CN113049714A - Method for detecting chlorogenic acid compound and application thereof - Google Patents

Method for detecting chlorogenic acid compound and application thereof Download PDF

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CN113049714A
CN113049714A CN202110318232.1A CN202110318232A CN113049714A CN 113049714 A CN113049714 A CN 113049714A CN 202110318232 A CN202110318232 A CN 202110318232A CN 113049714 A CN113049714 A CN 113049714A
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acid
lonicera
mass spectrometry
honeysuckle
chlorogenic acid
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张峰
冯峰
汪恩婷
仲维科
刘芳
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Chinese Academy of Inspection and Quarantine CAIQ
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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
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • 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
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
    • 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
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • 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

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Abstract

The invention discloses a method for detecting chlorogenic acid compounds and application thereof, wherein the method for detecting the chlorogenic acid compounds comprises the following steps: extracting a sample to be detected to obtain an extracting solution; and carrying out ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry detection on the extracting solution so as to obtain a qualitative and/or quantitative detection result of the chlorogenic acid compound. The method utilizes ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry for detection, has high analysis speed and high sensitivity, and can be used for species identification and quality evaluation of lonicera confusa and lonicera japonica.

Description

Method for detecting chlorogenic acid compound and application thereof
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for detecting chlorogenic acid compounds and application thereof.
Background
The Lonicera confusa is dried bud or flower with initial blossom of Lonicera macranthoides (L.macroanthoides hand. -Mazz.) of Caprifoliaceae, Lonicera fulvescens (L.fulvolota Hsu et S.C.Cheng), Lonicera hypoglauca (L.hygoglauca Miq.), or Lonicera confusa (L.convusa DC.); honeysuckle is a dried flower bud or a flower with an initial bloom of Lonicera Japonica Thunb of Caprifoliaceae, both of which have the effects of clearing away heat and toxic materials and dispelling wind and heat [1], and is widely used in the traditional Chinese medicine preparation, beverage and food industry, health care product industry and cosmetic industry [2-4] as a large number of medicine and food homologous products. Before the edition of Chinese pharmacopoeia 2005, honeysuckle and lonicera confusa are generally called as honeysuckle, but the edition of the pharmacopoeia 2005 revises the honeysuckle, combines lonicera macranthoides, lonicera fulva, lonicera hypoglauca and lonicera huananensis into lonicera confusa categories, and uses lonicera as the only source of honeysuckle and separates from lonicera confusa. The current research shows. The honeysuckle and the lonicera confusa have certain differences in the content, efficacy and market price of active ingredients due to differences in species, production places and the like, but the lonicera confusa is often mixed with the honeysuckle for sale in the health product market and traditional Chinese medicine processing at present, and in addition, the differences of the active ingredients of the honeysuckle and the lonicera confusa are not fully known at present. The active ingredients are the basis of the drug effect of the lonicera confusa and the honeysuckle. At present, the main medicinal components in lonicera confusa and honeysuckle comprise various compounds such as chlorogenic acid, flavone, saponin thereof and the like, but a reliable simultaneous analysis method is lacked aiming at the compounds.
Thus, methods for detecting chlorogenic acids are in need of improvement.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention aims to provide a method for preparing chlorogenic acid compounds, which has high analysis speed and high sensitivity and can be used for species identification and quality evaluation of lonicera confusa and lonicera japonica.
It should be noted that the present invention is completed based on the following work of the inventors:
researches show that the main active ingredients in the lonicera confusa and the honeysuckle comprise various compounds such as chlorogenic acid, flavonoid, saponin and the like. For the detection of these compounds, in "chinese pharmacopoeia" of 2020 edition, an analysis method by liquid chromatography was used, and these 3 types of compounds were detected by using an ultraviolet detector or an evaporative light scattering detector, respectively. In addition, some analysis methods such as gas chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry and the like are used in the literature, but most of the methods are also used for analyzing chlorogenic acids, flavonoids or saponin compounds in a single category, and the methods for simultaneously analyzing various compounds such as the chlorogenic acids, the flavonoids, the saponins and the like in the lonicera confusa and the lonicera japonica are not reported in the literature.
The inventor finds that the main active ingredients in the lonicera confusa and the honeysuckle can be simultaneously detected by adopting ultra-high performance liquid chromatography-tandem mass spectrometry (8 chlorogenic acid compounds (1-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid), 2 flavonoids (luteolin and luteolin), and 3 saponin compounds (dipsacoside B, lonicera grisea saponin B and swertiamarin))
Thus, according to one aspect of the invention, there is provided a method of detecting chlorogenic acids. According to an embodiment of the invention, the method comprises: extracting a sample to be detected to obtain an extracting solution; and carrying out ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry detection on the extracting solution so as to obtain a qualitative and/or quantitative detection result of the chlorogenic acid compound.
According to the method for detecting the chlorogenic acid compound, provided by the embodiment of the invention, the detection is carried out by using the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry, the analysis speed is high, the sensitivity is high, and the method can be used for species identification and quality evaluation of lonicera confusa and honeysuckle.
In addition, the method for detecting chlorogenic acid compounds according to the above embodiment of the present invention may further have the following additional technical features:
according to an embodiment of the invention, the extraction treatment is carried out with a 50% methanol solution.
According to the embodiment of the invention, in the extraction treatment, the ratio of the sample to be detected to the 50% methanol solution is 1 g: 500 mL.
According to an embodiment of the present invention, the extraction process includes:
mixing the sample to be tested with the 50% methanol solution so as to obtain a mixture;
carrying out ultrasonic treatment on the mixture to obtain an ultrasonic mixture; and
and (4) carrying out refrigerated centrifugation on the mixture after ultrasonic treatment so as to obtain the extracting solution.
According to the embodiment of the invention, the chromatographic conditions of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry are as follows: a chromatographic column: a C18 chromatography column; flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction volume: 5 μ L.
According to the embodiment of the invention, the C18 chromatographic column is an active ZORBAX extended C18 chromatographic column with the specification of 2.1X 100mm and 3.5 μm.
According to the embodiment of the invention, the chromatographic flow term of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry is A: acetonitrile, B: 0.1% aqueous formic acid.
According to the embodiment of the invention, the chromatography of the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry adopts gradient elution.
According to an embodiment of the invention, the conditions of the gradient elution are: 0-1.0 min, 8% A; 1.0-7.0 min, 8% -20% A; 7.0-10.5 min, 20-40% A; 10.5-10.6 min, 40% -95% A; 10.6-12.0 min, 95% A; 12.0-12.1 min, 95% -8% A; 12.1-14.0 min, 8% A.
According to the embodiment of the invention, the mass spectrum conditions of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrum are as follows: electrospray ionization source (ESI), negative ion mode, Multiple Reaction Monitoring (MRM); gas: nitrogen gas; collision gas (CAD) pressure: 5 kPa; gas (CUR) pressure: 30 kPa;
atomizing gas (GS1) pressure was 55 kPa; assist gas (GS2) pressure: 50 kPa; electrospray voltage (IS): -4.5 kV: desolventizing Temperature (TEM): 550 ℃; inlet voltage (EP): -10V; collision cell exit voltage (CXP): -15V.
According to an embodiment of the present invention, the chlorogenic acid compound is at least one selected from the group consisting of 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, luteolin, dipsacus asperoides B, lonicera macranthoides B and swertiamarin.
Further, according to another aspect of the present invention, the present invention provides a method for detecting lonicera confusa or lonicera japonica. According to an embodiment of the present invention, the method is performed using the aforementioned method for detecting chlorogenic acids.
The method for detecting the lonicera confusa or the honeysuckle in the embodiment of the invention can be used for species identification and quality evaluation of the lonicera confusa and the honeysuckle, can be used for accurately measuring active ingredients in the honeysuckle and the lonicera confusa and quickly identifying the lonicera confusa doped in the honeysuckle in some embodiments, and has the advantages of rapidness, accuracy and high sensitivity.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic diagram comparing the separation of 8 chlorogenic acids under 3 chromatographic columns according to one embodiment of the present invention, wherein 1 is 1-O-caffeoylquinic acid; 2 is 5-O-caffeoylquinic acid; 3 is 3-O-caffeoylquinic acid; 4 is 4-O-caffeoylquinic acid; 5 is 1,5-dicaffeoylquinic acid; 6 is 3,4-dicaffeoylquinic acid; 7 is 3,5-dicaffeoylquinic acid; 8 is 4,5-dicaffeoylquinic acid;
FIG. 2 shows a schematic of 13 compound extraction chromatography according to one embodiment of the present invention, wherein 1 is 1-O-caffeoylquinic acid; 2 is 5-O-caffeoylquinic acid; 3 is 3-O-caffeoylquinic acid; 4 is swertiamarin; 5 is 4-O-caffeoylquinic acid; 6 is 1,5-dicaffeoylquinic acid; 7 is luteolin; 8 is 3,4-dicaffeoylquinic acid; 9 is 3,5-dicaffeoylquinic acid; 10 is 4,5-dicaffeoylquinic acid; 11 is macranthoside B; 12 is luteolin; 13 is asperosaponin B;
fig. 3 is a schematic diagram showing the comparison of the contents of the main active ingredients in the honeysuckle and the lonicera confusa according to one embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
According to one aspect of the invention, there is provided a method of detecting chlorogenic acids. According to an embodiment of the invention, the method comprises: extracting a sample to be detected to obtain an extracting solution; and carrying out ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry detection on the extracting solution so as to obtain a qualitative and/or quantitative detection result of the chlorogenic acid compound.
According to the method for detecting the chlorogenic acid compound, provided by the embodiment of the invention, the detection is carried out by using the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry, the analysis speed is high, the sensitivity is high, and the method can be used for species identification and quality evaluation of lonicera confusa and honeysuckle.
According to the method for detecting chlorogenic acid compounds, 13 active ingredients in lonicera confusa and honeysuckle can be effectively separated within 14min, the respective linear relation is good in a linear range, the correlation coefficient is larger than 0.998, and the detection Limit (LOD) is as follows: 0.5-19.5 μ g/L, and a limit of quantitation (LOQ) of 1.4-65.1 μ g/L. The method has the advantages of high analysis speed and high sensitivity, and can be used for accurately determining the active ingredients in the honeysuckle and the lonicera confusa and quickly identifying the lonicera confusa doped in the honeysuckle.
According to the method for detecting chlorogenic acid compounds, disclosed by the invention, effective separation and accurate quantification of 13 active ingredient substances including 8 chlorogenic acid compounds in honeysuckle and lonicera confusa are realized. The method of the embodiment can be applied to the identification of the lonicera confusa and the honeysuckle and provides a means for the quality evaluation and the understanding of the structure-activity relationship of the lonicera confusa and the honeysuckle. The method of the embodiment is applied to the analysis of actual samples, and the result shows that the content of active ingredients in the honeysuckle and the lonicera confusa is obviously different, so that the method can be applied to the identification of the lonicera confusa and the honeysuckle and also provides a means for the quality evaluation and the understanding of the structure-activity relationship of the lonicera confusa and the honeysuckle.
The term "active ingredient" as used herein refers to a substance or mixture of substances that have been identified in lonicera confusa or lonicera japonica and that have pharmacological activity or other direct effects in the diagnosis, treatment, symptom relief, treatment or prevention of a disease or that can affect the body.
The term "Lonicera confusa" (Lonicera flors) as used herein refers to dried buds or blossomed flowers of Lonicera macranthoides hand-d. -mazz, Lonicera hypoglauca miq, Lonicera confusa dc, or Lonicera fulvotomosa Hsu et s.c. Flos Lonicerae is a large amount of medicinal and edible product, and its main active ingredients include chlorogenic acid, lonicera macranthoides saponin B, radix Dipsaci saponin B, etc.
The term "honeysuckle flower" (Lonicera japonica flors) as used herein refers to the dried bud or blossoming flower of Lonica japonica Thunb, a Lonica japonica, Caprifoliaceae. The honeysuckle is a bulk medicinal and edible product, and the main active ingredients comprise chlorogenic acid, galuteolin and the like. In the early Chinese pharmacopoeia, lonicera confusa is classified as the name of honeysuckle, and the lonicera confusa is not distinguished from the honeysuckle until the Chinese pharmacopoeia 2005, which also causes the phenomenon that the commercially available lonicera confusa and honeysuckle products are often mixed.
The term "chlorogenic acid compound" as used herein refers to a class of natural phenolic compounds of depside acids generated from Caffeic acid (Caffeic acid) and Quinic acid (Quinic acid, 1-hydroxyhexahydrogallic acid).
According to the method for detecting chlorogenic acid compounds provided by the embodiments of the invention, technical support can be provided for the research on the in vivo metabolism rule of the lonicera confusa or honeysuckle containing active ingredients taken by a patient.
According to an embodiment of the present invention, the extraction process is performed using a 50% methanol solution. Therefore, the method is favorable for fully extracting the active ingredients of the lonicera confusa and the honeysuckle and has high detection accuracy.
According to the embodiment of the invention, in the extraction treatment, the ratio of the sample to be detected to the 50% methanol solution is 1 g: 500 mL. Therefore, the proportion of the extraction solvent is appropriate, the active ingredients of the lonicera confusa and the honeysuckle can be conveniently and fully extracted, and the detection accuracy is high.
According to an embodiment of the present invention, the extraction process includes: mixing a sample to be detected with a 50% methanol solution to obtain a mixture; carrying out ultrasonic treatment on the mixture to obtain an ultrasonic mixture; and (4) freezing and centrifuging the mixture after ultrasonic treatment to obtain the extracting solution. Therefore, the method is favorable for fully extracting the active ingredients of the lonicera confusa and the honeysuckle and has high detection accuracy.
According to the embodiment of the invention, the chromatographic conditions of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry are as follows: a chromatographic column: a C18 chromatography column; flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction volume: 5 μ L. Therefore, the method is beneficial to more fully separating various active ingredients of the lonicera confusa and the honeysuckle and has good chromatographic peak shape.
The inventor researches and discovers that chlorogenic acid compounds coexist in various isomer forms, have the same primary characteristic fragment ions and secondary characteristic fragment ions, and cannot be characterized by the fragment ion pairs cracked only by mass spectrometry, and according to the embodiment of the invention, the inventor selects a C18 chromatographic column as an active ZORBAX extended C18 chromatographic column with the specification of 2.1X 100mm and 3.5 mu m. Experiments show that not only 8 chlorogenic acid compounds are well separated, but also 13 compounds are effectively separated within 14min, and the peak shapes are symmetrical and thin.
According to the embodiment of the invention, the chromatographic flow term of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry is A: acetonitrile, B: 0.1% aqueous formic acid. This flow therefore has a high degree of separation from the chlorogenic acid compound, and contributes to sufficient separation of the compound.
According to the embodiment of the invention, the chromatography of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry adopts gradient elution. Further, according to an embodiment of the present invention, the conditions of the gradient elution are: 0-1.0 min, 8% A; 1.0-7.0 min, 8% -20% A; 7.0-10.5 min, 20-40% A; 10.5-10.6 min, 40% -95% A; 10.6-12.0 min, 95% A; 12.0-12.1 min, 95% -8% A; 12.1-14.0 min, 8% A. Therefore, the chlorogenic acid compound has symmetrical peak shape without tailing and good separation degree.
The inventor finds that the response value of each compound in a negative ion mode is obviously superior to that in a positive ion mode, and selects an ESI-ionization mode. Aiming at the mass spectrum peak characteristics of chlorogenic acid compounds, taking two molecular ion peaks of [ M-H ] -and [ M + HCOO ] -as parent ions, and adjusting other parameters aiming at the parent ions and the specificity of compounds to be detected, according to the embodiment of the invention, the mass spectrum conditions of the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrum are as follows: electrospray ionization source (ESI), negative ion mode, Multiple Reaction Monitoring (MRM); gas: nitrogen gas; collision gas (CAD) pressure: 5 kPa; gas (CUR) pressure: 30 kPa; atomizing gas (GS1) pressure was 55 kPa; assist gas (GS2) pressure: 50 kPa; electrospray voltage (IS): -4.5 kV: desolventizing Temperature (TEM): 550 ℃; inlet voltage (EP): -10V; collision cell exit voltage (CXP): -15V. Thus, the sensitivity and accuracy of detection are high.
According to an embodiment of the present invention, the chlorogenic acid compound is at least one selected from the group consisting of 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, luteolin, dipsacus asperoides B, lonicera macranthoides B and swertiamarin.
The monitored ion pairs, Collision Energies (CE) and declustering voltages (DP) for the 13 active components according to the example of the present invention are shown in table 1.
TABLE 113 optimum Mass Spectrometry parameters for active ingredient Compounds
Figure BDA0002991676180000061
Figure BDA0002991676180000071
Is a quantitative ion pair
Further, according to another aspect of the present invention, the present invention provides a method for detecting lonicera confusa or lonicera japonica. According to an embodiment of the present invention, the method is performed using the aforementioned method for detecting chlorogenic acids.
The method for detecting the lonicera confusa or the honeysuckle in the embodiment of the invention can be used for species identification and quality evaluation of the lonicera confusa and the honeysuckle, can be used for accurately measuring active ingredients in the honeysuckle and the lonicera confusa and quickly identifying the lonicera confusa doped in the honeysuckle in some embodiments, and has the advantages of rapidness, accuracy and high sensitivity.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.
Example 1
In this embodiment, the method for detecting chlorogenic acid compounds according to the embodiment of the present invention is used to simultaneously analyze active ingredients in lonicera confusa and lonicera japonica, and specifically includes the following steps:
1. experimental part
1.1 instruments, reagents and materials
1.1.1 instruments
ExionLC AD 6500 ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometer (AB SCIEX, USA); analyst workstation (AB SCIEX, usa); analytical balance XP 105 (METTLER TOLEDO, Switzerland); Milli-Q Advantage A10 ultra pure water machine (Millipore, USA); a numerical control ultrasonic cleaner KQ-500DE (ultrasonic instruments Co., Ltd., Kunshan); high speed refrigerated centrifuge BECKMAN Allegra X-22R (BECKMAN COULTER, USA)
1.1.2 reagents
Methanol, acetonitrile, ethanol (chromatographically pure, Fisher, usa); formic acid, acetic acid (chromatographically pure, CNW technologies, germany); ammonium formate, ammonium acetate (LC/MS grade, Fisher company, USA).
1.1.3 materials
1-O-Caffeoylquinic acid (1-O-Caffeoylquinic acid) (CAS: 1241-87-8, purity ≥ 98%), available from Woji Biotechnology GmbH; 3-O-Caffeoylquinic acid (3-O-Caffeoylquinic acid) (CAS: 327-97-9, purity ≥ 96.6%) is available from Dr.Ehrenstontorfer GmbH, Germany; 3,5-Dicaffeoylquinic acid (3,5-Dicaffeoylquinic acid) (CAS: 2450-53-5), 3,4-Dicaffeoylquinic acid (3,4-Dicaffeoylquinic acid) (CAS: 14534-61-3), 4,5-Dicaffeoylquinic acid (4,5-Dicaffeoylquinic acid) (CAS: 32451-88-0), all from Beijing Bailingwei science and technology Limited, the purity is more than or equal to 98%; 5-O-Caffeoylquinic acid (5-O-Caffeoylquinic acid) (CAS: 906-33-2), 4-O-Caffeoylquinic acid (4-O-Caffeoylquinic acid) (905-99-7), 1,5-Dicaffeoylquinic acid (1,5-Dicaffeoylquinic acid) (CAS: 30964-13-7), all from SIGMA corporation of America, with purity of 98% or more; luteolin (Luteolin) (CAS: 491-70-3, purity is more than or equal to 94.4%), Luteolin (Luteolin-7-O-beta-D-glucoside) (CAS: 5373-11-5, purity is more than or equal to 93.5%), dipsacus asperoides saponin B (CAS: 33289-85-9, purity is more than or equal to 96.8%), lonicera macranthoides saponin B (CAS: 136849-88-2, purity is more than or equal to 97.2%), and swertiamarin (Swiamarine) (CAS: 17388-39-5, purity is more than or equal to 98.3%) were purchased from Chinese food and drug assay research institutes. 10 batches of lonicera confusa and honeysuckle samples are purchased from the market and subjected to species identification by the Linlingin researcher of medicinal plant institute of Chinese academy of medical sciences.
1.2 Experimental conditions
1.2.1 preparation of the solution
Standard stock solutions: accurately weighing 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, luteolin, radix Dipsaci saponin B, lonicera macranthoides saponin B and swertiamarin standard substances, respectively dissolving with methanol to obtain single standard stock solution with concentration of 1mg/mL, and storing at-20 deg.C.
Mixing standard solutions: taking the standard stock solutions of 13 active ingredient substances respectively, diluting with methanol to prepare a mixed standard solution intermediate solution of 10 mug/mL, and storing at 4 ℃. When used, the mixture is diluted to a required concentration by methanol-water (50:50, v/v) according to requirements.
1.2.2 sample preparation
Taking a proper amount of samples, crushing, sieving by a 40-mesh sieve, and uniformly mixing to prepare uniform samples for later use. According to 'Chinese pharmacopoeia' 2020 edition, 0.1g (accurate to 1mg) of a sample is precisely weighed into a 50mL centrifuge tube with scales, 50mL methanol-water (50:50, v/v) is added into the centrifuge tube, the centrifuge tube is sealed, the mixture is shaken up, and ultrasonic treatment (power 300W, frequency 40kHz) is carried out for 45 minutes. Cooling, supplementing to 50mL with methanol-water (50:50, v/v), shaking, and centrifuging at 8000r/min for 5 min; accurately sucking 1mL of supernatant, diluting with methanol-water (50:50, v/v) to 100mL, filtering with 0.22 μm filter membrane, and measuring.
1.2.3 chromatographic conditions
A chromatographic column: an agent ZORBAX extended C18 column (2.1X 100mm,3.5 μm); acetonitrile is used as a mobile phase (A), and 0.1% formic acid aqueous solution is used as a mobile phase (B); flow rate: 0.3 mL/min; column temperature: 30 ℃; sample introduction volume: 2 μ L. The gradient elution procedure was: 0-1.0 min, 8% A; 1.0-7.0 min, 8% -20% A; 7.0-10.5 min, 20-40% A; 10.5-10.6 min, 40% -95% A; 10.6-12.0 min, 95% A; 12.0-12.1 min, 95% -8% A; 12.1-14.0 min, 8% A.
1.2.4 Mass Spectrometry conditions
Electrospray ionization source (ESI), negative ion mode, Multiple Reaction Monitoring (MRM); the gases used in the experiment were all high purity nitrogen, the collision gas (CAD) pressure was 5kPa, the curtain gas (CUR) pressure/force was 30kPa, the atomizing gas (GS1) pressure was 55kPa, the auxiliary gas (GS2) pressure was 50kPa, the electrospray voltage (IS) -4.5kV, the desolventizing Temperature (TEM) was 550 ℃, the inlet voltage (EP) was-10V, and the collision cell outlet voltage (CXP) was-15V.
2 results and discussion
2.1 examination of the conditions of the experiment
2.1.1 Mass Spectrometry Condition inspection
This example examines the mass spectral response of each compound in different ionization modes for positive and negative ions. The result shows that the response value of each compound in the negative ion mode is obviously superior to that in the positive ion mode, and the ESI is selected-Ionization mode. When the total sweeping is carried out in the negative ion mode, except for lonicera macranthoides saponin B and swertiamarin, the quasi-molecular ion peak of 11 active ingredient compounds is mainly [ M-H ]]-Exists in the form of the extract, and the Lonicera macranthoides saponin B and the swertiamarin exist simultaneously [ M-H ]]–And [ M + HCOO]–Two molecular ion peaks. Investigating first-order fragment ionsIn response to sensitivity and stability, both substances were found to be [ M + HCOO ]]–Higher response and better stability, and respectively selecting [ M + HCOO]–m/z 1443.5、[M+HCOO]–m/z 419.1 is used as parent ion for collision induced dissociation of lonicera macranthoides saponin B and swertiamarin. And further performing secondary mass spectrum cracking on the primary mass spectrum structure of each compound, selecting two characteristic fragment ions with the highest abundance as qualitative ions and quantitative ions, and optimizing Collision Energy (CE) and declustering voltage (DP). The results show that 11 compounds can select two daughter ions to form two monitoring ion pairs, and chlorogenic acid and lonicera macranthoides saponin B2 compounds only have one monitoring ion pair due to the influence of a structure fracture mode in a negative ion mode, and the selected MRM mass spectrum parameters are shown in Table 1.
2.1.2 inspection of chromatographic conditions
Selection of chromatographic column: the 13 active ingredient substances selected in this example comprise 8 chlorogenic acid compounds, 2 saponins, 2 flavonoids, and 1 iridoid. Based on the advantage of accurate qualitative determination of the LC-MS technology, saponins, flavonoids and iridoids in the selected target can be accurately determined qualitatively and quantitatively by different molecular weights. However, chlorogenic acid compounds coexist in various isomer forms, have the same primary characteristic fragment and secondary characteristic fragment ions, and cannot be characterized only by fragment ion pairs cracked by mass spectrometry, so that the rapid and effective separation of 8 chlorogenic acid compounds is the key point of chromatographic condition optimization. This example compares Therom AcclaimTMPhenyl-1(2.1 × 150mm,3um), Thermo Hypersil GOLD aQ (2.1 × 100mm,1.9um), and active ZORBAX extended C18(2.1 × 100mm,3.5um)3 kinds of chromatographic columns have the separation effect on 8 kinds of chlorogenic acid compounds, and the separation effect of 8 kinds of chlorogenic acid is shown in figure 1. As a result, it was found that AcclaimTMPhenyl-1 Phenyl column (part a in fig. 1) has the ability to form p-p conjugate interaction, but 3-O-caffeoylquinic acid and 4-O-caffeoylquinic acid, and 3,4-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid can not be separated effectively under various mobile phase gradients. 3-O-caffeoyl when separated using Hypersil GOLD aQ chromatographic column (part b in FIG. 1)The quinic acid and the 4-O-caffeoyl quinic acid still cannot be separated, and the whole peak tailing is serious, so that the accuracy of the analysis result is influenced. Good peak shapes can be obtained through a ZORBAX extended C18 chromatographic column (part C in figure 1), wherein the separation degree R of 3-O-caffeoylquinic acid and 4-O-caffeoylquinic acid is 1.64, and the separation degree R of 3,4-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid is 1.21, so that not only 8 chlorogenic acid compounds are well separated, but also 13 compounds are effectively separated within 14min, the peak shapes are symmetrical and lean (shown in figure 2), and therefore, the Aglient ZORBAX extended C18 chromatographic column is selected.
Selection of mobile phase: in the embodiment, methanol-water and acetonitrile-water systems are respectively selected to examine the organic phase, and the result shows that when acetonitrile is used as the organic phase, the elution capacity is superior to that of methanol, the peak shape symmetry of each target object is better, and the separation degree of chlorogenic acid compounds is higher. Different electrolytes (0.1% formic acid, 0.1% acetic acid, 2.5mmol/L ammonium acetate, 2.5mmol/L ammonium formate) were added to the water, respectively, and the inorganic phase was investigated by the sensitivity and the degree of separation of the target using the same gradient. The results showed that when 2.5mmol/L ammonium acetate aqueous solution and 2.5mmol/L ammonium formate aqueous solution were used as inorganic phases, each peak had a distinct tail; and the target substance has symmetrical peak shape without tailing in 0.1% formic acid water and 0.1% acetic acid water solution, and has better separation degree. Because the macranthoides saponin B and swertiamarin are both in the form of [ M + HCOO]–As the parent ion for the collision induced dissociation, the two targets are found to have higher response value and stability than 0.1% acetic acid under the condition of 0.1% formic acid aqueous solution. Therefore, in this example, acetonitrile (a) -0.1% aqueous formic acid solution (B) was selected as a mobile phase system for separation.
2.2 methodological investigation
2.2.1 Linear relationship, detection Limit and quantification Limit
Taking series of mixed standard solutions of 13 active ingredient compounds, drawing a standard curve by taking a peak area (Y) as a vertical coordinate and a mass concentration (X, ng/mL) as a horizontal coordinate, and obtaining a linear regression equation/linear range and a correlation coefficient (r)2). According to 3 times signal-to-noise ratio (S/N) Determining a limit of detection (LOD) of the method,the limit of quantitation (LOQ) of the method was determined according to 10 times S/N and the results are shown in Table 2. As a result, the linear relationship between the concentrations of the target compounds in the respective concentration ranges of the curves was good (r)2Not less than 0.998), and the detection Limit (LOD) is as follows: 0.3-5.2 ng/mL, and the limit of quantitation (LOQ) is 1.2-16.7 ng/mL.
TABLE 213 Linear Range, Linear equation, correlation coefficient, detection limits and quantitation limits for active ingredient compounds
Figure BDA0002991676180000101
Figure BDA0002991676180000111
Y is peak area; x is mass concentration, ng/mL.
2.2.2 examination of repeatability, precision, recovery from spiking
Respectively weighing 18 parts of representative matrix samples of lonicera confusa and lonicera japonica, wherein each part is about 0.1g, respectively adding mixed standard solutions with concentration levels of 2.5, 5.0 and 10.0mg/g 3 according to the content level of each compound in an original sample, measuring 6 parts of each level by HPLC-MS/MS, recording peak area, and inspecting the standard adding recovery rate, precision and repeatability of each compound at 3 levels, wherein the results are shown in Table 3. The average recovery rate of the low-medium-high 3 addition levels in the lonicera confusa ranges from 76.0 to 108.2 percent, the relative standard deviation ranges from 0.5 to 6.1 percent (n is 6), the average recovery rate in the lonicera confusa ranges from 75.7 to 106.3 percent, and the relative standard deviation ranges from 0.3 to 8.2 percent (n is 6), and all the requirements of laboratory method control are met. The result proves that the method for detecting the chlorogenic acid compound is stable and reliable, and can be used for measuring 13 active ingredient substances in honeysuckle and lonicera confusa.
TABLE 313 recovery and precision of active ingredient spiked at 3 levels: (n＝6)
Figure BDA0002991676180000112
Figure BDA0002991676180000121
2.2.3 stability Studies
Adding 5.0mg/g mixed standard solution into a lonicera confusa sample, preparing a test solution, injecting samples for 0, 4, 8, 12, 24 and 48 hours respectively, and measuring the RSD of the content of 1-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, swertiamarin, 4-O-caffeoylquinic acid, 1,5-dicaffeoylquinic acid, luteolin, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, lonicera griseola saponin B, luteolin and dipsacus aspen B in the test solution is respectively as follows: 5.3%, 2.4%, 4.3%, 5.7%, 4.0%, 3.9%, 3.5%, 4.7%, 2.2%, 3.6%, 5.2%, 2.5%, 4.4%, indicating that the test solution is stable within 48 h.
2.3 sample determination
The active ingredients of 10 batches of samples, such as commercially available honeysuckle (nos. 1-4), lonicera confusa (nos. 5, 6-9), and honeysuckle products (honeysuckle granules (No. 10)), were measured, and the detailed results are shown in table 4.
TABLE 4 determination of 13 active ingredients in the samples
Figure BDA0002991676180000131
ND: not detected.
The results showed that 12 active ingredients were detected to varying degrees, except that 1-O-caffeoylquinic acid was not detected in 10 samples. The saponins (lonicera macranthoides saponin B and dipsacus asperoides saponin B) in the lonicera confusa are obviously higher than those in the honeysuckle, and the dipsacus asperoides saponin B belongs to the specific components of the lonicera confusa and can be used for the doping identification between the lonicera confusa and the honeysuckle. The content of chlorogenic acid compounds in the lonicera confusa is higher than that of honeysuckle in whole, and the chlorogenic acid compounds mainly exist in the forms of 3-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 4,5-dicaffeoylquinic acid, 4-O-caffeoylquinic acid, 3,4-dicaffeoylquinic acid and the like from high to low. The content of luteolin, luteolin and swertiamarin in the honeysuckle flower is higher than that of lonicera confusa. The content of the main active ingredients in flos Lonicerae and flos Lonicerae is shown in figure 3.
In addition, through analysis of a determination result, the product name of the honeysuckle is marked on the sample package No. 1, but a small amount of asperosaponin B is detected, the content of lonicera macranthoides saponin B is obviously higher than the determination average value of the honeysuckle and lower than the determination average value of the lonicera confusa, and the sample is judged to be the honeysuckle mixed with part of lonicera confusa for sale; sample No. 10 is a commercially available honeysuckle granule, but both the asperosaponin B and the lonicera macranthoides saponin B are detected, which indicates that the production raw material may be lonicera confusa or a doped product of lonicera confusa and lonicera confusa; meanwhile, analysis of No. 10 samples shows that active ingredients in the honeysuckle and the lonicera confusa are influenced by the process mode in the processing process to cause loss in different degrees, so that reference is provided for commercialization of the honeysuckle and the lonicera confusa products and exploration of effective ingredients. In conclusion, the lonicera confusa and the honeysuckle have large difference in the content of the active ingredients, and the lonicera confusa and the honeysuckle can be subjected to variety identification and quality evaluation by the method for detecting the chlorogenic acid compounds simultaneously.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims (10)

1. A method for detecting a chlorogenic acid compound, comprising:
extracting a sample to be detected to obtain an extracting solution; and
and carrying out ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry on the extracting solution so as to obtain a qualitative and/or quantitative detection result of the chlorogenic acid compound.
2. The method of claim 1, wherein the extraction process is performed using a 50% methanol solution.
3. The method according to claim 2, wherein in the extraction process, the ratio of the sample to be tested to the 50% methanol solution is 1 g: 500 mL.
4. The method of claim 3, wherein the extraction process comprises:
mixing the sample to be tested with the 50% methanol solution so as to obtain a mixture;
carrying out ultrasonic treatment on the mixture to obtain an ultrasonic mixture; and
and (4) carrying out refrigerated centrifugation on the mixture after ultrasonic treatment so as to obtain the extracting solution.
5. The method of claim 1, wherein the chromatographic conditions of ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry are as follows:
a chromatographic column: a C18 chromatography column;
flow rate: 0.3 mL/min;
column temperature: 30 ℃;
sample introduction volume: 5 μ L.
6. The method of claim 5, wherein the C18 column is an active ZORBAX extended C18 column with a specification of 2.1 x 100mm,3.5 μm.
7. The method of claim 5, wherein the chromatography flow term of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry is A: acetonitrile, B: 0.1% aqueous formic acid;
optionally, the chromatography of the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry adopts gradient elution,
optionally, the conditions of the gradient elution are: 0-1.0 min, 8% A; 1.0-7.0 min, 8% -20% A; 7.0-10.5 min, 20-40% A; 10.5-10.6 min, 40% -95% A; 10.6-12.0 min, 95% A; 12.0-12.1 min, 95% -8% A; 12.1-14.0 min, 8% A.
8. The method of claim 1, wherein the mass spectrometry conditions of the ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry are as follows:
electrospray ionization source (ESI), negative ion mode, Multiple Reaction Monitoring (MRM);
gas: nitrogen gas;
collision gas (CAD) pressure: 5 kPa;
air curtain air (CUR) pressure: 30 kPa;
atomizing gas (GS1) pressure was 55 kPa;
assist gas (GS2) pressure: 50 kPa;
electrospray voltage (IS): -4.5 kV:
desolventizing Temperature (TEM): 550 ℃;
inlet voltage (EP): -10V;
collision cell exit voltage (CXP): -15V.
9. The method according to claim 1, wherein the chlorogenic acid compound is at least one selected from the group consisting of 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, luteolin, teasaponin, loniceragenin B, and swertiamarin.
10. A method for detecting lonicera confusa or lonicera japonica thunb, which is carried out by the method for detecting chlorogenic acid compounds in any one of claims 1 to 9.
CN202110318232.1A 2021-03-25 2021-03-25 Method for detecting chlorogenic acid compound and application thereof Pending CN113049714A (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
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