CN114689764B - 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 multi-component quality detection method of a Erdong decoction extract, which belongs to the technical field of Chinese patent medicine production, and aims to detect the multi-component quality detection of the Erdong decoction extract by preparing a reference solution, researching the preparation of a test solution, measuring by high performance liquid chromatography, determining relative correction factor values, obtaining a linear regression equation of the relation between the component content and the peak area of the reference solution by the peak area of baicalin, substituting the peak area of baicalin measured by the test solution into the linear regression equation to obtain the content of baicalin in a sample, and multiplying correction factors by the peak area of baicalin to calculate the content of mangiferin, hyperin and glycyrrhizic acid.
Description
Technical Field
The invention relates to the technical field of Chinese patent medicine production, in particular to a multi-component quality detection method of a Erdong decoction extract.
Background
The Erdong decoction is from volume III of Qing dynasty Cheng Guopeng, has the functions of nourishing yin, promoting the production of body fluid, invigorating qi and clearing heat, and is used for treating diabetes. Original book records: the thirst and polydipsia are the upper and lower symptoms of the diabetes, the hunger and the middle and middle energizer, and the thirst and the urine like ointment. The three syndromes of the three syndromes are all dryness-heat accumulation. The method comprises the following steps: for upper energizer, it is indicated for lung-moistening, stomach-clearing and spleen-stomach-regulating herbs. The prescription and the preparation method are as follows: "Dongqian (heart removed), ophiopogon root three-money (heart removed), pollen one-money, baikal skullcap root one-money, rhizoma anemarrhenae one-money, licorice five-part, ginseng five-part and lotus leaf one-money. Decocting with water. "
According to examination papers such as dosage and preparation methods in the requirements of declaration materials (solicited opinion manuscripts) and the key information table (7 prescriptions) of ancient classical prescription, the modern decoction method of Erdong decoction can be known according to the examination results: 7.46g of asparagus, 11.19g of dwarf lilyturf tuber, 3.73g of baical skullcap root, 3.73g of common anemarrhena rhizome, 1.87g of ginseng, 3.73g of liquoric root, 3.73g of mongolian snakegourd root, 1.87g of lotus leaf and water decoction.
The Erdong decoction is used as a Chinese medicinal compound preparation, has more prescription medicinal materials (comprising 8 medicinal materials), and has very complex and various components. The prior patent document CN110658295A (method for measuring the finger print of lotus leaf decoction pieces in the Erdong decoction formulation, 20191023) discloses a method for identifying the finger print of lotus leaf in Erdong decoction, and the prior patent document CN101239150A (Erdong oral preparation and preparation method thereof, 20080813) also discloses a method for preparing the Erdong decoction into oral preparation by a conventional process. Other part of literature studies were based on standard decoction reference, erdong decoction particle extraction process study [ J ], zhang Guoqing, cheng Huagang, etc. However, the above reports only aim at the single-medicine fingerprint research and the preparation technology research in the formula of the Erdong decoction, but at present, no research report about the multi-component content of the Erdong decoction exists, and the research on the Erdong decoction is obviously incomplete, namely, the components of the Erdong decoction cannot be comprehensively evaluated, and the integral characteristics of the Chinese herbal compound preparation cannot be reflected.
The classical prescription is a compound prescription composed of a plurality of traditional Chinese medicines, and because of the complexity of the traditional Chinese medicines, single component or index evaluation is difficult to characterize the quality of the traditional Chinese medicines, and the method for controlling the quality of the traditional Chinese medicines by the plurality of components needs more reference substances. The traditional Chinese medicine chemical component reference substances have the defects of large separation difficulty or unstable monomer, difficult supply, high cost and the like, and have various limitations in practical application, so that the implementation of multi-component quality control has various difficulties and is difficult to popularize. The one-measurement multi-evaluation method is to measure one component (the reference substance is easy to obtain, cheap and effective) by an intrinsic function relation and a proportional relation method existing among the effective components of the traditional Chinese medicine so as to realize synchronous measurement of a plurality of components (the reference substance is difficult to obtain or difficult to supply). Thereby saving rare resources of traditional Chinese medicines, reducing the inspection cost, solving the problem of lack of reference substances in multi-component quantification and multi-index quality control of traditional Chinese medicines, and realizing multi-index synchronous quality control to reflect the quality of traditional Chinese medicines.
Disclosure of Invention
The invention aims to provide a multi-component quality detection method for a Erdong decoction extract, which solves the problems that the multi-component quality control method for a compound prescription of traditional Chinese medicine in the prior art needs more reference substances, the existing operation method has high cost and more quality control limitation, and has a plurality of difficulties in practical application.
The invention is realized by the following technical scheme:
a multi-component quality detection method of Erdong decoction extract comprises the following steps:
A. preparing a reference substance solution: taking appropriate amount of mangiferin, hyperin, baicalin and glycyrrhizic acid reference substance, adding methanol as solvent to obtain mixed reference substance solution containing mangiferin, hyperin, baicalin and glycyrrhizic acid reference substance;
B. sample solution preparation: adding methanol as solvent into the Erdong decoction control extract to obtain test solution;
C. high performance liquid chromatography assay: respectively sucking the reference substance solution in the step A and the sample solution to be tested in the step B, and injecting into a high performance liquid chromatograph to perform data measurement;
D. determination of relative correction factor values: according to the peak areas of mangiferin, hyperin, baicalin and glycyrrhizic acid measured by the reference substance solution, substituting the peak areas into a relative correction factor to calculate a formula: relative correction factor (f) =a s *C r /C s *A r Calculating the relative correction factors of mangiferin, hyperin and glycyrrhizic acid relative to baicalin by taking baicalin as an internal standard;
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 sample solution into the linear regression equation to obtain the content of the baicalin component in the sample, and multiplying correction factors by the baicalin peak area serving as a reference to calculate the content of mangiferin, hyperin and glycyrrhizic acid.
Further, in the step A, the specific preparation method of the reference substance solution comprises the following steps: taking appropriate amount of mangiferin, hyperin, baicalin and glycyrrhizic acid reference substances, precisely weighing, adding 70% methanol to obtain mixed solution containing mangiferin, hyperin and glycyrrhizic acid 50 μg per 1ml, and baicalin 100 μg per 1ml, and taking as mixed reference substance solution to obtain Erdong decoction reference substance solution.
Further, in the step B, the specific preparation method of the sample solution comprises the following steps: taking 0.5g of Erdong decoction control extract, precisely weighing, placing into a conical bottle with a plug, precisely adding 25mL of 70% methanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, weighing again, supplementing the reduced mass with 70% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate to obtain the Erdong decoction control extract sample solution.
Further, it is characterized in that: in the step C, the measurement conditions of the high performance liquid chromatograph are as follows:
chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the granularity is 5 mu m;
the column temperature is 30 ℃;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, and the 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;
2-12 min, 15-25% of mobile phase A and 85-75% of mobile phase B;
12-32 min, 25-50% of mobile phase A and 75-50% of mobile phase B;
32-40 min, 50-95% of mobile phase A and 50-5% of mobile phase B.
Further, the total content of mangiferin, hyperin, baicalin and glycyrrhizic acid is 0.805% -1.494%.
Further, the preparation method of the Erdong decoction control extract comprises the following steps:
a1: the preparation method comprises the steps of (1) taking decoction pieces according to the weight parts of 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;
b1: placing all decoction pieces obtained in the step a1 in a decocting container, adding 8 times of water, soaking for 30min, and decocting for 30min; adding 6 times of water for the second time, decocting for 30min, decocting for 2 times, filtering, mixing filtrates, and lyophilizing to obtain Erdong decoction control extract.
Compared with the prior art, the invention has the following advantages:
(1) According to the method, an internal function relation and a proportional relation method existing among the effective components of the traditional Chinese medicine are combined, one component (a reference substance is easy to obtain, low in cost and effective) is measured, so that synchronous measurement of a plurality of components (a reference substance is difficult to obtain or difficult to supply) is realized, meanwhile, a correction factor is obtained by the method, no relevant literature report exists at present, the content is calculated by using the result of the correction factor, so that rare resources of the traditional Chinese medicine are saved, the inspection cost is reduced, the problem of reference substance deficiency existing 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 by multiple indexes.
(2) In the invention, the high performance liquid chromatography is adopted, the detection conditions are designed independently, and the detection method is convenient and reliable by combining a one-measurement-multiple-evaluation method.
(3) According to the invention, the quality of the Erdong decoction control extract can be effectively controlled by measuring 4 components of mangiferin, hyperin, baicalin and glycyrrhizic acid, so that the stability of the quality of the Erdong decoction control extract is ensured, and the Erdong decoction control extract can be used for quality detection of Erdong decoction related preparations subsequently.
(4) In the invention, the HPLC multi-component detection method has the advantages of simple preparation of the sample, high precision, good stability, good repeatability and high accuracy. The method can make up the blank of the research of the Erdong decoction at the present stage and provides reference for the subsequent research of preparations such as Erdong decoction.
(5) In the invention, in the detection process of the Erdong decoction sample, an external standard method is used for measuring the peak area of baicalin and obtaining the content of the baicalin, and simultaneously measuring the peak areas of mangiferin, hyperin and glycyrrhizic acid, the content of mangiferin, hyperin and glycyrrhizic acid can be calculated and obtained according to the information and the relative correction factors among substances, and the detection is reliable and the time consumption is short.
Drawings
FIG. 1 is a UV absorption spectrum of mangiferin in example 2.
FIG. 2 is a graph showing the ultraviolet absorption spectrum of hyperin in example 2.
FIG. 3 is a ultraviolet absorption spectrum of baicalin in example 2.
FIG. 4 is a graph showing the ultraviolet absorption spectrum of glycyrrhizic acid in example 2.
Fig. 5 is a mobile phase investigation of scheme one of example 3.
Fig. 6 is a mobile phase investigation of scheme two in example 3.
Fig. 7 is a mobile phase investigation of scheme three in example 3.
FIG. 8 is a chromatogram of the specificity study in example 4.
FIG. 9 is a mangiferin standard graph in example 7.
FIG. 10 is a graph of hyperin standard curve in example 7.
FIG. 11 is a baicalin standard chart in example 7.
FIG. 12 is a standard graph of glycyrrhizic acid in example 7.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
High performance liquid chromatograph: waters2695-2998 type high performance liquid chromatograph, agilent 1260 type high performance liquid chromatograph, shimadzu LC-20AD type high performance liquid chromatograph;
an electronic balance: ME204E/02, MS205DU, XP26 (Metrele Tolyduo instruments Co., ltd.);
ultrapure water machine: cell type 1810A (Shanghai mueller scientific instruments limited);
ultrasonic cleaner: KQ5200DB model (600W, 40KHz; kunshan ultrasonic instruments Co., ltd.);
chromatographic column: agilent 5TC C18X 4.6mm, kromasil C18 μm 4.6X1250 mm, waters XB ridge C18.6X1250 mm.
Baicalin (national food and drug verification institute, lot number: 110715-201720, content of 93.5%);
mangiferin (Chinese food and drug verification institute, lot number: 111607-201704, content of 98.1%);
hyperin (Chinese food and drug assay institute, lot number 111521-201708, content of 95.1%);
glycyrrhizic acid (Sichuan Uygur Biotechnology Co., ltd., batch number: wkq19011705, content 98%);
acetonitrile and phosphoric acid are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity;
negative sample missing information: radix asparagi deficiency control extract (manufactured by Sichuan New green pharmaceutical technology development Co., ltd., lot number: EDT 200911);
radix Ophiopogonis deficiency control extract (manufactured by Sichuan New green pharmaceutical technology development Co., ltd., batch number: EDT 200912);
radix Trichosanthis deficiency control extract (manufactured by Sichuan New green pharmaceutical technology development Co., ltd., lot: EDT 200913);
radix Scutellariae deficiency control extract (manufactured by Sichuan green pharmaceutical technology development Co., ltd.; batch number: EDT 200914);
rhizoma anemarrhenae deficiency control extract (manufactured by Sichuan New green pharmaceutical technology development Co., ltd., batch number: EDT 200915);
lotus leaf deficiency control extract (manufactured by Sichuan green pharmaceutical technology development Co., ltd., batch number: EDT 200916);
the reference extract of radix Ginseng (manufactured by Sichuan green pharmaceutical technology development Co., ltd., batch number: EDT 200916);
radix Glycyrrhizae deficiency control extract (manufactured by Sichuan green pharmaceutical technology development Co., ltd.; batch number: EDT 200916);
erdong decoction control extract (manufactured by Sichuan New Green pharmaceutical technology development Co., ltd., lot number: EDT200901, EDT200902, EDT200903, EDT200904, EDT200905, EDT200906, EDT200907, EDT200908, EDT200909, and EDT 200910).
Example 1
The embodiment provides a multi-component quality detection method of a Erdong decoction extract, which comprises the following steps:
A. preparing a reference substance solution: taking appropriate amount of mangiferin, hyperin, baicalin and glycyrrhizic acid reference substances, precisely weighing, adding 70% methanol to prepare mixed solution containing mangiferin, hyperin and glycyrrhizic acid 50 μg per 1ml, and baicalin 100 μg per 1ml as mixed reference substance solution;
B. sample solution preparation: taking about 0.5g of Erdong decoction control extract, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 70% methanol, sealing, weighing, performing ultrasonic treatment (power 220W, frequency 50 kHz) for 30 minutes, weighing again, supplementing the lost mass with 70% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate;
C. high performance liquid chromatography assay: respectively sucking 10 μl of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for data measurement;
D. determination of relative correction factor values: according to the peak areas of mangiferin, hyperin, baicalin and glycyrrhizic acid measured by the reference substance solution, substituting the peak areas into a relative correction factor to calculate a formula: relative correction factor (f) =a s *C r /C s *A r A in the formula S Peak area for internal standard substance; a is that r Peak area as control; c (C) S Is the concentration of the internal standard substance; c (C) r Taking baicalin as an internal standard for the concentration of the reference substance, and respectively calculating to obtain relative correction factors of mangiferin, hyperin and glycyrrhizic acid relative to baicalin;
the relative retention time of mangiferin, hyperin and glycyrrhizic acid is calculated by taking baicalin reference substance and the corresponding peak of the retention time as S1 peak, and the relative retention time is within + -10% of the specified value (if the relative retention time deviates by more than 10%, the corresponding replaced reference substance is used for confirmation). 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 sample solution into the linear regression equation to obtain the content of the baicalin component in the sample, and multiplying correction factors by the baicalin peak area serving as a reference to calculate the content of mangiferin, hyperin and glycyrrhizic acid.
The data obtained in Table 1 and the relative correction factors were combined, and the contents of mangiferin, hyperin and glycyrrhizic acid were calculated by multiplying the correction factors by the peak areas of baicalin as a control.
Example 2
This example is a study of the chromatographic condition selection and the system suitability test-detection wavelength in step C of example 1.
Full-wavelength spectrum acquisition is carried out on the mangiferin, hyperin, baicalin and glycyrrhizic acid reference substance solution, and the result is shown in figures 1-4 through analysis of the spectrogram.
As can be seen from FIGS. 1-4, mangiferin and hyperin in the Erdong decoction control extract have good absorption at 360nm wavelength, baicalin and glycyrrhizic acid have good absorption at 254nm wavelength, and the detection wavelength is finally determined by comprehensively considering: 360nm for 0-16 min and 254nm for 16-40 min.
Example 3
This example shows the selection of chromatographic conditions and the investigation of chromatographic conditions in step C of example 1.
Three chromatographic conditions are designed by combining the prior common chromatographic conditions and the initial judgment of the material characteristics, and the specific conditions are as follows:
scheme one: acetonitrile as mobile phase a and 0.1% phosphoric acid water as mobile phase B, and gradient elution was performed as specified in table 2 below; the flow rate is 1.0mL per minute; the column temperature is 30 ℃; detection wavelength: 254nm, the result is shown in FIG. 5.
Table 2: scheme one gradient elution table
Under the chromatographic conditions, the peak time was late and the peaks were not separated, and the chromatographic conditions were examined again.
Scheme II: acetonitrile as mobile phase a and 0.1% phosphoric acid water as mobile phase B, and gradient elution was performed as specified in table 3 below; the flow rate is 1.0mL per minute; the column temperature is 30 ℃; detection wavelength: 254nm, the result is shown in FIG. 6.
Table 3: gradient elution table in scheme two
Under this chromatographic condition, the chromatographic peak separation degree and symmetry are not feasible, and the chromatographic condition is required to be optimized again.
Scheme III: octadecylsilane chemically bonded silica was used as a filler (column length: 250mm, inner diameter: 4.6mm, particle size: 5 μm), acetonitrile was used as mobile phase A, and 0.1% phosphoric acid water was used as mobile phase B, and gradient elution was performed as specified in Table 4 below; the flow rate is 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 three
From the above, the theoretical plate number, the degree of separation and the symmetry of the target peak are all better under the chromatographic condition of the scheme, so the chromatographic condition is selected as the mobile phase of the quality detection method of the Erdong decoction control extract.
In summary, the chromatographic conditions and system adaptations are: octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, granularity is 5 μm), acetonitrile is used as a mobile phase A, 0.1% phosphoric acid water is used as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 1.0ml per minute; the column temperature is 30 ℃; detection wavelength: 360nm for 0-16 min, 254nm for 16-40 min, and theoretical plate number is not lower than 5000 calculated by baicalin hydrochloride.
Example 4
This example is a methodological investigation-specific test of the invention.
Preparation of test solution: test solutions of the control extracts of Erdong decoction were prepared according to the experimental conditions set forth in example 1.
Preparation of a reference solution for a control: taking appropriate amount of mangiferin, hyperin, baicalin and glycyrrhizic acid reference substances, precisely weighing, adding methanol to obtain mixed solution containing mangiferin, hyperin and glycyrrhizic acid 50 μg per 1ml, and baicalin 100 μg per 1ml as mixed reference substance solution. In the preparation method of the reference substance solution, the reference substances are prepared into different concentrations, so that the peak area is convenient to calculate, and the content is convenient to calculate.
Preparation of negative control solution: an asparagus control extract negative solution was prepared according to the experimental conditions outlined above.
Preparation of negative control solution: the negative solution of the radix ophiopogonis control extract is prepared according to the experimental conditions set forth above.
Preparation of negative control solution: a negative solution of the control extract of trichosanthes kirilowii (trichosanthes kirilowii Maxim) was prepared according to the experimental conditions set forth above.
Preparation of negative control solution: according to the experimental conditions, preparing negative solution of radix Scutellariae lack control extract.
Preparation of negative control solution: negative solutions of the control extract of Anemarrhena asphodeloides were prepared according to the experimental conditions outlined above.
Preparation of negative control solution: according to the experimental conditions set forth above, a lotus leaf lack control extract negative solution is prepared.
Preparation of negative control solution: according to the experimental conditions set forth above, a negative solution of the reference extract of the ginseng-deficient is prepared.
Preparation of negative control solution: a licorice (licorice) deficient control extract negative solution was prepared according to the experimental conditions set forth above.
And detecting according to a proposed method. The results are shown in fig. 8, wherein 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 method has good specificity.
Example 5
This example is a methodology investigation-precision investigation of the embodiment of example 1.
The control solution was sampled 6 times continuously, the peak areas of the components were recorded, and the RSD values were calculated, and the results are shown in table 5.
Table 5: precision investigation results
From the results shown in Table 5, it was found that the peak area RSD values of the respective components in the precision examination were all less than 0.93%, and the precision of the apparatus was good.
Example 6
This example is a methodology-reproducibility study of the solution of example 1.
0.5g of the same sample (batch number: EDT 200901) is taken, 6 parts of the sample are precisely weighed, the same operator prepares a sample solution according to a planned method, the content of each component in the 6 parts of the sample is calculated, and the result is shown in Table 6.
Table 6: results of the repeatability experiments
The result shows that the RSD values of the contents of mangiferin, hyperin, 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 study of the embodiment of example 1.
And taking mother liquor of the mixed reference substance of mangiferin, hyperin, baicalin and glycyrrhizic acid, and then respectively diluting step by step to obtain corresponding mixed reference substance solution. 10 mu L of the solution is respectively injected into a liquid chromatograph, the peak area is obtained by analysis, the concentration (mu g/mL) is taken as an abscissa, the peak area (Y) is taken as an ordinate, and a response curve is drawn, and the results are shown in tables 7-10 and figures 9-12.
Table 7: analysis results of mangiferin standard curve
Table 8: hyperin standard curve analysis results
Table 9: baicalin standard curve analysis result
Table 10: glycyrrhizic acid standard curve analysis result
From the above examination results, it was found that each component exhibited a good linear relationship in the respective sample injection amount range, and the linear relationship examination results were as shown in table 11.
TABLE 11
Example 8
This example is a methodological investigation-stability experiment of the technical scheme of example 1.
About 0.5g of the same sample solution (batch No. EDT 200901) was taken, the sample solution was prepared, and the chromatographic peak areas of the respective components were measured at 0, 2h, 4h, 8h, 12h and 24h, respectively, and the results are shown in Table 12.
Table 12:
the results show that: under the experimental conditions, the peak area RSD values of all the components are qualified, and the stability of the sample solution is good within 24 hours.
Example 9
This example is a methodology of the protocol of example 1 for examining-sample recovery.
About 0.25g of a test sample (batch number: EDT 200901) with known content is taken, 6 parts are taken, precisely weighed, a certain amount of reference substances are respectively and precisely added, the preparation and measurement of a test sample solution are carried out according to a planned method, and the recovery rate is calculated, and the result is shown in Table 13.
Table 13: sample recovery rate experimental results
The result shows that the RSD value of the recovery rate result of each component content is qualified, and the method has good accuracy.
Example 10
The embodiment is a methodological investigation-an investigation by different instruments
Based on the experimental conditions set forth in the foregoing examples, the two winter soup control extracts (lot number EDT 200901) were precisely weighed, sample solutions were prepared, and measured on Waters2695-2998, agilent 1260, and Shimadzu LC-20AD type HPLC (columns were Agilent 5 TC-C18.times.4.6mm 5 μm), respectively, and the contents of the respective components were calculated. The results are shown in Table 14.
Table 14: investigation of results by different instruments
The results show that when the 3 instruments are used for detecting the test samples, the RSD values of the contents of the components are qualified, and the method has good durability.
Example 11
This example is a methodology-durability study.
The same test sample (batch No. EDT 200901) was tested using different brands of chromatographic columns (Waters XBridge C18.6X250 mm 5.0 μm, agilent 5 TC-C18.6X250 mm 5 μm, kromasil 100-5-C18.6X250 mm 5.0 μm) and the results are shown in Table 15.
Table 15: durability inspection results
The result shows that the RSD values of the contents of the components are qualified, and the chromatographic column has good durability.
Example 12
This example is a further examination and determination of the relative correction factor in example 1.
Calculating relative correction factors of components to be tested:
taking mixed reference substance solution, and carrying out sample injection for 1, 2, 5, 10, 15 and 20 mu L measurement, wherein baicalin is used as an internal standard, and relative correction factors of mangiferin, hyperin and glycyrrhizic acid are calculated respectively, and the calculation formula is as follows: relative correction factor (f) =a s *C r /C s *A r In which A S Peak area for internal standard substance; a is that r Peak area as control; c (C) S Is the concentration of the internal standard substance; c (C) r As a reference substanceIs a concentration of (3). The results are shown in tables 16 and 17.
Table 16: peak area data table for mixed reference substances
Table 17: relative correction factor calculation result table
Further, durability inspection needs to be performed on the relative correction factors obtained by the scheme.
3 different brands of chromatographic columns (Waters were examined using Agilent type 1260
C18 4.6X105 mm 5.0 μm, agilent 5 TC-C18.6X1250 mm 5 μm, kromasil 100-5-C18.6X1250 mm 5.0 μm); the effect of Waters e2695-2998, agilent 1260 and Shimadzu LC-20AD type high performance liquid chromatography on correction factors was examined by using an Agilent 5TC-C18 chromatographic column, and the results are shown in Table 18.
Table 18: relative correction factor reproducibility investigation
The results show that the relative positive factors of the components are well durable.
Determination of the relative correction factor:
referring to technical guidelines established by the one-test multiple evaluation method, each factor affecting the relative correction factor is examined, and in the case that the RSD value is less than 5%, the average value of the relative correction factors obtained by each test is taken, and the result is shown in table 19 below.
Table 19: summary of relatively positive factor results
As is clear from Table 19, the relative correction factors of mangiferin, hyperin and glycyrrhizic acid were finally determined to be 0.65, 0.55 and 1.42, respectively.
Positioning of the chromatograph to be measured: the positioning of each component to be measured was performed by calculating the relative retention time of each component to be measured chromatographic peak and baicalin chromatographic peak in different chromatographic instruments and different chromatographic columns, and the results are shown in table 20.
Table 20: relative retention time of the ingredients
The results show that the relative retention time fluctuation among chromatographic peaks of each component is small, and the RSD% value is less than 3%.
Comparing the measurement results of the multi-evaluation method with those of the external standard method:
the contents of mangiferin, hyperin, baicalin and glycyrrhizic acid in the Erdong decoction extract were calculated by external standard method and one-test-multiple evaluation method, and the measurement results are shown in Table 21.
Table 21: comparing the one-test multi-evaluation method with the external standard method
The result shows that the actual measurement value of the conventional external standard method has no significant difference with the content value calculated by the one-measurement-multiple-evaluation method, thereby indicating that the one-measurement-multiple-evaluation method is applicable to the quality evaluation of multiple index components of the Erdong decoction.
The result shows that the actual measurement range of the total amount of mangiferin, hyperin, 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 average value plus or minus 3 times SD is 1.060% -1.240%. According to the method of reasonably determining the upper limit and the lower limit of the quality requirement of the relevant quality control project in the quality standard in the declaration data requirement (solicited opinion manuscript) of the substance standard of the traditional Chinese medicine compound preparation of the ancient classical prescription, namely the fluctuation range of the content measurement is generally not more than 70% -130% of the mean value, the total content of baicalin, mangiferin, hyperin and glycyrrhizic acid of the product is obtained to be 0.805% -1.494%.
Example 13
This example is a further optimization based on the embodiment 1, and in the step B, the preparation method of the second winter soup control extract comprises the following steps:
a1: the preparation method comprises the steps of (1) taking decoction pieces according to the weight parts of 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;
b1: placing all decoction pieces obtained in the step a1 in a decocting container, adding 8 times of water, soaking for 30min, and decocting for 30min; adding 6 times of water for the second time, decocting for 30min, decocting for 2 times, filtering, mixing filtrates, and lyophilizing to obtain Erdong decoction control extract.
By adopting the preparation method of the Erdong decoction control extract in the embodiment, the obtained Erdong decoction control extract freeze-dried powder has stable property and more accurate content detection result.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (6)
1. A multi-component quality detection method of Erdong decoction extract is characterized by comprising the following steps:
A. preparing a reference substance solution: taking appropriate amount of mangiferin, hyperin, baicalin and glycyrrhizic acid reference substance, adding methanol as solvent to obtain mixed reference substance solution containing mangiferin, hyperin, baicalin and glycyrrhizic acid reference substance;
B. sample solution preparation: adding methanol as solvent into the Erdong decoction control extract to obtain test solution;
C. high performance liquid chromatography assay: respectively sucking the reference substance solution in the step A and the sample solution to be tested in the step B, and injecting into a high performance liquid chromatograph to perform data measurement;
D. determination of relative correction factor values: according to the peak areas of mangiferin, hyperin, baicalin and glycyrrhizic acid measured by the reference substance solution, substituting the peak areas into a relative correction factor to calculate a formula: relative correction factors (f) =as×cr/cs×ar, and baicalin is used As an internal standard, and relative correction factors of mangiferin, hyperin and glycyrrhizic acid relative to baicalin are calculated respectively;
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 control 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 multiplying correction factors by the peak area of baicalin serving as a reference to calculate the content of mangiferin, hyperin and glycyrrhizic acid;
in the step C, the measurement conditions of the high performance liquid chromatograph are as follows:
chromatographic column: octadecylsilane chemically bonded silica is used as a filler, the column length is 250mm, the inner diameter is 4.6mm, and the granularity is 5 mu m;
the column temperature is 30 ℃;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is carried out;
the gradient elution process comprises the following steps:
0-2 min, 10-15% of mobile phase A and 90-85% of mobile phase B;
2-12 min, 15-25% of mobile phase A and 85-75% of mobile phase B;
12-32 min, 25-50% of mobile phase A and 75-50% of mobile phase B;
32-40 min, 50-95% of mobile phase A and 50-5% of mobile phase B.
2. The method for detecting the multi-component quality of the Erdong decoction extract according to claim 1, wherein in the step A, the specific preparation method of the reference substance solution is as follows: taking appropriate amount of mangiferin, hyperin, baicalin and glycyrrhizic acid reference substances, precisely weighing, adding 70% methanol to obtain mixed solution containing mangiferin, hyperin and glycyrrhizic acid 50 μg per 1ml, and baicalin 100 μg per 1ml, and taking as mixed reference substance solution to obtain Erdong decoction reference substance solution.
3. The method for detecting the multi-component quality of the Erdong decoction extract according to claim 1, wherein 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 into a conical bottle with a plug, precisely adding 25mL of 70% methanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, weighing again, supplementing the reduced mass with 70% methanol, shaking uniformly, filtering, and collecting the subsequent filtrate to obtain the Erdong decoction control extract sample solution.
4. The method for multi-component quality detection of a Erdong decoction extract according to claim 1, 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.
5. The multi-component quality detection method of the Erdong decoction extract according to claim 1, which is characterized in that: in the step E, the total amount of the mangiferin, hyperin, baicalin and glycyrrhizic acid is measured to be 0.805% -1.494%.
6. The multi-component quality detection method of the Erdong decoction extract according to claim 1, wherein in the step B, the preparation method of the Erdong decoction control extract comprises the following steps:
a1: the preparation method comprises the steps of (1) taking decoction pieces according to the weight parts of 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;
b1: placing all decoction pieces obtained in the step a1 in a decocting container, adding 8 times of water, soaking for 30min, and decocting for 30min; adding 6 times of water for the second time, decocting for 30min, decocting for 2 times, filtering, mixing filtrates, and lyophilizing to obtain Erdong decoction control extract.
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| 壳聚糖絮凝沉降法与乙醇沉淀法对二冬汤精制的对比研究;陈以新;盛华刚;林桂涛;冯金鹰;;中成药(第04期);第612-615页 * |
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