CN110907579A - HPLC quantitative analysis method for aesculin and aesculetin in pulsatilla chinensis powder - Google Patents

HPLC quantitative analysis method for aesculin and aesculetin in pulsatilla chinensis powder Download PDF

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CN110907579A
CN110907579A CN201911224122.8A CN201911224122A CN110907579A CN 110907579 A CN110907579 A CN 110907579A CN 201911224122 A CN201911224122 A CN 201911224122A CN 110907579 A CN110907579 A CN 110907579A
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aesculetin
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aesculin
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赵玉丛
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Henan University of Animal Husbandry and Economy
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample

Abstract

The invention discloses an HPLC quantitative analysis method of aesculin and aesculetin in pulsatilla chinensis powder, which specifically comprises the following steps: the method comprises the following steps: establishing chromatographic conditions; step two: the extraction process comprises the following steps: accurately weighing Pulsatilla chinensis powder, placing into conical flask with stopper, accurately adding methanol, sealing the stopper, weighing, ultrasonically extracting, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and collecting the filtrate; step three: preparing a solution: comprises the preparation of a reference substance storage solution, a reference substance solution, a test sample solution, a negative reference solution and a reference medicinal material solution; step four: methodology investigation: the method comprises the following steps of specificity test, linear relation investigation, precision test and accuracy test; step five: and (3) sample determination: comprises the content determination of a home-made Chinese pulsatilla root powder sample and the content determination of the commercially available Chinese pulsatilla root powder. The invention has the technical effect of realizing accurate quantification of effective components of aesculin and aesculetin in the pulsatilla chinensis powder.

Description

HPLC quantitative analysis method for aesculin and aesculetin in pulsatilla chinensis powder
Technical Field
The invention relates to the technical field of determination of content of index components of medicaments, in particular to an HPLC quantitative analysis method of aesculin and aesculetin in pulsatilla chinensis powder.
Background
The pulsatilla chinensis powder recorded in the second part of the pharmacopoeia of the people's republic of China 2015 is a classic prescription of traditional Chinese veterinary medicine powder and consists of pulsatilla chinensis, coptis chinensis, phellodendron amurense and ash bark. Wherein the Chinese pulsatilla root is a monarch drug and has the functions of clearing away heat and toxic material, cooling blood and stopping dysentery; cortex Fraxini, cortex Phellodendri and Coptidis rhizoma are used as adjuvant drugs for clearing away heat and toxic materials, eliminating dampness and relieving dysentery. The 4 medicinal materials are combined to play the roles of clearing away heat and toxic material, cooling blood and stopping dysentery. The main treatment is as follows: dysentery due to toxic heat, dysentery due to damp-heat, red, white and scanty, fever and thirst, etc. In the standard of veterinary drug dictionary, only the microscopic identification method of the pulsatilla chinensis regel powder and the qualitative identification method of the coptis chinensis, the ash bark and the berberine hydrochloride are specified, and the effective components in the pulsatilla chinensis regel powder are not quantified.
However, the similarity can not be distinguished by adopting the traditional character identification and microscopic identification methods, so that the preparation has various problems in the using process, such as insufficient addition amount of medicinal materials, insufficient supplement and the like, even the main components of aesculin and aesculetin in the commercial pulsatilla chinensis powder can not be detected, and the like. As the quantitative analysis of the medicine is an important content for evaluating the quality of the traditional Chinese medicine preparation.
Therefore, it is very necessary to design an HPLC quantitative analysis method for aesculin and aesculetin in the pulsatilla chinensis powder in order to improve the quality standard of the pulsatilla chinensis powder.
Disclosure of Invention
The invention aims to provide an HPLC quantitative analysis method of aesculin and aesculetin in the pulsatilla chinensis powder to solve the problems in the background technology.
In order to realize the purpose, the invention provides the following technical scheme: an HPLC quantitative analysis method of aesculin and aesculetin in pulsatilla chinensis powder specifically comprises the following steps:
the method comprises the following steps: establishment of chromatographic conditions: changing the ratio of mobile phase acetonitrile-0.1% phosphoric acid aqueous solution from 8:92 to 12:88, and determining the chromatographic conditions as follows: a chromatographic column: waters Symmetry C18 column; mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution 12: 88; flow rate: 1.0 mL/min; wavelength: 334 nm; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; the theoretical plate number under the chromatographic condition is not less than 5000 according to the aesculetin peak;
step two: the extraction process comprises the following steps: precisely weighing 1.875g of Chinese pulsatilla root powder, placing into a conical flask with a stopper, precisely adding 50mL of methanol, sealing the stopper, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, and taking the subsequent filtrate;
step three: preparing a solution: the method comprises the following steps of preparing a reference substance storage solution, a reference substance solution, a test sample solution, a negative reference solution and a reference medicinal material solution:
the preparation method of the reference substance storage solution comprises the following steps: accurately weighing appropriate amount of aesculin and aesculetin reference substance, adding methanol to obtain mixed solution containing aesculetin 408 μ g and aesculetin 260 μ g per 1mL, and storing as reference substance stock solution;
the preparation method of the reference substance solution comprises the following steps: precisely measuring 5mL of a reference substance storage solution, adding methanol for stepwise dilution to obtain a series of mixed reference substance solutions with the concentrations of aesculin 25.5 mu g/mL, aesculetin 16.25 mu g/mL, aesculetin 51 mu g/mL, aesculetin 32.5 mu g/mL, aesculetin 102 mu g/mL, aesculetin 65 mu g/mL, aesculetin 204 mu g/mL, aesculetin 130 mu g/mL, aesculetin 408 mu g/mL and aesculetin 260 mu g/mL;
the preparation method of the test solution comprises the following steps: and (5) preparing according to the extraction process in the step two.
The preparation method of the negative control solution comprises the following steps: taking 1.875g of negative sample powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, sealing, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;
the preparation method of the reference medicinal material solution comprises the following steps: precisely weighing 1.875g of cortex Fraxini reference medicinal material powder, placing in a conical flask with a stopper, precisely adding 50mL of methanol, sealing, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and collecting the subsequent filtrate;
step four: methodology investigation: the method comprises the following steps of specificity test, linear relation investigation, precision test and accuracy test;
the specificity test specifically comprises the steps of respectively taking a proper amount of a reference substance solution, a test substance solution, a negative reference solution and a reference medicinal material solution, and determining according to the chromatographic conditions in the step one;
the linear relation is investigated by taking the reference substance solutions with the series of concentrations, determining according to the chromatographic conditions in the step one, and recording the peak area;
the precision test specifically comprises the steps of selecting mixed reference substance solutions with the concentrations of aesculin and aesculetin of 102 mu g/mL and 65 mu g/mL respectively, carrying out repeated sample injection for 6 times, recording peak areas, calculating relative standard deviation RSD, and carrying out precision measurement in a day;
the accuracy test specifically comprises the steps of taking 9 parts of pulsatilla chinensis bunge powder test samples of the same batch, wherein each part is about 0.5g and is divided into 3 groups, respectively adding a certain amount of aesculin and aesculetin reference substances, determining according to the chromatographic conditions in the step one and the extraction method in the step two, recording a chromatogram, and calculating the average recovery rate and RSD of the aesculetin and the aesculetin according to the following formulas:
percent recovery is (C-B)/a × 100%;
c is the actual measured quantity; b is the content of the known sample; a is the addition amount of the reference substance;
step five: and (3) sample determination: comprises the content determination of a self-made Chinese pulsatilla root powder sample and the content determination of the Chinese pulsatilla root powder sold in the market;
the content determination of the self-made pulsatilla chinensis bunge powder sample specifically comprises the steps of precisely weighing 1.875g of 3 batches of self-made pulsatilla chinensis bunge powder, processing according to the preparation method of the negative control solution in the third step and the chromatographic condition in the first step, recording the peak area, and calculating the content of the sample by using an external standard method;
the method comprises the steps of precisely weighing 1.875g of 8 batches of commercially available pulsatilla chinensis powder samples, processing according to the preparation method of the reference medicinal material solution in the third step, processing according to the chromatographic condition in the first step, and calculating the sample content by using an external standard method.
Preferably, the optimization method of the extraction process in the second step specifically comprises the following steps: performing ultrasonic extraction and heating reflux extraction, and replacing the heating reflux method specified in animal pharmacopoeia with the ultrasonic extraction method with higher extraction rate; the extraction time is compared for 20min, 40min and 60min (see table 2), and considering that the extraction contents of 40min and 60min are similar and both higher than 20min, the selection time is shortened from 60min specified in the animal pharmacopoeia to 40 min.
Preferably, the negative sample powder is a self-made ash-free medicinal material.
Preferably, the specific method for recording the peak area in the fourth step is to perform linear regression by using the concentration C of the control solution as an abscissa and the peak area a as an ordinate.
Preferably, the peak area is recorded and the relative standard deviation RSD is calculated in step four, and the precision within the day is determined by the following specific method: selecting aesculin 25.5 mu g/mL, aesculetin 16.25 mu g/mL, aesculetin 102 mu g/mL, aesculetin 65 mu g/mL, aesculetin 408 mu g/mL, aesculetin 260 mu g/mL, 3 mixed reference substance solutions with different concentrations, injecting 2 times per day for 3 days, recording peak area, calculating RSD, and performing day precision determination.
Compared with the prior art, the invention has the beneficial effects that: the HPLC quantitative analysis method for the aesculin and the aesculetin in the pulsatilla chinensis powder can realize accurate quantification of the effective components of the aesculetin and the aesculetin in the pulsatilla chinensis powder so as to improve the quality standard of the pulsatilla chinensis powder.
Drawings
FIG. 1 is a chromatogram of a mixed control of the examples;
FIG. 2 is a chromatogram of a test sample in an example;
FIG. 3 is a chromatogram of a negative sample in the example;
FIG. 4 is a chromatogram of an ash bark control drug in an example;
FIG. 5 is a standard curve of aesculin in the examples.
FIG. 6 is a standard curve of aesculetin in the examples.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: an HPLC quantitative analysis method of aesculin and aesculetin in pulsatilla chinensis powder specifically comprises the following steps:
the method comprises the following steps: establishment of chromatographic conditions: the chromatographic conditions under the item of ash bark (content determination) recorded in Chinese veterinary pharmacopoeia are taken as reference, the proportion of mobile phase acetonitrile-0.1% phosphoric acid aqueous solution is changed from 8:92 to 12:88, so that the retention time of the aesculin and the aesculetin is shortened, and the peak shapes of the aesculetin and the aesculetin are further improved. On the other hand, the proportion of acetonitrile is improved, so that the polarity of the mobile phase is reduced, and the damage to the chromatographic column is reduced. The chromatographic conditions were finally determined to be: a chromatographic column: waters symmetry C18 column; mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution 12: 88; flow rate: 1.0 mL/min; wavelength: 334 nm; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; the theoretical plate number under the chromatographic condition is not less than 5000 according to the aesculetin peak;
step two: the extraction process comprises the following steps: taking the extraction process under the item of ash bark [ preparation of test solution ] collected in the pharmacopoeia of Chinese beast (2015 edition two) as reference, the comparison of ultrasonic extraction and heating reflux extraction is carried out, and the results are shown in the content of aesculin and aesculetin of different extraction methods in table 1:
Figure BDA0002301663880000051
TABLE 1
The ultrasonic extraction method with higher extraction rate replaces the heating reflux method specified in the animal pharmacopoeia; the extraction time is compared for 20min, 40min and 60min, and the results are shown in table 2 for the content of aesculin and aesculetin at different extraction times:
Figure BDA0002301663880000052
TABLE 2
In view of the fact that the extraction contents of 40min and 60min are relatively similar and are both higher than 20min, the selection time is shortened from 60min specified in the animal pharmacopoeia to 40min, and therefore the analysis efficiency is improved. The finally determined extraction process is as follows: precisely weighing 1.875g of Chinese pulsatilla root powder, placing into a conical flask with a stopper, precisely adding 50mL of methanol, sealing the stopper, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, and taking the subsequent filtrate;
step three: preparing a solution: the method comprises the following steps of preparing a reference substance storage solution, a reference substance solution, a test sample solution, a negative reference solution and a reference medicinal material solution:
the preparation method of the reference substance storage solution comprises the following steps: accurately weighing appropriate amount of aesculin and aesculetin reference substance, adding methanol to obtain mixed solution containing aesculetin 408 μ g and aesculetin 260 μ g per 1mL, and storing as reference substance stock solution;
the preparation method of the reference substance solution comprises the following steps: precisely measuring 5mL of a reference substance storage solution, adding methanol for stepwise dilution to obtain a series of mixed reference substance solutions with the concentrations of aesculin 25.5 mu g/mL, aesculetin 16.25 mu g/mL, aesculetin 51 mu g/mL, aesculetin 32.5 mu g/mL, aesculetin 102 mu g/mL, aesculetin 65 mu g/mL, aesculetin 204 mu g/mL, aesculetin 130 mu g/mL, aesculetin 408 mu g/mL and aesculetin 260 mu g/mL;
the preparation method of the test solution comprises the following steps: and (5) preparing according to the extraction process in the step two.
The preparation method of the negative control solution comprises the following steps: taking 1.875g of negative sample powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, sealing, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;
the preparation method of the reference medicinal material solution comprises the following steps: precisely weighing 1.875g of cortex Fraxini reference medicinal material powder, placing in a conical flask with a stopper, precisely adding 50mL of methanol, sealing, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and collecting the subsequent filtrate;
step four: methodology investigation: the method comprises the following steps of specificity test, linear relation investigation, precision test and accuracy test;
the specificity test specifically comprises taking appropriate amount of reference solution, test solution, negative reference solution, and reference solution, respectively, and determining according to the chromatographic conditions in step one, wherein the results are shown in fig. 1-4, wherein fig. 1 is chromatogram of mixed reference, and 1 is aesculin, and 2 is aesculetin; FIG. 2 is a chromatogram of the sample, wherein 1 is aesculin and 2 is aesculetin; FIG. 3 is a chromatogram of a negative sample; FIG. 4 is chromatogram of cortex Fraxini reference medicinal material, wherein 1 is aesculin and 2 is aesculetin. As can be seen from FIGS. 1 and 4, the retention time of aesculin and aesculetin is 5.883min and 11.282 min; at this retention time, fig. 2 shows that the test sample has chromatographic peaks corresponding to aesculin and aesculetin, and the separation degrees are respectively: 4.4 parts of aesculin and 2.0 parts of aesculetin are completely separated; the negative control sample of figure 3 has no chromatographic peak at the corresponding retention times of aesculin and aesculetin; the result shows that other components in the pulsatilla chinensis bunge test sample do not interfere the determination of the aesculin and the aesculetin.
The linear relation is investigated by taking the reference substance solutions with the series of concentrations, determining according to the chromatographic conditions in the step one, and recording the peak area; linear regression was performed using the concentration (C) of the control solution as abscissa and the peak area (a) as ordinate, as shown in fig. 5 and 6, to obtain the regression equation a, aesculin 19700C +116000 (R0.99989, linear range: 25.5 μ g/mL to 408 μ g/mL), and aesculetin 34000C +51800 (R0.99996, linear range: 16.25 μ g/mL to 260 μ g/mL).
The precision test specifically comprises the steps of selecting mixed reference substance solutions with the concentrations of aesculin and aesculetin of 102 mu g/mL and 65 mu g/mL respectively, carrying out repeated sample injection for 6 times, recording peak areas, calculating relative standard deviation RSD, and carrying out precision measurement in a day; selecting mixed reference substance solutions with low (aesculin 25.5 mu g/mL and aesculetin 16.25 mu g/mL), medium (aesculetin 102 mu g/mL and aesculetin 65 mu g/mL) and high (aesculetin 408 mu g/mL and aesculetin 260 mu g/mL)3 different concentrations, injecting samples for 2 times every day for 3 days, recording peak areas, calculating RSD, and performing daytime precision determination. As a result: the daily precision of both the aesculin and the aesculetin is less than 0.30 percent, and the daily precision is less than 2 percent, which meets the requirement.
The accuracy test specifically comprises the steps of taking 9 parts of pulsatilla chinensis bunge powder test samples of the same batch, wherein each part is about 0.5g and is divided into 3 groups, respectively adding a certain amount of aesculin and aesculetin reference substances, determining according to the chromatographic conditions in the step one and the extraction method in the step two, recording a chromatogram, and calculating the average recovery rate and RSD of the aesculetin and the aesculetin according to the following formulas: the results are shown in Table 3.
Percent recovery is (C-B)/A × 100%
C is the actual measured quantity; b is the content of the known sample; a is the addition amount of the reference substance;
Figure BDA0002301663880000081
TABLE 3
As can be seen from Table 3, the average recovery rate of aesculin was 98.3% (RSD 1.80%); the average recovery rate of aesculetin is 102.8% (RSD 2.42%).
Step five: and (3) sample determination: comprises the content determination of a self-made Chinese pulsatilla root powder sample and the content determination of the Chinese pulsatilla root powder sold in the market;
the content determination of the self-made pulsatilla chinensis bunge powder sample specifically comprises the steps of precisely weighing 1.875g of 3 batches of self-made pulsatilla chinensis bunge powder, processing according to the preparation method of the negative control solution in the third step and the chromatographic condition in the first step, recording peak area, and calculating the content of the sample by using an external standard method, wherein the result is shown in the following table 4:
Figure BDA0002301663880000082
TABLE 4
The content of the aesculin in 3 batches of the self-made pulsatilla chinensis powder is 3874.870 mug-1, 3882.096 mug-1 and 3994.053 mug-1 respectively; the content of the aesculetin is 310.994 mug.g < -1 >, 315.998 mug.g < -1 > and 321.263 mug.g < -1 > respectively, and the reliability of the extraction process and the HPLC content determination method of the aesculetin and the aesculetin in the pulsatilla chinensis powder established by the test is verified.
The content of the commercial anemone powder is determined by taking 8 samples of the commercial anemone powder, and the sources of the samples are shown in the following table 8:
Figure BDA0002301663880000091
TABLE 8
Precisely weighing 1.875g of the crude drug, processing according to the preparation method of the reference drug solution in the third step, processing according to the chromatographic conditions in the first step, and calculating the content of the sample by using an external standard method. The results of the measurements are shown in tables 6 to 7 below.
Content of aesculin in commercial sample (n ═ 2)
Figure BDA0002301663880000101
TABLE 6
Content of aesculetin in commercial sample (n ═ 2)
Figure BDA0002301663880000102
TABLE 7
As can be seen from tables 6 and 7, the content of the aesculin and the aesculetin in 8 batches of the commercially available samples are different, except that the content of the aesculetin in the batch III sample and the content of the aesculetin in the batch II sample are higher than those of the self-made samples, the content of the aesculetin and the aesculetin in other batches of the commercially available samples are obviously lower than those of the self-made samples, particularly the content of the batches VI and VIII samples is not detected, and the content of the aesculetin and the aesculetin is not directly related to the price of the commercially available samples.
The HPLC quantitative analysis method for the aesculin and the aesculetin in the pulsatilla chinensis powder can realize accurate quantification of the effective components of the aesculetin and the aesculetin in the pulsatilla chinensis powder so as to improve the quality standard of the pulsatilla chinensis powder.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (5)

1. An HPLC quantitative analysis method of aesculin and aesculetin in the pulsatilla chinensis powder is characterized by comprising the following steps: the method specifically comprises the following steps:
the method comprises the following steps: establishment of chromatographic conditions: changing the ratio of mobile phase acetonitrile-0.1% phosphoric acid aqueous solution from 8:92 to 12:88, and determining the chromatographic conditions as follows: a chromatographic column: waters Symmetry C18 column; mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution 12: 88; flow rate: 1.0 mL/min; wavelength: 334 nm; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; the theoretical plate number under the chromatographic condition is not less than 5000 according to the aesculetin peak;
step two: the extraction process comprises the following steps: precisely weighing 1.875g of Chinese pulsatilla root powder, placing into a conical flask with a stopper, precisely adding 50mL of methanol, sealing the stopper, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, and taking the subsequent filtrate;
step three: preparing a solution: the method comprises the following steps of preparing a reference substance storage solution, a reference substance solution, a test sample solution, a negative reference solution and a reference medicinal material solution:
the preparation method of the reference substance storage solution comprises the following steps: accurately weighing appropriate amount of aesculin and aesculetin reference substance, adding methanol to obtain mixed solution containing aesculetin 408 μ g and aesculetin 260 μ g per 1mL, and storing as reference substance stock solution;
the preparation method of the reference substance solution comprises the following steps: precisely measuring 5mL of a reference substance storage solution, adding methanol for stepwise dilution to obtain a series of mixed reference substance solutions with the concentrations of aesculin 25.5 mu g/mL, aesculetin 16.25 mu g/mL, aesculetin 51 mu g/mL, aesculetin 32.5 mu g/mL, aesculetin 102 mu g/mL, aesculetin 65 mu g/mL, aesculetin 204 mu g/mL, aesculetin 130 mu g/mL, aesculetin 408 mu g/mL and aesculetin 260 mu g/mL;
the preparation method of the test solution comprises the following steps: and (5) preparing according to the extraction process in the step two.
The preparation method of the negative control solution comprises the following steps: taking 1.875g of negative sample powder, precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of methanol, sealing, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;
the preparation method of the reference medicinal material solution comprises the following steps: precisely weighing 1.875g of cortex Fraxini reference medicinal material powder, placing in a conical flask with a stopper, precisely adding 50mL of methanol, sealing, weighing, ultrasonically extracting for 40min, cooling, weighing again, supplementing the lost weight with methanol, shaking, filtering, and collecting the subsequent filtrate;
step four: methodology investigation: the method comprises the following steps of specificity test, linear relation investigation, precision test and accuracy test;
the specificity test specifically comprises the steps of respectively taking a proper amount of a reference substance solution, a test substance solution, a negative reference solution and a reference medicinal material solution, and determining according to the chromatographic conditions in the step one;
the linear relation is investigated by taking the reference substance solutions with the series of concentrations, determining according to the chromatographic conditions in the step one, and recording the peak area;
the precision test specifically comprises the steps of selecting mixed reference substance solutions with the concentrations of aesculin and aesculetin of 102 mu g/mL and 65 mu g/mL respectively, carrying out repeated sample injection for 6 times, recording peak areas, calculating relative standard deviation RSD, and carrying out precision measurement in a day;
the accuracy test specifically comprises the steps of taking 9 parts of pulsatilla chinensis bunge powder test samples of the same batch, wherein each part is about 0.5g and is divided into 3 groups, respectively adding a certain amount of aesculin and aesculetin reference substances, determining according to the chromatographic conditions in the step one and the extraction method in the step two, recording a chromatogram, and calculating the average recovery rate and RSD of the aesculetin and the aesculetin according to the following formulas:
percent recovery is (C-B)/a × 100%;
c is the actual measured quantity; b is the content of the known sample; a is the addition amount of the reference substance;
step five: and (3) sample determination: comprises the content determination of a self-made Chinese pulsatilla root powder sample and the content determination of the Chinese pulsatilla root powder sold in the market;
the content determination of the self-made pulsatilla chinensis bunge powder sample specifically comprises the steps of precisely weighing 1.875g of 3 batches of self-made pulsatilla chinensis bunge powder, processing according to the preparation method of the negative control solution in the third step and the chromatographic condition in the first step, recording the peak area, and calculating the content of the sample by using an external standard method;
the method comprises the steps of precisely weighing 1.875g of 8 batches of commercially available pulsatilla chinensis powder samples, processing according to the preparation method of the reference medicinal material solution in the third step, processing according to the chromatographic condition in the first step, and calculating the sample content by using an external standard method.
2. The HPLC quantitative analysis method of aesculin and aesculetin in Pulsatilla chinensis powder as claimed in claim 1, wherein the HPLC quantitative analysis method comprises the following steps: the optimization method of the extraction process in the step two specifically comprises the following steps: performing ultrasonic extraction and heating reflux extraction, and replacing the heating reflux method specified in animal pharmacopoeia with the ultrasonic extraction method with higher extraction rate; the extraction time is compared for 20min, 40min and 60min (see table 2), and considering that the extraction contents of 40min and 60min are similar and both higher than 20min, the selection time is shortened from 60min specified in the animal pharmacopoeia to 40 min.
3. The HPLC quantitative analysis method of aesculin and aesculetin in Pulsatilla chinensis powder as claimed in claim 1, wherein the HPLC quantitative analysis method comprises the following steps: the negative sample powder is a self-made ash-free medicinal material.
4. The HPLC quantitative analysis method of aesculin and aesculetin in Pulsatilla chinensis powder as claimed in claim 1, wherein the HPLC quantitative analysis method comprises the following steps: the specific method for recording the peak area in the fourth step is to perform linear regression by taking the concentration C of the reference solution as the abscissa and the peak area A as the ordinate.
5. The HPLC quantitative analysis method of aesculin and aesculetin in Pulsatilla chinensis powder as claimed in claim 1, wherein the HPLC quantitative analysis method comprises the following steps: recording peak areas in the fourth step and calculating relative standard deviation RSD, wherein the specific method for measuring the precision in the day comprises the following steps: selecting aesculin 25.5 mu g/mL, aesculetin 16.25 mu g/mL, aesculetin 102 mu g/mL, aesculetin 65 mu g/mL, aesculetin 408 mu g/mL, aesculetin 260 mu g/mL, 3 mixed reference substance solutions with different concentrations, injecting 2 times per day for 3 days, recording peak area, calculating RSD, and performing day precision determination.
CN201911224122.8A 2019-12-04 2019-12-04 HPLC quantitative analysis method for aesculin and aesculetin in pulsatilla chinensis powder Pending CN110907579A (en)

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