WO2021258526A1 - 一种灯盏细辛注射液的一测多评定量检测方法 - Google Patents

一种灯盏细辛注射液的一测多评定量检测方法 Download PDF

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WO2021258526A1
WO2021258526A1 PCT/CN2020/110370 CN2020110370W WO2021258526A1 WO 2021258526 A1 WO2021258526 A1 WO 2021258526A1 CN 2020110370 W CN2020110370 W CN 2020110370W WO 2021258526 A1 WO2021258526 A1 WO 2021258526A1
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acid
dicaffeoylquinic acid
scutellarin
ester
detection method
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PCT/CN2020/110370
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French (fr)
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林艳和
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云南生物谷药业股份有限公司
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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
    • 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

Definitions

  • the invention relates to a detection method for one measurement and multiple evaluations of Erigeron breviscapus injection, which belongs to the field of traditional Chinese medicine detection.
  • Erigeron breviscapus injection is a small-volume injection produced by Yunnan Biological Valley Pharmaceutical Co., Ltd., which is extracted and purified from the medicinal materials of breviscapine.
  • the main components are flavonoids and caffeic acid esters.
  • the main flavonoids are scutellarin and scutellarin, and the caffeic acid esters are mainly caffeic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid.
  • Dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, peprofen ester B and breviscapine ester are dicaffeic acid esters.
  • the current pharmacopoeia standards have the following shortcomings: 1.
  • the total amount of caffeic acid esters in the content determination item is determined by ultraviolet spectrophotometry at a wavelength of 305nm to determine the absorbance to calculate the total amount of caffeic acid esters.
  • caffeic acid ester in Dengzhanxixin injection there are other flavonoids, small molecule aromatic acids, ⁇ -pyrone and other phenolic compounds. These components have ultraviolet absorption at 305nm wavelength, so ultraviolet spectroscopy is used.
  • it When determining the total amount of caffeic acid esters by photometry, it not only detects the ultraviolet absorption of caffeic acid esters, but also includes the sum of ultraviolet absorption of flavonoids and other phenolic components.
  • the selection of ultraviolet spectrophotometry as a measurement method lacks specificity. 2.
  • the original standard method is not accurate enough.
  • the 305nm wavelength not only has the ultraviolet absorption of caffeic acid esters, but also includes the sum of the ultraviolet absorption of flavonoids and other phenolic components, and this wavelength is not all caffeic acid esters.
  • the maximum absorption wavelength in addition, with 1,3-O-dicaffeoylquinic acid as a control, 1,3-DQC is not in the breviscapine injection, this component exists in the root, the previous breviscapine injection Wild grasses are used. Wild grasses are harvested directly by uprooting, and now they are planted, using the first two harvests, so there is no such ingredient. The calculation did not introduce relative correction factors, only simple calculations were performed, and the obtained data lacked accuracy.
  • the chemical components of botanicals are complex, and the determination of the content of similar effective components is the consensus of the quality control of botanicals.
  • ultraviolet spectroscopy was used to quantitatively determine the same type of substance, or the external standard method was used to measure each single component and add up to obtain the content value of the same type of substance in the drug. In this way, it is not only difficult to obtain reference products, but also energy-consuming and time-consuming for long inspection cycles.
  • the one-test-multi-evaluation method uses a single or mixed reference solution to determine one component (easy to obtain, more stable chemical and physical properties) to achieve multiple components ( It is more difficult to obtain the reference substance, and the chemical and physical properties are relatively unstable.) Simultaneous determination. Better shorten the measurement time, save the reference substance, and reduce the cost of quality control.
  • the inventors provide a one-measure and multiple-rate detection method for breviscapine injection, wherein the detection method determines the content of total dicaffeate and scutellarin, and the method includes the following steps :
  • the preparation of the mixed reference substance, the reference substance of the tested substance is configured according to the concentration of each component in the breviscapine injection;
  • the inventors used a variety of reference substances to mix, the reference substances include: wild baicalin, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid One or more of nicotinic acid, 4,5-O-dicaffeoylquinic acid, peprofen ester B, and scutellarin ester.
  • the reference substances include: wild baicalin, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid
  • the inventor screened the diluent of the test solution, and screened the organic and inorganic solvents respectively, and finally determined that the EDTA sodium salt was ideally stable, and then performed the relevant concentration screening.
  • the concentration of EDTA sodium salt of 0.5-1.0mmol/L can ensure its stability within 48 hours, and the preferred concentration is 0.7mmol/L.
  • test solution accurately measure 2ml of Erigeron breviscapus injection and place it in a 10ml measuring flask, preferably add 0.6-0.8mmol/L ethylenediaminetetraacetate disodium solution to the mark, most preferably 0.7mmol/L ethyl acetate
  • the disodium diamine tetraacetate solution is diluted to the mark and shaken, filtered, and the subsequent filtrate is taken to obtain it.
  • the obtained test solution maintains a stable content within 48 hours.
  • Technical modification point 2 Introduce dual-wavelength measurement, select the detection wavelength of total dicaffeate at 326 ⁇ 328nm, and the detection wavelength of scutellarin at 334 ⁇ 336nm; preferably, the detection wavelength of total dicaffeate is 327nm and the detection wavelength of scutellarin. It is 335nm. According to methodological requirements, the number of theoretical plates should not be less than 5000 based on the peak of 3,5-O-dicaffeoylquinic acid.
  • the pharmacopoeia uses 1,3-O-dicaffeoylquinic acid as a reference substance, and it is determined by ultraviolet spectrophotometry.
  • the disadvantage is that at this wavelength, flavonoids and other phenolic components are also absorbed, and the test results are not accurate.
  • the resolution between each test peak should be in the range of about 1.5, while in the existing chromatography method, the resolution is less than 1.5, at about 1.0, and there are different chromatographic columns, and peaks The order will be reversed. If the peak is reversed, the relative retention time and relative correction factor will be reversed, and control extracts and external standard substances must be used, which will increase the cost and complexity of the test.
  • the method adopted in the pharmacopoeia chromatographic conditions and system suitability test with octadecylsilane bonded silica as filler; methanol-tetrahydrofuran-0.1% phosphoric acid solution (14:14:72) as mobile phase; detection wavelength is 335nm; The column temperature is 40°C.
  • the number of theoretical plates should not be less than 2500 based on the peak of wild baicalin.
  • Preparation of total caffeic acid ester reference substance solution Take about 10mg of 1,3-O-dicaffeoylquinic acid reference substance, accurately weigh it, and place it in a 10ml measuring flask, add 2ml of 0.01mol/L sodium bicarbonate solution, and ultrasonic treatment (Power 120W, frequency 40kHz) for 3 minutes, let cool, add water to the mark, shake well; accurately measure 1ml, place in a 100ml measuring flask, add water to the mark, shake well, and then get (each 1ml contains 1,3-O- Dicaffeoylquinic acid 10 ⁇ g).
  • Preparation of the test solution Precisely measure 1ml of this product, put it in a 200ml measuring flask, dilute to the mark with water, and shake well to get it. Determination method Take the reference solution and the test solution respectively, according to the ultraviolet-visible spectrophotometry (General Rule 0401), measure the absorbance at the wavelength of 305nm, calculate, and get.
  • This product contains 1,3-O-dicaffeoylquinic acid (C 25 H 24 O 12 ) per 1ml of total caffeic acid ester, which should be 2.0 ⁇ 3.0mg.
  • the pharmacopoeial detection method of breviscapine injection cannot clearly show the exact content of various dicaffeic acid esters. Only 1,3-O-dicaffeoylquinic acid is used as a control, and no relative correction is introduced in the calculation. Factors, only simple calculations, the data obtained lack accuracy.
  • the inventor avoids using a gradient elution method to separate multiple components. Using the isocratic elution method and the pharmacopoeia method, it is impossible to obtain a result with a resolution that meets the methodological requirements. Therefore, a chromatographic column with octadecylsilane-bonded silica gel as a filler is used; the flow rate is 0.8-1.2ml/min, and the mobile phase A is methanol:acetonitrile, preferably the ratio of methanol to acetonitrile in mobile phase A is 30:70 :
  • the mobile phase B is 0.1% trifluoroacetic acid, A:B is 16-20:84-80; the detection wavelength of total dicaffeic acid ester is 324-330nm, the detection wavelength of wild baicalin is 333-340nm, the number of theoretical plates According to the calculation of 3,5-O-dicaffeoylquinic acid peak, it should be no less than 5000, and the column temperature should be 35 ⁇
  • the present invention is extremely sensitive and stable after multiple sets of experiments.
  • the chromatographic column with the octadecylsilane-bonded silica gel as the filler Waters Sunfire C18 (5 ⁇ m, 4.6 ⁇ 250mm), Waters Sunfire C18 (5 ⁇ m, 4.6 ⁇ 150mm), Agilent ZORBAX SB-C18 (5 ⁇ m, 4.6 ⁇ 250mm), Shimadzu InertSustain-C18 (5 ⁇ m, 4.6 ⁇ 250mm), Shimadzu VP ODS-C18 (5 ⁇ m, 4.6 ⁇ 250mm), Agilent XDB-C18 (5 ⁇ m, 4.6 ⁇ 250mm) 150mm).
  • the column usage rate is strong.
  • the flow rate is 1.0ml/min
  • the detection wavelength of total dicaffeic acid ester is 327nm
  • the detection wavelength of scutellarin is 335nm
  • the number of theoretical plates is calculated based on the peak of 3,5-O-dicaffeoylquinic acid Should not be less than 5000.
  • the chromatographic condition of the invention adopts dual wavelengths, which saves time and cost.
  • the factor of the correction factor is further introduced, and the calculation method is as follows:
  • the total dicaffeic acid ester content is calculated with the peak area of the 3,5-O-dicaffeoylquinic acid reference substance as the control (3,5-dicaffeic acid is used as the standard for all dicaffeic acid, so as to calibrate the relative retention time And the correction factor, that is, only this standard product can be used to determine the sum of the content and relative retention time of all two coffees), respectively calculate the 3,4-O-dicaffeoylquinic acid, The content of 3,5-O-dicaffeoylquinic acid, feprofen ester B, 4,5-O-dicaffeoylquinic acid, and scutellarin ester, calculate the total amount of the above five substances, and use the components to be tested Determination of the relative retention time of the chromatographic peak and the chromatographic peak of 3,5-O-dicaffeoylquinic acid 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, peprofen ester B.
  • the correction factors are as follows:
  • the preferred method of the present invention is:
  • each 1ml solution contains the above 3,4-O-dicaffeoylquinic acid, 3,5 -O-dicaffeoylquinic acid, peperone ester B, 4,5-O-dicaffeoylquinic acid, scutellarin ester and scutellarin reference substance 50 ⁇ g, 30 ⁇ g ⁇ 3, 50 ⁇ g ⁇ 5, 60 ⁇ g ⁇ 6 , 100 ⁇ g ⁇ 10, 100 ⁇ g ⁇ 10 solution, shake well, pass through 0.45 ⁇ l filter head, take additional filtrate, and get it. See attached figure 1 for the determination spectrum.
  • test solution accurately measure 2ml of Erigeron breviscapus injection, put it in a 10ml measuring flask, add 0.7mmol/L ethylenediaminetetraacetic acid disodium solution to dilute to the mark, shake well, filter, and take Continue the filtrate to get.
  • test solution Preparation of the test solution. If the breviscapine injection is diluted with conventional water or alcohol, it is easy to be oxidized in the air due to its chemical composition of polyphenols, and the solution is exposed to the air. Carbon dioxide easily causes the pH of the solution to drop, making the solution unable to maintain a stable content within 48 hours, and it cannot pass the methodological verification. After adding the disodium ethylenediaminetetraacetate solution, the solution can maintain a stable content within 48 hours. After testing, it is found that adding 0.7mmol/L disodium ethylenediaminetetraacetate solution is the best, and it can successfully pass the methodological verification.
  • the choice of mobile phase is the key point in the present invention. Since the measured dicaffeic acid esters are the same as 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid
  • the isomers, and feprofen ester B and scutellarin ester are also isomers, and their structures are very similar. It is difficult to achieve better resolution. After hundreds of experiments, the ratio of hundreds of solutions.
  • Figure 2 Typical chromatogram of Erigeron breviscapus injection (327nm).
  • the detection indicators for content determination involve 6 chemical components, and their structure information is shown in Table 2;
  • the stability of the sample diluted with methanol, water and 0.7mmol/L disodium ethylenediaminetetraacetate solution was analyzed and compared. Among them, only the stability performance of 0.7mmol/L disodium ethylenediaminetetraacetate solution was analyzed. It was stable within 48 hours, so 0.7mmol/L disodium ethylenediaminetetraacetate solution was chosen as the solvent.
  • each 1ml solution contains the above-mentioned 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, feberyl ester B, and 4,5-O-dicaffeoylquinic acid.
  • Scutellarin and scutellarin reference substance 50 ⁇ g, 30 ⁇ g ⁇ 3, 50 ⁇ g ⁇ 5, 60 ⁇ g ⁇ 6, 100 ⁇ g ⁇ 10, 100 ⁇ g ⁇ 10 solution. It can be.
  • test solution accurately measure 2ml of this product, place it in a 10ml volumetric flask, add 0.7mmol/L disodium ethylenediaminetetraacetate solution to the mark, shake well, filter, take the additional filtrate, Immediately.
  • dicaffeic acid ester 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, peprofen B, 4,5-O-dicaffeoylquinic acid
  • the dicaffeic acid ester components such as scutellarin and scutellarin all have the maximum ultraviolet absorption around 327nm wavelength, so the final measurement wavelength is 327nm.
  • Scutellarin has the maximum ultraviolet absorption around 335, so the final measurement wavelength is 335nm.
  • the mobile phase is acetonitrile-0.1% phosphoric acid solution (17:83), which has obvious tailing phenomenon.
  • [A(methanol:acetonitrile 30:70)-B(0.1% trifluoroacetic acid)] (18:82) is the mobile phase at 40°C, flow rate 1.0ml/min, isocratic elution conditions, wild baicalin And the 5 dicaffeic acid esters to be tested components can be separated well, and the peaks are stable in the continuous analysis process, so the final choice is [A(methanol:acetonitrile 30:70)-B(0.1% trifluoroacetic acid)] ( 18:82) is the mobile phase.
  • octadecylsilane bonded silica as filler (waters Sunfire C18, 5 ⁇ m, 4.6 ⁇ 250mm) [or Agilent ZORBAX SB-C 18 (250mm ⁇ 4.6mm, 5 ⁇ m)]; use [A(methanol:acetonitrile 30: 70)-B (0.1% trifluoroacetic acid)] (18:82) is the mobile phase; the flow rate is 1.0ml/min; the detection wavelength of the total amount of caffeate ⁇ : 327nm, the detection wavelength of wild baicalin ⁇ : 335nm; column temperature is 40°C.
  • the number of theoretical plates should not be less than 5000 calculated based on the peak of 3,5-O-dicaffeoylquinic acid.
  • this study adopted a method of one test and multiple evaluations to achieve the use of only two reference substances (i.e. wild baicalin and 3,5-O-two). Caffeoylquinic acid), determination of multiple active ingredients.
  • f sx is the relative correction factor of the reference object and the component to be measured x
  • k sx is the relative slope ratio of the reference s and the component to be measured x
  • k x is the slope of the standard curve of a component to be tested
  • k s is the slope of the standard curve of the reference s
  • f sx is the relative correction factor of the reference object and the component to be measured x;
  • a s is the peak area of the reference object
  • W s is the concentration of the reference substance
  • a x is the peak area of a component to be measured x
  • W x is the concentration of a component x to be tested.
  • Relative retention time retention time of analyte peak / retention time of reference peak
  • Determination method take 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, feprofen B, 4,5-O-dicaffeoylquinic acid, scutellarin An appropriate amount of octyl ester and scutellarin reference substance, accurately weighed, to a 50ml measuring flask, add about 40ml methanol, shake and ultrasonic for 30 minutes, let cool, and then use methanol to make the volume to the mark, and each 1ml contains approximately 50 ⁇ g, 30 ⁇ g, 50 ⁇ g, 60 ⁇ g, 100 ⁇ g, 100 ⁇ g solutions, shake well, pass through a 0.45 ⁇ m filter, take the additional filtrate, and accurately pipette the mixed reference solution 2 ⁇ l, 4 ⁇ l, 6 ⁇ l, 8 ⁇ l, 10 ⁇ l, 15 ⁇ l, 20 ⁇ l, injection solution In the phase chromatograph, determine, each injection volume is injected twice consecutively, and calculate the average value of the peak area of each component
  • Preparation of mixed reference substance stock solution take 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, peprofen B, 4,5-O-dicaffeoylquinic acid, respectively Ning acid, scutellarin and scutellarin reference substance are appropriate, accurately weighed, to a 25ml measuring flask, add about an appropriate amount of methanol, shake and ultrasonic for 30 minutes, let cool, and then dilute to the mark with methanol to make each 1ml
  • the medium contains about 110 ⁇ g, 60 ⁇ g, 90 ⁇ g, 130 ⁇ g, 180 ⁇ g, 270 ⁇ g solution, shake well, pass through a 0.45 ⁇ m filter, take the filtrate, and get it.
  • the recovery rate meets the requirements of the required methodology.
  • the correction factor RSD% should be ⁇ 3.0%.
  • the correction factor RSD% should be ⁇ 3.0%.
  • the RSD% of the test results of different chromatographic columns (Batch of Dengzhanxixin injection 20191040) meets the requirements, and the RSD% should be ⁇ 8%.
  • the correction factor RSD% of column temperature at different levels meets the requirements.
  • the correction factor RSD% should be ⁇ 3.0%.
  • the RSD% of the test product (35-45°C) (Batch of Dengzhanxixin Injection 20191040) at different column temperatures meets the requirements, and the RSD% should be ⁇ 8%.
  • the correction factor RSD% of the three different levels of flow rate (0.8, 1.0, 1.2ml/min) meets the requirements, and the correction factor RSD% should be ⁇ 3.0%.
  • the correction factor RSD% of the three different levels of mobile phase ratios (18:82, 16:84, 20:80) meets the requirements, and the correction factor RSD% should be ⁇ 3.0%.
  • the RSD% of the test results (Batch of Erigeron breviscapus injection 20191040) of the test samples (18:82, 16:84, 20:80) with different mobile phase ratios should be ⁇ 8%.
  • the mixed reference substance external standard method and single standard correction factor method were used to determine 30 batches of breviscapine injection samples, and the relative deviation of each component was within the acceptable range. RSD%( ⁇ 5%)
  • the present invention has passed various methodological verifications, stable measurement results, stable relative peak positions, dual-wavelength measurement, ensuring stable results and high adaptability each time, introducing correction factors, one measurement, and accurate measurement of wild baicalin and total caffeic acid esters
  • the content of each ingredient better reflects the stability between batches, which is of great significance for the quality control of medicines.

Abstract

一种灯盏细辛注射液的一测多评定量检测方法，该方法以野黄芩苷和3,5-O-二咖啡酰奎宁酸为对照品，通过一测多评法定量灯盏细辛注射液中野黄芩苷和二咖啡酸酯类化合物3,5-O-二咖啡酰奎宁酸、3,4-O-二咖啡酰奎宁酸、4,5-O-二咖啡酰奎宁酸、飞蓬酯乙和灯盏细辛酯，并通过指纹图谱控制灯盏细辛注射液批间质量稳定性，该检测方法具有一测多评，测试结果准确稳定，节省测试时间及成本的优势。

Description

一种灯盏细辛注射液的一测多评定量检测方法 技术领域
本发明涉及一种灯盏细辛注射液的一测多评定量检测方法，属于中药检测领域。
技术背景
灯盏细辛注射液是云南生物谷药业股份有限公司生产的小容量注射液，由灯盏细辛药材提取纯化精制而成。其中主要成分为黄酮类和咖啡酸酯类成分。黄酮类成分主要为野黄芩苷和灯盏花甲素，咖啡酸酯类成分主要有咖啡酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，3，4-O-二咖啡酰奎宁酸，3，5-O-二咖啡酰奎宁酸，4，5-O-二咖啡酰奎宁酸，飞蓬酯乙和灯盏细辛酯。而其中主要的咖啡酸酯成分为二咖啡酸酯类成分。
现行药典标准有如下不足：1、含量测定项中咖啡酸酯类总量采用紫外分光光度法在305nm波长处测定吸收度，计算咖啡酸酯物质的总量。但是，灯盏细辛注射液中除了有咖啡酸酯，还有其他黄酮、小分子芳香酸，γ-吡喃酮等多种酚类化合物，这些成分在305nm波长均有紫外吸收，所以采用紫外分光光度法测定咖啡酸酯的总量时，不仅是检测咖啡酸酯成分的紫外吸收，还包括黄酮及其它酚类成分紫外吸收的总和，因此，选用紫外分光光度法作为测定方法缺乏专属性。2、原标准方法准确度不够，同上述理由，305nm波长处不仅有咖啡酸酯成分的紫外吸收，还包括黄酮及其它酚类成分紫外吸收的总和，且这个波长并不是所有咖啡酸酯成分的最大吸收波长，另外，以1，3-O-二咖啡酰奎宁酸作为对照，1，3-DQC在灯盏细辛注射液里没有，此成分存在于根中，以前的灯盏细辛注射液是用野生草，野生草采收是直接连根拔出来，现在种植的，用的是前两次收割品，因而没有此成分。计算时没有引入相对校正因子，仅进行简单的计算，所获得的数据缺乏准确性。
植物药化学成分复杂，同类药效成分的含量测定是植物药质量控制的共识。以往多用紫外光谱法定量测定同一类物质，或者用外标法测定每一个单一成分加和起来得到药品中同一类物质的含量数值。这样不但面临对照品难得，检验周期长还耗能、耗时。而一测多评法通过用单一或者混合的对照溶液，根据同一类有效成分间存在的内在函数关 系和比例关系法，测定一个成分(较易获得，化学物理性质较稳定)实现多个成分(较难获得对照品，化学物理性质相对不太稳定)同时测定。较好地缩短测定时间，节约对照品，降低质量控制成本。
发明内容
针对上述技术问题，发明人提供了一种灯盏细辛注射液的一测多评定量检测方法，其中，所述检测方法测定总二咖啡酸酯和野黄芩苷的含量，所述方法包括以下步骤：
1)混合对照品的制备，将所检测物质的对照品按照灯盏细辛注射液中各成分的浓度进行相应浓度配置；
2)供试品溶液配置，所述灯盏细辛注射液使用乙二胺四乙酸二钠溶液进行稀释配置；
3)色谱条件与系统适用性实验，以十八烷基硅烷键合硅胶为填充剂的色谱柱；流速为0.8-1.2ml/min，流动相A为甲醇：乙腈25-35：65-75，流动相B为0.05-0.4％的三氟乙酸，流动相A与流动相B的比例为16～20：84～80；总二咖啡酸酯的检测波长为324～330nm、野黄芩苷的检测波长为333～340nm，理论板数按3，5-O-二咖啡酰奎宁酸峰计算应不低于5000，柱温35～45℃。
4)分别精密吸取对照品溶液与供试品溶液，注入液相色谱仪，测定相关含量。
在本检测方法中，发明人采用了多种对照品进行混合，所述对照品包括：野黄芩苷、3，5-O-二咖啡酰奎宁酸、3，4-O-二咖啡酰奎宁酸、4，5-O-二咖啡酰奎宁酸、飞蓬酯乙和灯盏细辛酯的一种或多种。优选为野黄芩苷、3，5-O-二咖啡酰奎宁酸、3，4-O-二咖啡酰奎宁酸、4，5-O-二咖啡酰奎宁酸、飞蓬酯乙和灯盏细辛酯的混合。
在研发本发明的方法时：
技术难题1为，市售灯盏细辛注射液采用药典配置方法配置供试液时(水稀释或醇稀释)，溶液中野黄芩苷无法保证48小时稳定，即相应含量减少约10％。
为了能够保证供试液的稳定，发明人对于供试液的稀释液进行筛选，分别筛选过有机和无机溶剂，最终确定EDTA钠盐为理想的稳定，而后进行相关浓度筛选，当供试液中EDTA钠盐浓度为0.5-1.0mmol/L能够保证其48小时内稳定，其中优选浓度为0.7mmol/L。
供试品溶液的制备：精密量取灯盏细辛注射液2ml，置10ml量瓶中，优选加 0.6-0.8mmol/L乙二胺四醋酸二钠溶液稀释至刻度，最优选0.7mmol/L乙二胺四醋酸二钠溶液稀释至刻度摇匀，滤过，取续滤液，即得，所得供试液48小时内保持含量稳定。
技术修改点2：引入双波长测定，分别选用总二咖啡酸酯检测波长为326～328nm、野黄芩苷检测波长为334～336nm；优选总二咖啡酸酯检测波长为327nm、野黄芩苷检测波长为335nm，根据方法学要求，理论板数按3，5-O-二咖啡酰奎宁酸峰计算应不低于5000。
药典使用1，3-O-二咖啡酰奎宁酸为对照品，采用紫外分光光度法进行测定，存在缺点为：该波长下，黄酮及其它酚类成分也有吸收，测试结果准确度不高。
技术点3：根据方法学要求，各个测试峰之间分离度要在1.5左右范围，而现有色谱学方法中，分离度达不到1.5，在1.0左右，且存在换不同的色谱柱，出峰的顺序会颠倒的缺点。如果峰颠倒了，相对保留时间和相对校正因子都会颠倒，必须使用对照提取物和外标物质，倒增加检验的成本和复杂程度。
药典中采用方法：色谱条件与系统适用性试验以十八烷基硅烷键合硅胶为填充剂；以甲醇-四氢呋喃-0.1％磷酸溶液(14：14：72)为流动相；检测波长为335nm；柱温为40℃。理论板数按野黄芩苷峰计算应不低于2500。
总咖啡酸酯对照品溶液的制备取1，3-O-二咖啡酰奎宁酸对照品约10mg，精密称定，置10ml量瓶中，加0.01mol/L碳酸氢钠溶液2ml，超声处理(功率120W，频率40kHz)3分钟，放冷，加水至刻度，摇匀；精密量取1ml，置100ml量瓶中，加水至刻度，摇匀，即得(每1ml含1，3-O-二咖啡酰奎宁酸10μg)。供试品溶液的制备精密量取本品1ml，置200ml量瓶中，加水稀释至刻度，摇匀，即得。测定法分别取对照品溶液与供试品溶液，照紫外-可见分光光度法(通则0401)，在305nm波长处测定吸光度，计算，即得。本品每1ml含总咖啡酸酯以1，3-O-二咖啡酰奎宁酸(C 25H 24O 12)计，应为2.0～3.0mg。
正如技术背景所述，灯盏细辛注射液药典检测方法无法清晰显示各种二咖啡酸酯的确切含量，仅使用1，3-O-二咖啡酰奎宁酸作为对照，计算时没有引入相对校正因子，仅进行简单的计算，所获得的数据缺乏准确性。
发明人避免使用了梯度洗脱的方法来分离多种成分，采用等度洗脱这个方法，采用药典方法，无法得到分离度符合方法学要求的结果。因此采用了以十八烷基硅烷键合硅胶为填充剂的色谱柱；流速为0.8-1.2ml/min，流动相A为甲醇：乙腈，优选流动相A 的甲醇与乙腈的比例为30：70：优选流动相B为0.1％的三氟乙酸，A:B为16～20：84～80；总二咖啡酸酯检测波长为324～330nm、野黄芩苷检测波长为333～340nm，理论板数按3，5-O-二咖啡酰奎宁酸峰计算应不低于5000，柱温35～45℃。
特别强调，如果将流动相A中的两相进行拆分，则无法获得稳定的结果，因此本发明经过多组实验检测，极为灵敏和稳定。
本发明中检测方法，更有选地，所述十八烷基硅烷键合硅胶为填充剂的色谱柱：Waters Sunfire C18(5μm，4.6×250mm)、Waters Sunfire C18(5μm，4.6×150mm)、Agilent ZORBAX SB-C18(5μm，4.6×250mm)、岛津InertSustain-C18(5μm，4.6×250mm)、岛津VP ODS-C18(5μm，4.6×250mm)、Agilent Eclipse XDB-C18(5μm，4.6×150mm)中一种。色谱柱使用率强。
在本发明检测方法中，流速为1.0ml/min，总二咖啡酸酯检测波长为327nm、野黄芩苷检测波长为335nm，理论板数按3，5-O-二咖啡酰奎宁酸峰计算应不低于5000。
本发明色谱条件采用双波长，更节约时间和成本。
在本发明方法中，更引入了校正因子的因素，计算方法如下：
1)总二咖啡酸酯含量计算以3，5-O-二咖啡酰奎宁酸对照品的峰面积为对照(3，5-二咖作为所有二咖的标准，以此来标定相对保留时间和校正因子，也就是只用这一个标准品可以测定所有二咖的含量之和和相对保留时间)，分别按下表相对应的校正因子计算3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯的含量，计算上述五个物质的总量，用待测成分色谱峰与3，5-O-二咖啡酰奎宁酸色谱峰的相对保留时间确定3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯的峰位，其相对保留时间应在规定值的±10％范围之内，即得。
2)野黄芩苷，以野黄芩苷对照品的峰面积为对照，按外标法以峰面积计算。
所述校正因子如下：
Figure PCTCN2020110370-appb-000001
Figure PCTCN2020110370-appb-000002
本发明优选方法为：
(1)混合对照品的制备：分别取3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯以及野黄芩苷对照品适量，精密称定，至50ml量瓶中，加入约40ml甲醇(约为常用技术标示，可以为40ml±5ml)，振摇超声30分钟，放冷，再用甲醇定容至刻度，制成与供试品溶液浓度相当的混合对照品溶液，例如每1ml溶液中分别含上述3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯以及野黄芩苷对照品50μg、30μg±3、50μg±5、60μg±6、100μg±10、100μg±10的溶液，摇匀，过0.45μl滤头，取续滤液，即得。测定图谱见附图1。
(2)供试品溶液的制备：精密量取灯盏细辛注射液2ml，置10ml量瓶中，加0.7mmol/L乙二胺四醋酸二钠溶液稀释至刻度，摇匀，滤过，取续滤液，即得。
(3)色谱条件与系统适用性试验以十八烷基硅烷键合硅胶为填充剂；以[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)](16～20：84～80)为流动相；总二咖啡酸酯检测波长为324～330nm、野黄芩苷检测波长为333～340nm。理论板数按3，5-O-二咖啡酰奎宁酸峰计算应不低于5000。
(4)测定法：分别精密吸取对照品溶液与供试品溶液各5-20μl，注入液相色谱仪，测定。
然后根据测定方法计算二咖啡酸酯和野黄芩苷含量。
本发明的主要创新点
1.供试品溶液的制备，若取灯盏细辛注射液用常规的水或者醇稀释，由于其化学成分为多酚类物质，在空气中易被氧化，且溶液暴露在空气中，由于有二氧化碳，容易导致溶液pH下降，使得溶液不能48小时内保持含量上的稳定，无法通过方法学验证。加入乙二胺四醋酸二钠溶液后，溶液可在48小时内保持含量稳定，经过试验，发现加入0.7mmol/L乙二胺四醋酸二钠溶液为最佳，能顺利通过方法学验证。
2.流动相的选择在本发明里面是关键点。由于测定的二咖啡酸酯类成分中3，4-O-二咖啡酰奎宁酸，3，5-O-二咖啡酰奎宁酸，4，5-O-二咖啡酰奎宁酸为同分异构体，而飞 蓬酯乙和灯盏细辛酯也是同分异构体，且结构十分相似，要达到较好的分离度就成了难点。经过数百次实验，数百种溶液的配比。选取以[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)](16～20：84～80)为流动相，优选以[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)](17～29：83～81)为流动相；最优选以[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)](18：82)为流动相。这样等度的流动相，在不同的仪器和色谱柱上容易有相似的表现。
附图说明
图1混合对照品图谱(327nm)；
图2灯盏细辛注射液典型色谱图(327nm)。
具体实施方式
以下实施例对本发明做进一步的描述，但该实施例非用于限制本发明的保护范围。本发明优选的实验方法、色谱条件和溶剂提取方法，是经过筛选得到的，筛选过程如下：
1、对照品
本研究共使用6种对照物质，见下表1；
表1对照物质列表
Figure PCTCN2020110370-appb-000003
含量测定用检测指标涉及6个化学成分，其结构信息见表2；
表2灯盏细辛注射液中含量检测的成分信息
Figure PCTCN2020110370-appb-000004
2、溶液的制备
2.1溶媒的选择：
2.1.1对照品溶剂的选择
根据3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯等对照品的物理性质：可溶于甲醇、乙醇、DMSO等有机溶剂，野黄芩苷的物理性质：溶于碱和冰醋酸、吡啶，微溶于一般的有机溶媒，不溶于水。故对照品选择使用甲醇作为溶剂。
2.1.2供试品溶剂的选择
在配制供试品溶液时，分析比较了甲醇，水以及0.7mmol/L乙二胺四醋酸二钠溶液稀释样品的稳定性，其中只有0.7mmol/L乙二胺四醋酸二钠溶液的稳定性能在48小时内稳定，故而选择0.7mmol/L乙二胺四醋酸二钠溶液作为溶剂。
2.2最终溶液制备条件
2.2.1混合对照品溶液的制备：分别取3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯以及野黄芩苷对照品适量，精密称定，至50ml量瓶中，加入约40ml甲醇，振摇超声30分钟，放冷，再用甲醇定容至刻度，制每1ml中分别约含50μg、30μg、50μg、60μg、100μg、100μg的溶液，摇匀，过0.45μl滤头，取续滤液，即得。所述配置浓度范围与所测定的注射液浓度相匹配。例 如每1ml溶液中分别含上述3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯以及野黄芩苷对照品50μg、30μg±3、50μg±5、60μg±6、100μg±10、100μg±10的溶液。均可。
2.2.2对照品溶液的制备：分别取3，5-O-二咖啡酰奎宁酸和野黄芩苷对照品适量，精密称定，至50ml量瓶中，加入约40ml甲醇，振摇超声30分钟，放冷，再用甲醇定容至刻度，制成每1ml中分别约含30μg、100μg的溶液，摇匀后，滤过，取续滤液，即得；
2.2.3供试品溶液的制备：精密量取本品2ml，置10ml容量瓶中，加0.7mmol/L乙二胺四醋酸二钠溶液稀释至刻度，摇匀，滤过，取续滤液，即得。
【含量测定】项方法验证
1、二咖啡酸酯检测波长确认：3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸和灯盏细辛酯等二咖啡酸酯成分均在327nm波长左右具有最大紫外吸收，故最终选用测定波长为327nm。野黄芩苷在335左右具有最大紫外吸收，故最终选用测定波长为335nm。
2、流动相确认
由于二咖啡酸酯成分结构相近，且极性和野黄芩苷相比较小，HPLC色谱分离难度较大，因此采用更小极性的乙腈作为流动相成分，分析比较了乙腈-0.1％三氟乙酸溶液(18：82)、乙腈-0.1％磷酸溶液(17：83)、[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)]18：82为流动相，发现流动相为乙腈-0.1％三氟乙酸溶液(18：82)时，保留时间在连续分析过程中出现明显漂移，不适用。流动相为乙腈-0.1％磷酸溶液(17：83)存在明显拖尾现象。[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)](18：82)为流动相在柱温40℃，流速1.0ml/min，等度洗脱条件下，野黄芩苷和5个二咖啡酸酯待测成分能得到较好分离，且在连续分析过程中出峰稳定，故最终选用[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)](18：82)为流动相。
3、最终色谱条件
以十八烷基硅烷键合硅胶为填充剂(waters Sunfire C18，5μm，4.6×250mm)[或Agilent ZORBAX SB-C 18(250mm×4.6mm，5μm)]；以[A(甲醇：乙腈30：70)-B(0.1％三氟乙酸)](18：82)为流动相为流动相；流速为1.0ml/min；二咖啡酸酯总量检测波长λ：327nm、 野黄芩苷检测波长λ：335nm；柱温为40℃。理论板数按3，5-O-二咖啡酰奎宁酸峰计算应不低于5000。
为达到多指标含量控制的目的，同时节省对照品的使用，降低检测成本，本研究采用一测多评的方法，实现仅使用2个对照品(即野黄芩苷和3，5-O-二咖啡酰奎宁酸)，测定多个有效成分。
对灯盏细辛注射液中5种二咖啡酸酯成分含量分析测定结果进行了分析，发现3，5-O-二咖啡酰奎宁酸在对照品溶液中稳定，且目前已有国家标准物质供应，故选择3，5-O-二咖啡酰奎宁酸为本研究的化学单体对照物质。经过耐用性考察，在最后确定的色谱系统条件下，各色谱峰相对于3，5-O-二咖啡酰奎宁酸的相对保留时间稳定，因此采用相对保留时间定位各色谱峰。灯盏细辛注射液典型色谱图附图2。
4、相对校正因子的测定
采用校准曲线相对斜率法及浓度法，以3，5-O-二咖啡酰奎宁酸峰为对照，测定其它4种二咖啡酸酯指标成分相对于3，5-O-二咖啡酰奎宁酸的校正因子。
斜率法相对校正因子的计算过程
Figure PCTCN2020110370-appb-000005
f sx为参照物与待测成分x的相对校正因子
k sx为参照物s与待测成分x的相对斜率比值
k x为某待测成分x的标准曲线斜率
k s为参照物s的标准曲线斜率
浓度法相对校正因子的计算过程
Figure PCTCN2020110370-appb-000006
f sx为参照物与待测成分x的相对校正因子；
A s为参照物峰面积；
W s为参照物浓度；
A x为某待测成分x的峰面积；
W x为某待测成分x的浓度。
相对保留时间的计算过程
相对保留时间＝待测物峰保留时间/参照峰保留时间
校准曲线及斜率法测定校正因子(三份)
测定方法：分别取3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯以及野黄芩苷对照品适量，精密称定，至50ml量瓶中，加入约40ml甲醇，振摇超声30分钟，放冷，再用甲醇定容至刻度，制成每1ml中分别约含50μg、30μg、50μg、60μg、100μg、100μg的溶液，摇匀，过0.45μm滤头，取续滤液，分别精密吸取混合对照品溶液2μl、4μl、6μl、8μl、10μl、15μl、20μl，注入液相色谱仪中，测定，每个进样体积连续进样两次，计算各成分对照品溶液峰面积平均值。
在327nm的图谱中，绘制3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯等标准曲线。
在335nm的图谱中，绘制野黄芩苷标准曲线。
以进样量(μg)为横坐标，峰面积为纵坐标，绘制标准曲线(过原点)，计算回归方程、相关系数r(≥0.995)、线性范围。
确定各成分相对于3，5-O-二咖啡酰奎宁酸的校正因子，见表3。
表3.相对校正因子的确定
Figure PCTCN2020110370-appb-000007
5、保留时间
采用标准曲线法确定保留时间，见表4-5。
表4.各物质色谱峰保留时间(三条标准曲线)
Figure PCTCN2020110370-appb-000008
表5.各物质色谱峰相对保留时间
Figure PCTCN2020110370-appb-000009
6、含量测定
分别采用对照品外标法和校正因子法测定同一批次灯盏细辛注射液(20191040)样品，结果各成分相对偏差均在可接受范围内。见表6。
(如果采用药典方法，只测总咖含量，2-3mg/ml。没有确切的二咖，紫外法测定的包括单咖和其他杂质。)
表6.外标法与校正因子法结果比较   单位：μg/ml
Figure PCTCN2020110370-appb-000010
Figure PCTCN2020110370-appb-000011
7、稳定性试验
取混合对照品溶液STD191228、对照品溶液STD191228和供试品溶液(灯盏细辛注射液20191040)，从配制完毕开始，在稳定色谱条件下，每隔一定时间进样，比较各成分峰面积的变化，结果5个二咖啡酸酯成分和野黄芩苷在48小时内稳定。
8、回收率试验
混合对照品储备溶液的制备：分别取3，4-O-二咖啡酰奎宁酸、3，5-O-二咖啡酰奎宁酸、飞蓬酯乙、4，5-O-二咖啡酰奎宁酸、灯盏细辛酯以及野黄芩苷对照品适量，精密称定，至25ml量瓶中，加入约适量甲醇，振摇超声30分钟，放冷，再用甲醇定容至刻度，制每1ml中分别约含110μg、60μg、90μg、130μg、180μg、270μg的溶液，摇匀，过0.45μm滤头，取续滤液，即得。
精密移取已进行重复性试验测定结果批的灯盏细辛注射液(批号：20191040)9份约1ml，置10ml容量瓶中，再分别精密加入上述“混合对照品储备溶液”1ml、2ml、3ml(相当于1ml供试品溶液的50％、100％、150％)，各梯度各3份；再加0.7m mol/L EDTA溶液稀释至刻度，摇匀后，用0.45μm的微孔滤膜滤过，取续滤液，进样分析，记录色谱图。计算各成分的回收率。
回收率符合要求方法论要求。
9、耐用性试验(结论小节)
校正因子耐用性试验
检测波长(327nm、±1nm、±2nm)
结果表明检测波长耐用性符合要求(RSD≤3.0％)
溶液稳定性：校正因子RSD％应≤3.0％。校正因子RSD％应≤3.0％。
不同色谱柱对供试品检测结果(灯盏细辛注射液20191040批)的RSD％符合要求，RSD％应≤8％。
不同水平的(35-45℃)柱温的校正因子RSD％符合要求校正因子RSD％应≤3.0％。
不同柱温对供试品检测结果(35-45℃)(灯盏细辛注射液20191040批)的RSD％符合要求，RSD％应≤8％。
三个不同水平的流速(0.8，1.0，1.2ml/min)的校正因子RSD％符合要求，校正因子RSD％应≤3.0％。
不同流速(0.8，1.0，1.2ml/min)对供试品检测结果(灯盏细辛注射液20191040批)的RSD％符合要求，RSD％应≤8％。
三个不同水平的流动相比例(18：82、16：84、20：80)的校正因子RSD％符合要求，校正因子RSD％应≤3.0％。
各不同流动相比例(18：82、16：84、20：80)对供试品检测结果(灯盏细辛注射液20191040批)的RSD％应≤8％。
分别采用混合对照品外标法和单标校正因子法测定30批次灯盏细辛注射液样品，结果各成分相对偏差均在可接受范围内。RSD％(≤5％)
本发明通过各方面的方法学验证，测定结果稳定，相对峰位置稳定，双波长测定，保证每次结果稳定且适应度高，引入校正因子，一次测定，准确测定野黄芩苷及总咖啡酸酯各成分的含量，更好反应批次间的稳定性，对于药品的质量控制具有重要意义。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制，应当指出的是，对于本领域的技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改造，这些都属于本发明的保护范围，因此，本发明专利的保护范围以所附权利要求为准。

Claims (10)

  1. 一种灯盏细辛注射液的一测多评定量检测方法，其特征在于，所述检测方法同时测定总二咖啡酸酯和野黄芩苷的含量，所述方法包括以下步骤：
    1)混合对照品的制备，将所检测物质的对照品按照灯盏细辛注射液中各成分的浓度进行相应浓度配置；
    2)供试品溶液配置，所述灯盏细辛注射液使用乙二胺四乙酸二钠溶液进行稀释配置；
    3)色谱条件与系统适用性实验，以十八烷基硅烷键合硅胶为填充剂的色谱柱；流速为0.8-1.2ml/min，流动相A为甲醇：乙腈25-35：65-75，流动相B为0.05-0.4％的三氟乙酸，流动相A与流动相B的比例为16～20：84～80；总二咖啡酸酯的检测波长为324～330nm、野黄芩苷的检测波长为333～340nm，理论板数按3,5-O-二咖啡酰奎宁酸峰计算应不低于5000，柱温35～45℃。
    4)分别精密吸取对照品溶液与供试品溶液，注入液相色谱仪，测定相关含量。
  2. 根据权利要求1所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，所述混合对照品为野黄芩苷、3,5-O-二咖啡酰奎宁酸、3,4-O-二咖啡酰奎宁酸、4,5-O-二咖啡酰奎宁酸、飞蓬酯乙和灯盏细辛酯的混合物。
  3. 根据权利要求1所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，混合对照品的制备：分别取3,4-O-二咖啡酰奎宁酸、3,5-O-二咖啡酰奎宁酸、飞蓬酯乙、4,5-O-二咖啡酰奎宁酸、灯盏细辛酯以及野黄芩苷对照品适量，精密称定，至50ml量瓶中，加入约40ml甲醇，振摇超声，放冷，再用甲醇定容至刻度，制成与供试品溶液浓度相匹配的浓度范围的混合对照品溶液，摇匀，过0.45μl滤头，取续滤液，即得。
  4. 根据权利要求1所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，乙二胺四乙酸二钠溶液浓度为0.5-1.0mmol/L。
  5. 根据权利要求4所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，乙二胺四乙酸二钠溶液浓度为0.7mmol/L。
  6. 根据权利要求1所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，所述十八烷基硅烷键合硅胶为填充剂的色谱柱：Waters Sunfire C18 5μm，4.6×250mm、Waters Sunfire C18 5μm，4.6×150mm、Agilent ZORBAX SB-C18 5μm，4.6×250mm、 岛津InertSustain-C18 5μm，4.6×250mm、岛津VP ODS-C18 5μm，4.6×250mm、Agilent Eclipse XDB-C18 5μm，4.6×150mm中的一种。
  7. 根据权利要求1所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，流动相A为甲醇：乙腈，其比例为30：70：流动相B为0.1％的三氟乙酸，流动相A与流动相B的比例为18：82；总二咖啡酸酯检测波长为326～328nm，野黄芩苷检测波长为334～336nm。
  8. 根据权利要求1所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，流速为1.0ml/min，总二咖啡酸酯检测波长为327nm、野黄芩苷检测波长为335nm，理论板数按3,5-O-二咖啡酰奎宁酸峰计算应不低于5000。
  9. 根据权利要求1所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，含量测定方法：
    1)总二咖啡酸酯含量计算，以3,5-O-二咖啡酰奎宁酸对照品的峰面积为对照，分别按相对校正因子计算3,4-O-二咖啡酰奎宁酸、3,5-O-二咖啡酰奎宁酸、飞蓬酯乙、4,5-O-二咖啡酰奎宁酸、灯盏细辛酯的含量，计算上述五个物质的总量，用待测样品的色谱峰与3,5-O-二咖啡酰奎宁酸色谱峰的相对保留时间确定3,4-O-二咖啡酰奎宁酸、3,5-O-二咖啡酰奎宁酸、飞蓬酯乙、4,5-O-二咖啡酰奎宁酸、灯盏细辛酯的峰位，其相对保留时间应在规定值的±10％范围之内，即得；
    2)野黄芩苷，以野黄芩苷对照品的峰面积为对照，按外标法以峰面积计算。
  10. 根据权利要求9所述灯盏细辛注射液的一测多评定量检测方法，其特征在于，相对保留时间和相对校正因子如下：
    Figure PCTCN2020110370-appb-100001
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CN115452983A (zh) * 2022-09-08 2022-12-09 成都市药品检验研究院 用于测定呋塞米片中有关物质的含量的hplc主成分自身对照法

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