CN115901986B - Method for measuring contents of 6 components in ash bark formula particles by one-measurement-multiple-evaluation method - Google Patents
Method for measuring contents of 6 components in ash bark formula particles by one-measurement-multiple-evaluation method Download PDFInfo
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- 238000011156 evaluation Methods 0.000 title claims abstract description 23
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- 238000001514 detection method Methods 0.000 claims abstract description 13
- 239000012488 sample solution Substances 0.000 claims abstract description 12
- 238000004364 calculation method Methods 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 6
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 claims description 51
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 claims description 51
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
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- 239000013558 reference substance Substances 0.000 claims description 30
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- YDZWHGJRWMQCDP-NKILCQAGSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4ar,6ar,6bs,8as,12as,14ar,14br)-8a-carboxy-4,4,6a,6b,11,11,14b-heptamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3-hydroxy-4-[(2s,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-5-[(2s,3r,4 Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(O)=O)C(O)=O)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YDZWHGJRWMQCDP-NKILCQAGSA-N 0.000 claims description 29
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
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- 239000003814 drug Substances 0.000 abstract description 8
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- RODXRVNMMDRFIK-UHFFFAOYSA-N scopoletin Chemical compound C1=CC(=O)OC2=C1C=C(OC)C(O)=C2 RODXRVNMMDRFIK-UHFFFAOYSA-N 0.000 description 2
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- 241000116639 Ferula stylosa Species 0.000 description 1
- 241000565359 Fraxinus chinensis Species 0.000 description 1
- 241000588214 Fraxinus chinensis subsp. rhynchophylla Species 0.000 description 1
- 241000565391 Fraxinus mandshurica Species 0.000 description 1
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- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The application discloses a method for measuring the content of 6 components in ash bark formula particles by adopting a one-measurement-multiple-evaluation method, and belongs to the technical field of traditional Chinese medicine component detection. The method for measuring the content of 6 components in the ash bark formula particle by using the multi-evaluation method comprises the following steps: preparing a sample solution, preparing a reference solution, calculating a relative correction factor, calculating the content of a target compound, and analyzing the relative correction factor and the content of the target compound obtained by calculation. The method for measuring the content of 6 components in the ash bark formula particles by the one-measurement multi-evaluation method simplifies the quantitative detection method of the ash bark formula particles, saves the detection economic cost of quality control of the ash bark formula particles, and has more stable and controllable detection result.
Description
Technical Field
The application relates to a method for measuring the content of 6 ingredients in ash bark formula particles by a multi-evaluation method, and belongs to the technical field of traditional Chinese medicine ingredient detection.
Background
Cortex Fraxini is dried branch bark or dry bark of Fraxinus chinensis Fraxinus rhynchophylla Hance, fraxinus chinensis F.chinensis Roxb. Of Fraxinus of Oleaceae, fraxinus acutifolia Fraxinus szaboana Lingelsh. Or Fraxinus mandshurica F.stylosa Lingelsh. Of Fraxinaceae. Has effects of clearing heat, eliminating dampness, astringing, relieving dysentery, relieving leukorrhagia, and improving eyesight. It is mainly used for treating damp-heat diarrhea, leukorrhagia, itching, conjunctival congestion, swelling and pain due to liver heat, and nebula.
A multi-evaluation method (QAMS) is a quality control method which only measures a certain representative component (effective, cheap and easy to obtain) and can calculate the content of other effective components to be measured so that the calculated value and the actual value meet the quantitative methodology requirement. The one-measurement multi-evaluation method can better solve the bottleneck problem of lack of a reference substance in the quality control of the traditional Chinese medicine, and can better complete quantitative evaluation of unstable components.
At present, the related literature for evaluating the quality of Chinese medicinal materials by adopting a one-test-multiple-evaluation method is less, and particularly, the report of simultaneously measuring the contents of various different types of components in the ash bark formula particles by adopting the one-test-multiple-evaluation method is not seen. Although the UPLC method is reported to measure the contents of seven components (aesculin, salidroside, aesculin, syringin, aesculin, and scopoletin) in the ash bark formula particle simultaneously (see Mu Xiyan, etc., the UPLC method is used for measuring 7 components [ J ] in ash bark formula particle simultaneously, journal of Chinese experimental prescription, 2017,23 (11): 63-67 ]), the method has the advantages of multiple standard products, complex content measuring method and higher detection economic cost, and limits the application of controlling the quality in practical work by measuring the content of main active components of ash bark formula particle.
Disclosure of Invention
In order to solve the problems, a method for measuring the content of 6 components in the ash bark formula particles by a multi-evaluation method is provided, the quantitative detection method of the ash bark formula particles is simplified, the detection economic cost of quality control of the ash bark formula particles is saved, and the detection result is more stable and controllable.
The invention adopts the following technical scheme:
the method for measuring the content of 6 components in the ash bark formula particle by using the multi-evaluation method comprises the following steps:
(1) Preparation of test solution: taking appropriate amount of cortex Fraxini granule, grinding, adding methanol, ultrasonic treating, cooling, shaking, filtering, and collecting filtrate;
(2) Preparation of a control solution: accurately weighing appropriate amounts of fraxinin reference substance, syringin reference substance, fraxinin reference substance and caulis Akebiae phenethyl alcohol glycoside B reference substance, adding methanol, dissolving, and fixing volume to obtain mixed reference substance solution;
(3) Calculation of the relative correction factor: injecting the mixed reference substance solution in the step (2) into a high performance liquid chromatograph to obtain peak areas of aesculin and other components to be detected in the mixed reference substance solution, and respectively calculating relative correction factors of syringin, aesculin and akebia phenyl ethanol glycoside B by taking the aesculin as an internal reference substance;
(4) Calculating the content of the target compound: injecting the sample solution in the step (1) into a high performance liquid chromatograph to obtain peak areas of components to be detected in the sample solution, and respectively calculating the contents of aesculin, syringin, aesculin and akebia phenyl ethanol glycoside B by combining the relative correction factors in the step (3);
(5) Analyzing the relative correction factors and the content of the target compound obtained through the calculation in the steps (3) and (4).
Optionally, the conditions of high performance liquid chromatography in steps (3) and (4) are:
Chromatographic column: WATERS ATLANTIS T3 or Inertsil ODS-3; detection wavelength: 220nm; acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, elution was performed according to the following gradient elution procedure:
optionally, the flow rate is 0.9-1.1ml/min;
The column temperature is 29-31 ℃;
The theoretical plate number is not less than 2000 calculated according to the peak of aesculin.
Optionally, the chromatographic column has a specification of 4.6mm x 250mm,5 μm;
The instrument is Agilent 1260 high performance liquid chromatograph or Waters e2695 high performance liquid chromatograph.
Optionally, the relative retention times and relative correction factors of aesculin, syringin, aesculin and akebia phenylethanoid glycoside B are as follows:
optionally, the preparation of the control solution in step (2) is: precisely weighing appropriate amounts of aesculin, syringin reference substance, fraxinin reference substance, and caulis Akebiae phenethyl alcohol glycoside B reference substance, adding methanol to obtain mixed reference substance solutions with concentration of 1523.43 μg/ml,131.23 μg/ml,632.32 μg/ml,298.30 μg/ml,202.30 μg/ml,332.37 μg/ml, and diluting by multiple dilution method by 2, 4, 8, and 16 times respectively.
Optionally, the preparation of the sample solution in step (1) is: taking 0.3-0.7g of ash bark formula particles, grinding, placing into a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 30-60 minutes, cooling, shaking uniformly, filtering, and taking the subsequent filtrate.
Optionally, the time of the ultrasonic treatment is 30 minutes, the power of the ultrasonic treatment is 250W, and the frequency is 40kHz.
Optionally, the step (5) specifically includes: combining the high performance liquid chromatogram of the sample solution with the relative correction factors of syringin, fraxinin and akebia stem phenethyl alcohol glycoside B, and comparing the calculated value of the one-test multi-evaluation method with the measured value of the external standard method by a correlation coefficient method and a t-test method.
The beneficial effects of the application include, but are not limited to:
The method for measuring the content of 6 components in the ash bark formula particles by using the one-measurement multi-evaluation method is quick and accurate, greatly saves time and capital cost, and has higher reproducibility, stability and reliability; according to the method, aesculin is taken as an internal reference, relative correction factors between aesculin and syringin, aesculin and akebia stem phenethyl alcohol glycoside B are established, the contents of aesculin, syringin, aesculin and akebia stem phenethyl alcohol glycoside B are calculated through the relative correction factors, a comprehensive quality standard of the controllable aesculin formula granule is established, the detection result is more stable and controllable, and the safety and effectiveness of clinical medication are ensured; the method is simple, convenient, rapid, comprehensive, accurate, high in sensitivity and good in stability, and has good application prospect in multi-index quality control of the ash bark formula particles.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:
fig. 1 is a comparison of reflux and ultrasonic extraction in accordance with the present application.
Fig. 2 is a specific chromatogram according to the present application.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The raw materials or instruments used are conventional products available commercially without identifying the manufacturer.
1. Instrument and reagent
1.1 Instrument:
Agilent 1260 hplc, waters e2695 hplc, BSA224S-CW electronic balance (cerdos instruments (beijing) inc.), XS105 electronic balance (METTLER TOLEDO), KDM adjustable temperature electrothermal jacket (shandong, city Hua Lu electrothermal inc.).
1.2 Chromatography column:
Waters Atlantis T3，SN：01893110314080(4.6×250mm，5μm)。
Waters Atlantis T3，SN：01893110314030(4.6×250mm，5μm)。
Inertsil ODS-3，SN：21D0201379(4.6×250mm，5μm)。
1.3 reagents:
QinPijia extract control (lot number: 110740-201606, chinese food and drug inspection institute, purity: 92.4%).
Syringin reference (lot number: 111574-202106, purity: 94.3% of national food and drug verification institute).
A control of fraxinin (lot number: 170194-202205, shanghai Yongsheng biotechnology Co., ltd., purity: 98.0%).
Qinpi B extract reference (lot No. 110741-201708, purity 99.9%) of China food and drug inspection institute.
The fraxins reference (lot number: 111731-202103, purity: 97.9% of China food and drug inspection institute).
Akebia phenylethanoid glycoside B control (lot number: 130031-202201, shanghai Yongsheng biosciences Co., ltd., purity: 98.0%).
Freeze-dried powder of ash bark standard decoction ：180701、200401、20080011、18112003、201810012、20201201、13102511、201810011、190201、20180301、20201101、180601、20080403、18021303、20090301.
Cortex Fraxini granule (lot number 2010001, 2010002, 2010003, shandong Hongji Tang pharmaceutical Co., ltd.).
1.4 Reagent:
Acetonitrile and phosphoric acid are chromatographic purity, methanol is analytically pure, and water is purified water of the Chen's type.
2. The method for measuring the content of 6 components in the ash bark formula particle by using the multi-evaluation method comprises the following steps:
2.1 liquid chromatography conditions
Acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, elution was performed according to the gradient elution procedure of table 1:
TABLE 1
The flow rate is 1ml/min; detection wavelength: 220nm; the column temperature is 30 ℃; the theoretical plate number is not less than 2000 calculated according to the peak of aesculin.
2.2 Preparation of sample solutions
Taking 0.5g of ash bark formula particles, grinding, placing into a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 30 minutes (power 250W and frequency 40 kHz), cooling, shaking uniformly, filtering, and taking subsequent filtrate.
2.2.1 Extraction method investigation
Taking appropriate amount of cortex Fraxini granule (2010001), respectively heating and refluxing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 min to obtain test solution, and injecting into liquid chromatograph for measurement, and calculating the content of aesculin, syringin, aesculin, and akebia stem phenethyl alcohol glycoside B, wherein the result shows (see Table 2 and figure 1) that the content of each component to be tested is not greatly different, so that the selected extraction mode is ultrasonic extraction.
Table 2 extraction method investigation results
2.2.2 Extraction solvent investigation
The method comprises the steps of respectively preparing a test solution by adopting methanol, 30% methanol, 50% methanol and 70% methanol through ultrasonic, measuring the peak area of the test solution, calculating the contents of aesculin, syringin, aesculin, akebia stem phenethyl alcohol glycoside B, and obtaining the results (see table 3), wherein the extraction of each component to be detected is relatively complete when 50% methanol and 70% methanol are adopted for extraction, and finally, the extraction solvent is 50% methanol.
TABLE 3 comparison of different extraction solvents
2.2.3 Investigation of extraction time
The ultrasonic time is respectively set to be 30 minutes, 45 minutes and 60 minutes, a test solution is prepared, the peak area of the test is measured, the contents of aesculin, syringin, aesculin and akebia phenylethanoid glycoside B are calculated, and the result shows (see table 4) that the ultrasonic time has little influence on the contents of all components to be detected, so that the ultrasonic time is selected to be 30 minutes.
TABLE 4 extraction time investigation results
2.2.4 Investigation of the sample size
About 0.3g, 0.5g and 0.7g of ash bark formula particles are respectively taken, precisely weighed, a test solution is prepared, and is injected into a high performance liquid chromatograph for measurement, and the contents of ash bark extract, syringin, ash bark glycoside, ash bark extract and akebia stem phenylethanoid glycoside B are calculated, and the result shows (see table 5) that different sample amounts have little influence on the contents of the components to be measured, and finally the sample amount is selected to be 0.5g.
Table 5 sample weighing investigation results
2.3 Preparation of control solution
Precisely weighing appropriate amounts of aesculin, syringin reference substance, fraxinin reference substance, and caulis Akebiae phenethyl alcohol glycoside B reference substance, adding methanol to obtain mixed reference substance solutions with concentration of 1523.43 μg/ml,131.23 μg/ml,632.32 μg/ml,298.30 μg/ml,202.30 μg/ml,332.37 μg/ml, and diluting by multiple dilution method by 2, 4, 8, and 16 times respectively.
2.4 Methodological verification
2.4.1 Linear relationship investigation
And precisely sucking 10 mu l of each mixed reference substance solution under the item "2.3", injecting into an ultra-high performance liquid chromatograph, measuring the chromatographic peak area of each component to be measured, and carrying out linear regression by taking the peak area as an ordinate and the concentration as an abscissa, wherein the result is shown in Table 6.
Table 66 Linear relation and Linear Range of the ingredients
2.4.2 Specificity investigation
Taking a proper amount of auxiliary materials (dextrin) used by the ash bark formula particles, and preparing a negative control solution according to the preparation method of the test solution under the item 2.2; taking the sample solution of the ash bark prescription granule, the ash bark extract, the syringin, the ash bark glycoside, the ash bark extract, the akebia stem phenethyl alcohol glycoside B mixed reference substance solution, the negative control solution and the blank solvent to obtain the chromatogram thereof according to the chromatographic condition of 2.1 item, and the result shows that (see figure 2), the dextrin negative sample and the blank solvent have no chromatographic peak at the retention time of the ash bark extract, the syringin, the ash bark extract and the akebia stem phenethyl alcohol glycoside B, and the method has good specificity.
2.4.3 Repeatability test
0.5G of ash bark formula particles are taken, ground, precisely weighed, 6 parts are taken, a test solution is prepared according to the method under the item "2.2", and then the sample is sampled and measured according to the chromatographic condition under the item "2.1", and the result is shown in Table 7. The RSD of the content of the aesculin is 0.22%, the RSD of the content of the syringin is 0.15%, the RSD of the content of the aesculin is 0.22%, the RSD of the content of the aesculin is 0.97%, the RSD of the content of the aesculin is 2.59%, the RSD of the content of the akebia stem phenylethanoid glycoside B is 0.24%, the repeatability is good, and the analysis requirements are met.
TABLE 7 repeatability test
2.4.4 Intermediate precision
Different analysts use another high performance liquid chromatograph at different time to perform repeatability test, the result is shown in table 8, the RSD of the content of the aesculin in the ash bark formula granule is measured to be 0.37% by different instruments, the RSD of the syringin content is 0.21%, the RSD of the ash bark content is 0.68%, the RSD of the ash bark B content is 2.62%, the RSD of the ash bark content is 2.43%, and the RSD of the akebia stem phenylethanoid glycoside B content is 0.67%, which meet the analysis requirements.
TABLE 8 intermediate precision results
2.4.5 Recovery test
Taking about 0.25g of ash bark formula granule powder, precisely weighing, respectively adding appropriate amounts of ash bark extract, syringin, ash bark glycoside, ash bark extract and akebia stem phenylethanoid glycoside B reference substance solution, preparing a sample solution according to a method under the item of 2.2, carrying out sample injection according to a chromatographic condition under the item of 2.1, measuring the content of 6 components, and calculating the recovery rate. The results show (see tables 9-14) that the recovery rate of the aesculin is 98.17-101.17%, the recovery rate of the syringin is 95.85-100.36%, the recovery rate of the aesculin is 95.04-98.88%, the recovery rate of the aesculin is 98.35-101.52%, the recovery rate of the aesculin is 98.85-104.53%, the recovery rate of the akebia phenyl ethanol glycoside B is 92.67-100.09%, and the analysis requirements are met.
TABLE 9 fraxinin recovery test
TABLE 10 syringin recovery test
TABLE 11 test of recovery of Fraxini
Table 12 fraxinin recovery test
TABLE 13 fraxinin recovery test
TABLE 14 test of recovery of Phenylethanoid glycoside B
2.4.6 Stability investigation
The content of the components to be detected in the sample is measured by adopting repeated sample solutions for one time at 0h, 2h, 4h, 8h, 12h and 24h respectively, and the result shows (see table 15) that the RSD of the aesculin content is 0.26%, the RSD of the syringin content is 1.22%, the RSD of the aesculin content is 0.26%, the RSD of the aesculin content is 0.34%, the RSD of the aesculin content is 1.68%, the RSD of the akebia phenylethanoid glycoside B content is 0.65%, and the solution stability is good and meets the analysis requirement.
Table 15 sample stability test
2.5 Relative correction factor calculation
Taking the mixed reference solution under the item "2.3", injecting into a liquid chromatograph, measuring, taking fraxinin as an internal reference, and calculating relative correction factors of syringin, fraxinin and akebia stem phenethyl alcohol glycoside B, wherein the results are shown in Table 16.
Wherein A s is the peak area of the internal reference substance, W S is the concentration (or mass) of the internal reference substance, A k is the peak area of the component to be detected, and W k is the concentration (or mass) of the component k to be detected.
TABLE 16 relative correction factor
2.6 Relative correction factor reproducibility investigation
2.6.1 Different instruments and different chromatographic column surveys
The relative correction factors are measured by different instruments and different chromatographic columns respectively, the result is shown in table 17, and the measured relative correction factor RSD value is between 1.82% and 2.83%, which shows that the relative correction factors of the components to be measured have good reproducibility between different instruments and different chromatographic columns.
TABLE 17 influence of different instruments and different chromatographic columns on the relative correction factors of the components to be tested
2.6.2 Different flow Rate investigation
The relative correction factors are measured by adopting different flow rates respectively, and the result is shown in a table 18, wherein the measured relative correction factor RSD value is between 0.00% and 1.21%, which shows that the relative correction factors of the components to be measured have good reproducibility among different flow rates.
TABLE 18 influence of different flow rates on the relative correction factors of the components to be measured
2.6.3 Different column temperature investigation
The relative correction factors are measured by adopting different column temperatures respectively, the result is shown in a table 19, and the measured relative correction factor RSD value is between 0.00% and 1.06%, which shows that the relative correction factors of the components to be measured have good reproducibility among different column temperatures.
TABLE 19 influence of different column temperatures on the relative correction factors of the components to be tested
2.7 Positioning of chromatographic peaks of Components to be measured
And respectively calculating the relative retention value (r) and retention time difference (Deltat R) of each component to be detected and the reference substance aesculin, and examining the reproducibility of the components to be detected between different instruments and different chromatographic columns, wherein the results show that (tables 20-21) the relative retention time fluctuation is small, and finally, the average value of the relative retention time values of each component to be detected in different instruments and different chromatographic columns is selected as the peak positioning basis.
TABLE 20 relative retention times of various components to be tested in different instruments and different chromatographic columns
TABLE 21 retention time differences for various components to be tested in different instruments and different chromatographic columns
2.8 Comparison of the measurement results of the one-test-multiple-evaluation method and the external Standard method
Respectively taking 15 batches of freeze-dried powder of the cortex fraxini standard decoction and 3 batches of cortex fraxini formula particles, preparing a test solution according to the method under the item "2.2", and carrying out sample injection measurement according to the chromatographic condition under the item "2.1". The contents of aesculin, syringin, aesculin, and akebia phenylethanoid glycoside B were calculated by a one-test-multiple-evaluation method (QASM method) and an external standard method (ESM method), respectively, and the results are shown in Table 22. The content calculated by the external standard method and the one-measurement-multiple-evaluation method is compared by t test, and P is far more than 0.05, so that no obvious difference exists between the two contents measured by the two methods, and the relative error between the two contents is less than 3%, so that the established one-measurement-multiple-evaluation method has good accuracy, and can be used for measuring the content of ash bark formula particles and standard decoction.
TABLE 22 one-measurement-multiple-evaluation method and external standard method for measuring 6 component contents
The above description is only an example of the present application, and the scope of the present application is not limited to the specific examples, but is defined by the claims of the present application. Various modifications and variations of the present application will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.
Claims (4)
1. The method for measuring the content of 6 components in the ash bark formula particles by using the multi-evaluation method is characterized by comprising the following steps of:
(1) Preparation of test solution: taking 0.3-0.7g of ash bark formula particles, grinding, placing into a conical flask with a plug, adding 50ml of 50% methanol, performing ultrasonic treatment for 30 minutes at a power of 250W and a frequency of 40kHz, cooling, shaking, filtering, and collecting the subsequent filtrate;
(2) Preparation of a control solution: precisely weighing appropriate amounts of aesculin, syringin reference substance, fraxinin reference substance, and caulis Akebiae phenethyl alcohol glycoside B reference substance, adding methanol to obtain mixed reference substance solutions with concentration of 1523.43 μg/ml,131.23 μg/ml,632.32 μg/ml,298.30 μg/ml,202.30 μg/ml,332.37 μg/ml, and diluting by multiple dilution method by 2, 4, 8, and 16 times respectively;
(3) Calculation of the relative correction factor: injecting the mixed reference substance solution in the step (2) into a high performance liquid chromatograph to obtain peak areas of aesculin and other components to be detected in the mixed reference substance solution, and respectively calculating relative correction factors of syringin, aesculin and akebia phenyl ethanol glycoside B by taking the aesculin as an internal reference substance;
(4) Calculating the content of the target compound: injecting the sample solution in the step (1) into a high performance liquid chromatograph to obtain peak areas of components to be detected in the sample solution, and respectively calculating the contents of aesculin, syringin, aesculin and akebia phenyl ethanol glycoside B by combining the relative correction factors in the step (3);
(5) Analyzing the relative correction factors and the content of the target compounds obtained by the calculation in the steps (3) and (4);
wherein, the high performance liquid chromatography conditions in the steps (3) and (4) are as follows:
chromatographic column: WATERS ATLANTIS T3 or Inertsil ODS-3; detection wavelength: 220nm; acetonitrile as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, elution was performed according to the following gradient elution procedure:
；
The column temperature is 29-31 ℃;
the theoretical plate number is not lower than 2000 calculated according to the peak of aesculin;
The relative retention times and relative correction factors of aesculin, syringin, aesculin and akebia phenylethanoid glycoside B are as follows:
。
2. the method for measuring the contents of 6 ingredients in the ash bark formulation granule according to claim 1, wherein the flow rate is 0.9-1.1ml/min.
3. The method for measuring the content of 6 components in ash bark formula particles by a one-test-multiple-evaluation method according to claim 1, wherein the specification of the chromatographic column is 4.6mm multiplied by 250mm and 5 μm;
The instrument is Agilent 1260 high performance liquid chromatograph or Waters e2695 high performance liquid chromatograph.
4. The method for measuring the content of 6 ingredients in the ash bark formulation granule according to claim 1, wherein the step (5) specifically comprises: combining the high performance liquid chromatogram of the sample solution with the relative correction factors of syringin, fraxinin and akebia stem phenethyl alcohol glycoside B, and comparing the calculated value of the one-test multi-evaluation method with the measured value of the external standard method by a correlation coefficient method and a t-test method.
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| CN109001336A (en) * | 2018-09-20 | 2018-12-14 | 哈尔滨珍宝制药有限公司 | Qinpi Extracts characteristic spectrum, its method for building up and quality determining method |
| CN109917048A (en) * | 2019-04-18 | 2019-06-21 | 江阴天江药业有限公司 | A kind of construction method of the UPLC characteristic spectrum of bark of ash medicinal material, the characteristic spectrum constructed by this method and its application |
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| CN109001336A (en) * | 2018-09-20 | 2018-12-14 | 哈尔滨珍宝制药有限公司 | Qinpi Extracts characteristic spectrum, its method for building up and quality determining method |
| CN109917048A (en) * | 2019-04-18 | 2019-06-21 | 江阴天江药业有限公司 | A kind of construction method of the UPLC characteristic spectrum of bark of ash medicinal material, the characteristic spectrum constructed by this method and its application |
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