Quality detection method and application of phlegm-reducing and cough-stopping granules for children

Abstract

The invention belongs to the technical field of pharmaceutical analysis. The invention provides a quality detection method of children phlegm-reducing and cough-stopping granules, which comprises the following steps: sequentially carrying out ultrasonic extraction and filtration on children phlegm-reducing and cough-stopping granule samples and a solvent to obtain a children phlegm-reducing and cough-stopping granule sample solution to be detected; and measuring by adopting a high performance liquid chromatography, and calculating according to a standard curve model to obtain the contents of the four active components in a sample solution of the to-be-measured phlegm-eliminating and cough-stopping granules for children. The quality detection method provided by the invention can more comprehensively evaluate and control the overall quality of the phlegm-reducing and cough-stopping granules for children, control the content uniformity of mental drugs and toxic drugs, and ensure that the product is safe and effective; and methodology verifies that the detection method is strong in specificity, has good precision, repeatability, stability, linear relation and accuracy, is simple and convenient to operate, and saves cost.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a quality detection method and application of children's phlegm-relieving and cough-relieving granules. Background Art

[0002] Pediatric phlegm-relieving and cough-relieving granules are a compound preparation of Chinese and Western medicine made from ipecac tincture, mulberry bark fluid extract, platycodon fluid extract and ephedrine hydrochloride. They have the effect of removing phlegm and relieving cough, and are clinically used to treat children's lung heat cough, sputum and other symptoms.

[0003] Ipecac fluid extract is prepared by wetting ipecac coarse powder with dilute hydrochloric acid and dilute ethanol, then percolating with dilute ethanol as the solvent. Appropriate amounts of dilute hydrochloric acid, ethanol, and water are mixed, and then the ipecac fluid extract is added, followed by water to produce ipecac tincture. Existing methods use titration to determine the total alkaloid content of ipecac tincture. Morus alba and Platycodon grandiflorus fluid extracts are prepared by percolating Morus alba and Platycodon grandiflorus in a 55% ethanol solution, followed by percolation to produce a paste. Currently, there is no quality control method for these extracts. Ephedrine hydrochloride is a single chemical component, and its content is currently determined using high-performance liquid chromatography.

[0004] The current standard for pediatric phlegm-relieving and cough-relieving granules (WS3-B-1691-94) only uses thin-layer chromatography to identify ephedrine hydrochloride and ipecac tincture. The registered standards of some companies (WS3-B-1691-94-2 and WS3-B-1691-94-3) control the ephedrine hydrochloride content in them, but overall the current standards have a low level of control over preparations.

[0005] The chemical drug ephedrine hydrochloride in the prescription is a type of psychotropic drug, which is strictly controlled by the state; the mulberry bark fluid extract and platycodon grandiflorum fluid extract are derived from mulberry bark and platycodon grandiflorum respectively, which are commonly used Chinese medicinal materials with relatively good quality control; ipecac tincture is extracted and refined from the ipecac medicinal material, and the ipecac raw materials are mainly imported. It has certain toxicity, and overdose can cause nausea, vomiting, difficulty breathing, tachycardia, and in severe cases, death. Its toxic components and therapeutic components are alkaloid components mainly composed of emetine hydrochloride and cephaeline hydrochloride. The commonly used determination methods are titration and colorimetric methods to determine the total ipecac alkaloids, and there is a lack of precise control over the components.

[0006] Therefore, it is necessary to establish a more scientific and comprehensive quality inspection method based on the multiple active ingredients of Pediatric Huatan Zhike Granules that exert expectorant and antitussive effects, to provide a reference for the comprehensive evaluation and control of the intrinsic quality of Pediatric Huatan Zhike Granules, and to provide a strong basis for improving the quality standards of Pediatric Huatan Zhike Granules. Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies in the existing technology and provide a quality detection method and application of pediatric Huatan Zhike granules. The method can simultaneously and accurately and rapidly determine the content of psychotropic components, toxic components, and active ingredients (ephedrine hydrochloride, morusin A, emetine hydrochloride, and cephaeline hydrochloride) in pediatric Huatan Zhike granules. The method is a simple and feasible method for determining the content of multiple indicators of pediatric Huatan Zhike granules.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A quality testing method for pediatric phlegm-relieving and cough-relieving granules, comprising the following steps:

[0010] 1) ultrasonically extracting and filtering the pediatric Huatan Zhike granule sample and solvent in sequence to obtain a pediatric Huatan Zhike granule sample solution to be tested;

[0011] 2) The contents of ephedrine hydrochloride, morulin A, emetine hydrochloride, and cephaeline hydrochloride in the sample solution of the pediatric Huatan Zhike granules were determined by high performance liquid chromatography and calculated based on the standard curve model.

[0012] Preferably, the particle size of the pediatric phlegm-relieving and cough-relieving granule sample in step 1) is 200 to 1900 μm.

[0013] Preferably, the solvent in step 1) is phosphoric acid solution and / or acetonitrile, and the volume concentration of the phosphoric acid solution is 0.05-0.15%;

[0014] When the solvent is phosphoric acid solution and acetonitrile, the volume ratio of the phosphoric acid solution to the acetonitrile is 8 to 10:1.

[0015] Preferably, in step 1), the mass-to-volume ratio of the pediatric Huatan Zhike granules sample and the solvent is 4-6 g: 20-30 mL.

[0016] Preferably, the ultrasonic extraction time in step 1) is 15 to 45 minutes, the ultrasonic extraction power is 400 to 600 W, and the ultrasonic extraction frequency is 30 to 50 kHz.

[0017] Preferably, the filler for the high performance liquid chromatography determination in step 2) is a C18 chromatographic column, the column temperature is 25-30° C., and the detection wavelength is 200-340 nm.

[0018] Preferably, in step 2), the mobile phase A of the high performance liquid chromatography determination is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution; wherein the volume concentration of the phosphoric acid aqueous solution is 0.05-0.15%, and the flow rate is 0.8-1.2 mL / min.

[0019] Preferably, the elution gradient of the high performance liquid chromatography determination in step 2) is 0-12 min, 8% mobile phase A; 12-30 min, 8-35% mobile phase A; 30-45 min, 35-70% mobile phase A; 45-48 min, 70-8% mobile phase A; 48-50 min, 8% mobile phase A.

[0020] Preferably, the injection volume of the high performance liquid chromatography determination in step 2) is 8 to 12 μL.

[0021] The present invention also provides application of the quality detection method in quality evaluation and control of pediatric Huatan Zhike granules.

[0022] The beneficial effects of the present invention include the following:

[0023] 1) The present invention uses Xiaoer Huatan Zhike Granules as the research object and establishes a method for determining the contents of four active ingredients: ephedrine hydrochloride, morusin A, emetine hydrochloride, and cephaeline hydrochloride. These four ingredients detected are the pharmacological ingredients that exert the expectorant and antitussive effects of Xiaoer Huatan Zhike Granules, but also include psychotropic and toxic ingredients. Establishing these content determination methods can more comprehensively evaluate and control the overall quality of Xiaoer Huatan Zhike Granules, control the uniformity of the content of psychotropic and toxic drugs, and ensure product safety and effectiveness.

[0024] 2) The sample solution preparation method of the technical solution of the present invention is simple, rapid, and has good reproducibility. The quality detection method can quickly determine the contents of ephedrine hydrochloride, morusin A, emetine hydrochloride, and cephaeline hydrochloride in Xiaoer Huatan Zhike Granules with good durability. Methodological verification shows that the detection method has strong specificity, good precision, reproducibility, stability, linearity, and accuracy, is easy to operate, and is cost-effective.

[0025] 3) The quality detection method of the pediatric Huatan Zhike granules described in the present invention can not only evaluate the comprehensive quality indicators of the pediatric Huatan Zhike granules, but also evaluate the quality differences of the pediatric Huatan Zhike granules from different manufacturers, the quality consistency of the pediatric Huatan Zhike granules from different batches from the same manufacturer, and the quality control of each link in the production of the pediatric Huatan Zhike granules.

[0026] 4) The present invention makes up for the deficiencies of existing standards for Pediatric Huatan Zhike Granules. This method is applied to the quality control of Pediatric Huatan Zhike Granules and related preparations during the production process and after market launch, and to establish relevant quality inspection standards.

[0027] 5) The present invention also solves the problem of the lack of an accurate content determination method for the toxic components emetine and cephaeline in ipecac-containing drugs, and can be used for the accurate determination of the two alkaloid components in such drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The liquid phase spectra of the four reference solutions used in the specificity test in 2.5.2 of Example 1 are shown, wherein 1 is the liquid phase spectra of the ephedrine hydrochloride reference solution, 2 is the liquid phase spectra of the mulberryside A reference solution, 3 is the liquid phase spectra of the cecaine hydrochloride reference solution, and 4 is the liquid phase spectra of the emetine hydrochloride reference solution;

[0029] Figure 2 The liquid phase spectra of the test solution used in the specificity test of 2.5.2 in Example 1, wherein 1 is the liquid phase spectrum of the ephedrine hydrochloride test solution, 2 is the liquid phase spectrum of the mulberry sideroside A test solution, 3 is the liquid phase spectrum of the cecaine hydrochloride test solution, and 4 is the liquid phase spectrum of the cecaine hydrochloride test solution;

[0030] Figure 3 is the liquid phase spectrum of the negative sample solution of Example 1; wherein A is the liquid phase spectrum of the negative granules without medicinal materials, B is the liquid phase spectrum of the ephedrine hydrochloride negative control solution, C is the liquid phase spectrum of the Morus alba negative control solution, D is the liquid phase spectrum of the ipecac tincture negative control solution, and E is the liquid phase spectrum of the Platycodon grandiflorum negative control solution;

[0031] Figure 4 The liquid phase spectrum of the test solution at a column temperature of 25°C in Example 2;

[0032] Figure 5 The liquid phase spectra of the test solution of Example 4 under different chromatographic columns, wherein A is the liquid phase spectrum of the chromatographic column Agilent ZOBAX, and B is the liquid phase spectrum of the chromatographic column ShimNex CS;

[0033] Figure 6 The liquid phase spectrum of the test solution of Example 5 at different flow rates, where A is the flow rate of 0.8 mL min -1 Liquid phase spectrum, B is the flow rate of 1.2mL·min -1 Liquid phase spectrum;

[0034] Figure 7 The liquid phase spectrum of the test solution of Example 7 under different extraction conditions, wherein A is the liquid phase spectrum of the test solution under extraction condition 1, B is the liquid phase spectrum of the test solution under extraction condition 2, C is the liquid phase spectrum of the test solution under extraction condition 3, D is the liquid phase spectrum of the test solution under extraction condition 4, and E is the liquid phase spectrum of the test solution under extraction condition 5;

[0035] Figure 8 The liquid phase spectra of the test solution of Example 8 at different detection wavelengths, wherein A is the liquid phase spectrum of the test solution at a detection wavelength of 267 nm, and B is the liquid phase spectrum of the test solution at a detection wavelength of 203 nm. DETAILED DESCRIPTION

[0036] The present invention provides a method for quality detection of pediatric Huatan Zhike granules, comprising the following steps:

[0037] 1) ultrasonically extracting and filtering the pediatric Huatan Zhike granule sample and solvent in sequence to obtain a pediatric Huatan Zhike granule sample solution to be tested;

[0038] 2) The contents of ephedrine hydrochloride, morulin A, emetine hydrochloride, and cephaeline hydrochloride in the sample solution of the pediatric Huatan Zhike granules were determined by high performance liquid chromatography and calculated based on the standard curve model.

[0039] In the present invention, the particle size of the pediatric phlegm-relieving and cough-relieving granule sample in step 1) is preferably 200-1900 μm, more preferably 500-1500 μm, and even more preferably 800-1200 μm.

[0040] In the present invention, the solvent in step 1) is preferably a phosphoric acid solution and / or acetonitrile, and the volume concentration of the phosphoric acid solution is preferably 0.05 to 0.15%, more preferably 0.1%;

[0041] When the solvent is phosphoric acid solution and acetonitrile, the volume ratio of phosphoric acid solution to acetonitrile is preferably 8 to 10:1, more preferably 9:1.

[0042] In the present invention, the mass volume ratio of the pediatric Huatan Zhike granules sample and the solvent in step 1) is preferably 4-6 g:20-30 mL, more preferably 4.5-5.5 g:24-26 mL, and even more preferably 5 g:25 mL.

[0043] In the present invention, the time of ultrasonic extraction in step 1) is preferably 15 to 45 minutes, more preferably 20 to 40 minutes, and more preferably 30 minutes; the power of ultrasonic extraction is preferably 400 to 600 W, more preferably 450 to 550 W, and more preferably 500 W; the frequency of ultrasonic extraction is preferably 30 to 50 kHz, more preferably 35 to 45 kHz, and more preferably 40 kHz.

[0044] In the present invention, the filler for the high performance liquid chromatography determination in step 2) is preferably a C18 chromatographic column; the column temperature is preferably 25-30°C, more preferably 30°C; the detection wavelength is preferably 200-340nm, more preferably 250-300nm, and more preferably 260-280nm.

[0045] In the present invention, the mobile phase A for the high performance liquid chromatography determination in step 2) is preferably acetonitrile, and the mobile phase B is preferably an aqueous phosphoric acid solution; wherein the volume concentration of the aqueous phosphoric acid solution is preferably 0.05 to 0.15%, more preferably 0.08 to 0.12%, and more preferably 0.1%; the flow rate is preferably 0.8 to 1.2 mL / min, more preferably 0.9 to 1.1 mL / min, and more preferably 1 mL / min.

[0046] In the present invention, the elution gradient of the high performance liquid chromatography determination in step 2) is preferably 0-12 min, 8% mobile phase A; 12-30 min, 8-35% mobile phase A; 30-45 min, 35-70% mobile phase A; 45-48 min, 70-8% mobile phase A; 48-50 min, 8% mobile phase A.

[0047] In the present invention, the injection volume for the high performance liquid chromatography determination in step 2) is preferably 8 to 12 μL, more preferably 9 to 11 μL, and even more preferably 10 μL.

[0048] The present invention also provides application of the quality detection method in quality evaluation and control of pediatric Huatan Zhike granules.

[0049] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] 1. Instruments and Materials

[0052] Agilent 1260 high-performance liquid chromatograph; Agilent OpenLAB chromatography workstation (version 2.4); XS105DU electronic analytical balance (Mettler); KS-500XDS ultrasonic cleaner (Kunshan Jielimei Ultrasonic Instrument Co., Ltd.); Agilent ZOBAX; ShimNex CS; CAPCELLPAK C18 chromatographic column (250 mm × 4.6 mm, 5 μm).

[0053] Acetonitrile, chromatographic grade, produced by Thermo Fisher Scientific, 4 L, concentration >99.9%;

[0054] Methanol, chromatographic grade, produced by Thermo Fisher Scientific, 4 L, concentration >99.9%;

[0055] Water was ultrapure water, and other reagents were of analytical grade;

[0056] Ephedrine hydrochloride reference substance (batch number 171241-201809; purity 100.0%) was purchased from the China Food and Drug Administration.

[0057] Mulberoside A reference substance (batch number MUST-23120510; purity 99.9%) was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.

[0058] Emetine reference substance (batch number JOT-10757; purity 88.4%) and cephaeline reference substance (batch number JOT-11142; purity 98.4%) were purchased from Chengdu Pufeide Biotechnology Co., Ltd.

[0059] 2. Methods and Results

[0060] 2.1 Chromatographic conditions and system suitability test

[0061] Octadecylsilane bonded silica gel (250 mm × 4.6 mm, 5 μm) was used as the filler; acetonitrile was used as the mobile phase A, and 0.1% aqueous phosphoric acid solution was used as the mobile phase B. Gradient elution was performed (0–12 min, 8% mobile phase A; 12–30 min, 8–35% mobile phase A; 30–45 min, 35–70% mobile phase A; 45–48 min, 70–8% mobile phase A; 48–50 min, 8% mobile phase A); the detection wavelength was 210 nm; and the flow rate was 1.0 mL min. -1 ; Column temperature was 30℃; Injection volume was 10μL.

[0062] 2.2 Preparation of reference solution

[0063] Take appropriate amounts of ephedrine hydrochloride, mulberry glycoside A, cephaeline hydrochloride, and emetine hydrochloride reference substance, accurately weigh them, and dilute them with methanol to prepare a mixed solution containing 75.32 μg of ephedrine hydrochloride, 21.03 μg of mulberry glycoside A, 23.75 μg of cephaeline hydrochloride, and 10.78 μg of emetine hydrochloride per 1 mL.

[0064] 2.3 Preparation of test solution

[0065] Take an appropriate amount of pediatric phlegm and cough granules (batch number 22061122), grind it into powder, take 5 g, accurately weigh it, place it in a stoppered conical flask, accurately add a mixed solution of acetonitrile and phosphoric acid solution (in the mixed solution, the volume ratio of acetonitrile and phosphoric acid solution is 1:9, and the volume concentration of phosphoric acid solution is 0.1%) 25 mL, weigh the weight, and ultrasonically extract (the power of ultrasonic extraction is 500 W, and the frequency of ultrasonic extraction is 40 kHz) for 15 minutes. Take it out, let it cool, make up the lost weight with the above-mentioned mixed solution of acetonitrile and phosphoric acid, filter it, and take the filtrate as the test solution.

[0066] 2.4 Preparation of negative sample solution

[0067] Negative control solution: Referring to the preparation method of pediatric phlegm-relieving and cough-relieving granules in the "National Drug Standard" (WS3-B-1691-94), prepare negative granules of Morus alba fluid extract, ephedrine hydrochloride, ipecac tincture, and Platycodon grandiflorum fluid extract, as well as negative granules without medicinal materials (only excipients), and then prepare negative control solutions according to the preparation method of the test sample solution.

[0068] The details are as follows:

[0069] Ephedrine hydrochloride negative control solution: 1.0 mL of Platycodon grandiflorum fluid extract, 1.5 mL of Morus alba fluid extract, 6.0 mL of ipecac tincture, add 0.02 g of citric acid and 0.2 g of sodium citrate in aqueous solution, add to powdered sugar in sequence, mix well, make into granules, dry, spray into an appropriate amount of ethanol solution of edible essence, mix well, and make into 100 g.

[0070] Negative control solution of Morus alba fluid extract: 1.0 mL of Platycodon grandiflorus fluid extract, 0.037 g of ephedrine hydrochloride, 6.0 mL of ipecac tincture, add 0.02 g of citric acid and 0.2 g of sodium citrate in aqueous solution, add to powdered sugar in sequence, mix well, make into granules, dry, spray into an appropriate amount of ethanol solution of edible essence, mix well, and make into 100 g.

[0071] Ipecac tincture negative control solution: 1.0 mL of Platycodon grandiflorum fluid extract, 1.5 mL of Morus alba fluid extract, 0.037 g of ephedrine hydrochloride, 0.02 g of citric acid and 0.2 g of sodium citrate in aqueous solution, add to powdered sugar in sequence, mix well, make into granules, dry, spray into an appropriate amount of ethanol solution of edible essence, mix well, and make into 100 g.

[0072] Platycodon grandiflorum fluid extract negative control solution: 0.037g of ephedrine hydrochloride, 1.5mL of mulberry bark fluid extract, 6.0mL of ipecac tincture, add 0.02g of citric acid and 0.2g of sodium citrate in aqueous solution, add to powdered sugar in sequence, mix well, make into granules, dry, spray into an appropriate amount of ethanol solution of edible essence, mix well, and make into 100g.

[0073] 2.5 Methodological Investigation

[0074] 2.5.1 Linear Relationship Investigation

[0075] Accurately weigh appropriate amounts of ephedrine hydrochloride, morulin A, cephaeline hydrochloride, and emetine hydrochloride reference substances, dissolve them in methanol, and prepare a series of linear concentration mixed reference substance solutions. Inject 10 μL of the solution into the liquid chromatograph and determine the concentration according to the chromatographic conditions in "2.1". -1 ) as the abscissa (X) and the peak area as the ordinate (Y) to draw a standard curve. The results are shown in Table 1, which shows that ephedrine hydrochloride, moruloside A, cephaeline hydrochloride, and emetine hydrochloride have a good linear relationship with the peak area within their respective linear concentration ranges.

[0076] Table 1 Results of linear relationship investigation of four components

[0077] Element Regression equation r Linear range (μg / mL) Ephedrine hydrochloride y=21.329x+160.64 0.9949 7.53～376.6 Mulberoside A y=21.485x+43.65 0.9951 2.10～105.15 Cephaeline hydrochloride y=42.442x+94.872 0.9954 2.37～118.75 emetine hydrochloride y=47.239x+15.877 0.9999 1.07～53.9

[0078] 2.5.2 Specificity test

[0079] Take 10 μL of reference solution, test solution and negative sample solution respectively and inject them into liquid chromatograph. The corresponding spectrum is as follows: Figure 1 (wherein, 1 is the liquid phase spectrum of the ephedrine hydrochloride reference solution, 2 is the liquid phase spectrum of the mulberry glycoside A reference solution, 3 is the liquid phase spectrum of the cephaeline hydrochloride reference solution, and 4 is the liquid phase spectrum of the emetine hydrochloride reference solution). Figure 2 (wherein, 1 is the liquid phase spectrum of the ephedrine hydrochloride test solution, 2 is the liquid phase spectrum of the mulberry glycoside A test solution, 3 is the liquid phase spectrum of the cecaine hydrochloride test solution, and 4 is the liquid phase spectrum of the cecaine hydrochloride test solution) and Figure 3 (A is the liquid phase spectrum of the negative granules without medicinal materials, B is the liquid phase spectrum of the negative reference solution of ephedrine hydrochloride, C is the liquid phase spectrum of the negative reference solution of Morus alba, D is the liquid phase spectrum of the negative reference solution of ipecac tincture, and E is the liquid phase spectrum of the negative reference solution of Platycodon grandiflorum). Figures 1 to 3 It can be seen that: in the chromatogram of the test solution, a chromatographic peak appears at the corresponding peak time of the chromatogram of the reference solution, the chromatographic peak of the main component in the test solution is baseline-separated from the adjacent peak, and the separation is good; the negative sample solution has no interference at the same position of the chromatographic peak of the reference solution, indicating that the method has good specificity.

[0080] 2.5.3 Precision test

[0081] The reference solution prepared under item "2.2" was taken and injected five times continuously according to the chromatographic conditions of item "2.1". The RSDs of the peak areas of ephedrine hydrochloride, moruloside A, cephaeline hydrochloride and emetine hydrochloride were 0.70%, 2.34%, 0.56% and 0.83%, respectively, indicating that the precision of the instrument was good.

[0082] 2.5.4 Repeatability test

[0083] Take an appropriate amount of Xiaoer Huatan Zhike Granules (batch number 22061122), grind it into powder, take 5 portions at 5 g each, weigh them accurately, prepare the test solution according to item "2.3", and determine it according to the chromatographic conditions of item "2.1". The RSDs of the peak areas of ephedrine hydrochloride, moruloside A, cephaeline hydrochloride, and emetine hydrochloride were 0.46%, 2.88%, 1.13%, and 2.04%, respectively, indicating that the method has good reproducibility.

[0084] 2.5.5 Stability test

[0085] Take Xiaoer Huatan Zhike Granules (batch number 22061122) and prepare the test solution according to item "2.3". Samples were injected and measured at 0h, 4h, 8h, 24h, and 48h according to the chromatographic conditions of "2.1". The RSDs of ephedrine hydrochloride, moruloside A, cephaeline hydrochloride, and emetine hydrochloride were 2.26%, 0.79%, 1.58%, and 1.58%, respectively, indicating that the test solution had good stability within 48h.

[0086] 2.5.6 Sample recovery test

[0087] Six portions of 2.5 g of Xiaoer Huatan Zhike Granules (batch number 22061122) with known content of each component were taken and accurately weighed. The contents were placed in a stoppered conical flask. A 100% level of reference solution (containing 1.05 mg of ephedrine hydrochloride, 0.15 mg of mulberry glycoside A, 0.10 mg of cephaeline hydrochloride, and 0.040 mg of emetine hydrochloride) was accurately taken and added to the test solution (the solvent was a mixed solution of 25 mL of acetonitrile and phosphoric acid solution, in which the volume ratio of acetonitrile to phosphoric acid solution was 1:9 and the volume concentration of phosphoric acid solution was 0.1%). The weight was determined and ultrasonic extraction was performed (the power of ultrasonic extraction was 500 W and the frequency of ultrasonic extraction was 4 0kHz) for 15 min, removed, cooled, and replaced with a mixed solution of acetonitrile and phosphoric acid solution, wherein the volume ratio of acetonitrile to phosphoric acid solution was 1:9 and the volume concentration of phosphoric acid solution was 0.1% to make up for the reduced weight. Filtered, and the filtrate was measured according to the chromatographic conditions of "2.1". The recovery rate of each component and its RSD value were calculated. The results showed that the average recoveries of ephedrine hydrochloride, moruloside A, cephaeline hydrochloride, and emetine hydrochloride were 101.04%, 108.41%, 105.25%, and 99.52%, respectively, and the RSDs were 0.75%, 2.69%, 1.88%, and 2.14%, respectively, indicating excellent accuracy of the method.

[0088] 2.6 Content determination

[0089] Take 5 batches of samples from different manufacturers and batches, prepare the test solution according to "2.3", and measure them under the chromatographic conditions of "2.1". Calculate the content of each component in the sample. The results are shown in Table 2.

[0090] Table 2 Content determination results

[0091]

[0092]

[0093] As shown in Table 2, this result is not affected by other ingredients and can accurately measure the contents of ephedrine hydrochloride, morulin A, emetine hydrochloride, and cephaeline hydrochloride in each batch of pediatric phlegm-relieving and cough-relieving granules. It can be seen that there are significant differences in the contents of ingredients from different manufacturers. Therefore, it is necessary to establish content methods for these ingredients, especially for ephedrine hydrochloride, emetine hydrochloride, and cephaeline hydrochloride. It is necessary to establish a quality control method with precise upper and lower limits.

[0094] Compared to conventional single-ingredient testing techniques for Pediatric Huatan Zhike Granules, the method described in this invention employs a multi-index component content determination method. This method can strictly control the content of alkaloids in one class of psychotropic drugs, ephedrine hydrochloride and the toxic ipecac, allowing for a more comprehensive evaluation of the quality of Pediatric Huatan Zhike Granules. Furthermore, the components tested in this method are the active ingredients that underlie the efficacy of Pediatric Huatan Zhike Granules. By focusing on these active ingredients for quantitative testing, the method can more accurately control the intrinsic quality of Pediatric Huatan Zhike Granules, enabling a scientific evaluation of their efficacy.

[0095] In addition, the liquid chromatography conditions used in the present invention are suitable, the preparation method is relatively simple, the operation is easy, the cost is low, and it is more economical.

[0096] In summary, the present invention makes up for the shortcomings of the existing standards of pediatric phlegm-relieving and cough-relieving granules, provides a strong basis for further improving and enhancing the quality standards of pediatric phlegm-relieving and cough-relieving granules and related preparations, and provides an effective method for fully guiding the quality control of each link in the production of pediatric phlegm-relieving and cough-relieving granules and scientifically evaluating the efficacy and quality of the products.

[0097] Example 2

[0098] The test solution was prepared according to item "2.3". The column temperature in item "2.1" was changed to 25℃ for chromatographic conditions. The liquid phase spectrum of the test solution was as follows: Figure 4 shown.

[0099] Example 3

[0100] The column temperature in the chromatographic conditions described in Example 2 was modified to 30° C., and the other conditions were the same as in Example 2. The column temperature investigation results of Examples 2 and 3 are shown in Table 3.

[0101] Table 3 Results of investigation at different column temperatures

[0102]

[0103] Example 4

[0104] The chromatographic conditions were carried out according to item "2.1", and the test solution was prepared according to item "2.3". The test solution was investigated under different chromatographic columns. The results are shown in Table 4. The liquid phase chromatograms of the chromatographic column AgilentZOBAX and the chromatographic column ShimNex CS are shown in Figure 5 As shown, A is the liquid phase spectrum of the chromatographic column Agilent ZOBAX, and B is the liquid phase spectrum of the chromatographic column ShimNex CS.

[0105] Table 4 Results of investigation on different chromatographic columns

[0106]

[0107]

[0108] Example 5

[0109] The chromatographic conditions were carried out according to item "2.1". The test solution was prepared according to item "2.3". The test solution was investigated at different flow rates. The results are shown in Table 5. The flow rate was 0.8 mL min -1 and 1.2 mL·min -1 The liquid phase spectrum of Figure 6 As shown, where A is the flow rate 0.8 mL min -1 Liquid phase spectrum, B is the flow rate of 1.2mL·min -1 Liquid phase spectrum.

[0110] Table 5 Investigation results of different flow rates

[0111]

[0112] From Tables 3 to 5, Figures 4-6 The results showed that different brands of chromatographic columns (Agilent ZOBAX; ShimNex CS; CAPCELLPAK) had the same peak concentration when the column temperature was set at 25°C and 30°C, and the flow rate was 0.8, 1.0, and 1.2 mL min -1 At the same time, the parameters of the peak time, separation, peak shape, peak area and other parameters of the main components in the Children's Tanzhike Granules can meet the requirements and comply with the detection standards.

[0113] Example 6

[0114] The chromatographic conditions were carried out according to item "2.1", and the test solution was prepared according to item "2.3". The test solution was tested at different extraction times. The results are shown in Table 6.

[0115] Table 6 Results of investigation at different extraction times

[0116]

[0117] Example 7

[0118] The chromatographic conditions were carried out according to item "2.1", and the test solution was prepared according to item "2.3". The test solution was investigated under different extraction conditions. The results are shown in Table 7. The liquid chromatographic results of the test solution are shown in Table 7. Figure 7 As shown, A is the liquid phase spectrum of the test solution under extraction condition 1, B is the liquid phase spectrum of the test solution under extraction condition 2, C is the liquid phase spectrum of the test solution under extraction condition 3, D is the liquid phase spectrum of the test solution under extraction condition 4, and E is the liquid phase spectrum of the test solution under extraction condition 5;

[0119] Extraction conditions 1: Accurately take 5 g of the fine powder of this product, place it in a stoppered conical flask, accurately add 25 mL of a mixed solution of acetonitrile and 0.1% phosphoric acid (10:90), and sonicate (power 500 W, frequency 40 kHz) for 15 min. After removing, cool, filter, and use the filtrate as the test solution;

[0120] Extraction condition 2: Accurately take 5 g of the fine powder of this product and place it in a stoppered conical flask. Accurately add 25 mL of a 0.5% hydrochloric acid methanol solution. Ultrasonicate (power 500 W, frequency 40 kHz) for 15 min. Remove and cool, filter, and use the filtrate as the test solution.

[0121] Extraction condition formula 3: Take 5 g of fine powder of this product accurately, place it in a stoppered conical flask, add 50 ml of water, stopper it tightly, weigh it, ultrasonicate it (power 500 W, frequency 40 kHz) for 15 min, remove it, let it cool, filter it, add 1 ml of concentrated ammonia test solution, shake it with ether for 4 times, 30 ml each time, combine the extracts, add 1 ml of 5% hydrochloric acid ethanol solution, shake it well, let it stand for 30 min, evaporate it to dryness, dissolve the residue in water and transfer it to a 25 ml volumetric flask, add water to the scale, shake it well, filter it, and take the filtrate as the test solution;

[0122] Extraction condition 4: Take 5g of fine powder of this product accurately, place it in a stoppered conical flask, and add it accurately to a concentration of 0.05mol·L -1的 20 mL of hydrochloric acid solution was sealed and ultrasonically treated (power 500 W, frequency 40 kHz) for 15 min. After shaking, 2 mL of supernatant was accurately measured and added to a solid phase extraction column (with mixed cation exchange reverse phase adsorbent as filler, specification 6 mL·500 mg). -1 , pre-wash with 6 mL of methanol and water), elute with 6 mL of methanol, discard the eluent, and then elute with 9 mL of a freshly prepared acetonitrile-concentrated ammonia solution (volume ratio 95:5) mixed solution, collect the eluate and place it in a 2 mL volumetric flask, add the above mixed solution to dilute to the scale, shake well, and the test solution is obtained;

[0123] Extraction condition 5: Accurately take 5 g of the fine powder of this product, place it in a stoppered conical flask, accurately add 25 mL of methanol solution, and ultrasonically treat (power 500 W, frequency 40 kHz) for 15 minutes. After taking it out, cool it, filter it, and take the filtrate as the test solution.

[0124] Table 7 Results of investigation under different extraction conditions

[0125]

[0126]

[0127] It can be seen from Tables 6 and 7 that when the ultrasonic extraction is performed using a mixed solvent of acetonitrile-0.1% phosphoric acid (10:90), the peak area, i.e., the content, of the target component is the highest, and the separation is good, with few interfering peaks, making detection easy. In addition, when the ultrasonic extraction time is ≥15 min, the peak area of each component increases slightly, but the change is not large, indicating that the component has been basically extracted completely.

[0128] Example 8

[0129] The chromatographic conditions are as per item "2.1", and the test solution is prepared as per item "2.3". The test solution is tested at different detection wavelengths. The test results are as follows: Figure 8 As shown, A is the liquid phase spectrum of the test solution at a detection wavelength of 267 nm, and B is the liquid phase spectrum of the test solution at a detection wavelength of 203 nm.

[0130] Depend on Figure 8 It can be seen that: when the detection wavelength is 203-267nm, each target component peak has ultraviolet absorption, which meets the requirements for content determination; when the detection wavelength is 210nm, the peak shape and separation of each component peak are good, the ultraviolet absorption is strong, and there is no negative interference, which meets the requirements for content determination.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A quality detection method for children's phlegm-clearing and cough-relieving granules, characterized in that: The following steps are included: 1) ultrasonically extracting and filtering the pediatric Huatan Zhike granule sample and solvent in sequence to obtain a pediatric Huatan Zhike granule sample solution to be tested; 2) The contents of ephedrine hydrochloride, morulin A, emetine hydrochloride, and cephaeline hydrochloride in the sample solution of the pediatric Huatan Zhike granules were determined by high performance liquid chromatography and calculated based on the standard curve model.

2. The quality inspection method according to claim 1, characterized in that: Step 1) The particle size of the pediatric phlegm-relieving and cough-relieving granule sample is 200 to 1900 μm.

3. The quality inspection method according to claim 1 or 2, characterized in that: Step 1) The solvent is a phosphoric acid solution and / or acetonitrile, and the volume concentration of the phosphoric acid solution is 0.05-0.15%; When the solvent is phosphoric acid solution and acetonitrile, the volume ratio of the phosphoric acid solution to the acetonitrile is 8 to 10:

1.

4. The quality inspection method according to claim 3, characterized in that: Step 1) The mass volume ratio of the pediatric phlegm-relieving and cough-relieving granules sample and the solvent is 4-6 g: 20-30 mL.

5. The quality inspection method according to claim 4, characterized in that: The ultrasonic extraction time in step 1) is 15 to 45 minutes, the ultrasonic extraction power is 400 to 600 W, and the ultrasonic extraction frequency is 30 to 50 kHz.

6. The quality inspection method according to claim 5, characterized in that: Step 2) The filler for the high performance liquid chromatography determination is a C18 chromatographic column, the column temperature is 25-30° C., and the detection wavelength is 200-340 nm.

7. The quality inspection method according to claim 5 or 6, characterized in that: Step 2) The mobile phase A of the high performance liquid chromatography determination is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution; wherein the volume concentration of the phosphoric acid aqueous solution is 0.05-0.15%, and the flow rate is 0.8-1.2 mL / min.

8. The quality inspection method according to claim 7, characterized in that: Step 2) The elution gradient of the high performance liquid chromatography determination is 0-12 min, 8% mobile phase A; 12-30 min, 8-35% mobile phase A; 30-45 min, 35-70% mobile phase A; 45-48 min, 70-8% mobile phase A; 48-50 min, 8% mobile phase A.

9. The quality inspection method according to claim 8, characterized in that: The injection volume of the high performance liquid chromatography determination in step 2) is 8 to 12 μL.

10. Use of the quality detection method according to any one of claims 1 to 9 in the quality evaluation and control of Xiaoer Huatan Zhike Granules.

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