CN115420827B - Detection method for comprehensively controlling quality of phyllanthus urinaria - Google Patents

Abstract

The invention discloses a detection method for comprehensively controlling quality of phyllanthus urinaria, which is characterized in that a characteristic spectrum is established by an ultra-high performance liquid chromatography, ellagic acid, gallic acid and corilagin are used as reference peaks, index content detection and thin-layer detection are combined, and the phyllanthus urinaria formula particles, medicinal materials, decoction pieces, processed products thereof, preparation intermediates or preparation finished products are subjected to relatively comprehensive quality monitoring by an optimized detection method. The method is simple to operate, good in reproducibility, accurate and reliable, saves time, and provides a new analysis means for the internal quality control of the phyllanthus urinaria.

Description

Detection method for comprehensively controlling quality of phyllanthus urinaria

Technical Field

The invention relates to a detection method for quality of a traditional Chinese medicine preparation, in particular to a detection method for comprehensively controlling quality of phyllanthus urinaria formula particles.

Background

The Phyllanthus urinaria is of EuphorbiaceaeEuphorbiceace) Phyllanthus plant of Phyllanthus (also known as Citrus)Phyllanthus urinaria Linnacus) Is prepared by drying whole herb. As a traditional Chinese medicine, the traditional Chinese medicine is used for treating diarrhea, urinary tract infection, nephritis edema, icterohepatitis and other diseases, and is more widely used for treating tumor resistance, hepatitis B virus resistance, antibiosis and antiphlogosis, central nervous system diseases and the like.

In the prior literature, the quality of phyllanthus urinaria is mostly detected only by phyllanthus urinaria medicinal materials. For example, fan Shi et al employ reverse-phase high performance liquid chromatography to determine and compare fingerprints of Phyllanthus urinaria at different locations, different harvest times, and different locations (suitable, left-handed, rao Liqun. Comparative study of HPLC fingerprints of Phyllanthus urinaria [ J ]. North-northwest plant theory, 2016,36 (06): 1206-1215). Sun Min the content of gallic acid, protocatechuic aldehyde, corilagin, ellagic acid and quercetin was also determined by HPLC on the phyllanthus urinaria medicinal material, but the measurement time was 60 minutes, and the obtained chromatogram showed no significant peak separation of ellagic acid characteristic (Sun Min. HPLC method was used to simultaneously determine the content of gallic acid, protocatechuic aldehyde, corilagin, ellagic acid and quercetin in phyllanthus urinaria [ J ]. Qingdao medical health, 2012,44 (05): 325-328.). Summarizing the problems of long detection time, high detection cost, uneven chromatogram baseline and the like of the existing method for detecting the quality of the phyllanthus urinaria. And no related research on quality detection of phyllanthus urinaria formula particles, decoction pieces, and preparation products, preparation intermediates or preparation finished products thereof is available.

Disclosure of Invention

The invention aims to: the invention aims to provide a more comprehensive detection method for quality control of phyllanthus urinaria.

The technical scheme is as follows: the invention relates to a detection method for comprehensively controlling quality of phyllanthus urinaria, which comprises the following steps:

(1) Preparing a sample solution from a sample;

(2) Taking phyllanthus urinaria control medicinal material to prepare a control medicinal material solution; respectively preparing ellagic acid reference substance, gallic acid reference substance and corilagin reference substance to obtain reference substance solution;

(3) Detecting the sample solution, the reference substance solution and the reference medicinal material solution by using an ultra-high performance liquid chromatograph respectively to obtain corresponding maps;

(4) Analyzing and comparing the characteristic patterns of the reference substance solution, the reference medicinal material solution and the test substance solution, selecting 8 characteristic peaks corresponding to the retention time in the characteristic chromatogram of the reference medicinal material solution in the characteristic pattern of the test substance solution as common characteristic peaks, and constructing an ultra-high performance liquid characteristic pattern of phyllanthus urinaria; wherein, the characteristic peak 1 should correspond to the characteristic peak retention time of the gallic acid control solution, the characteristic peak 4 should correspond to the characteristic peak retention time of the corilagin control solution, and the characteristic peak 7 should correspond to the characteristic peak retention time of the ellagic acid control solution.

Preferably, the chromatographic conditions in the sample injection detection in the step (3) are as follows: octadecyl bonded silica gel column, mobile phase a: acetonitrile, mobile phase B:0.05% -0.2% phosphoric acid solution, and eluting according to the following elution gradient:

time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0～1	5	95
1～3	5→11	95→89
			3～7	11→13	89→87
7～12	13→23	87→77
			12～14	23→37	77→63
14～15	37→5	63→95
			15~20	5	95

The ultraviolet detector is adopted to detect the wavelength: 254nm; flow rate: 0.25-0.35 ml/min; column temperature: 35-45 ℃;

sample injection amounts of the sample solution, the reference medicinal material solution and the reference substance solution are 0.5-2 mu l.

Preferably, chromatographic conditions during sample injection detection are as follows: eclipse Plus C18 RRHD (2.1 mm. Times.100 mm,1.8 μm) column, mobile phase A: acetonitrile, mobile phase B:0.1% phosphoric acid solution, detection wavelength: 254nm; flow rate: 0.3 ml/min; column temperature: 40 ℃.

Preferably, in the ultra-high performance liquid phase characteristic spectrum of the phyllanthus urinaria in the step (4), the characteristic peak of the ellagic acid reference substance solution is taken as an S peak, the relative retention time of other characteristic peaks is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is: peak 2:0.58, peak 3:0.64, peak 5:0.78, peak 6:0.86, peak 8:1.29.

preferably, the sample solution and the reference medicinal material solution are prepared by using water, methanol or 10-75% methanol aqueous solution as the extraction solvent, wherein the volume of the extraction solvent is 15-50 ml, the extraction mode is heating reflux, and the extraction time is 30-90 minutes.

Preferably, the concentration of the reference substance solution is 30-60 mug/ml, and the extraction solvent is methanol aqueous solution with the volume concentration of 50%.

Preferably, step (3) further comprises: the content of ellagic acid is used as a content index component, and the content of ellagic acid in the sample solution is detected.

The content of ellagic acid is selected as the content index component because the content of ellagic acid is higher, compared with gallic acid and corilagin, the ellagic acid is relatively stable in the process of decoction, the transfer rate is in a normal range, and the gallic acid and corilagin are easy to be converted in the process of decoction, so that the transfer rate exceeds 100%, and the ellagic acid and corilagin are not suitable for being used as the content index of phyllanthus urinaria formula particles.

Preferably, the test sample is phyllanthus urinaria formula particles, medicinal materials, decoction pieces, and processed products, preparation intermediates or preparation finished products thereof.

Preferably, step (4) further comprises: and carrying out thin-layer detection on the test sample.

Preferably, the thin layer detection step includes:

adding organic solvent into phyllanthus urinaria formula particles, carrying out ultrasonic treatment, filtering, evaporating to dryness, and adding the organic solvent for dissolution to prepare a sample solution;

adding water into phyllanthus urinaria control medicinal material, heating and refluxing, filtering, evaporating to dryness, adding organic solvent, performing ultrasonic treatment, filtering, evaporating to dryness, and dissolving with organic solvent to obtain control medicinal material reference solution;

adding organic solvent into gallic acid reference substance to obtain gallic acid reference substance solution;

sucking the sample solution, the reference substance solution of the reference medicinal material and the gallic acid reference substance solution, respectively spotting on the same silica gel G thin layer plate, unfolding, taking out, airing, and developing by spraying the color developing agent.

Preferably, the organic solvent is methanol, the concentration of the reference substance solution is 1mg/ml, the developing agent is chloroform-ethyl acetate-formic acid=5:5:1, and the color-developing agent is 2% ferric trichloride ethanol solution.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:

(1) The method of the invention realizes the comprehensive detection of the quality standard of the phyllanthus urinaria for the first time.

(2) The invention adopts a set of chromatographic methods to realize qualitative and quantitative control of the phyllanthus urinaria. The characteristic spectrum of the phyllanthus urinaria and the content determination method of the active ingredients are established at the same time, and the method is more beneficial to the overall quality control of the phyllanthus urinaria.

(3) The invention establishes the characteristic spectrum by adopting an ultra-high performance liquid chromatograph (UPLC), totally determines 8 common characteristic peaks, has short analysis period, good common peak separation degree, stable base line, low detection cost and reliable recovery rate, can be used for quality detection and evaluation of phyllanthus urinaria formula particles, medicinal materials, decoction pieces and processed products thereof, preparation intermediates or preparation finished products, and has wider applicability.

(4) The invention establishes the organic combination of the characteristic spectrum, content measurement and thin layer identification method of the phyllanthus urinaria, wherein the characteristic spectrum and the thin layer identification belong to a qualitative method, the analysis result of the characteristic spectrum method has little artificial interference and high detection sensitivity, and is mainly suitable for analyzing medium-polarity and low-polarity components, and the thin layer identification method has simple operation and lower cost and is used for analyzing components with different polarities; the content determination belongs to a quantitative method, and ellagic acid is designated as a content index, so that quantitative analysis can be performed on main effective components; the combination of the two can carry out more comprehensive mass analysis on the phyllanthus urinaria.

Drawings

FIG. 1 is a UPLC profile of a phyllanthus urinaria formulation particle;

FIG. 2 is a UPLC profile of a phyllanthus intermediate;

FIG. 3 is a UPLC characteristic map of Phyllanthus urinaria;

FIG. 4 is a UPLC profile of phyllanthus urinaria decoction pieces;

FIG. 5 is a view of the characteristic spectrum of the phyllanthus urinaria formula particle at different detection wavelengths;

FIG. 6 is a graph of different gradient condition investigation of characteristic spectrum of phyllanthus urinaria formula particles;

FIG. 7 is a view of different extraction solvents for characteristic patterns of phyllanthus urinaria formula particles;

FIG. 8 is a view of different extraction modes of characteristic patterns of phyllanthus urinaria formula particles;

FIG. 9 is a view of different extraction times of characteristic spectrum of phyllanthus urinaria formula particles;

FIG. 10 is a graph of different extraction volumes of characteristic patterns of phyllanthus urinaria formula particles;

FIG. 11 is a characteristic spectrum-specific test of phyllanthus urinaria formula particles;

FIG. 12 is a phyllanthus urinaria formula particle characterization profile integrity test;

FIG. 13 is a view of different chromatographic columns of characteristic spectrum of phyllanthus urinaria formula particles;

FIG. 14 is a graph showing various column temperature studies of characteristic patterns of phyllanthus urinaria formula particles;

FIG. 15 is a graph of different flow rates for characteristic features of phyllanthus urinaria formula particles;

FIG. 16 is a full wave scan of ellagic acid;

FIG. 17 is a graph showing the linear relationship of ellagic acid controls;

FIG. 18 is a test specific for particle content detection of a phyllanthus urinaria formulation;

FIG. 19 is a TLC chart of thin layer detection of different spotting amounts of phyllanthus recipe particles, wherein 1: 1 μl of sample solution; 2: 2 μl of sample solution; 3: 3 μl of sample solution; 4: 4 μl of sample solution; s1: 1 μl of control medicinal material solution; s2: 2 μl of control medicinal material solution; s3: 3 μl of control medicinal material solution; s4: 4 μl of control medicinal material solution; t: gallic acid reference solution;

FIG. 20 is a TLC chart of a thin layer assay specific for phyllanthus urinaria formulation, wherein 1-3: a test solution; 4: a negative control solution; s: controlling the medicinal material solution; t: gallic acid reference solution;

FIG. 21 is a TLC chart of phyllanthus urinaria formula particles under different temperature conditions, wherein 1-3: a test solution; 4: a negative control solution; s: controlling the medicinal material solution; t: gallic acid reference solution;

FIG. 22 is a TLC chart of phyllanthus urinaria formula particles under different humidity conditions, wherein 1-3: a test solution; 4: a negative control solution; s: controlling the medicinal material solution; t: gallic acid reference solution;

FIG. 23 is a TLC chart of thin-layer plate phyllanthus formula particles of different manufacturers, wherein 1-3: a test solution; s: controlling the medicinal material solution; t: gallic acid reference solution; thin layer plate manufacturer: qingdao ocean chemical works (left), shanghai Ala Biochemical technology Co., ltd. (middle), qingdao Kang Yexin pharmaceutical silica gel desiccant Co., ltd. (right);

FIG. 24 is a thin layer identification chromatogram of different batches of phyllanthus urinaria formula particle samples, 1-3: a test solution; s: controlling the medicinal material solution; t: gallic acid reference solution.

Description of the embodiments

The technical scheme of the invention is further described below with reference to the accompanying drawings.

Example 1 establishment of phyllanthus urinaria feature map

1. Preparation of test solutions

Taking proper amount of phyllanthus urinaria formula particles, grinding, taking about 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 50ml of 50% methanol, sealing, weighing, heating and refluxing for 60 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.

2. Preparation of control solution

Taking 0.2g of phyllanthus urinaria control medicine, placing into a conical flask with a plug, adding 25ml of 30% methanol, heating and refluxing for 30 minutes, cooling, shaking uniformly, filtering, and taking the subsequent filtrate as a control medicine solution.

Respectively taking ellagic acid reference substance, gallic acid reference substance, and corilagin reference substance, precisely weighing, and adding 50% methanol to obtain solutions containing 50 μg reference substance per 1ml, and taking as reference substance solution.

3. Sample injection detection

Map conditions: eclipse Plus C18 RRHD (2.1 mm. Times.100 mm,1.8 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and performing gradient elution according to the specifications in Table 1; the flow rate is 0.3ml per minute; column temperature is 40 ℃; the detection wavelength was 254nm.

TABLE 1 gradient elution conditions

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0～1	5	95
1～3	5→11	95→89
			3～7	11→13	89→87
7～12	13→23	87→77
			12～14	23→37	77→63
14～15	37→5	63→95
			15~20	5	95

Precisely sucking each reference substance solution, each reference medicinal material solution and each sample solution 1 μl, and injecting into the ultra-high performance liquid chromatograph.

4. Atlas analysis

Obtaining a sample chromatogram with 8 characteristic peaks and a phyllanthus urinaria control medicinal material chromatogram, wherein 8 characteristic peaks in the two chromatograms correspond to each other, characteristic peak 1 corresponds to the characteristic peak retention time of the gallic acid control solution, characteristic peak 4 corresponds to the characteristic peak retention time of the corilagin control solution, and characteristic peak 7 corresponds to the characteristic peak retention time of the ellagic acid control solution. Calculating the relative retention time of other peaks by taking the characteristic peak 7 (S) as a reference peak, wherein the relative retention time is within +/-10% of a specified value, and the specified value is: peak 2:0.58, peak 3:0.64, peak 5:0.78, peak 6:0.86, peak 8:1.29. the characteristic spectrum of the phyllanthus urinaria formula granule is shown in figure 1.

In addition, the characteristic spectrum of the intermediate of the phyllanthus urinaria (shown in figure 2), the characteristic spectrum of the medicinal material of the phyllanthus urinaria (shown in figure 3), the characteristic spectrum of decoction pieces of the phyllanthus urinaria (shown in figure 4) are also measured by the method, and the retention time of characteristic peaks in the obtained characteristic spectrum is within the specified value range of the method, so that the method is proved to be also suitable for establishing the characteristic spectrum of the medicinal material of the phyllanthus urinaria, the decoction pieces and the processed products thereof, the preparation intermediate or the preparation finished product.

EXAMPLE 2 methodological investigation of the characteristic Spectrum of the phyllanthus urinaria formula particles

1. Determination of detection wavelength

The same batch of samples was taken to prepare a sample solution of the phyllanthus urinaria formulation particles by ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, and chromatograms at wavelengths of 220nm,254nm and 300nm were collected in the same manner as in example 1. The results are shown in FIG. 5.

The results show that the number of peaks is large at 254nm wavelength, the response is large, and the base line is stable, so 254nm is selected as the detection wavelength.

2. Determination of gradient elution conditions

The same batch of samples was taken to prepare a sample solution of the phyllanthus urinaria formulation particles, and the other methods were the same as in example 1, and gradient elution was performed according to the conditions shown in tables 2, 3 and 4, respectively. The results are shown in FIG. 6.

TABLE 2 method-gradient elution conditions

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0～8	5→13	95→87
8～12	13	87
			12～18	13→23	87→77
18～21	23→37	77→63
			21～22	37→5	63→95
22～25	5	95

TABLE 3 method two gradient elution conditions

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0～13	5→13	95→87
13～20	13→17	87→83
			20～21	17→24	83→76
21～24	24→27	76→73
			24～31	27→41	73→59
31～31.1	41→5	59→95
			31.1～33	5	95

TABLE 4 method three gradient elution conditions

Time (minutes)	Mobile phase a (%)	Mobile phase B (%)
			0～1	5	95
1～3	5→11	95→89
			3～7	11→13	89→87
7～12	13→23	87→77
			12～14	23→37	77→63
14～15	37→5	63→95
			15~20	5	95

The results show that the separation effect of the gradient elution according to the table 3 is good, the base line is stable, the peak shape is symmetrical, and the analysis time is moderate, so that the method selects the elution conditions specified in the table 3 for subsequent study.

3. Preparation of test solutions

3.1 investigation of different extraction solvents

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, 6 groups of parallel samples were prepared, water, 10% methanol, 30% methanol, 50% methanol, 75% methanol and 50ml of methanol were added to the extraction solvents, respectively, and the characteristic peak area/sample weight was calculated in the same manner as in example 1, and the results are shown in Table 5 and FIG. 7.

TABLE 5 comparison of extraction efficiency for different extraction solvents (peak area/sample size)

Extraction solvent	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5	Peak 6	Peak 7	Peak 8	Total peak area/sample size
										Water and its preparation method	27.123	9.915	4.241	66.973	4.074	1.680	97.409	3.090	214.51
10% methanol	26.573	10.086	4.259	68.776	4.034	1.655	96.830	3.130	215.34
										30% methanol	26.206	10.175	4.822	70.474	3.933	1.592	116.286	3.133	236.62
50% methanol	26.236	10.361	4.998	72.392	3.921	1.586	118.605	3.202	241.30
										75% methanol	26.109	10.374	4.777	72.308	3.933	1.575	121.551	3.240	243.87
Methanol	27.375	9.227	2.980	46.063	6.118	0.820	78.887	3.230	174.70

The results showed that, except for the lower extraction efficiency of methanol, the extraction results of the other different concentrations of methanol as the extraction solvent were closer, and the extraction solvent was determined to be 50% methanol in consideration of the solvent effect of a part of chromatographic peaks under the high concentration methanol extraction conditions.

3.2 investigation of different extraction methods

The same batch of samples are taken to prepare test sample solutions of phyllanthus urinaria formula particles, 3 groups of sample solutions are parallel, ultrasonic treatment (power is 250W, frequency is 40 kHz), shaking extraction and heating reflux are respectively adopted for the extraction method, and other methods are the same as in example 1, and characteristic peak-to-peak area/sample weighing amount is calculated, so that the results are shown in Table 6 and FIG. 8.

TABLE 6 comparison of extraction efficiency for different extraction modes (Peak area/sample size)

Extraction mode	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5	Peak 6	Peak 7	Peak 8	Total peak area/sample size
										Ultrasonic wave	26.204	10.527	5.040	71.770	13.454	1.700	82.299	3.175	214.17
Shaking machine	26.175	10.375	5.083	69.858	12.766	1.625	66.693	3.075	195.65
										Reflow process	26.196	10.369	4.649	71.736	13.401	1.668	113.878	3.174	245.07

The result shows that the extraction efficiency of the heating reflux is higher and the extraction mode is consistent with the content measurement extraction mode, so that the extraction method is determined to be the heating reflux.

3.3 investigation of different extraction times

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, and the extraction time was 30 minutes, 45 minutes, 60 minutes and 90 minutes respectively in parallel for 4 groups, and the characteristic peak area/sample weighing/50 extraction volume was calculated in the same manner as in example 1, and the results are shown in table 7 and fig. 9.

TABLE 7 comparison of extraction efficiency at different extraction times (peak area/sample size)

Extraction time	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5	Peak 6	Peak 7	Peak 8	Total peak area/sample size
										30min	26.339	10.529	5.027	72.410	13.697	1.643	112.603	3.206	245.46
45min	26.154	10.394	5.010	72.129	13.591	1.623	119.231	3.195	251.33
										60min	26.737	10.582	4.807	73.283	13.647	1.691	118.045	3.235	252.03
90min	26.164	10.393	4.850	72.021	13.554	1.751	120.976	3.185	252.89

The results show that the difference of the characteristic peak-to-peak area/the weighed sample in the phyllanthus urinaria formula particles is smaller in different times of heating reflux, and the extraction time is determined to be 60 minutes in order to unify the preparation mode of the sample in consideration of the requirement of heating reflux extraction for 60 minutes of ellagic acid content.

3.4 investigation of different extraction volumes

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, 3 groups of sample solutions were prepared in parallel, the volumes of the extracts were respectively 15ml, 25ml and 50ml, and the characteristic peak area/sample weight was calculated in the same manner as in example 1, and the results are shown in Table 8 and FIG. 10.

Table 8 comparison of extraction efficiency for different extraction volumes (peak area/sample size extraction volume/50)

Extraction volume	Peak 1	Peak 2	Peak 3	Peak 4	Peak 5	Peak 6	Peak 7	Peak 8	Total peak area/sample size/50 extraction volume
										15ml	25.940	10.413	4.608	71.685	13.670	1.634	95.585	3.143	226.68
25ml	25.967	10.419	4.262	71.920	13.707	1.637	111.441	3.169	242.52
										50ml	26.317	10.443	4.443	72.137	13.531	1.662	116.377	3.191	248.10

The results show that the extraction efficiency of 50ml and 25ml of extraction solvents are not greatly different, and the extraction volume is determined to be 50ml in consideration of the higher extraction efficiency of 50ml of content measurement, so that the preparation mode of a sample is unified.

4. Investigation of specificity

The same batch of samples were taken to prepare a test sample solution of phyllanthus urinaria formula particles, a negative solution of phyllanthus urinaria, a control medicinal material solution and a control solution, and the results are shown in fig. 11 by injecting the sample solution into an ultra-high performance liquid chromatograph under the chromatographic conditions of example 1.

The result shows that the negative solution has no chromatographic peak at the retention time of each characteristic peak in the chromatogram of the solution of the test sample, which indicates that the solvent and the auxiliary materials have no interference to the measurement of the phyllanthus urinaria formula particles, and the phyllanthus urinaria formula particles measured by the method have specificity.

5. Integrity inspection

The same batch of samples was used to prepare a sample solution of the phyllanthus urinaria formulation particles, and the elution time was extended under the chromatographic conditions of example 1 to examine whether the residual impurity peaks would affect the subsequent samples under the predetermined chromatographic conditions, and the results are shown in fig. 12.

The result shows that the chromatographic condition basically meets the principle of maximum information amount and has no influence on the analysis of subsequent samples.

6. Precision investigation

The same batch of samples was taken to prepare a sample solution of the phyllanthus urinaria formulation particles, and the sample solution was continuously sampled 6 times, and the characteristic peak retention time was recorded in the same manner as in example 1, and the relative retention time was calculated according to the method of example 1, and the results are shown in Table 9.

TABLE 9 precision experimental results (relative retention time)

Sequence number	Peak 1RRT	Peak 2RRT	Peak 3RRT	Peak 4RRT	Peak 5RRT	Peak 6RRT	Peak 7RRT	Peak 8RRT
									1	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
2	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									3	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
4	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									5	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
6	0.14	0.57	0.63	0.67	0.78	0.86	1.00	1.29
									Mean value of	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
RSD(%)	0.10	0.02	0.02	0.05	0.05	0.06	0.00	0.08
									Specified value	--	0.58	0.64	--	0.78	0.86	--	1.29
Specified range	--	0.52~0.64	0.58~0.70	--	0.70~0.86	0.77~0.95	--	1.16~1.42

The results show that the relative retention time of each characteristic peak has RSD less than 1% and the precision is good.

7. Intermediate precision investigation

The same batch of sample was taken to prepare test solutions of phyllanthus urinaria formulation particles, each 3 parts by each of the two laboratory workers A, B, operating on Agilent, thermo apparatus as in example 1, and recording the characteristic peak retention times, the relative retention times being calculated as in example 1, the results being shown in Table 10.

Table 10 intermediate precision investigation (relative retention time)

The results show that the RSD of the relative retention time of the characteristic peaks of the sample except for the peak 1 is less than 2%, and the intermediate precision test is good.

8. Stability investigation

The same batch of sample was used to prepare test solutions of phyllanthus urinaria formula particles, which were sampled at 0, 2,4, 8, 12 and 24 hours, and the characteristic peak retention times were recorded in the same manner as in example 1, and the relative retention times were calculated according to the method of example 1, and the results are shown in table 11.

TABLE 11 stability investigation (relative retention time)

Sample injection time (hours)	Peak 1RRT	Peak 2RRT	Peak 3RRT	Peak 4RRT	Peak 5RRT	Peak 6RRT	Peak 7RRT	Peak 8RRT
									0	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
2	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									4	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
8	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.28
									12	0.14	0.58	0.63	0.68	0.78	0.86	1.00	1.28
24	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									Mean value of	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
RSD(%)	0.24	0.08	0.11	0.16	0.15	0.14	0.00	0.14
									Specified value	--	0.58	0.64	--	0.78	0.86	--	1.29
Specified range	--	0.52~0.64	0.58~0.7	--	0.7~0.86	0.77~0.95	--	1.16~1.42

The results show that the relative retention time of each characteristic peak is less than 1% and the stability of the test solution is good within 24 hours.

9. Repeatability investigation

The same batch of samples was used to prepare test solutions of phyllanthus urinaria formulation particles, in parallel 6 groups, and the characteristic peak retention times were recorded in the same manner as in example 1, and the relative retention times were calculated according to the method of example 1, and the results are shown in Table 12.

TABLE 12 repeatability test (relative retention time)

Sequence number	Peak 1RRT	Peak 2RRT	Peak 3RRT	Peak 4RRT	Peak 5RRT	Peak 6RRT	Peak 7RRT	Peak 8RRT
									1	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
2	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									3	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
4	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									5	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
6	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									Mean value of	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
RSD(%)	0.07	0.05	0.05	0.05	0.05	0.05	0.00	0.04
									Specified value	--	0.58	0.64	--	0.78	0.86	--	1.29
Specified range	--	0.52~0.64	0.58~0.7	--	0.7~0.86	0.77~0.95	--	1.16~1.42

The results show that the reproducibility of the method is good.

10. Durability inspection

10.1 chromatography column inspection

The same batch of samples was used to prepare sample solutions of the phyllanthus urinaria formulation particles, using Eclipse Plus C18 RRHD (2.1 mm. Times.100 mm,1.8 μm), acclaim RSLC 120C 18 (2.1. Times.100 mm,2.2 μm), ACQUITY UPLC BEH C18 (2.1. Times.100 mm,1.7 μm) 3 columns, and the results are shown in FIG. 13.

As a result, the effect of different columns on peak 6 was large and the degree of separation was poor, so that the characteristic spectrum detection of the phyllanthus urinaria formula particles recommended the use of column Eclipse Plus C18 RRHD (2.1 mm. Times.100 mm,1.8 μm).

10.2 column temperature investigation

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, three column temperatures of 35 ℃ and 40 ℃ and 45 ℃ were used respectively, the other methods were the same as in example 1, the characteristic peak retention time was recorded, the relative retention time was calculated according to the method of example 1, and the results are shown in table 13 and fig. 14.

Table 13 column temperature investigation (relative retention time)

Column Wen	Peak 1RRT	Peak 2RRT	Peak 3RRT	Peak 4RRT	Peak 5RRT	Peak 6RRT	Peak 7RRT	Peak 8RRT
									35	0.14	0.58	0.65	0.69	0.80	0.87	1.00	1.26
40	0.14	0.57	0.63	0.67	0.77	0.86	1.00	1.29
									45	0.14	0.57	0.62	0.66	0.76	0.85	1.00	1.32
Mean value of	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									RSD(%)	2.48	1.46	2.02	1.84	2.28	1.33	0.00	2.52
Specified value	--	0.58	0.64	--	0.78	0.86	--	1.29
									Specified range	--	0.52~0.64	0.58~0.7	--	0.7~0.86	0.77~0.95	--	1.16~1.42

The results show that the column temperature is 35-45 ℃ and the measurement results of the sample meet the retention time requirement specified by the text, so that the durability is good.

10.3 flow Rate investigation

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, three flow rates of 0.25ml, 0.30ml and 0.35ml each minute were respectively adopted, the characteristic peak retention time was recorded in the same manner as in example 1, the relative retention time was calculated according to the method of example 1, and the results are shown in table 14 and fig. 15.

TABLE 14 flow rate investigation (relative retention time)

Flow rate ml/min	Peak 1RRT	Peak 2RRT	Peak 3RRT	Peak 4RRT	Peak 5RRT	Peak 6RRT	Peak 7RRT	Peak 8RRT
									0.25	0.15	0.60	0.66	0.70	0.81	0.87	1.00	1.25
0.30	0.14	0.57	0.63	0.67	0.77	0.86	1.00	1.29
									0.35	0.13	0.55	0.61	0.65	0.75	0.85	1.00	1.33
Mean value of	0.14	0.58	0.63	0.67	0.78	0.86	1.00	1.29
									RSD%	8.50	4.09	4.16	3.52	3.94	1.20	0.00	3.16
Specified value	--	0.58	0.64	--	0.78	0.86	--	1.29
									Specified range	--	0.52~0.64	0.58~0.7	--	0.7~0.86	0.77~0.95	--	1.16~1.42

The result shows that the flow rate is in the interval of 0.25 ml/min-0.35 ml/min, the measurement results of the sample are relatively stable, and all the flow rate meets the retention time requirement specified by the method, so that the durability is good.

11. Sample measurement

Test solutions were prepared by the method of example 1 from 3 batches of phyllanthus urinaria formula granule samples, 20 batches of phyllanthus urinaria medicinal materials and 20 batches of decoction pieces, and the test method was the same as that of example 1, and the results are shown in tables 15, 16 and 17.

TABLE 15 sample measurement results (relative retention time)

Sequence number	Peak 1RRT	Peak 2RRT	Peak 3RRT	Peak 4RRT	Peak 5RRT	Peak 6RRT	Peak 7RRT	Peak 8RRT
									1	0.11	0.55	0.60	0.65	0.74	0.85	1.00	1.33
2	0.11	0.55	0.60	0.65	0.74	0.85	1.00	1.33
									3	0.11	0.55	0.60	0.65	0.74	0.85	1.00	1.33
Average value of	0.11	0.55	0.60	0.65	0.74	0.85	1.00	1.33
									RSD(%)	0.05	0.11	0.10	0.03	0.05	0.05	0.00	0.04
Specified value	--	0.58	0.64	--	0.78	0.86	--	1.29
									Specified range	--	0.52~0.64	0.58~0.7	--	0.7~0.86	0.77~0.95	--	1.16~1.42

TABLE 16 determination of 20 samples of phyllanthus urinaria (relative retention time)

TABLE 17 determination of 20 samples of phyllanthus urinaria decoction pieces (relative retention time)

Example 3 determination of ellagic acid content in Phyllanthus Emblica formula particles

1. Inspection of control origin and purity

Ellagic acid is purchased from Chinese food and drug inspection institute under the number 111959-201903 for content measurement, and the content is 88.8%, and the ellagic acid is not treated before use.

Other methods and test conditions were the same as those of example 1, the method for establishing characteristic patterns of phyllanthus urinaria

2. Mass analysis

And calculating the content of chicoric acid, and carrying out quality analysis on the sample.

EXAMPLE 4 methodology review of ellagic acid content determination in phyllanthus urinaria formula particles

1. Determination of detection wavelength

The ellagic acid control solution was scanned for full wavelength and its ultraviolet absorbance was recorded, and the results are shown in fig. 16.

The results show that ellagic acid has larger absorption at a wavelength of about 254nm, and is comprehensively considered, so that 254nm is selected as the detection wavelength for determining the ellagic acid content.

2. Preparation of test solutions

2.1 investigation of different extraction solvents

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, 6 groups of samples were prepared in parallel, 2 parts of each group were prepared in parallel, 50ml of each of water, 10% methanol, 30% methanol, 50% methanol, 75% methanol and methanol was added to the extraction solvent, and the content of ellagic acid was calculated in the same manner as in example 1, and the results are shown in Table 18.

TABLE 18 comparison of different extraction solvents

The results showed that the extraction efficiencies of 30% methanol, 50% methanol and 75% methanol were relatively close, and the extraction efficiencies of water, 10% methanol and methanol were lower, and the extraction solvent was determined to be 50% methanol in consideration of the solvent effect of a part of the chromatographic peaks under the condition of high concentration methanol.

2.2 investigation of different extraction methods

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, 3 groups of samples were prepared in parallel, 2 parts of each group were prepared in parallel, the extraction method was respectively carried out by ultrasonic treatment (power 250W, frequency 40 kHz), shaking extraction and heating reflux, and the other methods were the same as in example 1, and the ellagic acid content was calculated, and the results are shown in Table 19.

TABLE 19 comparison of different extraction methods

The result shows that the extraction efficiency of the heating reflux is obviously higher than that of the other two extraction modes, so that the extraction method is determined to be the heating reflux.

2.3 investigation of different extraction times

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, 3 groups of samples were prepared in parallel, 2 parts of each group were prepared in parallel, the extraction time was 30 minutes, 45 minutes and 60 minutes respectively, and the ellagic acid content was calculated by the other methods as in example 1, and the results are shown in Table 20.

Table 20 comparison of different extraction times

The results showed that the ellagic acid content had tended to stabilize after 45 minutes of extraction, and the extraction time was determined to be 60 minutes from the viewpoint of extraction completion.

2.4 investigation of different extraction volumes

The same batch of samples were taken to prepare test solutions of phyllanthus urinaria formula particles, 3 groups of samples were arranged in parallel, 2 parts of each group were arranged in parallel, 15ml, 25ml and 50ml of the extract were added respectively, the content of ellagic acid was calculated by the other methods as in example 1, and the results are shown in Table 21.

TABLE 21 comparison of different extraction volumes

The result shows that the extraction efficiency of 50ml of the extraction solvent is high, and the extraction volume is determined to be 50ml by comprehensively considering that the area of partial characteristic peak-to-peak is too small in consideration of the continuous increase of the solvent.

3. Linearity of

Ellagic acid control solutions (concentration 178.67. Mu.g/ml) 0.1. Mu.l, 0.2. Mu.l, 0.5. Mu.l, 1.0. Mu.l, 2.0. Mu.l, 3.0. Mu.l were respectively precisely aspirated and injected into an ultra performance liquid chromatograph, and the other methods were the same as in example 1, and the ellagic acid regression equation was obtained by drawing a standard curve with the peak area integrated value as the ordinate and the sample injection amount (mug) as the abscissa, and the results are shown in Table 22, table 23, and FIG. 17.

Table 22 regression equation

Component (A)	Regression equation
		Ellagic acid	y = 30253x + 27.313 R² = 0.9998

TABLE 23 sample injection amount and peak area relationship of ellagic acid control

The result shows that the sample injection amount of ellagic acid is within the range of 0.01786656-0.53599680 mug, and the sample injection amount and the peak area value are in good linear relation.

4. Precision test

4.1 precision test of instruments

The same batch of samples was taken to prepare a sample solution of the phyllanthus urinaria formulation particles, and the sample solution was continuously sampled 6 times, and the peak area was recorded and the relative standard deviation was calculated by the other methods as in example 1, and the results are shown in Table 23.

Meter 23 Instrument precision test

Sequence number	Ellagic acid peak area
		1	1320.2952
2	1321.6700
		3	1315.3659
4	1313.9524
		5	1317.4967
6	1309.5050
		Mean value of	1316.3809
RSD%	0.34%

The result shows that the precision test of the instrument is good (RSD%. Ltoreq.2.0%).

4.2 repeatability test

The same batch of samples was taken to prepare 6 parallel solutions of the test sample of the phyllanthus urinaria formulation particles, the peak area was measured in the same manner as in example 1, and the content and RSD were calculated, and the results are shown in Table 24.

Table 24 results of repeatability test of samples

The results show that the repeatability test is good (RSD%. Ltoreq.2.0%).

4.3 intermediate precision test

The same batch of samples was taken to prepare a sample solution of the phyllanthus urinaria formula particles, 3 parts each was processed by two laboratory workers A, B, 1 μl was sampled on Thermo-UPLC and Agilent-UPLC, respectively, and the peak area value was measured and the content and RSD were calculated in the same manner as in example 1, and the results are shown in Table 25.

Intermediate precision test of Table 25

The results show that the intermediate precision is good.

5. Accuracy test

Three samples (11.3 mg/g ellagic acid content) of known content were taken and ground, about 0.1g was taken and 3 parts were taken in parallel, precisely weighed, and 0.1g of a control of 50%, 100% and 150% of each component contained in the samples was added respectively. Sample solutions were prepared as in example 1, 1 μl of each sample was introduced, and the recovery, average recovery, RSD, were calculated according to the following formula, and the results are shown in table 26.

TABLE 26 ellagic acid accuracy results

The result shows that the recovery rate of ellagic acid is 94.32% -99.38%, and the accuracy test result is good.

6. Specificity test

The auxiliary materials added in the phyllanthus urinaria formula particles are lactose, silicon dioxide and magnesium stearate. The influence of a negative sample of the phyllanthus urinaria on the measurement of the particle content of the phyllanthus urinaria formula is examined in the experiment.

Taking a negative sample of the phyllanthus urinaria, and preparing a negative sample solution according to a sample preparation method.

The sample solution, negative control solution and ellagic acid control solution of the phyllanthus urinaria formula particles were tested according to the chromatographic conditions of example 1, and the results are shown in figure 18.

The result shows that the negative chromatogram has no chromatographic peak at the retention time corresponding to the control, which indicates that the auxiliary materials and the solvent have no interference to the determination of the ellagic acid, and the determination of the ellagic acid content in the phyllanthus urinaria formula particles by the method has specificity.

7. Integrity test

The elution gradient was maintained at the highest acetonitrile ratio under the chromatographic conditions of example 1, the elution time was doubled, and whether or not the residual impurity peak had an influence on the subsequent sample under the predetermined chromatographic conditions was examined, and the results are shown in fig. 12.

The result shows that the elution time is doubled, no impurity peak exists, the chromatographic condition basically meets the principle of maximum information quantity, and the analysis of the subsequent sample is not influenced.

8. Durability test

8.1 stability test

The same batch of samples was taken to prepare sample solutions of phyllanthus urinaria formula particles, and samples were taken at 0, 2,4, 8, 12 and 24 hours, and the peak area value was measured in the same manner as in example 1, and the RSD was calculated, and the results are shown in table 27.

Table 27 stability test results

Sample injection time (hours)	Ellagic acid peak area
		0	1320.2952
2	1303.2777
		4	1301.0154
8	1297.5402
		12	1293.2272
24	1278.7814
		Mean value of	1299.023
RSD%	1.04%

The results show that the sample test solution has good stability within 24 hours.

8.2 investigation of different flow Rate

The same batch of samples was used to prepare test solutions of phyllanthus urinaria formula particles, three flow rates of 0.25ml, 0.30ml and 0.35ml per minute were used, respectively, and the ellagic acid content was calculated in the same manner as in example 1, and the results are shown in Table 28.

Table 28 investigation of different flow rates

The results show that the separation degree of ellagic acid chromatographic peaks is good, the content is similar and the durability is good under the three flow rates.

8.3 investigation of different column temperatures

The same batch of samples was used to prepare test solutions of phyllanthus urinaria formula particles, three column temperatures of 35℃and 40℃and 45℃were used, respectively, and the ellagic acid content was calculated in the same manner as in example 1, and the results are shown in Table 29.

Investigation of Table 29 different column temperatures

The results show that the separation degree of ellagic acid chromatographic peaks is good, the content is similar and the durability is good at the three column temperatures.

8.4 column chromatography investigation

The sample solution of the phyllanthus recipe particles was prepared from the same batch of samples, using Eclipse Plus C18 RRHD (2.1 mm. Times.100 mm,1.8 μm) and Acclaim RSLC 120C 18 (2.1. Times.100 mm,2.2 μm), ACQUITY UPLC BEH C18 (2.1. Times.100 mm,1.7 μm) 3 different columns, and the ellagic acid content was calculated in the same manner as in example 1, and the results are shown in Table 30.

Table 30 comparison of different chromatographic columns

The results show that the chromatographic columns of three different types have good separation effect on ellagic acid, and have small influence on the content results, and the method has universal adaptability.

9. Sample measurement

3 batches of phyllanthus urinaria formula particles, 20 batches of phyllanthus urinaria medicinal materials and 20 batches of decoction pieces are taken, a test solution is prepared according to the method of example 1, the test method is the same as that of example 1, the ellagic acid content in the samples is measured, and the experimental results are shown in tables 31, 32 and 33.

Table 31 determination of ellagic acid content in phyllanthus urinaria formulation particles

Batch of	Ellagic acid content (mg/g)
		1	11.2
2	10.8
		3	11.0

Determination of ellagic acid content in Table 32 Phyllanthus urinaria medicinal material

Sequence number	Ellagic acid content (%)
		1	0.69
2	0.68
		3	0.66
4	0.65
		5	0.69
6	0.58
		7	0.37
8	0.39
		9	0.35
10	0.36
		11	0.27
12	0.24
		13	0.42
14	0.46
		15	0.45
16	0.45
		17	0.43
18	0.47
		19	0.44
20	0.35

TABLE 33 determination of ellagic acid content in decoction pieces of Phyllanthus urinaria

Sequence number	Content (%)
		1	0.69
2	0.68
		3	0.66
4	0.65
		5	0.69
6	0.58
		7	0.37
8	0.39
		9	0.35
10	0.36
		11	0.27
12	0.24
		13	0.42
14	0.46
		15	0.45
16	0.45
		17	0.43
18	0.47
		19	0.44
20	0.35

Example 5 establishment of a thin layer identification method for phyllanthus urinaria formulation particles

1. Preparation of test solutions

Taking 0.2g of phyllanthus urinaria prescription granule, grinding, adding 20ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a sample solution.

2. Preparation of control solution

Taking 1g of phyllanthus urinaria control medicinal material, adding 50ml of water, heating and refluxing for 30 minutes, shaking uniformly, filtering, evaporating the filtrate to dryness, adding 20ml of methanol into the residue, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1ml of methanol into the residue to dissolve to obtain a control medicinal material solution.

Taking gallic acid reference substance, adding methanol to prepare 1mg solution per 1ml, and taking the solution as gallic acid reference substance solution.

3. Thin layer detection

Absorbing each 2 μl of the three solutions, respectively spotting on the same silica gel G thin layer plate, developing with chloroform-ethyl acetate-formic acid (5:5:1) as developing agent, taking out, air drying, and spraying with 2% ferric trichloride ethanol solution for color development.

EXAMPLE 6 methodological investigation of thin layer identification methods for phyllanthus urinaria formulation particles

1. Preparation of test solutions

Taking 0.2g of phyllanthus urinaria prescription granule, grinding, adding 20ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and adding 1ml of methanol into residues to dissolve the residues to obtain a sample solution.

2. Preparation of control solution

Taking 1g of phyllanthus urinaria control medicinal material, adding 50ml of water, heating and refluxing for 30 minutes, shaking uniformly, filtering, evaporating the filtrate to dryness, adding 20ml of methanol into the residue, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1ml of methanol into the residue to dissolve to obtain a control medicinal material solution.

Taking gallic acid reference substance, adding methanol to prepare 1mg solution per 1ml, and taking the solution as gallic acid reference substance solution.

Taking 0.2g of the negative sample of the phyllanthus urinaria, and preparing the negative sample with the preparation method of the test sample solution to prepare a negative control solution.

3. Investigation of different spotting volumes

Sucking the sample solution and the reference medicinal material solution, respectively spotting 1 mu l, 2 mu l, 3 mu l and 4 mu l on the same silica gel G thin layer plate, spreading with chloroform-ethyl acetate-formic acid (5:5:1) as a spreading agent, taking out, airing, spraying 2% ferric trichloride ethanol solution, and inspecting under sunlight. The results are shown in FIG. 19.

The results show that when the sample application amount of the sample solution and the control medicinal material solution is 2 mu l, the spots of the sample chromatogram and the control medicinal material chromatogram at the corresponding positions are clearly corresponding, and no other interference exists, so that the sample application amount of the sample solution and the control medicinal material solution is 2 mu l.

4. Specificity experiments

Sucking 3 different batches of sample solution, control medicinal material solution, negative control solution and gallic acid control solution prepared from phyllanthus urinaria formula particles, spotting on the same silica gel G thin layer plate, spreading with chloroform-ethyl acetate-formic acid (5:5:1) as developing agent, taking out, air drying, spraying 2% ferric chloride ethanol solution, and inspecting in sunlight. The results are shown in FIG. 20.

The result shows that the color spectrum of the phyllanthus urinaria formula particle to be tested shows spots with the same color on the corresponding positions as the color spectrum of the reference medicinal material and the color spectrum of the gallic acid reference substance, and the negative control is not interfered. The thin layer identification method has good specificity.

5. Durability test

5.1 investigation of different temperatures

The test solution, the control medicinal material solution and the gallic acid control solution prepared by sucking 3 different batches of phyllanthus urinaria formula particles are respectively detected under different temperature conditions according to the thin layer detection conditions of the example 5, and the result is shown in figure 21.

The results show that under different temperature conditions, the color spectrum of the phyllanthus urinaria formula particle to be tested and the color spectrum of the control medicinal material and the color spectrum of the gallic acid control product show spots with the same color at the corresponding positions, and the separation effect is good. The temperature has no influence on the thin layer identification of the phyllanthus urinaria formula particles, so that the thin layer identification method has good durability on different temperatures.

5.2 investigation of different humidity

The test solution, the control medicinal material solution and the gallic acid control solution prepared by sucking 3 different batches of phyllanthus urinaria formula particles are respectively detected under different humidity conditions according to the thin layer detection conditions of the example 5, and the result is shown in fig. 22.

The results show that under the condition of different humidity, the color spectrum of the phyllanthus urinaria formula particle to be tested and the color spectrum of the control medicine and the color spectrum of the gallic acid control product show spots with the same color at the corresponding positions, and the basic separation is achieved. The humidity has no influence on the thin-layer identification of the phyllanthus urinaria formula particles, so that the thin-layer identification method has good durability on different humidity.

5.3 inspection of different brands of lamina plates

The test solution, the control medicinal material solution and the gallic acid control solution prepared by sucking 3 different batches of phyllanthus urinaria formula particles are respectively detected on silica gel G thin-layer plates of different brands according to the thin-layer detection conditions of the example 5, and the result is shown in figure 23.

The results show that in different brands of silica gel G plates, the color spectrum of the phyllanthus urinaria formula particles shows spots with the same color on the corresponding positions of the color spectrum of the control medicine and the color spectrum of the gallic acid control substance, and only the main spot Rf value is different. Experimental results show that different brands of silica gel G plates have no influence on the thin-layer identification result of phyllanthus urinaria formula particles, and the Qingdao ocean chemical silica gel G thin-layer plates are adopted in methodology research in consideration of the actual thin-layer plate brand use condition in a laboratory.

6. Sample detection

Taking 3 different batches of phyllanthus urinaria formula particles, and preparing the sample solution according to the preparation method of the sample solution. The sample solution, the control drug solution and the gallic acid control solution were aspirated and spotted on the same silica gel G thin layer plate, respectively, and the results were examined according to the thin layer examination conditions of example 5, as shown in fig. 24.

The result shows that the fluorescence spots with the same color appear on the positions corresponding to the control medicine chromatogram and the gallic acid control product chromatogram in the sample chromatogram.

Claims (10)

1. The detection method for comprehensively controlling the quality of the phyllanthus urinaria is characterized by comprising the following steps of:

(1) Preparing a sample solution;

(2) Taking phyllanthus urinaria control medicinal material to prepare a control medicinal material solution; respectively preparing ellagic acid reference substance, gallic acid reference substance and corilagin reference substance to obtain reference substance solution;

(3) Detecting the sample solution, the reference substance solution and the reference medicinal material solution by using an ultra-high performance liquid chromatograph respectively to obtain corresponding maps;

(4) Analyzing and comparing the characteristic patterns of the reference substance solution, the reference medicinal material solution and the test substance solution, selecting 8 characteristic peaks corresponding to the retention time in the characteristic chromatogram of the reference medicinal material solution in the characteristic pattern of the test substance solution as common characteristic peaks, and constructing an ultra-high performance liquid characteristic pattern of phyllanthus urinaria; wherein, the characteristic peak 1 should correspond to the characteristic peak retention time of the gallic acid control solution, the characteristic peak 4 should correspond to the characteristic peak retention time of the corilagin control solution, and the characteristic peak 7 should correspond to the characteristic peak retention time of the ellagic acid control solution;

the chromatographic conditions during sample injection detection in the step (3) are as follows: octadecyl bonded silica gel column, mobile phase a: acetonitrile, mobile phase B:0.05 to 0.2 percent phosphoric acid solution, and the elution is carried out according to the following elution gradient:

time (minutes) Mobile phase a (%) Mobile phase B (%) 0～1 5 95 1～3 5→11 95→89 3～7 11→13 89→87 7～12 13→23 87→77 12～14 23→37 77→63 14～15 37→5 63→95 15~20 5 95

The ultraviolet detector is adopted to detect the wavelength: 254nm; flow rate: 0.25-0.35 ml/min; column temperature: 35-45 ℃;

sample injection amounts of the sample solution, the reference medicinal material solution and the reference substance solution are 0.5-2 mu l.

2. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 1, wherein the method comprises the following steps: in the ultra-high performance liquid phase characteristic spectrum of the phyllanthus urinaria in the step (4), the characteristic peak of the ellagic acid reference substance solution is taken as an S peak, the relative retention time of other characteristic peaks is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is: peak 2:0.58, peak 3:0.64, peak 5:0.78, peak 6:0.86, peak 8:1.29.

3. the method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 1, wherein the method comprises the following steps: the preparation of the sample solution and the control medicinal material solution uses water, methanol or methanol water solution with volume concentration of 10-75%, the volume of the extraction solvent is 15-50 ml, the extraction mode is heating reflux, and the extraction time is 30-90 minutes.

4. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 1, wherein the method comprises the following steps: the concentration of the reference substance solution is 30-60 mug/ml, and the extraction solvent is 50% methanol aqueous solution by volume concentration.

5. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 1, wherein the step (3) further comprises: the content of ellagic acid is used as a content index component, and the content of ellagic acid in the sample solution is detected.

6. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 1, wherein the method comprises the following steps: the test sample is phyllanthus urinaria medicinal material, and a preparation product, a preparation intermediate or a preparation finished product thereof.

7. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 1, wherein the method comprises the following steps: the test sample is phyllanthus urinaria formula granules or decoction pieces.

8. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 1, wherein the step (4) further comprises: and carrying out thin-layer detection on the test sample.

9. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 8, wherein the method comprises the following steps: the thin layer detection step comprises the following steps:

adding organic solvent into phyllanthus urinaria formula particles, carrying out ultrasonic treatment, filtering, evaporating to dryness, and adding the organic solvent for dissolution to prepare a sample solution;

adding water into phyllanthus urinaria control medicinal material, heating and refluxing, filtering, evaporating to dryness, adding organic solvent, performing ultrasonic treatment, filtering, evaporating to dryness, and dissolving with organic solvent to obtain control medicinal material reference solution;

adding organic solvent into gallic acid reference substance to obtain gallic acid reference substance solution;

sucking the sample solution, the reference substance solution of the reference medicinal material and the gallic acid reference substance solution, respectively spotting on the same silica gel G thin layer plate, unfolding, taking out, airing, and developing by spraying the color developing agent.

10. The method for detecting the quality of the fully controlled phyllanthus urinaria according to claim 9, wherein the method comprises the following steps: the organic solvent is methanol, the concentration of the reference substance solution is 1mg/ml, the developing agent is chloroform-ethyl acetate-formic acid=5:5:1, and the color developing agent is 2% ferric trichloride ethanol solution.