CN110736799B

CN110736799B - Quality detection method of traditional Chinese medicine children's cold-relieving granules

Info

Publication number: CN110736799B
Application number: CN201911066541.3A
Authority: CN
Inventors: 江玉娟; 吴世德; 贾美春; 潘瑞雪; 李琳; 王莉; 米凤雅; 许敏; 刘晓彤
Original assignee: SHANDONG MINGREN FURUIDA PHARMACEUTICAL CO Ltd
Current assignee: SHANDONG MINGREN FURUIDA PHARMACEUTICAL CO Ltd
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2022-06-10
Anticipated expiration: 2039-11-04
Also published as: CN110736799A

Abstract

The invention relates to a quality detection method of traditional Chinese medicine children's cold-relieving granules. The invention uses high performance liquid chromatography to measure the content of baicalin, wogonoside, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate in a traditional Chinese medicine children cold relieving preparation. The method realizes that the separation degree between each component to be detected and an adjacent peak in the chromatogram of the test sample is more than 1.5, and the negative control is not interfered, further ensures the quality safety and the comprehensive evaluation of the children's influenza relieving granules, and has the advantages of high practicability, strong operability, cost saving and the like.

Description

Quality detection method of traditional Chinese medicine children's cold-relieving granules

Technical Field

The invention relates to a quality detection method of traditional Chinese medicine children's cold-relieving granules, belonging to the technical field of medicines.

Background

The infantile influenza-relieving granules are not recorded in any pharmacopoeia, and the infantile influenza-relieving tablets are recorded in the first part of the 2015 th edition of Chinese pharmacopoeia. Has the effects of clearing away heat, relieving exterior syndrome, relieving sore throat and relieving cough. Can be used for treating common cold, fever, headache, nasal obstruction, cough, sneeze, and sore throat. At present, the children cold-relieving preparation is prepared from the following medicinal materials in parts by weight: 830 g of dyers woad leaf, 415 g of Chinese thorowax root, 415 g of baical skullcap root, 415 g of fineleaf schizonepeta herb, 250 g of platycodon root and 165 g of liquoric root. The children cold-relieving granule has good effect and obvious curative effect through clinical verification, and is clinically favored by patients.

The children's cold relieving granule is prepared from 6 Chinese medicinal materials including folium Isatidis, bupleuri radix, Scutellariae radix, herba Schizonepetae, radix Platycodi, and Glycyrrhrizae radix, wherein the main active ingredients of Scutellariae radix and Glycyrrhrizae radix are baicalin, wogonoside, baicalein, wogonin, liquiritin, and ammonium glycyrrhizinate. The quality control of the children's cold-relieving granules in the prior art mainly focuses on single components of single medicinal materials, and the methods have certain one-sidedness and cannot realize effective control of the whole quality of a prescription.

At present, the quality standard of children cold-relieving granules is loaded with identification tests of indirubin in folium isatidis, indirubin in radix scutellariae, radix bupleuri, platycodon grandiflorum and liquorice; the content measurement method only contains indirubin under the content measurement item. Besides the detection method and the limit, the content measurement of glycyrrhizic acid in liquorice is increased in the children cold-relieving tablet of the year 2015 edition in Chinese pharmacopoeia.

Chinese patent document CN106124656A (application No. 201610442800.8) discloses a detection method of a traditional Chinese medicine children's cold-relieving granule preparation, and provides a method for determining the content of folium isatidis and platycodon grandiflorum in a traditional Chinese medicine children's cold-relieving granule by using high performance liquid chromatography. This patent provides a method for detecting the content of a single component, which cannot detect the content of two components at the same time.

Chinese patent document CN104897787A (application No. 201410452554.5) discloses a method for simultaneously measuring six active ingredients in Niuhuang Ninggong tablets, which comprises preparing a mixed reference solution containing chrysophanol, emodin, liquiritin, phillyrin, baicalin and berberine hydrochloride; preparing a test solution of the Niuhuang Ninggong tablet; injecting reference solution and test solution respectively by high performance liquid chromatography under the same condition, and separating to check the contents of six components in NIUHUANGNING GONG tablet. Although the method can detect six active ingredients at one time, when the method is applied to the ingredient detection of the children's influenza relieving granules, the detection result is inaccurate because the ingredient structures of the children's influenza relieving granules are similar.

In addition to that, the component (baicalin) to be detected in the test solution cannot be completely separated from the adjacent peaks under the condition, no response peak is detected in the component ammonium glycyrrhizinate to be detected, the negative control is interfered, and the accuracy of the detection result still needs to be improved.

In conclusion, the traditional Chinese medicine product has various and complex components, the quality of the medicine is not accurately evaluated by a single component, the comprehensive evaluation of the quality of the traditional Chinese medicine is not facilitated, the consumption of a reference substance is high, the detection cost is high, and the detection operation difficulty is high in multi-index content measurement. In the prior art, a detection method capable of determining the contents of six components in the children's influenza relieving granules by one-test and multi-evaluation methods is unavailable, and the determination conditions and determination substances of one-test and multi-evaluation methods in other prior art are not suitable for the children's influenza relieving granules. Therefore, it is necessary to establish a one-test-multiple-evaluation content detection method suitable for children's influenza-relieving granules so as to achieve the purpose of comprehensively controlling the quality of the preparation by multiple components and multiple targets.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a quality detection method of a traditional Chinese medicine children's cold-relieving granule.

Summary of The Invention

The invention uses high performance liquid chromatography to perform one-test-multiple-evaluation content determination on baicalin, wogonoside, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate in a traditional Chinese medicine children cold-relieving preparation, and the method has the advantages of simple operation, accurate analysis data and capability of ensuring the safety, stability and controllability of product quality.

The technical scheme of the invention is as follows:

a detection method of traditional Chinese medicine children's cold-relieving granules comprises the following steps:

chromatographic conditions and system applicability test:

according to the high performance liquid chromatography of four parts 0512 of the year version of Chinese pharmacopoeia 2015:

performing gradient elution by using acetonitrile-0.1% (volume percentage concentration) phosphoric acid as a mobile phase, wherein the detection wavelength is 274nm, and the flow rate is 0.9-1.1 ml/min;

preparation of mixed control solution: weighing baicalin, wogonin, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate reference substances respectively, adding 70% methanol solution by volume percent for dissolving to obtain mixed reference substance stock solutions with determined mass concentration, respectively sucking the mixed reference substance stock solutions, and adding methanol to prepare mixed reference substance solutions with series concentrations for later use;

Preparing a test solution: taking 0.5g of the children's cold relieving granules, adding 25mL of 70% methanol solution, carrying out ultrasonic treatment, complementing the loss of ultrasonic treatment with 70% methanol solution, and filtering; taking 1mL of filtrate, diluting the filtrate to 10mL by using 70% methanol solution, and filtering the filtrate through a 0.22 mu m microporous filter membrane for later use;

the determination method comprises the following steps: and (4) respectively taking 20 mu L of the reference substance solution and the sample solution, injecting into a liquid chromatograph, and recording the chromatogram.

According to the invention, the high performance liquid chromatography column is a Waters Sun Fire C18 column with specification of 4.6mm × 250mm and 5 μm.

Preferably, according to the invention, the flow rate is 1.0 ml/min.

According to the invention, the preferable mass concentrations of the baicalin, the wogonoside, the baicalein, the wogonoside, the liquiritin and the ammonium glycyrrhizinate reference substances are 460.36-488.84 mu g/mL respectively^-1、92.07～97.77μg·mL^-1、66.27～

70.37μg·mL^-1、54.57～57.95μg·mL^-1、275.29～292.31μg·mL^-1、109.34～116.10μg·mL^-1(ii) a More preferably, the quality concentrations of baicalin, wogonoside, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate reference substances are 474.60 μ g/mL respectively^-1、94.92μg·mL^-1、68.32μg·mL^-1、56.26μg·mL^-1、283.80μg·mL^-1、112.72μg·mL^-1。

Preferably, in the gradient elution, the volume percentage of the phase A in the initial elution is 12%, and the volume percentage of the phase B in the initial elution is 88%;

When the elution time is 20 minutes, the volume percentage of the phase A is 27 percent, and the volume percentage of the phase B is 73 percent;

when the elution time is 40 minutes, the volume percentage of the phase A is 50 percent, and the volume percentage of the phase B is 50 percent; elution duration from 3 minutes to 43 minutes;

when the elution time is 44 minutes, the volume percentage of the phase A is 10 percent, and the volume percentage of the phase B is 90 percent; elution duration was 11 minutes to 55 minutes.

According to a preferred embodiment of the present invention, the ultrasonic treatment conditions are: the power is 250W, the frequency is 53kHz, and the ultrasonic treatment is carried out for 30 min.

The invention has the beneficial effects that:

the method for detecting the content of 6 components in the children's anti-influenza particles for the first time is realized, the current situation that single component detection and multi-index component content detection are insufficient is perfected, the separation degree between each component to be detected and an adjacent peak in a chromatogram of a sample is larger than 1.5, negative control is not interfered, the quality safety and the comprehensive evaluation of the children's anti-influenza particles are further ensured, and the detection method has the advantages of high practicability, strong operability, cost saving and the like.

Drawings

FIG. 1 is a high performance liquid chromatogram of a mixed control;

FIG. 2 is a high performance liquid chromatogram of a sample for measuring the content of each component to be measured in the children's influenza relieving granule;

FIG. 3 is a high performance liquid chromatogram of negative control for determining the content of children's influenza-relieving granules;

FIG. 4 is a linear graph of baicalin content measurement;

FIG. 5 is a linear graph of the content measurement of wogonoside, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate;

FIG. 6 is a high performance liquid chromatogram of a pediatric influenza relieving granule test solution before chromatographic condition improvement;

in fig. 1 to 3 and fig. 5 to 6, the ordinate represents voltage, unit: microvolts, abscissa time, unit: the method comprises the following steps of (1) taking minutes; in fig. 4, the ordinate is peak area, unit: mAu · s, abscissa concentration, unit: μ g/mL.

Detailed Description

The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1:

1. instrument, reagent and test sample

The instrument comprises the following steps: high performance liquid chromatograph: agilent 1260, G1311C infusion pump, G1329B autosampler system, G1315D diode array detector, Waters Empower3 workstation; METTLER TOLEDO XPE205 electronic balance

Comparison products: baicalin (batch No. 110715-201821, purity 95.4%), wogonin (batch No. 112002-201702, purity 98.5%), liquiritin (batch No. 111610-201607, purity 93.1%), baicalein (batch No. 111595-201808, purity 97.9%), wogonin (batch No. 111514-201706, purity 100%), ammonium glycyrrhizinate (batch No. 110731-201720, purity 97.7%)

A test sample: children's cold-relieving granule (Shandong Mingren Furuida pharmaceutical Co., Ltd.)

Batch number: 18105084, 18105080, 18105083, 18112401, 18112701, 18112901, 20190128, 20190129, 20190130, 19105028, 19105029, 19105030.

2. Chromatographic conditions

Measuring by high performance liquid chromatography (high performance liquid chromatography 0512 in the four parts of the 2015 edition of Chinese pharmacopoeia).

Waters Sun Fire C18(4.6 mm. times.250 mm, 5 μm) as a chromatographic column; acetonitrile-0.1% (v/v) phosphoric acid was used as a mobile phase, and gradient elution was performed according to the following table, with a detection wavelength of 274nm and a flow rate of 1.0 ml/min.

Linear gradiometer

Time (min)	A(％)	B(％)
			0	12	88
20	27	73
			40	50	50
43	50	50
			44	10	90
55	10	90

3. Preparation of mixed control: accurately weighing appropriate amount of baicalin, wogonoside, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate reference substances, and dissolving with 70% methanol to obtain mixed reference substance stock solutions with mass concentrations of 474.60 μ g/mL-1, 94.92 μ g/mL-1, 68.32 μ g/mL-1, 56.26 μ g/mL-1, 283.80 μ g/mL-1 and 112.72 μ g/mL-1. Accurately sucking appropriate amount of mixed reference stock solution, and adding methanol to obtain a series of mixed reference solutions.

4. Preparing a test solution: precisely weighing about 0.5g of the infantile common cold relieving granule, placing into a conical flask with a plug, precisely adding 25mL of 70% methanol solution, weighing, performing ultrasonic treatment (power 250W and frequency 53kHz) for 30min, weighing again, supplementing the loss with 70% methanol solution, and filtering. Precisely measuring 1mL of the subsequent filtrate, placing the subsequent filtrate in a 10mL measuring flask, diluting with 70% methanol solution, fixing the volume to a scale, and filtering through a 0.22-micrometer microporous filter membrane for later use.

5. System suitability test and negative interference test

Under the chromatographic conditions, 20ul of each of the reference solution, the test solution and the negative control solution is precisely measured, injected into a liquid chromatograph, and recorded in the chromatogram. The result shows that the separation degree between each component to be detected and the adjacent peak in the chromatogram of the test sample is more than 1.5, and the negative control is not interfered.

6. Preparation of standard curve, linear relation investigation and determination of quantitative limit

Precisely measuring 1ml, 3ml, 5ml, 7ml and 9ml of mixed reference substance storage solution, respectively placing in 10ml volumetric flasks, diluting to scale with methanol, shaking up, precisely sampling 20ul of each sample, and recording the peak area of chromatogram; the linear solution is diluted into a series of concentrations, each sample is precisely injected into 20ul, the signal-to-noise ratio of each component to be measured is recorded, the quantitative limit concentration is determined, and the result is shown in the following table, and the linear relation of the peak area (A) to the concentration (C) of the reference substance is good.

TABLE 1 Standard Curve results Table

1-1 baicalin standard curve result table

Serial number	Concentration (ug/ml)	Peak area (A)
			1	45.2768	2769334
2	135.8305	9470218
			3	226.3842	15740940
4	316.9379	22035850
			5	407.4916	27429555
Limit of quantification	0.102	6822

The regression equation: Y-68368.792X +9613.657 correlation coefficient: r is 0.9995

1-2 wogonin standard curve result table

Serial number	Concentration (ug/ml)	Peak area (A)
			1	9.3496	697897
2	28.0489	2205259
			3	46.7481	3652924
4	65.4473	5145531
			5	84.1466	6639838
Limit of quantification	0.067	5333

The regression equation: Y-79000.363X-20686.191 correlation coefficient: r 1.0000

1-3 Baicalein standard curve result table

The regression equation: Y-104186.748X-22483.614 correlation coefficient: r 1.0000

1-4 wogonin standard curve result table

Serial number	Concentration (ug/ml)	Peak area (A)
			1	5.6260	715635
2	16.8780	2130750
			3	28.1300	3514937
4	39.3820	4922452
			5	50.6340	6344525
Limit of quantification	0.054	5535

The regression equation: Y-125038.470X +6736.852 correlation coefficient: r 1.0000

1-5 Glycyrrhiza glycoside Standard Curve results Table

Serial number	Concentration (ug/ml)	Peak area (A)
			1	26.421780	992830
2	79.265340	2967684
			3	132.108900	4895172
4	184.952460	6845599
			5	237.796020	8831521
Limit of quantification	0.254	5491

The regression equation: Y-37062.057X +7960.003 correlation coefficient: r 1.0000

1-6 ammonium glycyrrhizinate standard curve result table

Serial number	Concentration (ug/ml)	Peak area (A)
			1	11.0127	31756
2	33.0382	115873
			3	55.0637	194955
4	77.0892	276686
			5	99.1147	357322
Limit of quantification	0.330	5842

The regression equation: Y-3610.082X-2113.462 correlation coefficient: r is 0.9995

7. Precision test table 3 ultrasonic temperature selection table

Precisely measuring 5ml of the mixed reference substance stock solution, placing the mixed reference substance stock solution into a 10ml volumetric flask, adding methanol to dilute the mixed reference substance stock solution to a scale, and shaking the mixed reference substance stock solution uniformly to obtain the reference substance solution. Precisely measuring 20ul of the mixed reference solution, injecting into a liquid chromatograph, continuously measuring for 6 times, recording peak area, and calculating relative standard deviation, wherein RSD of each component is less than 2%. The results show that the instrument precision is good.

TABLE 2 precision data Table

TABLE 2-1 precision data table of baicalin

TABLE 2-2 precision data table of wogonoside

TABLE 2-3 precision data table of baicalein

Tables 2-4 wogonin precision data tables

TABLE 2-5 precision data table of liquiritin

TABLE 2-6 ammonium glycyrrhizinate precision data sheet

8. Stability test

After the preparation of the test solution is finished, precisely sucking 20ul of the test solution, injecting the test solution into a liquid chromatograph, recording the peak area of the chromatograph, measuring the peak area once every 2 hours, inspecting the peak area for 8 hours, and calculating the relative standard deviation of the peak area. The result shows that the solution stability of each component to be tested in the test sample is good within 8 hours.

TABLE 3 stability data Table

TABLE 3-1 baicalin stability data Table

TABLE 3-2 stability data sheet of wogonoside

TABLE 3-3 Licorice glycoside stability data sheet

TABLE 3-4 baicalein stability data Table

Tables 3-5 wogonin stability data tables

TABLE 3-6 ammonium glycyrrhizinate stability data Table

9. Repeatability test

Taking the product, repeatedly measuring for 6 times, and calculating the content of each component in the sample. The result shows that the RSD of each component to be tested is less than 2 percent, and the analysis method has good repeatability.

TABLE 4 repeatability data sheet

10. Recovery test

Precisely weighing 6 parts of the same batch of samples, precisely adding a proper amount of each reference substance, determining the content of the reference substances by the function, and calculating the recovery rate, wherein the recovery rate of each component to be detected is shown in the following table. The result shows that the measuring method has accurate measuring result.

TABLE 5 recovery data sheet

Components	Baicalin	Wogonoside	Baicalein	Wogonin	Liquiritin	Ammonium glycyrrhizinate
							Recovery ratio of 1 to 1	98.17	103.6	98.01	96.24	96.45	100
Recovery ratio of 2%	99.18	100.01	95.77	95.09	100.01	103.29
							Recovery ratio-3	98.55	98.18	97.98	99.88	101.2	99.24
Recovery ratio-4	99.99	101.11	96.15	98.14	98.11	98.89
							Recovery rate-5	99.44	97.46	100.77	100.3	99.88	101.98
Recovery rate is 6 percent	99.61	102.9	99.43	97.38	97	102.48
							Average recovery rate%	99.16	100.54	98.02	97.84	98.78	100.98
RSD％	0.69	2.46	1.94	2.08	1.9	1.82

11. Determination of the relative correction factor f

Precisely sucking 20 μ l of mixed reference solution with each concentration under linear condition, injecting into liquid chromatograph, and recording corresponding chromatographic peak area. Taking baicalin as an internal reference substance, calculating f of wogonoside, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate, wherein the calculation formula is as follows:

f＝f_s/f_k＝W_sA_k/W_kA_s

in the formula: a. the_kIs the peak area of the internal reference substance, W_kIs the concentration of the internal reference substance, A_sThe peak area of a certain component to be measured, W_sThe relative correction factor f for each component for a given concentration of the component to be measured is shown in the following table

TABLE 6 correction factor for each component

12. Chromatographic peak location of each component to be measured

And positioning the chromatographic peak of the component to be detected by adopting the relative retention time, namely the retention time ratio between each component to be detected and baicalin, precisely absorbing 20 mu l of the mixed reference substance solution with each concentration under a linear condition, injecting the mixed reference substance solution into a liquid chromatograph, and calculating the relative retention time of the chromatographic peak of each component to be detected. The results are shown in the following table, and the RSD is less than 2%, which indicates that the positioning method has accurate measurement results.

TABLE 7 relative retention time Table for each component

Component to be tested	Wogonoside	Baicalein	Ammonium glycyrrhizinate	Wogonin	Liquiritin
						Relative retention time-1	1.191	1.381	1.475	1.663	0.644
Relative retention time-2	1.192	1.382	1.477	1.664	0.643
						Relative retention time-3	1.193	1.383	1.481	1.668	0.643
Relative Retention time-4	1.193	1.382	1.479	1.667	0.643
						Relative retention time-5	1.193	1.383	1.48	1.668	0.643
Average relative retention time	1.1924	1.3822	1.4784	1.666	0.6432
						RSD％	0.08	0.06	0.16	0.14	0.07

13. Durability test

Influence of different instruments and chromatographic columns on f: considering the influence of 2 liquid chromatographs (Agilent1260, Waters e2695) and 3 chromatographic columns (SunAire C18(A), ZORBAX C18(B), YMC C18(C)) on f, the RSD of 5 components f is less than 2%, which shows that different instruments and chromatographic columns have no significant influence on f of each component, and the results are shown in Table 8.

TABLE 8 data table of the effect of different instruments and chromatographic columns on f

Influence of different column temperatures on f: the influence of different column temperatures (27, 30 and 33 ℃) on f is examined, and the results show that the RSD of 5 components f is less than 2 percent, which shows that different column temperatures have no significant influence on the f of each component, and the results are shown in Table 9.

TABLE 9 influence data of different column temperatures on f

Column temperature/. degree.C	f_{Baicalin/wogonoside}	f_{Baicalin/baicalein}	f_{Baicalin/wogonin}	f_{Baicalin/liquiritin}	f_{Baicalin/ammonium glycyrrhizinate}
						27	0.81257	0.66541	0.57859	1.84145	18.88745
30	0.83195	0.67777	0.55593	1.86555	18.97124
						33	0.83195	0.68025	0.54962	1.85973	19.08147
Mean value (average)	0.82549	0.67448	0.56138	1.85558	18.98005
						RSD/％	1.36	1.18	1.91	0.68	0.51

14. Comparison of content measured by one-test-multiple-evaluation method and external standard method

Taking 12 batches of samples, preparing a test solution, precisely measuring 20ul of the test solution, injecting into HPLC, recording peak areas of various chromatographic peaks, quantitatively measuring 5 components and baicalin by adopting an external standard method, and then quantitatively calculating by using the established QAMS method to verify the accuracy of the QAMS method for multi-index evaluation in the children's influenza treating granules, wherein the results are shown in a table 10. The contents of the components measured by the 2 methods have no obvious difference, which shows that the established method has better accuracy.

TABLE 10 QAMS and ESM comparison of the results of determining the content of the children's anti-influenza granules (μ g/g)

15. Selection of index component

The children cold-relieving granule comprises folium Isatidis, bupleuri radix, herba Schizonepetae, Scutellariae radix, radix Platycodi, and Glycyrrhrizae radix. Wherein, the scutellaria is bitter and cold, has the effects of clearing heat and drying dampness, purging fire and removing toxicity, can clear heat of shaoyang, is mainly used for treating warm diseases, and is combined with the dyers woad leaf, the bupleurum and the fineleaf schizonepeta herb for mainly dispelling wind and heat, clearing heat and removing toxicity, clearing heat and cooling blood, and can reduce low fever and treat high fever; the liquorice has sweet and mild nature and taste, and has the effects of eliminating phlegm, relieving cough, detoxifying and relieving sore throat; the platycodon root is bitter and pungent in flavor, and has the effects of ventilating lung, relieving sore throat, eliminating phlegm and expelling pus; the liquorice and the platycodon root have exact effect of treating the swollen sore throat; according to the rational and legal prescription and medicine analysis of the ingredients in the prescription, the main drug effect ingredients to be detected are determined, and finally, a substance set to be subjected to combined detection is screened out.

The application sequentially carries out HPLC (high performance liquid chromatography) determination on main components of radix bupleuri, liquorice, radix scutellariae and platycodon grandiflorum, and inspects indexes such as maximum wavelength absorption, peak shape, separation degree and the like, wherein the radix scutellariae and liquorice components have larger absorption at 274nm, the radix bupleuri components (saikosaponin a, saikosaponin b and saikosaponin c) have larger absorption at 210nm, and the difference of detection wavelengths is larger; the repeatability of the platycodon grandiflorum components is poor under the detection wavelength of 274nm, comprehensive consideration is taken, the QAMS detection method for 6 components of baicalin, wogonoside, liquiritin, ammonium glycyrrhizinate, baicalein and wogonin is determined, and the selected indexes can meet the effective detection of the drug effect and can realize one-time rapid detection.

16. Determination of mobile phase

The 6 components measured by the method take the separation degree, the peak shape, the peak height and the like as investigation indexes, and different mobile phases are successively investigated by adopting HPLC gradient elution: the separation effect of acetonitrile-0.1% (v/v) phosphoric acid, acetonitrile-0.2% (v/v) phosphoric acid, methanol-0.1% (v/v) phosphoric acid, methanol-0.2% (v/v) phosphoric acid, acetonitrile-0.1% (v/v) acetic acid, and acetonitrile-0.2% (v/v) acetic acid systems, and when methanol-0.1% (v/v) phosphoric acid, methanol-0.2% (v/v) phosphoric acid, methanol-0.1% (v/v) phosphoric acid, and methanol-0.2% (v/v) phosphoric acid are used as a mobile phase, the deviation of the RSD (RSD) of the peak reproducibility of ammonium glycyrrhizinate is large; finally, a detection method for gradient elution by taking acetonitrile (A) -0.1% (v/v) phosphoric acid aqueous solution (B) as a mobile phase is determined, elution conditions are optimized, a better separation effect is achieved, and methodological investigation such as repeatability and precision meets requirements.

To sum up: the invention establishes a multiple evaluation method (QAMS) for measuring the content of baicalin, wogonin, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate in the children's influenza removing granules by taking the baicalin as an internal reference substance. The invention can be used for measuring the content of more components of the children's cold-relieving granules and comprehensively controlling the quality of the preparation.

Claims

1. A detection method of traditional Chinese medicine children cold-relieving granules is characterized by comprising the following steps:

chromatographic conditions and system applicability test:

according to the measurement of high performance liquid chromatography of four parts 0512 of the year version of Chinese pharmacopoeia 2015, a chromatographic column measured by the high performance liquid chromatography is a Waters Sun Fire C18 chromatographic column:

performing gradient elution by using acetonitrile-0.1 volume percent concentration phosphoric acid as a mobile phase, wherein the detection wavelength is 274nm, and the flow rate is 0.9-1.1 ml/min;

the volume percentage of the phase A which is initially eluted is 12 percent, and the volume percentage of the phase B is 88 percent in the gradient elution; when the elution time is 20 minutes, the volume percentage of the phase A is 27 percent, and the volume percentage of the phase B is 73 percent; when the elution time is 40 minutes, the volume percentage of the phase A is 50 percent, and the volume percentage of the phase B is 50 percent; elution duration from 3 minutes to 43 minutes; when the elution time is 44 minutes, the volume percentage of the phase A is 10 percent, and the volume percentage of the phase B is 90 percent; elution duration 11 min to 55 min;

the quality concentrations of the baicalin, the wogonoside, the baicalein, the wogonoside, the liquiritin and the ammonium glycyrrhizinate reference substance are 460.36-488.84 mug/mL respectively^-1、92.07～97.77µg·mL^-1、66.27～70.37µg·mL^-1、54.57～57.95µg·mL^-1、275.29～292.31µg·mL^-1、109.34～116.10µg·mL^-1；

Preparation of a test solution: taking 0.5g of the children's cold-relieving granules, adding 25mL of 70% methanol solution, carrying out ultrasonic treatment, complementing the amount of ultrasonic treatment loss by using 70% methanol solution, and filtering; taking 1mL of filtrate, diluting the filtrate to 10mL by using 70% methanol solution, and filtering the filtrate through a 0.22 mu m microporous filter membrane for later use;

2. The detection method according to claim 1, wherein the column specification measured by high performance liquid chromatography is 4.6mm x 250mm, 5 μm.

3. The assay of claim 1, wherein the flow rate is 1.0 ml/min.

4. The detection method of claim 1, wherein the mass concentrations of the baicalin, wogonoside, baicalein, wogonin, liquiritin and ammonium glycyrrhizinate control are 474.60 μ g-mL respectively^-1、94.92µg·mL^-1、68.32µg·mL^-1、56.26µg·mL^-1、283.80µg·mL^-1、112.72µg·mL^-1。

5. The detection method according to claim 1, wherein the sonication conditions are: 250W of power, 53kHz of frequency and 30min of ultrasonic treatment.