CN107192770B - Analytical method for identifying vitex negundo honey and syrup adulterated vitex negundo honey - Google Patents
Analytical method for identifying vitex negundo honey and syrup adulterated vitex negundo honey Download PDFInfo
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Abstract
The invention discloses an analysis method for identifying vitex negundo honey and syrup adulterated vitex negundo honey, in particular to a method combining ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry technology with metabonomics, which comprises the following steps: after the true vitex negundo honey sample and the syrup adulterated vitex negundo honey sample to be detected are respectively pretreated by adopting an organic solvent, the separation and the determination of chemical components in the pretreated sample are realized by applying an ultra-high performance liquid chromatography-quadrupole-orbital trap high-resolution mass spectrometry method, then UHPLC-MS original data of the obtained true vitex negundo honey sample and the vitex negundo honey sample to be detected are pretreated, and finally, the main component analysis is applied to distinguish the true vitex honey and the syrup adulterated vitex negundo honey. The invention comprehensively analyzes the vitex negundo honey and the syrup adulterated vitex negundo honey by combining multivariate statistical analysis after comprehensively acquiring the metabolite information of the vitex negundo honey and the syrup adulterated vitex negundo honey, and completes the detection of the syrup adulterated vitex negundo honey.
Description
Technical Field
The invention belongs to the technical field of food adulteration identification, and particularly relates to an analysis method for identifying vitex negundo honey and syrup adulterated vitex negundo honey by applying a metabolic omics technology combined by UHPLC-Q active.
Background
The honey is natural sweet substance obtained by fully brewing honey, secretion or honeydew collected by bees and self secretion, and has high nutritive value, wherein the vitex negundo is short for vitex negundo honey, also called vitex negundo honey. Is one of four famous honey; is also one of the most stable honey products per year in the bulk honey sources in China. Not only is an ideal nourishing nutritional food, but also has the effects of relieving cough, moistening lung, invigorating stomach, relaxing bowels, preventing heatstroke, clearing away heart-fire, tonifying body, benefiting qi and prolonging life.
However, because of the higher price and the lower yield of the honey, the honey becomes a main object for illegal vendor adulteration, and in order to improve the production enthusiasm of beekeepers, guarantee the benefits of consumers, support the fair competition of regular honey manufacturers, maintain the order of the honey market and promote the healthy development of the honey industry in China, the method has very important significance and value for finding out the marker metabolite of a real honey sample, and trying to establish a set of sensitive, efficient and accurate honey adulteration identification method.
High fructose corn syrup, starch syrup, rice syrup and the like are added into true honey: the method is the most common honey adulteration method at present, and because the fructose and glucose ratios of the syrups are very similar to the components in honey, all detection indexes after the syrups are mixed completely meet the national standard, great difficulty is caused to detection. The methods commonly used for identifying honey adulteration at present comprise a stable carbon isotope ratio analysis method (SCIRA), a thin-layer chromatography (TIC), a high-performance anion exchange chromatography (HPAEC-PAD) of a pulse current detector, a gas chromatography-mass spectrometry (GC-MS), a high-performance liquid chromatography (HPLC), a high-performance liquid isotope mass spectrometer (HPLC-IRMS), a Nuclear Magnetic Resonance (NMR) technology, a near infrared spectrum (NIRS) and the like. The existing method has a plurality of disadvantages for determining the adulteration of honey: the Stable Carbon Isotope Ratio Analysis (SCIRA) method is effective only for natural honey doped with C4 plant sugar, and is difficult to identify if honey is doped with saccharide components prepared from C3 plant starch such as rice, wheat, and soybean, or completely fake honey prepared from saccharide materials prepared from C3 plant starch such as rice, wheat, and soybean, and other materials. Kushnir et al determined that polysaccharides having a degree of polymerization of 12 to 19 were markers for honey-adulterated high fructose corn syrup and corn syrup using thin layer chromatography measurements of corn syrup adulterated honey. However, this method requires a complicated pretreatment process to remove glucose, fructose and a small amount of oligosaccharides from honey, resulting in a complicated operation of the detection method. The high performance anion exchange chromatography (HPAEC-PAD) method of pulsed amperometric detectors is more limited and hydrolysis of oligo-and polysaccharides in the previous stages is likely to result in false positive results because true honey also has oligosaccharides with a degree of polymerization of 3-6. High performance anion exchange chromatography (HPAEC-PAD) with pulsed current detectors requires complex pretreatment processes to remove monosaccharides and oligosaccharides, which are complex. The GC-MS method for determining the adulteration of the honey has certain defects, and a honey sample needs to be subjected to derivatization before detection. Nuclear Magnetic Resonance (NMR) techniques have low sensitivity, may be overlooked for metabolites of interest in honey, and nuclear magnetic resonance instruments are expensive and not suitable for routine monitoring. The near infrared spectrum (NIRS) also has its fatal drawbacks: 1. a large number of representative and chemically known samples are required to model. Thus, it is impractical to use near infrared for analysis of small batches of samples. 2. The model needs to be updated continuously, and the model changes due to the change of the state of the instrument or the change of the standard sample. 3. The models are not universal, and the models of all instruments are different, so that the use limitation is increased. 4. The modeling cost is high, and the test utilization degree is large.
The existing methods for identifying the quality of honey are GB14963-2011 and SN/T0852-: sensory properties to honey; the physical and chemical indexes are as follows: detecting the fructose glucose content, the sucrose content and the like; microbial indexes, pesticide residues, heavy metals, additives and the like are detected. Maltose, fructose syrup, cocoa powder, citric acid, allura red, caramel color, potassium sorbate, carrageenan, edible essence and the like in honey are measured by a plurality of enterprise standards. At present, various syrups adulterated with honey are very similar to honey in the aspects of physicochemical properties, component composition, content and flavor, so the method for measuring the quality of the honey cannot detect the honey adulterated with the syrup.
In conclusion, a rapid, effective, intuitive and obvious method for identifying the adulteration of the vitex negundo honey is absent in the prior art, and no related research for determining the marker metabolites of the vitex negundo honey by using the ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry combined metabonomics method is available.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an analysis method for identifying the vitex negundo honey and the syrup adulterated vitex negundo honey by applying the ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry technology in combination with a metabonomics method, and the analysis method can effectively identify the true vitex negundo honey and the syrup adulterated vitex negundo honey.
The technical scheme adopted by the invention is as follows:
the invention aims to provide an analysis method for distinguishing vitex negundo honey and syrup adulterated vitex negundo honey by applying an ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry technology in combination with a metabonomics method, which comprises the following steps:
after the true vitex negundo honey sample and the syrup adulterated vitex negundo honey sample to be detected are respectively pretreated by adopting an organic solvent, the separation and the determination of chemical components in the pretreated sample are realized by using an ultra-high performance liquid chromatography-quadrupole-orbital trap high-resolution mass spectrometry method, then UHPLC-MS original data of the obtained true vitex negundo honey sample and the vitex honey sample to be detected are pretreated, and finally, the true vitex negundo honey and the syrup adulterated vitex negundo honey are distinguished by using a Principal Component Analysis (PCA) model of a multivariate statistical analysis method.
The analysis method of the invention is not particularly limited to the syrup used by adulterated vitex negundo honey, and covers all kinds of C3 and C4 syrups used by adulteration at present, including: rice syrup, corn syrup, beet syrup, composite fructose-glucose syrup, special syrup for online purchased honey or other syrups.
In a preferred embodiment, the organic solvent used in the pretreatment of the sample in the present invention is a mixed solvent of methanol and water containing formic acid, wherein the volume fraction of formic acid is preferably 1%, and methanol and water are mixed in equal volumes to form the mixed solvent.
As a preferable scheme, the sample pretreatment process in the present invention includes: mixing the sample with an organic solvent until the sample is completely dissolved, performing ultrasonic treatment, centrifuging, and filtering with an organic filter membrane to obtain a sample capable of being detected on a computer. Wherein the ultrasonic time is 10-30 min, preferably 25 min; centrifugation conditions: centrifuging at 800-1200 rpm for 4-8 min, preferably at 1000rpm for 5 min; the aperture of the organic filter membrane is 0.20-0.25 μm, preferably 0.22 μm; in order to ensure that the metabolite extraction effect in the sample is better, the adding proportion of the sample and the organic solvent is 1 g: (15-25) mL.
In the whole pretreatment process, in order to ensure that the metabolite information of the honey sample and the syrup adulterated chaste tree fruit honey sample is obtained as much as possible, the extraction reagent preferably adopts a mixed solvent of methanol and water (containing a small amount of formic acid) to dissolve the test sample, the ultrasonic treatment is carried out, and the test can be carried out on a machine by centrifuging through an organic filter membrane, so that the pretreatment step is simple and the operation is easy.
Chromatographic separation and mass spectrometric data acquisition are performed simultaneously, and in order to separate and identify each compound, appropriate chromatographic and mass spectrometric analysis conditions must be selected.
Aiming at the characteristics of the vitex negundo honey sample and the syrup adulterated vitex negundo honey component, the invention inspects the influence of conditions such as mobile phase, gradient elution flow, column temperature, sample feeding amount and the like in the ultra-high performance liquid chromatography on the separation efficiency and the analysis speed, and finally obtains a group of ultra-high performance liquid chromatography conditions which enable the analysis sample to obtain the best separation effect by optimized screening.
As a preferred scheme, the conditions of the ultra-high performance liquid chromatography are as follows: bonding a silica gel column (C18 column) by using octadecyl; mobile phase: phase A: acetonitrile, phase B: ammonium acetate in water (preferably 10mM), gradient elution protocol: 0-2min 1% A,2-3.25min 1% -5% A, 3.25-4.25min 5% A,4.25-7.75min 5% -55% A,7.75-9.75min 55% -90% A,9.75-11.75min 90% A,11.75-12min 90% -1% A,12-15min 1% A. Flow rate: 0.2-0.5 mL/min (preferably 0.3mL/min), column temperature 30-37 deg.C (preferably 35 deg.C), and sample injection amount of 3 microliter.
Aiming at the characteristics of the vitex negundo honey sample and the components of syrup adulterated vitex negundo honey, the invention finally optimizes and screens to obtain a group of quadrupole-orbitrap high-resolution mass spectrum conditions which enable the detection effect to be accurate by investigating conditions such as resolution, gas flow rate, spray voltage and the like in order to improve the atomization and ionization conditions of the compound and improve the sensitivity.
As a preferred scheme, a Thermo Fisher Q exact Mass Spectrometer is selected for the quadrupole-orbitrap high-resolution mass spectrometry, and the positive spectrum conditions are as follows: resolution, 70000; sheath gas flow rate, 40 units; flow rate of auxiliary gas, 10 units; the reverse blowing airflow rate is 0 unit; spray voltage, 3.5 kV; capillary temperature, 320 ℃; auxiliary gas temperature, 350 ℃. Scanning range, m/z: 70-1050. Scanning mode: full scan Ms.
Qualitative/quantitative information can be determined for a number of endogenous compounds using metabolomics techniques. The information is shown as a plurality of signal peaks on the output spectrogram and is shown as chromatographic peaks with different retention times on the chromatographic mass spectrogram.
And preprocessing the UHPLC-MS original data of the obtained vitex negundo honey sample and the honey sample to be detected to obtain the retention time, peak height, peak area and mass-to-charge ratio data of each peak. Various software in the prior art can be used for preprocessing raw data, the types of the software are not particularly limited, and the software has a data processing function on a total ion flow diagram.
From the aspect of processing effect and convenience, as an optimal scheme, the UHPLC-MS raw data of the obtained chaste tree honey sample and the chaste tree honey sample to be detected are preprocessed by adopting Compound distributor software, wherein the preprocessing refers to the processing of extraction of chromatographic peaks, peak alignment, detection of noise-removing unknown substances and the like in the raw data of a total ion flow graph, and the retention time, peak height, peak area and mass-to-charge ratio data of each peak are obtained; and then displaying the distinguishing result in a PCA score chart form by a multivariate statistical analysis method.
The components distributor software is software developed by Saimer Feishale technologies, Inc. for processing LC-MS raw data.
In order to overcome the defect that the type of syrup with adulterated vitex negundo honey cannot be specifically identified in the prior art, the second purpose of the invention is to provide a method for screening the marked metabolites of true vitex negundo honey different from syrup based on the analysis method, the method can screen the different metabolites of the true vitex negundo honey and the syrup, and then only needs to further confirm the different metabolites, so that the trouble of counting and analyzing the metabolites in all samples is avoided, and the analysis accuracy and the analysis efficiency are improved; the research and analysis of differential metabolites provides important information for the deep research of the intrinsic differences of the samples; furthermore, the method provides a basis for establishing a general model for identifying true and false vitex negundo honey in the future, can be used for quickly identifying the sample type (which kind of syrup type and content of syrup adulteration) of the adulterated vitex honey, has short analysis time, and improves the accuracy and reliability of the identification result.
After the honey schizonepeta strip sample and the syrup are respectively pretreated by adopting an organic solvent, the separation and the determination of chemical components in the pretreated sample are realized by applying the ultra-high performance liquid chromatography-quadrupole-orbital trap high-resolution mass spectrometry method, then the UHPLC-MS original data of the obtained honey schizonepeta strip sample and the syrup are pretreated, finally, the main component analysis is carried out by applying a multivariate statistical analysis method to carry out differential compound screening, and the marker metabolite is determined and identified.
And (2) preprocessing the UHPLC-MS raw data of the chaste tree honey sample and the syrup by adopting compound distributor software, wherein the preprocessing refers to the processing of extraction, peak alignment, noise removal, detection of unknown substances and the like of chromatographic peaks in the raw data of the total ion flow diagram to obtain retention time, peak height, peak area and mass-to-charge ratio data of each peak, and a PCA score map and a load map are obtained by a principal component analysis model of a multivariate statistical analysis method, so that the marked metabolites are screened out, and then the marked metabolites are identified.
The PCA score map and the load map can be obtained by performing PCA analysis in the Compounds discover software, which is a technical means known to those skilled in the art and will not be described herein again.
Aiming at the characteristics of the vitex negundo honey components, the method for screening differential metabolites by adopting the load map is a simple method, and potential marker metabolites are screened by setting Ratio more than 20 or less than 0.5 and P value less than 0.01 when the load map is obtained. Wherein the Ratio is the Ratio of the peak areas of the metabolite in the two groups.
Through detection of an ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry method, the metabolites in the vitex negundo honey are found to be very many, in order to find the metabolite with the largest difference between the metabolites, the Ratio in the PCA model is set to be larger than 20, the Ratio in the PCA model is the Ratio of the peak area of a certain metabolite in the vitex negundo honey to the peak area of the metabolite in the syrup, and through the setting, the metabolites with large difference between the metabolites can be rapidly screened.
Removing false positive ions of the screened potential marker metabolites, wherein the false positive ions are substances which can not be extracted in a total ion flow graph; obtaining information of the marked metabolites under the cation mode, wherein the information comprises a molecular formula, accurate mass number of molecular ions, retention time and the like, the maximum deviation of the accurate mass number is within 5ppm, and the deviation of the retention time is within 0.2 minute; similarly, according to the obtained molecular formula, extracting the molecular ions of the potential marker metabolites in an anion mode, and speculating the potential marker metabolites according to the accurate mass number of molecular ion peak units in an anion-cation mode, so as to finally obtain the chaste tree flower honey marker metabolites different from the syrup.
When the marker metabolite is identified, fragment ions of the marker metabolite are searched by setting three energy levels of a high-energy collision pool of a high-energy collision induced fragmentation technique (HCD) through a secondary mass spectrum of a quadrupole-orbitrap high-resolution mass spectrum, so that the identification and analysis of the marker metabolite of the schizonepeta honey sample are realized.
In order to overcome the defect that the type of the syrup with the adulterated vitex honey cannot be specifically identified in the prior art, the third purpose of the invention is to obtain the vitex honey marker metabolite which is different from the rice syrup based on the screening method, wherein the marker metabolite consists of the following compounds: phenylalanine, leucine, pantothenic acid, p-coumaric acid, cinnamic acid, chrysin and tyrosine. The marker metabolite can be used for identifying the adulterated chaste tree fruit honey mixed with rice syrup.
Compared with the prior art, the invention has the beneficial effects that:
(1) almost all metabolite information in the vitex honey and the syrup can be obtained by combining a metabonomics method with an ultra-high performance liquid chromatography tandem mass spectrometry technology.
The analysis method of the invention is different from the previous method for measuring adulteration of the vitex negundo honey, and only one or more conventional compounds are qualitatively and quantitatively detected to judge the adulteration, which can cause lawless persons to adjust the adulteration technology according to the latest honey adulteration method. The method of the invention comprehensively analyzes the vitex negundo honey and the syrup adulterated vitex negundo honey by combining multivariate statistical analysis after comprehensively acquiring the metabolite information of the vitex negundo honey and the syrup adulterated vitex negundo honey, establishes a model and completes the detection of the syrup adulterated vitex negundo honey.
The analytical method can effectively distinguish the vitex negundo honey and the syrup adulterated vitex negundo honey, the result is displayed in the form of the PCA score chart, a person skilled in the art can intuitively distinguish the true and false conditions of the vitex negundo honey and does not need to perform related verification and analysis, the true and false conclusion can be obtained, and the detection result is accurate and reliable.
(2) The pretreatment method of the vitex negundo honey sample is simple and quick, and the requirement on the operation technology of detection personnel after the method is established is low.
In the whole pretreatment process, in order to ensure that the metabolite information of the vitex negundo honey sample and the syrup adulterated vitex negundo honey sample is obtained as much as possible, a mixed solvent (containing formic acid) of methanol and water is used for extracting a reagent to dissolve a test sample, the test sample can be subjected to machine detection by centrifuging through an organic filter membrane after ultrasonic treatment, and the pretreatment step is simple and easy to operate.
(3) After the detection model is established, the method can be used for batch detection of the syrup adulterated chaste tree fruit honey sample.
And (3) carrying out total ion current diagram chromatographic peak extraction, peak alignment, detection of unknown substances and other differential analysis on the chaste tree honey sample and the syrup adulterated chaste tree honey sample by using the original data after the detection on the computer through company discover software, and finally displaying the distinguishing result in a PCA (principal component analysis) diagram form. After the detection method is established, the unknown syrup adulterated chaste tree fruit honey sample and chaste tree fruit honey can be distinguished according to the same flow. Experiments prove that an inappropriate pretreatment method cannot extract endogenous metabolites of true honey to the maximum extent, and detection results which are not easy to distinguish can be generated, so that the detection results are inaccurate.
(4) Compared with other detection methods, the method does not need qualitative and quantitative determination of one or some target substances, and is a macroscopic non-target type distinguishing method.
(5) The vitex negundo linn marker metabolite which is different from rice syrup and can be obtained by the screening method provided by the invention is composed of the following compounds: phenylalanine, leucine, pantothenic acid, p-coumaric acid, cinnamic acid, chrysin and tyrosine. The marker metabolite of the vitex chinensis honey which is different from other syrups can be obtained by adopting the same screening method as the invention. According to the obtained vitex negundo honey marker metabolites different from different syrups, a set of general model for identifying syrup adulterated vitex honey of different types and different adulteration contents can be established, and the types and the adulteration contents of the adulterated syrups can be rapidly known through the general model; and the establishment of the general model has very important significance and value for quickly, efficiently, sensitively and accurately identifying the adulterated chaste tree fruit honey.
Drawings
FIG. 1 is a graph of PCA scores for Vitex agnus-castus with 1%, 5%, 10% rice syrup, 1% corn syrup, 1% sugar beet syrup, 1% honey specialty syrup, and 1% adulterated Vitex agnus-castus.
Fig. 2 is a total ion flow graph of a chaste tree honey sample and a F55 rice syrup sample in a cation mode.
FIG. 3 is a plot of the PCA scores of the Vitex honey sample set versus the syrup sample set.
Fig. 4 is a chart of chaste tree honey sample set versus syrup sample set loading.
Figure 5 is a graph of chaste tree honey sample set versus syrup sample set loading after screening.
Fig. 6A, 6B and 6C are phenylalanine fragment ion mass spectra.
Fig. 7A and 7B are leucine fragment ion mass spectra.
FIGS. 8A and 8B are ion mass spectra of pantothenate fragments.
Fig. 9A, 9B, 9C and 9D are mass spectra of p-coumaric acid fragment ions.
Fig. 10A, 10B and 10C are cinnamic acid fragment ion mass spectra.
FIG. 11 is a chrysin fragment ion mass spectrum.
Fig. 12A and 12B are tyrosine fragment ion mass spectra.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
Interpretation of terms:
the multivariate statistical analysis method is a general term of a class of methods for processing multivariate statistical data based on multivariate statistical distribution, and is an important branch with rich theoretical results and numerous application methods in statistics. The commonly used multivariate statistical analysis method mainly comprises the following steps: multivariate regression analysis, cluster analysis, discriminant analysis, principal component analysis, factor analysis, correspondence analysis, canonical correlation analysis, and the like. The invention mainly adopts a principal component analysis method.
Instruments and equipment:
ACQUITY UPLC BEH C18 analytical column U.S. Waters corporation (2.1X 75mm,1.7 μm);
thermo Scientific Q active Thermo Scientific Ultimate3000 Thermo Fisher company, USA;
SIGMA 3-18K high speed refrigerated centrifuge SIGMA corporation, Germany;
Milli-Q-A-11 ultrA pure Water Millipore;
ningbo Xinzhi Biotech Co., Ltd., ultrasonic cleaning machine;
IKA MS 3basic vortex mixer IKA;
materials and reagents:
collecting vitex negundo honey from different beekeepers;
syrup Shandong province food and drug inspection institute;
ultrapure water Milli-Q-A-11;
acetonitrile Sammer Feishell science, Inc.;
methanol Sammer Feishel technologies, Inc.;
anhydrous formic acid Tianjin, Kemiou Chemicals, Inc.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
(1) The principle of the method of the invention is as follows:
after a true vitex negundo honey sample and a syrup adulterated vitex negundo honey sample are treated by the same pretreatment method, a UHPLC serial Thermo Fisher Q active Mass Spectrometer instrument is used for realizing the separation and the determination of chemical components in the sample, and Compunds discover software is used for extracting chromatographic peaks, aligning the peaks and performing library search analysis on unknown Compounds on original data obtained by the instrument. And distinguishing true vitex negundo honey from syrup adulterated vitex negundo honey by applying multivariate statistical analysis PCA analysis.
(2) Sample pretreatment
Weighing 1g of vitex negundo linn honey sample (the repetition frequency of one sample is 6 times), adding 20mL of extractant (the extractant is the equal volume mixture of methanol containing 1% formic acid and ultrapure water), vortex mixing until the vitex negundo linn sample is completely dissolved, placing the centrifuge tube in an ultrasonic cleaning machine for ultrasonic treatment for 25 minutes, centrifuging at 1000rpm for 5 minutes, and filtering the supernatant by using a 0.22 mu m organic filter membrane for sampling.
Respectively weighing 0.99g, 0.95g and 0.90g of chaste tree fruit honey samples (the repetition frequency of one sample is 6 times) in a 50mL centrifuge tube, respectively weighing 0.1g, 0.5g and 1g of syrup samples in a 100mL conical flask, adding 19mL of extracting agent (the extracting agent is methanol containing 1% formic acid and ultrapure water which are equal in volume) into the chaste tree fruit honey samples, adding 10mL of extracting agent into the syrup samples, uniformly mixing and dissolving, then respectively taking 1mL of syrup, adding into the chaste tree fruit honey samples to prepare artificial adulterated chaste tree fruit honey, vortex mixing until the honey samples are completely dissolved, placing the centrifuge tube into an ultrasonic cleaning machine for 25 minutes by ultrasonic treatment, centrifuging at 1000rpm for 5 minutes, and filtering the supernatant by using an organic filter membrane with the diameter of 0.22 mu m to prepare a supernatant.
(3) A UHPLC serial Thermo Fisher Q active Mass Spectrometer instrument is used for realizing the separation and the determination of chemical components in a sample.
Chromatographic conditions are as follows:
phase A: acetonitrile, phase B: 10mM ammonium acetate in water, gradient elution protocol: 0-2min 1% A,2-3.25min 1% -5% A, 3.25-4.25min 5% A,4.25-7.75min 5% -55% A,7.75-9.75min 55% -90% A,9.75-11.75min 90% A,11.75-12min 90% -1% A,12-15min 1% A. Flow rate: 0.3mL/min, the column temperature of 35 ℃, and the sample injection amount of 3 microliters.
Mass spectrum conditions:
and (3) positive spectrum condition: resolution, 70000; sheath gas flow rate, 40 units; flow rate of auxiliary gas, 10 units; the reverse blowing airflow rate is 0 unit; spray voltage, 3.5 kV; capillary temperature, 320 ℃; auxiliary gas temperature, 350 ℃. Scanning range, m/z: 70-1050. Scanning mode: full Ms.
(4) Data processing and multivariate statistical analysis:
and preprocessing UHPLC-MS raw data of the obtained chaste tree honey sample and the syrup adulterated chaste tree honey sample by adopting Compunds distributor software, wherein the preprocessing refers to the processing of extraction of chromatographic peaks, alignment of peaks, noise elimination, detection of unknown substances and the like in the raw data of a total ion flow graph, and then displaying a distinguishing result in a PCA scoring graph form through a multivariate statistical analysis method.
The PCA score chart is a distribution chart of a PCA model, and the PCA analysis is established on the basis of the same data set X, and after the first principal component of the PCA is calculated through a projection method, the score t of each sample point on the first principal component can be obtained1And then obtaining the score t on the second principal component of each sample point2Such as fig. 1-3. The score of each sample in each principal component is the space coordinate of each sample in the calculated mathematical model, and naturally, the specific position of each sample in the model is determined, and the distribution of each sample in the mathematical model space is directly reflected.
(5) And (3) result display:
the samples are vitex negundo honey (the number of the adopted samples is 12, the samples are collected from different beekeepers, and each sample is subjected to 6 times of test repetition), the syrup is rice syrup, corn syrup, beet syrup and special syrup for online purchased honey, and the operation is carried out according to the processes of (1) to (4), so that the separation of the vitex negundo honey, 1% adulterated rice syrup, 5% 10% rice syrup, 1% corn syrup, 1% beet syrup and special syrup for 1% online purchased honey, adulterated vitex negundo honey is obtained.
As shown in FIG. 1, in the PCA score chart, 1r, 5r, 10r, 1c, 1b and 1h respectively represent adulterated vitex negundo honey adulterated with 1%, 5% and 10% rice syrup, adulterated vitex honey adulterated with 1% corn syrup, adulterated vitex honey adulterated with 1% beet syrup and adulterated vitex honey adulterated with 1% honey special syrup. The aggregation and dispersion degree of each sample can be seen from the PCA score chart, each point represents a sample, wherein, the vitex honey comprises 12 samples (each sample is repeated for six times, only one of the samples is selected and is illustrated in figure 1), all the 12 sample points are relatively concentrated on the upper right part of the coordinate axis in figure 1, which indicates that the 12 true vitex honey samples contain metabolites with very close composition and concentration, the intra-group difference is small, and the sample points of the vitex honey adulterated with other syrup are very far away, which indicates that the composition and concentration of the metabolites contained in the true vitex honey samples are greatly different from the syrup adulterated honey, and the method can effectively distinguish the true vitex honey from the syrup adulterated honey, and the result is very obvious; 1r, 5r, 10r, 1c, 1b and 1h all comprise 3 samples (six replicates of each sample, only one of which is selected and illustrated in fig. 1), three points are shown in fig. 1, and it can be seen from the figure that some of the syrup adulterated chaste tree honey can be clearly distinguished, for example, the sample points of the two groups of 1r, 5r, 1c and 10r, 1h and 1b are far away, and can be effectively distinguished. While some syrup adulterated chaste tree fruit honey can not be effectively distinguished, as shown in figure 1, 1r, 5r and 1c sample points are approximately concentrated on the upper left part; the 10r, 1h and 1b sample points are all concentrated at the lower right part, the sample points are very close to each other and cannot be effectively distinguished, the difference between the 10r and the 1h and 1b is large, at least from the adulteration content, the difference is still large, but the 10r and the 1h and 1b cannot be effectively distinguished, and from this point, even if two different samples adopt the metabonomics method, the metabonomics method cannot be effectively distinguished, namely, for the two samples, the skilled person cannot effectively expect whether the metabonomics method can be effectively distinguished.
In conclusion, as can be seen from fig. 1, the method can well distinguish true vitex negundo honey from adulterated vitex negundo honey mixed with 1%, 5% and 10% rice syrup, adulterated vitex negundo honey mixed with 1% corn syrup, adulterated vitex negundo honey mixed with 1% beet syrup and adulterated vitex negundo honey mixed with 1% honey special syrup.
Example 2
A method for screening vitex chinensis honey marker metabolites based on UHPLC serial Thermo Fisher Q active Mass Spectrometer combined with a metabonomics method comprises the following steps:
(1) sample pretreatment
1g of vitex negundo honey sample is put into a 50mL centrifuge tube, 20mL of an extracting agent (the extracting agent is methanol containing 1% formic acid and ultrapure water which are mixed in the same volume) is added, vortex mixing is carried out until the vitex negundo honey sample is completely dissolved, the centrifuge tube is placed into an ultrasonic cleaning machine for ultrasonic treatment for 25 minutes, centrifugation is carried out for 5 minutes at 1000rpm, and supernate is filtered by a 0.22 mu m organic filter membrane to prepare for loading.
1g of the syrup sample is put into a 50mL centrifuge tube, 20mL of an extractant (the extractant is methanol containing 1% formic acid and ultrapure water which are mixed in equal volume) is added, vortex mixing is carried out until the syrup sample is completely dissolved, the centrifuge tube is placed into an ultrasonic cleaning machine for 25 minutes by ultrasound, centrifugation is carried out for 5 minutes at 1000rpm, and the supernatant is filtered by a 0.22 mu m organic filter membrane to prepare a sample.
The syrup is not particularly limited, and all kinds of C3 and C4 syrups used in the current adulteration are covered, including: rice syrup, corn syrup, beet syrup, composite fructose-glucose syrup, special syrup for online purchased honey or other syrups.
(2) A UHPLC serial Thermo Fisher Q active Mass Spectrometer instrument is used for realizing the separation and the determination of chemical components in a sample. Wherein the chromatographic conditions were the same as in example 1 and the mass spectrometric conditions were as follows:
primary mass spectrum conditions:
and (3) negative spectrum condition: resolution, 70000 (FWHM); sheath gas, 40 units; auxiliary gas, 10 units; back blowing, 0 unit; spray voltage, 2.8 kV; capillary temperature, 320 ℃; auxiliary gas temperature, 350 ℃. Scanning range, m/z: 70-1050. Scanning mode: full Ms.
And (3) positive spectrum condition: resolution, 70000 (FWHM); sheath gas, 40 units; auxiliary gas, 10 units; back blowing, 0 unit; spray voltage, 3.5 kV; capillary temperature, 320 ℃; auxiliary gas temperature, 350 ℃. Scanning range, m/z: 70-1050. Scanning mode: full Ms.
Secondary mass spectrum conditions:
and (3) positive spectrum condition: resolution, 175000 (FWHM); sheath gas, 40 units; auxiliary gas, 10 units; back blowing, 0 unit; spray voltage, 3.5 kV; capillary temperature, 320 ℃; auxiliary gas temperature, 350 ℃. Scanning range, m/z: 70-1050. HCD high energy collision cell energy, 50, 100, 150. Scanning mode: full Ms.
(3) Data processing and multivariate statistical analysis
And (3) preprocessing the UHPLC-MS raw data of the chaste tree honey sample and the syrup by adopting compound scanner software, wherein the preprocessing comprises the steps of extracting total ion current diagram chromatographic peaks, aligning the peaks, removing noise, detecting unknown substances and the like, obtaining a PCA score chart and a load chart by a multivariate statistical analysis method, and further screening out the marked metabolites.
The PCA score plot and the load plot are two distribution plots obtained by PCA model analysis. The loading map represents the distribution of the detected variables (e.g., mass to charge ratio) corresponding to the distribution and location of the samples in the score map.
The specific application and operation are as follows:
samples were vitex honey (12 samples were used, collected from different beekeepers, 6 replicates per sample) and F55 rice syrup (12 samples were used, 6 replicates per sample):
after mass spectrometry detection, a total ion flow graph of a vitex negundo honey sample and an F55 rice syrup sample is obtained, as shown in fig. 2, the vitex negundo honey sample is also obviously different from the F55 rice syrup in retention time of 2-7 minutes and 9-13 minutes in a cation mode, and the total peak output number of the vitex negundo honey total ion flow graph is greater than that of the F55 rice syrup total ion flow graph, which indicates that the vitex negundo honey may contain more metabolites than the syrup. The search for specific different substances requires further analysis. PCA analysis was performed in the compounddiscover software, fig. 3 is a plot of PCA scores for the chaste honey sample group and the syrup sample group, in the score chart, the schizonepeta honey sample group (the point of the right half part) and the syrup sample group (the point of the left half part) can be well separated on the first main component, which shows that the schizonepeta honey sample and the syrup sample have great difference in metabolite, the method also provides experimental data basis for the next step of experiments to find the marker metabolite of the vitex negundo honey sample, and also proves the feasibility of the test method for identifying the true and false vitex negundo honey, because the current method for adulteration of honey is to adulterate by adding syrup, the syrup sample group used in the research comprises rice syrup, corn syrup, beet syrup, composite fructose-glucose syrup and special syrup for online purchased honey, and all kinds of C3 and C4 syrups used in the current adulteration are covered. The PCA score chart obtained by data analysis in the research method can completely separate a chaste tree honey sample group from a syrup sample group, and also illustrates the feasibility of the test method for distinguishing chaste tree honey from fake chaste tree honey, metabolites of the chaste tree honey sample group and the syrup sample group are greatly different, FIG. 4 is a load chart of the chaste tree honey sample group and the syrup sample group, each point in the load chart represents one metabolite (metabolite of all samples) extracted from the chaste tree honey and the syrup sample group through compound discover software analysis, 26 shows that the metabolite extracted from the chaste tree honey sample group is more than that extracted from the syrup sample group, and the marker metabolite of the chaste tree honey sample group is searched by screening the metabolite on the load chart of FIG. 4. Generally speaking, the screened substances with the Ratio value of > 2 or < 0.5 and the P value of < 0.01 can be used as differential metabolites, wherein the Ratio value of each metabolite is the Ratio of the average peak areas of the metabolites in the two groups, but the number of the extracted metabolites is huge, and the research aims at searching for the substances with the maximum difference, in the differential metabolite searching step, the Ratio value is set to be > 20 or < 0.5, and the P value is less than 0.01 to search for the marker metabolites in the chaste tree honey sample group. For example, fig. 5 is a screened load map, the load map needs to be analyzed in combination with a PCA score map, the corresponding positions of each group on the PCA score map correspond to the group attribution of points on the load map, and the chastetree fruit sample group in the PCA score map of fig. 3 is located in the right half of the first principal component, so that the differential metabolites of the chastetree fruit sample group can be searched from the screened points in the right half corresponding to the load map of fig. 5. Removing false positive ions of the screened substances, wherein the false positive ions are the substances which can not be extracted in a total ion flow diagram, obtaining the information of the marker metabolites under a cation mode, including molecular formulas, accurate molecular ion mass numbers, retention time and the like, the maximum deviation of the accurate mass numbers is within 5ppm, the deviation of the retention time is within 0.2 min, extracting the molecular ions of the markers under an anion mode according to the obtained molecular formulas, and speculating the markers according to the accurate molecular ion peak unit mass numbers under the anion and cation modes, and finally obtaining the possible marker metabolites of the Jingtiao honey sample, such as phenylalanine, leucine, pantothenic acid, p-coumaric acid, cinnamic acid, chrysin and tyrosine. The complete information of the marker metabolites of the vitex negundo sample is shown in Table 1.
Wherein, the anion and cation mode conditions in the invention are as follows: the chromatographic conditions are the same, and the mass spectrometry conditions include positive and negative spectrum conditions.
TABLE 1 Vitex negundo honey sample marker metabolite molecular ion information table
In the table, the superscript 1 is [ M + NH ]4]2 is [ M + H-H2O]+
Identification of vitex negundo honey marker metabolite
And in order to further determine the seven substances, fragment ions of the marker metabolites are searched by setting three energy levels of an HCD high-energy collision pool through a secondary mass spectrum, so that the qualitative analysis of the found marker metabolites of the schizonepeta honey sample is realized. As shown in table 1, in the fragment ion information table of the vitex negundo sample marker metabolite, at least one fragment ion is found in each marker metabolite under three energies of the set HCD high-energy collision cell, and then two molecular ions and one fragment ion in the positive and negative ion mode of each vitex negundo sample metabolite are divided into 5 points, which already meets the requirement that the qualification of an analyte in the european union at least reaches more than 4 points, one molecular ion is divided into 1 point, and one fragment ion in the high-resolution mass spectrum is divided into 3 points. The found markers can be characterized, and finally the marked metabolites of the schizonepeta honey sample are determined to be phenylalanine, leucine, pantothenic acid, p-coumaric acid, cinnamic acid, chrysin and tyrosine. FIGS. 6A to 12B are fragment ion mass spectra obtained by a Vitex honey marker metabolite HCD high-energy collision cell under three different set energies, wherein three fragment ions are obtained by phenylalanine in total, and the fragment ions with the mass-nucleus ratio of 120.08075 are obtained when the HCD energy is set to be 50, and the fragment ions are obtained by losing one carboxyl group from phenylalanine molecule ions; when the HCD energy is set to be 100, fragment ions with the mass-nucleus ratio of 103.05440 are obtained, and the fragment ions are obtained after a carboxyl group and an amino group are lost from phenylalanine molecule ions; a fragment ion having a nucleus ratio of 79.05478, a fragment ion of a benzene ring group in phenylalanine, was obtained at an HCD energy of 150. Leucine obtains two fragment ions as shown in fig. 7A and 7B, and the fragment ion with the mass-to-nucleus ratio of 86.09689 is obtained when the HCD energy is set to 50, and is obtained after the leucine molecular ion loses one carboxyl group; HCD energy setting 100 yielded a fragment ion with a mass-to-nucleus ratio of 69.07057, and a fragment ion obtained after loss of one carboxyl and amino group for leucine. Pantothenic acid obtained a fragment ion with loss of one hydroxyl and alanine group at HCD energy set at 50 with a proton to nuclear ratio of 116.03441, fig. 8A and 8B. P-coumaric acid obtained 4 fragment ions only when the HCD energy was set to 50 as shown in fig. 9A, 9B, 9C and 9D, the proton-nuclear ratios were 120.05233, 109.06503, 95.04947 and 73.02895, respectively, and the fragment ions were obtained after one carboxyl group was lost, the fragment ions obtained after molecular ions were cleaved from a double bond, the fragment ions of a phenol group and the fragment ions of an acrylic group. 10A, 10B cinnamic acid obtained two fragment ions at HCD setting 50, respectively fragment ion nucleus ratio 91.05464 after breakage at acrylic acid branched double bond, fragment ion nucleus ratio 104.05780 is fragment ion with one carboxyl group removed; fig. 10C shows the fragment ion obtained when the HCD was set at 150, the exact mass number was 79.05458, and the fragment ion was a benzene ring group. See fig. 11, fragment ions with a nucleus ratio of 147.04364 were obtained when HCD energy was set at 50, and are fragment ions of chrysin molecular ions that lost one benzene ring and two hydroxyl groups. Tyrosine obtained two and one fragment ions respectively when HCD energy was set to 50 and 150 as shown in fig. 12A and 12B, fragment ions with a proton nucleus ratio of 136.07545 were fragment ions with one carboxyl group removed, 119.04916 were fragment ions with one carboxyl group and one amino group removed, and 95.04958 were phenol cluster fragment ions.
TABLE 2 Vitex agnus-castus mark metabolite fragment ion information table
The difference of the metabolites of the vitex negundo honey and the rice syrup is mainly analyzed, and the marker metabolite different from other syrups can be researched by adopting the same method as the embodiment.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A method for screening true vitex negundo honey marker metabolites different from syrup is characterized by comprising the following steps:
respectively pretreating a vitex negundo honey sample and syrup by adopting an organic solvent, then separating and determining chemical components in the pretreated sample by using an ultra-high performance liquid chromatography-quadrupole-orbital trap high-resolution mass spectrometry method, then preprocessing UHPLC-MS original data of the obtained vitex negundo honey sample and syrup, finally screening differential compounds by using a principal component analysis model of a multivariate statistical analysis method, and determining and identifying a marker metabolite;
the conditions of the ultra-high performance liquid chromatography are as follows: bonding a silica gel column (C18 column) by using octadecyl; mobile phase: phase A: acetonitrile, phase B: ammonium acetate aqueous solution, gradient elution procedure: 0-2min 1% A,2-3.25min 1% -5% A, 3.25-4.25min 5% A,4.25-7.75min 5% -55% A,7.75-9.75min 55% -90% A,9.75-11.75min 90% A,11.75-12min 90% -1% A,12-15min 1% A; flow rate: 0.2-0.5 mL/min, and the column temperature is 30-37 ℃;
the syrup comprises rice syrup, corn syrup, beet syrup, composite fructose-glucose syrup, special syrup for online purchased honey or other syrups;
the organic solvent adopted in the sample pretreatment is a mixed solvent of methanol containing formic acid and water;
the volume fraction of formic acid is 1%, and methanol and water are mixed in equal volume to form a mixed solvent;
preprocessing UHPLC-MS original data of the obtained vitex negundo honey sample and the syrup adulterated vitex negundo honey sample by adopting Comounds distributor software, wherein the preprocessing refers to the extraction, peak alignment, noise removal and unknown detection processing of chromatographic peaks in the original data of a total ion flow diagram, and retention time, peak height, peak area and mass-to-charge ratio data of each peak are obtained; then displaying the distinguishing result in the form of a PCA score chart by a multivariate statistical analysis method;
removing false positive ions of the screened potential marker metabolites, wherein the false positive ions are substances which can not be extracted in a total ion flow graph; obtaining information of a marker metabolite under a cation mode, wherein the information comprises a molecular formula, the accurate mass number of molecular ions and retention time, the maximum deviation of the accurate mass number is within 5ppm, and the deviation of the retention time is within 0.2 min; similarly, extracting the molecular ions of the potential marker metabolites in an anion mode according to the obtained molecular formula, and speculating the potential marker metabolites according to the accurate mass number of molecular ion peak units in an anion-cation mode, so as to finally obtain the true vitex chinensis honey marker metabolites different from the syrup;
the screened vitex chinensis honey marker metabolite different from rice syrup consists of the following compounds: phenylalanine, leucine, pantothenic acid, p-coumaric acid, cinnamic acid, chrysin and tyrosine.
2. The analytical method of claim 1, wherein: mixing a sample with an organic solvent until the sample is completely dissolved, performing ultrasonic treatment, centrifuging, and filtering with an organic filter membrane to obtain a sample which can be detected on a computer; wherein the ultrasonic time is 10-30 min; centrifugation conditions: centrifuging at 800-1200 rpm for 4-8 min, wherein the aperture of the organic filter membrane is 0.20-0.25 μm; the adding ratio of the sample to the organic solvent is 1 g: (15-25) mL.
3. The analytical method of claim 2, wherein: centrifuging at 1000rpm for 5min, and ultrasonic treating for 25min to obtain organic filter membrane with pore diameter of 0.22 μm.
4. The analytical method of claim 1, wherein: the concentration of the ammonium acetate aqueous solution is 10mM, the flow rate is 0.3mL/min, the column temperature is 35 ℃, and the sample injection amount is 3 microliters.
5. The analytical method of claim 1, wherein: the mass spectrum conditions are as follows: resolution, 70000; sheath gas flow rate, 40 units; flow rate of auxiliary gas, 10 units; the reverse blowing airflow rate is 0 unit; spray voltage, 3.5 kV; capillary temperature, 320 ℃; the temperature of the auxiliary gas is 350 ℃; scanning range, m/z: 70-1050; scanning mode: and (4) full scanning.
6. The method of claim 1, further comprising: potential marker metabolites were screened by setting Ratio > 20 or < 0.5, and P value < 0.01 when obtaining the loading map.
7. The method of claim 1, further comprising: when the marker metabolite is identified, fragment ions of the marker metabolite are searched by setting three energy levels of a high-energy collision pool of a high-energy collision induced fragmentation technique (HCD) through a secondary mass spectrum of a quadrupole-orbitrap high-resolution mass spectrum, so that the identification and analysis of the marker metabolite of the schizonepeta honey sample are realized.
8. Use of the Vitex negundo L.var.negundo L.var.miq.var.
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