A characteristic spectrum construction method and quality detection method of a lung detoxification granule

Abstract

The present application relates to the field of medicine, and more particularly to a characteristic chromatogram construction method and quality detection method of Qingfei Paidu Granules. The high performance liquid chromatography is used, and the chromatographic conditions include: a C18 silica bonded silica gel chromatographic column, acetonitrile as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, and a detection wavelength of 210 nm. The characteristic chromatogram construction method proposed in the present application characterizes alkaloids and organic acid characteristic components, increases the specificity identification of alkaloids characteristic components of four medicines, i.e. Ephedra, Bitter Apricot Kernel, Immature Bitter Orange Fruit and Tangerine Peel, and increases the specificity identification of organic acid components of two medicines, i.e. Coltsfoot Flower and Purpleflowered Aster, so as to comprehensively reflect the types and quantities of chemical components contained in Qingfei Paidu Granules, make up for the deficiency of simple index components in quality control, and be more scientific, rigorous and comprehensive.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a method for constructing a characteristic spectrum of lung-clearing and detoxifying granules and a method for quality testing. Background Technology

[0002] The Lung-Clearing and Detoxifying Decoction originates from a combination of classic Chinese medicine formulas, including Ma Xing Shi Gan Tang, She Gan Ma Huang Tang, Xiao Chai Hu Tang, and Wu Ling San, and has a mild nature. The main ingredients are: Ephedra 9g, Prepared Licorice Root 6g, Prepared Bitter Apricot Kernel 9g, Gypsum 15-30g (decocted first), Cinnamon Twig 9g, Alisma Rhizome 9g, Polyporus Rhizome 9g, Atractylodes Rhizome 9g, Poria 15g, Bupleurum Root 16g, Scutellaria Root 6g, Prepared Pinellia Rhizome 9g, Fresh Ginger 9g, Aster Root 9g, Coltsfoot Flower 9g, She Gan 9g, Asarum 6g, Dioscorea Rhizome 12g, Immature Bitter Orange 6g, Tangerine Peel 6g, and Patchouli 9g.

[0003] Lung-Clearing and Detoxifying Granules are a compound preparation based on the "Lung-Clearing and Detoxifying Decoction." This granule formulation retains the advantages of the decoction—rapid absorption and rapid action—while overcoming its disadvantages such as inconvenience in preparation, large dosage, and susceptibility to mold. Its main functions and indications are "dispelling cold and dampness, regulating the lungs and detoxifying. Used for epidemic diseases caused by cold-dampness toxins. Symptoms include fever and chills, body aches, fatigue and heaviness in the limbs; or cough with little phlegm, shortness of breath; or loss of taste, poor appetite, nausea and vomiting, and incomplete bowel movements; pale or swollen tongue with a greasy coating, and a slippery or soft pulse." With the large-scale production and widespread use of Lung-Clearing and Detoxifying Granules in the future, the supervision and control of its quality will become increasingly important and necessary.

[0004] In recent years, fingerprinting technology has become an efficient and convenient method for quality inspection and monitoring of traditional Chinese medicine (TCM) components. Fingerprinting technology utilizes high-performance liquid chromatography (HPLC) to analyze TCM extracts. By identifying specific peaks and their combinations that characterize specific components in the chromatogram, a unique fingerprint pattern is formed, enabling quality inspection and monitoring of TCM preparations. However, current technologies for quality inspection and monitoring of TCM using fingerprinting, and for establishing TCM preparation methods based on this method, generally require the exploration and establishment of characteristic fingerprint peaks for multiple medicinal ingredients. Qingfei Paidu Decoction is a combination and adjustment of four ancient formulas, containing over 20 medicinal ingredients, with potentially hundreds of detectable active components. Therefore, if the constructed characteristic chromatogram identifies too few characteristic components and their corresponding medicinal ingredients, a comprehensive assessment and identification of the quality of Qingfei Paidu granules cannot be achieved. However, if each of the different active ingredients in the finished product particles is identified individually and its characteristic fingerprint peaks are determined, the identification process would be too cumbersome and inconvenient, and the testing cost would be too high to be widely applied in actual production.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for constructing a characteristic spectrum of lung-clearing and detoxifying granules and a method for quality detection.

[0007] This invention provides a method for constructing the characteristic chromatogram of lung-clearing and detoxifying granules, employing high-performance liquid chromatography (HPLC). The chromatographic conditions include: a C18 silane-bonded silica column; acetonitrile as mobile phase A; 0.4% phosphoric acid solution as mobile phase B; and a detection wavelength of 210 nm. The gradient elution conditions are: from 0 to 7 minutes, the volume percentage of mobile phase A to mobile phase B gradually changes from 1.5:98.5 to 5:95; from 7.01 to 9 minutes, the volume percentage of mobile phase A to mobile phase B gradually changes from 5:95 to 6:94; and so on. From 0.01 to 18 minutes, the volume percentage of mobile phase A to mobile phase B gradually changes from 6:94 to 9:91 at a constant rate; from 18.01 to 36 minutes, the volume percentage of mobile phase A to mobile phase B gradually changes from 9:91 to 11:89 at a constant rate; from 36.01 to 38 minutes, the volume percentage of mobile phase A to mobile phase B remains at 11:89; from 38.01 to 40 minutes, the volume percentage of mobile phase A to mobile phase B gradually changes from 11:89 to 95:5 at a constant rate; the theoretical plate number calculated based on the ephedrine hydrochloride peak should not be less than 10,000.

[0008] Optionally, the column has a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm, preferably a Welch Ultimate AQ C18 column.

[0009] Optionally, the chromatographic conditions include: a flow rate of 0.8 to 1.2 mL / min, preferably 1.0 mL / min; a column temperature of 20 to 30 °C, preferably 20 °C; and an injection volume of 5 μL to 15 μL, preferably 5 μL.

[0010] Optionally, the method for constructing the feature map of lung-clearing and detoxifying granules includes the following steps:

[0011] S1. A reference solution was prepared using ephedrine hydrochloride reference standard with a diluent, which was an aqueous solution containing 0.05-0.2% phosphoric acid and 50-100% methanol by volume.

[0012] S2. Extract the lung-clearing and detoxifying granules test sample to obtain an extract. The extraction includes: dissolving the lung-clearing and detoxifying granules test sample in a 50-100% (v / v) methanol aqueous solution, sonicating for 10-30 minutes, filtering, drying and redissolving, using solid-phase extraction, first eluting with water and discarding the water, then eluting with a 20-40% (v / v) methanol aqueous solution, collecting the eluent, and filtering to obtain the extract.

[0013] S3. Analyze the extract and reference solution under chromatographic conditions to obtain characteristic chromatograms.

[0014] Optionally, in S1, the reference solution contains 0.1 mg of ephedrine hydrochloride reference standard per 1 mL. The diluent is an aqueous solution containing 0.1% phosphoric acid and 80% methanol by volume.

[0015] Optionally, in step S2, the Qingfei Paidu Granules test sample is dissolved in an 80% (v / v) methanol-water solution. The ultrasonic treatment time is 20 minutes; preferably, the ultrasonic power is 500W and the frequency is 40kHz. When eluting with water, the volume-to-mass ratio of water to the Qingfei Paidu Granules test sample is 2mL:1g. When eluting with a 4% (v / v) methanol-water solution, the volume-to-mass ratio of the 40% methanol-water solution to the Qingfei Paidu Granules test sample is 5mL:1g.

[0016] Optionally, the method for constructing the feature map of lung-clearing and detoxifying granules specifically includes the following steps:

[0017] S1. Take ephedrine hydrochloride reference standard, add 0.1% phosphoric acid and 80% methanol aqueous solution to prepare a solution containing 0.1 mg of ephedrine hydrochloride reference standard per 1 mL, and use it as a reference solution.

[0018] S2. Take 1.0g of Qingfei Paidu Granules as the test sample, add 25mL of 80% methanol aqueous solution, sonicate for 20 minutes, filter, dry and redissolve in 5mL of water, pass through a C18 solid phase extraction column, elute with 2mL of water, discard the aqueous solution, elute with 5mL of 40% methanol aqueous solution, collect the eluent, filter to obtain the extract.

[0019] S3. Analyze the extract and reference solution under chromatographic conditions to obtain characteristic chromatograms.

[0020] Optionally, a standard characteristic spectrum was constructed using the standard of Qingfei Paidu Granules as the test sample. The eight characteristic peaks in the standard characteristic spectrum were named peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and peak 8 in order of elution time. The compound of peak 1 is synephrine, the compound of peak 2 is adenosine, the compound of peak 3 is guanosine, the compound of peak 4 is ephedrine hydrochloride, the compound of peak 5 is pseudoephedrine hydrochloride, the compound of peak 6 is L-amygdaloid, the compound of peak 7 is amygdaloid, and the compound of peak 8 is chlorogenic acid.

[0021] This invention also proposes a quality testing method for lung-clearing and detoxifying granules. The method involves constructing a characteristic spectrum of the sample to be tested using the above method, with the peak of the reference sample as the S peak. The relative retention time of each characteristic peak in the characteristic spectrum of the sample to be tested relative to the S peak is calculated. If the relative retention time is within ±9% of a specified value, the product is deemed to be of qualified quality. Specifically, the specified retention time values ​​are: peak 1 (0.303), peak 2 (0.428), peak 3 (0.539), peak 4 (1.000), peak 5 (1.049), and peak 6 (1). The retention time values ​​for peaks 7 and 8 are specified as 1.770 and 1.894, respectively. Preferably, peak 1 is used to identify Citrus aurantium and Citrus reticulata in the raw materials of Qingfei Paidu Granules; peaks 4 and 5 are used to identify Ephedra sinica in the raw materials of Qingfei Paidu Granules; peaks 6 and 7 are used to identify Prunus armeniaca in the raw materials of Qingfei Paidu Granules; peak 8 is used to identify Tussilago farfara and Aster tataricus in the raw materials of Qingfei Paidu Granules; peak 2 is used to identify the remaining Chinese medicinal herbs in the raw materials of Qingfei Paidu Granules except for gypsum and ginger; and peak 3 is used to identify the remaining Chinese medicinal herbs in the raw materials of Qingfei Paidu Granules except for Cinnamomum cassia, gypsum, and ginger.

[0022] The present invention also proposes the application of the above-mentioned quality testing method as a quality standard for lung-clearing and detoxifying granules.

[0023] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0024] This invention proposes a characteristic spectrum construction method that addresses the problem of limited variety and attribution of characteristic components in the original standard characteristic spectrum. It proposes a characteristic spectrum for highly polar components in Qingfei Paidu granules, characterizing alkaloids and organic acids. This includes specific identification of alkaloids from four herbs: ephedra, bitter apricot kernel, immature bitter orange, and tangerine peel; and specific identification of organic acids from two herbs: coltsfoot flower and aster. Ephedrine and pseudoephedrine have anti-asthmatic and anti-inflammatory effects; amygdalin has antitussive and antiasthmatic effects; and synephedrine has bronchodilatory effects. These alkaloids are all major active ingredients in Qingfei Paidu granules. Chlorogenic acid also has antibacterial and anti-inflammatory effects and is an important auxiliary component in Qingfei Paidu granules. Therefore, the characteristic spectrum constructed by this invention can effectively reflect the variety and quantity of major active ingredients in Qingfei Paidu granules, compensating for the shortcomings of simply identifying index components in quality control.

[0025] This invention employs UPLC-Q-TOF-MS to qualitatively analyze the chemical components contained in the UPLC-MS spectrum of Qingfei Paidu Granules samples, thereby enabling accurate control and evaluation of this traditional Chinese medicine preparation. Attached Figure Description

[0026] Figure 1 The standard feature map is constructed using the method described in this embodiment of the invention.

[0027] Figure 2 The UV 210 nm chromatogram of the lung-clearing and detoxifying granules;

[0028] Figure 3 Total ion chromatogram (positive ions) for Qingfei Paidu Granules (TIC);

[0029] Figure 4 The chromatogram of the positive test sample solution of ephedra slices;

[0030] Figure 5 The chromatogram of the positive test sample solution of Asarum sieboldii slices;

[0031] Figure 6 The chromatogram of the positive test sample solution of cinnamon twig slices;

[0032] Figure 7 The chromatogram of the positive test sample solution of patchouli slices;

[0033] Figure 8 The chromatogram of the positive test sample solution of Atractylodes macrocephala slices;

[0034] Figure 9 The chromatogram of the positive test sample solution of yam slices;

[0035] Figure 10 The chromatogram of the positive test sample solution of gypsum slices;

[0036] Figure 11 The chromatogram of the positive test sample solution of Bupleurum chinense slices;

[0037] Figure 12 The chromatogram of the positive test sample solution of blanched bitter almond slices;

[0038] Figure 13 Chromatogram of a positive test sample solution of coltsfoot flower slices;

[0039] Figure 14 The chromatogram of the positive test sample solution of Aster tataricus slices;

[0040] Figure 15 The chromatogram of the positive test sample solution of Belamcanda chinensis slices;

[0041] Figure 16 Chromatogram of a positive test sample solution of ginger and pinellia slices;

[0042] Figure 17 The chromatogram of the positive test sample solution of ginger slices;

[0043] Figure 18 The chromatogram of the positive test sample solution of Citrus aurantium slices;

[0044] Figure 19 The chromatogram of the positive test sample solution of dried tangerine peel slices;

[0045] Figure 20 The chromatogram of the positive test sample solution of Polyporus umbellatus decoction pieces;

[0046] Figure 21 The chromatogram of the positive test solution of Poria cocos slices;

[0047] Figure 22 The chromatogram of the positive test sample solution of Alisma plantago-aquatica slices;

[0048] Figure 23 Chromatogram of a positive test sample solution of Scutellaria baicalensis slices;

[0049] Figure 24 The chromatogram of the positive test sample solution of prepared licorice root slices;

[0050] Figure 25 For identification of the reference sample;

[0051] Figure 26 The original chromatograms of the characteristic chromatograms of 14 batches of Qingfei Paidu Granules standard products;

[0052] Figure 27 Chromatograms of the reference standard and the test sample;

[0053] Figure 28 The graph is for examining the extraction method;

[0054] Figure 29 The chromatogram for investigating the extraction solvent;

[0055] Figure 30 For the time-based analysis of the graph;

[0056] Figure 31 A chromatogram showing the volume of water elution;

[0057] Figure 32 The chromatogram is for the elution volume of 40% methanol.

[0058] Figure 33 This is a full-wavelength scan spectrum;

[0059] Figure 34 , Figure 35 The spectrum is for different detection wavelengths;

[0060] Figure 36 The chromatogram for the column analysis is shown.

[0061] Figure 37 The spectrum is for the investigation of the mobile phase;

[0062] Figure 38The graph shows the column temperature.

[0063] Figure 39 A graph showing the flow velocity;

[0064] Figure 40 The chromatogram shows the injection volume.

[0065] Figure 41 The graphs are of the blank mobile phase and blank solvent.

[0066] Figure 42 The chromatogram for the column analysis is shown.

[0067] Figure 43 A chromatogram for investigating elution conditions;

[0068] Figure 44 This represents the results of the intermediate precision assessment.

[0069] In the chromatogram above, the horizontal axis represents time, and the vertical axis represents intensity. Detailed Implementation

[0070] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0071] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0072] This invention proposes a method for constructing characteristic chromatograms of lung-clearing and detoxifying granules, mainly targeting the simultaneous characterization of highly polar pharmacological components of the granules, such as alkaloids and organic acids. Liquid chromatography was employed, with the following chromatographic conditions: a C18 silane-bonded silica column, acetonitrile as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, and a detection wavelength of 210 nm; gradient elution conditions: 0–7 minutes, the volume percentage of mobile phase A and mobile phase B gradually changes from 1.5:98.5 to 5:95; 7.01–9 minutes, the volume percentage of mobile phase A and mobile phase B gradually changes from 5:95 to 6:94; 9.01–18 minutes, the mobile phase… The volume percentage of mobile phase A to mobile phase B gradually changes from 6:94 to 9:91 at a constant rate; from 18.01 to 36 minutes, the volume percentage of mobile phase A to mobile phase B gradually changes from 9:91 to 11:89 at a constant rate; from 36.01 to 38 minutes, the volume percentage of mobile phase A to mobile phase B remains at 11:89; from 38.01 to 40 minutes, the volume percentage of mobile phase A to mobile phase B gradually changes from 11:89 to 95:5 at a constant rate; the theoretical plate number, calculated based on the ephedrine hydrochloride peak, should not be less than 10,000.

[0073] This invention proposes a method for constructing characteristic chromatograms of highly polar medicinal components in lung-clearing and detoxifying granules. It adds characteristic chromatograms of highly polar components, primarily targeting alkaloids and organic acids. This includes the specific identification of alkaloids from four herbs (Ephedra, bitter almond, immature bitter orange, and tangerine peel) and the specific identification of organic acids from two herbs (Tussilago farfara and Aster tataricus). Due to the high polarity of alkaloids and organic acids, achieving the required chromatographic peak separation for characteristic chromatograms is extremely difficult and causes significant damage to ordinary C-18 chromatographic columns. Therefore, the applicant, through in-depth research and screening, selected the most suitable chromatographic conditions for characterizing polar components by examining the chromatographic column, mobile phase composition, and pH value. Furthermore, the compounds in the characteristic chromatograms of this invention were analyzed by UPLC-Q-TOF-MS and all were verified with reference standards, ensuring greater scientific rigor and comprehensiveness.

[0074] As an improvement of this invention, the column has a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm, preferably a Welch Ultimate AQ C18 column.

[0075] As an improvement to this embodiment of the invention, the chromatographic conditions include: a flow rate of 0.8 to 1.2 mL / min, preferably 1.0 mL / min; a column temperature of 20 to 30°C, preferably 20°C; and an injection volume of 5 μL to 15 μL, preferably 5 μL.

[0076] As an improvement to an embodiment of the present invention, the method for constructing the feature map of lung-clearing and detoxifying granules specifically includes the following steps:

[0077] S1. A reference solution was prepared using ephedrine hydrochloride reference standard with a diluent, which was an aqueous solution containing 0.05-0.2% phosphoric acid and 50-100% methanol by volume.

[0078] S2. Extract the lung-clearing and detoxifying granules test sample to obtain an extract. The extraction includes: dissolving the lung-clearing and detoxifying granules test sample in a 50-100% (v / v) methanol aqueous solution, sonicating for 10-30 minutes, filtering, drying and redissolving, using solid-phase extraction, first eluting with water and discarding the water, then eluting with a 40% (v / v) methanol aqueous solution, collecting the eluent, and filtering to obtain the extract.

[0079] S3. Analyze the extract and reference solution under chromatographic conditions to obtain characteristic chromatograms.

[0080] As an improvement to an embodiment of the present invention, in S1, each 1 mL of the reference solution contains 0.1 mg of ephedrine hydrochloride reference standard.

[0081] As an improvement to an embodiment of the present invention, in S1, the diluent is an aqueous solution containing 0.1% phosphoric acid and 80% methanol by volume.

[0082] As an improvement to an embodiment of the present invention, in S2, the lung-clearing and detoxifying granules test sample are dissolved in an 80% methanol aqueous solution by volume.

[0083] As an improvement to this embodiment of the invention, in S2, the ultrasonic treatment time is 20 minutes; preferably, the ultrasonic treatment power is 500W and the frequency is 40kHz.

[0084] As an improvement to this embodiment of the invention, in S2, when water is used for elution, the volume-to-mass ratio of water to the lung-clearing and detoxifying granules test sample is 2 mL: 1 g.

[0085] As an improvement to this embodiment of the invention, in S2, when eluting with a 40% methanol aqueous solution, the volume-to-mass ratio of the 40% methanol aqueous solution to the lung-clearing and detoxifying granules test sample is 5 mL: 1 g.

[0086] As a specific implementation of this invention, the construction method includes the following steps:

[0087] S1. Take ephedrine hydrochloride reference standard, add 0.1% phosphoric acid and 80% methanol aqueous solution to prepare a solution containing 0.1 mg of ephedrine hydrochloride reference standard per 1 mL, and use it as a reference solution.

[0088] S2. Take 1.0g of Qingfei Paidu Granules as the test sample, add 25mL of 80% methanol aqueous solution, sonicate for 20 minutes, filter, dry and redissolve in 5mL of water, pass through a C18 solid phase extraction column, elute with 2mL of water, discard the aqueous solution, elute with 5mL of 40% methanol aqueous solution, collect the eluent, filter to obtain the extract.

[0089] S3. Analyze the extract and reference solution under chromatographic conditions to obtain characteristic chromatograms.

[0090] As an improvement of this invention, a standard characteristic spectrum was constructed using the standard of Qingfei Paidu Granules as the test sample. The eight characteristic peaks in the standard characteristic spectrum were named peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and peak 8 in sequence according to the elution time. The compound of peak 1 is synephrine, the compound of peak 2 is adenosine, the compound of peak 3 is guanosine, the compound of peak 4 is ephedrine hydrochloride, the compound of peak 5 is pseudoephedrine hydrochloride, the compound of peak 6 is L-amygdaloid, the compound of peak 7 is amygdaloid, and the compound of peak 8 is chlorogenic acid.

[0091] This invention also relates to a quality testing method for lung-clearing and detoxifying granules. The method involves constructing a characteristic spectrum of the sample to be tested using the above method, with the peak of the reference sample as the S peak. The relative retention times of each characteristic peak in the characteristic spectrum of the sample to be tested relative to the S peak are calculated. If the relative retention time is within ±9% of a specified value, the product is deemed to be of qualified quality. Specifically, the specified retention times are: peak 1 (0.303), peak 2 (0.428), peak 3 (0.539), peak 4 (1.000), peak 5 (1.049), peak 6 (1.730), peak 7 (1.770), and peak 8 (1.894). Peak 1 is used to identify Citrus aurantium and Citrus reticulata peel in the raw materials of Qingfei Paidu Granules; peaks 4 and 5 are used to identify Ephedra sinica in the raw materials of Qingfei Paidu Granules; peaks 6 and 7 are used to identify Prunus armeniaca in the raw materials of Qingfei Paidu Granules; peak 8 is used to identify Tussilago farfara and Aster tataricus in the raw materials of Qingfei Paidu Granules; peak 2 is used to identify the remaining Chinese medicinal herbs in the raw materials of Qingfei Paidu Granules except for gypsum and ginger; peak 3 is used to identify the remaining Chinese medicinal herbs in the raw materials of Qingfei Paidu Granules except for Cinnamomum cassia, gypsum, and ginger. Ephedrine and pseudoephedrine have anti-asthmatic and anti-inflammatory effects; amygdalin has antitussive and antiasthmatic effects; synephrine has bronchodilatory effects. These alkaloid components are the main active ingredients of Qingfei Paidu Granules. Chlorogenic acid also has antibacterial and anti-inflammatory effects and is an important auxiliary ingredient in Qingfei Paidu Granules. Therefore, the quality detection method of this embodiment of the invention detects the quality of Qingfei Paidu Granules from the perspective of efficacy, which is more significant.

[0092] As an improvement to this invention, the above-mentioned feature map construction method can be used in combination with the feature map construction method disclosed in the applicant's previous invention patent application number 202110772465.9. The quality detection method of this invention utilizes high-performance liquid chromatography to detect highly polar active ingredients. By establishing multiple feature maps, multiple categories of components can be qualitatively identified simultaneously, which can more comprehensively reflect the types and quantities of chemical components contained in the Qingfei Paidu granules. This allows for a more comprehensive analysis of drug quality and improves the quality control system of Qingfei Paidu granules.

[0093] This invention also relates to the application of the above quality testing method as a quality standard for Qingfei Paidu Granules. The quality testing method of this invention is scientific, rigorous, and comprehensive, with strong specificity, clear direction, simplicity, low equipment requirements, high precision, good reproducibility and stability. Furthermore, it compensates for the deficiencies of the original standard characteristic spectrum, enabling better control of product quality and ensuring the safety and effectiveness of clinical medication. Therefore, it can be used as a quality standard for Qingfei Paidu Granules.

[0094] The instruments and reagents used in the following examples are:

[0095] Instruments: High-performance liquid chromatograph (HPLC): Huapu S6000, Thermo U-3000, Agilent Technologies 1260 Infinity II; UPLC-Q-TOF-MS: HPLC system: Agilent 1260 HPLC system, including autosampler, binary pump, column oven, online degassing device, and DAD detector; mass spectrometer: Agilent G6510AMS Q-TOF; CNC ultrasonic cleaner: KQ-500DE, 500W, 40kHz, Kunshan Ultrasonic Instrument Co., Ltd.; Analytical balance: BT25S, BS2202S, and BS124S, Sartorius; EYELA rotary evaporator: N-1100; EYELA water bath: SB-1000, Shanghai Ailang Instrument Co., Ltd.

[0096] Reagents: Methanol, chromatographic grade, Lot 213476, F22M47201, Fisher Scientific; Acetonitrile, chromatographic grade, batch number 211030426002, Tianjin Biaoshiqi Technology Development Co., Ltd.; Water, Millipore Clear-D, prepared using an integrated ultrapure water system; Phosphoric acid, analytical grade, batch number C12198142, MACKLIN.

[0097] Standards: Ephedrine hydrochloride, batch number 171241-201809, purity 100.0%; Pseudoephedrine hydrochloride, batch number 171237-201510, purity 99.8%; Amygdalin, batch number 110820-202109, purity 93.1%; Adenosine, batch number 110879-200202, all purchased from the China National Institutes for Food and Drug Control; Synephrine, batch number MUST-14010310, purity ≥98%; Chlorogenic acid, batch number MUST-14031401, purity ≥98%, all purchased from Chengdu Mansite Biotechnology Co., Ltd.; Guanosine, batch number 131007, purity >98%, purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd.

[0098] Example 1

[0099] This embodiment illustrates the method for constructing the feature map of Qingfei Paidu Granules:

[0100] 1. Preparation of reference solution: Take an appropriate amount of ephedrine hydrochloride reference standard, accurately weigh it, and add 80% methanol (containing 0.1% phosphoric acid) to prepare a solution containing 0.1 mg per 1 mL.

[0101] 2. Preparation of the test solution: Take the lung-clearing and detoxifying granules as the test sample, mix well, grind finely, take about 1.0 g, accurately weigh, place in a stoppered conical flask, accurately add 25 mL of 80% methanol, sonicate (power 500W, frequency 40kHz) for 20 minutes, remove, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, and pass through a C18 solid-phase extraction column (Waters). C18-E, 500mg / 3mL, was prewashed with 10mL of methanol and 10mL of water, eluted with 2mL of water, and the aqueous solution was discarded. Then, it was eluted with 5mL of 40% methanol. The 40% methanol eluent was collected, shaken well, filtered, and the filtrate was collected to obtain the final product.

[0102] 3. Determination method: Accurately pipette 5 μL of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram after 40 minutes.

[0103] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (25 cm column length, 4.6 mm inner diameter, 5 μm particle size, Welch Ultimate AQ-C18 column); acetonitrile was used as mobile phase A, and 0.4% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 1.0 mL / min; the column temperature was 20 °C; and the detection wavelength was 210 nm. The theoretical plate number, calculated based on the ephedrine hydrochloride peak, should be no less than 10,000.

[0104] Table 1

[0105] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～7 1.5→5 98.5→95 7～9 5→6 95→94 9～18 6→9 94→91 18～36 9→11 91→89 36～38 11 89 38～40 11→95 89→5

[0106] The standard characteristic spectrum was constructed using the lung-clearing and detoxifying granules standard according to the above method, as follows: Figure 1 As shown.

[0107] Depend on Figure 1 It can be seen that the eight characteristic peaks in the standard characteristic spectrum are named peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and peak 8 in order of elution time; the compound of peak 1 is synephrine, the compound of peak 2 is adenosine, the compound of peak 3 is guanosine, the compound of peak 4 is ephedrine hydrochloride, the compound of peak 5 is pseudoephedrine hydrochloride, the compound of peak 6 is L-amygdaloid, the compound of peak 7 is amygdaloid, and the compound of peak 8 is chlorogenic acid.

[0108] Example 2

[0109] This example illustrates the use of UPLC-Q-TOF-MS to establish the chemical structure of each peak in the characteristic spectrum of Qingfei Paidu Granules:

[0110] 1. The preparation of the test solution is the same as in Example 1;

[0111] 2. Determination method: Accurately pipette 5 μL of the test solution and inject it into UPLC-Q-TOF-MS for determination.

[0112] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (column length 25 cm, inner diameter 4.6 mm, particle size 5 μm, Welch Ultimate AQ-C18 column); acetonitrile was used as mobile phase A, and 0.5% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 2; the flow rate was 1.0 mL per minute; the column temperature was 25 °C; and the detection wavelength was 210 nm. The theoretical plate number, calculated based on the ephedrine hydrochloride peak, should not be less than 10,000.

[0113] Table 2: Gradient Elution Table

[0114] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～7 1.5→5 98.5→95 7～9 5→6 95→94 9～18 6→9 94→91 18～42 9→10 91→90 42～45 10→95 90→5

[0115] The mass spectrometry parameters for UPLC-Q-TOF-MS analysis are as follows:

[0116] Ion Source Type: Dual ESI; Scan Mode: Positive ion mode and negative ion mode; Scan Range: 60-2000 m / z (MS), 40-1500 m / z (MS / MS); Scan Rate: 4.00 (MS), 3.00 (MS / MS); Cone Gas Flow Rate: 11.0 l / min; Vaporization Temperature: 300℃; Octuple RF Amplitude: 750 Vpp; Cone Voltage: 65.0 Volt.

[0117] The UV 210nm chromatogram and TIC total ion chromatogram (positive ions) were obtained as follows: Figure 2 and Figure 3 As shown.

[0118] Table 3: UPLC-Q-TOF-MS data of each compound in the lung-clearing and detoxifying granules.

[0119]

[0120]

[0121]

[0122] Example 3

[0123] This example illustrates the investigation into the attribution of characteristic chromatographic peaks of Qingfei Paidu Granules:

[0124] Following the preparation process of Qingfei Paidu Granules, 21 medicinal herbs were prepared as decoctions: Ephedra, Asarum, Cinnamon Twig, Patchouli, Atractylodes Macrocephala, Dioscorea, Gypsum, Bupleurum, Prepared Bitter Almond, Coltsfoot Flower, Aster, Belamcanda Rhizome, Ginger-processed Pinellia, Fresh Ginger, Immature Bitter Orange, Tangerine Peel, Polyporus, Poria, Alisma, Scutellaria, and Prepared Licorice. Positive test solutions for each herb were then prepared according to the method in Example 1. Under the chromatographic conditions of Example 1, the positive test solutions and Qingfei Paidu Granules standard solutions were injected separately to determine the correlation of chromatographic peaks in the characteristic chromatograms. The chromatograms are shown below. Figures 4 to 24 , Figure 25 This is the reference standard identification diagram. See Table 4 for details of the chromatographic peak assignments.

[0125] Table 4: Results of the investigation into the attribution of chromatographic peaks

[0126] Chromatographic peak numbering source compound 1 Fructus Aurantii Immaturus and Pericarpium Citri Reticulatae Synephrin 2 Except for gypsum and ginger, all have adenosine 3 Except for cinnamon twigs, gypsum, and ginger, all have Guanosine 4 Ephedra Ephedrine hydrochloride 5 Ephedra pseudoephedrine hydrochloride 6 Blanched bitter almonds L-Amygdalin 7 Blanched bitter almonds amygdalin 8 Coltsfoot, Asters chlorogenic acid

[0127] The results showed that among the eight chromatographic peaks, two peaks were specific to Ephedra slices, two peaks were specific to bitter almond slices, peak 1 was shared by Citrus aurantium and Citrus reticulata, peak 8 was shared by Tussilago farfara and Aster tataricus, peak 2 was present in all varieties except Gypsum and Zingiber officinale, and peak 3 was present in all varieties except Cinnamomum cassia, Gypsum, and Zingiber officinale.

[0128] Example 4

[0129] This embodiment illustrates the method for establishing the quality standards of Qingfei Paidu Granules:

[0130] Characteristic chromatograms of 14 batches of Qingfei Paidu Granules standard were detected according to the method described in the examples. Results are shown below. Figure 26 , Figure 1 See Table 5.

[0131] Table 5: Detection Results of Characteristic Chromatographic Profiles of 14 Batches of Qingfei Paidu Granules Standard Samples

[0132]

[0133]

[0134] Results Analysis: The detection results of the characteristic chromatograms of 14 batches of standard samples were analyzed. Typical chromatographic peaks appearing in the characteristic chromatograms of each batch of test samples were taken as common peaks, totaling 8 common peaks. Peak 4, representing ephedrine hydrochloride, was designated as the reference peak. The relative retention times of each characteristic peak and the S peak were calculated. The average relative retention times of the 8 characteristic peaks of the 14 batches of Qingfei Paidu Granules were 0.303 (peak 1), 0.428 (peak 2), 0.539 (peak 3), 1.000 (peak S), 1.049 (peak 5), 1.730 (peak 6), 1.770 (peak 7), and 1.894 (peak 8). This time was established as the specified value. According to the results of multi-batch sample testing and intermediate precision studies, the characteristic chromatogram of the test sample should present 8 characteristic peaks, with the peak corresponding to the reference peak designated as the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and their relative retention times should be within ±9% of the specified value. Within this range, the characteristic peaks of the sample chromatograms obtained using different models of analytical instruments all met the requirements.

[0135] Example 5

[0136] This example illustrates the selection of a reference point:

[0137] Take the ephedrine hydrochloride reference solution and detect it according to the detection method in Example 1. The corresponding chromatogram is shown in [Figure 1]. Figure 27 .

[0138] Figure 27 The chromatographic peak shape and separation effect of the ephedrine hydrochloride reference standard were both good, so ephedrine hydrochloride was selected as the characteristic chromatographic reference.

[0139] Example 6

[0140] This example is used to evaluate the extraction method of the standard solution:

[0141] 1. Two extraction methods to be investigated: ultrasonic and reflux extraction.

[0142] The method is as follows: Take the standard of lung-clearing and detoxifying granules, mix well, grind finely, take about 1.0g, accurately weigh two portions, place them in stoppered conical flasks, accurately add 25mL of 80% methanol, sonicate one portion (power 500W, frequency 40kHz) for 20 minutes, and heat the other portion under reflux for 20 minutes, remove, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 5mL of water, and pass through a C18 solid-phase extraction column (Waters). C18-E, 500 mg / 3 mL, pre-washed sequentially with 10 mL each of methanol and water, eluted with 2 mL of water, discarding the aqueous solution, then eluted with 5 mL of 40% methanol, collecting the 40% methanol eluent, shaking well, filtering, and taking the filtrate as the final product. Each extraction method was performed in duplicate. Chromatograms are shown below. Figure 28 .

[0143] The results showed that the number and area of ​​chromatographic peaks obtained by the two extraction methods were not significantly different, so ultrasonic extraction was the appropriate choice.

[0144] 2. Examine the extraction solvent:

[0145] The study aimed to investigate the effects of different solvents: 50% methanol, 80% methanol, and methanol. The method was as follows: The standard of Qingfei Paidu Granules was mixed, ground finely, and approximately 1.0 g was accurately weighed into three portions. Each portion was placed in a stoppered conical flask, and 25 mL of the corresponding solvent was accurately added. The mixture was ultrasonically treated (500 W, 40 kHz) for 20 minutes. Afterward, the mixture was removed, cooled, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 5 mL of water and passed through a C18 solid-phase extraction column (Waters). C18-E, 500 mg / 3 mL, pre-washed sequentially with 10 mL each of methanol and water, eluted with 2 mL of water, discarding the aqueous solution, then eluted with 5 mL of 40% methanol, collecting the 40% methanol eluent, shaking well, filtering, and taking the filtrate as the final product. Two replicates were performed for each extraction solvent. See chromatogram below. Figure 29 .

[0146] The results showed that 80% methanol had a good extraction effect on each chromatographic peak in the Qingfei Paidu Granules standard, and was easy to filter with a stable baseline. Therefore, 80% methanol was selected as the extraction solvent.

[0147] 3. Examination of extraction time

[0148] The study aimed to investigate the effects of different extraction times of 10 min, 20 min, and 30 min. The method was as follows: The lung-clearing and detoxifying granules standard was mixed, ground finely, and approximately 1.0 g was accurately weighed into three portions. Each portion was placed in a stoppered conical flask, and 25 mL of 80% methanol was accurately added. The mixture was ultrasonically treated (500 W power, 40 kHz frequency) for the corresponding time. Afterward, the mixture was removed, cooled, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 5 mL of water and then passed through a C18 solid-phase extraction column (Waters). C18-E, 500 mg / 3 mL, pre-washed sequentially with 10 mL each of methanol and water, eluted with 2 mL of water, discarding the aqueous solution, then eluted with 5 mL of 40% methanol, collecting the 40% methanol eluent, shaking well, filtering, and taking the filtrate as the final product. Two replicates were performed for each extraction time. See chromatogram below. Figure 32 .

[0149] The results showed that different extraction times had little effect on the number and area of ​​chromatographic peaks in the characteristic chromatograms, so a relatively suitable extraction time of 20 min was selected.

[0150] 4. Examination of water washing volume

[0151] The study aimed to investigate the effects of different water elution volumes of 1 mL, 2 mL, and 3 mL. The method was as follows: The lung-clearing and detoxifying granules standard was mixed, ground finely, and approximately 1.0 g was accurately weighed into three portions. Each portion was placed in a stoppered conical flask, and 25 mL of 80% methanol was accurately added. The mixture was ultrasonically treated (500 W power, 40 kHz frequency) for the appropriate time. Afterward, the mixture was removed, cooled, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 5 mL of water and passed through a C18 solid-phase extraction column (Waters). C18-E, 500 mg / 3 mL, pre-washed sequentially with 10 mL each of methanol and water, eluted with the corresponding volume of water, discarding the aqueous solution, then eluted with 5 mL of 40% methanol, collecting the 40% methanol eluent, shaking well, filtering, and taking the filtrate as the final product. Two replicates were performed for each water eluent volume. See chromatogram below. Figure 31 .

[0152] The results showed that the baseline was not stable when the elution volume was 1 mL, and the peak area was small when the elution volume was 3 mL. Taking all factors into consideration, a water elution volume of 2 mL was selected.

[0153] 5. Investigation of the elution volume of 40% methanol

[0154] The elution volume of 40% methanol was to be investigated. The method was as follows: Take the Qingfei Paidu Granules standard, mix well, grind finely, take approximately 1.0 g, place in a stoppered conical flask, accurately add 25 mL of 80% methanol, sonicate (500 W power, 40 kHz frequency) for the appropriate time, remove, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of water, and pass through a C18 solid-phase extraction column (Waters). C18-E, 500 mg / 3 mL, was pre-washed sequentially with 10 mL each of methanol and water, then eluted with 2 mL of water, the aqueous solution was discarded, followed by elution with 5 mL of 40% methanol. Another 5 mL of 40% methanol was added for further elution, and the 40% methanol eluent was collected, shaken well, filtered, and the filtrate was taken as the final product. This procedure was repeated in duplicate. The chromatogram is shown below. Figure 32 .

[0155] The results showed that almost no chromatographic peaks were detected in the added 5 mL of 40% methanol eluent, so a 5 mL volume of 40% methanol eluent was sufficient.

[0156] Example 5

[0157] This example is used to illustrate chromatographic conditions and system suitability:

[0158] 1. Selection of detection wavelength

[0159] A diode array detector was used to perform full-wavelength detection on the Qingfei Paidu Granules standard, and three-dimensional spectra were collected. (See attached image) Figure 33 Spectra were also collected at wavelengths of 210nm, 215nm, 220nm, 230nm, 254nm, 280nm, 320nm, and 360nm, as shown in [reference needed]. Figure 34 , Figure 35 .

[0160] The test results showed that there were more chromatographic peaks and larger peak areas at a wavelength of 210 nm, so 210 nm was selected as the detection wavelength for the characteristic spectrum.

[0161] 2. Examination of the chromatographic column

[0162] Three different batches of Welch Ultimate AQ-C18 (4.6 × 250 mm, 5 μm) columns were investigated. Chromatograms are shown below. Figure 36 The results showed that the separation effects were all good.

[0163] 3. Investigation of the mobile phase

[0164] Three mobile phases—acetonitrile-water, acetonitrile-0.2% phosphoric acid solution, and acetonitrile-0.4% phosphoric acid solution—were investigated, and the gradient elution table is shown in Table 1. The chromatograms are shown below. Figure 37 .

[0165] The results showed that the acetonitrile-0.4% phosphoric acid solution had a good separation effect on each chromatographic peak, so the acetonitrile-0.4% phosphoric acid solution was selected as the mobile phase.

[0166] 4. Examination of column temperature

[0167] Three column temperatures (20℃, 25℃, and 30℃) were selected for investigation; the chromatograms are shown below. Figure 38 .

[0168] The results showed that the separation of each chromatographic peak was better when the column temperature was 20℃, so the column temperature of 20℃ was selected.

[0169] 5. Flow velocity assessment

[0170] Three flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min were selected for investigation. The chromatograms are shown below. Figure 39 .

[0171] The results showed that when the flow rate was 1.0 mL / min, the retention time of each chromatographic peak was moderate, the separation effect was good, and the peak shape was good. Therefore, the flow rate of 1.0 mL / min was selected.

[0172] 6. Examination of injection volume

[0173] Three injection volumes of 5 μL, 10 μL, and 15 μL were selected for investigation. The chromatograms are shown below. Figure 40 .

[0174] The results showed that when the injection volume was 5 μL, the separation of each chromatographic peak was good and the peak shape was good. Therefore, the injection volume of 5 μL was selected.

[0175] 7. Specificity Examination

[0176] The interference of blank mobile phase and blank solvent on the test solution was investigated. The chromatogram is shown below. Figure 41 The results showed that the blank mobile phase and blank solvent did not interfere with the characteristic spectra of the test solution.

[0177] 8. Chromatographic column investigation

[0178] Three chromatographic columns were investigated: Welch Ultimate AQ-C18 (4.6 × 250 mm, 5 μm), CAPCELL PAK AQ-C18 (4.6 × 250 mm, 3 μm), and Phenomenex Luna(2)C18 (4.6 × 250 mm, 5 μm). Chromatograms are shown below. Figure 42 .

[0179] The results showed that the Welch Ultimate AQ-C18 column provided better separation.

[0180] 9. Investigation of elution conditions

[0181] Considering the complexity of the sample solution, a gradient elution method was adopted, and the elution conditions were optimized. The elution conditions are shown in Table 6, and the chromatograms are shown in [Table 6]. Figure 43 .

[0182] Table 6: Comparison of Gradient Conditions

[0183]

[0184]

[0185] The results showed that elution condition 3 had a good separation effect on all chromatographic peaks, and the retention time of the chromatographic peaks was moderate and the overall distribution was uniform.

[0186] Example 6

[0187] This example is used to illustrate the results of the methodological investigation:

[0188] 1. Precision test

[0189] Take the Qingfei Paidu Granules standard (batch number 2108009), construct the characteristic spectrum according to the method in Example 1, inject the sample 6 times consecutively, detect the characteristic spectrum of the sample, use ephedrine hydrochloride as a reference, calculate the relative retention time, and the results are shown in Table 7.

[0190] Table 7: Precision test results

[0191] serial number 1 2 3 4 5 6 7 8 1 0.312 0.438 0.546 1.000 1.049 1.733 1.773 1.897 2 0.310 0.435 0.544 1.000 1.049 1.732 1.772 1.897 3 0.309 0.434 0.543 1.000 1.049 1.732 1.773 1.897 4 0.307 0.432 0.542 1.000 1.049 1.732 1.772 1.897 5 0.305 0.430 0.541 1.000 1.049 1.732 1.773 1.897 6 0.302 0.429 0.540 1.000 1.049 1.732 1.772 1.897 mean 0.307 0.433 0.543 1.000 1.049 1.732 1.773 1.897 RSD (%) 1.12 0.78 0.38 0.00 0.01 0.02 0.02 0.01

[0192] The results show that the method of the present invention has good precision.

[0193] 2. Stability test

[0194] The Qingfei Paidu Granules standard (batch number 2108009) was used to construct a characteristic spectrum according to the method in Example 1. The characteristic spectrum was detected at 0, 1.5, 3, 6, 12 and 20 hours. The relative retention time was calculated with ephedrine hydrochloride as a reference. The results are shown in Table 8.

[0195] Table 8: Stability Test Results

[0196]

[0197]

[0198] The results showed that the test solution had good stability and met the detection requirements of the characteristic spectrum.

[0199] 3. Repeatability test

[0200] Take the Qingfei Paidu Granules standard (batch number 2108009) and prepare 6 test samples according to the preparation method of the test sample solution in Example 1. Detect the characteristic chromatograms and calculate the relative retention time with ephedrine hydrochloride as a reference. The results are shown in Table 9.

[0201] Table 9: Results of Repeatability Testing

[0202] serial number 1 2 3 4 5 6 7 8 1 0.302 0.429 0.540 1.000 1.049 1.732 1.772 1.897 2 0.303 0.428 0.540 1.000 1.049 1.732 1.772 1.896 3 0.303 0.428 0.540 1.000 1.049 1.731 1.771 1.896 4 0.302 0.429 0.540 1.000 1.049 1.730 1.771 1.896 5 0.303 0.427 0.539 1.000 1.049 1.730 1.771 1.896 6 0.303 0.428 0.540 1.000 1.049 1.730 1.771 1.896 mean 0.303 0.428 0.540 1.000 1.049 1.731 1.771 1.896 RSD (%) 0.11 0.12 0.07 0.00 0.01 0.04 0.04 0.03

[0203] The results show that the method of the present invention has good repeatability and meets the detection requirements of feature maps.

[0204] 4. Intermediate precision test

[0205] The Qingfei Paidu Granules standard (batch number 2108009) was used. On different dates, different analysts prepared the test sample and reference solution according to the method in Example 1. The solutions were analyzed using three analytical instruments: Huapu S6000, Thermo U-3000, and Agilent Technologies 1260 Infinity II. The experimental results are shown in [Figure 1]. Figure 44 See Table 10.

[0206] Table 10: Results of intermediate precision testing

[0207] instrument 1 2 3 4 5 6 7 8 Huapu 0.303 0.428 0.540 1.000 1.049 1.731 1.771 1.896 Thermo 0.320 0.434 0.535 1.000 1.048 1.703 1.743 1.870 Agilent 0.329 0.401 0.514 1.000 1.049 1.735 1.776 1.896 RSD (%) 4.27 4.13 2.64 0.00 0.04 1.01 0.99 0.80

[0208] The results show that the method of the present invention has good applicability.

[0209] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a feature map of lung-clearing and detoxifying granules, characterized in that, The method employs high-performance liquid chromatography and includes the following steps: S1. Take ephedrine hydrochloride reference standard, add an aqueous solution of 0.1% phosphoric acid and 80% methanol by volume to prepare a solution containing 0.1 mg of ephedrine hydrochloride reference standard per 1 mL, and use it as a reference solution. S2. Take 1.0g of Qingfei Paidu Granules as test sample, add 25 mL of 80% methanol aqueous solution, sonicate for 20 minutes, filter, dry and redissolve in 5 mL of water, pass through a C18 solid phase extraction column, elute with 2 mL of water, discard the aqueous solution, elute with 5 mL of 40% methanol aqueous solution, collect the eluent, filter to obtain the extract; S3. The extract and the reference solution are analyzed under the following chromatographic conditions to obtain the characteristic chromatograms; The chromatographic conditions include: The chromatographic column was a C18 silane-bonded silica gel column, with acetonitrile as mobile phase A and 0.4% phosphoric acid solution as mobile phase B, and the detection wavelength was 210 nm. The gradient elution conditions were as follows: 0–7 minutes, the volume percentage of mobile phase A to mobile phase B gradually changed from 1.5:98.5 to 5:95; 7.01–9 minutes, the volume percentage of mobile phase A to mobile phase B gradually changed from 5:95 to 6:94; 9.01–18 minutes, the volume percentage of mobile phase A to mobile phase B gradually changed from 6:94 to 9:91; 18.01–36 minutes, the volume percentage of mobile phase A to mobile phase B gradually changed from 9:91 to 11:89; 36.01–38 minutes, the volume percentage of mobile phase A to mobile phase B remained at 11:89; 38.01–40 minutes, the volume percentage of mobile phase A to mobile phase B gradually changed from 11:89 to 95:5; the theoretical plate number, calculated based on the ephedrine hydrochloride peak, should be no less than 10,000. The chromatographic column has a length of 25 cm, an inner diameter of 4.6 mm, and a particle size of 5 μm. The flow rate was 0.8 ~ 1.2 mL / min, the column temperature was 20 ~ 30℃, and the injection volume was 5 μL ~ 15 μL; A standard characteristic spectrum was constructed using the standard of the lung-clearing and detoxifying granules as the test sample. The eight characteristic peaks in the standard characteristic spectrum were named peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and peak 8 in sequence according to the elution time. The compound of peak 1 is synephrine, the compound of peak 2 is adenosine, the compound of peak 3 is guanosine, the compound of peak 4 is ephedrine hydrochloride, the compound of peak 5 is pseudoephedrine hydrochloride, the compound of peak 6 is L-amygdaloid, the compound of peak 7 is amygdaloid, and the compound of peak 8 is chlorogenic acid.

2. The construction method according to claim 1, characterized in that, The chromatographic column used was a Welch Ultimate AQC18 column.

3. The construction method according to claim 1, characterized in that, Chromatographic conditions include: The flow rate was 1.0 mL / min; The column temperature is 20℃; The injection volume was 5 μL.

4. The construction method according to claim 1, characterized in that, In S2, the power of the ultrasonic treatment is 500W and the frequency is 40 kHz.

5. A method for quality testing of lung-clearing and detoxifying granules, characterized in that, The characteristic spectrum of the test sample is constructed according to the method described in any one of claims 1 to 4. The peak of the reference sample is taken as the S peak. The relative retention time of each characteristic peak in the characteristic spectrum of the test sample with the S peak is calculated. If the relative retention time is within ±9% of the specified value, the product is judged to be of qualified quality. Among them, the specified retention time values ​​are 0.303 for peak 1, 0.428 for peak 2, 0.539 for peak 3, 1.000 for peak 4, 1.049 for peak 5, and 1.73 for peak 6. The retention time values ​​for peaks 0 and 7 are 1.770 and 1.894, respectively. Peak 1 is used to identify Citrus aurantium and Citrus reticulata in the raw materials of Qingfei Paidu Granules. Peaks 4 and 5 are used to identify Ephedra in the raw materials of Qingfei Paidu Granules. Peaks 6 and 7 are used to identify Alpinia officinarum in the raw materials of Qingfei Paidu Granules. Peak 8 is used to identify Tussilago farfara and Aster tataricus in the raw materials of Qingfei Paidu Granules. Peak 2 is used to identify the Chinese medicinal herbs in the raw materials of Qingfei Paidu Granules except for Gypsum and Zingiber officinale. Peak 3 is used to identify the Chinese medicinal herbs in the raw materials of Qingfei Paidu Granules except for Cinnamomum cassia, Gypsum, and Zingiber officinale.

6. The quality testing method as described in claim 5, used as a quality standard for lung-clearing and detoxifying granules.

Images