CN114577926A

CN114577926A - Fingerprint detection method of traditional Chinese medicine composition for ventilating lung and removing toxicity

Info

Publication number: CN114577926A
Application number: CN202210032961.5A
Authority: CN
Inventors: 张伯礼; 刘清泉; 高秀梅; 张俊华; 宋新波; 张磊; 王毅; 杨丰文; 郑文科; 王涛; 王跃飞; 张晗; 黄宇虹; 王苹; 刘二伟; 刘岱琳; 张静泽; 黄明
Original assignee: Shandong Buchang Pharmaceuticals Co ltd
Current assignee: Shandong Buchang Pharmaceuticals Co ltd
Priority date: 2021-01-19
Filing date: 2022-01-12
Publication date: 2022-06-03
Also published as: CN114184696A; CN112858515A

Abstract

The invention provides a fingerprint detection method of a traditional Chinese medicine composition for diffusing lung and removing toxicity, wherein the traditional Chinese medicine composition for diffusing lung and removing toxicity mainly comprises raw ephedra, bitter apricot seed, raw gypsum, raw coix seed, atractylis lancea, patchouli, artemisia apiacea, polygonum cuspidatum, verbena, dry reed root, semen lepidii, pummelo peel and raw liquorice; the detection method of the lung ventilating and toxin removing composition comprises the following steps: a thin-layer identification method of naringin, emodin, physcion, polydatin and ephedrine hydrochloride; determination method for ephedrine hydrochloride, pseudoephedrine hydrochloride, naringin and glycyrrhizic acid content. The invention establishes characteristic fingerprint spectrums of 12 index components of verbascoside, viscapine-2, polydatin, verbascoside, naringin, rhoifolin, glycyrrhizic acid and emodin, thereby ensuring the quality controllability of the batch of the traditional Chinese medicines and the stability of the internal quality components. The fingerprint detection method provides a powerful guarantee for the safety, effectiveness and quality controllability of the lung-ventilating and toxin-vanquishing granules.

Description

Fingerprint detection method of traditional Chinese medicine composition for ventilating lung and removing toxicity

Technical Field

The invention belongs to the field of Chinese patent medicine content component detection quality analysis, and particularly relates to a fingerprint detection method of a traditional Chinese medicine composition for dispersing lung qi and removing toxicity.

Background

Coronaviruses are a small part of a large family of viruses, three of the seven classes of coronaviruses known to man include: SARS-CoV, Middle East respiratory syndrome virus (MERS-CoV) and novel coronavirus (SARS-CoV 2), which have high killing power to human. A novel coronavirus pneumonia (Corona Virus Desase 2019, COVID-19) caused by SARS-CoV2 Virus is spreading worldwide.

Among them, the clinical symptoms of COVID-19 are usually manifested as fever, dry cough and hypodynamia, and a few patients are accompanied by nasal obstruction, watery nasal discharge, pharyngalgia, myalgia, diarrhea and other symptoms. Severe patients often develop dyspnea and/or hypoxemia after one week of onset, and severe patients can rapidly progress to acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, hemorrhagic coagulation dysfunction, multiple organ failure and the like. The traditional Chinese medicine plays an important role in treating novel coronavirus pneumonia diseases. According to clinical observation, the prescription has obvious effects on improving fever, cough, suffocated asthma and hypodynamia of patients with new coronary pneumonia, and particularly has unique advantages in preventing and reducing severe cases of patients with mild symptoms. Because the traditional Chinese medicine composition for dispersing lung qi and removing toxicity has a plurality of medicinal materials and is extracted by water decoction, the traditional Chinese medicine composition has quite complex chemical components, and the curative effect of the traditional Chinese medicine is mostly a comprehensive expression of multiple components and multiple action ways. The quality control of Chinese herbal compound is also advancing to deeper level continuously with the acceleration of the modernization process of Chinese herbal medicine, and various new technologies and methods are developed, which are helpful for further clarifying the quality monitoring and the action mechanism of Chinese herbal medicine and its compound preparation. At present, the quality evaluation research of the traditional Chinese medicine oral preparation can be roughly divided into 4 modes: the quality control is carried out on a plurality of effective components or main index components based on the quality control of the Chinese medicinal preparation with multiple indexes; secondly, the fingerprint spectrum of the traditional Chinese medicine preparation product is measured and controlled based on the quality control of the traditional Chinese medicine preparation of the fingerprint spectrum; in-vitro evaluation, the dissolution rate or the biological value is detected to evaluate the difference of various dosage forms; and fourthly, evaluating the medicine effect, namely researching the components and the metabolic components of the traditional Chinese medicine preparation in the blood. The invention aims at the index components of the main medicinal materials in the other party and the corresponding quality control method based on the medicinal material composition of the lung-ventilating and toxin-vanquishing granules and the quality control method research to be developed. Therefore, the invention aims to establish a qualitative and quantitative detection method which is strong in specificity, scientific, simple and convenient, and can ensure the quality and the curative effect of the traditional Chinese medicine compound preparation.

Disclosure of Invention

The invention aims to solve the technical problem of providing a fingerprint detection method of a traditional Chinese medicine composition for ventilating the lung and removing toxicity, wherein the detection method not only comprises a thin-layer qualitative identification method of naringin, emodin, physcion, polydatin and ephedrine hydrochloride, but also establishes a determination method of multi-index content components of ephedrine hydrochloride, pseudoephedrine hydrochloride, naringin and glycyrrhizic acid. The detection method has good specificity and stability and strong durability, and can effectively ensure the stability and controllability of the quality of the medicine in the industrial production process. The invention also provides a fingerprint detection method of the traditional Chinese medicine composition for dispersing lung qi and removing toxicity, which can improve the internal quality control standard of the traditional Chinese medicine composition for dispersing lung qi and removing toxicity, and further effectively ensure the safety and effectiveness of the medicine.

The technical scheme of the invention is as follows:

a fingerprint detection method of a traditional Chinese medicine composition for ventilating the lung and removing toxicity comprises the following steps:

preparing a reference substance solution: mixing appropriate amount of radix Knoxiae verbascoside, viscapine-2, polydatin, acteoside, naringin, rhoifolin, glycyrrhizic acid, and emodin with methanol to obtain mixed reference solution;

preparing a test solution: taking the traditional Chinese medicine composition, grinding, weighing, adding 60-80% methanol, carrying out ultrasonic treatment, cooling, weighing again, supplementing the weight loss with pure water, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition;

the chromatographic detection conditions are as follows: the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, acetonitrile is used as a mobile phase A, a 0.1% formic acid solution is used as a mobile phase B, and gradient elution is carried out according to the following specified proportion: the elution time is 0min, the proportion of the mobile phase A is 5%, the proportion of the mobile phase B is 95%, the elution time is 5min, the proportion of the mobile phase A is 10%, the proportion of the mobile phase B is 90%, the elution time is 10-12 min, the proportion of the mobile phase A is 13%, the proportion of the mobile phase B is 87%, the elution time is 18min, the proportion of the mobile phase A is 19%, the proportion of the mobile phase B is 81%, the elution time is 21min, the proportion of the mobile phase A is 21%, the proportion of the mobile phase B is 79%, the elution time is 29min, the proportion of the mobile phase A is 40%, the proportion of the mobile phase B is 60%, the elution time is 35min, the proportion of the mobile phase A is 30%, and the proportion of the mobile phase B is 70%; the detection wavelength is 230-270 nm; the flow rate is 0.1-0.6 ml/min; the column temperature is 24-32 ℃;

(4) respectively and precisely sucking the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring.

Preferably, the concentration of the mixed reference product solution in the preparation of the reference product solution is: 250-450 mu g/ml of verbascoside, 30-60 mu g/ml of viscapine-2, 200-400 mu g/ml of polydatin, 50-100 mu g/ml of verbascoside, 350-450 mu g/ml of naringin, 10-50 mu g/ml of rhoifolin, 80-120 mu g/ml of glycyrrhizic acid and 200-250 mu g/ml of emodin.

Preferably, the concentration of the mixed reference product solution in the preparation of the reference product solution is: 365 mu g/ml of verbascoside, 373 mu g/ml of verbascoside, 244 mu g/ml of visanin, 300 mu g/ml of polydatin, 85 mu g/ml of verbascoside, 453 mu g/ml of naringin, 25 mu g/ml of rhoifolin, 100 mu g/ml of glycyrrhizic acid and 223 mu g/ml of emodin.

Preferably, the step of preparing the test solution comprises the step of preparing the methanol with the concentration of 70 percent.

Preferably, the chromatographic detection conditions of the step three are as follows: the model of the octadecylsilane chemically bonded silica chromatographic column is as follows: ACQUITY UPLC BEH C₁₈、ACQUITY UPLC Shield RP ₁₈、ACQUITY HSS T3。

Preferably, the chromatographic detection conditions of the step three are as follows: the model of the octadecylsilane chemically bonded silica chromatographic column is as follows: waters ACQUITY UPLC BEH C₁₈。

Preferably, the chromatographic detection conditions of the step three are as follows: the detection wavelength is 200-300 nm; the flow rate is 0.4 ml/min; the column temperature was 30 ℃.

Preferably, there are 10 fingerprint spectrum peaks generated according to the detection method, where the spectrum peak 1 is: verbena glycoside, chromatographic peak 2 of polydatin, chromatographic peak 3 of verbascoside, chromatographic contrast peak 4 of naringin, chromatographic peak 5 of rhoifolin, chromatographic peak 7 of emodin-8-O-beta-D-pyranoside, chromatographic peak 9 of glycyrrhizic acid, and chromatographic peak 10 of emodin.

Preferably, the fingerprint chromatogram peak generated according to the detection method presents 10 characteristic peaks, the peak corresponding to the reference substance peak 4 is an S peak, and the relative retention time of each characteristic peak and the S peak is calculated to be within ± 10% of a specified value, wherein the retention time of the chromatogram peak 1 is 0.43, the retention time of the chromatogram peak 2 is 0.65, the retention time of the chromatogram peak 3 is 0.92, the retention time of the chromatogram control peak 4 is 1.00, the retention time of the chromatogram peak 5 is 1.02, the retention time of the chromatogram peak 6 is 1.37, the retention time of the chromatogram peak 7 is 1.40, the retention time of the chromatogram peak 8 is 1.62, the relative retention time of the chromatogram peak 9 is 1.67, and the retention time of the chromatogram peak 10 is 1.84.

The fingerprint detection method provided by the invention is applied to quality detection or authenticity identification of the traditional Chinese medicine composition.

The traditional Chinese medicine composition for dispersing lung qi and removing toxicity comprises 13 traditional Chinese medicines, including 6g of raw ephedra herb, 15g of bitter apricot seed, 30g of gypsum, 30g of raw coix seed, 10g of atractylis lancea, 15g of patchouli, 12g of sweet wormwood herb, 20g of giant knotweed rhizome, 30g of verbena, 30g of dry reed rhizome, 15g of pepperweed seed, 15g of pummelo peel and 10g of raw liquorice. The lung ventilating and toxin counteracting composition can be added with pharmaceutic adjuvants to prepare a pharmaceutically allowable common medicine oral preparation, such as: capsule, tablet, oral liquid, pill, granule, etc.

The specific technical scheme of the invention is as follows:

a detection method of a Chinese medicinal composition for dispersing lung qi and removing toxic substance comprises thin layer identification method of naringin, emodin, physcion, polydatin, and ephedrine hydrochloride, and content determination method of ephedrine hydrochloride, pseudoephedrine hydrochloride, naringin and glycyrrhizic acid; the method for measuring the content of naringin and glycyrrhizic acid is high performance liquid chromatography, and chromatographic conditions and system applicability tests comprise the following steps: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase A: 0.05-0.15% formic acid aqueous solution, mobile phase B: acetonitrile, gradient elution procedure as follows: the elution time is 0-35 min, the proportion of the mobile phase A is 95% -30%, the proportion of the mobile phase B is 5% -70%, and the detection wavelength is as follows: 254 nm.

Preferably, the content of naringin and glycyrrhizic acid measured in mobile phase A: 0.1% aqueous formic acid, mobile phase B: acetonitrile, gradient elution procedure as follows: the elution time is 0min, the proportion of the mobile phase A is 95%, the proportion of the mobile phase B is 5%, the elution time is 5min, the proportion of the mobile phase A is 90%, the proportion of the mobile phase B is 10%, the elution time is 10-12 min, the proportion of the mobile phase A is 87%, the proportion of the mobile phase B is 13%, the elution time is 18min, the proportion of the mobile phase A is 81%, the proportion of the mobile phase B is 19%, the elution time is 21min, the proportion of the mobile phase A is 79%, the proportion of the mobile phase B is 21%, the elution time is 29min, the proportion of the mobile phase A is 60%, the proportion of the mobile phase B is 40%, the elution time is 35min, the proportion of the mobile phase A is 30%, and the proportion of the mobile phase B is 70%.

As a further preferred aspect of the present invention, the method for measuring the naringin and glycyrrhizic acid content comprises the following steps:

preparing a reference product solution: taking a proper amount of naringin and ammonium glycyrrhetate reference substances, precisely weighing, and preparing a mixed solution containing 0.6-1.0 mg of naringin and 0.1-0.3 mg of ammonium glycyrrhetate per 1mL by using methanol to obtain the naringin/ammonium glycyrrhetate reference substance;

preparing a test solution: taking the traditional Chinese medicine composition, grinding, precisely weighing, precisely adding 60-80% of methanol, weighing, carrying out ultrasonic treatment, cooling, weighing again, complementing the loss weight with 60-80% of methanol, shaking up, centrifuging for 8-12 min at the rotating speed of 11000-15000 r/min, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition;

and thirdly, precisely sucking the reference substance solution and the test solution respectively, injecting the reference substance solution and the test solution into a liquid chromatograph, and measuring to obtain the test solution.

Preferably, the thin layer identification method in the detection method is any one or combination of more of the following items (1) to (4):

(1) the identification method of the naringin comprises the following steps: taking the powder of the invention, adding methanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, and dissolving with methanol to obtain a sample solution; adding methanol into a naringin reference substance to prepare a solution containing 0.5-1.5 mg of naringin per 1mL of naringin reference substance as a reference substance solution; and (3) sucking the two solutions, respectively dropping the two solutions on the same high-efficiency silica gel G thin-layer plate, and mixing the two solutions with ethyl acetate-butanone-formic acid-water in a proportion of 8-12: 4-8: 1-3: 0.8-1.2 of developing agent, developing, taking out, airing, spraying 3-7% aluminum trichloride ethanol solution, heating at 105 ℃ for 0.5-1.5 minutes, placing under an ultraviolet lamp of 365nm for inspection, and displaying fluorescent spots with the same color in the chromatogram of the test sample at the position corresponding to the chromatogram of the reference sample;

(2) the identification method of the emodin or the physcion comprises the following steps: taking the powder of the invention, adding methanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, dissolving the filtrate by using methanol, taking an emodin reference substance or an physcion reference substance, and adding methanol to prepare solutions containing 0.2-0.8 mg of the reference substance in each 1mL as reference substance solutions; absorbing a test solution and a reference solution, respectively dropping the test solution and the reference solution on the same silica gel G thin-layer plate, taking an upper layer solution of 30-60 ℃ petroleum ether-ethyl formate-formic acid 13-17: 4-6: 0.8-1.2 as a developing agent, developing, taking out, drying in the air, and viewing under an ultraviolet lamp 365nm to display fluorescent spots with the same color on the positions corresponding to the reference chromatogram in the test chromatogram;

(3) the identification method of the polydatin comprises the following steps: taking the powder of the invention, grinding, adding methanol, carrying out ultrasonic treatment, filtering, and taking the filtrate as a test solution; adding methanol into polydatin reference substance to obtain reference solution; performing thin-layer chromatography test, respectively dropping the two solutions on the same silica gel G thin-layer plate, pre-saturating the solution in a developing cylinder by using ethyl acetate-methanol-trichloromethane-water (23-28: 7-11: 6-10: 3-5) as a developing agent, developing, taking out, drying, spraying 8-12% potassium hydroxide methanol solution, inspecting under ultraviolet light 254nm, and displaying fluorescent spots with the same color on the positions corresponding to the reference substance chromatogram in the test substance chromatogram;

(4) the identification method of ephedrine hydrochloride comprises the following steps: taking the powder of the invention, adding methanol, carrying out ultrasonic treatment for 20-40 minutes, filtering, evaporating filtrate to dryness, dissolving with methanol, taking ephedrine hydrochloride reference substance, adding methanol to prepare a solution containing 0.8-1.2 mg per 1mL, taking the solution as a reference substance solution, sucking the two solutions, respectively dropping the two solutions on the same silica gel G thin layer plate, taking chloroform-methanol-concentrated ammonia test solution 18-22: 4-6: 0.4-0.6 as a developing agent, developing, taking out, airing, spraying ninhydrin test solution, heating at 102-107 ℃ until spots are clearly developed, and displaying the same red spots in the chromatogram of the test substance at the positions corresponding to the chromatogram of the reference substance chromatogram.

Preferably, the determination method of ephedrine hydrochloride and pseudoephedrine hydrochloride content is high performance liquid chromatography, and the chromatographic conditions and system applicability test are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the mobile phase is as follows: acetonitrile-0.1-0.3% phosphoric acid, the proportion is: 2-4: 95-99, detection wavelength: 207 nm.

Preferably, the mobile phase in the detection method is: acetonitrile-0.12% phosphoric acid, the proportion is: 3:97.

Preferably, the octadecylsilane chemically bonded silica chromatographic column is of the type: COSMOSIL 5C18-MS-II, SHIMADZU VP-ODS, YMC-Pack ODS-A.

Preferably, the method for measuring the content of ephedrine hydrochloride and pseudoephedrine hydrochloride in the detection method comprises the following steps:

preparing a reference product solution: accurately weighing appropriate amount of ephedrine hydrochloride reference substance and pseudoephedrine hydrochloride reference substance, and adding 60-80% methanol to obtain mixed solution containing 30-50 μ g of each 1 mL;

preparing a test solution: taking the traditional Chinese medicine, grinding, precisely weighing, placing in a conical flask with a plug, adding an aqueous solution, carrying out ultrasonic treatment, cooling, weighing again, complementing the weight loss by water, shaking up, centrifuging for 2-8 min at the rotating speed of 3000-8000 r/min, taking the supernatant, adding concentrated ammonia test solution, shaking up and extracting by ether for 1-4 times, adding a hydrochloric acid ethanol solution, shaking up, placing, drying by distillation, dissolving the residue by adding 50-80% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine.

preparing a reference product solution: precisely weighing appropriate amount of ephedrine hydrochloride reference substance and pseudoephedrine hydrochloride reference substance, and adding 70% methanol to obtain mixed solutions each containing 40 μ g of ephedrine hydrochloride and pseudoephedrine hydrochloride in each 1 mL;

preparing a test solution: taking the traditional Chinese medicine, grinding, precisely weighing, placing in a conical flask with a plug, precisely adding an aqueous solution, carrying out ultrasonic treatment, cooling, weighing again, complementing the weight loss by water, shaking up, centrifuging for 4-7 min at the rotating speed of 5000-7000 r/min, taking the supernatant, adding concentrated ammonia test solution, shaking up and extracting for 3 times by using diethyl ether, adding 5% ethanol hydrochloride solution, shaking up, placing, drying by distillation, dissolving the residue by adding 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine.

In order to highlight the innovation of the technical scheme of the content detection method of the traditional Chinese medicine composition, the following part of experiments screened in the experimental chromatographic condition exploration process are provided.

Firstly, content determination index component and method selection

Because the traditional Chinese medicine composition for dispersing lung and detoxifying has a plurality of components, main active ingredients related to the treatment efficacy of the traditional Chinese medicine are selected as much as possible for detection in index component detection control. Wherein ephedrine hydrochloride, pseudoephedrine hydrochloride, naringin and glycyrrhizic acid are selected as index components of content determination, and the following considerations are taken into account: (1) the pummelo peel has the effects of dispelling cold, eliminating dampness, regulating qi and eliminating phlegm, wherein the naringin is a representative compound of flavonoid components in the pummelo peel, has the effects of diminishing inflammation, relieving cough and relieving asthma, is the main pharmacological action of the prescription for diffusing the lung and relieving the toxin, and is stable in component, high in content and easy to identify in the composition for diffusing the lung and relieving the toxin; (2) the ephedra herb has the effects of inducing sweat, relieving exterior syndrome, freeing lung, relieving asthma, activating collaterals with pungent and warm natured drugs, and inducing diuresis to alleviate edema, is a main monarch drug in the composition for freeing lung and detoxifying drugs, and the index component for determining the content of ephedra herb is the total amount of ephedrine hydrochloride and pseudoephedrine hydrochloride specified in the 2015 version of Chinese pharmacopoeia; (3) glycyrrhizic acid has various pharmacological actions such as anti-tumor, antivirus, antiserum, immunoregulation, cardiovascular disease treatment, antioxidation and the like, and is one of the most important medicinal active ingredients reflecting the quality of medicinal materials in the liquorice.

② investigation test of partial index content of Chinese medicinal composition for dispersing lung qi and removing toxicity

2.1 determination of ephedrine hydrochloride and pseudoephedrine hydrochloride content

Ephedrine hydrochloride and pseudoephedrine hydrochloride are the effective components of main monarch drug ephedra in the prescription, the literature is consulted, the content measurement usually adopts an HPLC method or an HPLC method of pre-column derivation, and the specific scheme is determined according to the chromatographic conditions in the literature and by combining the actual situation.

2.2 selection of chromatographic conditions

2.2.1 wavelength screening

Performing 190-800 nm full-wavelength scanning on ephedrine hydrochloride and pseudoephedrine hydrochloride reference solution by using a DAD detector to respectively obtain spectrograms of ephedrine hydrochloride and pseudoephedrine hydrochloride, and determining the content determination wavelength as follows according to the spectrogram result: 207 nm.

2.2.2 Mobile phase screening

2.2.2.1 preparation of samples

Preparing a test solution: taking a proper amount of extracting solution with the batch number XX200306T1, centrifuging at 13000 r/min for 10min, and taking supernatant to obtain the traditional Chinese medicine.

Preparation of a reference solution: accurately weighing appropriate amount of ephedrine hydrochloride reference and pseudoephedrine hydrochloride reference, and adding methanol to obtain mixed solutions each containing 40 μ g per 1 mL.

The determination method comprises the following steps: 10mL of the sample solution was aspirated and injected into the high performance liquid chromatography, and the chromatogram information was recorded as follows according to the following method.

2.2.2.2 chromatographic conditions

The chromatographic conditions are as follows: the chromatographic column was Hypersil Gold C18 (250X 4.6mm, 5 μm), the mobile phase was acetonitrile (A) and 0.1% phosphoric acid solution (B), the elution gradient was as shown in the following table, the flow rate was 1mL/min, the column temperature was 30 ℃, the sample volume was 10 μ L, and the detection wavelength was 207 nm.

TABLE S1 gradient of elution

And (2) chromatographic conditions II: the chromatographic column is Hypersil Gold C₁₈(250X 4.6mm, 5 μm), acetonitrile (A) and 0.1% phosphoric acid solution (containing 0.1% triethylamine) (B) are adopted as mobile phases, the elution gradient is shown in the following table, the flow rate is 1mL/min, the column temperature is 30 ℃, the sample injection amount is 10 μ L, and the detection wavelength is 207 nm.

TABLE S2 gradient of elution

And (3) chromatographic conditions are as follows: the chromatographic column is Hypersil Gold C₁₈(250X 4.6mm, 5 μm), flowThe phases adopt acetonitrile (A) and 0.1 percent phosphoric acid solution-0.1 percent triethylamine (B), the elution gradient is shown in the following table, the flow rate is 1mL/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, and the detection wavelength is 207 nm.

TABLE S3 gradient of elution

And thirdly, the chromatographic conditions of the test results can effectively separate ephedrine hydrochloride and pseudoephedrine hydrochloride from nearby chromatographic peaks, so that the chromatographic conditions III are determined to be used as a method for measuring the contents of the ephedrine hydrochloride and the pseudoephedrine hydrochloride in the traditional Chinese medicine composition for ventilating the lung and detoxifying, and based on the scheme, the gradient and the type of a mobile phase and a chromatographic column are changed for optimization.

2.2.2.3 chromatographic conditions

Content determination: herba Ephedrae is determined by high performance liquid chromatography (China pharmacopoeia 2020 edition general regulation 0512).

Chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler (chromatographic column length of 25cm, inner diameter of 4.6mm, particle size of 5 μm); acetonitrile-0.12% phosphoric acid solution (containing 0.1% triethylamine) (3: 97) is used as a mobile phase; the detection wavelength was 207 nm. The number of theoretical plates is not less than 2000 calculated by ephedrine hydrochloride peak.

Preparation of reference solution ephedrine hydrochloride reference and pseudoephedrine hydrochloride reference are weighed precisely, and 70% methanol is added to obtain mixed solution containing 40 μ g of each 1 ml.

2.2.2.4 method for preparing test sample

According to the property that ephedrine hydrochloride and pseudoephedrine hydrochloride are easily dissolved in organic solvent with lower polarity under alkaline condition, the sample is extracted, and the specific operation is as follows.

Precisely weighing about 1g of the traditional Chinese medicine composition (batch number: 210206), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of water, sealing the plug, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss by water, shaking up, placing in a centrifuge tube, centrifuging (6000 rpm) for 5min, precisely weighing 10ml of supernatant, adding 0.3ml of concentrated ammonia test solution, shaking up and extracting for 4 times by diethyl ether, 15ml each time, combining diethyl ether solutions, adding 0.3ml of 5% ethanol hydrochloride solution, shaking up, placing for 30min, evaporating to dryness, adding a proper amount of 70% methanol into residues for dissolving, transferring to a 10ml measuring flask, adding 70% methanol to scale, shaking up, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine composition.

Another 10ml of supernatant is precisely measured, the supernatant is placed in a centrifuge tube, 0.3ml of concentrated ammonia test solution is added, dichloromethane is used for shaking extraction for 4 times, 15ml of the supernatant is used for each time, the mixed solution is placed in a centrifuge, centrifugation is carried out for 5 minutes (6000 revolutions per minute), dichloromethane solutions are combined, 0.3ml of 5% hydrochloric acid ethanol solution is added, shaking is carried out evenly, the mixed solution is placed for 30min and dried by distillation, a proper amount of 70% methanol is added into residues to dissolve the residues, the residues are transferred to a 10ml measuring flask, 70% methanol is added to the scale mark, shaking is carried out evenly, filtering is carried out, and a subsequent filtrate is taken, thus obtaining the product.

The above test samples were taken and measured under "2.2.2.3 chromatographic conditions". According to the experimental results, a suitable extraction solvent is selected.

2.2.2.5 sample-loading recovery preliminary experiment

Because the operation steps are more, the method provides reference for the accuracy determination of the later-stage method and carries out sample-adding recovery pre-experimental research, and the specific operation is as follows.

Taking about 0.5g of the Chinese medicinal composition (batch number: 210206) for ventilating the lung and removing toxicity, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of ephedrine hydrochloride and pseudoephedrine hydrochloride mixed solution (solvent is water, concentration is 21.68 μ g/ml of ephedrine hydrochloride and 20.30 μ g/ml of pseudoephedrine hydrochloride), sealing, weighing, ultrasonic treating for 30min, cooling, weighing again, supplementing lost weight with water, shaking, placing into a centrifuge tube, centrifuging for 5min (6000 rpm), precisely measuring supernatant 10ml, adding concentrated ammonia solution 0.3ml, shaking with diethyl ether for 4 times (15ml each time), mixing diethyl ether solutions, adding 5% ethanol solution of hydrochloric acid 0.3ml, shaking, standing for 30min, evaporating, dissolving residue with 70% methanol, transferring to 10ml measuring flask, adding 70% methanol to scale, shaking, filtering, and collecting filtrate.

The above test sample was taken and measured under the "2.2.1.1 chromatographic conditions".

2.2.3 pretreatment method of sample

2.2.3.1 comparative results of extraction solvent

The extraction results of the anhydrous ether and the dichloromethane show that ephedrine hydrochloride and pseudoephedrine hydrochloride can be effectively enriched by the extraction of the anhydrous ether and the extraction of the dichloromethane, the ether extraction content is slightly higher than that of the extraction of the dichloromethane, the dichloromethane extraction emulsification phenomenon is seriously difficult to delaminate in the operation process, the centrifugal operation needs to be added, and the delamination is obviously easy to operate by the extraction of the anhydrous ether, so the ether is considered as an extraction solvent to be superior to the dichloromethane.

2.2.3.2 sample loading and recovery preliminary experiment results

The results show that the recovery rate of ephedrine hydrochloride in the prepared test sample is 92% and the recovery rate of pseudoephedrine hydrochloride is 92% after the extraction with anhydrous ether, the recovery rate of ephedrine hydrochloride in the prepared test sample is 96% and the recovery rate of pseudoephedrine hydrochloride is 94% after the extraction with dichloromethane, and the recovery rates meet the requirements of the 2020 edition of Chinese pharmacopoeia.

TABLE S4 Pre-test results of Anhydrous Ether extraction, sample application and recovery

TABLE S5 Pre-experimental results of methylene chloride extraction, sample loading and recovery

2.2.3.3 summary

In earlier stage research, after chromatographic columns of different manufacturers are replaced, interference peaks can appear near chromatographic peaks of ephedrine hydrochloride and pseudoephedrine hydrochloride, so that the durability of the chromatographic columns is poor, the pretreatment method of a test sample is improved, and compared with extraction results of anhydrous ether and dichloromethane, although the sample adding recovery pre-experiment results meet the regulations of Chinese pharmacopoeia 2020 edition, dichloromethane is not easy to delaminate, centrifugal operation needs to be added, and the operation is more complicated than that of the anhydrous ether.

2.2.4 examination of the method of preparing the test article

2.2.4.1 redissolving solvent investigation

Examining the type of the redissolving solvent of the residue, referring to the anhydrous ether extraction method under the item of 2.2.2.4 test sample preparation method, respectively selecting pure water, 30% methanol, 50% methanol, 70% methanol and methanol as the residue for sample preparation, collecting the test sample according to the 2.2.2.3 chromatographic condition, comparing the redissolving effect and result of methanol with different concentrations, and selecting the optimal solvent.

2.2.4.2 ultrasonic Power investigation

Precisely weighing about 1g of a Chinese medicinal composition (batch No. 210206) for dispersing lung qi and removing toxicity, placing the composition into a conical flask with a plug, precisely adding 25ml of water, sealing the plug, weighing, performing ultrasonic treatment for 30min by respectively using 50% of rated power, 75% of rated power and 100% of rated power, cooling, weighing again, complementing the lost weight with water, filtering, taking subsequent filtrate, preparing a test solution, inspecting the influence of ultrasonic waves with different powers on a detection result, collecting the test according to a chromatographic condition in a chromatographic condition of 2.2.1.1, and selecting the optimal ultrasonic power.

2.2.4.3 ultrasonic time survey

Precisely weighing about 1g of a Chinese medicinal composition (batch number: 210206) for dispersing lung qi and removing toxicity, placing into a conical flask with a plug, precisely adding 25ml of water, sealing the plug, weighing, performing ultrasonic treatment for 20min, 30min and 40min respectively, cooling, weighing again, supplementing the lost weight with water, filtering, taking the subsequent filtrate, preparing a test solution, examining the influence of different ultrasonic time on the detection result, collecting the test according to the chromatographic condition in the chromatographic condition of 2.2.1.1, and selecting the optimal ultrasonic time.

2.2.4.4 examination of number of times of extraction with dehydrated ether

Examining the extraction frequency of the anhydrous ether, the preparation method of the test sample refers to the anhydrous ether extraction method under the item '2.2.2.4 test sample preparation method', 15ml of anhydrous ether is used for extraction for 5 times, the anhydrous ether layer extracted each time is taken to respectively prepare test sample solutions, the test sample is collected according to the chromatographic condition in the '2.2.2.3 chromatographic condition', and the extraction frequency of the anhydrous ether (15ml) is screened.

Extracting with 45ml anhydrous ether for 2 times, extracting with 30ml anhydrous ether for 2 times, collecting anhydrous ether layer to obtain sample solution, and comparing extraction effects according to experiment results.

2.2.4.5 inspection of the volume of ammonia solution added

Precisely weighing about 1g of Chinese medicinal composition (lot number: 210206), placing into a conical flask with a plug, precisely adding 25ml of water, sealing, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with water, shaking, placing into a centrifuge tube, centrifuging (6000 rpm) for 5min, precisely weighing 10ml of supernatant, measuring pH, adding concentrated ammonia solution to 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml, 0.8ml, 0.9ml, 1.0ml and 1.5ml, and measuring pH as follows.

2.2.5 results of the experiment

2.2.5.1 examination of redissolving solvent

The redissolution results of different solvents are shown in table S6, and the results show that, except for poor redissolution peak pattern of methanol and low peak area, redissolution of other solvents has no significant difference in the results, considering that more fat-soluble components are contained in the residue in actual operation, 70% methanol of the most soluble solvent residue is selected as the solvent.

TABLE S6 comparison of the redissolving effect of methanol at different concentrations

2.2.5.2 ultrasonic power examination result

The results of the different ultrasonic power tests are shown in table S7, and according to the experimental results, the optimal ultrasonic power is determined to be 100% of the rated power, i.e. 520W, 40 kHz. Meanwhile, the results show that different ultrasonic powers have little influence on the extraction result, so that the ultrasonic powers are not specially regulated.

TABLE S7 comparison of different ultrasonic Power effects

2.2.5.3 ultrasonic time examination result

The examination results of different ultrasonic time are shown in a table S8, the results show that different ultrasonic powers have great influence on the extraction result, and the optimal ultrasonic time is determined to be 20min according to the experimental results.

TABLE S8 comparison of results from different ultrasound time surveys

2.2.5.4 examination of extraction frequency of anhydrous ether

The results of examining the number of times of extraction with different volumes of anhydrous ether are shown in tables S9, S10 and S11, and the results show that the extraction with 15ml of anhydrous ether for three times can completely extract ephedrine hydrochloride and pseudoephedrine hydrochloride, and the extraction with 45ml of anhydrous ether and 30ml of anhydrous ether for the second time shows that a small amount of ephedrine hydrochloride and pseudoephedrine hydrochloride still exist, so 15ml of ether is selected for extraction for three times.

TABLE S915 ml comparison of results of examination of number of times of extraction with anhydrous diethyl ether

TABLE S1030 ml comparison of results of number of times of extraction with dehydrated ether

TABLE S1145 ml comparison of results of examination of number of times of extraction with anhydrous ether

2.2.5.5 test result of volume of concentrated ammonia solution

The volume of the concentrated ammonia solution with different volumes is examined and the result is shown in the table S12, and the result shows that the effect is best when 1ml of concentrated ammonia solution is added, so that the volume of the added ammonia solution is determined to be 1 ml.

TABLE S12 comparison of results of different volume ammonia test solutions

2.2.5.65% ethanol hydrochloride addition volume investigation result

The results of the examination of the different volumes of 5% hydrochloric acid ethanol are shown in the table S13, and the results show that the best effect is achieved by adding 1ml of 5% hydrochloric acid ethanol, so that the volume of the 5% hydrochloric acid ethanol is determined to be 1 ml.

TABLE S comparison of the results of the volume investigation of the 135% ethanol hydrochloride addition

2.2.4.7 examination of reaction time

The results of the examination of the reaction time after the addition of 5% ethanol hydrochloride are shown in Table S14, and the results show that the effect is best when the mixture is allowed to stand for 30min after the addition of 5% ethanol hydrochloride, so that the standing time is determined to be 30 min.

TABLE S reaction time examination results after addition of 145% ethanol hydrochloride

The detection method of the invention adopts the technical scheme with the following beneficial effects:

1. the detection method provided by the invention adds multi-index component content detection, and can control the internal quality of the product so as to ensure that the content of effective components meets the drug standard. The detection method is scientific, reasonable and feasible, and effective component detection is carried out on the main components of the ephedra, the pummelo peel, the liquorice and the like in the prescription, so that the clinical curative effect of the traditional Chinese medicine composition for dispersing lung qi and detoxifying is ensured.

2. The detection method selects 4 components of ephedrine hydrochloride, pseudoephedrine hydrochloride, naringin and glycyrrhizic acid in the lung-ventilating and toxin-vanquishing composition as content determination index components, and the components are index components with related drug effects, so that the product quality can be effectively controlled in the industrial production of products, the product quality control program is simplified, and the production cost is saved.

3. The invention relates to a multi-component fingerprint detection method, and establishes characteristic fingerprints of 12 index components of verbascoside, viscapine-2, polydatin, verbascoside, naringin, rhoifolin, glycyrrhizic acid and emodin in lung-ventilating and toxin-vanquishing granules, thereby ensuring the quality controllability of the batches of the traditional Chinese medicines and the stability of the internal quality components. The experimental result shows that the precision result shows that the relative retention time of the characteristic peak and the relative peak area RSD are both less than 2 percent, which shows that the precision is good. The reproducibility result shows that the relative retention time of the characteristic peak and the relative peak area RSD are both less than 2 percent, and the RSD is both less than 2 percent, which indicates that the repeatability is good. The stability result shows that the relative retention time of the characteristic peak and the relative peak area RSD are both less than 2%, which indicates that the test article has good stability within 24 h. The fingerprint detection method provides a powerful guarantee for the safety, effectiveness and quality controllability of the lung-ventilating and toxin-vanquishing granules.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. The test sample is a traditional Chinese medicine composition for ventilating the lung and removing toxicity.

FIG. 1-naringin specificity thin layer identification diagram, wherein, firstly, naringin is used as reference; ② pummelo peel reference medicinal materials; ③ XF-HJH200325 (negative extract of exocarpium citri rubrum in the absence); fourthly, XX200325T1 (sample solution of a test article);

FIG. 2 is a diagram of thin-layer identification of emodin and physcion durability, including emodin reference substance; ② emodin monomethyl ether reference substance; ③ and polygonum cuspidatum as reference medicinal materials; fourthly, XF-HZ200325 (lacking the negative extract of giant knotweed medicinal material); fifthly, XX200325T 1;

FIG. 3 is a thin-layer identification diagram of polydatin specificity, including polydatin reference substance; ② lot number 210206 particles; ③, batch No. 2021 granule (negative of lacking giant knotweed rhizome);

FIG. 4 is a thin-layer identification diagram of ephedrine hydrochloride, which shows the reference substance of ephedrine hydrochloride; ② herba Ephedrae reference medicinal material; ③ XF-MH 200325; fourthly, XX200325T1 (sample solution of a test article);

FIG. 5-a thin-layer identification diagram of naringin of pilot products, namely, a naringin reference substance; ② XZ200323Z1 (sample solution of a test sample); ③ XZ200324Z1 (sample solution of the test sample); (iv) XZ200324Z2 (sample solution of a test sample);

FIG. 6 is the identification chart of emodin and physcion thin layers of pilot products; firstly, emodin reference substances; ② emodin monomethyl ether reference substance; ③ XZ200323Z1 (sample solution of the test sample); (iv) XZ200324Z1 (sample solution of a test sample); XZ200324Z2 (sample solution of the test sample);

FIG. 7-thin-layer polydatin identification chart for three batches of Chinese medicinal composition with effects of dispersing lung qi and removing toxic substances; firstly, a polydatin reference substance; ② 210204 particles (sample solution of test sample); ③ batch 210205 particle (sample solution of test sample); (iv) lot number 210206 particles (sample solution of test sample);

FIG. 8 is a diagram showing the identification of ephedrine hydrochloride thin layer of the pilot product; firstly, ephedrine hydrochloride reference substance; ② XZ200323Z1 (sample solution of a test sample); ③ XZ200324Z1 (sample solution of the test sample); fourthly, XZ200324Z2 (sample solution of a test sample);

FIG. 9 is a chromatogram of the content determination of ephedrine hydrochloride and pseudoephedrine hydrochloride; (wherein the chromatographic peak 1 is ephedrine hydrochloride, the chromatographic peak 1 is pseudoephedrine hydrochloride, and the chromatographic peak 2 is A-lung-ventilating and toxin-vanquishing traditional Chinese medicine composition (batch number: 210206); B-Ephedra sinica Stapf-deficient (batch number: 2103); mixing C-ephedrine hydrochloride and pseudoephedrine hydrochloride;

FIG. 10-repeatability test chromatogram (content chromatogram of naringin and ammonium glycyrrhizinate at 254 nm);

fig. 11-a comparative characteristic spectrum of the Chinese medicinal composition for ventilating the lung and removing toxicity, wherein 10 characteristic peaks in the characteristic spectrum are respectively peak 1: verbascoside; peak 2: polydatin; peak 3: verbascoside; peak 4 (S): naringin; peak 5: rhoifolin; peak 7: emodin-8-O- β -D-pyranoside; peak 9: glycyrrhizic acid; peak 10: emodin is added.

FIG. 12 is a chromatogram of a characteristic spectrum reproducibility test of the lung-ventilating and toxin-vanquishing granule.

Detailed Description

The present invention is further illustrated by the following exemplary embodiments in order that the practice of the invention may be more fully understood. Unless defined otherwise, technical or scientific terms used herein in the specification and claims of the present patent application shall have the ordinary meaning as understood by those of ordinary skill in the art to which the present invention belongs.

Example 1 thin layer identification method for detecting index component of the present invention

1 Instrument and reagent

The test instrument: analytical balance (Saedolis, SQP QUINTIX224-1CN, Germany), oven (BINDER, FD115, Germany), Thermo Ultimate3000 high performance liquid chromatograph (with DAD detector, temperature-controlled column oven, temperature-controlled autosampler, USA), Thermo Ultimate3000 high performance liquid chromatograph (with VWD detector, temperature-controlled column oven, temperature-controlled autosampler, USA), high-speed desktop centrifuge (SIGMA, 1-14, Germany), ultrapure water meter (CASSCADA, PALL, USA), ACQUITY UPLC H-ClASS Plus (Waters, USA), ultrasonic cleaner (Tianjin Authence, AS205 BT, China), water bath (Jumbo, TW20, Germany), ultraviolet light (Shanghai Ugaku industries, Ltd., 203L-18-127, China). And (3) testing the sample: the traditional Chinese medicine composition (batch number: XZ200323Z1, XZ200324Z1, XZ200324Z2, TRT200302, TRT200303 and TRT200304) for releasing lung and removing toxin is prepared by pilot test and pilot test amplification production according to the preparation method of the traditional Chinese medicine composition for releasing lung and removing toxin. Comparison products: ephedrine hydrochloride (batch No. 171241-201809, purity: 100%), pseudoephedrine hydrochloride (batch No. 171237-201510, purity: 99.8%), naringin (batch No. 110722-201815, purity 91.7%), ammonium glycyrrhizinate (batch No. 110731-201720, purity 97.7%), emodin (batch No. 110756-201913, purity 96.0%), physcion (batch No. 110758-201817, purity: 99.2%) and amygdalin (batch No. 110820-201808, purity: 88.2%) were purchased from China food and drug testing institute; martensin (lot: 7504) and Bidensenin (lot: 7505) were purchased from Shanghai Shidande Standard technical services, Inc.

Test reagents: acetonitrile (Tianjin Bohai Chemical reagent Co., Ltd., 20200102), triethylamine (Tianjin Mao Chemical reagent Co., Ltd., 20200102), phosphoric acid (Tianjin Feng boat Chemical reagent Co., Ltd., 20190715), acetonitrile (Fisher Chemical, 190888) formic acid (Shanghai city reagent Co., Ltd., 2019-1-2), methanol (Tianjin Bohai Chemical reagent Co., Ltd., 191130) ethyl acetate (Tianjin Kancoded Chemical reagent Co., Ltd., 180918), acetone (Tianjin Bohai Chemical reagent Co., Ltd., 181126), glacial acetic acid (Tianjin Kancoded Co., Ltd., 181011), petroleum ether (Tianjin Bohai Chemical reagent Co., Ltd., 20200228), ethyl formate (Tianjin Bohai Chemical reagent Co., Ltd., 20200102), formic acid (Tianjin east Chemical reagent Co., Ltd.), trichloromethane (Tianjin Chemical reagent supply and marketing Co., second division, 181102), aqueous ammonia (Tianjin Feng boat Chemical reagent Co., Ltd., 20180806).

2 methods and results

2.1 test sample preparation

2.1.1 sample preparation of test articles

The preparation method comprises the steps of taking 300g of ephedra, 1000g of giant knotweed, 750g of bitter almond, 1500g of verbena, 1500g of gypsum, 1500g of reed rhizome, 1500g of coix seed, 750g of lepidium seed, 500g of bran-fried rhizoma atractylodis, 750g of exocarpium citri grandis, 750g of pogostemon cablin, 500g of liquorice and 600g of sweet wormwood, adding 4 times of water, soaking for 30 minutes, heating to boil, wherein the decocting time is 40 minutes, decocting filtrate with water, and concentrating the filtrate to obtain a traditional Chinese medicine extract (60 ℃) with the relative density of 1.02-1.10;

the preparation method comprises the steps of taking the Chinese medicinal extract, carrying out spray drying, preparing dry paste powder, adding a proper amount of lactose and mannitol (lactose: mannitol 2: 1), uniformly mixing, carrying out dry granulation, and preparing 1000g of granules.

2.1.2 negative sample preparation:

according to the preparation method of the sample for the test sample of the whole formula in 2.1.1, the corresponding medicinal materials are deducted, and the negative extract (batch number: XF-HJH200325) of exocarpium citri grandis, the negative extract (XF-HZ200325) of polygonum cuspidatum and the negative extract (XF-MH200325) of ephedra are respectively prepared.

2.1.3 preparation of reference medicinal material extract

Taking 1g of exocarpium Citri Grandis reference medicinal material powder, adding 10mL of methanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, dissolving with 3mL of methanol to obtain exocarpium Citri Grandis reference medicinal material solution; taking 1g of rhizoma Polygoni Cuspidati reference medicinal material powder, adding 10mL of methanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, dissolving with 3mL of methanol to obtain rhizoma Polygoni Cuspidati reference medicinal material solution; taking 1g of herba Ephedrae control medicinal material powder, adding 10mL of methanol, performing ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, dissolving with 3mL of methanol to obtain herba Ephedrae control medicinal material solution.

2.1.4 preparation of control solutions

Weighing appropriate amount of naringin reference substance, emodin reference substance, physcion reference substance, polydatin reference substance, and ephedrine hydrochloride reference substance, and adding methanol to obtain naringin reference substance solution containing 1mg per 1 mL; 1mL of emodin reference solution containing 1mg of emodin; every 1mL contains 1mg of physcion reference solution; 1mL of ephedrine hydrochloride reference solution containing 1mg of ephedrine hydrochloride is added into each 1mL of the solution; each 1mL of the control solution contains 0.2mg polydatin.

2.2 specificity test for thin layer identification

2.2.1 Naringin specificity

The test method comprises the following steps: according to a test of 0502 of the four ministerial rules of Chinese pharmacopoeia of thin layer chromatography 2015 edition, 2 microlitres of a naringin reference solution, a pummelo peel reference medicinal material solution, a complete formula sample solution (XX200325T1) and a pummelo peel lacking negative sample solution (XF-HJH200325) are respectively absorbed and respectively placed on the same high-efficiency silica gel G thin-layer plate, ethyl acetate-acetone-glacial acetic acid-water (8:4:0.3:1) is taken as a developing agent, the developing agent is taken out, the drying agent is dried, a 5% aluminum trichloride ethanol solution is sprayed, the heating agent is heated for 1 minute at 105 ℃, and the test is carried out under an ultraviolet lamp (365 nm).

And (3) test results: as shown in FIG. 1, after color development, the sample is observed at 365nm, and spots with the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatogram of the control solution; in the chromatogram of the reference medicinal material, spots with the same color appear at the corresponding positions of the chromatogram of the reference substance; in the negative sample chromatogram, no fluorescent spot with the same color is observed at the position corresponding to the control chromatogram; therefore, the method can be used as an identification method of the index component naringin of the pummelo peel in the lung ventilating and toxin removing composition.

2.2.2 emodin and Physcion specialties

The test method comprises the following steps: performing thin-layer chromatography 2015 edition Chinese pharmacopoeia general rule 0502 test, sucking 4 μ L of sample solution (XX200325T1) of a test sample, 4 μ L of rhizoma Polygoni Cuspidati control medicinal material solution, 4 μ L of negative sample solution lacking rhizoma Polygoni Cuspidati (XF-HZ200325), and 1 μ L of control solution of emodin and physcion, respectively dropping on the same silica gel G thin-layer plate, developing with petroleum ether (30-60 deg.C) -ethyl formate-formic acid (15:5:1) upper layer solution as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).

And (3) test results: as shown in FIG. 2, the test piece was examined under an ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the emodin and the physcion reference solution; in the chromatogram of the rhizoma Polygoni Cuspidati reference material, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference substance; in the chromatogram of the negative sample lacking the giant knotweed rhizome, no fluorescent spot with the same color is seen at the position corresponding to the chromatogram of the reference substance, so the method can be used as the identification method of the index components emodin and physcion in the giant knotweed rhizome in the composition for ventilating the lung and detoxifying.

2.2.3 Polydatin specificity

The test method comprises the following steps: the test method comprises the following steps: taking 1g of the product, grinding, adding 10ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, and taking the filtrate as a test solution. Taking polydatin control, adding methanol to obtain solution containing 0.2mg per 1ml as control solution. Performing thin layer chromatography (general rule 0502 of 2020 version of Chinese pharmacopoeia), sucking 2 μ l of the above two solutions, respectively dropping on the same silica gel G thin layer plate, pre-saturating in developing cylinder for 15 min with ethyl acetate-methanol-chloroform-water (26:9.5:8:4) as developing agent, developing, taking out, air drying, spraying with 10% potassium hydroxide methanol solution, and inspecting under ultraviolet light (254 nm).

And (3) test results: as shown in fig. 3, the test chromatogram shows fluorescence spots of the same color at the corresponding positions of the control chromatogram; in the negative sample chromatogram, no fluorescent spot with the same color is observed at the position corresponding to the control chromatogram; therefore, the method can be used as an identification method of polydatin serving as an index component of the giant knotweed rhizome in the traditional Chinese medicine composition for ventilating the lung and removing toxicity.

2.2.4 ephedrine hydrochloride specificity

The test method comprises the following steps: according to thin-layer chromatography of 0502 test of the four ministerial rules of the Chinese pharmacopoeia 2015 edition, a sample solution of a sample to be tested, a solution of a herba ephedrae control drug, a solution of a negative sample lacking herba ephedrae (XF-MH200325) and a solution of ephedrine hydrochloride control are respectively absorbed and respectively spotted on the same silica gel G thin-layer plate, a chloroform-methanol-concentrated ammonia test solution (20:5:0.5) is used as a developing agent, the thin-layer plate is developed, taken out, dried, sprayed with a ninhydrin test solution, and heated at 105 ℃ until spots are clearly developed.

And (3) test results: as shown in FIG. 4, spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the ephedrine hydrochloride control solution after the color development; in the chromatogram of the herba Ephedrae reference medicinal material solution, spots with the same color appear at the positions corresponding to the chromatogram of ephedrine hydrochloride reference; in the chromatogram of the negative sample without the ephedra, no fluorescent spot with the same color is seen at the position corresponding to the chromatogram of the ephedrine hydrochloride control; therefore, the method can be used as an identification method of ephedrine hydrochloride serving as an index component of ephedra in the composition for ventilating the lung and removing toxicity.

2.3 thin layer identification of target Components

2.3.1 thin layer identification of Naringin

Weighing three batches of XZ200323Z2, XZ200324Z1 and XZ200324Z2 granules, respectively 1g, adding 10mL of methanol, carrying out ultrasonic treatment for 30min, filtering, evaporating filtrate to dryness, and dissolving with 3mL of methanol to obtain a sample solution.

Taking naringin reference substance, adding methanol to obtain naringin solution containing 1mg per 1mL, and using as reference substance solution.

Testing thin layer chromatography according to 0502 of the four parts of the national pharmacopoeia 2015 edition, sucking up four solutions of 2 μ L, respectively dropping on the same high performance silica gel G thin layer plate, developing with ethyl acetate-acetone-glacial acetic acid-water (8:4:0.3:1) as developing agent, taking out, air drying, spraying with 5% aluminum trichloride ethanol solution, heating at 105 deg.C for 1min, and inspecting under ultraviolet lamp (365 mn).

As shown in FIG. 5, the thin layer was developed and then observed under an ultraviolet lamp (365nm), and the chromatogram of the test sample showed fluorescent spots of the same color at the positions corresponding to the chromatogram of the naringin control.

2.3.2 thin-layer identification of emodin and physcion

Taking emodin reference substance and physcion reference substance, and adding methanol to obtain emodin reference substance solution and physcion reference substance solution each containing 1mg per 1mL as reference substance solutions.

According to thin-layer chromatography of 0502 test of the general guidelines of the four parts of the national pharmacopoeia 2015 edition, 4 mul of each sample solution and 1 mul of each reference solution are absorbed and respectively spotted on the same silica gel G thin-layer plate, the upper layer solution of petroleum ether (30-60 ℃) -ethyl formate-formic acid (15:5:1) is used as a developing agent, the developing agent is developed, taken out, dried and placed under an ultraviolet lamp (365nm) for inspection.

As shown in FIG. 6, the chromatogram of the sample showed fluorescence spots of the same color at the positions corresponding to the chromatogram of the emodin and the physcion control.

2.3.3 thin layer identification of Polydatin

Taking 1g of the traditional Chinese medicine composition for ventilating the lung and relieving toxicity, grinding, adding 10ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, and taking the filtrate as a test solution. Taking polydatin control, adding methanol to obtain solution containing 0.2mg per 1ml as control solution. Performing thin layer chromatography (general rule 0502 of 2020 version of Chinese pharmacopoeia), sucking 2 μ l of the above two solutions, respectively dropping on the same silica gel G thin layer plate, pre-saturating in developing cylinder for 15 min with ethyl acetate-methanol-chloroform-water (26:9.5:8:4) as developing agent, developing, taking out, air drying, spraying with 10% potassium hydroxide methanol solution, and inspecting under ultraviolet light (254 nm). The test chromatogram shows fluorescent spots of the same color at the positions corresponding to those of the control chromatogram. As shown in FIG. 7, spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the polydatin control solution.

2.3.4 thin layer identification of ephedrine hydrochloride

Taking ephedrine hydrochloride reference substance, adding methanol to make into 1mg solution per 1mL as reference substance solution.

According to thin layer chromatography of 0502 test of the four ministerial rules of pharmacopoeia 2015, absorbing ephedrine hydrochloride reference solution and test solution 5 μ L each, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-concentrated ammonia solution (20:5:0.5) as developing agent, taking out, air drying, spraying ninhydrin solution, and heating at 105 deg.C until the spots are clearly developed. As shown in FIG. 8, the test chromatogram showed spots of the same color at the positions corresponding to those of the control chromatogram.

EXAMPLE 2 determination of contents of target components of ephedrine hydrochloride and pseudoephedrine hydrochloride according to the present invention

2.1 instruments and reagents

The test instrument: high performance liquid chromatography (Saimer Feishell technologies, Inc., model ULtimate 3000), one-hundred-thousandth analytical balance (Sedoris, model SQP QUINTIX224-1 CN); ultra pure water instruments (PALL, model casscada); small bench top centrifuges (SIGMA, 1-14 gauge).

Reagent: the control substance ephedrine hydrochloride (batch number: 171241-201809, purity: 100%), pseudoephedrine hydrochloride (batch number: 171237-201510, purity: 99.8%), purchased from China institute for testing food and drug; the test reagent is as follows: acetonitrile (batch No. 20200102, Tianjin Bohai chemical reagent Co., Ltd.), phosphoric acid (Tianjin Feng boat chemical reagent science and technology Co., Ltd., 20190715), triethylamine (batch No. 20200302, Tianjin Ma Mao chemical reagent factory), and ultrapure water were prepared by a pure water instrument.

2.2 test procedures

Specifically, in the present invention, the determination method of ephedrine hydrochloride and pseudoephedrine hydrochloride content is as follows:

2.2.1 chromatographic conditions and System suitability test

Octadecylsilane chemically bonded silica is used as filler (the length of a chromatographic column is 25cm, the inner diameter is 4.6mm, and the particle size is 5 μm); acetonitrile-0.12% phosphoric acid solution (containing 0.1% triethylamine) (3: 97) is used as a mobile phase; the detection wavelength was 207 nm. The number of theoretical plates is not less than 2000 calculated by ephedrine hydrochloride peak.

2.2.2 preparation of control solutions

Accurately weighing appropriate amount of ephedrine hydrochloride reference substance and pseudoephedrine hydrochloride reference substance, and adding 70% methanol to obtain mixed solution containing 40 μ g of each 1 ml.

2.2.3 preparation of test solutions

Precisely weighing about 1g of the traditional Chinese medicine, placing the traditional Chinese medicine into a conical flask with a plug, precisely adding 25ml of water, sealing the plug, weighing the weight, carrying out ultrasonic treatment (power 520W and frequency 40kHz) for 20min, cooling, weighing the weight again, supplementing the reduced weight with water, shaking up, placing the mixture into a centrifuge tube, centrifuging (6000 revolutions per minute) for 5min, precisely weighing 10ml of supernate, adding 1ml of concentrated ammonia test solution, shaking up and extracting for 3 times by using diethyl ether and 15ml each time, combining the diethyl ether solution, adding 1ml of 5% ethanol hydrochloride solution, shaking up, placing the mixture for 30min, drying by distillation, adding a proper amount of 70% methanol into residues for dissolving, transferring the residues into a 10ml measuring flask, adding 70% methanol to scale marks, shaking up, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine.

2.2.4 assay: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

2.3 methodological Studies

Methodological research is carried out according to the 2015 edition Chinese pharmacopoeia medicine quality standard analysis method verification guiding principle (Tong rule 9101).

2.3.1 precision

The precision degree refers to the closeness between the results of multiple sampling measurements of the same uniform sample under specified conditions, and mainly includes repeatability, period precision degree and reproducibility. Precision is generally expressed in terms of deviation, standard deviation, or relative standard deviation.

2.3.1.1 precision of period

Taking a reference substance of a mixture of ephedrine hydrochloride and pseudoephedrine hydrochloride, adopting a chromatographic condition in a '2.2.1 chromatographic condition', repeatedly injecting 6 needles, calculating the relative standard deviation of the peak areas of the ephedrine hydrochloride and the pseudoephedrine hydrochloride, and the result is shown in table 1, wherein the result shows that the RSD value of the injection peak area of the ephedrine hydrochloride 6 needle is 0.9 percent, the RSD value of the injection peak area of the pseudoephedrine hydrochloride 6 needle is 1.0 percent, which shows that the instrument precision is good, and the reference substance meets the requirement of the verification guiding principle of a medicine quality standard analysis method (the four-part rule of contents 9101 in 2020 edition of Chinese pharmacopoeia).

TABLE 1 determination of ephedrine hydrochloride and pseudoephedrine hydrochloride content precision test results

2.3.1.2 repeatability

Taking a proper amount of a traditional Chinese medicine composition (batch number: 210206) for diffusing the lung and detoxifying, preparing 6 parts of a test sample according to a test sample preparation method under the item of 2.2.3 test sample preparation method investigation, respectively adopting a chromatographic condition in a chromatographic condition of 2.1.1, carrying out liquid phase analysis, analyzing the content RSD values of ephedrine hydrochloride and pseudoephedrine hydrochloride in the test sample, and the result is shown in table 2, wherein the result shows that the content RSD value of ephedrine hydrochloride in the 6 parts of test sample is 1.5%, the content RSD value of pseudoephedrine hydrochloride is 0.9%, the repeatability is good, and the requirement of a verification guiding principle of a medicine quality standard analysis method is met (the four-part general rule 9101 in 2020 edition of Chinese pharmacopoeia).

TABLE 2 determination of ephedrine hydrochloride and pseudoephedrine hydrochloride content repeatability test results

2.3.2 accuracy

Accuracy refers to the degree to which the results of the assay using this method are close to true or reference values, typically expressed as recovery (%). Taking a sample of the lung-ventilating and toxin-vanquishing traditional Chinese medicine composition (batch number: 210206), adding a reference substance according to the ratio of the amount of the reference substance to the amount of components to be determined in the test substance to be 0.5:1, 1:1, 1:1.5 respectively, carrying out sample addition (n is 9), preparing a test substance, carrying out liquid phase analysis according to 2.2.1 medium chromatographic conditions, and averaging the test results, wherein the results are shown in table 3 and table 4, and the results show that the sample addition recovery test has the average recovery rate of ephedrine hydrochloride of 90.1%, the RSD of 3.2%, the average recovery rate of pseudoephedrine hydrochloride of 90.7% and the RSD of 2.8%, and meet the requirement of a verification guiding principle of a medicine quality standard analysis method (9101 in the 2020 version of Chinese pharmacopoeia on the fourth Tongtao rule.

TABLE 3 ephedrine hydrochloride loading recovery test data

TABLE 4 Pseudoephedrine hydrochloride loading recovery test data

2.3.3 Linearity and Range

Taking a proper amount of ephedrine hydrochloride and pseudoephedrine hydrochloride reference substances, precisely weighing, and adding 70% methanol to prepare a mixed standard solution containing 304.00 μ g/ml of ephedrine hydrochloride and 313.37 μ g/ml of pseudoephedrine hydrochloride per 1 ml.

The standard solution was aspirated, and the standard solutions having concentrations of 1%, 10%, 25%, 50%, 75%, and 100% of the concentration of the mixed standard solution were diluted, respectively, and subjected to liquid phase analysis under the chromatography conditions in "2.2.1 chromatography conditions". Taking the peak area as the abscissa X and the concentration as the ordinate Y, fitting an ephedrine hydrochloride standard curve as follows: Y0.4064X +0.7156(n 6), R²The standard curve of ephedrine hydrochloride is 1: 0.3608X +0.7000(n 6), R ²1. The results show that the ephedrine hydrochloride is 30.4 ng-3040.0 ng, the pseudoephedrine hydrochloride is 31.3 ng-3133.7 ng, and the linear relation is good.

2.3.4 specificity

About 1g of ephedra-lacking negative particles (batch number: 2103) are taken, the test sample is prepared according to the test sample preparation method under the item of 2.2.3 test sample preparation method investigation, the 2.2.1 chromatographic condition is respectively adopted for liquid phase analysis, the result is shown in figure 9, and the result shows that ephedrine hydrochloride and pseudoephedrine hydrochloride have no interference peak at the position of the lacking negative, and the specificity of content determination is good.

2.3.5 stability

Taking a traditional Chinese medicine composition (batch number: 210206) for dispersing lung and detoxifying, preparing a test sample according to a test sample preparation method under the item of 2.2.3 test sample preparation method investigation, respectively adopting a chromatographic condition in a 2.2.1 chromatographic condition to carry out liquid phase analysis, respectively carrying out sample injection measurement for 0h, 3h, 6h, 9h, 12h and 24h, taking a peak area as an index, investigating the stability of the sample in 24h, and the result is shown in table 5, wherein the result shows that the peak area RSD of ephedrine hydrochloride in 24h of the test sample is 0.8%, the peak area RSD of pseudoephedrine hydrochloride is 0.9%, and the stability is good.

TABLE 5 stability test results of the test articles

EXAMPLE 3 measurement of naringin and glycyrrhizic acid content in accordance with the present invention

3.1 instruments and reagents

The test instrument: ultra high performance liquid chromatography (Waters, model ACQUITY H-CLASSPLUS), one hundred thousand analytical balance (Sedolis, model SQP); ultra pure water instruments (PALL, model casscada); small bench top centrifuges (SIGMA, 1-14 gauge).

Reagent: reference products naringin (batch number: 110722-201815, purity 91.7%), ammonium glycyrrhizinate (batch number: 110731-201720, purity 97.7%), and emodin (batch number: 110756-201913, purity 96.0%) were purchased from China institute for testing and testing food and drug; the test reagent is as follows: acetonitrile (batch No. 190888, Fisher Chemical), formic acid (batch No. 2019-1-2, reagent one of Shanghai city), and ultrapure water were prepared by a pure water meter.

3.2 test procedure

Specifically, in the invention, the method for measuring the content of naringin and glycyrrhizic acid comprises the following steps:

3.2.1 chromatographic conditions and System Adaptation

Chromatography column Waters ACQUITY UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1 percent formic acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 254 nm; the flow rate is 0.4 ml/min; the column temperature is 30 ℃; the amount of sample was 2. mu.l.

TABLE 8 gradient elution procedure for mobile phases A and B

3.2.2 preparation of control solutions

Taking appropriate amount of naringin and ammonium glycyrrhizinate as reference substances, precisely weighing, and preparing into mixed solution containing naringin 0.8mg and ammonium glycyrrhizinate 0.2mg per 1mL with methanol (weight of glycyrrhizic acid is ammonium glycyrrhizinate weight/1.0207).

3.2.3 preparation of test solutions

Taking the products with different dosages, mixing uniformly, taking a proper amount, grinding, taking about 1g, precisely weighing, precisely adding 25mL of 70% methanol, weighing, performing ultrasonic treatment (power 520W and frequency 40kHz) for 30min, cooling, weighing again, complementing the loss weight with 70% methanol, shaking uniformly, centrifuging (rotation speed of 13000 r/min) for 10min, taking supernate, filtering, and taking subsequent filtrate.

3.3 methodological Studies

3.3.1 precision

Precision refers to the closeness of the results of multiple sampling measurements of the same homogeneous sample under defined conditions, including reproducibility, duration precision and reproducibility, wherein the company has only one laboratory and therefore has no investigation on the reproducibility among different laboratories. Precision is generally expressed in terms of deviation, standard deviation, or relative standard deviation.

3.3.1.1.1 repeatability

Preparing 6 groups of test solution repeatedly from XZ200323Z1 granule, and performing liquid phase analysis according to the chromatographic condition under the content determination item of naringin and glycyrrhizic acid. The results are shown in table 9, the detection method has good reproducibility, the RSD values are 0.50% of naringin and 0.19% of ammonium glycyrrhizinate respectively, no significant difference is found, and the RSD values meet the verification guiding principle of the quality standard analysis method of 9101 medicines in the four-part general rules of Chinese pharmacopoeia 2015.

TABLE 9 results of the repeatability tests

3.3.1.2 precision

Taking a reference solution under the items of measuring the content of naringin and glycyrrhizic acid, adopting chromatographic conditions under the items of measuring the content of naringin and glycyrrhizic acid, repeatedly injecting samples for 6 needles, and calculating the relative standard deviation of the peak areas of naringin and ammonium glycyrrhizinate, wherein the result is shown in table 10, the RSD value of the peak area of the sample injection of the naringin 6 needle is 0.12%, and the RSD value of the peak area of the sample injection of the ammonium glycyrrhizinate 6 needle is 0.38%, which shows that the precision of the instrument is good, and meets the verification guidance principle requirement of the quality standard analysis method of 9101 medicine in the four-part general rules of pharmacopoeia 2015.

TABLE 10 results of precision test

3.3.2 accuracy

Accuracy refers to the degree to which the results of the assay using this method are close to true or reference values, typically expressed as recovery (%). Taking a granule sample of the lot number XZ200323Z1, adding a reference substance according to the ratio of the reference substance amount to the component amount to be measured in the sample of 1.5:1, 1:1 and 0.5:1 respectively to prepare a sample, carrying out liquid phase analysis according to the chromatographic conditions under the items of measuring the contents of naringin and glycyrrhizic acid, carrying out a sample loading recovery rate experiment (n is 9), and averaging the test results.

TABLE 11 Naringin sample application recovery test data

TABLE 12 ammonium glycyrrhizinate sample recovery test data

The results are shown in tables 11 and 12, and the average recovery rate of naringin in the sample recovery test is 101.1%, and the RSD is 3.3%; the average recovery rate of the ammonium glycyrrhetate is 102.8 percent, the RSD is 4.0 percent, the method conforms to the relevant regulation of 2015 edition Chinese pharmacopoeia, and the method has good accuracy.

3.3.3 Linear Range

The control mother liquor was diluted to 100%, 80%, 50%, 40%, 25%, 20%, 2% of the original concentration, respectively, and subjected to liquid phase measurement. The results show that the regression equation Y of naringin is 5480.1X-2950.5, R²Good linearity, ranging from 40.7ng to 2035.7ng, where S/N is 1320.74 when the naringin amount is 40.7ng (minimum). Glycyrrhizic acid regression equation Y2527.4X-4738.4, R²0.9993, good linearity, range from 10.4ng to 492.5ng, and 219.30S/N when ammonium glycyrrhizinate amount is 10.4ng (minimum amount).

3.3.4 specificity

Taking a test solution, mixing with a standard solution, carrying out liquid phase analysis on a negative control solution according to chromatographic conditions in the chromatographic condition selection of naringin and glycyrrhizic acid content determination, and investigating the specificity.

The result shows that no interference peak is detected at the corresponding position of the negative control sample chromatogram map by naringin (284nm) and ammonium glycyrrhetate (254nm), and the specificity is good.

3.3.5 stability

Stability examination is carried out on the test solution of the lung ventilating and toxin removing composition for 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h, and the results are shown in tables 13 and 14, and the results show that the RSD value of the naringin peak area is 1.50%, the RSD value of the ammonium glycyrrhizinate peak area is 0.70%, the test solution is stable in 24h, and the requirement of system adaptability is met.

TABLE 13 naringin 24h stability test System adaptability

TABLE 14 ammonium glycyrrhizinate 24h stability test System adaptability

In conclusion, the method is verified by methodology that the naringin linear regression equation: 5480.1X-2950.5, R²Good linearity, ranging from 40.7ng to 2035.7ng, at 0.9994, where when the naringin amount is 40.7ng (minimum), S/N is 1320.74; glycyrrhizic acid regression equation Y-2527.4X-4738.4, R²The linearity is good at 0.9993, the range is 10.6 ng-502.7 ng, and when the quantity of ammonium glycyrrhizinate is 10.6ng (minimum quantity), the S/N is 219.30. Repeatability tests naringin RSD (n ═ 6) was 0.19%, ammonium glycyrrhizinate RSD (n ═ 6) was 0.50%. Precision test naringin RSD (n ═ 6) was 0.12%, ammonium glycyrrhizinate RSD (n ═ 6) was 0.38%, indicating good instrument precision. In the accuracy test, sample-adding recovery test investigation is carried out, and the result shows that the average recovery rate of the naringin is 101.1 percent and the RSD (n is 9) is 3.3 percent; the average recovery rate of ammonium glycyrrhetate was 102.8%, and RSD (n ═ 9) was 4.0%. And (3) observing the stability of the lung ventilating and toxin removing composition test solution for 24h, wherein the result shows that the test solution is stable within 24 h.

Example 4 Lung ventilating and toxin removing granule feature map study

4.1 study of chemical Components of Lung-ventilating and toxin-vanquishing granule

4.1.1 instruments and reagents for experiments

4.1.1.1 Experimental Instrument

An experimental instrument: UPLC-TOF system: acquisty H-class type high performance liquid chromatograph (Waters, USA), XEVO G2-XS QTOF type time-of-flight mass spectrometer, equipped with an electrospray ion source (Waters, USA); model 1-14 ultra high speed centrifuge (SIGMA, Germany); SQP type electronic balance (Sartorius, germany).

4.1.1.2 reagent for experiment

Lung ventilating and toxin removing granules (210204); acetonitrile (chromatographically pure, Fisher, usa); formic acid (chromatographically pure, Merck, usa); distilled water (drochen).

4.1.2 UPLC-Q-TOF analysis method

4.1.2.1 sample of test article

Taking a proper amount of lung ventilating and toxin removing granules (batch number: 210206), grinding, taking about 1g, precisely weighing, precisely adding 25ml of 70% methanol, sealing, weighing, carrying out ultrasonic treatment (power 390W, frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine composition.

4.1.2.2 chromatographic conditions

The column was Watt's acid UPLC BEH C18(100mm × 2.1mm i.d.,1.7 μm); taking 0.1% formic acid solution as phase A and acetonitrile as phase B; the column temperature is 30 ℃; the detection wavelength is 254 nm; the amount of sample was 2. mu.l. The gradient elution procedure is shown in the following table.

TABLE 15 Lung-ventilating and toxin-vanquishing granule gradient elution procedure

4.1.2.2.3 method of mass spectrometry

UPLC-TOF-MS time of flight LC-MS: a positive and negative ion scanning mode; the scanning range is m/z 100-1500; the air curtain air is 50L/h; the desolventizing gas is 800L/h; ion source temperature 100 ℃ (positive)/100 ℃ (negative); the temperature of the desolvation gas is 500 ℃ (positive)/500 ℃ (negative); the taper hole voltage is 30V (positive)/40V (negative); the ion source voltage is 2.0KV (positive)/3.5 KV (negative).

4.1.3 UPLC-Q-TOF analysis results

Total ion flux chromatogram of the lung-ventilating and toxin-vanquishing granules in a UPLC-QTOF-MS positive and negative ion mode identifies 34 components respectively including ephedrine, methylephedrine, kaempferol-3-O-rhamnoside (ephedra herb), polydatin, catechin (polygonum cuspidatum), quercetin-3-O-beta-D-glucose-7-O-beta-D-gentiobioside (semen lepidii), 3, 4-2-hydroxycinnamic acid (rhizoma phragmitis), amygdalin (semen armeniacae amarae), pennyroyal, verbascoside, 3, 4-dihydroverbascoside (verbena officinalis), neoliquiritin, liquiritin, liquiritigenic acid (liquorice), and a reference substance by accurate molecular weight, secondary mass spectrum fragment ion analysis, chemical components of each medicinal material in a reference document and a database and comparison with a reference substance, New Zealand vitexin-2, 6-methoxy-7-hydroxycoumarin (licorice/exocarpium Citri Grandis), marigold-3-O-glucoside, ferulaldehyde (herba Artemisiae Annuae), verbascoside (herba Verbenae/herba Pogostemonis), naringin, rhoifolin (exocarpium Citri Grandis), isoliquiritin glucoside, isoliquiritin (licorice), tansymidin, emodin-8-O-beta-D pyranoside (rhizoma Polygoni Cuspidati), 4-hydroxywogonin (licorice), eupatorium alcohol (herba Artemisiae Annuae), atractyloide A, wogonin, limonin (rhizoma Atractylodis), glycyrrhizic acid (licorice), vitexin (herba Agastaches rugosus), atractyloide III (rhizoma Atractylodis), and emodin (rhizoma Polygoni Cuspidati).

Chromatographic peaks with better separation degree and higher peak height are selected as common peaks in the chromatogram, so the 12 peaks are tentatively characterized as lung ventilating and toxin relieving particles.

TABLE 16 analysis of the main chemical components of the Lung-ventilating and toxicity-removing granule

4.2 preparation of control of No. 9 peak among characteristic peaks

4.2.1 Lung ventilating and toxin removing granules for experiment

The lung-ventilating and toxin-vanquishing granule (batch No. 210206) is prepared by Shandong Tan Siji (pharmaceutical products of Shandong province) through pilot and pilot scale production according to the preparation method of lung-ventilating and toxin-vanquishing.

4.2.2 reagents for assays

Acetonitrile (Tianjin Bohai chemical reagent, Inc., 20201208, analytical purity), formic acid (reagent one factory, Shanghai, 2019-1-2, chromatographic purity).

4.2.3 test apparatus

Analytical balance (Sidolisi, SQP QUINTIX224-1CN, Germany), liquid chromatograph (Waters, Prep150B, USA), ultra-pure water apparatus (CASSCADA, PALL, USA), ultrasonic cleaner (Tianjin Ottone, AS20500BT, China), UPLC-G2XS-TOF system: acquisty ultra type high performance liquid chromatograph (Waters, USA), G2XS-QTOF type time-of-flight mass spectrometer equipped with electrospray ion source (Waters, USA)

4.2.4 chromatographic conditions for the preparation of samples

Grinding appropriate amount of Lung ventilating and toxin removing granule (batch No. 210206), precisely weighing about 1g, and precisely adding pure water10ml, sealing, weighing, carrying out ultrasonic treatment (power 390W, frequency 40kHz) for 30 minutes, shaking up, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine composition. The following chromatographic conditions were used for the measurement. The chromatographic conditions are as follows: chromatographic column WatersSunAire Prep C₁₈OBD (250 mm. times.30 mm, 4.5 μm); acetonitrile is used as a mobile phase A, 0.1% formic acid solution is used as a mobile phase B, and gradient elution is carried out according to the following specified proportion: the elution time is 0-30 min, the proportion of the mobile phase A is 30%, the proportion of the mobile phase B is 70%, and the detection wavelength is 254 nm; the flow rate is 26 ml/min; the column temperature is 30 ℃; the sample size was 5 ml.

4.2.5 structural analysis of control

The sample was a yellow powder (methanol). ESI-MS M/z 431[ M-H ]]-. Bonding of¹H-NMR and¹³C-NMR spectrum of the compound of formula C₂₁H₂₀O₁₀。¹In the H-NMR (DMSO) spectrum, a methyl hydrogen signal delta of 2.40(3H, s, CH) can be observed in the high field region₃) (ii) a The hydrogen signals on 6 sugars are 3.23-3.73 (6H, m, H); hydroxyl hydrogen signal on one saccharide 4.61(1H, t, J ═ 2.4Hz, -OH); one sugar top hydrogen signal 5.06(1H, d, J ═ 7.8Hz, -H); four benzene ring proton signals [ δ H7.46 (1H, s), 7.27(1H, s), 7.15(1H, s), 6.99(1H, s)]。

¹³C-nmr (dmso) spectra showed a total of 19 carbon signals with 13 carbon signals δ: 21.6, 108.3, 114.5, 119.2, 124.2, 131.9, 136.8, 146.9, 160.9, 161.6, 164.4, 182.1 and 186.4 are carbon signals on anthraquinone aglycone; in addition, a set of carbon signals 100.9, 77.4, 76.6, 73.1, 69.3, 60.7 were observed for the carbon signals on pyranose. The compound is determined to be an anthraquinone glycoside compound connected with pyranose by combining the analysis.

The NMR data of the sample is consistent with that of emodin-8-O-beta-D-glucopyranoside in the literature, and the sample with the characteristic peak number 9 is determined to be the emodin-8-O-beta-D-glucopyranoside.

4.3 feature Peak identification in feature map

4.3.1 Lung ventilating and toxin removing granules for experiment

4.3.2 reagents for testing

The verbascoside (batch No. 112059-202001 with purity not less than 98%) was purchased from China institute for testing and testing food and drug; the polydatin (batch number: 110758-; verbascoside (batch number: 111530-201914, purity is more than or equal to 98%) reference substance is purchased from China institute for testing and testing food and drug; naringin (batch number: 110722 and 201815, purity is more than or equal to 98%) reference substance is purchased from China food and drug testing research institute; the rhoifolin (batch number: 111919-; glycyrrhizic acid (batch number: 5559, purity ≥ 98%) reference substance was purchased from Shanghai Shidande Standard technology service, Inc.; the reference substance of emodin (batch number: 110756-201913, purity is more than or equal to 98%) is purchased from China institute for food and drug testing, and emodin-8-O-beta-D-glucopyranoside (self-made in laboratories, purity is more than or equal to 98%).

Methanol (Tianjin Bohai chemical reagent Co., Ltd., 20201208, analytical purification), formic acid (Shanghai reagent I, 2019-1-2, chromatographic purification), acetonitrile (Fisher, 204142, chromatographic purification), formic acid (Tokyo Kaisha, 3C7ON-NJ, chromatographic purification), distilled water (Drech), and n-butanol (Tianjin Bohai chemical reagent Co., Ltd., 20201208, analytical purification).

4.3.3 test apparatus

Analytical balance (sidoris, SQP QUINTIX224-1CN, germany), Thermo Vanquish high performance liquid chromatograph (equipped with DAD detector, temperature-controlled column oven, temperature-controlled autosampler, usa), ultra-pure water meter (casscada, PALL, usa), ultrasonic cleaner (tianjin oxter siang, AS20500BT, china), UPLC-G2XS-TOF system: acquisty ultra-type high performance liquid chromatography (Waters, USA), G2XS-TOF type time-of-flight mass spectrometry, equipped with an electrospray ion source (Waters, USA).

4.3.4 characteristic map method establishment

The chromatographic conditions are as follows: chromatographic column Waters ACQUITY UPLC BEH C₁₈(100 mm. times.2.1 mm, 1.7 μm); with the secondNitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, according to the following ratio for gradient elution: the elution time is 0min, the proportion of the mobile phase A is 5%, the proportion of the mobile phase B is 95%, the elution time is 5min, the proportion of the mobile phase A is 10%, the proportion of the mobile phase B is 90%, the elution time is 10-12 min, the proportion of the mobile phase A is 13%, the proportion of the mobile phase B is 87%, the elution time is 18min, the proportion of the mobile phase A is 19%, the proportion of the mobile phase B is 81%, the elution time is 21min, the proportion of the mobile phase A is 21%, the proportion of the mobile phase B is 79%, the elution time is 29min, the proportion of the mobile phase A is 40%, the proportion of the mobile phase B is 60%, the elution time is 35min, the proportion of the mobile phase A is 30%, and the proportion of the mobile phase B is 70%; the detection wavelength is 254 nm; the flow rate is 0.4 ml/min; the detection wavelength is 254 nm; the flow rate is 0.4 ml/min; the column temperature is 30 ℃; the amount of sample was 2. mu.l.

Preparation of reference solutions: taking appropriate amount of radix Knoxiae verbascoside, viscapine-2, polydatin, verbascoside, naringin, rhoifolin, glycyrrhizic acid, and emodin, precisely weighing, and adding methanol to obtain solution containing 365 μ g, 373 μ g, 44 μ g, 300 μ g, 85 μ g, 453 μ g, 25 μ g, 100 μ g, and 223 μ g per 1 ml.

Preparation of a test solution: taking a proper amount of the product, grinding, taking about 1g, precisely weighing, precisely adding 25ml of 70% methanol, sealing, weighing, carrying out ultrasonic treatment (with the power of 390W and the frequency of 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with pure water, shaking up, filtering, and taking the subsequent filtrate to obtain the product.

The determination method comprises precisely sucking 2 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

10 characteristic peaks should be presented in the characteristic map of the test sample, the peak corresponding to the reference peak is the S peak, the relative retention time of each characteristic peak and the S peak is calculated, and should be within +/-10% of the specified value, the specified value of the retention time is: 0.43 (peak 1), 0.65 (peak 2), 0.92 (peak 3), 1.00[ peak 4(S) ], 1.02 (peak 5), 1.37 (peak 6), 1.40 (peak 7), 1.62 (peak 8), 1.67 (peak 9), 1.84 (peak 10).

4.3.5 characteristic Pattern chromatogram Condition inspection

Taking a proper amount of lung ventilating and toxin removing granules (batch number: 210206), grinding, taking about 1g, precisely weighing, precisely adding 25ml of 70% methanol, sealing, weighing, carrying out ultrasonic treatment (power 390W, frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 70% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine composition. The following chromatographic conditions were used for the measurement. The chromatographic conditions are as follows: chromatographic column Waters ACQUITY UPLC BEH C₁₈(100 mm. times.2.1 mm, 1.7 μm); acetonitrile is used as a mobile phase A, 0.1% formic acid solution is used as a mobile phase B, the elution ratio of the chromatographic mobile phase refers to the chromatographic condition established by the 4.3.4 characteristic map method, and the detection wavelength is 254 nm; the flow rate is 0.4 ml/min; the column temperature is 30 ℃; the amount of sample was 2. mu.l. By adopting a full-wavelength scanning spectrum and a characteristic map, the test result shows that more chromatographic peaks are reflected under the wavelength of 254nm, so that the detection wavelength is determined to be 254 nm.

4.3.6 identification of common peaks

Respectively taking 10 batches of lung-ventilating and toxin-vanquishing particles (batch numbers: 210203, 210204, 210205, 210206, 210207, 210208, 210209, XZ210206T4, XZ210206T5 and XZ210206T6), grinding, taking about 1g of the fine powder, precisely weighing, precisely adding 25ml of 70% methanol, sealing, weighing, carrying out ultrasonic treatment (power 390W and frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the loss weight by 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition. The measurement was carried out under the chromatographic conditions under "4.3.4". It can be known that 10 batches of the peaks 1 to 12 of the granules for releasing lung and relieving toxicity are common peaks, so that the peaks 1 to 12 can be determined as characteristic peaks of the granules for releasing lung and relieving toxicity.

4.3.7 characteristic peak source attribution

Taking 12 batches of single medicine negative lung-ventilating and toxin-vanquishing particles (batch numbers: XX210221T3, XX210221T4, XX210218T1, XX210221T1, XX210221T5, XX210220T4, XX210220T2, XX210220T5, XX210221T5, XX210220T3, XX210221T2 and 2103), grinding, taking about 1g, precisely weighing, precisely adding 25ml of 70% methanol, sealing, weighing, ultrasonically treating (power 390W and frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate. The measurement was carried out under the chromatographic conditions under the term "1.2.1". The characteristic spectrum of the lung-ventilating and toxin-vanquishing granules in 12 batches shows that

peaks

1, 2 and 5 are from verbena, peaks 3, 6 and 7 are from pummelo peel, peaks 4, 8, 9 and 12 are from giant knotweed, and peaks 10 and 11 are from liquorice.

Through preliminary analysis of common characteristic peaks of 10 batches of samples, the chromatographic peak separation degree of the stigmatisin of peak 1 is poor, certain uncertainty exists in detection of subsequent characteristic peaks, the chromatographic peak area of peak 3 is small, and the reproducibility difference among batches is large, so that the subsequent detection is not facilitated, the peak 1 and the peak 3 are removed from 12 characteristic peaks, and the other 10 characteristic peaks are reserved and used as the characteristic peak assessment indexes of the lung-ventilating and toxin-vanquishing particles.

4.4 study of feature Pattern methodology

4.4.1 examination of extraction solvent

Taking Lung-freeing and toxin-vanquishing granules (batch number: 210206), based on the test sample preparation method under item 4.3.4, respectively adopting pure water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 100% methanol as extraction solvents to prepare the test sample, taking pure water as a solvent to dissolve 1g of Lung-freeing and toxin-vanquishing granules in 25ml, extracting with equal volume of water saturated n-butanol for 1 time, taking an n-butanol layer, evaporating to dryness, and re-dissolving with 25ml of 70% methanol. The method is characterized in that determination is carried out according to chromatographic conditions under the item of '2.3 characteristic map mobile phase and elution gradient investigation', the influence of pure water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 100% methanol as extraction solvents on the characteristic peak extraction result of the lung-ventilating and toxicity-removing particles is investigated, the result shows that the extraction of pure water, 10% methanol, 30% methanol, 50% methanol, 70% methanol and 100% methanol has no significant influence on the experimental result, but the areas of the No. 1 peak and the No. 2 peak are reduced after the extraction of water saturated n-butyl alcohol, the extraction of 70% methanol is adopted when content determination is carried out, and 70% methanol is selected as the extraction solvent to avoid waste and unnecessary operation.

Taking lung-ventilating and toxin-vanquishing particles (batch number: 210206), carrying out sample preparation and liquid phase experiment according to the chromatographic condition of '4.3.4 item', repeatedly injecting a sample for 6 needles, taking naringin as an S peak, calculating the relative retention time and relative peak area RSD value of other characteristic peaks and S peaks, and showing that the relative retention time and relative peak area RSD of the characteristic peaks are less than 2% in the results shown in tables 17-18, thereby showing that the precision is good.

TABLE 17 precision test results (relative retention time) of Lung ventilating and toxicity removing granule feature map

TABLE 18 precision test results (relative peak area) of characteristic spectrum of Xuanfei Baidu granules

4.4.3 reproducibility

Taking lung-ventilating and toxin-vanquishing particles (batch number: 210206), preparing a test sample according to the chromatographic condition under the item of 4.3.4, preparing 6 test samples, performing a liquid phase experiment, taking naringin as an S peak, calculating the relative retention time of other characteristic peaks and the S peak and the RSD value of the relative peak area, and showing that the results are shown in tables 19-20, and the results show that the relative retention time of the characteristic peaks and the RSD value of the relative peak area are both less than 2%, which indicates that the repeatability is good.

TABLE 19 reproduction test results (relative retention time) of characteristic spectra of Lung-ventilating and toxin-vanquishing granules

TABLE 20 precision test results (relative peak area) of Lung ventilating and toxicity removing characteristic chromatogram

4.4.4 stability

Taking lung-ventilating and toxin-vanquishing particles (batch number: 210206), carrying out sample preparation and liquid phase experiment according to the chromatographic condition under item 4.3.4, carrying out sample injection measurement according to the chromatographic condition under item 4.3.4 respectively at 0, 3, 6, 9, 12, 15, 18, 21 and 24 hours, and recording the retention time and peak area of an identification peak, wherein the results are as follows. And (3) calculating the relative retention time and relative peak area RSD values of other characteristic peaks and the S peak by taking the kynurenic acid as the S peak, wherein the results are shown in tables 21-22, and the results show that the relative retention time and relative peak area RSD of the characteristic peaks are both less than 2%, which indicates that the stability of the test sample is good within 24 h.

TABLE 21 stability test results (relative Retention time) of Lung ventilating and toxin removing granules

TABLE 22 stability test results (relative peak area) of Lung-ventilating and toxin-vanquishing granule

4.4.5 chromatographic column durability test

Selecting Lung-ventilating and toxin-vanquishing granules (batch No. 210206), pressingThe samples were prepared according to the sample preparation method under the item of "4.4.1 concentration investigation of extraction solvent", the durability of the chromatographic columns of different types was investigated, and the system adaptability test was performed, and the information of the chromatographic columns is shown in table 23. The results show that the different chromatographic columns have larger difference on the characteristic spectrum separation effect of the lung ventilating and toxin removing granules, and the No. 12 peak of the chromatographic column is not eluted at 35min, and the results show that Waters ACQUITY UPLC BEH C₁₈The chromatographic column has better effect than other chromatographic columns, so the currently specified chromatographic column model is Waters ACQUITY UPLC BEH C₁₈A chromatographic column.

TABLE 23 characteristic chromatogram determination of lung ventilating and toxin removing granule chromatographic column information

4.4.6 different operator durability Studies

Taking lung ventilating and toxin removing granules (batch number: 210206), preparing a test sample according to a test sample preparation method under the item of 4.4.1 extraction solvent concentration inspection, inspecting the durability of operators of different types, and carrying out a system adaptability test. The result shows that the method is simple and feasible to operate.

Based on the method, the characteristic spectrum of the lung ventilating and toxin removing granules is determined, 12 characteristic peaks are screened, and the relative retention time of the characteristic peaks and the relative peak area RSD are less than 2% in a precision experiment through methodological investigation, so that the precision is good. In a reproducibility experiment, the relative retention time of the characteristic peak and the relative peak area RSD are both less than 2%, which indicates that the reproducibility is good. In the test of the stability of the test sample, the relative retention time of the characteristic peak and the relative peak area RSD are both less than 2 percent, which indicates that the test sample has good stability within 24 hours. Inspecting chromatographic columns of different manufacturers, finding that the chromatographic columns have large influence on the separation effect, and determining the type of the chromatographic columns as ACQUITY UPLC BEH C₁₈。

Finally, it should be noted that: the present invention is not intended to be limited to the embodiments shown above, which are intended to be illustrative, instructional and not restrictive. Those skilled in the art, having the benefit of this disclosure, will appreciate that many variations, equivalents, and modifications are possible which remain within the spirit and scope of the invention.

Claims

1. A fingerprint detection method of a traditional Chinese medicine composition for ventilating the lung and removing toxicity is characterized by comprising the following steps:

2. The fingerprint detection method according to claim 1, wherein the concentration of the mixed reference substance solution in the preparation of the reference substance solution is: 250-450 mu g/ml of verbascoside, 30-60 mu g/ml of viscapine-2, 200-400 mu g/ml of polydatin, 50-100 mu g/ml of verbascoside, 350-450 mu g/ml of naringin, 10-50 mu g/ml of rhoifolin, 80-120 mu g/ml of glycyrrhizic acid and 200-250 mu g/ml of emodin.

3. The detection method according to claim 2, wherein the concentration of the mixed control solution in the preparation of the reference substance solution is: 365 mu g/ml of verbascoside, 373 mu g/ml of verbascoside, 244 mu g/ml of visanin, 300 mu g/ml of polydatin, 85 mu g/ml of verbascoside, 453 mu g/ml of naringin, 25 mu g/ml of rhoifolin, 100 mu g/ml of glycyrrhizic acid and 223 mu g/ml of emodin.

4. The method for detecting according to claim 1, wherein the concentration of methanol in the preparation of the test solution is 70%.

5. The fingerprint detection method of claim 1, wherein the chromatographic detection conditions of step three are as follows: the model of the octadecylsilane chemically bonded silica chromatographic column is as follows: ACQUITY UPLC BEH C₁₈、ACQUITY UPLC Shield RP ₁₈、ACQUITY HSS T3。

6. The fingerprint detection method of claim 5, wherein the chromatographic detection conditions of step three are as follows: the model of the octadecylsilane chemically bonded silica chromatographic column is as follows: waters ACQUITY UPLC BEH C₁₈。

7. The fingerprint detection method of claim 1, wherein the chromatographic detection conditions of step three are as follows: the detection wavelength is 200-300 nm; the flow rate is 0.4 ml/min; the column temperature was 30 ℃.

8. The fingerprint detection method according to claim 1, wherein the fingerprint chromatogram peak generated according to the detection method presents 10 characteristic peaks, wherein the chromatogram peak 1 is: verbena glycoside, chromatographic peak 2 of polydatin, chromatographic peak 3 of verbascoside, chromatographic contrast peak 4 of naringin, chromatographic peak 5 of rhoifolin, chromatographic peak 7 of emodin-8-O-beta-D-pyranoside, chromatographic peak 9 of glycyrrhizic acid, and chromatographic peak 10 of emodin.

9. The fingerprint detection method according to claim 8 wherein the fingerprint chromatogram peak generated according to the detection method exhibits 10 characteristic peaks, the peak corresponding to the reference substance peak 4 is an S peak, and the relative retention time of each characteristic peak to the S peak is calculated to be within ± 10% of the prescribed value, wherein the retention time of chromatogram peak 1 is 0.43, the retention time of chromatogram peak 2 is 0.65, the retention time of chromatogram peak 3 is 0.92, the retention time of chromatogram control peak 4 is 1.00, the retention time of chromatogram peak 5 is 1.02, the retention time of chromatogram peak 6 is 1.37, the retention time of chromatogram peak 7 is 1.40, the retention time of chromatogram peak 8 is 1.62, the relative retention time of chromatogram peak 9 is 1.67, and the retention time of chromatogram peak 10 is 1.84.

10. The fingerprint detection method of claim 1, wherein the detection method is applied to quality detection or authenticity identification of the traditional Chinese medicine composition.