CN112067708B - One-measurement-multiple-evaluation quantity detection method for erigeron breviscapus injection - Google Patents
One-measurement-multiple-evaluation quantity detection method for erigeron breviscapus injection Download PDFInfo
- Publication number
- CN112067708B CN112067708B CN202010579280.1A CN202010579280A CN112067708B CN 112067708 B CN112067708 B CN 112067708B CN 202010579280 A CN202010579280 A CN 202010579280A CN 112067708 B CN112067708 B CN 112067708B
- Authority
- CN
- China
- Prior art keywords
- dicaffeoylquinic acid
- erigeron
- erigeron breviscapus
- solution
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- 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
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Quality & Reliability (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to a one-test-multiple evaluation quantity detection method for erigeron breviscapus injection, which takes scutellarin and 3, 5-O-dicaffeoylquinic acid as reference substances, quantifies 3, 5-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, erigeron pivoxil and erigeron breviscapus in the erigeron breviscapus injection by a one-test-multiple evaluation method, and controls the batch quality stability of the erigeron breviscapus injection by a fingerprint.
Description
Technical Field
The invention relates to a one-test-multiple-evaluation-quantity detection method for erigeron breviscapus injection, belonging to the field of detection of traditional Chinese medicines.
Technical Field
The erigeron breviscapus injection is a small-volume injection produced by Yunnan biological grain pharmaceutical industry GmbH, and is prepared by extracting, purifying and refining erigeron breviscapus. Wherein the main ingredient is flavonoid and caffeic acid ester. The flavonoids mainly comprise scutellarin and breviscapine, and the caffeic acid esters mainly comprise caffeic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and erigeron breviscapus. Wherein the main caffeate component is a dicaffeonate component.
The current pharmacopoeia standards have the following disadvantages: 1. the total amount of caffeic acid esters in the content determination item is measured by ultraviolet spectrophotometry at 305nm wavelength to calculate total amount of caffeic acid esters. However, erigeron breviscapus injection contains caffeic acid ester, other flavone, small molecular aromatic acid, gamma-pyrone and other phenolic compounds, and the components all have ultraviolet absorption at the wavelength of 305nm, so when the total amount of caffeic acid ester is measured by adopting an ultraviolet spectrophotometry, the ultraviolet absorption of the caffeic acid ester component is not only detected, but also the sum of the ultraviolet absorption of the flavone and other phenolic components is included, and therefore, the ultraviolet spectrophotometry is not special for the measuring method. 2. The original standard method has insufficient accuracy, and for the reasons mentioned above, the ultraviolet absorption of caffeic acid ester components is not only available at the wavelength of 305nm, but also includes the sum of the ultraviolet absorption of flavone and other phenolic components, and this wavelength is not the maximum absorption wavelength of all caffeic acid ester components, and in addition, 1, 3-DQC is not available in the erigeron breviscapus injection by using 1, 3-O-dicaffeoylquinic acid as a control, this component is present in the root, the erigeron breviscapus injection before is wild grass, the wild grass harvest is directly extracted from the root, the erigeron breviscapus injection is planted now, the previous two harvests are used, and thus this component is not available. Relative correction factors are not introduced in the calculation, only simple calculation is carried out, and the obtained data lack accuracy.
The chemical components of the plant medicine are complex, and the content measurement of the same kind of effective components is the consensus of the quality control of the plant medicine. In the past, the same substance is quantitatively measured by a multi-purpose ultraviolet spectroscopy method, or each single component is measured by an external standard method and added to obtain the content value of the same substance in the medicine. Therefore, the method is not only difficult to obtain the reference substance, but also energy-consuming and time-consuming in the inspection period. And the single-measurement and multi-evaluation method realizes the simultaneous measurement of a plurality of components (a reference substance is difficult to obtain and the chemical and physical properties are relatively unstable) by measuring one component (the component is easy to obtain and the chemical and physical properties are relatively stable) by using a single or mixed control solution according to the internal functional relationship and the proportional relation method existing among the same type of effective components. The determination time is better shortened, the reference substance is saved, and the quality control cost is reduced.
Disclosure of Invention
In view of the above technical problems, the inventors provide a one-measurement-multiple-evaluation-amount detection method for erigeron breviscapus injection, wherein the detection method is used for measuring the content of total dicaffeonate and scutellarin, and the method comprises the following steps:
1) preparing a mixed reference substance, and preparing the reference substance of the detected substances according to the concentration of each component in the erigeron breviscapus injection at corresponding concentration;
2) preparing a test solution, wherein the erigeron breviscapus injection is prepared by diluting an ethylene diamine tetraacetic acid disodium solution;
3) chromatographic conditions and system applicability experiments, namely a chromatographic column using octadecylsilane chemically bonded silica as a filler; the flow rate is 0.8-1.2ml/min, and the mobile phase A is methanol: 25-35 parts of acetonitrile: 65-75 percent, the mobile phase B is 0.05-0.4 percent of trifluoroacetic acid, and the proportion of the mobile phase A to the mobile phase B is 16-20: 84-80 parts; the detection wavelength of the total dicaffeonate is 324-330 nm, the detection wavelength of the scutellarin is 333-340 nm, the number of theoretical plates is not lower than 5000 according to the peak of 3, 5-O-dicaffeoylquinic acid, and the column temperature is 35-45 ℃.
4) Precisely sucking the reference solution and the sample solution, respectively, injecting into a liquid chromatograph, and measuring the related content.
In the detection method, the inventor adopts a plurality of reference substances to mix, and the reference substances comprise: one or more of scutellarin, 3, 5-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, erigeron ester B and erigeron breviscapus. Preferably scutellarin, 3, 5-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, erigeron ester B and erigeron breviscapus.
In developing the method of the invention:
the technical problem 1 is that when a test solution (diluted by water or alcohol) is prepared by adopting a pharmacopoeia preparation method for a commercially available erigeron breviscapus injection, scutellarin in the solution cannot be guaranteed to be stable for 48 hours, namely, the corresponding content is reduced by about 10 percent.
In order to ensure the stability of the test solution, the inventor screens the diluent of the test solution, respectively screens organic and inorganic solvents, finally determines that the EDTA sodium salt is ideal and stable, and then screens related concentrations, wherein the EDTA sodium salt concentration in the test solution is 0.5-1.0mmol/L, and the concentration is preferably 0.7mmol/L, and can ensure the stability of the EDTA sodium salt within 48 hours.
Preparation of a test solution: precisely measuring 2ml of herba Erigerontis injection, placing into a 10ml measuring flask, preferably adding 0.6-0.8mmol/L disodium EDTA solution to dilute to scale, most preferably 0.7mmol/L disodium EDTA solution to dilute to scale, shaking, filtering, and collecting the filtrate to obtain the final product, wherein the content of the obtained test solution is stable within 48 hr.
Technical modification point 2: introducing dual-wavelength measurement, wherein the detection wavelength of total dicaffeonate is 326-328 nm, and the detection wavelength of scutellarin is 334-336 nm; preferably, the detection wavelength of total dicaffeonate is 327nm, the detection wavelength of scutellarin is 335nm, and the number of theoretical plates is not less than 5000 according to the peak of 3, 5-O-dicaffeoylquinic acid according to the methodological requirements.
The pharmacopoeia uses 1, 3-O-dicaffeoylquinic acid as a reference substance and adopts an ultraviolet spectrophotometry for determination, and the defects are as follows: at this wavelength, flavones and other phenolic components are also absorbed, and the accuracy of the test results is not high.
Technical point 3: according to the requirement of methodology, the separation degree between each test peak is about 1.5, while in the existing chromatographic method, the separation degree is not 1.5 and is about 1.0, and the defects that the order of peak emergence is reversed by changing different chromatographic columns exist. If the peaks are reversed, both the relative retention time and the relative correction factor will be reversed, necessitating the use of control extracts and external standard substances, which in turn increases the cost and complexity of the assay.
The method adopted in pharmacopoeia is as follows: octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; methanol-tetrahydrofuran-0.1% phosphoric acid solution (14: 14: 72) is used as a mobile phase; the detection wavelength is 335 nm; the column temperature was 40 ℃. The number of theoretical plates is not less than 2500 calculated by scutellarin peak.
Preparation of Total caffeate reference solution about 10mg of 1, 3-O-dicaffeoylquinic acid reference substance is precisely weighed, placed in a 10ml measuring flask, and added with 02ml of 01mol/L sodium bicarbonate solution, carrying out ultrasonic treatment (power 120W and frequency 40kHz) for 3 minutes, cooling, adding water to the scale, and shaking up; precisely measuring 1ml, placing into a 100ml measuring flask, adding water to the scale, and shaking to obtain the final product (each 1ml contains 10 μ g of 1, 3-O-dicaffeoylquinic acid). Preparing the test solution by precisely measuring 1ml of the product, placing the product in a 200ml measuring flask, adding water to dilute the product to a scale, and shaking up the product to obtain the test solution. The determination method comprises respectively taking reference solution and sample solution, measuring absorbance at 305nm wavelength by ultraviolet-visible spectrophotometry (general rule 0401), and calculating. The product contains 1, 3-O-dicaffeoylquinic acid (C) per 1ml total caffeate25H24O12) The amount of the active ingredient is 2.0-3.0 mg.
As the technical background shows, the detection method of the erigeron breviscapus injection dictionary cannot clearly show the exact content of various dicaffeonates, only 1, 3-O-dicaffeoylquinic acid is used as a reference, no relative correction factor is introduced during calculation, only simple calculation is carried out, and the obtained data lacks accuracy.
The inventor avoids using a gradient elution method to separate a plurality of components, adopts an isocratic elution method and a pharmacopoeia method, and cannot obtain a result with the separation degree meeting the requirements of methodology. Therefore, a chromatographic column using octadecylsilane chemically bonded silica as a filler is adopted; the flow rate is 0.8-1.2ml/min, and the mobile phase A is methanol: acetonitrile, preferably mobile phase a, in a ratio of methanol to acetonitrile of 30: 70: preferably, the mobile phase B is 0.1% of trifluoroacetic acid, and the ratio of A to B is 16-20: 84-80 parts; the detection wavelength of the total dicaffeonate is 324-330 nm, the detection wavelength of the scutellarin is 333-340 nm, the number of theoretical plates is not lower than 5000 calculated according to the peak of 3, 5-O-dicaffeoylquinic acid, and the column temperature is 35-45 ℃.
Particularly, if two phases in the mobile phase A are separated, stable results cannot be obtained, so that the method is extremely sensitive and stable through multiple sets of experimental detection.
In the detection method of the invention, more preferably, the chromatographic column using octadecylsilane chemically bonded silica as a filler is: waters Sunfire C18(5 μm, 4.6X 250mm), Waters Sunfire C18(5 μm, 4.6X 150mm), Agilent ZORBAX SB-C18(5 μm, 4.6X 250mm), Shimadzu Inertsustain-C18(5 μm, 4.6X 250mm), Shimadzu VP ODS-C18(5 μm, 4.6X 250mm), Agilent Eclipse XDB-C18(5 μm, 4.6X 150 mm). The chromatographic column has high utilization rate.
In the detection method, the flow rate is 1.0ml/min, the detection wavelength of the total dicaffeonate is 327nm, the detection wavelength of the scutellarin is 335nm, and the number of theoretical plates is not less than 5000 according to the peak of 3, 5-O-dicaffeoylquinic acid.
The chromatographic condition of the invention adopts double wavelengths, thus saving time and cost.
In the method, a factor of a correction factor is introduced, and the calculation method is as follows:
1) calculating the total dicaffeonate content by taking the peak area of a 3, 5-O-dicaffeoylquinic acid reference substance as a reference (3, 5-dicaffeoylquinic acid is used as a standard of all dicaffeoylquinic acids to calibrate relative retention time and correction factors, namely, only one reference substance can be used for measuring the sum of the content of all dicaffeoylquinic acids and the relative retention time), calculating the content of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid and erigeron breviscaput according to the correction factors corresponding to the following table respectively, calculating the total amount of the five substances, and determining the relative retention time of the chromatographic peak of the component to be measured and the chromatographic peak of the 3, 5-O-dicaffeoylquinic acid to determine the content of the 3, 4-O-dicaffeoylquinic acid, The peak positions of 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid and erigeron breviscapus are within the range of +/-10% of the specified value, and the preparation method is obtained.
2) And (4) using the peak area of the scutellarin reference substance as a reference, and calculating by using the peak area according to an external standard method.
The correction factors are as follows:
the preferred method of the invention is as follows:
(1) preparation of mixed control: respectively taking appropriate amount of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and scutellarin as reference substances, precisely weighing, adding about 40ml of methanol (about common technical indication, which can be 40ml + -5 ml) into a 50ml measuring flask, shaking and ultrasonic treating for 30 min, cooling, and diluting to desired volume with methanol to obtain mixed reference substance solution with concentration equivalent to that of the sample solution, wherein each 1ml of solution contains the above 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and scutellarin as reference substances, such as 50 μ g + -3, 4-O-dicaffeoylquinic acid, 3, 5-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, and scutellarin as reference substances, 30 μ g + -, Shaking the solution of 60 μ g + -6, 100 μ g + -10, shaking, and filtering with 0.45 μ l filter head to obtain the final filtrate. The measured spectrum is shown in figure 1.
(2) Preparation of a test solution: precisely measuring 2ml of herba Erigerontis injection, placing into a 10ml measuring flask, adding 0.7mmol/L disodium ethylene diamine tetraacetate solution to dilute to scale, shaking, filtering, and collecting the filtrate.
(3) Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; using [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid) ] (16-20: 84-80) as a mobile phase; the detection wavelength of the total dicaffeonate is 324-330 nm, and the detection wavelength of the scutellarin is 333-340 nm. The number of theoretical plates is not less than 5000 calculated according to the peak of 3, 5-O-dicaffeoylquinic acid.
(4) The determination method comprises the following steps: precisely sucking 5-20 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Then calculating the contents of dicaffeonate and scutellarin according to the measuring method.
Main innovation of the invention
1. If the erigeron breviscapus injection is diluted by conventional water or alcohol, the erigeron breviscapus injection is easy to oxidize in the air due to chemical components of polyphenols, and the solution is exposed in the air, so that the pH value of the solution is easy to reduce due to carbon dioxide, and the solution cannot keep the content stable within 48 hours and cannot pass methodology verification. After the disodium ethylene diamine tetraacetate solution is added, the solution can keep the content stable within 48 hours, and tests show that the addition of 0.7mmol/L disodium ethylene diamine tetraacetate solution is the best solution and can successfully pass methodology verification.
2. The choice of mobile phase is a key point within the present invention. Because the 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid and 4, 5-O-dicaffeoylquinic acid in the measured dicaffeoylquinic acid ester components are isomers, the erigeron pivoxil B and the erigeron breviscapus are also isomers, and the structures are very similar, the difficulty is created in achieving better separation degree. Through hundreds of experiments, the proportion of hundreds of solutions is improved. Selecting [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid) ] (16-20: 84-80) as a mobile phase, and preferably selecting [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid) ] (17-29: 83-81) as a mobile phase; most preferably, [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid) ] (18: 82) is used as the mobile phase. Such an isocratic mobile phase tends to behave similarly on different instruments and columns.
Drawings
FIG. 1 Mixed control spectra (327 nm);
FIG. 2 typical chromatogram of herba Erigerontis injection (327 nm).
Detailed Description
The invention is further described in the following examples, which are not intended to limit the scope of the invention. The preferred experimental method, chromatographic conditions and solvent extraction method of the invention are obtained by screening, and the screening process is as follows:
1. reference substance
A total of 6 control substances were used in this study, as shown in table 1 below;
TABLE 1 list of reference substances
The detection index for content measurement relates to 6 chemical components, and the structural information is shown in table 2;
TABLE 2 ingredient information of content detection in erigeron breviscapus injection
2. Preparation of the solution
2.1 selection of vehicle:
2.1.1 selection of control solvents
According to the physical properties of the reference substances such as 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and the like: can be dissolved in organic solvents such as methanol, ethanol, DMSO and the like, and the physical properties of the scutellarin are as follows: soluble in alkali, glacial acetic acid and pyridine, slightly soluble in common organic solvent and insoluble in water. Therefore, methanol was selected as the solvent for the control.
2.1.2 selection of test sample solvent
When a test solution is prepared, analysis compares the stability of a sample diluted by methanol, water and 0.7mmol/L disodium ethylene diamine tetraacetate solution, wherein the stability of the 0.7mmol/L disodium ethylene diamine tetraacetate solution is stable within 48 hours, so that the 0.7mmol/L disodium ethylene diamine tetraacetate solution is selected as a solvent.
2.2 Final solution preparation conditions
2.2.1 preparation of Mixed control solutions: respectively taking appropriate amounts of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and scutellarin as reference substances, precisely weighing, adding about 40ml of methanol into a 50ml measuring flask, shaking and ultrasonically treating for 30 minutes, cooling, adding methanol to a constant volume to a scale, preparing solutions respectively containing 50 mug, 30 mug, 50 mug, 60 mug, 100 mug and 100 mug in each 1ml, shaking uniformly, filtering by 0.45 mug filter head, and taking subsequent filtrate. The configured concentration range is matched with the determined concentration of the injection. For example, 1ml of the solution contains the above 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester, 4, 5-O-dicaffeoylquinic acid, and erigeron breviscapus, and scutellarin control substance 50. mu.g, 30. mu.g. + -. 3, 50. mu.g. + -. 5, 60. mu.g. + -. 6, 100. mu.g. + -. 10, and 100. mu.g. + -. 10, respectively. All can be used.
2.2.2 preparation of control solutions: respectively taking appropriate amount of 3, 5-O-dicaffeoylquinic acid and scutellarin as reference substances, precisely weighing, adding about 40ml of methanol into a 50ml measuring flask, shaking and ultrasonically treating for 30 minutes, cooling, then adding methanol to a constant volume to scale, preparing into solutions containing about 30 mug and 100 mug in each 1ml, shaking uniformly, filtering, and taking the subsequent filtrate; 2.2.3 preparation of test solutions: precisely measuring 2ml of the product, placing the product in a 10ml volumetric flask, adding 0.7mmol/L disodium ethylene diamine tetraacetate solution to dilute to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
[ CONTENT DETERMINATION ] METHOD VERIFICATION
1. Dicaffeate detection wavelength confirmed: the dicaffeonate components such as 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and the like have maximum ultraviolet absorption at the wavelength of about 327nm, so that the finally selected measuring wavelength is 327 nm. Scutellarin has maximum ultraviolet absorption at about 335nm, so the final measurement wavelength is 335 nm.
2. Mobile phase identification
Because the dicaffeonate component has a similar structure, and the polarity and the scutellarin phase are relatively small, and the HPLC chromatographic separation difficulty is relatively large, acetonitrile with smaller polarity is adopted as a mobile phase component, and the analysis compares acetonitrile-0.1% trifluoroacetic acid solution (18: 82), acetonitrile-0.1% phosphoric acid solution (17: 83), and [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid) ] 18: 82 was a mobile phase and it was found that when the mobile phase was acetonitrile-0.1% trifluoroacetic acid solution (18: 82), the retention time drifted significantly during the continuous analysis and was not suitable. The mobile phase is acetonitrile-0.1% phosphoric acid solution (17: 83), and has obvious tailing phenomenon. [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid) ] (18: 82) is used as a mobile phase, under the conditions of column temperature of 40 ℃, flow rate of 1.0ml/min and isocratic elution, scutellarin and 5 dicaffeonate components to be detected can be well separated, and a peak is stable in the continuous analysis process, so [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid) ] (18: 82) is finally used as the mobile phase.
3. Final chromatographic conditions
Octadecylsilane chemically bonded silica as filler (waters Sunfire C18, 5 μm, 4.6X 250mm) [ or Agilent ZORBAX SB-C18(250mm×4.6mm，5μm)](ii) a With [ A (methanol: acetonitrile 30: 70) -B (0.1% trifluoroacetic acid)](18: 82) the mobile phase is the mobile phase; the flow rate is 1.0 ml/min; dicaffeate total amount detection wavelength λ: 327nm, scutellarin detection wavelength lambda: 335 nm; the column temperature was 40 ℃. The number of theoretical plates is not less than 5000 calculated according to the peak of 3, 5-O-dicaffeoylquinic acid.
In order to achieve the purpose of multi-index content control, save the use of reference substances and reduce the detection cost, the research adopts a method of one test and multiple evaluation to realize the determination of a plurality of effective components by using only 2 reference substances (namely scutellarin and 3, 5-O-dicaffeoylquinic acid).
The analysis and determination results of the content analysis of the 5 dicaffeonate components in the erigeron breviscapus injection are analyzed, and the 3, 5-O-dicaffeoylquinic acid is found to be stable in a reference substance solution and is supplied with the national standard substances at present, so that the 3, 5-O-dicaffeoylquinic acid is selected as the chemical monomer reference substance in the research. Through durability examination, under the finally determined chromatographic system conditions, the relative retention time of each chromatographic peak relative to 3, 5-O-dicaffeoylquinic acid is stable, so that the relative retention time is adopted to locate each chromatographic peak. Typical chromatogram of herba Erigerontis injection is shown in figure 2.
4. Determination of relative correction factor
And (3) measuring the correction factors of other 4 dicaffeonate index components relative to the 3, 5-O-dicaffeoylquinic acid by adopting a calibration curve relative slope method and a concentration method and taking a 3, 5-O-dicaffeoylquinic acid peak as a reference.
Calculation process of slope method relative correction factor
fsxIs the phase of the reference substance and the component x to be measuredFor the correction factor
ksxIs the relative slope ratio of the reference substance s and the component x to be measured
kxThe slope of a standard curve of a certain component x to be measured
ksAs the slope of a standard curve of a reference s
Calculation process of concentration method relative correction factor
fsxRelative correction factors of a reference object and a component x to be detected are obtained;
Asas reference peak area;
Wsis the reference concentration;
Axthe peak area of a certain component x to be measured;
Wxis the concentration of a certain component x to be measured.
Calculation process of relative retention time
Relative retention time (peak retention time of the analyte/peak retention time of the reference peak)
Calibration curve and slope method for measuring correction factor (three parts)
The determination method comprises the following steps: respectively taking appropriate amounts of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and scutellarin reference substances, precisely weighing, adding about 40ml of methanol into a 50ml measuring flask, shaking for 30 minutes, cooling, fixing the volume to scale with methanol to prepare solutions respectively containing 50 mug, 30 mug, 50 mug, 60 mug, 100 mug and 100 mug in each 1ml, shaking uniformly, filtering through a 0.45 mug filter head, taking subsequent filtrate, respectively and precisely sucking and mixing 2 mug, 4 mug, 6 mug, 8 mug, 10 mug, 15 mug and 20 mug of the reference substance solution, injecting into a liquid chromatograph, measuring, continuously injecting samples for two times for each sample volume, and calculating the peak area of each component reference substance solution.
And drawing standard curves of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and the like in a spectrum of 327 nm.
Drawing a scutellarin standard curve in a graph with the wavelength of 335 nm.
And (3) drawing a standard curve (passing through an origin) by taking the sample amount (mu g) as a horizontal coordinate and the peak area as a vertical coordinate, and calculating a regression equation, a correlation coefficient r (more than or equal to 0.995) and a linear range.
The correction factors for each ingredient relative to 3, 5-O-dicaffeoylquinic acid were determined, see table 3.
TABLE 3 determination of relative correction factors
5. Retention time
Retention times were determined using standard curve methods, see tables 4-5.
TABLE 4 chromatographic peak retention time for each substance (three standard curves)
TABLE 5 relative retention time of chromatographic peaks for each substance
6. Determination of content
The erigeron breviscapus injection (20191040) samples of the same batch are respectively measured by adopting a reference substance external standard method and a correction factor method, and the relative deviation of each component is within an acceptable range. See table 6.
(if the pharmacopoeia method is used, only the total caffeine content is measured, 2-3 mg/ml. no exact second caffeine, measured by the UV method includes single caffeine and other impurities.)
Table 6. units for comparison of results of external standard method and correction factor method: mu.g/ml
7. Stability test
Mixing reference solution STD191228, reference solution STD191228 and test solution (herba Erigerontis injection 20191040), injecting sample at regular intervals under stable chromatography condition, comparing peak area changes of each component, and making 5 dicaffeonate components and scutellarin stable within 48 hr.
8. Recovery test
Preparation of mixed control stock solutions: respectively taking appropriate amounts of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester, 4, 5-O-dicaffeoylquinic acid, erigeron breviscapus and scutellarin as reference substances, precisely weighing, adding appropriate amount of methanol into a 25ml measuring flask, shaking and ultrasonically treating for 30 minutes, cooling, adding methanol to a constant volume to a scale, preparing solutions respectively containing 110 microgram, 60 microgram, 90 microgram, 130 microgram, 180 microgram and 270 microgram in each 1ml, shaking uniformly, filtering with a 0.45 microgram filter head, and taking a subsequent filtrate.
Precisely transferring about 1ml of herba Erigerontis injection (lot number: 20191040)9 parts of the test result, placing in 10ml volumetric flask, and precisely adding 1ml, 2ml and 3ml (corresponding to 50%, 100% and 150% of 1ml test solution) of the above "mixed reference substance stock solution", respectively, and each gradient is 3 parts; then adding 0.7m mol/L EDTA solution to dilute to scale, shaking up, filtering with 0.45 μm microporous membrane, collecting the filtrate, analyzing by sample injection, and recording chromatogram. The recovery of each component was calculated.
The recovery rate meets the requirements of the required methodology.
9. Durability test (conclusion section)
Calibration factor durability test
Detection wavelength (327nm, +/-1 nm, +/-2 nm)
The result shows that the durability of the detection wavelength meets the requirement (RSD is less than or equal to 3.0%)
Solution stability: the RSD percent of the correction factor is less than or equal to 3.0 percent. The RSD percent of the correction factor is less than or equal to 3.0 percent.
The RSD% of the test results (20191040 batches of erigeron breviscapus injection) of the test samples by different chromatographic columns meets the requirements, and the RSD% is less than or equal to 8%.
The correction factor RSD% of the column temperature at different levels (35-45 ℃) meets the requirement that the correction factor RSD% is less than or equal to 3.0%.
The RSD% of the test result (35-45 deg.C) of the test sample (20191040 batches of erigeron breviscapus injection) meets the requirement, and the RSD% is less than or equal to 8%.
The correction factors RSD% of the flow rates (0.8, 1.0 and 1.2ml/min) of three different levels meet the requirement, and the correction factor RSD% is less than or equal to 3.0%.
The RSD% of the test result (20191040 batches of erigeron breviscapus injection) of the test sample meets the requirement at different flow rates (0.8, 1.0 and 1.2ml/min), and the RSD% is not more than 8%.
The correction factors RSD% of the mobile phase proportion (18: 82, 16: 84 and 20: 80) of three different levels meet the requirement, and the correction factor RSD% is less than or equal to 3.0%.
The RSD% of the test results (20191040 batches of erigeron breviscapus injection) of the test samples in different mobile phase proportions (18: 82, 16: 84 and 20: 80) is less than or equal to 8 percent.
Respectively adopting a mixed reference substance external standard method and a single-standard correction factor method to measure 30 batches of erigeron breviscapus injection samples, and obtaining the result that the relative deviation of each component is within an acceptable range. RSD% (≦ 5%)
The method verifies through methodology in various aspects that the determination result is stable, the relative peak position is stable, double-wavelength determination is carried out, the stability of each result and high adaptability are ensured, correction factors are introduced, the content of each component of the scutellarin and the total caffeic acid ester is accurately determined by one-time determination, the stability among reaction batches is better, and the method has important significance for the quality control of medicines.
The above-mentioned embodiments only express a few embodiments of the present invention, and the description is specific and detailed, but it should not be understood as the limitation of the patent scope of the present invention, it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the concept of the present invention, and these all fall into the protection scope of the present invention, therefore, the protection scope of the present invention is subject to the appended claims.
Claims (6)
1. A one-measurement-multiple-evaluation quantity detection method for erigeron breviscapus injection is characterized in that the detection method simultaneously measures the contents of total dicaffeonate and scutellarin, and the method comprises the following steps:
1) preparing a mixed reference substance, namely preparing the reference substance of the detected substance according to the concentration of each component in the erigeron breviscapus injection, wherein the mixed reference substance is a mixture of scutellarin, 3, 5-O-dicaffeoylquinic acid, 3, 4-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, erigeron pivoxil and erigeron breviscapus;
2) preparing a test solution, wherein the erigeron breviscapus injection is prepared by diluting 0.5-1.0mmol/L disodium ethylene diamine tetraacetate solution;
3) chromatographic conditions and system applicability experiments, namely a chromatographic column using octadecylsilane chemically bonded silica as a filler; the flow rate was 1.0ml/min, mobile phase a was methanol: 30 parts of acetonitrile: 70, mobile phase B is 0.1% trifluoroacetic acid, the ratio of mobile phase a to mobile phase B is 18: 82; the detection wavelength of the total dicaffeonate is 327nm, the detection wavelength of the scutellarin is 335nm, the number of theoretical plates is not lower than 5000 calculated according to the peak of 3, 5-O-dicaffeoylquinic acid, and the column temperature is 35-45 ℃;
4) precisely sucking the reference solution and the sample solution, respectively, injecting into a liquid chromatograph, and measuring the related content.
2. The method for detecting the one-measurement-multiple evaluation quantity of the erigeron breviscapus injection as claimed in claim 1, wherein the preparation of the mixed reference substance comprises the following steps: respectively taking appropriate amount of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester, 4, 5-O-dicaffeoylquinic acid, octyl erigeron breviscapus and scutellarin as reference substances, precisely weighing, adding about 40ml of methanol into a 50ml measuring flask, shaking for ultrasonic treatment, cooling, adding methanol to constant volume to scale, making into mixed reference substance solution with concentration range matched with that of the sample solution, shaking, passing through 0.45 μm filter head, and taking the subsequent filtrate.
3. The method for detecting the one-test-multiple evaluation amount of the erigeron breviscapus injection as claimed in claim 1, wherein the concentration of the disodium ethylene diamine tetraacetate solution is 0.7 mmol/L.
4. The one-test-multiple-evaluation-amount detection method for the erigeron breviscapus injection as claimed in claim 1, wherein the chromatographic column using octadecylsilane chemically bonded silica as a filler is as follows: waters Sunfire C185 μm, 4.6 × 250mm, Waters Sunfire C185 μm, 4.6 × 150mm, Agilent ZORBAX SB-C185 μm, 4.6 × 250mm, Shimadzu Inertsustain-C185 μm, 4.6 × 250mm, Shimadzu ODS-C185 μm, 4.6 × 250mm, Agilent Eclipse XDB-C185 μm, 4.6 × 150 mm.
5. The method for detecting the one-measurement-multiple evaluation quantity of the erigeron breviscapus injection as claimed in claim 1, wherein the content determination method comprises the following steps:
1) calculating the content of the total dicaffeonate, respectively calculating the contents of 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid and erigeron thin octyl ester according to relative correction factors by taking the peak area of a 3, 5-O-dicaffeoylquinic acid reference substance as a reference, calculating the total amount of the five substances, determining the peak positions of the 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, erigeron ester B, 4, 5-O-dicaffeoylquinic acid and erigeron thin octyl ester according to the relative retention time of the chromatographic peak of a sample to be detected and the chromatographic peak of the 3, 5-O-dicaffeoylquinic acid, and the relative retention time of the peak positions of the 3, 4-O-dicaffeoylquinic acid, 5-O-dicaffeoylquinic acid and the eri, obtaining the product;
2) and (4) using the peak area of the scutellarin reference substance as a reference, and calculating by using the peak area according to an external standard method.
6. The method for detecting the one-measurement-multiple evaluation amount of the erigeron breviscapus injection as claimed in claim 5, wherein the relative retention time and the relative correction factor are as follows:
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010579280.1A CN112067708B (en) | 2020-06-23 | 2020-06-23 | One-measurement-multiple-evaluation quantity detection method for erigeron breviscapus injection |
| PCT/CN2020/110370 WO2021258526A1 (en) | 2020-06-23 | 2020-08-21 | Quantitative detection method for erigeron breviscapus injection by means of quantitative analysis of multi-components by single marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010579280.1A CN112067708B (en) | 2020-06-23 | 2020-06-23 | One-measurement-multiple-evaluation quantity detection method for erigeron breviscapus injection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112067708A CN112067708A (en) | 2020-12-11 |
| CN112067708B true CN112067708B (en) | 2021-06-01 |
Family
ID=73656424
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010579280.1A Active CN112067708B (en) | 2020-06-23 | 2020-06-23 | One-measurement-multiple-evaluation quantity detection method for erigeron breviscapus injection |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112067708B (en) |
| WO (1) | WO2021258526A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114577946B (en) * | 2022-03-10 | 2024-05-14 | 山东省食品药品检验研究院 | Method for detecting processing degree of honeysuckle and application thereof |
| CN114609295A (en) * | 2022-03-28 | 2022-06-10 | 中国林业科学研究院林产化学工业研究所 | High performance liquid chromatography analysis method for quinic acid content in tala enzymolysis waste liquid |
| CN114910586B (en) * | 2022-04-21 | 2024-04-05 | 上海医药工业研究院有限公司 | Method for determining caffeic acid and its derivatives content in Monascus purpureus extract |
| CN114755344B (en) * | 2022-04-21 | 2024-03-29 | 上海医药工业研究院有限公司 | Method for determining dicaffeoylquinic acid content in Monascus purpureus extract |
| CN115452983A (en) * | 2022-09-08 | 2022-12-09 | 成都市药品检验研究院 | HPLC main component self-control method for determining content of related substances in furosemide tablets |
| CN116818922A (en) * | 2022-12-30 | 2023-09-29 | 中国中医科学院中药研究所 | Method for quantifying multiple compounds with the same chromophore using a single standard |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1187356A (en) * | 1997-01-08 | 1998-07-15 | 大连弘丰制药厂 | Stable prescription and process of fleabane extract injection |
| CN1565465A (en) * | 2003-06-26 | 2005-01-19 | 天津药物研究院 | Injection preparation containing breviscapine active component and its preparation method |
| CN1830451A (en) * | 2005-03-08 | 2006-09-13 | 阿尔贝拉医药控股(通化)有限公司 | Preparation method of erigeron breviscapus glucese injection |
| CN102650626A (en) * | 2011-10-17 | 2012-08-29 | 云南施普瑞生物工程有限公司 | Standard fingerprint of fleabane phenol bulk drug and preparation, establishment method and application |
| CN102914609A (en) * | 2012-11-15 | 2013-02-06 | 昆明振华制药厂有限公司 | Method for simultaneously detecting contents of four active ingredients in herba erigerontis preparation |
| CN107782835A (en) * | 2017-12-08 | 2018-03-09 | 云南农业大学 | A kind of method of efficient liquid phase detection fleabane flower composition |
| CN108078920A (en) * | 2018-02-27 | 2018-05-29 | 云南玉药生物制药有限公司 | A kind of Breviscapini injection preparation process of stabilization |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104586911B (en) * | 2014-04-21 | 2016-01-27 | 林艳和 | Pharmaceutical composition containing caffeic acid ester and lamp-dish flower acetic and its preparation method and application |
-
2020
- 2020-06-23 CN CN202010579280.1A patent/CN112067708B/en active Active
- 2020-08-21 WO PCT/CN2020/110370 patent/WO2021258526A1/en active Application Filing
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1187356A (en) * | 1997-01-08 | 1998-07-15 | 大连弘丰制药厂 | Stable prescription and process of fleabane extract injection |
| CN1565465A (en) * | 2003-06-26 | 2005-01-19 | 天津药物研究院 | Injection preparation containing breviscapine active component and its preparation method |
| CN1830451A (en) * | 2005-03-08 | 2006-09-13 | 阿尔贝拉医药控股(通化)有限公司 | Preparation method of erigeron breviscapus glucese injection |
| CN102650626A (en) * | 2011-10-17 | 2012-08-29 | 云南施普瑞生物工程有限公司 | Standard fingerprint of fleabane phenol bulk drug and preparation, establishment method and application |
| CN102914609A (en) * | 2012-11-15 | 2013-02-06 | 昆明振华制药厂有限公司 | Method for simultaneously detecting contents of four active ingredients in herba erigerontis preparation |
| CN107782835A (en) * | 2017-12-08 | 2018-03-09 | 云南农业大学 | A kind of method of efficient liquid phase detection fleabane flower composition |
| CN108078920A (en) * | 2018-02-27 | 2018-05-29 | 云南玉药生物制药有限公司 | A kind of Breviscapini injection preparation process of stabilization |
Non-Patent Citations (3)
| Title |
|---|
| HPLC 同时测定灯盏细辛提取物中3种有效成分的含量;王舒;《中国实验方剂学杂志》;20151031;第21卷(第19期);第72-74页 * |
| 灯盏细辛注射液调控ROS/RNS-MMPs-TJs信号通路保护脑缺血致大鼠BBB损伤的机制研究;刘光丽;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20190115(第01期);第E057-433页 * |
| 高效液相色谱法测定灯盏细辛提取物中两种有效成分的含量;李春平等;《北方药学》;20161231;第13卷(第10期);第8-9页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021258526A1 (en) | 2021-12-30 |
| CN112067708A (en) | 2020-12-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112067708B (en) | One-measurement-multiple-evaluation quantity detection method for erigeron breviscapus injection | |
| CN101126752B (en) | Method for simultaneously determining multiple polyphenolic compound content of fruit | |
| CN109596751A (en) | A kind of Mailuo Ning oral liquid component detection method of clearing heat and nourishing yin promoting blood circulation | |
| Cai et al. | Rapid quantification of flavonoids in propolis and previous study for classification of propolis from different origins by using near infrared spectroscopy | |
| Kulić et al. | A detailed view on the proanthocyanidins in Ginkgo extract EGb 761 | |
| CN102078403A (en) | Detection method of Chinese traditional medicine preparation of oral liquid for treating infant cough with lung heat | |
| CN113484430B (en) | Method for measuring related substances of L-alanine isopropyl ester hydrochloride by adopting high performance liquid chromatography | |
| Chen et al. | Determination of marker constituents in radix Glycyrrhizae and radix Notoginseng by near infrared spectroscopy | |
| CN108254471A (en) | The detection method of 5 hydroxymethyl furfural and furfural content in xylo-oligosaccharide | |
| CN114527218A (en) | Method for detecting components of scutellaria baicalensis | |
| CN112611821B (en) | High performance liquid chromatography analysis method for calcium gluconate related substances | |
| CN112903860B (en) | Detection method of related substance MPEG in benzonatate | |
| CN112858530A (en) | Eucommia ulmoides leaf and eucommia ulmoides male flower extract and method for detecting multi-index components in products thereof | |
| CN112858531A (en) | Method for establishing HPLC (high performance liquid chromatography) characteristic spectrum of eucommia leaves and extract thereof | |
| CN105372369A (en) | Danhong injection fingerprint determination method | |
| CN113702514A (en) | Method for determining atorvastatin calcium related impurity I | |
| CN112162052A (en) | Method for determining multiple residues of veterinary drugs in aquatic products | |
| CN115575541B (en) | Method for simultaneously measuring multiple active ingredients in silver Huang Erchen mixture | |
| CN115598261B (en) | Method for measuring related substances in raw material and preparation of clopidodine hydrochloride | |
| CN109100456B (en) | Method for simultaneously determining content of 3 fat-soluble vitamins in multivitamin injection | |
| Baranowska et al. | Development and validation of a UHPLC method for the determination of flavonoids in red wine | |
| CN106290686A (en) | HPLC MS MS measures the method for WANGLAOJI LIANGCHA component content | |
| CN113030329B (en) | One-test-multiple-evaluation method for determining content of 6 gingerol components in ginger juice brown sugar | |
| CN108956835A (en) | A kind of fingerprint atlas detection method of the antipyretic oral drugs of clearing | |
| Wei et al. | Determination of AF and AFG in Red Ginseng by High Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD). |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |