A quality detection method for Xiaoer Yanbian granules

Abstract

The present invention belongs to the technical field of drug analysis. The present invention provides a quality detection method for pediatric pharyngeal flat granules, comprising the following steps: extracting and filtering the pediatric pharyngeal flat granule sample and an organic solvent in sequence, evaporating the filtrate, then dissolving the residue with an organic solvent and filtering to obtain the pediatric pharyngeal flat granule sample solution to be tested; determining by high performance liquid chromatography, and calculating the contents of 9 active ingredients in the pediatric pharyngeal flat granule sample solution to be tested based on a standard curve model. The quality detection method for pediatric pharyngeal flat granules of the present invention can more comprehensively evaluate and control the overall quality of pediatric pharyngeal flat granules, ensure the efficacy of the product; can quickly determine the contents of 9 active ingredients in pediatric pharyngeal flat granules, and has good durability. Moreover, according to methodological verification, the detection method has strong specificity, good precision, repeatability, stability, linear relationship and accuracy, is easy to operate, and saves costs.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, in particular to a quality detection method for children's pharyngeal flat granules. Background Art

[0002] Pediatric Throat Blister Granules are composed of eight herbs: honeysuckle, belamcanda vine, hollyhock, Scrophularia ningpoensis, Platycodon grandiflorum, Ophiopogon japonicus, artificial bezoar and borneol. They have the effects of clearing heat and relieving sore throat, detoxifying and relieving pain. They are clinically used for throat paralysis, sore throat and other diseases caused by excessive heat in the lungs and stomach in children.

[0003] In the prescription, honeysuckle and belamcanda are both the main medicines. Honeysuckle clears away heat and detoxifies, and dispels wind-heat. Its main active ingredients are phenolic acids such as chlorogenic acid, 5-caffeoylquinic acid, and 4-caffeoylquinic acid. Belamcanda clears away heat and detoxifies, and eliminates phlegm and relieves sore throat. Its main active ingredients are isoflavones such as belamcandarin and irisin. Olea europaea relieves sore throat and relieves pain. Scrophularia clears heat and cools blood. Platycodon opens and promotes lung qi. Ophiopogon nourishes yin and moistens dryness. The four medicines are matched together and serve as assistant medicines. The main active ingredient of Olea europaea is the alkaloid ingredient bamatine hydrochloride, the main active ingredient of Scrophularia is cinnamic acid and harpagoside. Artificial bezoar and borneol wake up the mind and open the orifices, and serve as adjuvant medicines.

[0004] The current quality standard for Children's Pharyngeal Granules is the 2020 edition of the "Pharmacopoeia of the People's Republic of China" and the national drug standard YBZ200562007-2014. The quality control items of the two are completely consistent. Chlorogenic acid is used as the indicator component for content determination, which is poorly representative. In addition, there is no content determination item for another main ingredient, Belamcanda chinensis. Using only a single component as an indicator cannot fully reflect the quality of the preparation.

[0005] Therefore, it is necessary to establish a more scientific and comprehensive quality inspection method based on the multiple active ingredients of Pediatric Throat Granules that clear heat, relieve sore throat, detoxify and relieve pain, to provide a reference for the comprehensive evaluation and control of the intrinsic quality of Pediatric Throat Granules, and to provide a strong basis for improving the quality standards of Pediatric Throat Granules. Summary of the Invention

[0006] The purpose of the present invention is to provide a quality detection method for pediatric pharyngeal flat granules in response to the deficiencies in the existing technology. The method can simultaneously and accurately and quickly determine the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside and bamipine hydrochloride in pediatric pharyngeal flat granules. The method is a simple and feasible method for determining the contents of multiple index components of pediatric pharyngeal flat granules.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for detecting the quality of pediatric pharyngeal granules, comprising the following steps:

[0009] 1) extracting and filtering the pediatric pharyngeal granule sample and the organic solvent in sequence, evaporating the filtrate to dryness, and then dissolving the residue with the organic solvent and filtering to obtain the pediatric pharyngeal granule sample solution to be tested;

[0010] 2) HPLC determination was performed, and the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and palmatine hydrochloride in the test Xiaoer Yanbian granule sample solution were calculated based on the standard curve model;

[0011] The extraction in step 1) is ultrasonic extraction or reflux extraction.

[0012] Preferably, the particle size of the pediatric pharyngeal flat granule sample in step 1) is 170-190 μm.

[0013] Preferably, the organic solvent in step 1) is methanol and / or ethanol, and the volume concentration of the organic solvent is ≧50%.

[0014] Preferably, in step 1), the mass-to-volume ratio of the pediatric pharyngeal flat granule sample and the organic solvent is 1-3 g:40-60 mL.

[0015] Preferably, the extraction time in step 1) is 30 to 60 minutes, the extraction power is 400 to 600 W, and the extraction frequency is 30 to 50 kHz.

[0016] 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 240-340 nm.

[0017] 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 0.5-1.5 mL / min.

[0018] Preferably, the elution gradient of the high performance liquid chromatography determination in step 2) is 0-20 min, 8% mobile phase A; 20-48 min, 8-39% mobile phase A; 48-60 min, 39-75% mobile phase A; 60-62 min, 75-8% mobile phase A; 62-65 min, 8% mobile phase A.

[0019] Preferably, the injection volume of the high performance liquid chromatography determination in step 2) is 8 to 12 μL.

[0020] The beneficial effects of the present invention include the following:

[0021] 1) The present invention uses Xiaoer Yanbian Granules as the research object and establishes a method for determining the contents of 9 active ingredients, including chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and bamipine hydrochloride. The 9 ingredients detected are the medicinal ingredients that enable Xiaoer Yanbian Granules to exert the effects of clearing heat, relieving sore throat, detoxifying, and relieving pain. Establishing the method for determining the contents of these ingredients can more comprehensively evaluate and control the overall quality of Xiaoer Yanbian Granules and ensure the efficacy of the product.

[0022] 2) The sample solution preparation method of the technical solution of the present invention is simple and has good repeatability; the quality detection method can quickly determine the content of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside and bamipine hydrochloride in pediatric pharyngeal granules with good durability; and, as verified by methodology, the detection method has strong specificity, good precision, repeatability, stability, linearity and accuracy, is easy to operate and cost-effective.

[0023] 3) The present invention makes up for the shortcomings of the existing standards for pediatric pharyngeal granules. This method is applied to the quality control of pediatric pharyngeal granules and related preparations during the production process and after market launch, and establishes relevant quality inspection standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The liquid phase spectra of the 9 reference solutions used in the specificity test of 2.5.2 in Example 1, wherein 1 is the liquid phase spectrum of the 5-caffeoylquinic acid reference solution, 2 is the liquid phase spectrum of the chlorogenic acid reference solution, 3 is the liquid phase spectrum of the 4-caffeoylquinic acid reference solution, 4 is the liquid phase spectrum of the belamcandide reference solution, 5 is the liquid phase spectrum of the bamipine hydrochloride reference solution, 6 is the liquid phase spectrum of the harpagoside reference solution, 7 is the liquid phase spectrum of the cinnamic acid reference solution, 8 is the liquid phase spectrum of the tectoriusin reference solution, and 9 is the liquid phase spectrum of the tectoriusin reference solution;

[0025] Figure 2 The liquid phase spectrum 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 test solution of 5-caffeoylquinic acid, 2 is the liquid phase spectrum of the test solution of chlorogenic acid, 3 is the liquid phase spectrum of the test solution of 4-caffeoylquinic acid, 4 is the liquid phase spectrum of the test solution of belamcandide, 5 is the liquid phase spectrum of the test solution of bamipine hydrochloride, 6 is the liquid phase spectrum of the test solution of harpagoside, 7 is the liquid phase spectrum of the test solution of cinnamic acid, 8 is the liquid phase spectrum of the test solution of tectorius flavonoids, and 9 is the liquid phase spectrum of the test solution of tectorius flavonoids;

[0026] Figure 3It 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 negative control solution of honeysuckle, C is the liquid phase spectrum of the negative control solution of Scrophularia, D is the liquid phase spectrum of the negative control solution of Belamcanda, and E is the liquid phase spectrum of the negative control solution of Oleander;

[0027] Figure 4 The liquid phase spectrum of the test solution at a column temperature of 25°C in Example 2;

[0028] 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 CAPCELL PAK, and B is the liquid phase spectrum of the chromatographic column Agilent ZORBAX;

[0029] 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;

[0030] Figure 7 The liquid phase spectra of the test solution of Example 7 in different solvents, wherein A is the liquid phase spectrum at a volume concentration of 50% methanol, B is the liquid phase spectrum at a volume concentration of 50% ethanol, C is the liquid phase spectrum at a volume concentration of 70% methanol, D is the liquid phase spectrum at a volume concentration of 70% ethanol, E is the liquid phase spectrum under methanol, and F is the liquid phase spectrum under ethanol. DETAILED DESCRIPTION

[0031] The present invention provides a method for detecting the quality of pediatric pharyngeal granules, comprising the following steps:

[0032] 1) extracting and filtering the pediatric pharyngeal granule sample and the organic solvent in sequence, evaporating the filtrate to dryness, and then dissolving the residue with the organic solvent and filtering to obtain the pediatric pharyngeal granule sample solution to be tested;

[0033] 2) HPLC determination was performed, and the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and palmatine hydrochloride in the test Xiaoer Yanbian granule sample solution were calculated based on the standard curve model;

[0034] The extraction in step 1) is ultrasonic extraction or reflux extraction.

[0035] In the present invention, the particle size of the pediatric pharyngeal flat granule sample in step 1) is preferably 170-190 μm, more preferably 175-185 μm, and even more preferably 180 μm.

[0036] In the present invention, the organic solvent in step 1) is preferably methanol and / or ethanol, and the volume concentration of the organic solvent is preferably ≧50%, more preferably ≧70%.

[0037] In the present invention, the mass volume ratio of the pediatric pharyngeal flat granule sample and the organic solvent in step 1) is preferably 1-3 g:40-60 mL, more preferably 1.5-2.5 g:45-55 mL, and more preferably 2 g:50 mL.

[0038] In the present invention, the extraction time in step 1) is preferably 30 to 60 min, more preferably 40 to 50 min, and more preferably 45 min; the extraction power is preferably 400 to 600 W, more preferably 450 to 550 W, and more preferably 500 W; the extraction frequency is preferably 30 to 50 kHz, more preferably 35 to 45 kHz, and more preferably 40 kHz.

[0039] 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 240-340 nm, more preferably 260-320 nm, and more preferably 280-300 nm.

[0040] 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 0.5 to 1.5 mL / min, more preferably 0.8 to 1.2 mL / min, and more preferably 1 mL / min.

[0041] In the present invention, the elution gradient of the high performance liquid chromatography determination in step 2) is preferably 0-20 min, 8% mobile phase A; 20-48 min, 8-39% mobile phase A; 48-60 min, 39-75% mobile phase A; 60-62 min, 75-8% mobile phase A; 62-65 min, 8% mobile phase A.

[0042] 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.

[0043] 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.

[0044] Example 1

[0045] 1. Instruments and Materials

[0046] Agilent 1260 high-performance liquid chromatograph; Agilent Open LAB chromatography workstation (version 2.4); XS105DU electronic analytical balance (Mettler); KS-500XDS ultrasonic cleaner (Kunshan Jielimei Ultrasonic Instrument Co., Ltd.); Agilent ZORBAX; ShimNex CS; CAPCELL PAK C18 chromatographic column (250 mm × 4.6 mm, 5 μm).

[0047] Acetonitrile, chromatographic grade, produced by Thermo Fisher Scientific, 4 L, concentration >99.9%;

[0048] Methanol, chromatographic grade, produced by Thermo Fisher Scientific, 4 L, concentration >99.9%;

[0049] Water was ultrapure water, and other reagents were of analytical grade.

[0050] Chlorogenic acid reference substance (batch number 110753-202119; purity 96.3%), belamcandide reference substance (batch number 111632-202304; purity 98.7%), cinnamic acid reference substance (batch number 110786-202305; purity 99.8%), and harpagoside reference substance (batch number 111730-202110; purity 96.8%) were all purchased from the China Food and Drug Inspection Institutes.

[0051] 5-Caffeoylquinic acid reference substance (batch number MUST-23120510; purity 99.9%) and 4-caffeoylquinic acid reference substance (batch number MUST-24011920; purity 99.29%) were purchased from Chengdu Munster Company.

[0052] Iridacetin reference substance (batch number M23IB000611; purity 98.0%) and irisacetin sub-reference substance (batch number Y30N10H104711; purity 98.0%) were purchased from Shanghai Yuanye Company.

[0053] Bamatin hydrochloride reference substance (batch number 23030960; purity 99.4%) was purchased from Tanmo Quality Inspection Company.

[0054] 2. Methods and Results

[0055] 2.1 Chromatographic conditions and system suitability test

[0056] Octadecylsilane bonded silica gel 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 (0-20 min, 8% A; 20-48 min, 8-39% A; 48-60 min, 39-75% A; 60-62 min, 75-8% A; 62-65 min, 8% A); the detection wavelength was 290 nm; and the flow rate was 1.0 mL min. -1 ; Column temperature was 25℃; Injection volume was 10μL.

[0057] 2.2 Preparation of reference solution

[0058] Take appropriate amount of chlorogenic acid reference substance, 5-caffeoylquinic acid reference substance, 4-caffeoylquinic acid reference substance, belamcandide reference substance, tectoriusin reference substance, tectoriusin reference substance, cinnamic acid reference substance, harpaoside reference substance, and palmatine hydrochloride reference substance, accurately weigh them, and dilute them with methanol to make a solution containing 40.94 μg of chlorogenic acid, 16.67 μg of 5-caffeoylquinic acid, 16.30 μg of 4-caffeoylquinic acid, 1.82 μg of belamcandide, 1.99 μg of tectoriusin, 1.92 μg of tectoriusin, 1.04 μg of cinnamic acid, 2.04 μg of harpaoside, and 1.59 μg of palmatine hydrochloride per 1 mL.

[0059] 2.3 Preparation of test solution

[0060] Take an appropriate amount of pediatric pharyngeal flat granules (batch number 22864007), grind it into powder, pass it through a No. 5 sieve (particle size 180μm±7.6μm), take 2g, accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of methanol with a volume concentration of 50%, weigh the weight, and ultrasonically extract (the power of ultrasonic extraction is 500W, and the frequency of ultrasonic extraction is 40kHz) for 45min. Take it out, let it cool, make up the lost weight with 50% volume concentration of methanol, filter it, and take the filtrate as the test solution.

[0061] 2.4 Preparation of negative sample solution

[0062] Negative control solution: Referring to the preparation method of pediatric pharyngeal granules in the "Chinese Pharmacopoeia" (2020 edition), prepare negative granules of honeysuckle, belamcanda vine, cyperus rotundus, and Scrophularia ningpoensis and negative granules without medicinal materials (only excipients), and then prepare the negative control solution according to the preparation method of the test solution.

[0063] The details are as follows:

[0064] Negative control solution of Jinguolan: 10.94g of honeysuckle, 6.25g of belamcanda, 7.81g of platycodon, 7.81g of scrophulariae, and 7.81g of ophiopogon are decocted twice with 100mL of water, the first time for 2.5h and the second time for 1.5h. Filter, combine the filtrates, and concentrate under reduced pressure to a relative density of 1.32-1.35 (50℃). Add 70-80g of sucrose, an appropriate amount of dextrin and 0.031g of artificial bezoar, mix well, and make into granules. Dry at 70℃ for 2h, add 0.016g of borneol, mix well, and make into 100g.

[0065] Scrophulariaceae negative control solution: 10.94g of honeysuckle, 6.25g of belamcanda, 7.81g of platycodon, 7.81g of ophiopogon, 7.81g of thyme, add 100mL of water and boil twice, the first time for 2.5h, the second time for 1.5h, filter, combine the filtrates, and concentrate under reduced pressure to a relative density of 1.32-1.35 (50℃), add 70-80g of sucrose, an appropriate amount of dextrin and 0.031g of artificial bezoar, mix well, make into granules, dry at 70℃ for 2h, add 0.016g of borneol, mix well, and make into 100g.

[0066] Belamcanda negative control solution: 10.94g of honeysuckle, 7.81g of cyperus rotundus, 7.81g of platycodon grandiflorus, 7.81g of Scrophularia ningpoensis, and 7.81g of ophiopogon japonicus are decocted twice with 100mL of water, the first time for 2.5h and the second time for 1.5h. Filter, combine the filtrates, and concentrate under reduced pressure to a relative density of 1.32-1.35 (50°C). Add 70-80g of sucrose, an appropriate amount of dextrin and 0.031g of artificial bezoar, mix well, and make into granules. Dry at 70°C for 2h, add 0.016g of borneol, mix well, and make into 100g.

[0067] Honeysuckle negative control solution: 7.81g of Cyperus rotundus, 6.25g of Belamcanda chinensis, 7.81g of Platycodon grandiflorus, 7.81g of Scrophularia ningpoensis, and 7.81g of Radix Ophiopogonis are decocted twice with 100mL of water, the first time for 2.5h and the second time for 1.5h. Filter, combine the filtrates, and concentrate under reduced pressure to a relative density of 1.32-1.35 (50°C). Add 70-80g of sucrose, an appropriate amount of dextrin, and 0.031g of artificial bezoar, mix well, and make into granules. Dry at 70°C for 2h, add 0.016g of borneol, mix well, and make into 100g.

[0068] 2.5 Methodological Investigation

[0069] 2.5.1 Linear Relationship Investigation

[0070] Accurately weigh appropriate amounts of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and palmatine hydrochloride reference substances, dissolve them in methanol, and prepare a series of linear concentrations of mixed reference substance solutions. Inject 10 μL of the solution into the liquid chromatograph and determine the concentration according to the chromatographic conditions in item "2.1". The concentration of the reference substance (μg·mL -1 ) as the abscissa (X) and the peak area as the ordinate (Y), and the results are shown in Table 1, which show that chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and palmatine hydrochloride have a good linear relationship with the peak area within their respective linear concentration ranges.

[0071] Table 1 Results of the linear relationship investigation of 9 components

[0072]

[0073]

[0074] 2.5.2 Specificity test

[0075] 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 、 Figure 2 and Figure 3 As shown. 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.

[0076] 2.5.3 Precision test

[0077] Take the reference solution prepared under "2.2" and perform the determination six times in succession according to the chromatographic conditions of "2.1". The RSDs of the peak areas of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside and palmatine hydrochloride were 0.25%, 0.46%, 0.32%, 1.44%, 0.57%, 0.42%, 2.27%, 0.61% and 1.08%, respectively, indicating that the precision of the instrument is good.

[0078] 2.5.4 Repeatability test

[0079] Take Xiaoer Yanbian Granules (batch number 22864007), grind it into powder, take 6 portions at 2 g / portion, 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 chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and palmatine hydrochloride were 0.52%, 0.36%, 0.41%, 0.76%, 0.53%, 1.25%, 2.09%, 1.06%, and 3.04%, respectively, indicating that the method has good repeatability.

[0080] 2.5.5 Stability test

[0081] Take Xiaoer Yanbian Granules (batch number 22864007) and prepare the test solution according to item "2.3". Samples were injected and measured according to the chromatographic conditions of "2.1" at 0h, 8h, 12h, 16h and 24h, respectively. The RSDs of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside and palmatine hydrochloride were 0.47%, 0.60%, 0.33%, 2.59%, 2.08%, 0.94%, 1.69%, 2.85% and 1.89%, respectively, indicating that the test solution had good stability within 24h.

[0082] 2.5.6 Sample recovery test

[0083] Take 6 portions of Xiaoer Yanbian Granules (batch number 22864007) with known content of each component, 1 g each, accurately weighed, placed in a stoppered conical flask, and accurately took 100% level (containing chlorogenic acid 0.84 mg, 5-caffeoylquinic acid 0.68 mg, 4-caffeoylquinic acid 0.53 mg, belamcandide 0.040 mg, tectorius flavonoids 0.045 mg, tectorius flavonoids 0.031 mg, cinnamic acid 0.019 mg, harpagoside 0.022 mg, bamipine hydrochloride 0.025 mg) of the reference solution and added it to the test solution (solvent is 50 mL of methanol with a volume concentration of 50%), weighed, ultrasonically extracted (ultrasonic extraction power is 500 W, ultrasonic extraction frequency is 40 kHz) for 45 min, taken out, cooled, and used The reduced weight was made up with methanol at a volume concentration of 50%, and the mixture was filtered. The filtrate was taken and determined according to the chromatographic conditions of item "2.1". The recovery rate of each component and its RSD value were calculated. As a result, the average recoveries of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and bamipine hydrochloride were 104.07%, 102.78%, 103.02%, 102.54%, 90.40%, 92.05%, 97.03%, 91.19%, and 111.38%, respectively, and the RSDs were 0.26%, 0.56%, 0.18%, 2.34%, 0.80%, 1.59%, 0.41%, 1.87%, and 3.59%, respectively, indicating that the method has excellent accuracy.

[0084] 2.6 Content determination

[0085] Take 5 batches of samples with different batch numbers, prepare the test solution according to "2.3", and measure it under the chromatographic conditions of "2.1". Calculate the content of each component in the sample. The results are shown in Table 2.

[0086] Table 2 Content determination results

[0087]

[0088]

[0089] As shown in Table 2, the results are not affected by other components and can accurately measure the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside and palmatine hydrochloride in each batch of Xiaoer Yanbian Granules.

[0090] Compared to conventional single-ingredient testing techniques for Pediatric Yanbian Granules, the method described in this invention utilizes a multi-index component content determination method, increasing the number of tested components from one to nine, enabling a more comprehensive evaluation of the quality of Pediatric Yanbian Granules. Furthermore, the nine components tested in this method serve as the underlying pharmacological agents for Pediatric Yanbian Granules' heat-clearing, sore throat-relieving, detoxifying, and analgesic effects. By focusing on these active ingredients for quantitative testing, we can more accurately control the intrinsic quality of Pediatric Yanbian Granules' therapeutic effects and scientifically evaluate their efficacy.

[0091] 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.

[0092] In summary, the present invention makes up for the shortcomings of the existing standards for pediatric pharyngeal flat granules, provides a strong basis for further improving and enhancing the quality standards of pediatric pharyngeal flat granules and related preparations, and provides an effective method for fully guiding the quality control of each link in the production of pediatric pharyngeal flat granules and scientifically evaluating the efficacy and quality of the products.

[0093] Example 2

[0094] The test solution was prepared according to item "2.3" and the chromatographic conditions were carried out according to item "2.1". The liquid phase spectrum of the test solution was as follows: Figure 4 shown.

[0095] Example 3

[0096] 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.

[0097] Table 3 Results of investigation at different column temperatures

[0098]

[0099]

[0100] Example 4

[0101] 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 spectra of the chromatographic column Agilent ZORBAX and the chromatographic column CAPCELL PAK are shown in Figure 5 As shown, A is the liquid phase spectrum of the chromatographic column CAPCELL PAK, and B is the liquid phase spectrum of the chromatographic column Agilent ZORBAX.

[0102] Table 4 Results of investigation on different chromatographic columns

[0103]

[0104]

[0105] Example 5

[0106] 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.

[0107] Table 5 Investigation results at different flow rates

[0108]

[0109]

[0110] From Tables 3 to 5, Figures 5-6 The results showed that different brands of chromatographic columns (Agilent ZORBAX; ShimNex CS; CAPCELL PAK, when the column temperature was set at 25℃ and 30℃, and the flow rate was 0.8, 1.0, and 1.2 mL·min -1 At the same time, the parameters of the main components of the Children's Pharyngeal Granules, such as peak time, separation, peak shape, peak area, etc., can meet the requirements and comply with the detection standards.

[0111] Example 6

[0112] 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.

[0113] Table 6 Results of investigation at different extraction times

[0114]

[0115] Example 7

[0116] 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 under different extraction solvents. The results are shown in Table 7.

[0117] Table 7 Results of investigation on different extraction solvents

[0118]

[0119] It can be seen from Tables 6 and 7 that when ultrasonic extraction is performed with methanol at a volume concentration of 50%, 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 easier. In addition, when the ultrasonic extraction time is ≥ 30 min, the peak area of ​​each component increases slightly, but the change is not large, indicating that the component has been basically extracted completely. After 45 min of ultrasonication, the content of each component reaches its highest and tends to be stable.

[0120] 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 the scope of protection of the present invention.

Claims

1. A method for quality inspection of children's pharyngeal granules, characterized in that: The following steps are included: 1) Extracting and filtering the pediatric pharyngeal granule sample and the organic solvent in sequence, evaporating the filtrate to dryness, and then dissolving the residue with the organic solvent and filtering to obtain the pediatric pharyngeal granule sample solution to be tested; 2) HPLC was used to determine the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcandide, tectoriusin, tectoriusin, cinnamic acid, harpagoside, and palmatine hydrochloride in the Xiaoer Yanbian granule sample solution according to the standard curve model; Step 1) The extraction is ultrasonic extraction or reflux extraction; Step 1) the organic solvent is methanol and / or ethanol, and the volume concentration of the organic solvent is ≥50%; Step 1) The extraction time is 30-60 min, the extraction power is 400-600 W, and the extraction frequency is 30-50 kHz; Step 2) The HPLC method is performed using a C18 column, a column temperature of 25-30°C, and a detection wavelength of 240-340 nm. 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 0.5-1.5 mL / min; Step 2) The elution gradient of the high performance liquid chromatography determination is 0-20 min, 8% mobile phase A; 20-48 min, 8-39% mobile phase A; 48-60 min, 39-75% mobile phase A; 60-62 min, 75-8% mobile phase A; 62-65 min, 8% mobile phase A.

2. The quality inspection method according to claim 1, characterized in that: Step 1) The particle size of the pediatric pharyngeal flat granule sample is 170-190 μm.

3. The quality inspection method according to claim 2, characterized in that: Step 1) The mass volume ratio of the pediatric pharyngeal flat granule sample and the organic solvent is 1-3 g: 40-60 mL.

4. The quality inspection method according to claim 3, characterized in that: The injection volume of the high performance liquid chromatography determination in step 2) is 8-12 μL.

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