CN107102077A - A kind of analysis method for differentiating acacia honey and the adulterated acacia honey of syrup - Google Patents
A kind of analysis method for differentiating acacia honey and the adulterated acacia honey of syrup Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The present invention discloses a kind of analysis method for differentiating acacia honey and the adulterated acacia honey of syrup, specifically applies ultra performance liquid chromatography quadrupole bar track trap high resolution mass spectrum technology combination metabolism group method, including：By true acacia honey sample and organic solvent is respectively adopted with the adulterated acacia honey sample of syrup to be detected to carry out after pre-treatment, separation and the measure to the chemical composition in the sample after pre-treatment are realized using ultra performance liquid chromatography quadrupole bar track trap high resolution mass spectrum method, then the UHPLC MS initial data for obtaining true acacia honey sample and acacia honey sample to be measured is pre-processed, true acacia honey and the adulterated acacia honey of syrup are distinguished in finally application Multielement statistical analysis method principal component analysis.The method of the present invention is after a comprehensive acquisition is carried out to the metabolin information of acacia honey and the adulterated acacia honey of syrup, with reference to multi-variate statistical analysis, the adulterated acacia honey of acacia honey and syrup to be analyzed comprehensively, the detection to the adulterated acacia honey of syrup is completed.
Description
Technical field
The invention belongs to food adulteration authentication technique field, and in particular to associated with one kind application UHPLC-Q Exactive
Metabonomic technology differentiates the analysis method of acacia honey and the adulterated acacia honey of syrup.
Background technology
Honey is the nectar, secretion or honeydew of honeybee herborization, after being mixed with itself secretion, through fully brewageing and
Into natural sweet substance, honey is of high nutritive value, and its main component is fructose and glucose, and both contents are total to account for 70%,
Also there are mineral matter, protein, amino acid, enzyme and vitamin etc. in honey in addition to glucide, be the natural of ideals of human being
Food.Informal dress honey for heart disease, hypertension, tuberculosis, illness in eye, hepatopathy, dysentery, constipation, anaemia, the nervous system disease,
Gastric duodenal ulcer disease, which is waited, good medical assistance effect.External application can also treat scald, skin care and preventing and treating
Frostbite.Wherein, acacia honey is in water white, and transparence, color and luster is limpid, and mouthfeel is fresh and sweet, faint yellow, there is the distinctive fragrant of sophora flower,
With sophora flower micro-perfume taste, crystallization is difficult, fructose ratio is high, fructose is easily absorbed by the body utilizations, and suitable all groups, are first-class
Honey.
But it is due to the higher price of honey and relatively low yield so that it is main that honey turns into illegal retailer adulterated one
Object, in recent years, domestic and international market constantly expand to the demand of honey, and the requirement to its quality is also improved constantly.But
It is that the phenomenon of honey agricultural chemicals, residue of veterinary drug and adulteration happens occasionally for a long time, and particularly honey adulteration occupies honey
The 20%~30% of market, or even some regional adulterated honey account for 50% of honey product or so, greatly compromise beekeeper, consumption
Person and the interests of normal honey manufacturing enterprise, have had a strong impact on the market order of honey product and the export trade of China's honey.
Under the driving of interests, some trade companies and producer mix syrup etc. to honey, manufacture adulterated honey, some producers are even with grape
The false honey of the alternative materials such as sugar, glucose syrup, fructose syrup, honey essence manufacture, is put on market in the form of natural honey, cheated
Consumer is deceived to obtain sudden huge profits.At home, honey fraud situation is on the rise, and delicately packed, cheap false honey is
Openly and legally enter some large supermarkets and market, it may be said that honey fraud scale, specialization.This savage phenomenon
Behind come from huge economic interests because the honey of actual production much can not meet the demand in market, while adulterated
Happy material price is again well below the real price of honey.But honey component is complicated, internal composition changes of contents scope is big
Make it that adulterated fraud is easy etc. feature, also cause honey quality detection technique to encounter very big challenge.These all give illegal point
Son provides the opportunity of honey adulteration.
In order to improve the enthusiasm for production of beekeeper, the interests of consumer be ensured, in order to support normal honey manufacturing enterprise
Fair competition, the order for safeguarding honey market, promote the sound development of China's honey industry, therefore for finding real honey sample
The significant metabolin of product, thus attempt to set up a set of sensitive, efficient, accurate honey adulteration authentication method have it is extremely important
Meaning and value.
Fructose syrup, starch syrup, rice syrup etc. are mixed in true honey：This is honey adulteration hand the most frequently used at present
Section, because fructose and glucose ratio and the composition in honey of these syrup are closely similar, items Testing index is complete after incorporation
Meet national standard entirely, very big difficulty is caused to detection.Being usually used in the method that honey adulteration identified at present has stably
Carbon isotope ratio analytic approach (SCIRA), thin-layered chromatography (TIC), the high performance anion exchange chromatography of pulse current detector
Method (HPAEC-PAD), gas chromatography combined with mass spectrometry technology (GC-MS), high performance liquid chromatography (HPLC), efficient liquid phase isotope
Mass spectrograph GC-MS (HPLC-IRMS), nuclear magnetic resonance technique (NMR) and near infrared spectrum (NIRS) etc..Existing method
There are many drawbacks for determining honey adulteration：Stable carbon isotope method for analyzing ratio (SCIRA) this method is only to natural honey
Middle incorporation C4 plant sugars are effective, if the carbohydrate content that incorporation is prepared using C3 plant starch such as paddy rice, wheat, soybean in honey
Or the complete false honey that the glucide and other materials prepared using C3 plant starch such as paddy rice, wheat, soybean is prepared, then it is difficult to
Differentiate.Kushnir etc. determines the polysaccharide that the degree of polymerization is 12 to 19 using the adulterated honey of tlc determination corn syrup
For honey adulteration high-fructose corn syrup and the mark of corn syrup.But this method needs complicated pretreatment process
Glucose, fructose and a small amount of oligosaccharides in honey are removed, causes detection method complex operation.Efficient the moon of pulse current detector
The limitation of ion-exchange chromatography (HPAEC-PAD) method is bigger, and the hydrolysis of oligosaccharides and polysaccharide is likely to cause in early stage
False positive results, because also there is the presence of degree of polymerization 3-6 oligosaccharides in true honey.The high-efficiency anion of pulse current detector is handed over
Colour changing spectrometry (HPAEC-PAD) then needs the pretreatment process of complexity to remove monose and oligosaccharides, and pretreatment process is complicated.GC-
MS methods, which determine honey adulteration, also certain defect, needs to perform the derivatization honey sample before detection.Nuclear magnetic resonance technique
(NMR) sensitivity is low, may be ignored for our metabolins of interest in honey, and nuclear magnetic resonance apparatus price is high
It is expensive, it is not particularly suited for daily monitoring.Near infrared spectrum (NIRS) also has the shortcomings that its is fatal：1. need a large amount of representative and change
Sample known to value sets up model.So, unactual is just seemed to the analysis near-infrared of small lot sample.2. model is needed
Constantly update, due to instrument state change or standard sample change, model will also change therewith.3. model is obstructed
With the model of every instrument is different from, and increases the limitation used.4. it is high to model capital, test expenditure is big.
The existing method identified honey quality such as GB14963-2011 and SN/T 0852-2012：To honey
Sensory properties；Physical and chemical index is such as：Detected in terms of pyroglutamate content, cane sugar content；Microbiological indicator, the residual beast of agriculture
Residual, heavy metal is also detected in terms of additive.Many company standards then to the maltose in honey, fruit glucose syrup,
Cocoa power, citric acid, lures red, burnt sugar coloring, potassium sorbate, carragheen, flavoring essence etc. to be measured.At present to honey adulteration
Various syrup, in physicochemical property, composition composition and content and local flavor aspect are closely similar with honey therefore above-mentioned to honey
The method that quality is measured can not detect the adulterated honey of syrup.
In summary, lack in currently available technology it is a kind of it is adulterated to acacia honey identified it is quick effectively and directly perceived bright
Aobvious method, does not also determine acacia with ultra performance liquid chromatography-quadrupole rod-track trap high resolution mass spectrum joint metabolism group method
The relevant research of the significant metabolin of honey.
The content of the invention
In view of the shortcomings of the prior art, the present invention provides a kind of application ultra performance liquid chromatography-quadrupole rod-track trap high score
Distinguish that mass-spectrometric technique combination metabolism group method differentiates the analysis method of acacia honey and the adulterated acacia honey of syrup, the analysis method can
Effectively differentiate true acacia honey and the adulterated acacia honey of syrup.
The technical solution adopted by the present invention is as follows：
First purpose of the present invention is to provide a kind of application ultra performance liquid chromatography-quadrupole rod-track trap high-resolution matter
Spectral technology combination metabolism group method differentiates the analysis method of acacia honey and the adulterated acacia honey of syrup, comprises the following steps：
It is respectively adopted true acacia honey sample organic solvent carried out before and with the adulterated acacia honey sample of syrup to be detected and locates
After reason, realized using ultra performance liquid chromatography-quadrupole rod-track trap high resolution mass spectrum method to the change in the sample after pre-treatment
The separation studied point and measure, then to obtaining the UHPLC-MS initial data of true acacia honey sample and acacia honey sample to be measured
Pre-processed, finally distinguish true acacia honey using Multielement statistical analysis method principal component analysis (PCA) model and syrup is adulterated
Acacia honey.
The analysis method of the present invention is not particularly limited for the syrup used in adulterated acacia honey, covers current adulterated institute
C3, C4 syrup all kinds, including：Rice syrup, corn syrup, sugar beet molasses, compound fructose syrup, net purchase honey are special
With syrup or other syrup.
As a kind of preferred scheme, the organic solvent used in the present invention in sample pre-treatments be the methanol containing formic acid and
The mixed solvent of water, wherein, it is preferred that the volume fraction of formic acid is 1%, and first alcohol and water mixes molten for isometric be mixed to form
Agent.
As a kind of preferred scheme, the sample pretreatment process in the present invention includes：By sample mixed with organic solvent to
Sample is completely dissolved, ultrasound, centrifugation, excessively organic filter membrane, the sample for the detection that obtains being available on the machine.Wherein, ultrasonic time be 10~
30min, preferably 25min；Centrifugal condition：800~1200rpm centrifuges 4~8min, preferably 1000rpm centrifugations 5min；Organic filter membrane
Aperture be 0.20~0.25 μm, preferably 0.22 μm；To ensure that metabolin extraction effect is preferably in sample, sample and organic solvent
Adding proportion be 1g：(15~25) mL.
In whole pretreatment process, in order to ensure acquisition honey sample as much as possible and the adulterated acacia honey sample of syrup
Metabolin information, what extracts reagent was preferentially selected is that the mixed solvent (contain a small amount of formic acid) of first alcohol and water is carried out to test sample
Machine testing can be gone up by centrifuging organic filter membrane after dissolving, ultrasound, and pre-treatment step is simple, it is easy to operate.
The separation of chromatogram and the collection of mass spectrometric data are to carry out simultaneously, in order that each compound is separated and reflected
It is fixed, it is necessary to select suitable chromatogram and mass spectral analysis condition.
The characteristics of present invention is for acacia honey sample and syrup adulterated acacia honey component, has investigated ultra performance liquid chromatography
In the influence of the condition to separative efficiency and analyze speed, final optimization pass such as mobile phase, gradient elution flow, column temperature and sample size
Screening obtains one group so that analysis sample obtains the ultra performance liquid chromatography condition of optimal separation effect.
As a kind of preferred scheme, ultra performance liquid chromatography condition is：Using octadecyl silane post (C18 posts)；
Mobile phase：A phases：Acetonitrile, B phases：Ammonium acetate aqueous solution (preferred concentration is 10mM), gradient elution flow：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 velocity：0.2~
0.5mL/min (preferably 0.3mL/min), 30~37 DEG C of column temperature (preferably 35 DEG C), 3 microlitres of sample size.
The characteristics of present invention is for acacia honey sample and syrup adulterated acacia honey component, for improve compound atomization and
Ionization situation, improves sensitivity, by being investigated to conditions such as resolution ratio, gas flow rate, spray voltages, final optimization pass screening
One group is obtained so that the accurate quadrupole rod of Detection results-track trap high resolution mass spectrum condition.
As a kind of preferred scheme, quadrupole rod-track trap high resolution mass spectrum selects Thermo Fisher Q Exactive
Mass Spectrometer, positive spectral condition is：Resolution ratio, 70000；Sheath gas, 40 units；Secondary air speed, 10 lists
Position；Blowback gas velocity, 0 unit；Spray voltage, 3.5kV；Capillary temperature, 320 DEG C；Aid in temperature degree, 350 DEG C.Scan model
Enclose, m/z：70-1050.Surface sweeping pattern：Full Ms (full scan).
The qualitative/quantitative information of many endogenous compounds can be measured to using metabonomic technology.These information
Many signal peaks are shown as on the spectrogram of output, different retention times are shown as on chromatographic mass spectrometry figure and chromatographic peak occur.
Obtained acacia honey sample and the UHPLC-MS initial data of honey sample to be measured is pre-processed, each is obtained
Retention time, peak height, peak area and the mass-to-charge ratio data at peak.At present can be using various software of the prior art for original number
According to being pre-processed, the species for software is not particularly limited, and these softwares have data processing to total ion current figure
Function.
For treatment effect and convenience, as a kind of preferred scheme, to obtained acacia honey sample and acacia to be measured
The UHPLC-MS initial data of sweet sample is pre-processed using Compounds Discoverer softwares,
The pretreatment refers to the extraction to the chromatographic peak in total ion current figure initial data, peak alignment, removes noise, unknown material
The processing such as detection, obtain retention time, peak height, peak area and the mass-to-charge ratio data at each peak；Then multivariate statistics point is passed through
The form of analysis method PCA shot charts is shown to distinguishing result.
The type of the adulterated syrup of acacia honey can not be specifically identified in the prior art in order to overcome, of the invention second
Purpose is to provide a kind of method that the significant metabolin of true acacia honey for being different from syrup is screened based on above-mentioned analysis method, the party
Method can filter out the difference metabolin of true acacia honey and syrup, then only these difference metabolins need to be carried out further true
Recognize, it is to avoid the trouble of statistics and analysis is carried out to the metabolin in all samples, precision of analysis and analysis is so improved
Efficiency；To the research and analysis of otherness metabolin important information is provided to further investigate the inherent difference of sample；Further,
This also differentiates that the universal model of true and false acacia honey provides the foundation to set up later, the sample adulterated available for quick discriminating acacia honey
Category type (adulterated is which kind of syrup type and the adulterated content of syrup), not only analysis time is short, also improves discriminating knot
The accuracy and reliability of fruit.
Organic solvent is respectively adopted to acacia honey sample and syrup to carry out after pre-treatment, using described ultra high efficiency liquid phase color
Spectrum-quadrupole rod-track trap high resolution mass spectrum method realizes the separation and measure to the chemical composition in the sample after pre-treatment, so
The UHPLC-MS initial data for obtaining acacia honey sample and syrup is pre-processed afterwards, finally using Multielement statistical analysis method
Principal component analysis carry out difference screening compound, it is determined that with appraisal mark metabolin.
Use Compounds Discoverer soft in obtained acacia honey sample and the UHPLC-MS initial data of syrup
Part is pre-processed, the pretreatment refer to the extraction to the chromatographic peak in total ion current figure initial data, peak alignment, go noise,
Detection of unknown material etc. is handled, and obtains retention time, peak height, peak area and the mass-to-charge ratio data at each peak, and pass through polynary system
Meter analysis method principal component model obtains PCA shot charts and load diagram, filters out significant metabolin, and then appraisal mark
Property metabolin.
PCA shot charts and load diagram can carry out PCA analyses in Compounds Discoverer softwares and obtain, and this is this
Technological means known to art personnel, then this repeats no more.
The present invention is a kind of simple method using load diagram screening otherness metabolin, when obtaining load diagram, this hair
It is bright by setting Ratio ＞ 20 or ＜ 0.5, P value ＜ 0.01, to screen potential significant metabolin.Wherein, the Ratio is to be somebody's turn to do
The ratio of peak area of the metabolin in two groups.
By the detection of ultra performance liquid chromatography-quadrupole rod-track trap high resolution mass spectrum method, the generation in acacia honey is found
Thank that thing is very more, for the ease of finding the metabolin that both differences are maximum, the Ratio in pca model is set as greatly by the present invention
Ratio in 20, the present invention refers to the peak of this kind of metabolin in the peak area and syrup of certain metabolin in acacia honey
The ratio of area, by such setting, can quickly screen the larger metabolin of both othernesses.
Carry out the exclusion of false positive ion, the false positive ion first to the potential significant metabolin screened
To return to the material extracted in total ion current figure less than chromatographic peak；Obtain the significant metabolin information under cation mode, bag
Molecular formula is included, molecular ion exact mass number and retention time etc., the maximum deviation of exact mass number retain within 5ppm
Time deviation is in 0.2 minute；Likewise, according to the molecular formula of acquisition, to potential significant metabolism under ion mode
The molecular ion of thing is extracted, and according to molecular ion peak unit exact mass number under zwitterion pattern to potential mark
Property metabolin speculated, it is final to obtain the significant metabolin of acacia honey for being different from syrup.
In appraisal mark metabolin, set high energy by the second order mses of quadrupole rod-track trap high resolution mass spectrum and touch
Hit three, the energetic encounter pond energy level of inducing lysis technology (HCD) to find the fragment ion of significant metabolin, so that real
Now to the identification and analysis of the significant metabolin of acacia honey sample.
The type of the adulterated syrup of acacia honey can not be specifically identified in the prior art in order to overcome, of the invention the 3rd
Purpose is the significant metabolin of the acacia honey for being different from rice syrup obtained based on above screening technique, the significant metabolism
Thing have following compound group into：Tryptophan, leucine, tyrosine, proline, retinoic acid, p-Coumaric Acid, abscisic acid, cinnamic acid,
Chrysin.The significant metabolin can be used to differentiate the adulterated acacia honey of rice syrup.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) it can be realized to acacia honey and syrup with metabolism group method combination ultra performance liquid chromatography tandem mass spectrum technology
In most metabolin information obtained.
The analysis method of the present invention be different from before the method being measured adulterated to acacia honey, just for a certain or
Several Conventional compounds progress qualitative and quantitative detections of person are adulterated to judge, this may result in criminal and can be put into effect according to newest
Honey adulteration method adulterated technology is adjusted.And the method for the present invention is to acacia honey and the adulterated acacia honey of syrup
Metabolin information carry out one comprehensively obtain after, with reference to multi-variate statistical analysis, the adulterated acacia honey of acacia honey and syrup is entered
Row analysis comprehensively, sets up model, completes the detection to the adulterated acacia honey of syrup.
Acacia honey and the adulterated acacia honey of syrup can effectively be distinguished using the analysis method of the present invention, as a result with PCA scores
The form displaying of figure, those skilled in the art can intuitively differentiate its true and false situation, without carrying out associated verification and analysis again,
True and false conclusion can be obtained, testing result is accurately and reliably.
(2) acacia honey sample-pretreating method simple and fast, method requires relatively low after setting up to testing staff's operating technology.
In whole pretreatment process, in order to ensure acquisition acacia honey sample as much as possible and the adulterated acacia honey sample of syrup
The metabolin information of product, extracts reagent is that the mixed solvent (contain formic acid) of first alcohol and water dissolves to test sample, super
Organic filter membrane was centrifuged after sound can go up machine testing, and pre-treatment step is simple, it is easy to operate.It is inappropriate by experimental verification
Pre-treating method can not extract the endogenous metabolites of true honey to greatest extent, it will cause the detection knot for being not easy to distinguish
Really so that testing result is inaccurate.
(3) after detection model is set up, batch detection is carried out available for the adulterated acacia honey sample of syrup.
Initial data after upper machine testing is mixed acacia honey sample and syrup by Compounds Discoverer softwares
False acacia honey sample carries out the extraction of total ion current figure chromatographic peak, and peak aligns, and the difference analysis such as detection of unknown material finally leads to
The form for crossing multi-variate statistical analysis PCA figures is shown to distinguishing result.After this detection method is set up, mixed for unknown syrup
False acacia honey sample can be operated with the differentiation of acacia honey by identical flow.
(4) it is better than other detection methods, it is not necessary to which qualitative, quantitative measure is carried out to a certain or some targeting substances, is a kind of
The method that the non-targeted species of macroscopic view is distinguished.
(5) the significant metabolin of the acacia honey for being different from rice syrup that can be obtained by the screening technique of the present invention,
The significant metabolin have following compound group into：It is tryptophan, leucine, tyrosine, proline, retinoic acid, p-Coumaric Acid, de-
Fall acid, cinnamic acid, Chrysin.Being different from the significant metabolin of the acacia honey of other syrup can use and identical of the present invention sieve
Choosing method is studied.The significant metabolin of acacia honey for being different from different syrup according to these obtained, can set up
A set of universal model for differentiating variety classes and the adulterated acacia honey of syrup of different adulterated contents, passes through the universal model, energy
It is enough quickly to know adulterated syrup species and adulterated content；And quick, efficient, sensitive after being established as of logical model,
Carry out differentiating that adulterated acacia honey has very important significance and is worth exactly.
Brief description of the drawings
Fig. 1 is acacia honey with adulterated 1%, 5%, 10% rice syrup, 1% corn syrup, 1% sugar beet molasses, 1% honey
The PCA shot charts of syrup dedicated adulterated acacia honey.
Fig. 2 is acacia honey sample and F55 rice syrup sample total ion current figures under cation mode.
Fig. 3 is acacia honey sample group and syrup sample group PCA shot charts.
Fig. 4 is acacia honey sample group and syrup sample group load diagram.
Fig. 5 is acacia honey sample group and syrup sample group load diagram after screening.
Fig. 6 is tryptophan fragment ion mass spectrogram.
Fig. 7 A and Fig. 7 B are leucine fragment ion mass spectrograms.
Fig. 8 A, Fig. 8 B and Fig. 8 C are tyrosine fragment ion mass spectrograms.
Fig. 9 is proline fragment ion mass spectrogram.
Figure 10 A and Figure 10 B are retinoic acid fragment ion mass spectrograms.
Figure 11 A, Figure 11 B and Figure 11 C are p-Coumaric Acid fragment ion mass spectrograms.
Figure 12 A, Figure 12 B and Figure 12 C are abscisic acid fragment ion mass spectrograms.
Figure 13 A, Figure 13 B and Figure 13 C are cinnamic acid fragment ion mass spectrograms.
Figure 14 is Chrysin fragment ion mass spectrogram.
Embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the present invention.Unless another
Indicate, all technologies used herein and scientific terminology are with usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation and/or combinations thereof.
Term is explained：
Multielement statistical analysis method is built upon the class processing multivariate statistics data method in multivariate statistics distributed basis
General name, be the important branch with abundant theoretical result and numerous application processes in statistics.Conventional multivariate statistics point
Analysis method mainly includes：Multiple regression analysis, clustering, discriminant analysis, principal component analysis, factorial analysis, correspondence analysis, allusion quotation
Type correlation analysis etc..The present invention mainly uses PCA.
Instrument and equipment：
ACQUITY UPLC BEH C18 analytical columns Waters, US (2.1 × 75mm, 1.7 μm)；
Thermo Scientific Q ExactiveThermoScientificTM Ultimate3000 U.S. Thermo
Fisher companies；
SIGMA companies of SIGMA 3-18K high speed freezing centrifuges Germany；
MiLLi-Q-A-11 ultrapure water machines Millipore Corp.；
Supersonic wave cleaning machine Xin Zhi bio tech ltd, Ningbo；
IKA MS 3basic turbine mixer IKA companies；
Material and reagent：
Collected at acacia honey difference beekeeper；
Syrup Shandong Province food and medicine examines institute；
Ultra-pure water MiLLi-Q-A-11；
Acetonitrile Thermo Fisher Scientific Inc.；
Methanol Thermo Fisher Scientific Inc.；
Anhydrous formic acid Tianjin Kermel Chemical Reagent Co., Ltd..Can be more clear in order that obtaining those skilled in the art
Understand technical scheme to Chu, technical scheme is described in detail below with reference to specific embodiment.
Embodiment 1
(1) Method And Principle of the invention：
After being handled with identical pre-treating method true acacia honey sample and the adulterated acacia honey sample of syrup, using UHPLC
Thermo Fisher Q Exactive Mass Spectrometer instruments of connecting realize the separation to chemical composition in sample
With measure, the extraction that initial data carries out chromatographic peak, peak alignment are obtained to instrument using Compounds Discoverer softwares
And the storehouse of searching of unknown compound is analyzed.True acacia honey and the adulterated acacia honey of syrup are distinguished using multi-variate statistical analysis PCA analyses.
(2) sample pre-treatments
1g acacia honeys sample (number of repetition of a sample is 6 times) is weighed in 50mL centrifuge tubes, 20mL is added and extracts
Agent (extractant is that the methanol containing 1% formic acid is mixed in equal volume with ultra-pure water), vortex mixed to acacia honey sample is completely dissolved, will
Centrifuge tube is placed in supersonic cleaning machine ultrasound 25 minutes, and 1000rpm is centrifuged 5 minutes, by supernatant with 0.22 μm of organic filter membrane mistake
Filter is in case loading.
Weigh 0.99g respectively, 0.95g, 0.90g acacia honeys sample (number of repetition of a sample is 6 times) in 50mL from
In heart pipe, 0.1g is weighed respectively, and 0.5g, 1g syrup samples add 19mL extractants in 50mL centrifuge tubes in acacia honey sample
10mL extractants are added in (extractant is that the methanol containing 1% formic acid is mixed in equal volume with ultra-pure water), syrup sample, dissolving is mixed
Afterwards, then respectively take 1mL to be added in acacia honey sample and do artificial adulterated acacia honey, vortex mixed to honey sample is completely dissolved,
Centrifuge tube is placed in supersonic cleaning machine ultrasound 25 minutes, 1000rpm is centrifuged 5 minutes, by supernatant with 0.22 μm of organic filter membrane
Filtering is in case loading.
(3) application UHPLC series connection Thermo Fisher Q Exactive Mass Spectrometer instrument realizations pair
The separation of chemical composition and measure in sample.
Chromatographic condition：
Positive spectral condition is：A phases：Acetonitrile, B phases：10mM ammonium acetate aqueous solutions, gradient elution flow：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 velocity：0.3mL/min,
35 degrees Celsius of column temperature, 3 microlitres of sample size.
Mass Spectrometry Conditions：
Resolution ratio, 70000；Sheath gas, 40 units；Secondary air speed, 10 units；Blowback gas velocity, 0 list
Position；Spray voltage, 3.5kV；Capillary temperature, 320 DEG C；Aid in temperature degree, 350 DEG C.Scanning range, m/z：70-1050.Surface sweeping
Pattern：Full Ms.
(4) data processing and multi-variate statistical analysis：
The UHPLC-MS initial data of the adulterated acacia honey sample of acacia acacia honey sample and syrup to obtaining is used
Compounds Discoverer softwares are pre-processed, and the pretreatment refers to the extraction for carrying out total ion current figure chromatographic peak, peak
Align, then the difference analysis such as detection of unknown material is tied by the form of Multielement statistical analysis method PCA shot charts to distinguishing
Fruit is shown.
PCA shot charts are a kind of distribution maps of pca model, because PCA analyses are built upon same data set X bases
On, after projecting method calculates PCA first principal components, score t of each sample spot in first principal component can be obtained1,
The score t in second principal component of each sample spot is obtained again2, such as Fig. 1~Fig. 3.Each sample is in each principal component
Score is exactly its space coordinate in the mathematical modeling of calculating, naturally also just determines its particular location in a model, and
Directly reflect distribution situation of each sample in mathematical modeling space.
(5) achievements exhibition：
Sample is that (the sample number of use is 15 to acacia honey, is collected at different beekeepers, and each sample is repeated 6 times examination
Test), syrup is that rice syrup, corn syrup, sugar beet molasses, net purchase honey are syrup dedicated, is carried out according to the flow of (1)~(4)
Operation, obtain acacia honey with adulterated 1%, 5%, 10% rice syrup, 1% corn syrup, 1% sugar beet molasses, 1% honey is special
The differentiation of the adulterated acacia honey of syrup.
Aggregation, the dispersion degree of each sample are can be seen that from PCA shot charts, each point represents a sample, wherein,
Acacia honey includes 15 samples (each six repetitions of sample, only have chosen wherein once illustrating in Fig. 1 for the sample),
The bottom left section of the whole Relatively centralizeds of 15 sample spots in Fig. 1, this illustrates the metabolism that this 15 true acacia honey samples contain
Thing is constituted and concentration is very close to group difference is smaller, and the sample spot distance of the acacia honey adulterated with other syrup is very big, and this says
The metabolome that bright true acacia honey sample contains distinguishes larger into the adulterated honey of concentration and syrup, can be effective using this method
True acacia honey and the adulterated honey of syrup are distinguished, this result is very apparent；1r, 5r, 10r, 1c, 1h include 3 samples,
1b includes 2 samples (each six repetitions of sample, only have chosen wherein once illustrating in Fig. 1 for the sample), in Fig. 1
In be shown three points (1r, 5r, 10r, 1c, 1h) or two points (1b), it can be seen that the adulterated ocean of some syrup
Chinese scholartree honey can clearly distinguish, such as 1r, 5r and 1c, two groups of 10r, 1h, 1b sample spot away from degree greatly, can be effective
Distinguish.And the adulterated acacia honey of some syrup can not be distinguished effectively, as shown in figure 1,10r, 1h, 1b sample spot are all concentrated on
Lower right-most portion, and sample spot is apart from close, it is impossible to directly perceived effectively to distinguish, and 10r and 1h, 1b differ greatly, at least from mixing
From the point of view of false content, difference is still than larger, but 10r and 1h, 1b but intuitively can not be distinguished effectively, from this point as can be seen that
Also not necessarily can effectively it be distinguished using the metabolism group method even if the larger sample of two species diversity.
To sum up, can be obtained by Fig. 1, this method can by true acacia honey with adulterated 1%, 5%, the adulterated ocean of 10% rice syrup
Chinese scholartree honey, the adulterated acacia honey of adulterated 1% corn syrup, the adulterated acacia honey of adulterated 1% sugar beet molasses, adulterated 1% honey is special
The adulterated acacia honey of syrup is distinguished well.
Embodiment 2
Based on UHPLC series connection Thermo Fisher Q Exactive Mass Spectrometer combinations metabolism group side
The method that method screens the significant metabolin of acacia honey, comprises the following steps：
(1) sample pre-treatments
By 1g acacia honeys sample in 50mL centrifuge tubes, add 20mL extractants (extractant for the methanol containing 1% formic acid with
Ultra-pure water is mixed in equal volume), vortex mixed to acacia honey sample is completely dissolved, and centrifuge tube is placed in into ultrasound 25 in supersonic cleaning machine
Minute, 1000rpm is centrifuged 5 minutes, by supernatant with 0.22 μm of organic membrane filtration in case loading.
By 1g syrup samples in 50mL centrifuge tubes, adding 20mL extractants, (extractant is the methanol containing 1% formic acid with surpassing
Pure water is mixed in equal volume), vortex mixed to syrup sample is completely dissolved, and centrifuge tube is placed in into 25 points of ultrasound in supersonic cleaning machine
Clock, 1000rpm is centrifuged 5 minutes, by supernatant with 0.22 μm of organic membrane filtration in case loading.
Wherein, the syrup is not particularly limited, and covers C3, C4 syrup all kinds used adulterated at present, including：
Rice syrup, corn syrup, sugar beet molasses, compound fructose syrup, net purchase honey is syrup dedicated or other syrup.
(2) application UHPLC series connection Thermo Fisher Q Exactive Mass Spectrometer instrument realizations pair
The separation of chemical composition and measure in sample.Wherein, chromatographic condition is identical with embodiment 1, and Mass Spectrometry Conditions are as described below：
First mass spectrometric condition：
Negative spectral condition：Resolution ratio, 70000 (FWHM)；Sheath gas, 40 units；Aid in gas, 10 units；Blowback air, 0
Unit；Spray voltage, 2.8kV；Capillary temperature, 320 DEG C；Aid in temperature degree, 350 DEG C.Scanning range, m/z：70-1050.Sweep
Surface model：Full Ms.
Positive spectral condition：Resolution ratio, 70000 (FWHM)；Sheath gas, 40 units；Aid in gas, 10 units；Blowback air, 0
Unit；Spray voltage, 3.5kV；Capillary temperature, 320 DEG C；Aid in temperature degree, 350 DEG C.Scanning range, m/z：70-1050.Sweep
Surface model：Full Ms.
Second order mses condition：
Positive spectral condition：Resolution ratio, 175000 (FWHM)；Sheath gas, 40 units；Aid in gas, 10 units；Blowback air, 0
Unit；Spray voltage, 3.5kV；Capillary temperature, 320 DEG C；Aid in temperature degree, 350 DEG C.Scanning range, m/z：70-1050.
HCD energetic encounters pond energy, 50,100,150.Surface sweeping pattern：Full Ms.
(3) data processing and multi-variate statistical analysis
Use Compounds Discoverer soft in obtained acacia honey sample and the UHPLC-MS initial data of syrup
Part is pre-processed, and the pretreatment refers to the extraction for carrying out total ion current figure chromatographic peak, and noise, the inspection of unknown material are removed in peak alignment
The processing such as survey, and PCA shot charts and load diagram are obtained by Multielement statistical analysis method, and then filter out significant metabolin.
PCA shot charts and load diagram are two kinds of distribution maps that pca model analysis is obtained.Load diagram illustrates detected change
Measure distribution and position of the variable distribution in the distribution situation of (such as mass-to-charge ratio), load diagram with sample in shot chart corresponding.
Concrete application and operation are as follows：
Sample is that (the sample number of use is 15 to acacia honey, is collected at different beekeepers, and each sample is repeated 6 times examination
Test) and F55 rice syrups (the sample number of use is 11, and each sample is repeated 6 times experiment)：
Flow according to (1)~(3) is operated, after Mass Spectrometer Method, obtains acacia honey sample and F55 rice syrups
The total ion current figure of sample, as shown in Fig. 2 under cation mode, acacia honey sample and F55 rice syrups total ion current figure are all
There is obvious difference, the chromatographic peak that the total ion current figure of acacia honey sample goes out under cation mode is more more rich, also illustrates
Honey sample contains a greater variety of metabolins than syrup sample in addition to sugar.
Fig. 3 is acacia honey sample group and syrup sample group PCA shot charts, as seen from the figure acacia honey sample group (left side
Point point) can be achieved with being kept completely separate on first principal component with syrup sample group (point of right half part), illustrate acacia honey and
The metabolome of syrup is expressed as soft by Compounds Discoverer into each point existed in very big difference, Fig. 4
Metabolin (metabolins of all samples) in acacia honey and syrup that part analysis is extracted, extracts altogether 580 kinds of metabolins,
By setting Ratio ＞ 20 or ＜ 0.5, P value ＜ 0.01, the 580 kinds of metabolins extracted are screened, acacia honey and sugar is found
The significant metabolin of the otherness metabolin of slurry, i.e. acacia honey.Fig. 5 is the otherness material after screening, passes through correspondence
Fig. 3 PCA shot charts, find the significant metabolin of acacia honey in the point that first principal component left-hand component is filtered out, and lead to
Cross and bring original total ion current figure progress false positive ion rejecting into, because carrying out the exact mass number of molecular ion under cation mode
Matching, eventually finds the potential significant metabolin of acacia honey：Tryptophan, leucine, tyrosine, proline, retinoic acid, to perfume (or spice)
Beans acid, abscisic acid, cinnamic acid, Chrysin.Table 1 is the significant metabolin molecular ion information table of acacia honey.Wherein exact mass
Number deviation is within 5ppm, and retention time maximum deviation is in 0.2 minute.
Wherein, the anions and canons mode condition in the present invention：Chromatographic condition is identical, Mass Spectrometry Conditions include positive spectral condition and
Negative spectral condition.
The significant metabolin molecular ion information table of the acacia honey of table 1
Subscript 1 is [M+NH in table4]+, subscript 2 is [M+H-H2O]+
The identification of the significant metabolin of acacia honey
Qualitative, HCD energetic encounters pond setting 50,100,150 3 energy are carried out to the potential significant metabolin of acacia honey
Amount is lower to carry out two grades of fragmentations, finds the fragment ion of the significant metabolin of acacia honey.Table 2 is broken for the significant metabolin of acacia honey
Piece ion information table.In the nine significant metabolins of acacia honey found, each metabolin at least have found a fragment from
Son, it may be determined that the significant metabolin of acacia honey be tryptophan, leucine, tyrosine, proline, retinoic acid, p-Coumaric Acid,
Abscisic acid, cinnamic acid, Chrysin.Fig. 6~Figure 14 is the significant metabolin fragment ion mass spectrogram of acacia honey.Wherein tryptophan exists
Two fragment ions are obtained when HCD energy is set as 50 and see Fig. 6, karyoplasmic ratio is respectively 159.09142,118.06522,
159.09142 be that tryptophan modules ion loses the fragment ion after a carboxyl, and 118.06522 be to fall to deaminize propionic acid side chain
Fragment ion afterwards.Leucine obtains a fragment ion such as Fig. 7 A and Fig. 7 B when HCD energy is set as 50 and 150 respectively,
The ion that karyoplasmic ratio is 86.09692 is to fall the fragment ion formed after carboxyl, and the ion that karyoplasmic ratio is 69.07056 is to fall carboxyl
With the fragment ion formed after amino.Tyrosine just obtains three fragment ion Fig. 8 A and Fig. 8 B, matter core when HCD is set as 50
Than being respectively 136.07545,119.01908,109.06507, respectively it is to lose the fragment ion formed after carboxyl, loses carboxylic
The fragment ion formed after base and amino, loses the fragment ion after aminocarbonyloxymethyl group；Obtained when HCD is set as 100
Fragment ion such as Fig. 8 C that one karyoplasmic ratio is 95.04950, are phenolic groups fragment ions.Proline is only in the setting of HCD energy
For 100 when obtain a karyoplasmic ratio be 72.04499, lose such as Fig. 9 of the fragment ion after carboxyl.Retinoic acid is set as 50 in HCD
A fragment ion such as Figure 10 A and Figure 10 B are obtained with each when 150, karyoplasmic ratio is respectively 83.08620,240.23172, is hexamethylene
Alkenyl group fragment ion and the fragment ion formed at the nearest double bond of c-terminus after fracture.P-Coumaric Acid is set as in HCD
Three fragment ions such as Figure 11 A, Figure 11 B and Figure 11 C are obtained when 50, the ion that karyoplasmic ratio is 79.05478 is benzene radicals fragment
Ion, 91.05460 fragment ion to be broken to form from phenyl ring at side chain double bond, 104.05782 be to fall shape after a carboxyl
Into fragment ion.Abscisic acid obtains two fragment ions when energy is set as 50 and sees figure Figure 12 A and Figure 12 B, and karyoplasmic ratio is
137.09534 and 111.04428,137.09534 be to fall the fragment ion formed after long-chain branch and hydroxyl, and 111.04428 be length
The fragment ion of branched group formation；HCD energy is set as that it is 61.02914 aceticoceptor fragment ions that karyoplasmic ratio is obtained when 150
Such as Figure 12 C.Cinnamic acid obtains three fragment ions in HCD energy setting 50 and sees Figure 13 A, Figure 13 B and Figure 13 C, and karyoplasmic ratio is
79.05478 be benzene radicals fragment ion, and 91.05460 be the fragment ion broken to form from phenyl ring at side chain double bond,
104.05782 be to fall the fragment ion formed after a carboxyl.Chrysin obtains a fragment only when HCD energy is set as 50
Ion such as Figure 14, karyoplasmic ratio is 147.04382, to fall the fragment ion of phenyl ring and two hydroxyl formation.
The significant metabolin fragment ion information table of the acacia honey of table 2
The present embodiment, which is mainly, to be analyzed the metabolin difference of acacia honey and rice syrup, is different from other syrup
Significant metabolin can be used and studied with the present embodiment identical method.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. one kind application ultra performance liquid chromatography-quadrupole rod-track trap high resolution mass spectrum technology combination metabolism group method differentiates
The analysis method of acacia honey and the adulterated acacia honey of syrup, it is characterized in that, comprise the following steps：
By true acacia honey sample and organic solvent is respectively adopted with the adulterated acacia honey sample of syrup to be detected to carry out after pre-treatment,
Using ultra performance liquid chromatography-quadrupole rod-track trap high resolution mass spectrum method realize to the chemistry in the sample after pre-treatment into
The separation divided and measure, are then carried out to the UHPLC-MS initial data for obtaining true acacia honey sample and acacia honey sample to be measured
Pretreatment, finally using Multielement statistical analysis method principal component analysis（PCA）Model distinguishes true acacia honey and the adulterated acacia of syrup
Honey.
2. analysis method as claimed in claim 1, it is characterized in that：The syrup includes rice syrup, corn syrup, beet sugar
Slurry, compound fructose syrup, net purchase honey is syrup dedicated or other syrup.
3. analysis method as claimed in claim 1, it is characterized in that：The organic solvent used in sample pre-treatments is containing formic acid
First alcohol and water mixed solvent；It is preferred that, the volume fraction of formic acid is 1%, and first alcohol and water mixes molten for isometric be mixed to form
Agent；
It is preferred that, sample pretreatment process includes：Sample is mixed to sample with organic solvent and is completely dissolved, ultrasound, centrifugation, mistake
Organic filter membrane, the sample for the detection that obtains being available on the machine；Wherein, ultrasonic time is 10 ~ 30min, preferably 25min；Centrifugal condition：800~
1200rpm centrifuges 4 ~ 8min, preferably 1000 rpm centrifugations 5min；The aperture of organic filter membrane is 0.20~0.25 μm, preferably
0.22μm；The adding proportion of sample and organic solvent is 1g：（15~25）mL.
4. analysis method as claimed in claim 1, it is characterized in that：Ultra performance liquid chromatography condition is：A phases：Acetonitrile, B phases：
Ammonium acetate aqueous solution, gradient elution flow：The A of 0-2min 1% A, 2-3.25min 1%-5% A, 3.25-4.25min 5%,
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 velocity：0.2 ~ 0.5mL/min, 30 ~ 37 DEG C of column temperature；It is preferred that, the ammonium acetate aqueous solution
Concentration be 10 mM, flow velocity is 0.3mL/min, 35 DEG C of column temperature, 3 microlitres of sample size.
5. analysis method as claimed in claim 1, it is characterized in that：Positive spectral condition is：Resolution ratio, 70000；Sheath gas, 40
Individual unit；Secondary air speed, 10 units；Blowback gas velocity, 0 unit；Spray voltage, 3.5Kv；Capillary temperature, 320
℃；Aid in temperature degree, 350 DEG C；Scanning range, m/z：70-1050；Surface sweeping pattern：Full scan.
6. analysis method as claimed in claim 1, it is characterized in that：By obtained acacia honey sample and the adulterated acacia honey of syrup
The UHPLC-MS initial data of sample is pre-processed using Compounds Discoverer softwares, and the pretreatment refers to total
The extraction of chromatographic peak in ion stream figure initial data, peak alignment, the detection process for removing noise, unknown material, obtain each peak
Retention time, peak height, peak area and mass-to-charge ratio data；Then by the form of Multielement statistical analysis method PCA shot charts to area
Point result is shown.
7. a kind of method for screening the significant metabolin of true acacia honey for being different from syrup, it is characterized in that, comprise the following steps：
Organic solvent is respectively adopted to acacia honey sample and syrup to carry out after pre-treatment, using any one of claim 1 ~ 6 institute
Ultra performance liquid chromatography-the quadrupole rod stated-track trap high resolution mass spectrum method realize to the chemistry in the sample after pre-treatment into
The separation divided and measure, are then pre-processed to the UHPLC-MS initial data for obtaining acacia honey sample and syrup, finally should
Carry out difference screening compound with Multielement statistical analysis method principal component model, it is determined that with appraisal mark metabolin；
It is preferred that, it is potential significant to screen by setting Ratio ＞ 20 or ＜ 0.5, P value ＜ 0.01 when obtaining load diagram
Metabolin.
8. method as claimed in claim 7, it is characterized in that：Vacation is carried out first to the potential significant metabolin screened
The exclusion of positive ions, the false positive ion is to return to the material extracted in total ion current figure less than chromatographic peak；Obtain sun from
Significant metabolin information under subpattern, including molecular formula, molecular ion exact mass number and retention time etc., accurate matter
The maximum deviation of number is measured within 5ppm, retention time deviation is in 0.2 minute；Likewise, according to the molecular formula of acquisition, in the moon
The molecular ion of potential significant metabolin is extracted under ion mode, and according to molecular ion under zwitterion pattern
Peak unit exact mass number speculates to potential significant metabolin, final to obtain the true acacia honey mark for being different from syrup
Property metabolin；
It is preferred that, in appraisal mark metabolin, high energy is set by the second order mses of quadrupole rod-track trap high resolution mass spectrum
Collision-induced cracking technique（HCD）Three, energetic encounter pond energy level find the fragment ion of significant metabolin so that
Realize the identification and analysis to the significant metabolin of acacia honey sample.
9. the significant metabolin of the acacia honey for being different from rice syrup obtained using the method screening described in claim 7 or 8,
It is characterized in that, the significant metabolin have following compound group into：Tryptophan, leucine, tyrosine, proline, retinoic acid,
P-Coumaric Acid, abscisic acid, cinnamic acid, Chrysin.
10. the significant metabolin of the acacia honey for being different from rice syrup described in claim 9 is differentiating the adulterated ocean of rice syrup
Application in Chinese scholartree honey.
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| CN107807192A (en) * | 2017-10-13 | 2018-03-16 | 南昌大学 | The method that microwave plasma torch tandem mass spectrum differentiates honey adulteration |
| CN107807192B (en) * | 2017-10-13 | 2021-02-09 | 南昌大学 | Method for identifying honey adulteration by microwave plasma torch tandem mass spectrometry |
| CN109142596A (en) * | 2018-08-27 | 2019-01-04 | 福建出入境检验检疫局检验检疫技术中心 | A kind of recognition methods of honey or royal jelly authenticity |
| CN111965280A (en) * | 2020-08-14 | 2020-11-20 | 安徽农业大学 | Detection method for rapidly determining sugar mixing ratio of Dangshan pear syrup |
| CN112285219A (en) * | 2020-09-04 | 2021-01-29 | 中国农业科学院蜜蜂研究所 | Authenticity evaluation method for acacia honey and application of authenticity evaluation method in adulteration identification |
| CN113933353A (en) * | 2021-10-15 | 2022-01-14 | 聊城大学 | Method for rapidly identifying adulteration of honey |
| CN113820434A (en) * | 2021-11-23 | 2021-12-21 | 中国农业科学院蜜蜂研究所 | Method for identifying maturity of acacia honey |
| WO2023093731A1 (en) * | 2021-11-23 | 2023-06-01 | 中国农业科学院蜜蜂研究所 | Method for identifying degree of maturity of acacia honey |
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