CN114689764A - Multi-component quality detection method for Erdong decoction extract - Google Patents
Multi-component quality detection method for Erdong decoction extract Download PDFInfo
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Abstract
The invention discloses a method for detecting the quality of multiple components of an Erdong decoction extract, which belongs to the technical field of Chinese patent medicine production, and comprises the steps of obtaining a linear regression equation of the relation between the component content and the peak area of baicalin by the aid of the peak area of baicalin in a reference solution, researching the preparation of a test solution, measuring by high performance liquid chromatography, and determining relative correction factor values, substituting the peak area of the baicalin measured by the test solution into the linear regression equation to obtain the content of the baicalin component in a sample, and calculating the content of mangiferin, hyperoside and glycyrrhizic acid by taking the peak area of the baicalin as a reference and multiplying the reference by the correction factor respectively, so that the purpose of detecting the quality of the multiple components of the Erdong decoction extract is achieved, and the detection is convenient and reliable, and the cost is low.
Description
Technical Field
The invention relates to the technical field of Chinese patent medicine production, and particularly relates to a method for detecting the quality of multiple components of an Erdong decoction extract.
Background
Erdong decoction is originated from volume III of "medical Xin Wu" of Qing Dynasty Peng, has effects of nourishing yin, promoting fluid production, invigorating qi and clearing heat, and can be used for treating diabetes. The original book records: the thirst and polydipsia are the upper part of the body, the food depletion and hunger are the middle part of the body, and the thirst and urine like ointment are the lower part of the body. The syndrome of three types of disease is also caused by accumulation of dryness-heat. The major method comprises the following steps: for the upper energizer, it is indicated for moistening lung and clearing stomach, and it is indicated in Erdong decoction. The prescription and the preparation method are as follows: ' Tian Dong Er Qian (removing heart), Mai Dong san Qian (removing heart), Fa Hua Yi Qian, Huang Qin Yi Qian, Zhi mu Yi Qian, gan Cao Wu, ren Shen Wu and Ne Ye Yi Qian. Decocting with water. "
According to examination results such as dosage and preparation method in the requirements of declaration data of material standard of traditional Chinese medicine compound preparation of ancient classical famous prescription (solicited for comments and drafts) and the key information table of ancient classical famous prescription (7 prescriptions), the modern decoction method of the Erdong decoction can be known as follows according to the examination results: 7.46g of radix asparagi, 11.19g of radix ophiopogonis, 3.73g of scutellaria baicalensis, 3.73g of rhizoma anemarrhenae, 1.87g of ginseng, 3.73g of liquorice, 3.73g of radix trichosanthis, 1.87g of lotus leaves and decocted with water for oral administration.
The Erdong decoction is a traditional Chinese medicine compound preparation, has more medicinal materials (consisting of 8 medicinal materials) in a prescription and has very complex and various components. In addition, in the prior art, CN110658295A (fingerprint determination method of lotus leaf decoction pieces in Erdong decoction formula, 20191023) discloses a method for identifying a fingerprint of lotus leaf in Erdong decoction, and in the prior art, CN101239150A (oral preparation of Erdong and preparation method thereof, 20080813) also discloses a method for preparing an oral preparation from Erdong decoction by a conventional process. Other part of literature research is based on the research on the extraction process of Erdong decoction granules by reference of standard decoction [ J ], Zhangguoqing, Shenghuagang and the like. However, the above reports only aim at the research of fingerprint spectrum of single medicine and the research of preparation process in the prescription of the Erdong decoction, but no research report about the content of multiple components of the Erdong decoction exists at present, and the research on the Erdong decoction is obviously incomplete, i.e. the components of the Erdong decoction can not be comprehensively evaluated, and the integral characteristics of the compound Chinese medicine preparation can not be reflected.
The classical famous prescription is a compound prescription consisting of a plurality of traditional Chinese medicines, and due to the complexity of multiple components of the traditional Chinese medicines, the quality of the traditional Chinese medicines is difficult to characterize by single component or index evaluation, and the method for controlling the quality of the multiple components needs more reference substances. The traditional Chinese medicine chemical component reference substance has a plurality of limitations in practical application due to factors such as high separation difficulty or unstable monomer, difficult supply, high cost and the like, so that the implementation of multi-component quality control has a plurality of difficulties and is difficult to popularize. The one-test-multiple-evaluation method realizes synchronous measurement of multiple components (difficult to obtain reference substances or difficult to supply) by measuring one component (the reference substances are easy to obtain, cheap and effective) through an internal functional relation and a proportional relation method existing among effective components of the traditional Chinese medicines. Thereby saving rare resources of the traditional Chinese medicine, reducing the inspection cost, not only solving the problem of lack of reference substances in multi-component quantitative and multi-index quality control of the traditional Chinese medicine, but also realizing multi-index synchronous quality control reaction on the quality of the traditional Chinese medicine.
Disclosure of Invention
The invention aims to provide a method for detecting the quality of multiple components of Erdong decoction extract, which solves the problems that the prior method for controlling the quality of multiple components of a compound prescription of traditional Chinese medicine needs more reference substances, the prior operation method has high cost and more quality control limitations and has more difficulties in practical application, combines the prior one-test and multi-evaluation method, measures one component by the internal functional relationship and the proportional relation method existing among the effective components of the traditional Chinese medicine to realize the synchronous measurement of the multiple components, meanwhile, a correction factor is obtained by using the method, no relevant literature report exists at present, the content is calculated by using the correction factor result, so that rare resources of the traditional Chinese medicine are saved, the inspection cost is reduced, the problem of lack of reference substances in multi-component quantitative and multi-index quality control of the traditional Chinese medicine is solved, and the quality of the traditional Chinese medicine is synchronously controlled and reacted by using multiple indexes.
The invention is realized by the following technical scheme:
a method for detecting the quality of multiple components of Erdong decoction extract comprises the following steps:
A. preparation of a reference solution: taking appropriate amount of mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substance, adding solvent methanol, and making into mixed reference substance solution containing mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substance;
B. preparing a test solution: adding methanol as solvent into Erdong decoction as control extract to obtain test solution;
C. high performance liquid chromatography determination: respectively sucking the reference substance solution in the step A and the test substance solution in the step B, injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, and performing data measurement;
D. relative correction factor value determination: substituting the peak areas of mangiferin, hyperoside, baicalin and glycyrrhizic acid measured by the reference solution into a relative correction factor to calculate a formula: relative correction factor (f) is As*Cr/Cs*ArTaking baicalin as an internal standard, and respectively calculating to obtain relative correction factors of mangiferin, hyperoside and glycyrrhizic acid relative to baicalin;
E. obtaining a linear regression equation of the relation between the component content and the peak area according to the baicalin peak area of the reference solution, substituting the baicalin peak area measured by the test solution into the linear regression equation to obtain the content of the baicalin component in the sample, and respectively multiplying the baicalin peak area serving as a reference by a correction factor to calculate the content of mangiferin, hyperoside and glycyrrhizic acid.
Further, in the step a, the specific preparation method of the reference solution is as follows: taking appropriate amount of mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substance, precisely weighing, adding 70% methanol to obtain mixed solution containing mangiferin, hyperoside, and glycyrrhizic acid 50 μ g each per 1ml, and baicalin 100 μ g per 1ml, and making into mixed reference substance solution to obtain Erdong decoction reference substance solution.
Further, in the step B, the specific preparation method of the sample solution is as follows: taking 0.5g of Erdong decoction control extract, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 70% methanol, sealing, weighing, ultrasonically treating for 30 minutes, weighing again, supplementing lost mass with 70% methanol, shaking, filtering, and taking the subsequent filtrate to obtain Erdong decoction control extract sample solution.
Further, it is characterized in that: in the step C, the determination conditions of the high performance liquid chromatograph are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m;
the column temperature is 30 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent phosphoric acid water solution, and gradient elution is carried out.
Further, in the step C, the detection wavelength of the high performance liquid chromatograph: 360nm for 0-16 min, and 254nm for 16-40 min.
Further, the gradient elution process is as follows:
0-2 min, 10-15% of mobile phase A and 90-85% of mobile phase B;
for 2-12 min, the content of the mobile phase A is 15-25%, and the content of the mobile phase B is 85-75%;
12-32 min, wherein the mobile phase A is 25-50%, and the mobile phase B is 75-50%;
32-40 min, the mobile phase A is 50-95%, and the mobile phase B is 50-5%.
Furthermore, the total amount of mangiferin, hyperoside, baicalin and glycyrrhizic acid should be 0.805% -1.494%.
Further, the preparation method of the Erdong decoction control extract comprises the following steps:
a 1: taking the drinking tablets according to the parts by weight of the raw materials: 7.46 parts of asparagus, 11.19 parts of dwarf lilyturf tuber, 3.73 parts of baical skullcap root, 3.73 parts of common anemarrhena rhizome, 1.87 parts of ginseng, 3.73 parts of liquoric root, 3.73 parts of mongolian snakegourd root and 1.87 parts of lotus leaf;
b 1: putting all decoction pieces obtained in the step a1 into a decocting container, adding 8 times of water into the decoction pieces, soaking for 30min, and decocting for 30 min; adding 6 times of water for the second time, decocting for 30min for 2 times, filtering, mixing filtrates, and lyophilizing to obtain Erdong decoction as control extract.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) in the invention, one component (a reference substance is easy to obtain, cheap and effective) is determined by combining a one-test-multiple-evaluation method in the prior art and through an internal functional relation and a proportional relation method existing among effective components of the traditional Chinese medicine, so that a plurality of components (the reference substance is difficult to obtain or is difficult to supply) are synchronously determined.
(2) According to the invention, the high performance liquid chromatography is adopted, the detection conditions are independently designed, and the one-test-multiple-evaluation method is combined, so that the detection method is convenient and reliable.
(3) According to the invention, the quality of the Erdong decoction control extract can be effectively controlled by measuring the 4 components of mangiferin, hyperoside, baicalin and glycyrrhizic acid, the quality stability of the Erdong decoction control extract is ensured, and the Erdong decoction control extract is used for quality detection of the Erdong decoction related preparation after defecation.
(4) According to the HPLC multi-component detection method, the preparation of the test sample is simple, and the method is high in precision, good in stability, good in repeatability and high in accuracy. Can make up the blank of the research on the Erdong decoction at the present stage and provide reference for the follow-up research of preparations such as the Erdong decoction and the like.
(5) In the detection process of the Erdong decoction sample, the peak area of baicalin is measured by using an external standard method and the content of the baicalin is obtained, and meanwhile, the peak areas of mangiferin, hyperoside and glycyrrhizic acid are measured, so that the content of the mangiferin, hyperoside and glycyrrhizic acid can be calculated according to the information and the relative correction factors among substances, the detection is reliable, and the consumed time is short.
Drawings
Fig. 1 is a uv absorption spectrum of mangiferin of example 2.
FIG. 2 is the UV absorption spectrum of hyperin in example 2.
FIG. 3 is a diagram showing the ultraviolet absorption spectrum of baicalin in example 2.
FIG. 4 is a UV absorption spectrum of glycyrrhizic acid in example 2.
FIG. 5 is a diagram of mobile phase investigation of the first embodiment of example 3.
FIG. 6 is a mobile phase examination chart of the second embodiment in example 3.
Fig. 7 is a mobile phase examination diagram of the third embodiment in example 3.
FIG. 8 is a chromatogram for the specificity test in example 4.
Fig. 9 is a graph of the mangiferin standard in example 7.
FIG. 10 is the standard graph of hyperin in example 7.
FIG. 11 is the standard curve of baicalin in example 7.
FIG. 12 is a graph showing the standard curve of glycyrrhizic acid in example 7.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
A high performance liquid chromatograph: waters model 2695-2998 high performance liquid chromatograph, Agilent model 1260 high performance liquid chromatograph, and Shimadzu model LC-20AD high performance liquid chromatograph;
an electronic balance: ME204E/02, MS205DU, XP26 (Mettler-Tollido instruments, Inc.);
an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);
an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);
a chromatographic column: agilent 5TC 18250X 4.6mm, Kromasil C185 μm 4.6X 250mm, Waters Xbridge C184.6X 250 mm.
Baicalin (China institute for testing and testing food and drug, batch No. 110715-201720, content is 93.5%);
mangiferin (China institute for testing food and drug, batch No.: 111607-201704, content is 98.1%);
hyperin (China institute for testing and drug testing, lot number: 111521-;
glycyrrhizic acid (Vickqi Biotech Co., Ltd., Sichuan province, lot number wkq19011705, content 98%);
acetonitrile and phosphoric acid are chromatographically pure, water is ultrapure water, and other reagents are analytically pure;
negative sample information: radix asparagi lachniae control extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200911);
radix Ophiopogonis deficiency control extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200912);
lack radix Trichosanthis reference extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200913);
lack radix Scutellariae control extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200914);
lack of rhizoma anemarrhenae control extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200915);
a lotus leaf deficiency control extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200916);
a control extract of ginseng lacking (produced by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200916);
lack of radix Glycyrrhizae control extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No. EDT 200916);
erdong decoction reference extract (prepared by Sichuan New Green pharmaceutical science and technology development Co., Ltd., batch No.: EDT200901, EDT200902, EDT200903, EDT200904, EDT200905, EDT200906, EDT200907, EDT200908, EDT200909, EDT 200910).
Example 1
The embodiment provides a method for detecting the quality of multiple components of an Erdong decoction extract, which comprises the following steps:
A. preparation of a reference solution: precisely weighing appropriate amount of mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substances, and adding 70% methanol to obtain mixed solution containing mangiferin, hyperoside, and glycyrrhizic acid 50 μ g each per 1ml and baicalin 100 μ g per 1ml as mixed reference solution;
B. preparing a test solution: precisely weighing about 0.5g of Erdong decoction as a control extract, placing the Erdong decoction in a conical flask with a plug, precisely adding 25mL of 70% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 220W and frequency 50kHz) for 30 minutes, weighing again, complementing the loss mass with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the extract;
C. high performance liquid chromatography determination: respectively sucking 10 μ L of the reference solution and sample solution, injecting into high performance liquid chromatograph, and performing data measurement;
D. relative correction factor value determination: substituting the peak areas of mangiferin, hyperoside, baicalin and glycyrrhizic acid measured by the reference solution into a relative correction factor to calculate a formula: relative correction factor (f) is As*Cr/Cs*ArA in the formulaSPeak area of the internal standard substance; a. therPeak area for control; cSIs the concentration of the internal standard substance; crTaking baicalin as an internal standard for the concentration of a reference substance, and respectively calculating to obtain relative correction factors of mangiferin, hyperoside and glycyrrhizic acid relative to baicalin;
taking baicalin as reference, and taking the peak corresponding to the retention time as S1 peak, calculating the relative retention time of mangiferin, hyperoside and glycyrrhizic acid, wherein the relative retention time is within + -10% of the specified value (if the relative retention time is deviated by more than 10%, the corresponding substituted control should be confirmed as standard). The relative retention times and correction factors are shown in table 1 below:
table 1:
E. obtaining a linear regression equation of the relation between the component content and the peak area according to the baicalin peak area of the reference solution, substituting the baicalin peak area measured by the test solution into the linear regression equation to obtain the content of the baicalin component in the sample, and respectively multiplying the baicalin peak area serving as a reference by a correction factor to calculate the content of mangiferin, hyperoside and glycyrrhizic acid.
And (3) combining the data and the relative correction factors obtained in the table 1, taking the peak area of the baicalin as a reference, and respectively multiplying the peak area by the correction factors to calculate the contents of the mangiferin, the hyperoside and the glycyrrhizic acid.
Example 2
This example is a study of chromatographic condition selection and system suitability test-detection wavelength in step C of example 1.
Collecting the reference solutions of mangiferin, hyperoside, baicalin and glycyrrhizic acid by full wavelength spectrum, and analyzing the spectrograms to obtain the results shown in FIGS. 1-4.
As can be seen from fig. 1-4, mangiferin and hyperoside in the Erdong decoction control extract have better absorption at a wavelength of 360nm, and baicalin and glycyrrhizic acid have better absorption at a wavelength of 254nm, and the detection wavelength is finally determined by comprehensive consideration: 360nm for 0-16 min and 254nm for 16-40 min.
Example 3
This example is a study of chromatographic condition selection and system suitability test-chromatographic conditions in step C of example 1.
Three chromatographic conditions are designed according to the commonly used chromatographic conditions and the initial judgment on the material characteristics of the chromatographic conditions, and the specific conditions are as follows:
the first scheme is as follows: acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid water is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table 2; the flow rate was 1.0mL per minute; the column temperature is 30 ℃; detection wavelength: 254nm, the results are shown in FIG. 5.
Table 2: gradient elution table in scheme I
Under the chromatographic conditions, the peak emergence time is late and the peaks are not separated, and the chromatographic conditions are examined again.
Scheme II: acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid water is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table 3; the flow rate was 1.0mL per minute; the column temperature is 30 ℃; detection wavelength: 254nm, and the results are shown in FIG. 6.
Table 3: gradient elution table in scheme II
Under the chromatographic condition, chromatographic peak separation degree and symmetry are not feasible, and the chromatographic condition is optionally optimized.
The third scheme is as follows: octadecylsilane chemically bonded silica was used as a filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm), acetonitrile was used as a mobile phase A, 0.1% phosphoric acid water was used as a mobile phase B, and gradient elution was performed as specified in Table 4 below; the flow rate was 1.0mL per minute; the column temperature is 30 ℃; detection wavelength: 360nm for 0-16 min and 254nm for 16-40 min. The results are shown in FIG. 7.
Table 4: gradient elution table in scheme III
From the above, the number of target peak theoretical plates, the separation degree and the symmetry are all better under the chromatographic conditions of the scheme, so the chromatographic conditions are selected as the mobile phase of the quality detection method of the Erdong decoction reference extract.
In summary, the chromatographic conditions and system adaptations were: using octadecylsilane chemically bonded silica as filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm), acetonitrile as mobile phase A, and 0.1% phosphoric acid water as mobile phase B, and performing gradient elution according to the specification in the following table; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; detection wavelength: 360nm in 0-16 min and 254nm in 16-40 min, and the number of theoretical plates is not less than 5000 calculated according to baicalin hydrochloride.
Example 4
This example is a methodological examination of the invention-a specificity test.
Preparation of a test solution: test solutions of Erdong decoction control extract were prepared according to the experimental conditions set forth in example 1.
Preparation of control reference solutions: taking appropriate amount of mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substances, precisely weighing, adding methanol to obtain mixed solution containing mangiferin, hyperoside, and glycyrrhizic acid 50 μ g each per 1ml, and baicalin 100 μ g each per 1ml, and making into mixed reference solution. In the preparation method of the reference substance solution, the reference substance is prepared into different concentrations, so that the peak area and the content can be conveniently calculated.
Preparation of negative control solution: according to the experimental conditions set up above, a negative solution of the asparagus fern lacking control extract is prepared.
Preparation of negative control solution: according to the experimental conditions set up above, the dwarf lilyturf tuber lacking control extract negative solution is prepared.
Preparation of negative control solution: according to the experimental conditions set up above, negative solution of control extract of radix Trichosanthis is prepared.
Preparation of negative control solution: preparing the scutellaria baicalensis-lacking control extract negative solution according to the established experimental conditions.
Preparation of negative control solution: according to the experimental conditions set up above, a negative solution of the anemarrhena asphodeloides bunge control extract is prepared.
Preparation of negative control solution: negative solution of the control extract of the lotus leaf lacking is prepared according to the experimental conditions set forth above.
Preparation of negative control solution: negative solutions of the ginseng-deficient control extract were prepared according to the experimental conditions set forth above.
Preparation of negative control solution: the negative solution of the control extract lacking licorice (licorice) was prepared according to the experimental conditions set forth above.
The detection is carried out according to a proposed method. The results are shown in fig. 8, where peak 1: mangiferin; peak 2: hyperin; peak 3: baicalin; peak 4: glycyrrhizic acid.
The result shows that the negative control solution has no interference to the measurement of the peak to be measured, and the specificity of the method is good.
Example 5
This example is a methodology of the technical solution of example 1-precision.
The control solution was sampled 6 times continuously, the peak area of each component was recorded, and the RSD value was calculated, the results are shown in table 5.
Table 5: results of precision investigation
As shown in Table 5, the results showed that the peak areas RSD of the respective components in the precision measurement were all less than 0.93%, and the precision of the instrument was good.
Example 6
This example is a methodology-repeatability examination of the protocol of example 1.
Taking 0.5g of the same test sample (batch number: EDT200901), precisely weighing 6 parts, preparing a test sample solution by the same operator according to a proposed method, and calculating the content of each component in the 6 parts of test sample, wherein the results are shown in Table 6.
Table 6: results of repeatability experiments
The result shows that the RSD values of the contents of mangiferin, hyperoside, baicalin and glycyrrhizic acid in the Erdong decoction are all qualified, and the method has good repeatability.
Example 7
This example is a methodology-linear relationship examination of the technical solution of example 1.
Taking mangiferin, hyperoside, baicalin and glycyrrhizic acid mixed reference substance mother liquor, and respectively diluting step by step to obtain corresponding mixed reference substance solution. The solutions 10. mu.L were injected into a liquid chromatograph, and analyzed to obtain peak areas, and response curves were plotted with the concentration (. mu.g/mL) as abscissa and the peak area (Y) as ordinate, and the results are shown in tables 7 to 10 and FIGS. 9 to 12.
Table 7: analysis result of mangiferin standard curve
Table 8: analysis result of hyperin standard curve
Table 9: baicalin standard curve analysis result
Table 10: glycyrrhizic acid standard curve analysis result
From the above results, it was found that each component exhibited a good linear relationship in the range of the respective sample amount, and the results of the linear relationship examination are shown in table 11.
TABLE 11
Example 8
This example is a stability test, a methodology study of the technical solution of example 1.
About 0.5g of the same test solution (batch number: EDT200901) was precisely weighed to prepare a test solution, and the chromatographic peak areas of the components were measured at 0 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours, respectively, and the results are shown in Table 12.
Table 12:
the results show that: under the experimental condition, the peak area RSD values of all the components are qualified, and the stability of the test solution is good within 24 hours.
Example 9
This example is a methodological study of the technical solution of example 1-sample recovery.
Taking about 0.25g of a test sample (batch number: EDT200901) with known content, precisely weighing 6 parts, precisely adding a certain amount of reference substances respectively, preparing and measuring a test sample solution according to a proposed method, and calculating the recovery rate, wherein the results are shown in Table 13.
Table 13: experimental results of sample recovery
The result shows that the RSD value of the content recovery rate result of each component is qualified, and the method has good accuracy.
Example 10
This example is an approach to methodology-investigation of different instruments
Based on the experimental conditions set forth in the foregoing examples, the Erdong decoction control extract (lot No. EDT200901) was precisely weighed, and the test solutions were prepared and measured on Waters e2695-2998, Agilent 1260 and Shimadzu LC-20AD HPLC (Agilent 5 TC-C18250X 4.6mm 5 μm chromatography columns), and the content of each component was calculated. The results are shown in Table 14.
Table 14: investigation results of different instruments
The results show that when the 3 instruments are used for detecting the test sample, the RSD value of each component content is qualified, and the method has good durability.
Example 11
This example is a review of methodological-durability.
The same test sample (batch No. EDT200901) was tested using columns of different brands (Waters XBridge C184.6X 250mm 5.0. mu.m, Agilent 5 TC-C184.6X 250mm 5. mu.m, Kromasil 100-5-C184.6X 250mm 5.0. mu.m) and the results are shown in Table 15.
Table 15: durability examination result
The result shows that the RSD value of each component content is qualified, and the durability of the chromatographic column of the method is good.
Example 12
This example is a further examination and determination of the relative correction factors in example 1.
Calculating relative correction factors of the components to be detected:
taking mixed reference substance solution, injecting 1, 2, 5, 10, 15, 20 μ L for determination, with baicalin as internal standard and baicalin as internal standard, respectivelyCalculating relative correction factors of mangiferin, hyperoside and glycyrrhizic acid, wherein the calculation formula is as follows: relative correction factor (f) is As*Cr/Cs*ArIn the formula ASPeak area of the internal standard substance; a. therPeak area for control; cSIs the concentration of the internal standard substance; crIs the concentration of the control. The results are shown in tables 16 and 17.
Table 16: data sheet for peak area of mixed control
Table 17: relative correction factor calculation result table
Further, durability examination needs to be carried out on the relative correction factor obtained by the scheme.
Using Agilent model 1260, 3 different brands of columns were examined separately (Waters)C184.6X 250mm 5.0 μm, Agilent 5 TC-C184.6X 250mm 5 μm, Kromasil 100-5-C184.6X 250mm 5.0 μm); the effects of Waters e2695-2998, Agilent 1260 and Shimadzu LC-20AD high performance liquid chromatography systems on the correction factors were examined by using Agilent 5TC-C18 chromatographic columns, and the results are shown in Table 18.
Table 18: relative correction factor reproducibility study
The results show that the relative correction factors of the respective components are good in durability.
Determination of the relative correction factor:
the factors affecting the relative correction factors are examined with reference to technical guidelines established by the one-test-multiple-evaluation method, and the average value of the relative correction factors obtained in each test is taken under the condition that the RSD value is less than 5%, and the result is shown in the following table 19.
Table 19: summary of relative correction factor results
As can be seen from table 19, the relative correction factors of mangiferin, hyperoside, and glycyrrhizic acid were finally determined to be 0.65, 0.55, and 1.42, respectively.
Positioning of a chromatogram to be detected: the components to be measured are positioned by calculating the relative retention time of the chromatographic peak of each component to be measured and the chromatographic peak of baicalin in different chromatographic instruments and different chromatographic columns, and the results are shown in a table 20.
Table 20: relative retention time of ingredients
The results show that the relative retention time fluctuation among chromatographic peaks of all the components is small, and the RSD% values of all the components are less than 3%.
Comparing the measurement results of the one-test multi-evaluation method and the external standard method:
the contents of mangiferin, hyperoside, baicalin and glycyrrhizic acid in the Erdong decoction extract were calculated by an external standard method and a one-test-multiple evaluation method, and the measurement results are shown in Table 21.
Table 21: comparing the results of one-test and multi-evaluation methods with those of external standard method
The result shows that the content value calculated by the conventional external standard method and the content value calculated by the one-test-multiple-evaluation method have no significant difference, so that the one-test-multiple-evaluation method is feasible to be applied to the quality evaluation of the multi-index components of the Erdong decoction.
The result shows that the actual measurement range of the total amount of mangiferin, hyperoside, baicalin and glycyrrhizic acid in the Erdong decoction is 1.15%, the average value is 1.145% -1.154%, the SD value is 0.003, and the range of 70% -130% of the average value of the total content is 0.805% -1.494%; the limit range of the mean plus or minus 3 times SD is 1.060 percent to 1.240 percent. According to the method for reasonably determining the upper and lower limits of the quality requirements of related quality control items in the quality standard in the declaration data requirements (survey of comments) of the material basis of ancient classical famous prescription Chinese medicinal compound preparations, namely the fluctuation range of content measurement does not exceed 70-130% of the mean value, the total content of baicalin, mangiferin, hyperoside and glycyrrhizic acid in the product is 0.805-1.494%.
Example 13
The present embodiment is further optimized based on embodiment 1, and in the step B, the preparation method of the overwintering decoction control extract comprises the following steps:
a 1: taking the drinking tablets according to the parts by weight of the raw materials: 7.46 parts of asparagus cochinchinensis, 11.19 parts of radix ophiopogonis, 3.73 parts of scutellaria baicalensis, 3.73 parts of rhizoma anemarrhenae, 1.87 parts of ginseng, 3.73 parts of liquorice, 3.73 parts of trichosanthes root and 1.87 parts of lotus leaf;
b 1: putting all decoction pieces obtained in the step a1 into a decocting container, adding 8 times of water into the decoction pieces, soaking for 30min, and decocting for 30 min; adding 6 times of water for the second time, decocting for 30min for 2 times, filtering, mixing filtrates, and lyophilizing to obtain Erdong decoction as control extract.
By adopting the preparation method of the Erdong decoction comparison extract in the embodiment, the lyophilized powder of the Erdong decoction comparison extract is stable in property, and the result obtained by content detection is more accurate.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Claims (8)
1. A method for detecting the quality of multiple components of Erdong decoction extract is characterized by comprising the following steps:
A. preparation of a reference solution: taking appropriate amount of mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substances, adding solvent methanol, and making into mixed reference substance solution containing mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substances;
B. preparing a test solution: adding methanol as solvent into Erdong decoction as control extract to obtain test solution;
C. high performance liquid chromatography determination: respectively sucking the reference substance solution in the step A and the test substance solution in the step B, injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph, and performing data measurement;
D. relative correction factor value determination: substituting the peak areas of mangiferin, hyperoside, baicalin and glycyrrhizic acid measured by the reference solution into a relative correction factor to calculate a formula: relative correction factor (f) = as*Cr/Cs*ArTaking baicalin as an internal standard, and respectively calculating to obtain relative correction factors of mangiferin, hyperoside and glycyrrhizic acid relative to baicalin;
E. obtaining a linear regression equation of the relation between the component content and the peak area according to the baicalin peak area of the reference solution, substituting the baicalin peak area measured by the test solution into the linear regression equation to obtain the content of the baicalin component in the sample, and respectively multiplying the baicalin peak area serving as a reference by a correction factor to calculate the content of mangiferin, hyperoside and glycyrrhizic acid.
2. The method for detecting the quality of the multiple components of the Erdong decoction extract as claimed in claim 1, wherein in the step A, the specific preparation method of the reference solution is as follows: taking appropriate amount of mangiferin, hyperoside, baicalin, and glycyrrhizic acid reference substance, precisely weighing, adding 70% methanol to obtain mixed solution containing mangiferin, hyperoside, and glycyrrhizic acid 50 μ g each per 1ml, and baicalin 100 μ g per 1ml, and making into mixed reference substance solution to obtain Erdong decoction reference substance solution.
3. The method for detecting the quality of the multiple components of the Erdong decoction extract as claimed in claim 1, wherein in the step B, the specific preparation method of the test solution is as follows: taking 0.5g of the Erdong decoction reference extract, accurately weighing, placing in a conical flask with a plug, accurately adding 25mL of 70% methanol, sealing the plug, weighing, carrying out ultrasonic treatment for 30 minutes, weighing again, complementing the lost mass with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the Erdong decoction reference extract test solution.
4. The method for detecting the quality of the multiple components of the Erdong decoction extract as claimed in claim 1, wherein the method comprises the following steps: in the step C, the determination conditions of the high performance liquid chromatograph are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m;
the column temperature is 30 ℃;
flow rate: 1.0 mL/min;
sample introduction amount: 10 mu L of the solution;
the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent phosphoric acid water solution, and gradient elution is carried out.
5. The method for detecting the quality of the multiple components of the Erdong decoction extract as claimed in claim 4, wherein in the step C, the detection wavelength of the high performance liquid chromatograph: 360nm for 0-16 min and 254nm for 16-40 min.
6. The method for detecting the quality of the multiple components of the Erdong decoction extract as claimed in claim 5, wherein the gradient elution process comprises:
0-2 min, 10-15% of mobile phase A and 90-85% of mobile phase B;
for 2-12 min, the content of the mobile phase A is 15-25%, and the content of the mobile phase B is 85-75%;
12-32 min, wherein the mobile phase A is 25-50%, and the mobile phase B is 75-50%;
32-40 min, the mobile phase A is 50-95%, and the mobile phase B is 50-5%.
7. The method for detecting the quality of the multiple components of the Erdong decoction extract as claimed in claim 1, wherein the method comprises the following steps: in step E, the total amount of mangiferin, hyperoside, baicalin, and glycyrrhizic acid should be measured to be 0.805% -1.494%.
8. The method for detecting the quality of the multiple components of the Erdong decoction extract as claimed in claim 1, wherein the preparation method of the Erdong decoction control extract in the step B comprises the following steps:
a 1: taking the drinking tablets according to the parts by weight of the raw materials: 7.46 parts of asparagus, 11.19 parts of dwarf lilyturf tuber, 3.73 parts of baical skullcap root, 3.73 parts of common anemarrhena rhizome, 1.87 parts of ginseng, 3.73 parts of liquoric root, 3.73 parts of mongolian snakegourd root and 1.87 parts of lotus leaf;
b 1: putting all decoction pieces obtained in the step a1 into a decocting container, adding 8 times of water into the decoction pieces, soaking for 30min, and decocting for 30 min; adding 6 times of water for the second time, decocting for 30min for 2 times, filtering, mixing filtrates, and lyophilizing to obtain Erdong decoction as control extract.
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