CN115032310A - Method for detecting 6 dianthrone compounds in polygonum multiflorum - Google Patents
Method for detecting 6 dianthrone compounds in polygonum multiflorum Download PDFInfo
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- 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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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
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Abstract
The invention provides a detection method of 6 dianthrone compounds in polygonum multiflorum, and belongs to the technical field of chemical analysis and detection. The invention adopts liquid chromatography-mass spectrometry to simultaneously detect 6 dianthrone compounds of trans-emodin dianthrone, cis-emodin dianthrone, trans-emodin-physcion dianthrone, cis-emodin-physcion dianthrone, trans-emodin-physcion dianthrone and cis-emodin-physcion dianthrone in polygonum multiflorum. The 6 dianthrone compounds detected by the invention simultaneously contain free dianthrone and a product obtained by hydrolyzing dianthrone glycoside, are more scientific and reasonable when used as toxicity indexes of polygonum multiflorum, simultaneously make up the defect that the conventional polygonum multiflorum medicinal material lacks the quality control indexes of toxic components, and lay a foundation for the quality control of polygonum multiflorum medicinal material.
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a detection method of 6 dianthrone compounds in polygonum multiflorum.
Background
Polygonum multiflorum is specified as the dry root tuber of Polygonum multiflorum Thunb, a plant of Polygonaceae in the first part of the "Chinese pharmacopoeia" 2020 edition. The prepared fleece-flower root is a processed product of the fleece-flower root, and has the effects of nourishing liver and kidney, benefiting essence and blood, blackening beard and hair, strengthening muscles and bones, eliminating turbidity and reducing fat. The prepared fleece-flower root has a long medicinal history, and is widely applied to prescription drugs, non-prescription drugs and health care products as a good tonifying drug. The polygonum multiflorum has complex chemical components, and mainly comprises stilbene, anthraquinone, dianthrone, polyphenol, naphthalene and other chemical components. In recent years, adverse reactions such as polygonum multiflorum hepatotoxicity and the like are reported to attract extensive attention. Earlier researches find that dianthrone components in polygonum multiflorum have certain hepatotoxicity: trans-emodin-emodin dianthrones and cis-emodin-emodin dianthrones show certain toxic effects in embryonic development and hepatotoxicity evaluation models of zebra fish, and the cis-emodin-physcidin dianthrones show obvious hepatotoxicity in a human liver HepG2 cell model, which indicates that the components may be potential hepatotoxic components in fleece-flower roots.
According to the theory of traditional Chinese medicine, the processing has the effects of reducing toxicity and enhancing efficacy. The key of the change of the chemical components and the proportion of the polygonum multiflorum and the change of the biological activity of the polygonum multiflorum is the important production process for determining the quality of the prepared polygonum multiflorum. Earlier studies show that dianthrone in polygonum multiflorum has certain hepatotoxicity and the content of dianthrone in polygonum multiflorum is reduced or disappeared after processing, which indicates that it may be one of the mechanisms of polygonum multiflorum processing attenuation. Xuwen uses high resolution mass spectrometry (Xuwen. application of LC/MS technology in analysis of two Chinese medicinal components [ D ]. Guangzhou: Guangzhou university of traditional Chinese medicine, 2015) to confirm that dianthranone glycoside component in Polygoni Multiflori radix is emodin (10 → 10 ') emodin or mother nucleus of emodin (10 → 10') physcion. Therefore, it is necessary to increase the detection methods of 6 dianthrone compounds, i.e., trans-emodin dianthrone, cis-emodin-phytin dianthrone, trans-phytin dianthrone and cis-phytin dianthrone in polygonum multiflorum.
Disclosure of Invention
In view of this, the present invention provides a method for detecting 6 dianthrone compounds in polygonum multiflorum. The detection method can simultaneously detect 6 dianthrone compounds of trans-emodin dianthrone, cis-emodin dianthrone, trans-emodin-physcion dianthrone, cis-emodin-physcion dianthrone, trans-physcion dianthrone and cis-physcion dianthrone, and has the advantages of high sensitivity, good precision, high stability, good repeatability and good recovery rate.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a detection method of 6 dianthrone compounds in polygonum multiflorum, which comprises the following steps:
sequentially carrying out alcohol extraction, acid hydrolysis, extraction, organic phase concentration and redissolution on a polygonum multiflorum sample to obtain a test sample solution;
performing liquid chromatography-mass spectrometry on the test solution to obtain a chromatogram of the test;
the chromatographic conditions of the liquid chromatography-mass spectrometry analysis comprise:
a chromatographic column: WatersACQUITYLC CSH C18;
mobile phase: a is acetonitrile, B is formic acid aqueous solution with mass fraction of 0.1%;
flow rate: 0.3 mL/min;
the gradient elution procedure was: 0-4 min: the volume ratio of A is increased from 45% to 60%; 4-14 min: the volume ratio of A is kept at 60%; 14-16 min: the volume ratio of A is increased from 60% to 70%; 16-21 min: the volume ratio of A is kept at 70%; 21-24 min: the volume ratio of A is increased from 70% to 95%; 24-30 min: the volume ratio of A is kept at 45%;
column temperature: 25-35 ℃;
sample introduction amount: 1.0-3.0 μ L;
the mass spectrum conditions of the liquid chromatography-mass spectrometry analysis comprise:
electrospray ion source, negative ion mode, scanning mode: multiple Reaction Monitoring (MRM), temperature: 150 ℃, capillary voltage: 3.0KV, solvent removal temperature: 350 ℃, desolvation gas: nitrogen, flow rate: 650L/min;
comparing the chromatogram of the test sample with a predetermined standard spectrogram to determine the species of dianthrone compounds in the polygonum multiflorum sample;
the dianthrone compounds include: trans-emodin dianthrone, cis-emodin dianthrone, trans-emodin-physcion dianthrone, cis-emodin-physcion dianthrone, trans-physcion dianthrone, and cis-physcion dianthrone.
Preferably, the ethanol extraction solvent comprises an ethanol solution, and the volume fraction of the ethanol solution is 40-60%.
Preferably, the dosage ratio of the alcohol extraction solvent to the polygonum multiflorum sample is 40-60 mL: 0.5-1.5 g.
Preferably, the alcohol extraction comprises ultrasonic extraction, and the time of the ultrasonic extraction is 20-40 min.
Preferably, the solvent for acid hydrolysis comprises a hydrochloric acid solution, and the mass fraction of the hydrochloric acid solution is 8-12%.
Preferably, the dosage ratio of the solvent for acid hydrolysis to the polygonum multiflorum sample is 5-15 mL: 0.1-0.3 g.
Preferably, the temperature of the acid hydrolysis is 40-60 ℃, and the time is 20-40 min.
Preferably, the solvent of the extraction comprises ethyl acetate.
Preferably, the dosage ratio of the redissolved solvent to the polygonum multiflorum sample is 25mL: 0.1-0.3 g.
Preferably, the method further comprises the following steps after the chromatogram of the test sample is obtained: and calculating the content of the 6 dianthrones in the polygonum multiflorum by using a standard equation, wherein the standard equation is an equation with the mass concentration of the 6 dianthrone compounds as an independent variable and a peak area as a dependent variable.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts liquid chromatography-mass spectrometry to simultaneously detect 6 dianthrone compounds of trans-emodin dianthrone, cis-emodin dianthrone, trans-emodin-physcion dianthrone, cis-emodin-physcion dianthrone, trans-emodin-physcion dianthrone and cis-emodin-physcion dianthrone in polygonum multiflorum. The invention selects acetonitrile and formic acid water solution with mass concentration of 0.1% as mobile phase, so that the 6 kinds of dianthrone compounds which are difficult to separate in polygonum multiflorum have good separation degree. The detection method provided by the invention is simple to operate, can realize rapid qualitative and quantitative analysis of the 6 dianthrone compounds, and has the advantages of high sensitivity, good precision, high stability, good repeatability and good recovery rate.
Furthermore, the method adopts ethanol solution to carry out ethanol extraction on the dianthrone compounds in the polygonum multiflorum under the ultrasonic condition, and has high extraction effect rate. In the invention, after alcohol extraction, hydrochloric acid solution is further adopted for hydrolysis, so that the glycosidic bond of the dianthrone glycoside is broken to obtain corresponding free dianthrone. The 6 dianthrone compounds detected by the invention simultaneously contain free dianthrone and a product obtained by hydrolyzing dianthrone nucleotide, are more scientific and reasonable when used as toxicity indexes of polygonum multiflorum, simultaneously make up the defect that the conventional polygonum multiflorum medicinal material lacks the quality control indexes of toxic components, and lay a foundation for the quality control of the polygonum multiflorum medicinal material.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a structural diagram of 6 dianthrone compounds, wherein 1 is trans-emodin dianthrone, 2 is cis-emodin-dianthrone, 3 is trans-emodin-physcion dianthrone, 4 is cis-emodin-physcion dianthrone, 5 is trans-physcion dianthrone, and 6 is cis-physcion dianthrone;
fig. 2 is a TIC diagram sequentially showing chromatograms of 6 dianthrone compounds in polygonum multiflorum from top to bottom, and an ion flow diagram is extracted: 509.0>254.1, extracting an ion flow diagram: 509.1>254.0, extracting an ion flow diagram: 523.1>254.0, extracting an ion flow diagram: 523.1>254.0, extracting an ion flow diagram: 537.1> 522.2;
FIG. 3 shows the separation effect of different mobile phases, wherein A is acetonitrile-water solution gradient elution solvent system, and B is methanol-water solution gradient elution solvent system;
FIG. 4 shows the hydrolysis effect of dilute hydrochloric acid solution and sulfuric acid solution, wherein A is dilute hydrochloric acid solution hydrolysis and B is sulfuric acid solution hydrolysis.
Detailed Description
The invention provides a detection method of 6 dianthrone compounds in polygonum multiflorum, which comprises the following steps:
sequentially carrying out alcohol extraction, acid hydrolysis, extraction, organic phase concentration and redissolution on a polygonum multiflorum sample to obtain a test sample solution;
performing liquid chromatography-mass spectrometry on the test solution to obtain a chromatogram of the test;
the chromatographic conditions of the liquid chromatography-mass spectrometry analysis comprise:
a chromatographic column: WatersACQUITYLC CSH C18;
mobile phase: a is acetonitrile, B is formic acid aqueous solution with mass fraction of 0.1%;
flow rate: 0.3 mL/min;
the gradient elution procedure was: 0-4 min: the volume ratio of A is increased from 45% to 60%; 4-14 min: the volume ratio of A is kept at 60%; 14-16 min: the volume ratio of A is increased from 60% to 70%; 16-21 min: the volume ratio of A is kept at 70%; 21-24 min: the volume ratio of A is increased from 70% to 95%; 24-30 min: the volume ratio of A is kept at 45%;
column temperature: 25-35 ℃;
sample introduction amount: 1.0-3.0 μ L;
the mass spectrum conditions of the liquid chromatography-mass spectrometry analysis comprise:
electrospray ion source, negative ion mode, scanning mode: multiple Reaction Monitoring (MRM), temperature: 150 ℃, capillary voltage: 3.0KV, solvent removal temperature: 350 ℃, desolvation gas: nitrogen, flow rate: 650L/min;
comparing the chromatogram of the test sample with a predetermined standard spectrogram to determine the species of dianthrone compounds in the polygonum multiflorum sample;
the dianthrone compounds include: trans-emodin dianthrone, cis-emodin dianthrone, trans-emodin-physcion dianthrone, cis-emodin-physcion dianthrone, trans-physcion dianthrone, and cis-physcion dianthrone.
In the present invention, reagents, consumables and equipment used are commercially available in the art unless otherwise specified.
The method comprises the steps of sequentially carrying out alcohol extraction, acid hydrolysis, extraction, organic phase concentration and redissolution on a polygonum multiflorum sample to obtain a test solution.
Preferably, the polygonum multiflorum sample is crushed to obtain polygonum multiflorum medicinal powder, the polygonum multiflorum medicinal powder is sieved by a No. 4 sieve, and the part under the sieve is subjected to alcohol extraction.
In the invention, the ethanol extraction solvent preferably comprises an ethanol solution, and the volume fraction of the ethanol solution is preferably 40-60%, and more preferably 50%. The invention has no special requirement on the container for alcohol extraction, such as a conical bottle with a plug.
In the invention, the dosage ratio of the alcohol extraction solvent to the polygonum multiflorum sample is preferably 40-60 mL to 0.5-1.5 g, and more preferably 45-55 mL to 0.9-1.1 g.
In the invention, the alcohol extraction preferably comprises ultrasonic extraction, and the ultrasonic extraction time is preferably 20-40 min, and more preferably 30 min. The present invention has no special requirements on the temperature of the ultrasonic extraction.
In the present invention, the power of the ultrasound is preferably 100W, and the frequency is preferably 40 kHz.
In the present invention, it is preferable to weigh the entire container after adding the extraction solvent, and to monitor whether the extraction solvent is lost during the ultrasonic extraction process.
According to the invention, the solution and the container after ultrasonic extraction are preferably naturally cooled to room temperature, then the weight of the whole container is weighed, and the lost weight is complemented by the extraction solvent, so that the loss of the extraction solvent in the ultrasonic extraction process is compensated, and the accuracy of the detection result is ensured.
In the present invention, after the weight loss is made up with the extraction solvent, the extracted solution is preferably shaken up and filtered to obtain a filtrate. The invention has no special requirements on the filtering mode and adopts the mode which is usually used by the technical personnel in the field.
In the present invention, after obtaining the filtrate, the filtrate is preferably evaporated to dryness to obtain a residue.
In the present invention, it is preferable that the residue is dissolved in a solvent for acid hydrolysis and then hydrolyzed.
In the invention, the solvent for acid hydrolysis preferably comprises a hydrochloric acid solution, and the mass fraction of the hydrochloric acid solution is preferably 8-12%, and more preferably 9.5-10.5%.
In the invention, the dosage ratio of the solvent for acid hydrolysis to the polygonum multiflorum sample is 5-15 mL: 0.1-0.3 g, and more preferably 10mL:0.2 g.
In the invention, the temperature of the acid hydrolysis is preferably 40-60 ℃, more preferably 50 ℃, and the time is preferably 20-40 min, more preferably 30 min. In the present invention, the acid hydrolysis is preferably carried out in a water bath.
In the present invention, the acid hydrolysis breaks the glycosidic bond of bound dianthrone in polygonum multiflorum to give free dianthrone. The method can simultaneously determine the content of the total dianthrone including the free dianthrone and the combined dianthrone, and the total dianthrone is more scientific and reasonable when used as the toxicity index of the polygonum multiflorum.
In the present invention, the acid hydrolysis is preferably performed after cooling, and then the extraction is performed, and the cooling method is not particularly required, and can be performed in a manner commonly used by those skilled in the art.
In the present invention, the solvent for extraction preferably comprises ethyl acetate, the number of times of extraction is preferably 2, and the volume ratio of the solvent for extraction to the solvent for acid hydrolysis is preferably 3: 1. The invention has no special requirements on the extraction device, such as a separating funnel.
In the present invention, it is preferable that the organic phase obtained by the extraction is concentrated and then the redissolution is carried out. The present invention has no special requirements for the concentration mode, and the method is realized by adopting the mode commonly used by the technical personnel in the field.
In the invention, the dosage ratio of the redissolved solvent to the polygonum multiflorum sample is preferably 25mL: 0.1-0.3 g, more preferably 25mL:0.2g, and the redissolved solvent is preferably the same as the extracted solvent, and is not repeated herein.
After the redissolution is completed, the invention preferably filters the obtained solution by using a 0.22 mu m microporous filter membrane to obtain the test solution.
After the test solution is obtained, carrying out liquid chromatography-mass spectrometry on the test solution to obtain a chromatogram of the test;
the chromatographic conditions of the liquid chromatography-mass spectrometry analysis comprise:
a chromatographic column: WatersACQUITYLC CSH C18;
mobile phase: a is acetonitrile, B is formic acid aqueous solution with mass fraction of 0.1%;
flow rate: 0.3 mL/min;
the gradient elution procedure was: 0-4 min: the volume ratio of A is increased from 45% to 60%; 4-14 min: the volume ratio of A is kept at 60%; 14-16 min: the volume ratio of A is increased from 60% to 70%; 16-21 min: the volume ratio of A is kept at 70%; 21-24 min: the volume ratio of A is increased from 70% to 95%; 24-30 min: the volume ratio of A is kept at 45%;
the column temperature is preferably 25-35 ℃, and more preferably 30 ℃;
the sampling amount is preferably 1.0-3.0 muL, and more preferably 2.0 muL;
the mass spectrum conditions of the liquid chromatography-mass spectrometry analysis comprise:
electrospray ion source, negative ion mode, scanning mode: multiple reaction detection (MRM), temperature: 150 ℃, capillary voltage: 3.0KV, solvent removal temperature: 350 ℃, desolvation gas: nitrogen, flow rate: 650L/min.
In the present invention, the chromatography column: the specification of WatersACQUITYLC CSH C18 is preferably 2.1X 100mm, 1.7 μm.
In the invention, each compound in the 6 dianthrones compounds preferably selects a quantitative ion (Product ion 1) and a qualitative ion (Product ion 2), specific compound ion pairs, transmission voltages and collision energy are shown in table 1, 1 is trans-emodin dianthrone, 2 is cis-emodin-dianthrone, 3 is trans-emodin-phytin-anthrathrone, 4 is cis-emodin-phytyl-anthrathrone, 5 is trans-emodin-phytyl-anthrathrone, and 6 is cis-emodin-phytyl-emodin-anthrathrone.
TABLE 1 MRM parameters of 6 dianthrones in Polygonum multiflorum
After the test chromatogram is obtained, comparing the test chromatogram with a preset standard chromatogram to determine the type of dianthrone compounds in the polygonum multiflorum sample;
the dianthrone compounds include: trans-emodin dianthrone, cis-emodin dianthrone, trans-emodin-physcion dianthrone, cis-emodin-physcion dianthrone, trans-physcion dianthrone, and cis-physcion dianthrone.
After the chromatogram of the test sample is obtained, the method preferably further comprises the following steps: and calculating the content of the 6 dianthrones in the polygonum multiflorum by using a standard equation, wherein the standard equation is an equation with the mass concentration of the 6 dianthrone compounds as an independent variable and a peak area as a dependent variable.
The method for obtaining the standard equation is not particularly limited in the present invention, and methods known to those skilled in the art can be used.
In the invention, the calculation of the content of the 6 dianthrone compounds in the polygonum multiflorum by using a standard equation is preferably an external standard method.
In order to further illustrate the present invention, the following detailed description of the detection method provided by the present invention is made with reference to the drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Test solution: taking 1.0g of polygonum multiflorum medicinal material powder (screened by a No. 4 sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of ethanol solution with 50% of volume fraction, weighing, ultrasonically extracting (with the power of 100W and the frequency of 40kHz) for 30 minutes, taking out, placing to room temperature, weighing again, complementing the lost weight with the ethanol solution with 50% of volume fraction, shaking up, and filtering. Precisely measuring 10mL of continuous filtrate, recovering the solvent until the continuous filtrate is dry, precisely adding 10mL of dilute hydrochloric acid solution (234 mL of hydrochloric acid is taken and diluted with water until 1000mL is uniformly mixed) into residues for dissolving, carrying out water bath at 50 ℃ for 30 minutes, taking out, immediately cooling, placing in a separating funnel, washing a container with ethyl acetate, adding washing liquor into the separating funnel, shaking and extracting for 2 times with ethyl acetate, 30mL each time, separately taking and combining the ethyl acetate solution, recovering the solvent until the solvent is dry, adding 50 volume percent ethanol solution into the residues for dissolving, transferring to a 25mL volumetric flask, adding 50 volume percent ethanol solution to scale, shaking uniformly, filtering with a 0.22 mu m microporous filter membrane, and taking the continuous filtrate to obtain a sample solution.
Standard curve solution: accurately weighing comparison products of trans-emodin-physcion (1), cis-emodin-physcion (2), trans-emodin-physcion (3), cis-emodin-physcion (4), trans-physcion (5) and cis-physcion (6), adding 50% volume fraction ethanol solution to dissolve the comparison products to prepare 6 comparison product solutions containing 79.70(1), 79.87(2), 78.34(3), 79.56(4), 7.25(5) and 7.85(6) mu g per 1mL, and then gradually diluting the comparison product solutions to obtain a standard curve solution.
And (3) determination: and injecting the test solution and the standard curve solution into an ultra-high performance liquid chromatography-tandem quadrupole mass spectrometer to obtain a detection result.
The chromatographic conditions include: a chromatographic column: waters ACQUITY UPLC CSH C18 (2.1X 100mm, 1.7 μm); mobile phase: a is acetonitrile, B is formic acid aqueous solution with mass fraction of 0.1%; flow rate: 0.3 mL/min; the gradient elution procedure was: 0-4 min: the volume ratio of A is increased from 45% to 60%; 4-14 min: the volume ratio of A is kept at 60%; 14-16 min: the volume ratio of A is increased from 60% to 70%; 16-21 min: the volume ratio of A is kept at 70%; 21-24 min: the volume ratio of A is increased from 70% to 95%; 24-30 min: the volume ratio of A is kept at 45%; column temperature: 30 ℃; sample introduction amount: 2 mu L of the solution;
the mass spectrometry conditions include: electrospray ion source, negative ion mode, scanning mode: multiple reaction detection (MRM), temperature: 150 ℃, capillary voltage: 3.0KV, solvent removal temperature: 350 ℃, desolvation gas: nitrogen, flow rate: 650L/min;
a quantitative ion (Product ion 1) and a qualitative ion (Product ion 2) are selected for each compound, ion pairs, transmission voltages and collision energy of specific compounds are shown in table 1, a linear regression equation, a correlation coefficient, a linear range, a quantitative limit and a detection limit of 6 dianthrone compounds are obtained, the ratio (signal-to-noise ratio) of the height of a detection signal of the qualitative ion to the height of noise of a base line is equal to 3:1 and is the detection limit, and the ratio (signal-to-noise ratio) of the height of the detection signal of the quantitative ion to the height of noise of the base line is equal to 10:1 and is the quantitative limit, and the result is shown in table 2.
TABLE 2 Linear regression equation, correlation coefficient, linear range, quantitative limit and detection limit for 6 dianthrones in Polygonum multiflorum
| No. | Regression equation | R 2 | Range(ng/ml) | LOD(ng/ml) | LOQ(ng/ml) |
| 1 | Y＝437.66x+3967.5 | 0.9911 | 0.83～423.36 | 0.41 | 0.83 |
| 2 | Y＝363.94x+1279.5 | 0.9971 | 0.66～339.39 | 0.33 | 0.66 |
| 3 | Y＝642.03x+3708.2 | 0.9926 | 0.69～350.65 | 0.34 | 0.69 |
| 4 | Y＝477.46x+3564 | 0.9948 | 0.90～458.26 | 0.45 | 0.90 |
| 5 | Y＝37.473x–2.0684 | 0.9997 | 0.84～429.20 | 0.42 | 0.84 |
| 6 | Y＝45.271x+8.0834 | 0.9999 | 0.45～230.69 | 0.23 | 0.45 |
As can be seen from Table 2, the content of 6 dianthrones in Polygonum multiflorum has good correlation coefficient (R) in linear range 2 Not less than 0.99), and the method has relatively low detection limit and quantification limit, which indicates that the method has high sensitivity and accuracy.
FIG. 2 is a chromatogram of 6 dianthrone compounds in Polygonum multiflorum under an ultra-high performance liquid chromatography-mass spectrometry Multiple Reaction Monitoring (MRM) mode in example 1, wherein 1 is trans-emodin dianthrone, 2 is cis-emodin dianthrone, 3 is trans-emodin-phytin dianthrone, 4 is cis-emodin-phytin dianthrone, 5 is trans-phytin dianthrone, and 6 is cis-phytin dianthrone, and it can be seen from FIG. 2 that by using the detection method of the present invention, the separation degree of 6 dianthrone compounds is good, and the peaks are symmetrical, and the content of 6 dianthrone compounds in Polygonum multiflorum can be simultaneously determined.
Example 2 determination of the content of 6 dianthrones in different batches of Polygonum Multiflorum medicinal materials
20 batches of polygonum multiflorum medicinal material samples in different batches are taken and tested according to the method described in example 1, and the test results are shown in table 3.
Table 320 batches of Polygonum multiflorum medicinal material contains 6 dianthrone compounds (unit: mu g/g)
As can be seen from Table 3, in 20 lots of Polygonum multiflorum medicinal materials of different batches, the total content of trans-emodin dianthrone (1), cis-emodin-dianthrone (2), trans-emodin-physcion dianthrone (3), cis-emodin-physcion dianthrone (4), trans-physcion dianthrone (5) and cis-physcion dianthrone (6) is very low; the content of the compounds 1 and 2 in most polygonum multiflorum medicinal material samples is obviously higher than that of the compounds 4-6; the total content range of the 6 dianthrone compounds in 20 batches of polygonum multiflorum medicinal materials is 3.58-34.49 mu g/g.
Example 3 methodological validation
1. Precision test
The precision of the method is measured by the daily and daytime precision.
Selecting a polygonum multiflorum medicinal material sample PMR-19, preparing and measuring a test solution according to the method described in example 1, continuously repeating the sample injection for 6 times within 24h, continuously repeating the sample injection for 3 times every day for 72h, comparing the Relative Standard Deviation (RSD) of the corresponding peak area and retention time, and calculating the Relative Standard Deviation (RSD) of the peak area as shown in Table 5.
As can be seen from Table 5, the Relative Standard Deviation (RSD) of the area of the peak in the day of the sample to be measured in Polygonum multiflorum is less than 2%, and the Relative Standard Deviation (RSD) of the area of the peak in the day is less than 12%, indicating that the method has good precision.
2. Repeatability test
Taking 6 parts of polygonum multiflorum medicinal material sample PMR-19, preparing and measuring the test solution according to the method described in example 1 to obtain the content of 6 dianthrone compounds in each sample, and calculating the Relative Standard Deviation (RSD) of the content of 6 dianthrone compounds, wherein the calculation results are shown in Table 5.
As can be seen from Table 5, the Relative Standard Deviation (RSD) range of the contents of 6 dianthrone compounds in the polygonum multiflorum medicinal material sample is 3.41-6.12%, which shows that the method has good repeatability.
3. Stability test
Selecting a polygonum multiflorum medicinal material sample PMR-19, preparing and measuring a test solution according to the method described in example 1, taking 24h as a period, injecting samples once in 0, 2, 4, 6, 8, 10, 12, 18 and 24h respectively, calculating the Relative Standard Deviation (RSD) of the peak area, wherein the raw data is shown in Table 4, and the summary result is shown in Table 5.
As can be seen from tables 4 and 5, the Relative Standard Deviation (RSD) ranges of the peak areas of the 6 dianthrone compounds in the polygonum multiflorum sample are 5.32-6.98%, and the peak areas do not exceed 8.0%, which indicates that the method has good stability.
Table 4 stability test raw data
TABLE 5 precision, repeatability and stability of 6 dianthrone compounds in Polygonum multiflorum
4. Sample application recovery test
Selecting 6 parts of polygonum multiflorum medicinal material samples with known 6 dianthrone compound contents, precisely weighing 0.5g, adding 6 dianthrone compound reference substance solutions, wherein the contents and the standard addition amounts of the 6 dianthrone compounds in polygonum multiflorum are shown in table 6, preparing and measuring the sample solution according to the method in the embodiment 1 in the subsequent steps to obtain a test result, and calculating the standard addition recovery rate according to the standard addition recovery rate formula. The recovery rate is (C1-C2)/M × 100%, wherein C1 is the total amount of the target component measured after the labeling is added, C2 is the amount of the target component in the fleece-flower root sample, and M is the amount of the added dianthrone compound standard product.
As can be seen from Table 6, the average standard addition recovery rate of the 6 dianthrone compounds is within the range of 77.37-105.99%, which shows that the method has better recovery rate.
TABLE 6 results of recovery of 6 dianthrones from Polygonum multiflorum
Comparative example 1 examination of method for extracting test solution
This comparative example examined the effect of extraction with different extraction solvents during the extraction of the test solution, and the test solution was prepared and measured as described in example 1. A polygonum multiflorum medicinal material sample PMR-19 is selected for testing, in the comparative example, water, an ethanol solution with the volume fraction of 30%, an ethanol solution with the volume fraction of 50%, an ethanol solution with the volume fraction of 70%, an ethanol solution with the volume fraction of 95% and methanol are respectively selected for carrying out experiments, and the test results are shown in Table 7.
Meanwhile, the experiment inspects the effects of different ultrasonic time, and finally determines that the optimal ultrasonic extraction time is 30 min.
Table 7 test results for different extraction solvents
As can be seen from Table 7, the 6 dianthrone compounds obtained by ultrasonic extraction with 50% volume ethanol solution for 30min have high relative peak areas and high extraction efficiency.
Comparative example 2 selection of mobile phase
In the comparative example, the separation effect of different mobile phases is considered, and the preparation and the measurement of the test solution are carried out according to the method in the example 1, the polygonum multiflorum medicinal material sample PMR-19 is selected in the comparative example, and acetonitrile-water solution and methanol-water solution gradient elution solvent systems are firstly considered, and the result is shown in figure 3, wherein A is the acetonitrile-water solution gradient elution solvent system, and B is the methanol-water solution gradient elution solvent system.
As can be seen from fig. 3, when methanol-water is used as a mobile phase for elution, the compounds 5 and 6 cannot be detected, so an acetonitrile-water solution gradient elution solvent system is selected, formic acid is added into a water phase to improve the peak shape, and acetonitrile-0.1% formic acid water solution is used as the gradient elution solvent system, and the peak shape and the separation degree of the obtained spectrum are both good, as shown in fig. 2, so that the acetonitrile-0.1% formic acid water solution gradient elution solvent system is finally selected as the mobile phase system of the experiment.
Comparative example 3 selection of hydrolysis method
This comparative example examined the effect of different hydrolysis temperatures and the test solutions were prepared and tested as described in example 1. This comparative example compares four different hydrolysis temperatures of 30 deg.C, 50 deg.C, 70 deg.C and 95 deg.C, and the test results are shown in Table 8.
TABLE 8 Peak areas of 6 dianthrones in samples
| Hydrolysis temperature (. degree.C.) | 1 | 2 | 3 | 4 | 5 | 6 |
| 30 | 21993 | 18478 | 28267.5 | 28256 | 1820 | 1427 |
| 50 | 48420 | 39640 | 58177 | 57131 | 4647.5 | 3382 |
| 70 | 44702.5 | 37101.5 | 60944 | 60431.5 | 4485.5 | 3349 |
| 95 | 34444.5 | 28828.5 | 43292.5 | 39714.5 | 2653.5 | 1825 |
As can be seen from Table 8, the peak areas of 6 dianthrone compounds in Polygonum multiflorum obtained by hydrolysis at 50 ℃ are larger, which indicates that the dianthrone compounds to be detected have less loss and better hydrolysis efficiency when hydrolyzed at 50 ℃.
Meanwhile, the hydrolysis effects of the dilute hydrochloric acid solution and the sulfuric acid solution are considered in the comparative example, as shown in fig. 4, wherein a is hydrolysis of the dilute hydrochloric acid solution (the preparation method is the same as that in example 1), B is hydrolysis of the sulfuric acid solution, and the concentration of the sulfuric acid solution is 2.5mol/L (obtained by diluting 13.6mL of concentrated sulfuric acid to 100 mL).
As can be seen from fig. 4, when 6 dianthrones are hydrolyzed by using a sulfuric acid solution, a part of the dianthrone compounds are not detected, and a hydrochloric acid solution is finally selected to hydrolyze and combine the dianthrones; the experiment also inspects the extraction effects of two extraction solvents of dichloromethane and ethyl acetate, and the result shows that the peak area of ethyl acetate extraction is larger.
According to the embodiment and the comparative example, the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometer is adopted, an electrospray anion tandem mass spectrometry Multiple Reaction Monitoring (MRM) mode is adopted, automatic optimization comprises transmission voltage collision energy, and characteristic fragments are searched for rapid qualitative and quantitative analysis of dianthrone compounds in the polygonum multiflorum medicinal material; the detection method disclosed by the invention is simple to operate, has the advantages of high sensitivity, good precision, high stability, good repeatability and good recovery rate, can realize simultaneous determination of dianthrone components in polygonum multiflorum, makes up for the defect that the existing polygonum multiflorum medicinal material lacks of toxic component quality control indexes, and lays a foundation for quality control of polygonum multiflorum medicinal material.
Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, and not all embodiments, and one can also obtain other embodiments without inventive work according to the embodiments, and these embodiments all belong to the protection scope of the present invention.
Claims (10)
1. A detection method for 6 dianthrone compounds in polygonum multiflorum is characterized by comprising the following steps:
sequentially carrying out alcohol extraction, acid hydrolysis, extraction, organic phase concentration and redissolution on a polygonum multiflorum sample to obtain a test sample solution;
performing liquid chromatography-mass spectrometry on the test solution to obtain a chromatogram of the test solution;
the chromatographic conditions of the liquid chromatography-mass spectrometry analysis comprise:
a chromatographic column: waters acquisition UPLC CSH C18;
mobile phase: a is acetonitrile, B is formic acid aqueous solution with mass fraction of 0.1%;
flow rate: 0.3 mL/min;
the gradient elution procedure was: 0-4 min: the volume ratio of A is increased from 45% to 60%; 4-14 min: the volume ratio of A is kept at 60%; 14-16 min: the volume ratio of A is increased from 60% to 70%; 16-21 min: the volume ratio of A is kept at 70%; 21-24 min: the volume ratio of A is increased from 70% to 95%; 24-30 min: the volume ratio of A is kept at 45%;
column temperature: 25-35 ℃;
sample introduction amount: 1.0-3.0 μ L;
the mass spectrum conditions of the liquid chromatography-mass spectrometry analysis comprise:
electrospray ion source, negative ion mode, scanning mode: multiple Reaction Monitoring (MRM), temperature: 150 ℃, capillary voltage: 3.0KV, solvent removal temperature: 350 ℃, desolvation gas: nitrogen, flow rate: 650L/min;
comparing the chromatogram of the test sample with a predetermined standard spectrogram to determine the species of dianthrone compounds in the polygonum multiflorum sample;
the dianthrone compounds include: trans-emodin dianthrone, cis-emodin dianthrone, trans-emodin-physcion dianthrone, cis-emodin-physcion dianthrone, trans-physcion dianthrone, and cis-physcion dianthrone.
2. The detection method according to claim 1, wherein the alcohol extraction solvent comprises an ethanol solution, and the volume fraction of the ethanol solution is 40-60%.
3. The detection method according to claim 1 or 2, wherein the dosage ratio of the alcohol-extracted solvent to the polygonum multiflorum sample is 40-60 mL: 0.5-1.5 g.
4. The detection method as claimed in claim 1, wherein the alcohol extraction comprises ultrasonic extraction, and the time of the ultrasonic extraction is 20-40 min.
5. The detection method according to claim 1, wherein the solvent for acid hydrolysis comprises a hydrochloric acid solution, and the mass fraction of the hydrochloric acid solution is 8-12%.
6. The detection method according to claim 1 or 5, wherein the dosage ratio of the solvent for acid hydrolysis to the polygonum multiflorum sample is 5-15 mL: 0.1-0.3 g.
7. The detection method according to claim 1 or 5, wherein the temperature of the acid hydrolysis is 40 to 60 ℃ and the time is 20 to 40 min.
8. The detection method of claim 1, wherein the extracted solvent comprises ethyl acetate.
9. The detection method according to claim 1, wherein the dosage ratio of the redissolved solvent to the polygonum multiflorum sample is 25mL: 0.1-0.3 g.
10. The detection method according to claim 1, further comprising the following steps after obtaining the chromatogram of the sample: and calculating the content of the 6 dianthrone compounds in the polygonum multiflorum by using a standard equation, wherein the standard equation is an equation with the mass concentration of the 6 dianthrone compounds as an independent variable and a peak area as a dependent variable.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112964819A (en) * | 2021-02-05 | 2021-06-15 | 中国食品药品检定研究院 | Method for detecting dianthrone compounds in polygonum multiflorum |
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