CN107102077B - A kind of analysis method identifying acacia honey and the adulterated acacia honey of syrup - Google Patents
A kind of analysis method identifying acacia honey and the adulterated acacia honey of syrup Download PDFInfo
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- CN107102077B CN107102077B CN201710269820.4A CN201710269820A CN107102077B CN 107102077 B CN107102077 B CN 107102077B CN 201710269820 A CN201710269820 A CN 201710269820A CN 107102077 B CN107102077 B CN 107102077B
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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 identifying acacia honey and the adulterated acacia honey of syrup, specifically apply ultra performance liquid chromatography-quadrupole rod-orbit trap high resolution mass spectrum technology combination metabolism group method, include: by true acacia honey sample and with the adulterated acacia honey sample of syrup to be detected be respectively adopted organic solvent carry out pre-treatment after, the separation and measurement to the chemical component in the sample after pre-treatment are realized using ultra performance liquid chromatography-quadrupole rod-orbit 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, finally true acacia honey and the adulterated acacia honey of syrup are distinguished using Multielement statistical analysis method principal component analysis.Method of the invention is in conjunction with multi-variate statistical analysis, to be analyzed comprehensively acacia honey and the adulterated acacia honey of syrup after the metabolin information to acacia honey and the adulterated acacia honey of syrup carries out a comprehensive acquisition, complete the detection to the adulterated acacia honey of syrup.
Description
Technical field
The invention belongs to food adulteration authentication technique fields, and in particular to associated with a kind of application UHPLC-Q Exactive
The analysis method of metabonomic technology identification acacia honey and the adulterated acacia honey of syrup.
Background technique
Honey is the nectar, secretion or honeydew of honeybee herborization, after being mixed with itself secretion, sufficiently made and
At natural sweet substance, honey nutritive value is high, and main component is fructose and glucose, and the two content is total to account for about 70%,
Also there are minerals, protein, amino acid, enzyme and vitamin etc. in honey other than glucide, is the natural of ideals of human being
Food.Informal dress honey for heart disease, hypertension, tuberculosis, eye disease, hepatopathy, dysentery, constipation, anaemia, the nervous system disease,
Gastric duodenal ulcer disease etc. has good medical assistance to act on.External application can also treat scald, skin care and prevention and treatment
Frostbite.Wherein, acacia honey is in water white, and transparence, color is limpid, and mouthfeel is fresh and sweet, faint yellow, there is the distinctive fragrant of sophora flower,
It with sophora flower micro-perfume taste, is not easy to crystallize, fructose ratio is high, and fructose is easily absorbed by the body utilizations, is suitble to all groups, is first-class
Honey.
But due to the higher price of honey and lower yield so that honey become illegal retailer adulterated one it is main
Object, in recent years, domestic and international market constantly expand the demand of honey, and the requirement to its quality is also continuously improved.But
It is that the phenomenon that honey pesticide, residue of veterinary drug and adulteration happens occasionally for a long time, and especially honey adulteration occupies honey
The 20%~30% of market in addition some adulterated honey in area account for 50% of honey product or so, greatly compromise beekeeper, consumption
The interests of person and normal honey manufacturing enterprise have seriously affected the market order of honey product and the export trade of China's honey.
Under the driving of interests, some trade companies and producer manufacture adulterated honey, some producers are even with grape to honey incorporation syrup etc.
The alternative materials such as sugar, glucose syrup, fructose syrup, honey essence manufacture false honey, are launched in the form of natural honey, hole
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 be derived from huge economic interests because the honey of actual production much can not meet the demand in market, while adulterated
Happy substance price is again well below the real price of honey.However honey component is complicated, internal composition changes of contents range is big
Etc. features make it is adulterated fake be easy, but also honey quality detection technique encounters 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, ensure the interests of consumer, 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, to attempt to establish a set of sensitive, efficient, accurate honey adulteration identification method with extremely important
Meaning and value.
Fructose syrup, starch syrup, rice syrup etc. are mixed in true honey: this is most common honey adulteration hand
Section, since the fructose and glucose ratio of these syrup and the ingredient in honey are closely similar, every Testing index is complete after incorporation
Meet national standard entirely, causes very big difficulty to detection.Being usually used in the method that honey adulteration is identified has stabilization at present
Carbon isotope ratio analytic approach (SCIRA), thin-layered chromatography (TIC), pulse current detector high performance anion exchange chromatography
Method (HPAEC-PAD), gas chromatography combined with mass spectrometry technology (GC-MS), high performance liquid chromatography (HPLC), efficient liquid phase isotope
Mass spectrograph joint technology (HPLC-IRMS), nuclear magnetic resonance technique (NMR) and near infrared spectrum (NIRS) etc..Existing method
For measurement honey adulteration, there are many drawbacks: stable carbon isotope method for analyzing ratio (SCIRA) this method is only to natural honey
Middle incorporation C4 plant sugar is effective, if the carbohydrate content that incorporation is prepared using C3 plants starch such as rice, wheat, soybean in honey
Or the complete false honey prepared using the glucide of the C3 plants starch such as rice, wheat, soybean preparation and other substances, then it is difficult to
Identify.Kushnir etc. determines that the degree of polymerization is 12 to 19 polysaccharide using the adulterated honey of tlc determination corn syrup
For the marker of honey adulteration high-fructose corn syrup and corn syrup.But this method needs complicated pretreatment process
Glucose, fructose and a small amount of oligosaccharides in honey are removed, causes detection method complicated for operation.The efficient yin of pulse current detector
The limitation of ion-exchange chromatography (HPAEC-PAD) method is bigger, is likely to cause to the hydrolysis of oligosaccharides and polysaccharide in early period
False positive results, because also there is the presence of the oligosaccharides of degree of polymerization 3-6 in true honey.The high-efficiency anion of pulse current detector is handed over
Colour changing spectrometry (HPAEC-PAD) needs complicated pretreatment process then to remove monosaccharide and oligosaccharides, and pretreatment process is complicated.GC-
MS method measurement honey adulteration also has 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 establishes model.In this way, just seeming not practical to the analysis near-infrared of small lot sample.2. model needs
It constantly updates, since 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 that honey quality is identified such as GB14963-2011 and SN/T 0852-2012: to honey
Sensory properties；Physical and chemical index is such as: pyroglutamate content, cane sugar content etc. are detected；Microbiological indicator, the residual beast of agriculture
Residual, heavy metal, additive etc. is also detected.Many company standards then in honey maltose, fruit glucose syrup,
Cocoa power, citric acid, the red, burnt sugar coloring of temptation, potassium sorbate, carragheen, edible essence etc. are measured.At present to honey adulteration
Various syrup, in physicochemical property, at being grouped as with content and flavor in terms of it is closely similar with honey therefore above-mentioned to honey
The method that quality is measured can not detect the adulterated honey of syrup.
In conclusion lacking adulterated identified fast and effective of a kind of pair of acacia honey and intuitive bright in currently available technology
Aobvious method, also without measuring acacia with ultra performance liquid chromatography-quadrupole rod-orbit trap high resolution mass spectrum joint metabolism group method
The related research of the significant metabolin of honey.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of application ultra performance liquid chromatography-quadrupole rod-orbit trap high score
Distinguish that mass-spectrometric technique combination metabolism group method identifies the analysis method of acacia honey and the adulterated acacia honey of syrup, which can
Effectively identify true acacia honey and the adulterated acacia honey of syrup.
The technical solution adopted by the invention is as follows:
The first purpose of the invention is to provide a kind of application ultra performance liquid chromatography-quadrupole rod-orbit trap high-resolution matter
The analysis method of spectral technology combination metabolism group method differentiation acacia honey and the adulterated acacia honey of syrup, comprising the following steps:
Place before organic solvent carries out is respectively adopted by true acacia honey sample and with the adulterated acacia honey sample of syrup to be detected
After reason, realize using ultra performance liquid chromatography-quadrupole rod-orbit trap high resolution mass spectrum method to the change in the sample after pre-treatment
The separation and measurement studied point, then to obtaining the UHPLC-MS initial data of true acacia honey sample Yu acacia honey sample to be measured
It is pre-processed, finally distinguishes true acacia honey using Multielement statistical analysis method principal component analysis (PCA) model and syrup is adulterated
Acacia honey.
Analysis method of the invention is not particularly limited syrup used in adulterated acacia honey, covers current adulterated institute
C3, C4 syrup all kinds, comprising: rice syrup, corn syrup, sugar beet molasses, compound fructose syrup, online shopping honey are special
With syrup or other syrup.
As a preferred embodiment, the organic solvent used in sample pre-treatments in the present invention be the methanol containing formic acid and
The mixed solvent of water, wherein preferred, the volume fraction of formic acid is 1%, and first alcohol and water is molten to be mixed to form mixing in equal volume
Agent.
As a preferred embodiment, the present invention in sample pretreatment process include: sample is 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 is centrifuged 4~8min, and preferably 1000rpm is centrifuged 5min；Organic filter membrane
Aperture be 0.20~0.25 μm, preferably 0.22 μm；To guarantee that metabolin extraction effect is preferable in sample, sample and organic solvent
Adding proportion be 1g:(15~25) mL.
In entire pretreatment process, in order to guarantee the adulterated acacia honey sample of acquisition honey sample and syrup as much as possible
Metabolin information, extract that reagent preferentially selects is that the mixed solvent (containing a small amount of formic acid) of first alcohol and water carries out test sample
Dissolution, machine testing can be gone up by being centrifuged organic filter membrane after ultrasonic, and pre-treatment step is simple, easily operated.
The separation of chromatography and the acquisition of mass spectrometric data carry out simultaneously, in order to make each compound be separated and be reflected
It is fixed, it is necessary to select suitable chromatography 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 conditions such as mobile phase, gradient elution process, column temperature and sample volume to separative efficiency and analyze the influence of speed, final optimization pass
Screening obtains one group and analysis sample is made to obtain the ultra performance liquid chromatography condition of optimal separation effect.
As a preferred embodiment, ultra performance liquid chromatography condition are as follows: use octadecyl silane column (C18 column)；
Mobile phase: A phase: acetonitrile, B phase: ammonium acetate aqueous solution (preferred concentration 10mM), gradient elution process: 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 volume.
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
It obtains one group and makes the accurate quadrupole rod of detection effect-orbit trap high resolution mass spectrum condition.
As a preferred embodiment, quadrupole rod-orbit trap high resolution mass spectrum selects Thermo Fisher Q Exactive
Mass Spectrometer, positive spectral condition are as follows: 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；Auxiliary temperature degree, 350 DEG C.Scan model
It encloses, m/z:70-1050.Surface sweeping mode: 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, and different retention times are shown as on chromatographic mass spectrometry figure and chromatographic peak occur.
The UHPLC-MS initial data of obtained acacia honey sample and honey sample to be measured is pre-processed, is obtained each
Retention time, peak height, peak area and the mass-to-charge ratio data at peak.Various software in the prior art can be used at present for original number
According to being pre-processed, the type of software is not particularly limited, these softwares have data processing to total ion current figure
Function.
For treatment effect and convenience, as a preferred embodiment, to obtained acacia honey sample and acacia to be measured
The UHPLC-MS initial data of sweet sample is pre-processed using Compounds Discoverer software,
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 pass through multivariate statistics point
The form of analysis method PCA shot chart is shown to result is distinguished.
In order to overcome the type that can not specifically identify the adulterated syrup of acacia honey in the prior art, of the invention second
Purpose is to provide one kind, and method screens the method for being different from the significant metabolin of true acacia honey of syrup, the party based on above-mentioned analysis
Method can filter out the difference metabolin of true acacia honey and syrup, then need to only carry out to these difference metabolins further true
Recognize, avoids the trouble for carrying out statistics and analysis to the metabolin in all samples, improve precision of analysis and analysis in this way
Efficiency；Important information is provided to the research and analysis of otherness metabolin to further investigate the inherent difference of sample；Further,
This universal model that the true and false acacia honey of identification is established after being also provides the foundation, and can be used for quickly identifying the adulterated sample of 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 identification knot
The accuracy and reliability of fruit.
After organic solvent progress pre-treatment is respectively adopted to acacia honey sample and syrup, using the ultra high efficiency liquid phase color
Spectrum-quadrupole rod-orbit trap high resolution mass spectrum method realizes the separation and measurement to the chemical component in the sample after pre-treatment, so
The UHPLC-MS initial data for obtaining acacia honey sample and syrup is pre-processed afterwards, finally applies Multielement statistical analysis method
Principal component analysis carries out difference screening compound, determining and appraisal mark metabolin.
It is soft using Compounds Discoverer to the UHPLC-MS initial data of obtained acacia honey sample and 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,
The processing such as detection of unknown material obtains retention time, peak height, peak area and the mass-to-charge ratio data at each peak, and passes through polynary system
Meter analysis method principal component model obtains PCA shot chart and load diagram, filters out significant metabolin, and then appraisal mark
Property metabolin.
PCA shot chart and load diagram can carry out PCA in Compounds Discoverer software and analyze to obtain, this is this
Technological means well known to the technical staff of field, 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 set Ratio > 20 or < 0.5, P value < 0.01, come screen potentially 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-orbit trap high resolution mass spectrum method, the generation in acacia honey is found
It is very more to thank to object, for the ease of finding the two maximum metabolin of difference, the Ratio in pca model is set as big 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 can quickly screen the biggish metabolin of otherness of the two by such setting.
Carry out the exclusion of false positive ion, the false positive ion first to the potential significant metabolin screened
To return to the substance extracted in total ion current figure less than chromatographic peak；Obtain the significant metabolin information under cation mode, packet
Molecular formula, molecular ion exact mass number and retention time etc. are included, the maximum deviation of exact mass number retains 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 object extracts, and according to molecular ion peak unit exact mass number under zwitterion mode 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, high energy is set by quadrupole rod-orbit trap high resolution mass spectrum second order ms and is touched
Three, the energetic encounter pond energy level of inducing lysis technology (HCD) is hit to find the fragment ion of significant metabolin, thus real
Now to the identification and analysis of the significant metabolin of acacia honey sample.
In order to overcome the type that can not specifically identify the adulterated syrup of acacia honey in the prior art, third of the invention
Purpose is the significant metabolin of acacia honey for being different from rice syrup obtained based on the above screening technique, the significant metabolism
Object is made of following compound: tryptophan, leucine, tyrosine, proline, retinoic acid, p-Coumaric Acid, abscisic acid, cinnamic acid,
Chrysin.The significant metabolin can be used to identify the adulterated acacia honey of rice syrup.
Compared with prior art, the beneficial effects of the present invention are:
(1) it may be implemented with metabolism group method combination ultra performance liquid chromatography tandem mass spectrometry to acacia honey and syrup
In most metabolin information obtained.
Analysis method of the 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 will lead to criminal can be according to newest appearance
Honey adulteration method adulterated technology is adjusted.And method of the invention is to acacia honey and the adulterated acacia honey of syrup
Metabolin information carry out one it is comprehensive obtain after, in conjunction with multi-variate statistical analysis, to acacia honey and the adulterated acacia honey of syrup into
Row analysis comprehensively, establishes 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 analysis method of the invention, as a result with PCA score
The form of figure shows that those skilled in the art can intuitively differentiate its true and false situation, no longer needs to carry out associated verification and analysis,
Available true and false conclusion, testing result are accurate and reliable.
(2) acacia honey sample-pretreating method is simple and fast, and method requires testing staff's operating technology after establishing lower.
In entire pretreatment process, in order to guarantee the adulterated acacia honey sample of acquisition acacia honey sample and syrup as much as possible
The metabolin information of product, extract reagent is that the mixed solvent (containing formic acid) of first alcohol and water dissolves test sample, is surpassed
Organic filter membrane was centrifuged after sound can go up machine testing, and pre-treatment step is simple, easily operated.It is inappropriate by experimental verification
Pre-treating method cannot extract the endogenous metabolites of true honey to the maximum extent, it will cause the detection knot for being not easy to distinguish
Fruit, so that testing result is inaccurate.
(3) after detection model is established, can be used for carrying out batch detection to the adulterated acacia honey sample of syrup.
Initial data after upper machine testing mixes acacia honey sample with syrup by Compounds Discoverer software
False acacia honey sample carries out the extraction of total ion current figure chromatographic peak, peak alignment, and the difference analysis such as detection of unknown material finally lead to
The form for crossing multi-variate statistical analysis PCA figure is shown to result is distinguished.After this detection method is established, unknown syrup is mixed
False acacia honey sample can be operated with the differentiation of acacia honey by identical process.
(4) it is better than other detection methods, does not need to carry out qualitative, quantitative measurement to a certain or certain targeting substances, is a kind of
The method that the non-targeted type of macroscopic view is distinguished.
(5) the significant metabolin of acacia honey that screening technique through the invention can obtain be different from rice syrup,
The significant metabolin is made of following compound: tryptophan, tyrosine, proline, retinoic acid, p-Coumaric Acid, takes off leucine
Fall acid, cinnamic acid, Chrysin.Sieve same as the present invention can be used in the significant metabolin for being different from the acacia honey of other syrup
Choosing method is studied to obtain.The significant metabolin of acacia honey for being different from different syrup according to these obtained, can establish
The universal model of a set of adulterated acacia honey of syrup for identifying variety classes and different adulterated contents, passes through the universal model, energy
It is enough quickly to know adulterated syrup type and adulterated content；And quick, efficient, sensitive after being established as of logical model,
It accurately carries out identifying adulterated acacia honey and has very important significance and be worth.
Detailed description of the invention
Fig. 1 is acacia honey and adulterated 1%, 5%, 10% rice syrup, 1% corn syrup, 1% sugar beet molasses, 1% honey
The PCA shot chart of syrup dedicated adulterated acacia honey.
Fig. 2 is acacia honey sample and F55 rice syrup sample total ion current figure under cation mode.
Fig. 3 is acacia honey sample group and syrup sample group PCA shot chart.
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.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has 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 specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or their combination.
Term is explained:
Multielement statistical analysis method is built upon a kind of processing multivariate statistics data method in multivariate statistics distributed basis
General name, be the important branch with abundant theoretical result and numerous application methods in statistics.Common multivariate statistics point
Analysis method specifically includes that multiple regression analysis, clustering, discriminant analysis, principal component analysis, factorial analysis, correspondence analysis, allusion quotation
Type correlation analysis etc..The present invention mainly uses Principal Component Analysis.
Instrument and equipment:
ACQUITY UPLC BEH C18 analytical column Waters, US (2.1 × 75mm, 1.7 μm)；
Thermo Scientific Q ExactiveThermoScientificTM Ultimate3000 U.S. Thermo
Fisher company；
SIGMA company, SIGMA 3-18K high speed freezing centrifuge Germany；
MiLLi-Q-A-11 ultrapure water machine Millipore Corp.；
Supersonic wave cleaning machine Xin Zhi bio tech ltd, Ningbo；
IKA MS 3basic turbine mixer IKA company；
Material and reagent:
It is collected at acacia honey difference beekeeper；
Syrup Shandong Province food and medicine examines institute；
Ultrapure water MiLLi-Q-A-11；
Acetonitrile Thermo Fisher Scientific Inc.；
Methanol Thermo Fisher Scientific Inc.；
Anhydrous formic acid Tianjin Kermel Chemical Reagent Co., Ltd..In order to enable those skilled in the art can be more clear
Chu technical solution of the present invention is understood, below with reference to the specific embodiment technical solution that the present invention will be described in detail.
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 instrument of connecting realizes the separation to chemical component in sample
With measurement, the extraction that initial data carries out chromatographic peak, peak alignment are obtained to instrument using Compounds Discoverer software
And the library 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 analysis.
(2) sample pre-treatments
1g acacia honey sample (number of repetition of a sample is 6 times) is weighed in 50mL centrifuge tube, 20mL is added and extracts
Agent (extractant is that the methanol containing 1% formic acid mixes in equal volume with ultrapure water), vortex mixed to acacia honey sample is completely dissolved, will
Centrifuge tube is placed in ultrasound 25 minutes in supersonic cleaning machine, and 1000rpm is centrifuged 5 minutes, by 0.22 μm of organic filter membrane mistake of supernatant
Filter is in case loading.
Weigh 0.99g respectively, 0.95g, 0.90g acacia honey sample (number of repetition of a sample is 6 times) in 50mL from
In heart pipe, 0.1g, 0.5g are weighed respectively, and 19mL extractant is added in 50mL centrifuge tube in 1g syrup sample in acacia honey sample
(extractant mix in equal volume with ultrapure water for the methanol containing 1% formic acid) is added 10mL extractant in syrup sample, mixes and dissolve
Afterwards, then respectively it taking 1mL to be added in acacia honey sample and does artificial adulterated acacia honey, vortex mixed to honey sample is completely dissolved,
Centrifuge tube is placed in ultrasound 25 minutes in supersonic cleaning machine, 1000rpm is centrifuged 5 minutes, by 0.22 μm of organic filter membrane of supernatant
Filtering is in case loading.
(3) using UHPLC series connection Thermo Fisher Q Exactive Mass Spectrometer instrument realization pair
The separation and measurement of chemical component in sample.
Chromatographic condition:
Positive spectral condition are as follows: A phase: acetonitrile, B phase: 10mM ammonium acetate aqueous solution, gradient elution process: 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 volume.
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；Auxiliary temperature degree, 350 DEG C.Scanning range, m/z:70-1050.Surface sweeping
Mode: Full Ms.
(4) data processing and multi-variate statistical analysis:
The UHPLC-MS initial data of the adulterated acacia honey sample of obtained acacia acacia honey sample and syrup is used
Compounds Discoverer software is pre-processed, which refers to the extraction for carrying out total ion current figure chromatographic peak, peak
Then alignment, the difference analysis such as detection of unknown material are tied differentiation by way of Multielement statistical analysis method PCA shot chart
Fruit is shown.
PCA shot chart is a kind of distribution map of pca model, since PCA analysis is built upon the same basis data set X
On, after projecting method calculates PCA first principal component, score t of the available each sample spot in first principal component1,
The score t in second principal component of each sample spot is obtained again2, such as FIG. 1 to FIG. 3.Each sample is in each principal component
Score is exactly its space coordinate in the mathematical model of calculating, naturally also just determines its specific location in a model, and
Directly reflect distribution situation of each sample in mathematical model space.
(5) achievements exhibition:
Sample is that (the sample number of use is 15 to acacia honey, is collected from different beekeepers, and each sample is repeated 6 times examination
Test), syrup is that rice syrup, corn syrup, sugar beet molasses, online shopping honey are syrup dedicated, is carried out according to the process of (1)~(4)
Operation, obtains acacia honey and adulterated 1%, 5%, 10% rice syrup, 1% corn syrup, 1% sugar beet molasses, and 1% honey is dedicated
The differentiation of the adulterated acacia honey of syrup.
It can be seen that aggregation, the dispersion degree of each sample from PCA shot chart, each point represents a sample, wherein
Acacia honey includes 15 samples (six repetitions of each sample, only have chosen wherein once being illustrated in Fig. 1 for the sample),
15 sample spots are all relatively concentrated in the bottom left section in Fig. 1, this illustrates the metabolism that this 15 true acacia honey samples contain
Object composition and concentration are very close to group difference is smaller, and the sample spot of the acacia honey adulterated with other syrup distance is very big, this says
The metabolome that bright true acacia honey sample contains is larger at distinguishing with concentration and the adulterated honey of 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 (six repetitions of each sample, only have chosen wherein once being illustrated 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 from the figure that the ocean that some syrup are adulterated
Chinese scholartree honey can be apparent distinguish, for example 1r, 5r and 1c, two groups of 10r, 1h, 1b of sample spot are big far from degree, can be effective
It distinguishes.And the adulterated acacia honey of some syrup can not be distinguished effectively, as shown in Figure 1,10r, 1h, 1b sample spot all concentrate on
Lower right-most portion, and sample spot intuitively can not be distinguished effectively apart from close, and 10r and 1h, 1b differ greatly, at least from mixing
From the point of view of false content, difference or bigger, but 10r and 1h, 1b but intuitively can not be distinguished effectively, from this point as can be seen that
It not necessarily can effectively be distinguished the biggish sample of two species diversity uses the metabolism group method.
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 are dedicated
The adulterated acacia honey of syrup is distinguished well.
Embodiment 2
Based on UHPLC series connection Thermo Fisher Q Exactive Mass Spectrometer combination metabolism group side
The method of the method screening significant metabolin of acacia honey, comprising the following steps:
(1) sample pre-treatments
By 1g acacia honey sample in 50mL centrifuge tube, be added 20mL extractant (extractant be methanol containing 1% formic acid with
Ultrapure water mixes in equal volume), vortex mixed to acacia honey sample is completely dissolved, and centrifuge tube is placed in ultrasound 25 in supersonic cleaning machine
Minute, 1000rpm is centrifuged 5 minutes, by 0.22 μm of organic membrane filtration of supernatant in case loading.
By 1g syrup sample in 50mL centrifuge tube, 20mL extractant is added, and (extractant is the methanol containing 1% formic acid and surpasses
Pure water mixes in equal volume), vortex mixed to syrup sample is completely dissolved, and centrifuge tube is placed in 25 points of ultrasound in supersonic cleaning machine
Clock, 1000rpm are centrifuged 5 minutes, by 0.22 μm of organic membrane filtration of supernatant in case loading.
Wherein, the syrup is not particularly limited, and covers C3, C4 syrup all kinds used adulterated at present, comprising:
Rice syrup, corn syrup, sugar beet molasses, compound fructose syrup, online shopping honey is syrup dedicated or other syrup.
(2) using UHPLC series connection Thermo Fisher Q Exactive Mass Spectrometer instrument realization pair
The separation and measurement of chemical component in sample.Wherein, chromatographic condition is identical as in 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；Assist gas, 10 units；Blowback air, 0
Unit；Spray voltage, 2.8kV；Capillary temperature, 320 DEG C；Auxiliary temperature degree, 350 DEG C.Scanning range, m/z:70-1050.It sweeps
Surface model: Full Ms.
Positive spectral condition: resolution ratio, 70000 (FWHM)；Sheath gas, 40 units；Assist gas, 10 units；Blowback air, 0
Unit；Spray voltage, 3.5kV；Capillary temperature, 320 DEG C；Auxiliary temperature degree, 350 DEG C.Scanning range, m/z:70-1050.It sweeps
Surface model: Full Ms.
Second order ms condition:
Positive spectral condition: resolution ratio, 175000 (FWHM)；Sheath gas, 40 units；Assist gas, 10 units；Blowback air, 0
Unit；Spray voltage, 3.5kV；Capillary temperature, 320 DEG C；Auxiliary temperature degree, 350 DEG C.Scanning range, m/z:70-1050.
HCD energetic encounter pond energy, 50,100,150.Surface sweeping mode: Full Ms.
(3) data processing and multi-variate statistical analysis
It is soft using Compounds Discoverer to the UHPLC-MS initial data of obtained acacia honey sample and syrup
Part is pre-processed, which refers to that noise, the inspection of unknown material are gone in the extraction for carrying out total ion current figure chromatographic peak, peak alignment
The processing such as survey, and PCA shot chart and load diagram are obtained by Multielement statistical analysis method, and then filter out significant metabolin.
PCA shot chart and load diagram are two kinds of distribution maps that pca model is analyzed.Load diagram illustrates change detected
The distribution situation of (such as mass-to-charge ratio) is measured, the variable distribution in load diagram is corresponding with the distribution of sample in shot chart and position.
Concrete application and operation are as follows:
Sample is that (the sample number of use is 15 to acacia honey, is collected from different beekeepers, and each sample is repeated 6 times examination
Test) and F55 rice syrup (the sample number of use is 11, and each sample is repeated 6 times test):
It is operated according to the process of (1)~(3), after Mass Spectrometer Method, obtains acacia honey sample and F55 rice syrup
The total ion current figure of sample, as shown in Fig. 2, acacia honey sample and F55 rice syrup total ion current figure are all under cation mode
There is apparent difference, the chromatographic peak that the total ion current figure of acacia honey sample goes out under cation mode is more richer, also illustrates
Honey sample contains a greater variety of metabolins than syrup sample other than sugar.
Fig. 3 is acacia honey sample group and syrup sample group PCA shot chart, 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 at there is very big difference, each point in Fig. 4
The metabolin (metabolins of all samples) in acacia honey and syrup that part analysis extracts, 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 are found
The otherness metabolin of slurry, the i.e. significant metabolin of acacia honey.Fig. 5 is the otherness substance after screening, passes through correspondence
The PCA shot chart of Fig. 3 finds the significant metabolin of acacia honey in the point that first principal component left-hand component filters out, and leads to
It crosses and brings 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 acacia honey potentially significant metabolin: 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 present invention in anions and canons mode condition: chromatographic condition is identical, Mass Spectrometry Conditions include positive spectral condition and
Negative spectral condition.
The significant metabolin molecular ion information table of 1 acacia honey of table
Subscript 1 is [M+NH in table4]+, subscript 2 is [M+H-H2O]+
The identification of the significant metabolin of acacia honey
To acacia honey, potentially significant metabolin carries out qualitative, 50,100,150 3 energy of HCD energetic encounter pond setting
Amount is lower to carry out second level fragmentation, finds the fragment ion of the significant metabolin of acacia honey.Table 2 is the broken of 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, can determine acacia honey significant metabolin 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 sees that Fig. 6, karyoplasmic ratio are 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 branch
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 the fragment ion formed after falling 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 the fragment ion formed after losing carboxyl, loses carboxylic
The fragment ion formed after base and amino, the fragment ion after losing aminocarbonyloxymethyl group；The acquisition when HCD is set as 100
Fragment ion such as Fig. 8 C that one karyoplasmic ratio is 95.04950, is phenolic groups fragment ion.Proline is only set in HCD energy
It is 72.04499 that a karyoplasmic ratio is obtained when being 100, such as Fig. 9 of the fragment ion after losing carboxyl.Retinoic acid is set as 50 in HCD
With a fragment ion such as Figure 10 A and Figure 10 B are respectively obtained when 150, it is hexamethylene that karyoplasmic ratio, which is respectively 83.08620,240.23172,
Alkenyl group fragment ion and the fragment ion formed after fracture from the nearest double bond of c-terminus.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 break to form from the branch double bond from phenyl ring, 104.05782 be to fall shape after a carboxyl
At fragment ion.Abscisic acid obtains two fragment ions when energy is set as 50 and sees that figure Figure 12 A and Figure 12 B, karyoplasmic ratio are
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 that branched group is formed；HCD energy is set as obtaining karyoplasmic ratio when 150 being 61.02914 aceticoceptor fragment ions
Such as Figure 12 C.Cinnamic acid obtains three fragment ions in HCD energy setting 50 and sees that Figure 13 A, Figure 13 B and Figure 13 C, karyoplasmic ratio are
79.05478 be benzene radicals fragment ion, and 91.05460 be the fragment ion broken to form from branch double bond from phenyl ring,
104.05782 be to fall the fragment ion formed after a carboxyl.Chrysin only obtains a fragment when HCD energy is set as 50
Ion such as Figure 14, karyoplasmic ratio 147.04382, to fall the fragment ion that phenyl ring and two hydroxyls are formed.
The significant metabolin fragment ion information table of 2 acacia honey of table
The present embodiment mainly analyzes the metabolin difference of acacia honey and rice syrup, is different from other syrup
Significant metabolin can be used method identical with the present embodiment and be studied.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. a kind of application ultra performance liquid chromatography-quadrupole rod-orbit trap high resolution mass spectrum technology combination metabolism group method identifies
The analysis method of acacia honey and the adulterated acacia honey of syrup, characterized in that the following steps are included:
Before organic solvent progress sample is respectively adopted by true acacia honey sample and with the adulterated acacia honey sample of syrup to be detected
After reason, realize using ultra performance liquid chromatography-quadrupole rod-orbit trap high resolution mass spectrum method to the change in the sample after pre-treatment
The separation and measurement studied point, then to obtaining the UHPLC-MS initial data of true acacia honey sample Yu acacia honey sample to be measured
It is pre-processed, finally distinguishes true acacia honey using Multielement statistical analysis method principal component analysis (PCA) model and syrup is adulterated
Acacia honey；
Wherein, the organic solvent used in sample pre-treatments is the mixed solvent of the first alcohol and water containing formic acid, the volume of formic acid
Score is 1%, and first alcohol and water is to be mixed to form mixed solvent in equal volume；
Ultra performance liquid chromatography condition are as follows: A phase: acetonitrile, B phase: ammonium acetate aqueous solution, gradient elution process: 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, 30~37 DEG C of column temperature；
Mass Spectrometry Conditions are as follows: use ortho-spectrum, resolution ratio 70000,40 units of sheath gas, secondary air 10 units of speed, blowback
0 unit of gas velocity, spray voltage 3.5Kv, assist 350 DEG C of temperature degree, scanning range 70-1050m/ by 320 DEG C of capillary temperature
Z, surface sweeping mode are full scan.
2. analysis method as described in claim 1, it is characterized in that: the syrup includes rice syrup, corn syrup, beet sugar
Slurry, compound fructose syrup, online shopping honey is syrup dedicated or other syrup.
3. analysis method as described in claim 1, it is characterized in that: sample pretreatment process includes: by sample and organic solvent
It mixes to sample and is completely dissolved, ultrasound, centrifugation, excessively organic filter membrane, the sample for the detection that obtains being available on the machine；Wherein, ultrasonic time is
10~30min；Centrifugal condition: 800~1200rpm is centrifuged 4~8min；The aperture of organic filter membrane is 0.20~0.25 μm；Sample
Adding proportion with organic solvent is 1g:15~25mL.
4. analysis method as claimed in claim 3, it is characterized in that: sample pretreatment process includes: by sample and organic solvent
It mixes to sample and is completely dissolved, ultrasound, centrifugation, excessively organic filter membrane, the sample for the detection that obtains being available on the machine；Wherein, ultrasonic time is
25min；Centrifugal condition: 1000rpm is centrifuged 5min；The aperture of organic filter membrane is 0.22 μm.
5. analysis method as described in claim 1, it is characterized in that: ultra performance liquid chromatography condition are as follows: the ammonium acetate is water-soluble
The concentration of liquid be 10mM, flow velocity 0.3mL/min, 35 DEG C of column temperature, 3 microlitres of sample volume.
6. analysis method as described in claim 1, it is characterized in that: by obtained acacia honey sample and the adulterated acacia honey sample of syrup
The UHPLC-MS initial data of product is pre-processed using Compounds Discoverer software, the pretreatment refer to always from
The extraction of chromatographic peak in subflow figure initial data, peak alignment, the detection processing for removing noise, unknown material, obtain the guarantor at each peak
Stay time, peak height, peak area and mass-to-charge ratio data；Then to differentiation by way of Multielement statistical analysis method PCA shot chart
As a result it is shown.
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