Quality detection method of infantile pharyngeal flat granules

Abstract

The invention belongs to the technical field of pharmaceutical analysis. The invention provides a quality detection method of infantile pharyngeal flat granules, which comprises the following steps: sequentially extracting and filtering an infantile pharyngeal flat granule sample and an organic solvent, evaporating the filtrate to dryness, dissolving residues by using the organic solvent, and filtering to obtain an infantile pharyngeal flat 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 nine active components in a sample solution of the child pharyngeal flat granules to be measured. According to the quality detection method of the infantile pharyngeal flat granules, the overall quality of the infantile pharyngeal flat granules can be more comprehensively evaluated and controlled, and the curative effect of the product is ensured; the content of nine active ingredients in the infantile pharyngeal flat granules can be rapidly determined, and the durability is good. Moreover, 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 invention relates to the technical field of drug analysis, and in particular to a quality detection method for children's pharyngeal flat granules. Background Art

[0002] Pediatric Throat Bulbus Granules are composed of eight herbs: honeysuckle, belamcanda vine, olive, Scrophularia ningpoensis, Platycodon grandiflorum, Ophiopogon japonicus, artificial bezoar and borneol. It has the effects of clearing heat and relieving sore throat, detoxifying and relieving pain. It is 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 chinensis 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 chinensis clears away heat and detoxifies, and eliminates phlegm and relieves sore throat. Its main active ingredients are isoflavones such as belamcanda glycosides and irisin. Olea europaea relieves sore throat and relieves pain. Scrophularia ningpoensis clears heat and cools blood. Platycodon grandiflorum invigorates the lungs, and Ophiopogon japonicus nourishes yin and moistens dryness. The four medicines are matched together and serve as ministerial medicines. The main active ingredient of Olea europaea is the alkaloid ingredient bamipine hydrochloride, and the main active ingredients of Scrophularia ningpoensis are cinnamic acid and harpagoside. Artificial bezoar and borneol are refreshing and invigorating, and they are all adjuvants.

[0004] The current quality standard for Children's Yanbian 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. The content determination uses chlorogenic acid as the indicator component, which is poorly representative, and 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 Children's Throat Bile Granules that can clear away heat, relieve sore throat, detoxify and relieve pain, so as to provide a reference for the comprehensive evaluation and control of the intrinsic quality of Children's Throat Bile Granules and provide a strong basis for improving the quality standards of Children's Throat Bile Granules. Summary of the invention

[0006] The purpose of the present invention is to provide a quality detection method for children's pharyngeal flat granules in view of the deficiencies in the prior art. The method can simultaneously accurately and quickly determine the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcanda glycosides, tectorius flavonoids, tectorius flavonoids, cinnamic acid, harpagosides and bamipine hydrochloride in children's pharyngeal flat granules. The method is a simple and feasible method for determining the contents of multiple index components of children's 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 flat 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) Determine by high performance liquid chromatography, and calculate the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcanda glycosides, tectorius flavonoids, tectorius flavonoids, cinnamic acid, harpagosides and bamipine hydrochloride in the sample solution of the pediatric pharyngeal flat granules to be tested 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 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, the mobile phase A for the HPLC determination in step 2) 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 determined by the high performance liquid chromatography 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 takes the Children's 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, belamcanda glycosides, tectorius flavonoids, tectorius quinoids, cinnamic acid, harpagosides and bamipine hydrochloride. The 9 ingredients detected are the medicinal ingredients of the Children's Yanbian Granules that play the effects of clearing away heat, relieving sore throat, detoxifying and relieving pain. Establishing the content determination method can more comprehensively evaluate and control the overall quality of the Children's Yanbian Granules and ensure the product efficacy.

[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 contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, irisin, irisin, cinnamic acid, harpagoside and bamipine hydrochloride in the pediatric pharyngeal flat granules, and has good durability; and, after methodological verification, the detection method has strong specificity, good precision, repeatability, stability, linear relationship and accuracy, is easy to operate, and saves cost.

[0023] 3) The present invention makes up for the deficiencies of 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 9 reference solutions used in the specificity test of 2.5.2 in Example 1, wherein 1 is the liquid phase spectrum of 5-caffeoylquinic acid reference solution, 2 is the liquid phase spectrum of chlorogenic acid reference solution, 3 is the liquid phase spectrum of 4-caffeoylquinic acid reference solution, 4 is the liquid phase spectrum of belamcanda glycoside reference solution, 5 is the liquid phase spectrum of bamipine hydrochloride reference solution, 6 is the liquid phase spectrum of harpagoside reference solution, 7 is the liquid phase spectrum of cinnamic acid reference solution, 8 is the liquid phase spectrum of irisin reference solution, and 9 is the liquid phase spectrum of irisin sub-reference solution;

[0025] Figure 2 It is 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 belamcanda glycosides, 5 is the liquid phase spectrum of the test solution of bamipine hydrochloride, 6 is the liquid phase spectrum of the test solution of harpagosides, 7 is the liquid phase spectrum of the test solution of cinnamic acid, 8 is the liquid phase spectrum of the test solution of irisin, and 9 is the liquid phase spectrum of the test solution of irisin sub-

[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 reference solution of Honeysuckle, C is the liquid phase spectrum of the negative reference solution of Scrophularia, D is the liquid phase spectrum of the negative reference solution of Belamcanda chinensis, and E is the liquid phase spectrum of the negative reference solution of Olea europaea;

[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 quality detection of pediatric pharyngeal flat 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) Determine by high performance liquid chromatography, and calculate the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcanda glycosides, tectorius flavonoids, tectorius flavonoids, cinnamic acid, harpagosides and bamipine hydrochloride in the sample solution of the pediatric pharyngeal flat granules to be tested 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%, and 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 to 30°C, more preferably 30°C; the detection wavelength is preferably 240 to 340 nm, more preferably 260 to 320 nm, and more preferably 280 to 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 determined by the high performance liquid chromatography 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 of 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope 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%), belamcanda glycoside 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 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 irisin sub-reference substance (batch number: Y30N10H104711; purity: 98.0%) were purchased from Shanghai Yuanye Company.

[0053] Bamipine 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 filler; acetonitrile was used as mobile phase A, 0.1% volume concentration of phosphoric acid aqueous solution was used as mobile phase B, gradient elution (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); detection wavelength was 290 nm; flow rate was 1.0 mL min -1 ; Column temperature was 25°C; Injection volume was 10 μL.

[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, belamcanda glycoside reference substance, irisin reference substance, irisin sub-reference substance, cinnamic acid reference substance, harpaoside reference substance, and bamipine 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 belamcanda glycoside, 1.99 μg of irisin, 1.92 μg of irisin sub-reference substance, 1.04 μg of cinnamic acid, 2.04 μg of harpaoside, and 1.59 μg of bamipine hydrochloride per 1 mL.

[0059] 2.3 Preparation of test solution

[0060] Take an appropriate amount of Children's Throat 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, put it in a stoppered conical flask, accurately add 50mL of 50% methanol by volume, weigh it, and perform ultrasonic extraction (the power of ultrasonic extraction is 500W, and the frequency of ultrasonic extraction is 40kHz) for 45min. Take it out, cool it, make up the lost weight with 50% methanol by volume, 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 chinensis, hollyhock olive, 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 Jin Guolan: 10.94g of honeysuckle, 6.25g of belamcanda, 7.81g of platycodon, 7.81g of scrophularia, 7.81g of ophiopogon, 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.

[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 reference solution: 10.94g of honeysuckle, 7.81g of Olea cochinchinensis, 7.81g of Platycodon grandiflorum, 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.

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

[0068] 2.5 Methodological Review

[0069] 2.5.1 Investigation of linear relationship

[0070] Weigh the appropriate amount of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, irisin, irisin, cinnamic acid, harpagoside, and palmatine hydrochloride respectively, dissolve in methanol and prepare a series of mixed reference solutions with linear concentrations. Inject 10 μL of the solution into the liquid chromatograph and determine according to the chromatographic conditions of "2.1". -1 ) as the abscissa (X) and the peak area as the ordinate (Y), and the results are shown in Table 1, indicating that chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcanda glycosides, tectorius flavonoids, tectorius flavonoids, cinnamic acid, harpagosides, and bamipine hydrochloride showed a good linear relationship with the peak area within their respective linear concentration ranges.

[0071] Table 1 Results of the linear relationship investigation of the nine 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 the liquid chromatograph. The corresponding spectrum is as follows: Figure 1 , Figure 2 and Figure 3 As shown. Figure 1 to Figure 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 degree 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 make 6 consecutive injections according to the chromatographic conditions of "2.1". The RSDs of the peak areas of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, irisin, tectorius, cinnamic acid, harpagoside and bamipine 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, belamcanda glycoside, tectorius flavonoids, tectorius flavonoids, cinnamic acid, harpagoside, and bamipine 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), prepare the test solution according to item "2.3", and inject and measure 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, belamcanda glycoside, tectorius flavonoids, tectorius flavonoids, 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 has 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, accurately take 100% level (containing chlorogenic acid 0.84 mg, 5-caffeoylquinic acid 0.68 mg, 4-caffeoylquinic acid 0.53 mg, irradicandin 0.040 mg, tectorum cyanobacterium 0.045 mg, tectorum cyrtonema 0.031 mg, cinnamic acid 0.019 mg, harpagoside 0.022 mg, bamipine hydrochloride 0.025 mg) of the reference substance solution and add it to the test solution (the solvent is 50 mL of methanol with a volume concentration of 50%), weigh the weight, and perform ultrasonic extraction (ultrasonic extraction power is 500 W, ultrasonic extraction frequency is 40 kHz) for 45 min, take out, cool, and use The reduced weight was supplemented 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, irisin, irisin, 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", measure under the chromatographic conditions of "2.1", calculate the content of each component in the sample, and the results are shown in Table 2.

[0086] Table 2 Content determination results

[0087]

[0088]

[0089] It can be seen from Table 2 that the results are not interfered by other components, and can accurately measure the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcanda glycosides, irisin, irisin, cinnamic acid, harpagoside, and bamipine hydrochloride in each batch of pediatric pharyngeal granules.

[0090] Compared with the conventional single component testing technology of children's pharyngeal granules, the method described in the present invention is a multi-index component content determination method, and the number of detected components has been increased from 1 to 9, which can more comprehensively evaluate the quality of children's pharyngeal granules. At the same time, the 9 components tested by the method described in the present invention are the material basis of the efficacy of children's pharyngeal granules in clearing heat and relieving throat, detoxification and analgesia. Focusing on the above active ingredients for quantitative testing can more accurately control the intrinsic quality of children's pharyngeal granules to exert their therapeutic effects and scientifically evaluate the efficacy of the product.

[0091] In addition, the liquid chromatography conditions used in the present invention are suitable, the preparation method is relatively simple, the operation is simple, 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 granules, provides a strong basis for further improving and enhancing the quality standards of pediatric pharyngeal granules and related preparations, fully guides the quality control of each link in the production of pediatric pharyngeal granules, and provides an effective method for scientifically evaluating product efficacy and quality.

[0093] Example 2

[0094] The test solution is prepared according to item "2.3" and the chromatographic conditions are carried out according to item "2.1". The liquid phase spectrum of the test solution is 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", and 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 is as follows 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 of different flow rates

[0108]

[0109]

[0110] From Tables 3 to 5, Figures 5-6 The results show that different brands of chromatographic columns (Agilent ZORBAX; ShimNex CS; CAPCELL PAK, when the column temperature is set at 25℃, 30℃, and the flow rate is 0.8, 1.0, 1.2mL·min -1 At the same time, various parameters such as peak time, separation, peak shape, peak area, etc. of the main components in the Children's Pharyngeal Granules 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 investigated 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 investigated 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 using methanol with 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, which is easy to detect. 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 the 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 principle 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 flat 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) Determine by high performance liquid chromatography, and calculate the contents of chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, belamcanda glycosides, tectorius flavonoids, tectorius flavonoids, cinnamic acid, harpagosides and bamipine hydrochloride in the sample solution of the pediatric pharyngeal flat granules to be tested based on the standard curve model; The extraction in step 1) is ultrasonic extraction or reflux extraction.

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 1 or 2, characterized in that: Step 1) The organic solvent is methanol and / or ethanol, and the volume concentration of the organic solvent is ≧50%.

4. The quality inspection method according to claim 3, 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.

5. The quality inspection method according to claim 4, characterized in that: The extraction time of step 1) is 30 to 60 minutes, the extraction power is 400 to 600 W, and the 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 HPLC determination is a C18 chromatographic column, the column temperature is 25-30°C, and the detection wavelength is 240-340nm.

7. The quality inspection method according to claim 5 or 6, characterized in that: Step 2) The mobile phase A of the HPLC 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.

8. The quality inspection method according to claim 7, characterized in that: Step 2) The elution gradient determined by the high performance liquid chromatography 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; 62-65min, 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.

Images