of tangerine peel cough preparations.

[0006] The first objective of this invention is to provide a method for constructing a characteristic spectrum of a tangerine peel cough preparation, which includes the following steps:

[0007] 1) Preparation of the test solution:

[0008] Weigh out the tangerine peel cough preparation, dissolve it in a 50-100% methanol solution, filter it, and take the filtrate to obtain the test solution.

[0009] (2) Measurement

[0010] The test solution was injected into a high-performance liquid chromatograph for determination, and the characteristic chromatogram of the tangerine peel cough preparation was obtained.

[0011] The chromatographic conditions are as follows:

[0012] The chromatographic column was a C18 column. The mobile phase was methanol as mobile phase A and an acidic aqueous solution with pH 2.0~3.5 as mobile phase B. Gradient elution was used: 0-80 min, mobile phase A 10-90%, mobile phase B 90-10%, flow rate 0.7~1.2 ml / min, column temperature 25~35℃, detection wavelength: 0.00~19.00 min 207±2 nm, 19.01~50.00 min 320±2 nm, 50.01~80.00 min 250±2 nm.

[0013] The characteristic peaks of the described tangerine peel cough preparation are as follows: amygdalin, relative retention time 0.47~0.52 min; naringin, relative retention time 0.95~1.05 min; rosin, relative retention time 1.05~1.17 min; hesperidin hydrate, relative retention time 1.13~1.25 min; isohesperidin, relative retention time 1.29~1.43 min; schisandrol A, relative retention time 1.51~1.68 min; and characteristic components of licorice, relative retention time 1.79~1.99 min.

[0014] Preferably, the tangerine peel cough preparation includes oral liquid, decoction, syrup, granules, capsules, pills, tablets or powder.

[0015] Preferably, the acidic aqueous solution is a 0.1% phosphoric acid solution.

[0016] Preferably, the chromatographic conditions are as follows:

[0017] The chromatographic column was a C18 column. The mobile phase consisted of methanol (mobile phase A) and 0.1% phosphoric acid solution (mobile phase B), using gradient elution: 0-80 min, mobile phase A 10-90%, mobile phase B 90-10%, flow rate 1.0 mL / min, column temperature 35℃, and detection wavelengths: 0.00-19.00 min 207 nm, 19.01-50.00 min 320 nm, 50.01-80.00 min 250 nm.

[0018] Preferably, the dissolution by adding a methanol solution with a volume fraction of 50-100% refers to the dissolution by adding a methanol solution with a volume fraction of 50%.

[0019] The tangerine peel cough preparation contains the following raw materials: tangerine peel, bitter almond, pinellia or water pinellia, stemona, cynanchum, poria, schisandra and licorice.

[0020] The second objective of this invention is a detection method for tangerine peel cough preparations. This method involves using the tangerine peel cough preparation to be tested as a sample and performing the determination according to the steps in the method for constructing the characteristic chromatogram of tangerine peel cough preparations. A common pattern is generated using traditional Chinese medicine chromatographic fingerprint similarity software. Then, the characteristic chromatogram of the tangerine peel cough preparation is compared with the common pattern of the same preparation to evaluate the similarity of the traditional Chinese medicine chromatographic fingerprint. If the similarity is greater than or equal to 0.9, it is considered to be the tangerine peel cough preparation.

[0021] A third objective of this invention is to provide a method for determining the content of characteristic components in a tangerine peel cough syrup preparation, comprising the following steps:

[0022] (1) Preparation of reference solution:

[0023] Take appropriate amounts of amygdalin, naringin, and rhubarb glycoside reference standards, dissolve them in methanol, and the solution is obtained.

[0024] (2) Preparation of the test solution:

[0025] Weigh out the tangerine peel cough preparation, dissolve it in a 50-100% methanol solution, filter it, and take the filtrate to obtain the test solution.

[0026] (3) Measurement

[0027] Pipe the reference standard and test solution and inject them into the high performance liquid chromatograph for determination;

[0028] The chromatographic conditions are as follows:

[0029] The chromatographic column was a C18 column. The mobile phase was methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was used: 0-80 min, mobile phase A 10-90%, mobile phase B 90-10%, flow rate 1.0 ml / min, column temperature 35℃. The detection wavelengths were: 0.00~19.00 min 207±2 nm, 19.01~50.00 min 320±2 nm, 50.01~80.00 min 250±2 nm. Simultaneously, 283±2 nm and 338±2 nm were collected from 0.00~80.00 min as the detection wavelengths for naringin and arbutin.

[0030] Compared with existing technologies, this method achieves the following technological breakthroughs and innovations:

[0031] (1) This invention establishes for the first time a detection method that can simultaneously identify four main medicinal materials in a tangerine peel cough preparation. High-performance liquid chromatography with wavelength switching technology enables online separation and detection of characteristic components from multiple medicinal material sources. The characteristic spectrum contains seven characteristic peaks, corresponding to amygdalin, naringin, rosin, hesperidin hydrate, isohesperidin, schisandrol A, and licorice-related components. Among them, four characteristic components belong to tangerine peel, one to amygdalin, one to schisandra, and one to licorice. Through the characterization of multiple medicinal material components, a systematic and comprehensive evaluation of the overall quality of the tangerine peel cough preparation is achieved.

[0032] (2) This characteristic spectrum simultaneously meets the requirements for the determination of the content of key active ingredients. With a single injection, based on the high coverage characteristic spectrum, the content determination of three key components, including the principal drug Citrus reticulata and the slightly toxic bitter almond, is completed simultaneously, realizing the organic combination of qualitative identification and quantitative analysis.

[0033] (3) This method employs a one-step linear gradient elution procedure, resulting in high reproducibility. It does not impose limitations on the brand or model of the chromatographic column, greatly improving the precision, repeatability, applicability, and robustness of the method. Methanol is selected as the organic phase, which, compared to acetonitrile used in the current method for preparing citrus sputum expectorant, has lower toxicity and is also environmentally friendly. The method provides systematic methodological validation of analytical conditions such as sample solution preparation, mobile phase system, detection wavelength, elution procedure, column temperature, and flow rate. Attached Figure Description

[0034] Figure 1 This is an examination of the detection wavelength by the photodiode array detector;

[0035] Figure 2 It is a reference solution of amygdalin, peak 1: amygdalin; Figure 3It is a naringin reference solution, peak 2: naringin; Figure 4 It is a reference solution of rhubarb glycoside, peak 3: rhubarb glycoside; Figure 5 It is a reference solution of hesperidin hydrate, peak 4: hesperidin hydrate; Figure 6 It is a reference solution of isohesperidin, peak 5: isohesperidin; Figure 7 It is a schisandrol A reference solution, peak 6: schisandrol A.

[0036] Figure 8 The selection of the chromatographic column - Juhong Tan Ke Granules (A: Welch; B: Mid-Spectrum Red; C: HITACHI); Peak 1: Amygdalin; Peak 2: Naringin; Peak 3: Rhus glycoside; Peak 4: Hesperidin hydrate; Peak 5: Isohesperidin; Peak 6: Schisandrol A;

[0037] Figure 9 The selection of the chromatographic column - Juhong Tan Ke Jian Gao (A: Welch; B: Mid-spectrum Red; C: HITACHI); Peak 1: Amygdalin; Peak 2: Naringin; Peak 3: Rhododendronin; Peak 4: Hesperidin hydrate; Peak 5: Isohesperidin; Peak 6: Schisandrol A;

[0038] Figure 10 These are the characteristic chromatograms of 10 batches of Tangerine Peel Cough Relief Paste (A: C24A025; B: C24A024; C: C24A023; D: C24A022; E: C24A021; F: C24A020; G: C24A019; H: C24A018; I: C24A017; J: C24A016). Peak 1: amygdalin; Peak 2: naringin; Peak 3: rosin; Peak 4: hesperidin hydrate; Peak 5: isohesperidin; Peak 6: schisandrol A.

[0039] Figure 11 The characteristic spectra of three batches of Juhong Tan Ke Granules (A: C24A001; B: C24A002; C: C24A003) show the following peaks: Peak 1: amygdalin; Peak 2: naringin; Peak 3: rosin; Peak 4: hesperidin hydrate; Peak 5: isohesperidin; Peak 6: schisandrol A.

[0040] Figure 12 The characteristic chromatograms of three batches of Juhong Tan Ke Oral Liquid (A: 202403904; B: 202402905; C: 202403915) show the following peaks: Peak 1: amygdalin; Peak 2: naringin; Peak 3: anaposide; Peak 4: hesperidin hydrate; Peak 5: isohesperidin; Peak 6: schisandrol A.

[0041] Figure 13This is an evaluation of the similarity of the HPLC characteristic chromatograms of Tangerine Peel Cough Relief Paste. R: Reference fingerprint chromatogram of Tangerine Peel Cough Relief Paste; S1~S10: Batch numbers: C24A016, C24A017, C24A018, C24A019, C24A020, C24A021, C24A022, C24A023, C24A024, C24A025;

[0042] Figure 14 This is a comparison of the similarity of HPLC characteristic chromatograms between 3 batches of decoction extract and 4 batches of decoction extract (R: control characteristic chromatogram; S1~S7: A: decoction extract L24R010, B: decoction extract L24R009, C: decoction extract L24R008, D: decoction extract K25A001).

[0043] E: Decoction J25A001, F: Decoction F25A002, G: Decoction F25A001)

[0044] Figure 15 This is a comparison of the similarity of HPLC characteristic chromatograms of three batches of granule extract and three batches of granules (R: reference characteristic chromatogram; S1~S6: A: granule extract A25R008, B: granule extract A25R007, C: granule extract A25R005, D: granules K25A005, E: granules K25A004, F: granules K25A002). Detailed Implementation

[0045] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in other ways. Any person skilled in the art can make equivalent changes to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention are within the protection scope of the present invention.

[0046] Experimental preparation

[0047] 1. Instruments and reagents

[0048] (1) Instruments: 1 / 100,000 electronic analytical balance (MS105DU, Mettler Toledo, Switzerland); CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water system (Simplicity, Millipore, USA); Dionex Ultimate 3000 high performance liquid chromatograph (Dionex, USA, LPG-3400SDN single quaternary pump, WPS-3000SL autosampler, TCC-3000RS column oven, DAD-3000 detector, Chromeleon 7.2 data processing software); Agilent 1260 high performance liquid chromatograph (G1311B quaternary pump, G1316A column oven, G1329B autosampler, G1315D DAD detector, OpenLab Control Panel data processing software); chromatographic column: ①Welch XB-C18 (4.6×250mm, 5μm, SN211604931); ② Mid-spectrum Red RD-C18 (4.6×250mm, 5μm, SN018023563); ③ Hitachi High-Tech LaChrom C 18 (4.6×250mm, 5μm, SN28F5I-042).

[0049] (2) Reagents and reagents

[0050] Experimental reagents: Methanol (Tianjin Damao Chemical Reagent Factory, 20240918, analytical grade); ultrapure water.

[0051] Reagents used in liquid chromatography: methanol (Fisher Chemical, F25P26207, chromatographic grade); phosphoric acid (Aladdin, C2127420, chromatographic grade).

[0052] (3) Reference standard

[0053] Table 1. List of Reference Standards

[0054] (4) Sample

[0055] Table 2 Sample Sources

[0056] (5) Medicinal materials: Citrus reticulata peel, bitter almond, Cynanchum paniculatum, Pinellia ternata, Pinellia ternata with ginger water, Stemona japonica, Poria cocos, Schisandra chinensis, and Glycyrrhiza uralensis, provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd.

[0057] (6) Excipients: Citric acid, sodium benzoate, ethylparaben (4-hydroxybenzoate), dextrin, sucrose, and honey, provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd.

[0058] Example 1: Construction of the Feature Mapping Method

[0059] 1. Preparation of solutions of test samples, reference standards, and reference medicinal materials.

[0060] (1) Preparation of reference solutions: Take appropriate amounts of amygdalin, naringin, arugula glycoside, hesperidin hydrate, isohesperidin, schisandrol A, isoimperatorin, glycyrrhizin and naringin reference standards respectively, and add methanol to prepare reference stock solutions.

[0061] (2) Preparation of the medicinal material solution:

[0062] Preparing the raw material for Citrus reticulata: Take about 0.5g of Citrus reticulata powder (passed through a No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of methanol, weigh it, heat it in a water bath at 80°C under reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, accurately measure 5ml of the filtrate, place it in a 50ml volumetric flask, add 50% methanol to the mark, shake well, and collect the filtrate to obtain the product.

[0063] Other herbal solutions: According to the prescription ratio, take 10g of bitter almond, 3g of prepared pinellia (or 3g of ginger-infused pinellia), 5g of cynanchum paniculatum, 3g of stemona japonica, 1g of licorice, 3g of poria cocos, and 2g of schisandra chinensis, accurately weigh them, and place them in different round-bottom flasks. Add 240ml of ultrapure water, place the flasks on a heating mantle, heat under reflux, start timing when boiling, maintain a gentle boil, decoct for 30 minutes, filter, and collect the filtrate; add 200ml of ultrapure water to the residue, continue heating under a heating mantle for reflux for 15 minutes, start timing when boiling, maintain a gentle boil, filter, and collect the filtrate. Combine the first two filtrates, measure the total filtrate volume, accurately measure 50ml of the total filtrate from each herb (bitter almond, etc.), place them in different stoppered conical flasks, add 50ml of methanol to each, shake well, sonicate (power 500W, frequency 40KHz) for 30 minutes, filter, and collect the filtrate.

[0064] (3) Preparation of the test solution:

[0065] Juhong Tan Ke Granules: Take 10 sachets of granules, mix them well, grind them into a fine powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol solution, weigh it, sonicate for 30 minutes (power 500W, frequency 40kHz), take it out, let it cool, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0066] Juhong Tanke Decoction Extract: Take about 1.0 g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, ultrasonically treat it for 30 minutes (power 500 W, frequency 40 kHz), take it out, let it cool, weigh it again, make up the lost weight with 50% methanol, shake well, filter, and take the continuous filtrate, that is, obtain it.

[0067] 2. Optimization and Selection of Chromatographic Conditions

[0068] (1) Mobile phase: Use acetonitrile solution with 0.1% acetic acid by mass fraction - acetic acid solution with 0.1% acetic acid by mass fraction as mobile phase (1) and methanol - phosphoric acid solution with 0.1% acetic acid by mass fraction as mobile phase (2) to explore the conditions. The results show that both mobile phase systems can effectively separate the target components. Considering the advantages of methanol in terms of cost, toxicity, and environmental friendliness, and its excellent system applicability and method durability while ensuring good chromatographic behavior of the target components (especially flavonoids and coumarin compounds), which can meet the requirements for comprehensive quality evaluation of the complex system of Juhong Tanke preparations. And when 0.1% phosphoric acid solution is used as the aqueous phase of the mobile phase, the baseline is better than that of 0.1% acetic acid solution. Considering comprehensively, select mobile phase (2), that is, methanol - 0.1% phosphoric acid system as the mobile phase system of this study. The determined mobile phase and gradient are as follows: Use methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 3 and Table 4. The determination time for each sample injection is about 85 minutes, the flow rate is 1.0 ml / minute, and the column temperature is 35°C.

[0069] Table 3 Mobile Phase Elution Gradient

[0070] Table 4 Column Washing and Equilibration Gradient

[0071] (2) Detection wavelength: In the [Characteristic Chromatogram] item of Juhong Tanke Liquid in the Chinese Pharmacopoeia (Volume I) of 2025 Edition, the detection wavelength of the characteristic chromatogram is 320 nm, and all 6 characteristic peaks come from the monarch drug Exocarpium Citri Grandis. Analyzing the prescriptions of Juhong Tanke preparations (granules, decoction extracts), in addition to the monarch drug Exocarpium Citri Grandis, there are multiple characteristic components in multiple medicinal materials in Juhong Tanke preparations, and the characteristic components of other medicinal materials cannot be detected by a single detection wavelength of 320 nm.

[0072] The methanol solutions of amygdalin reference substance, rhoifolin reference substance, naringin reference substance, and schisandrin reference substance were used to measure the ultraviolet absorption spectra. The results showed that the maximum absorption wavelength of amygdalin reference substance was 207.50 nm; the maximum absorption wavelength of rhoifolin was 338.60 nm; the maximum absorption wavelength of naringin was 283.91 nm. In order to detect as many characteristic components of other medicinal materials as possible, through experiments such as condition exploration and methodology confirmation, the detection wavelengths of this experiment were: 207 nm, 320 nm, and 250 nm were switched at different times in the same channel as the detection wavelengths for the determination of the characteristic chromatogram (see Table 5). Amygdalin was detected at 207 nm, the characteristic components of the monarch drug, Exocarpium Citri Grandis, were detected at 320 nm; schisandrin was detected at 250 nm, that is: the characteristic components of Prunus armeniaca were detected at 207 nm, the characteristic components of Exocarpium Citri Grandis were detected at 320 nm, and the characteristic components of Schisandra chinensis were detected at 250 nm. A photodiode array detector was used to examine the chromatograms at different wavelengths. See Figure 1 .

[0073] Table 5 Detection wavelengths

[0074] (3) Selection of chromatographic column: According to the characteristic chromatogram item of Juhong Tanke Ye in Part I of the Chinese Pharmacopoeia (2025 Edition) and the structural characteristics and physicochemical properties of each raw material medicinal herb in the Juhong Tanke preparation, a C18 column was selected for separation. Three types of chromatographic columns, Welch XB-C18 (4.6×250 mm, 5 μm), Zhongpu Red C18 (4.6×250 mm, 5 μm), and Hitachi High-Tech C18 (4.6×250 mm, 5 μm), were respectively investigated. The elution conditions were as follows: methanol was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was carried out according to the regulations in Table 3 and Table 4, the determination of each sample took about 85 minutes, the flow rate was 1.0 ml / min, the column temperature was 35°C, and the detection wavelength was as shown in Table 5. All chromatographic peaks could achieve basic separation ( Figure 8 and Figure 9 ).

[0075] (4) Determination of the characteristic chromatogram chromatographic conditions: Octadecylsilane chemically bonded silica gel was used as the filler (Welch XB-C18 (4.6×250 mm, 5 μm)); methanol was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was carried out according to the regulations in Table 3 and Table 4, wavelength switching was carried out according to the detection wavelengths in Table 5, the determination of each sample took about 85 minutes, the flow rate was 1.0 ml / min, the column temperature was 35°C. The following experiments in this example and the experiments in Examples 2, 3, and 4 used these conditions.

[0076] The determination of the reference substance also used these conditions, Figure 2It is a reference solution of amygdalin, peak 1: amygdalin; Figure 3 It is a naringin reference solution, peak 2: naringin; Figure 4 It is a reference solution of rhubarb glycoside, peak 3: rhubarb glycoside; Figure 5 It is a reference solution of hesperidin hydrate, peak 4: hesperidin hydrate; Figure 6 It is a reference solution of isohesperidin, peak 5: isohesperidin; Figure 7 It is a schisandrol A reference solution, peak 6: schisandrol A.

[0077] 3. Methodological Research

[0078] The HPLC chromatographic conditions of the optimized tangerine peel cough preparation were studied. Unless otherwise noted, the chromatographic columns used in the methodological studies were Welch XB-C18 (4.6×250mm, 5μm).

[0079] (1) Specificity test:

[0080] Blank solvent: 50% methanol solution, injection will yield a blank solvent chromatogram.

[0081] Negative control solution:

[0082] ① Preparation of negative control solution for bitter almond deficiency: According to the prescription ratio, take the other herbs for which bitter almond deficiency is lacking, including 30g of Citrus reticulata peel, 3g of Pinellia ternata (or Pinellia ternata with ginger water), 5g of Cynanchum paniculatum, 3g of Stemona japonica, 1g of Glycyrrhiza uralensis, 3g of Poria cocos, and 2g of Schisandra chinensis. Place them in a round-bottom flask, add water, and decoct twice. For the first decoction, add 240ml of ultrapure water, place the flask on a heating mantle, heat under reflux, start timing when boiling, maintain a gentle boil, and decoct for 30 minutes. Filter and collect the filtrate. For the second decoction, add 200ml of ultrapure water to the residue, continue heating under a heating mantle under reflux for 15 minutes, start timing when boiling, maintain a gentle boil, filter, and collect the filtrate. Combine the two filtrates, measure the total volume, and precisely add 50ml of the water extract of each herb and 50ml of methanol to a beaker. Mix well, then sonicate (500W power, 40KHz frequency) for 30 minutes. Filter and collect the filtrate. ② Preparation of negative control solution for citrus desiccant deficiency: According to the prescription ratio, take the other herbs for citrus desiccant deficiency, including 10g of bitter almond, 3g of prepared pinellia (or ginger-infused pinellia), 5g of cynanchum paniculatum, 3g of stemona japonica, 1g of licorice, 3g of poria cocos, and 2g of schisandra chinensis. Accurately weigh these ingredients and place them in a round-bottom flask. Add water and decoct twice. For the first decoction, add 240ml of ultrapure water, place the flask on a heating mantle, heat under reflux, start timing when boiling, maintain a gentle boil, and decoct for 30 minutes. Filter and collect the filtrate. For the second decoction, add 200ml of ultrapure water to the residue, continue heating under reflux for 15 minutes, start timing when boiling, maintain a gentle boil, filter, and collect the filtrate. Combine the two filtrates and measure the total volume. Accurately add 50ml of the aqueous extract of each herb and 50ml of methanol to a beaker, mix thoroughly, and sonicate (500W power, 40KHz frequency) for 30 minutes. Filter and collect the subsequent filtrate. ③ Preparation of negative control solution for herbs lacking Schisandra chinensis and Pinellia ternata: According to the prescription ratio, take the other herbs lacking Schisandra chinensis and Pinellia ternata (or Pinellia ternata with ginger water), and take 30g of Citrus reticulata peel, 10g of bitter apricot kernel, 5g of Cynanchum paniculatum, 3g of Stemona japonica, 1g of Glycyrrhiza uralensis, and 3g of Poria cocos. Accurately weigh them and place them in a round-bottom flask. Add water and decoct twice. For the first decoction, add 240ml of ultrapure water, place the flask on an electric heating mantle, heat under reflux, start timing when boiling, keep it at a gentle boil, decoct for 30 minutes, filter, and collect the filtrate. For the second time, add 200ml of ultrapure water to the above residue, continue to heat under reflux for 15 minutes on an electric heating mantle, start timing when boiling, maintain a gentle boil, filter, collect the filtrate, combine the two filtrates, measure the total volume, and precisely add 50ml of the water extract of each of the added medicinal materials and 50ml of methanol to a beaker, mix well, and then sonicate (power 500W, frequency 40KHz) for 30 minutes, filter, and collect the subsequent filtrate to obtain the final product.④ Preparation of negative control solution for licorice-deficient herbs: According to the prescription ratio, take the other herbs lacking licorice, namely 30g of Citrus reticulata peel, 10g of bitter almond, 3g of prepared Pinellia ternata (or ginger-infused Pinellia ternata), 5g of Cynanchum paniculatum, 3g of Stemona japonica, 3g of Poria cocos, and 2g of Schisandra chinensis. Accurately weigh them and place them in a round-bottom flask. Add water and decoct twice. For the first decoction, add 240ml of ultrapure water, place the flask on a heating mantle, heat under reflux, start timing when boiling, maintain a gentle boil, and decoct for 30 minutes. Filter and collect the filtrate. For the second decoction, add 200ml of ultrapure water to the above residue, continue heating under reflux for 15 minutes, start timing when boiling, maintain a gentle boil, filter, and collect the filtrate. Combine the two filtrates and measure the total volume. Accurately add 50ml of the water extract of each herb and 50ml of methanol to a beaker, mix well, and sonicate (500W power, 40KHz frequency) for 30 minutes. Filter and collect the subsequent filtrate.

[0083] ⑤ Preparation of excipient solution:

[0084] Juhong Tan Ke Granules Excipient Solution: Accurately weigh approximately 1.0g of sucrose, 0.1g of dextrin, and 0.01g of citric acid, and place them in the same stoppered conical flask. Accurately add 25ml of 50% methanol, weigh, and sonicate for 30 minutes (500W power, 40kHz frequency). Remove, cool, and weigh again. Make up the lost weight with 50% methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0085] Juhong (Citrus reticulata) cough syrup excipient solution: Take approximately 3.5g of sucrose, 0.5g of honey, 0.01g of citric acid, 0.02g of sodium benzoate, and 0.005g of ethylparaben, accurately weigh them, and place them in the same stoppered conical flask. Accurately add 25ml of 50% methanol, weigh, and sonicate for 30 minutes (500W power, 40kHz frequency). Remove, cool, and weigh again. Make up the weight loss with 50% methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0086] Juhong Tan Ke Granules, Juhong Tan Ke Decoction, raw medicinal materials, and reference standards were prepared according to the methods under "Sample Preparation" and analyzed under the conditions of "Determination of Chromatographic Conditions for Characteristic Chromatography". The results showed that the identification of the seven common chromatographic peaks was not affected by factors such as solvents and excipients, and had good specificity.

[0087] The characteristic chromatographic method for tangerine peel cough preparation established in this study can simultaneously detect four medicinal materials. Among them, amygdalin detected at 207 nm belongs to bitter almond, naringin, rosin, hesperidin hydrate, and isohesperidin detected at 320 nm belong to tangerine peel, schisandrol A detected at 250 nm belongs to schisandra, and peak 7 belongs to licorice.

[0088] (2) Identification and attribution of common characteristic peaks and determination of characteristic peaks

[0089] Compared with the reference solutions, the granule and decoction test solutions all showed corresponding chromatographic peaks with amygdalin reference solutions, naringin reference solutions, rhubarb glycoside reference solutions, schisandrol A reference solutions, hesperidin hydrate reference solutions, and isohesperidin reference solutions.

[0090] Compared with the raw medicinal materials used:

[0091] ① Both the granules and the decoction showed two chromatographic peaks at 8 minutes and 57 minutes, respectively, corresponding to the added medicinal material Schisandra chinensis, and their ultraviolet absorption spectra were also consistent. Figure 1 Simultaneously, the chromatographic peak at 8 minutes corresponds to the added medicinal material, ginger-infused Pinellia ternata, and the ultraviolet absorption spectrum... Figure 1 The chromatographic peak at retention time of 8 minutes is presumably a component shared by Schisandra chinensis and Pinellia ternata. Based on UV absorption spectroscopy analysis, this component exhibits maximum absorption at 284 nm, suggesting it may be a flavonoid. Compared to the negative control solution lacking Schisandra chinensis and Pinellia ternata, this peak still exists in the chromatogram of the negative sample, suggesting it may be a shared component of multiple herbs in the formula or a shared component generated during preparation. To ensure the specificity and accuracy of the characteristic chromatogram identification and to avoid including non-specific chromatographic peaks in quality control standards, after comprehensive evaluation, this peak is temporarily excluded from the shared characteristic peaks of the Juhong Tan Ke preparation. The chromatographic peak at 57 minutes has the same retention time and UV absorption spectrum as the Schisandrin A reference solution; therefore, this peak is identified as Schisandrin A, originating from the added Schisandra chinensis herb.

[0092] ② Both the granules and the decoction showed a chromatographic peak at 17 minutes that corresponded to the bitter almond herb used in the treatment, and the ultraviolet absorption spectrum showed a similar peak. Figure 1 The chromatographic peak has the same retention time and UV absorption spectrum as the amygdalin reference solution. Compared with the negative control solution lacking bitter almonds, this peak was not present in the chromatogram of the negative sample; therefore, the peak is identified as amygdalin, originating from the bitter almond material used in the treatment.

[0093] ③ The granules and decoction showed four chromatographic peaks at 35 minutes, 40 minutes, 42 minutes, and 47 minutes, respectively, corresponding to the added medicinal material, Citrus reticulata peel, and their ultraviolet absorption spectra were consistent. Figure 1The chromatographic peak with a retention time of 35 minutes is consistent with the retention time and UV absorption spectrum of the naringin reference solution, therefore, this peak is identified as naringin; the chromatographic peak with a retention time of 40 minutes is consistent with the retention time and UV absorption spectrum of the arugula reference solution, therefore, this peak is identified as arugula; the chromatographic peak with a retention time of 42 minutes is consistent with the retention time and UV absorption spectrum of the hesperidin hydrate reference solution, therefore, this peak is identified as hesperidin hydrate; the chromatographic peak with a retention time of 47 minutes is consistent with the retention time and UV absorption spectrum of the isohesperidin reference solution, therefore, this peak is identified as isohesperidin; compared with the negative control solution lacking Citrus reticulata, these four chromatographic peaks are not present in the chromatogram of this negative sample, therefore, the above four components are identified as originating from the raw material Citrus reticulata.

[0094] ④ Both the granules and the decoction showed a chromatographic peak at 67 minutes corresponding to the added medicinal material, licorice, and their ultraviolet absorption spectra were similar. Figure 1 Therefore, it is determined that the source of this component is licorice.

[0095] ⑤ The UV absorption spectrum of the glycyrrhizin reference solution shows that glycyrrhizin has a maximum absorption peak at 276 nm. In both granules and decoction samples, the peak area of ​​glycyrrhizin is less than 0.01, indicating extremely low content, making it difficult to use as a stable and significant characteristic signal. Furthermore, licorice is used as an adjuvant, and its components are already reflected in the characteristic spectra. Current experimental methods effectively characterize the overall chemical characteristics of the preparation, meeting quality control requirements. Therefore, glycyrrhizin is not listed as a separate identified ingredient.

[0096] According to the prescription of the Citrus reticulata cough preparation, Citrus reticulata is the principal drug in the prescription, and naringin is one of the main effective components of Citrus reticulata. Furthermore, the 2025 edition of the Chinese Pharmacopoeia (Part I) uses naringin as the component for content determination of Citrus reticulata. In the Citrus reticulata cough syrup in the Chinese Pharmacopoeia, naringin is used as the reference peak S. Therefore, in this characteristic chromatographic method validation, the naringin peak is tentatively set as the reference peak S peak.

[0097] In summary, the characteristic spectrum of the tangerine peel cough preparation established in this experiment identified seven common characteristic peaks, covering four medicinal materials in the prescription: tangerine peel, bitter almond, schisandra, and licorice. Among these seven characteristic peaks, the chemical structures of six components were confirmed: amygdalin (peak 1), naringin (peak 2), rhubarb glycoside (peak 3), hesperidin hydrate (peak 4), isohesperidin (peak 5), and schisandrol A (peak 6). These components are the main active or indicative components of each medicinal material, providing a material basis for the qualitative identification of the characteristic spectrum.

[0098] (3) Precision test

[0099] Take the same granule test solution (batch number: C24A003) and the decoction test solution (batch number: C24A018), and inject them six times consecutively under the above chromatographic conditions (elution conditions: methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Tables 3 and 4, each sample determination time approximately 85 minutes, flow rate 1.0 ml / min, column temperature 35℃, detection wavelength see Table 5). Record the chromatograms, and calculate the relative retention times of the common peaks of each chromatogram using naringin peak 2 as a reference. The RSD values ​​of the relative retention times of the common peaks of each granule chromatogram are 0.00~0.02%, and the RSD values ​​of the relative retention times of the common peaks of each decoction chromatogram are 0.00~0.12%. There is no significant change in the relative retention times of the main chromatographic peaks, indicating good precision.

[0100] (4) Repeatability test

[0101] Six parallel aliquots of the same particulate test solution (batch number: C24A003) and decoction test solution (batch number: C24A018) were prepared according to the method described in the "Preparation of Test Solution" section. The solutions were injected under the chromatographic conditions described above (elution conditions: methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Tables 3 and 4, approximately 85 minutes per sample determination, flow rate 1.0 ml / min, column temperature 35℃, detection wavelength as shown in Table 5). Chromatograms were recorded, and the relative retention times of the common peaks in each chromatogram were calculated using naringin (peak 2) as a reference. The RSD values ​​of the relative retention times of the common peaks in the particulate chromatograms were 0.00–0.02%, and the RSD values ​​of the common peaks in the decoction chromatograms were 0.00–0.04%. No significant changes were observed in the relative retention times of the main chromatographic peaks, indicating good repeatability.

[0102] (5) Stability test

[0103] Take the same granule test solution (batch number: C24A003) and decoction test solution (batch number: C24A018). Inject the tangerine peel cough granules at 0, 2, 6, 8, 12, 24, 36, and 48 hours; inject the tangerine peel cough decoction at 0, 4, 6, 8, 12, 28, and 48 hours. The chromatographic conditions are: methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the specifications in Tables 3 and 4, approximately 85 minutes per injection, flow rate 1.0 ml / min, column temperature 35℃, and detection wavelengths as shown in Table 5. Record the chromatograms. Using peak 2 (naringin) as a reference, calculate the relative retention times of the common peaks in each chromatogram. The RSD values ​​of the relative retention times of the common peaks in the chromatograms of the granules were 0.00–0.19%, and the RSD values ​​of the relative retention times of the common peaks in the chromatograms of the decoction were 0.00–0.12%. The relative retention times of the main chromatographic peaks did not change significantly, indicating that the granules and decoction samples had good stability at room temperature for 48 hours.

[0104] (6) Intermediate precision

[0105] The effects of different analysts, different analysis dates, and different equipment on precision were investigated.

[0106] Different analysts took the same batch of Citrus reticulata granules (batch number: C24A003) and Citrus reticulata decoction (batch number: C24A018), and the same analyst took the same batch of Citrus reticulata granules (batch number: C24A003) and Citrus reticulata decoction (batch number: C24A018) on different analysis dates. They prepared the solutions according to the method described in the "Preparation of Test Solution" section, and analyzed them separately on the same analytical instrument under the chromatographic conditions described above. The same analyst took the same batch of Citrus reticulata granules (batch number: C24A003) and Citrus reticulata decoction (batch number: C24A018); and prepared the solutions according to the method described in the "Preparation of Test Solution" section. The preparation operations under the "Preparation" section were performed using different analytical devices and chromatographic conditions as described above. Using naringin (peak 2) as a reference, the relative retention times of the common peaks in each chromatogram were calculated. The results showed that, under varying conditions of different analysts, analysis dates, and equipment, the RSD values ​​of the relative retention times of the common peaks in the particle chromatograms ranged from 0.00 to 0.22, and the RSD values ​​of the relative retention times of the common peaks in the decoction chromatograms ranged from 0.00 to 0.31%. The relative retention times of the main chromatographic peaks did not change significantly, indicating that the method has good precision and that random variations do not affect its precision.

[0107] (7) Durability

[0108] Take the same batch of Juhong Tan Ke Granules (batch number: C24A003) and Juhong Tan Ke Decoction (batch number: C24A018), and use three types of chromatographic columns: Welch XB-C18 (4.6×250mm, 5μm), Zhongpu Red C18 (4.6×250mm, 5μm), and Hitachi High-Tech C18 (4.6×250mm, 5μm). The chromatographic conditions are: methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the specifications in Tables 3 and 4, the determination time for each sample is about 85 minutes, the flow rate is 1.0 ml / min, the column temperature is 35℃, and the detection wavelength is shown in Table 5. Determine the characteristic chromatograms of Juhong Tan Ke Granules and Juhong Tan Ke Decoction, and record the chromatograms. All chromatographic peaks can be basically separated. Using naringin peak No. 2 as a reference, the relative retention time of the common peaks of each chromatogram is calculated. The RSD values ​​of the relative retention times of the common peaks in the chromatograms of the particles ranged from 0.00 to 0.80%, and the RSD values ​​of the relative retention times of the common peaks in the chromatograms of the decoction ranged from 0.00 to 0.98%. The relative retention times of the main chromatographic peaks did not change significantly, indicating that the method has good robustness.

[0109] (8) Establishment of the specified value of relative retention time

[0110] After investigation using experiments with significant random variations (different chromatographic columns and intermediate precision), the retention times of the naringin peak in the chromatogram of the test sample were found to be the same as those of the corresponding reference peak. Using the naringin peak as the S peak, the relative retention times of the other seven peaks were calculated. The average relative retention times of the characteristic peaks of the Juhong Tan Ke Granules were 0.497 (peak 1), 1.112 (peak 3), 1.190 (peak 4), 1.365 (peak 5), 1.594 (peak 6), and 1.890 (peak 7); the average relative retention times of the characteristic peaks of the Juhong Tan Ke Decoction were 0.495 (peak 1), 1.113 (peak 3), 1.190 (peak 4), 1.366 (peak 5), 1.596 (peak 6), and 1.895 (peak 7).

[0111] Table 6. Assignment of common characteristic peaks of Juhong Tan Ke Granules

[0112] Table 7. Changes in relative retention time under different conditions - Juhong Tan Ke Granules

[0113] Table 8. Assignment of common characteristic peaks in Tangerine Peel Cough Syrup

[0114] Table 9. Changes in relative retention time under different conditions - Tangerine Peel Cough Syrup

[0115] II. Construction of Feature Maps

[0116] During the experiment, 10 batches of Citrus reticulata cough decoction, 3 batches of Citrus reticulata cough granules, and 3 batches of Citrus reticulata cough oral liquid were tested. The sources are shown in Table 2. The preparation method for Citrus reticulata cough oral liquid was as follows: 1.0 mL of Citrus reticulata cough oral liquid was accurately transferred to a 10 mL volumetric flask, 50% methanol was added to the mark, shaken well, filtered, and the filtrate was collected as the test solution. Citrus reticulata cough decoction and Citrus reticulata cough granules were prepared according to the method described in the "Preparation of Test Solution" section. The determination was performed according to the prescribed method (using methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Tables 3 and 4, the determination time for each sample was approximately 85 minutes, the flow rate was 1.0 mL / min, the column temperature was 35℃, and the detection wavelength is shown in Table 5).

[0117] Table 10. Characteristic chromatograms of 10 batches of Tangerine Peel Cough Syrup - Retention Time

[0118] Table 11 Characteristic Spectra of 10 Batches of Tangerine Peel Cough Syrup - Retention Time

[0119] Table 12 Characteristic Spectra of 22 Batches of Tangerine Peel Cough Syrup - Relative Retention Time

[0120] Table 13 Characteristic Spectra of 10 Batches of Tangerine Peel Cough Syrup - Average Relative Retention Time

[0121] Table 14 Characteristic spectrum of three batches of Tangerine Peel Cough Granules - Retention Time

[0122] Table 15 Characteristic spectrum of three batches of Tangerine Peel Cough Granules - relative retention time

[0123] Table 16 Characteristic spectrum of three batches of Tangerine Peel Cough Granules - Average relative retention time

[0124] Table 17 Characteristic Spectra of Three Batches of Tangerine Peel Cough Syrup - Retention Time

[0125] Table 18 Characteristic Spectra of Three Batches of Tangerine Peel Cough Syrup - Relative Retention Time

[0126] Table 19 Characteristic Spectra of Three Batches of Tangerine Peel Cough Syrup - Average Relative Retention Time

[0127] Example 2

[0128] Similarity evaluation results of different dosage forms of tangerine peel cough syrup preparation

[0129] Take samples of the following batches of tangerine peel cough syrup: C24A016, C24A017, C24A018, C24A019, C24A020, C24A021, C24A022, C24A023, C24A024, and C24A025; take samples of the following batches of tangerine peel cough syrup: C24A001, C24A002, and C24A003; take samples of the following batches: 2... Citrus reticulata extracts 02403915, 202403904, and 202402905 were prepared as test solutions and analyzed (using methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Tables 3 and 4, the analysis time for each sample was approximately 85 minutes, the flow rate was 1.0 ml / min, the column temperature was 35℃, and the detection wavelength is shown in Table 5), and the chromatograms were recorded. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" version 2012.1 was used to generate a common pattern (R) from the characteristic chromatograms of 10 batches of Citrus reticulata extract, and its similarity was evaluated with 3 batches of Citrus reticulata extract granules and 3 batches of Citrus reticulata extract oral solution. The results showed that the similarity between 10 batches of Juhong Tan Ke decoction was above 0.98; the similarity between the common pattern (R) generated by the 10 batches of Juhong Tan Ke decoction and the 3 batches of Juhong Tan Ke granules ranged from 0.82 to 0.84; and the similarity between the common pattern (R) generated by the 10 batches of Juhong Tan Ke decoction and Juhong Tan Ke oral liquid ranged from 0.76 to 0.79. These results demonstrate that the similarity evaluation validated the distinguishing ability of the characteristic fingerprinting method, effectively identifying differences in characteristic chemical components among different dosage forms and manufacturers, providing a scientific basis for the quality control, process optimization, and consistency evaluation of Juhong Tan Ke preparations. Figure 10 , 11 12).

[0130] Example 3:

[0131] Construction of content determination method

[0132] Based on the characteristic mapping method, the content of key pharmacodynamic components of the tangerine peel cough preparation was determined by multi-channel acquisition wavelength.

[0133] 1. Basis for selecting the components to be measured

[0134] Exocarpium Citri Grandis is the monarch drug in the prescription. The chemical components of Exocarpium Citri Grandis mainly include flavonoids, volatile oils, polysaccharides, coumarins, inorganic elements, etc. Exocarpium Citri Grandis and its various chemical components have good antitussive effects, can reduce the frequency of coughing, and prolong the cough latency period. Among them, flavonoids have effects such as relieving cough, reducing phlegm, relieving asthma, and antiviral. In "Geographical Indication Product Exocarpium Citri Grandis (DB 4409 / T 06 - 2019)", the contents of total flavonoids, naringin, rhoifolin, etc. are used as quality control indicators for Exocarpium Citri Grandis, and it is stipulated that the contents of naringin and rhoifolin shall not be less than 5.0% and 0.2% respectively; amygdalin is the main chemical component and active ingredient of bitter almonds, and its content shall not be less than 3.0%. Amygdalin has effects such as relieving cough and asthma, anti-inflammatory and analgesic, anti-tumor, and antioxidant. Therefore, the determination of the content of Exocarpium Citri Grandis calculated by naringin and rhoifolin and the content of bitter almonds calculated by amygdalin in the standard is of great significance for the quality control of Juhong Tanke preparations.

[0135] Naringin is a unique bioactive component in Exocarpium Citri Grandis. In the first part of the Chinese Pharmacopoeia 2025 edition, the content of naringin in Exocarpium Citri Grandis medicinal materials was determined by HPLC method. Therefore, this standard studied the determination of naringin content by HPLC method.

[0136] 2. Selection of chromatographic conditions

[0137] (1)Mobile phase: Same as the characteristic chromatogram item in Example 1

[0138] (2)Detection wavelength

[0139] Take the methanol solutions of amygdalin reference substance, rhoifolin reference substance, and naringin reference substance to measure the ultraviolet absorption spectra. The results show that the maximum absorption wavelength of amygdalin reference substance is 207.50 nm; the maximum absorption wavelength of rhoifolin is 338.60 nm; the maximum absorption wavelength of naringin is 283.91 nm. According to the national metrological verification regulation, the maximum allowable error of the wavelength indication of the diode array detector is ±2 nm. In this experiment, 207 nm, 338 nm, and 283 nm were selected as the detection wavelengths for the above three components.

[0140] (3)Selection of column temperature

[0141] In this experiment, the separation effects of the test samples at column temperatures of 30 °C and 35 °C were compared. When the column temperature was 35 °C, the asymmetry of each chromatographic peak was better and the resolution was better. Therefore, 35 °C was selected as the detection column temperature.

[0142] 3. Preparation of samples

[0143] Preparation of mixed reference solution: Take appropriate amounts of amygdalin, naringin and arugula glycoside reference standards, weigh them accurately, and add methanol to prepare a mixed solution containing 40 μg amygdalin, 200 μg naringin and 6 μg arugula glycoside per 1 ml.

[0144] Preparation of the test solution: Same as in Example 1.

[0145] 4. Methodological Validation

[0146] (1) System suitability test

[0147] Under these chromatographic conditions, the reference solution and the test solutions of Juhong Tan Ke Granules (batch number: C24A003) and Juhong Tan Ke Decoction (batch number: C24A018) were injected into the liquid chromatograph and determined according to the following method (chromatographic conditions: methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Tables 3 and 4, the determination time for each sample was approximately 85 minutes, the flow rate was 1.0 ml / min, the column temperature was 35℃, and the detection wavelength is shown in Table 5). The system suitability test results for amygdalin, naringin, and rhubarb glycoside showed that the chromatographic peaks were well separated and could be baseline separated from the preceding and following peaks. The theoretical plate number and asymmetry met the requirements for content determination, and the system can be used for content determination tests.

[0148] (2) Linear range

[0149] Amygdalin reference solution: An appropriate amount of amygdalin reference standard was accurately weighed and dissolved in methanol to prepare concentrations of 4.384 μg / ml, 8.769 μg / ml, 21.92 μg / ml, 43.84 μg / ml, 87.69 μg / ml, and 109.6 μg / ml (25-fold concentration). The solution was measured at 207 nm. Regression analysis was performed using the peak area integral value A of amygdalin against the concentration C of the amygdalin reference standard. The regression equation was: y = 0.1708x - 0.0241, R² = 1. The results showed that within the concentration range of 4.384–109.6 μg / ml, the concentration and peak area of ​​amygdalin exhibited a good linear relationship. Rhus glycoside reference solution: Accurately weigh an appropriate amount of rhus glycoside reference standard and add methanol to prepare concentrations of 1.158 μg / ml, 2.317 μg / ml, 5.792 μg / ml, 11.58 μg / ml, 23.17 μg / ml, and 28.96 μg / ml, respectively (25-fold concentration). The solution was measured at 338 nm. Regression analysis was performed using the peak area integral value A of rhus glycoside against the concentration C of the rhus glycoside reference standard. The regression equation was: y = 0.426x - 0.034, R² = 0.9998. The results show that within the concentration range of 1.158–28.96 μg / ml, the concentration and peak area of ​​rhus glycoside exhibit a good linear relationship. Naringin reference solution: Accurately weigh an appropriate amount of naringin reference standard and add methanol to prepare concentrations of 13.57 μg / ml, 33.92 μg / ml, 67.85 μg / ml, 169.6 μg / ml, 339.2 μg / ml, and 407.1 μg / ml, respectively (30-fold concentration). Measured at 283 nm, the peak area integral value A of naringin was used to perform regression analysis on the concentration C of naringin reference standard. The regression equation was: y = 0.2962x - 0.2319, R² = 0.9999. The results show that naringin concentration and peak area have a good linear relationship within the concentration range of 13.57~407.1 μg / ml. The R² of the linear regression of amygdalin reference standard... 2 =1, linear regression R of laccoside reference standard 2 =0.9998, linear regression R of naringin reference standard 2 =0.9999, indicating that the method has good linearity.

[0150] (3) Precision test

[0151] Amygdalin, arugula glycoside, and naringin reference standards were prepared according to the method described in the "Preparation of Reference Solution" section. Juhong Tan Ke Granules (batch number: C24A003) and Juhong Tan Ke Decoction (batch number: C24A018) were prepared according to the method described in the "Preparation of Test Solution" section. 10 μl of each solution was accurately measured and injected six times consecutively into the liquid chromatograph under the chromatographic conditions described above. The peak areas of amygdalin, arugula glycoside, and naringin in the reference standards and test samples were recorded, and the RSD was calculated. The results showed that the RSD% of the peak area of ​​amygdalin reference standard was 0.13%, and the RSD% of the peak areas of amygdalin in Juhong Tan Ke Granules and Juhong Tan Ke Decoction were 0.15% and 0.13%, respectively. The RSD% of the peak area of ​​arugula glycoside reference standard was 0.09%, while the RSD% of the peak area of ​​arugula glycoside in Juhong Tan Ke Granules and Juhong Tan Ke Decoction were 1.21% and 0.09%, respectively. The RSD of the peak area of ​​naringin reference standard was 0.10%, while the RSD% of the peak area of ​​naringin in Juhong Tan Ke Granules and Juhong Tan Ke Decoction were 0.06% and 0.10%, respectively. This indicates that the instrument has good precision.

[0152] (4) Repeatability test

[0153] Juhong Tan Ke Granules (batch number: C24A003) and Juhong Tan Ke Decoction (batch number: C24A018) were prepared according to the method under "Preparation of Test Solution". Six parallel samples were prepared and the contents were determined under the above chromatographic conditions. The average contents and RSD% of amygdalin, naringin, and rhubarb glycoside were calculated. The results showed that the RSD% of the average contents of amygdalin in the granules and decoction were 1.27% and 0.59%, respectively; the RSD% of the average contents of rhubarb glycoside in the granules and decoction were 2.40% and 1.09%, respectively; and the RSD% of the average contents of naringin in the granules and decoction were 1.32% and 0.37%, respectively, indicating that the method had good repeatability.

[0154] (5) Stability test

[0155] Prepare test solutions of Citrus reticulata granules (batch number: C24A003) and Citrus reticulata decoction (batch number: C24A018) according to the method under "Preparation of Test Solutions". Take one portion each of the reference solutions (amygin reference solution: 43.84 μg / ml, arbutin reference solution: 11.58 μg / ml, naringin reference solution: 67.85 μg / ml) and test solutions under the above chromatographic conditions at room temperature for 0-48 hours, accurately pipette 10 μl into the liquid chromatograph, record the chromatogram, and calculate the RSD of the peak area of ​​each reference solution and test solution. The results showed that the peak area RSD% of the tangerine peel cough granules, tangerine peel cough decoction, and reference solutions of amygdalin, rosin, and naringin were all between 0.12% and 2.61% after being stored at room temperature for 48 hours. The results indicated that the granules, decoction, and reference solutions were stable at room temperature for 48 hours.

[0156] (6) Recovery rate

[0157] Accurately weigh approximately 0.5 g of the following products (batch number: C24A003) and the following products (batch number: C24A018) containing amygdalin, anaposide, and naringin, respectively. Perform nine parallel tests, adding low, medium, and high concentrations of amygdalin, anaposide, and naringin reference solutions to each product. Perform the tests according to the procedure described under the test sample section and calculate the recovery rate. The average recoveries of amygdalin, rosin, and naringin in the test solution of Juhong Tan Ke Granules were 101.19%, 106.65%, and 107.75%, respectively, with RSDs ranging from 1.43% to 2.67%. The average recoveries of amygdalin, rosin, and naringin in the test solution of Juhong Tan Ke Decoction were 102.21%, 103.39%, and 107.45%, respectively, with RSDs ranging from 0.79% to 1.34%, indicating that the method has good recovery rates.

[0158] (7) Intermediate precision

[0159] The effects of different analysts, different analysis dates, and different equipment on precision were investigated.

[0160] Different analysts took the same batch of Citrus reticulata granules (batch number: C24A003) and Citrus reticulata decoction (batch number: C24A018), and the same analyst took the same batch of Citrus reticulata granules (batch number: C24A003) and Citrus reticulata decoction (batch number: C24A018) on different analysis dates. They prepared the solutions according to the method described in the "Preparation of Test Solution" section, and analyzed them separately on the same analytical instrument under the chromatographic conditions described above. The same analyst also took the same batch of Citrus reticulata granules (batch number: C24A003) and Citrus reticulata decoction (batch number: C24A018), prepared the solutions according to the method described in the "Preparation of Test Solution" section, and analyzed them separately on different analytical instruments under the chromatographic conditions described above. The contents of amygdalin, rhubarb glycoside, and naringin, as well as the relative mean deviation (RAD), were calculated. The results showed that, under different analysts, different analysis dates, and different equipment, the RAD values ​​of amygdalin content in granules and decoction were 0.50–1.05% and 0.57–1.95%, respectively; the RAD values ​​of rhubarb glycoside content in granules and decoction were 0.68–1.22% and 0.17–1.67%, respectively; and the RAD values ​​of naringin content in granules and decoction were 0.65–1.01% and 0.68–1.61%, respectively. The results indicated that the method had good precision, and random variations did not affect the precision of the method.

[0161] (9) Durability

[0162] Juhong Tan Ke Granules (batch number: C24A003) and Juhong Tan Ke Decoction (batch number: C24A018) were prepared according to the "Preparation of Test Solution". The contents of amygdalin, arugula glycoside and naringin were determined by Welch XB-C18 (4.6×250mm, 5μm), Spectrum Red-C18 (4.6×250mm, 5μm) and Hitachi High-Tech LaChrom C18 (4.6×250mm, 5μm) columns, respectively. The relative standard deviations (RSD) of the contents of amygdalin, arugula glycoside and naringin were calculated.

[0163] The results showed that the RSD value of amygdalin was 2.86% in Juhong Tan Ke Granules and 2.25% in Juhong Tan Ke Decoction; the RSD value of rosin was 2.07% in Juhong Tan Ke Granules and 7.23% in Juhong Tan Ke Decoction; and the RSD value of naringin was 0.88% in Juhong Tan Ke Granules and 1.57% in Juhong Tan Ke Decoction.

[0164] (10) Test results of different dosage forms and batches

[0165] Take 10 batches of tangerine peel cough decoction, 3 batches of tangerine peel cough granules, and 3 batches of tangerine peel cough oral liquid. The preparation method for the tangerine peel cough oral liquid is as follows: accurately transfer 1.0 mL of the tangerine peel cough oral liquid into a 10 mL volumetric flask, add 50% methanol to the mark, shake well, filter, and collect the filtrate as the test solution. The tangerine peel cough decoction and tangerine peel cough granules are prepared according to the method under "Preparation of Test Solution". Determine the results according to the prescribed method.

[0166] Ten batches of Tangerine Peel Cough Syrup contained amygdalin at levels of 0.8225–1.0109 mg / g, naringin at levels of 4.3536–4.8492 mg / g, and rhubarb glycoside at levels of 0.0187–0.0209 mg / g. Three batches of Tangerine Peel Cough Syrup Granules contained amygdalin at levels of 0.9479–0.9558 mg / g, naringin at levels of 5.0957–5.3700 mg / g, and rhubarb glycoside at levels of 0.0195–0.0200 mg / g.

[0167] The amygdalin content of the three batches of Juhong Tan Ke Oral Liquid was 0.5007~0.5293 mg / g, the naringin content was 2.7233~3.3030 mg / g, and the rosin content was 0.0153~0.0213 mg / g.

[0168] Example 4

[0169] Monitor the quality transfer of the active ingredients in the tangerine peel cough syrup preparation during the production process.

[0170] By utilizing the characteristic spectrum and content determination method of the constructed tangerine peel cough preparation, the raw materials, intermediates and finished products were tested. The transfer of the main indicator components between raw materials, extracts and decoctions / granules was compared to study the transfer law of the main active ingredients of the drug, determine whether the main indicator components are stably transferred, verify the stability of the current process, comprehensively monitor the drug production process, and comprehensively and objectively evaluate the quality of the tangerine peel cough preparation throughout its entire life cycle, thus effectively ensuring the quality of the drug.

[0171] 1. Instruments and reagents, see Example 1.

[0172] Samples: A total of 7 batches of quality transfer samples were tested. Each batch included medicinal materials, extract (intermediate), and granules / decoction (finished product). The batch correspondence is shown in the table below. Each batch will be referred to as "batch-number" in the following text.

[0173] Table 20. List of Corresponding Batch Numbers of Three Batches of Tangerine Peel Cough Granules

[0174] Table 21. List of Corresponding Batch Numbers of 4 Batches of Tangerine Peel Cough Relief Paste

[0175] 2. Solution preparation

[0176] (1) Preparation of the test solution:

[0177] Preparation of the test solution of Citrus reticulata: Take about 0.5g of Citrus reticulata powder (passed through a No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of methanol, weigh it, heat it in a water bath under reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, accurately measure 5ml of the filtrate, place it in a 50ml volumetric flask, add 50% methanol to the mark, shake it well, and the solution is ready.

[0178] Preparation of bitter almond test solution: Take about 0.25g of bitter almond powder (passed through a No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of methanol, stopper tightly, weigh it, sonicate (power 250W, frequency 50kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with methanol, shake well, filter it, accurately measure 5ml of the filtrate, place it in a 50ml volumetric flask, dilute to the mark with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0179] Preparation of intermediate test solution: Heat the intermediate extract in a water bath to 90°C, stir until homogeneous, cool, and then take about 0.6g of the extract, weigh accurately, place in a stoppered conical flask, add 100ml of 50% methanol solution accurately, weigh, sonicate for 30 minutes (power 500W, frequency 40kHz), remove, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0180] Preparation of test solution for Citrus Reticulatae Praeparata Granules: Take 10 sachets of granules, mix them well, grind them into a fine powder, take about 1.0g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 50% methanol solution, weigh it, sonicate for 30 minutes (power 500W, frequency 40kHz), take it out, let it cool, weigh it again, make up the weight loss with 50% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0181] Preparation of test solution for tangerine peel cough decoction: Take about 1.0g of the decoction, weigh it accurately, place it in a stoppered conical flask, add 25ml of 50% methanol accurately, weigh it, sonicate for 30 minutes (power 500W, frequency 40kHz), remove it, let it cool, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0182] (2) Preparation of reference solution:

[0183] Accurately weigh appropriate amounts of amygdalin, naringin, and rhubarb glycoside reference standards, respectively, and prepare stock solutions of the reference standards separately with methanol. The concentrations of amygdalin reference standard are: 0.0043–0.0851 mg / ml; naringin reference standard concentrations are: 0.0206–0.4121 mg / ml; and rhubarb glycoside reference standard concentrations are: 0.0012–0.0233 mg / ml.

[0184]

Feature Map

[0185] Using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Tables 3 and 4. The measurement time for each sample was approximately 85 minutes, the flow rate was 1.0 ml / min, the column temperature was 35℃, and the detection wavelength is shown in Table 5. Characteristic spectra of the test solution were determined.

[0186] The results showed that the transfer of the seven characteristic peak components between the raw medicinal materials, intermediates, and finished products was stable during the production process of granules and decoctions. The overall similarity of the fingerprint spectra of three batches of decoction extract and four batches of decoction extract, and three batches of granule extract and three batches of granules was calculated. The results showed that the similarity of all common peaks in the decoctions was greater than 0.989, and there was a good correlation between the three batches of decoction extract and four batches of decoction extract; the similarity of all common peaks in the granules was greater than 0.981, and there was a good correlation between the three batches of granule extract and three batches of granules. Figure 13 and Figure 14 ).

[0187] [Content Determination]

[0188] Using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Tables 3 and 4. Each sample determination took approximately 85 minutes, with a flow rate of 1.0 ml / min and a column temperature of 35℃. Detection wavelengths are shown in Table 5. Determination was performed according to the prescribed method at wavelengths of 207 nm (for amygdalin), 338 nm (for naringin), and 283 nm (for astragaloside). Regression analysis was performed between the corresponding peak area integral values ​​A and the concentration C of the reference standard, and the content of each component was calculated using a linear regression equation. The transfer rate was also calculated. The results showed that the linear correlation index R was greater than 0.999, demonstrating a good correlation between the concentration and peak area of ​​each component within a certain range. The linear concentration range and equation are shown in Table x.

[0189] Table 22 Linear Range for Mass Transfer Content Determination

[0190] Table 23 Transfer rate of amygdalin in each batch of raw medicinal materials and intermediates

[0191] Table 24 Transfer rate of naringin in each batch of raw medicinal materials and intermediates

[0192] Table 25 Transfer rate of rhubarb glycosides in each batch of raw medicinal materials and intermediates

[0193] Table 26. Transfer rates (%) of the three main components in seven batches of samples.

[0194] In summary, the transfer rates of amygdalin, naringin, and rhubarb glycoside remained relatively stable throughout the production process from raw materials to finished product, demonstrating that the current process has no significant impact on the quality transfer at each step. Given that amygdalin is a characteristic component of bitter almonds and naringin is a characteristic component of Citrus reticulata peel, and that the transfer of both is stable, it is recommended that these two components be included in the quality standards and production process monitoring system to achieve comprehensive control over the production process.

Claims

1. A method for constructing a characteristic spectrum of a tangerine peel cough preparation, characterized in that, Includes the following steps: (1) Preparation of the test solution: Weigh out the tangerine peel cough preparation, dissolve it in a 50-100% methanol solution, filter it, and take the filtrate to obtain the test solution. (2) Measurement The test solution was injected into a high-performance liquid chromatograph for determination, and the characteristic chromatogram of the tangerine peel cough preparation was obtained. The chromatographic conditions are as follows: The chromatographic column was a C18 column. The mobile phase was methanol as mobile phase A and an acidic aqueous solution with pH 2.0~3.5 as mobile phase B. Gradient elution was used: 0-80 min, mobile phase A 10-90%, mobile phase B 90-10%, flow rate 0.7~1.2 ml / min, column temperature 25~35℃, detection wavelength: 0.00~19.00 min 207±2 nm, 19.01~50.00 min 320±2 nm, 50.01~80.00 min 250±2 nm. The characteristic peaks of the described tangerine peel cough preparation are as follows: amygdalin, relative retention time 0.47~0.52 min; naringin, relative retention time 0.95~1.05 min; rosin, relative retention time 1.05~1.17 min; hesperidin hydrate, relative retention time 1.13~1.25 min; isohesperidin, relative retention time 1.29~1.43 min; schisandrol A, relative retention time 1.51~1.68 min; and characteristic components of licorice, relative retention time 1.79~1.99 min.

2. The construction method according to claim 1, characterized in that, The tangerine peel cough preparations include oral liquids, decoctions, syrups, granules, capsules, pills, tablets, or powders.

3. The construction method according to claim 1, characterized in that, The acidic aqueous solution is a 0.1% phosphoric acid solution.

4. The construction method according to claim 1, characterized in that, The chromatographic conditions are as follows: The chromatographic column was a C18 column. The mobile phase was methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was used: 0-80 min, mobile phase A 10-90%, mobile phase B 90-10%, flow rate 1.0 ml / min, column temperature 35℃, and detection wavelength: 0.00~19.00 min 207 nm, 19.01~50.00 min 320 nm, 50.01~80.00 min 250 nm.

5. The construction method according to claim 1, characterized in that, The addition of a 50-100% volume fraction methanol solution for dissolution refers to the addition of a 50% volume fraction methanol solution for dissolution.

6. The construction method according to claim 1, characterized in that, The tangerine peel cough preparation contains the following raw materials: tangerine peel, bitter almond, pinellia or water pinellia, stemona, cynanchum, poria, schisandra and licorice.

7. A method for detecting a tangerine peel cough syrup preparation, characterized in that, The method involves using the tangerine peel cough preparation to be tested as the test sample and performing the determination according to the steps in the method for constructing the characteristic chromatogram of the tangerine peel cough preparation as described in claim 1. A common pattern is generated using the software for similarity of traditional Chinese medicine chromatographic fingerprinting. Then, the characteristic chromatogram of the tangerine peel cough preparation is compared with the common pattern of the same preparation to evaluate the similarity of traditional Chinese medicine chromatographic fingerprinting. If the similarity is greater than or equal to 0.9, it is considered to be the tangerine peel cough preparation.

8. A method for determining the content of characteristic components in a tangerine peel cough syrup preparation, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take appropriate amounts of amygdalin, naringin, and rhubarb glycoside reference standards, dissolve them in methanol, and the solution is obtained. (2) Preparation of the test solution: Weigh out the tangerine peel cough preparation, dissolve it in a 50-100% methanol solution, filter it, and take the filtrate to obtain the test solution. (3) Measurement Pipe the reference standard and test solution and inject them into the high performance liquid chromatograph for determination; The chromatographic conditions are as follows: The chromatographic column was a C18 column. The mobile phase was methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was used: 0-80 min, mobile phase A 10-90%, mobile phase B 90-10%, flow rate 1.0 ml / min, column temperature 35℃. The detection wavelengths were: 0.00~19.00 min 207±2 nm, 19.01~50.00 min 320±2 nm, 50.01~80.00 min 250±2 nm. Simultaneously, 283±2 nm and 338±2 nm were collected from 0.00~80.00 min as the detection wavelengths for naringin and arbutin.

9. The determination method according to claim 8, characterized in that, The addition of a 50-100% volume fraction methanol solution for dissolution refers to the addition of a 50% volume fraction methanol solution for dissolution.

10. The determination method according to claim 8, characterized in that, The chromatographic conditions are as follows: The chromatographic column was a C18 column. The mobile phase was methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Gradient elution was used: 0-80 min, mobile phase A 10-90%, mobile phase B 90-10%, flow rate 1.0 ml / min, column temperature 35℃. The detection wavelengths were: 0.00~19.00 min 207 nm, 19.01~50.00 min 320 nm, 50.01~80.00 min 250 nm. Simultaneously, 283±2 nm and 338±2 nm were collected from 0.00~80.00 min as the detection wavelengths for naringin and arbutin.

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