CN107192770A - A kind of analysis method for differentiating chaste honey and the adulterated chaste honey of syrup - Google Patents
A kind of analysis method for differentiating chaste honey and the adulterated chaste honey of syrup Download PDFInfo
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- CN107192770A CN107192770A CN201710269827.6A CN201710269827A CN107192770A CN 107192770 A CN107192770 A CN 107192770A CN 201710269827 A CN201710269827 A CN 201710269827A CN 107192770 A CN107192770 A CN 107192770A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N2030/146—Preparation by elimination of some components using membranes
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Abstract
The present invention discloses a kind of analysis method for differentiating chaste honey and the adulterated chaste 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 chaste honey sample and organic solvent is respectively adopted with the adulterated chaste 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 chaste honey sample and chaste honey sample to be measured is pre-processed, true chaste honey and the adulterated chaste honey of syrup are distinguished in finally application Multielement statistical analysis method principal component analysis.The present invention is after a comprehensive acquisition is carried out to the metabolin information of chaste honey and the adulterated chaste honey of syrup, with reference to multi-variate statistical analysis, the adulterated chaste honey of chaste honey and syrup to be analyzed comprehensively, the detection to the adulterated chaste 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 chaste honey and the adulterated chaste 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, wherein, chaste honey is the abbreviation of twigs of the chaste tree anthophorids honey, is also chaste tree nectar.It is four big
One of name honey；It is also one of annual sweet product most surely received in the large nectar source of China.Preferable tonic nutritious food is not only, and
It can be cleared away heart-fire with relieving cough and moistening lung, invigorating stomach and relaxing bowels, heatstroke prevention, and have the effect of to mend body QI invigorating, promote longevity.
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 order to improve the enthusiasm for production of beekeeper, the interests of consumer be ensured, in order to support the justice of normal honey manufacturing enterprise
The order in honey market is competed, safeguarded, promotes the sound development of China's honey industry, therefore for finding real honey sample
Significant metabolin, so that attempting to set up a set of sensitive, efficient, accurate honey adulteration authentication method has very important meaning
Justice 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 chaste honey identified it is quick effectively and directly perceived bright
Aobvious method, does not also determine the twigs of the chaste tree 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 chaste honey and the adulterated chaste honey of syrup, the analysis method can
Effectively differentiate true chaste honey and the adulterated chaste 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 chaste honey and the adulterated chaste honey of syrup, comprises the following steps：
It is respectively adopted true chaste honey sample organic solvent carried out before and with the adulterated chaste 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 chaste honey sample and chaste honey sample to be measured
Pre-processed, finally distinguish true chaste honey using Multielement statistical analysis method principal component analysis (PCA) model and syrup is adulterated
Chaste honey.
The analysis method of the present invention is not particularly limited for the syrup used in adulterated chaste 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 chaste 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 chaste honey sample and syrup adulterated chaste 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 chaste honey sample and syrup adulterated chaste 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.Scan 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 chaste 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 chaste honey sample and the twigs of the chaste tree to be measured
The UHPLC-MS initial data of sweet sample is pre-processed using Compounds Discoverer softwares, and the pretreatment refers to pair
The processing such as the extraction of the chromatographic peak in total ion current figure initial data, peak alignment, the detection for removing noise unknown material, obtains each peak
Retention time, peak height, peak area and mass-to-charge ratio data；Then the form pair of Multielement statistical analysis method PCA shot charts is passed through
Result is distinguished to be shown.
Wherein, Compounds Discoverer softwares are a kind of processing that Thermo Fisher Scientific Inc. develops
The software of LC-MS initial data.
The type of the adulterated syrup of chaste 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 chaste 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 chaste 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 chaste honey provides the foundation to set up later, the sample adulterated available for quick discriminating chaste 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 chaste 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 chaste 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 chaste 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 characteristics of for chaste honey component, the present invention is a kind of simple side using load diagram screening otherness metabolin
Method, when obtaining load diagram, the present invention is potential significant to screen by setting Ratio ＞ 20 or ＜ 0.5, P value ＜ 0.01
Metabolin.Wherein, the Ratio is 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 chaste 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 chaste 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 chaste 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 chaste honey sample.
The type of the adulterated syrup of chaste 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 chaste honey for being different from rice syrup obtained based on above screening technique, the significant metabolism
Thing have following compound group into：Phenylalanine, leucine, pantothenic acid, p-Coumaric Acid, cinnamic acid, Chrysin, tyrosine.The mark
Property metabolin can be used to differentiate the adulterated chaste honey of rice syrup.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) it can be realized to chaste 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 chaste 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 chaste honey and the adulterated chaste honey of syrup
Metabolin information carry out one comprehensively obtain after, with reference to multi-variate statistical analysis, the adulterated chaste honey of chaste honey and syrup is entered
Row analysis comprehensively, sets up model, completes the detection to the adulterated chaste honey of syrup.
Chaste honey and the adulterated chaste 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) chaste 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 chaste honey sample as much as possible and the adulterated chaste 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.
(3) after detection model is set up, batch detection is carried out available for the adulterated chaste honey sample of syrup.
Initial data after upper machine testing is mixed chaste honey sample and syrup by Compounds Discoverer softwares
False chaste 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 chaste honey sample can be operated with the differentiation of chaste honey by identical flow.By experimental verification, inappropriate pre-treatment side
Method can not extract the endogenous metabolites of true honey to greatest extent, it will cause the testing result for being not easy to distinguish so that
Testing result is inaccurate.
(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 chaste 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：Phenylalanine, leucine, pantothenic acid, p-Coumaric Acid, cinnamic acid, Chrysin, junket
Propylhomoserin.The significant metabolin for being different from the chaste honey of other syrup can be using being studied with identical screening technique of the present invention
Obtain.The significant metabolin of chaste honey for being different from different syrup according to these obtained, can set up a set of discriminating not of the same race
The universal model of the adulterated chaste honey of syrup of class and different adulterated contents, by the universal model, can quickly know adulterated
Syrup species and adulterated content；And it is quick, efficient, sensitive after being established as of logical model, differentiated exactly
Adulterated chaste honey has very important significance and is worth.
Brief description of the drawings
Fig. 1 is chaste 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 chaste honey.
Fig. 2 is chaste honey sample and F55 rice syrup sample total ion current figures under cation mode.
Fig. 3 is chaste honey sample group and syrup sample group PCA shot charts.
Fig. 4 is chaste honey sample group and syrup sample group load diagram.
Fig. 5 is chaste honey sample group and syrup sample group load diagram after screening.
Fig. 6 A, Fig. 6 B and Fig. 6 C are phenylalanine fragment ion mass spectrograms.
Fig. 7 A and Fig. 7 B are leucine fragment ion mass spectrograms.
Fig. 8 A and Fig. 8 B are pantothenic acid fragment ion mass spectrograms.
Fig. 9 A, Fig. 9 B, Fig. 9 C and Fig. 9 D are p-Coumaric Acid fragment ion mass spectrograms.
Figure 10 A, Figure 10 B and Figure 10 C are cinnamic acid fragment ion mass spectrograms.
Figure 11 is Chrysin fragment ion mass spectrogram.
Figure 12 A and Figure 12 B are tyrosine fragment ion mass spectrograms.
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)；
The Thermo Scientific Q Exactive Thermo ScientificTM 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 chaste 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..
In order that technical scheme can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes technical scheme in detail.
Embodiment 1
(1) Method And Principle of the invention：
After being handled with identical pre-treating method true chaste honey sample and the adulterated chaste 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 chaste honey and the adulterated chaste honey of syrup are distinguished using multi-variate statistical analysis PCA analyses.
(2) sample pre-treatments
1g chaste 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 chaste 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 chaste 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 100mL conical flasks in chaste 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 chaste honey sample and do artificial adulterated chaste 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：
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, column temperature 35 is Celsius
Degree, 3 microlitres of sample size.
Mass Spectrometry Conditions：
Positive spectral condition：Resolution ratio, 70000；Sheath gas, 40 units；Secondary air speed, 10 units；Blowback air-flow
Speed, 0 unit；Spray voltage, 3.5kV；Capillary temperature, 320 DEG C；Aid in temperature degree, 350 DEG C.Scanning range, m/z：70-
1050.Scan pattern：Full Ms.
(4) data processing and multi-variate statistical analysis：
The UHPLC-MS initial data of the adulterated chaste honey sample of chaste honey sample and syrup to obtaining uses Compounds
Discoverer softwares are pre-processed, and the pretreatment refers to the extraction to the chromatographic peak in total ion current figure initial data, peak
Alignment, removes noise, and the detection of unknown material etc. is handled, then by the form of Multielement statistical analysis method PCA shot charts to distinguishing
As a result it 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 12 to chaste 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 chaste honey with adulterated 1%, 5%, 10% rice syrup, 1% corn syrup, 1% sugar beet molasses, 1% net purchase honey
The differentiation of syrup dedicated adulterated chaste honey.
As a result as shown in figure 1, in PCA shot charts, what 1r, 5r, 10r, 1c, 1b, 1h were represented respectively is adulterated 1%, 5%,
The adulterated chaste honey of 10% rice syrup, the adulterated chaste honey of adulterated 1% corn syrup, the adulterated chaste tree of adulterated 1% sugar beet molasses
Bar honey, the syrup dedicated adulterated twigs of the chaste tree honey of adulterated 1% honey.From PCA shot charts can be seen that each sample aggregation, from
The degree of dissipating, each point represents a sample, wherein, chaste honey includes 12 samples, and (each six repetitions of sample only have chosen
The sample is wherein once illustrated in Fig. 1), the right side of the reference axis of the whole Relatively centralizeds of 12 sample spots in Fig. 1
Upper part, this illustrate the composition and concentration of the metabolin that this 12 true chaste honey samples contain very close to, group difference is smaller, and
The sample spot distance of adulterated chaste honey is very big with other syrup, this illustrate the metabolin that true chaste honey sample contains composition and
Concentration distinguishes larger with the adulterated honey of syrup, and true chaste honey and the adulterated honey of syrup can be effectively distinguished using this method, this
As a result it is very apparent；1r, 5r, 10r, 1c, 1b, 1h include 3 samples, and (each six repetitions of sample, only have chosen this
Sample is wherein once illustrated in Fig. 1), three points are shown in Fig. 1, it can be seen that some syrup
Adulterated chaste honey can be distinguished clearly, such as 1r, 5r, 1c and 10r, two groups of 1h, 1b sample spot it is big away from degree,
Can effectively it distinguish.And the adulterated chaste honey of some syrup can not be distinguished effectively, as shown in figure 1,1r, 5r, 1c sample spot are big
Cause concentrates on upper left；10r, 1h, 1b sample spot all concentrate on lower right-most portion, and sample spot is apart from close, it is impossible to have
Effect difference, and 10r and 1h, 1b differ greatly, at least from the point of view of adulterated content, difference still than larger, but 10r and
1h, 1b but can not be distinguished effectively, from this point as can be seen that also being differed using the metabolism group method even if two kinds of different samples
Surely can effectively it distinguish, that is, this is for two kinds of samples, and those skilled in the art also can not be effectively expected using metabolism
Can the method that group is learned effectively be distinguished.
To sum up, can be obtained by Fig. 1, this method can by true chaste honey with adulterated 1%, 5%, the adulterated chaste tree of 10% rice syrup
Bar honey, the adulterated chaste honey of adulterated 1% corn syrup, the adulterated chaste honey of adulterated 1% sugar beet molasses, adulterated 1% honey is special
The adulterated chaste 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 chaste honey, comprises the following steps：
(1) sample pre-treatments
By 1g chaste 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 chaste 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
Retouch pattern：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
Retouch pattern：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.Scan pattern：Full Ms.
(3) data processing and multi-variate statistical analysis
Use Compounds Discoverer soft in obtained chaste 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 12 to chaste 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 12, and each sample is repeated 6 times experiment)：
After Mass Spectrometer Method, the total ion current figure of chaste honey sample and F55 rice syrup samples is obtained, as shown in Fig. 2
Also have significantly with F55 rice syrups in retention time 2-7 minutes and 9-13 minutes chaste honey samples under cation mode
Difference, chaste honey total ion current figure appearance sum is more than F55 rice syrup total ion current figures appearance sum, illustrates chaste honey and sugar
Slurry, which is compared, may contain more metabolins.The searching of specific difference material also needs to be further analyzed.In Compounds
PCA analyses are carried out in Discoverer softwares, Fig. 3 is the PCA shot charts of chaste honey sample group and syrup sample group, in shot chart
In, chaste honey sample group (point of right half part) just can be real on first principal component with syrup sample group (point of left-half)
Now separate well, illustrate that chaste honey sample has very big difference with syrup sample metabolin, this also tests next for us
The significant metabolin work that step finds chaste honey sample provides experimental data foundation, also demonstrates the test method of the present invention
Feasibility for differentiating true and false chaste honey, because the conventional method of honey adulteration is faked by mixing syrup at present, this
It is special that the syrup sample group of research institute includes rice syrup, corn syrup, sugar beet molasses, compound fructose syrup, net purchase honey
Syrup, covers C3, C4 syrup all kinds used adulterated at present.The PCA shot charts that the data analysis of this research method is obtained can
So that chaste honey sample group and syrup sample group to be kept completely separate, it also illustrate that test method can be by chaste honey and the false twigs of the chaste tree
There is very big difference in the feasibility that honey makes a distinction, chaste honey sample group, Fig. 4 is chaste honey sample with syrup sample group metabolin
Each point in the load diagram of group and syrup sample group, figure, which is represented, passes through Compounds Discoverer software analysis from the twigs of the chaste tree
Honey and a kind of metabolin (metabolins of all samples) extracted in syrup sample group, from 26 it can be seen that chaste honey sample group
The metabolin of extraction will be more than syrup sample group, be screened by the metabolin on the load diagram to Fig. 4, to find chaste honey
The significant metabolin of sample group.The material that Ratio values ＞ 2 or ＜ 0.5, P value ＜ 0.01 are screened as a rule is set just
Can be as difference metabolin, the Ratio values of each of which metabolin are average peak area of the metabolin in two groups
Ratio, but it is huge in view of the metabolin number extracted, and the purpose of research is to find the maximum material of difference, in difference generation
In the searching step for thanking to thing, Ratio values are set to ＞ 20 or ＜ 0.5, P value ＜ 0.01 significant to find chaste honey sample group
Metabolin.Such as Fig. 5 is the load diagram after screening, and load diagram needs to combine PCA shot charts to be analyzed, PCA shot charts
The corresponding position of upper each group, corresponding is exactly that the group of the last point of load diagram belongs to, the chaste honey sample in Fig. 3 PCA shot charts
This group is located at the right half part of first principal component, then in the load diagram after the screening of corresponding diagram 5, it is possible to from right half filtered out
It is partial select in find the otherness metabolin of chaste honey sample group.False positive ion is carried out first to the material filtered out
Exclude, false positive ion obtains the mark under cation mode to return to the material extracted in total ion current figure less than chromatographic peak
Property metabolin information, including molecular formula, molecular ion exact mass number and retention time etc., the maximum deviation of exact mass number
Within 5ppm, retention time deviation is in 0.2 minute, the same molecular formula according to acquisition, to mark under ion mode
The molecular ion of will thing is extracted, and mark is entered according to molecular ion peak unit exact mass number under zwitterion pattern
Row speculate, the possible significant metabolin of chaste honey sample finally obtained have phenylalanine, leucine, pantothenic acid, p-Coumaric Acid,
Cinnamic acid, Chrysin, tyrosine.If table 1 is the significant metabolin complete information of chaste honey sample.
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 chaste honey sample of table 1
Subscript 1 is [M+NH in table4]+, subscript 2 is [M+H-H2O]+
The identification of the significant metabolin of chaste honey
According to the first mass spectrometric information that the pattern of sweeping is provided entirely, the possible significant metabolin of chaste honey sample is found out, in order to
This seven kinds of materials are further determined that, set HCD energetic encounters three, pond energy level to find mark by second order mses
The fragment ion of property metabolin, so as to realize the qualitative analysis of the significant metabolin of chaste honey sample to finding.If table 1 is chaste tree
The fragment ion information table of the significant metabolin of bar honey sample, under three, the HCD energetic encounters pond energy of setting, every kind of mark
Property metabolin all have found at least one fragment ion, then two under every kind of significant metabolin negative ions pattern of chaste honey
Individual molecular ion and a fragment ion are scored at 5 points, met European Union an analyte it is qualitative at least to reach 4 points with
On requirement, a molecular ion obtains 1 point, and one fragment ion of high resolution mass spectrum obtains 3 points.The mark found can be carried out
It is qualitative, the final significant metabolin for determining chaste honey sample is phenylalanine, leucine, pantothenic acid, p-Coumaric Acid, cinnamic acid,
Chrysin, tyrosine.Fig. 6 A~Figure 12 B are three different energy of the significant metabolin HCD energetic encounters pond of chaste honey in setting
The lower fragment ion mass spectrogram obtained of amount, wherein phenylalanine have three fragment ions of acquisition altogether and see Fig. 6 A, Fig. 6 B and Fig. 6 C,
HCD energy is set as obtaining the fragment ion that karyoplasmic ratio is 120.08075 when 50, is that phenylalanine molecular ion loses a carboxylic
The fragment ion that base is obtained；The fragment ion that karyoplasmic ratio is 103.05440 is obtained when HCD energy is set as 100, is phenylpropyl alcohol ammonia
Acid molecule ion loses the fragment ion obtained after a carboxyl and amino；Karyoplasmic ratio is obtained when HCD energy is set as 150 is
The fragment ion of benzene radicals in 79.05478 fragment ion, phenylalanine.Leucine obtains two fragment ions as schemed
7A and Fig. 7 B, the fragment ion that karyoplasmic ratio is 86.09689 is obtained when HCD energy is set as 50, is that leucine molecular ion loses
Remove the fragment ion obtained after a carboxyl；HCD energy sets 100 acquisition karyoplasmic ratios as 69.07057 fragment ion, is bright
Propylhomoserin loses the fragment ion obtained after a carboxyl and amino.Pantothenic acid obtains one when HCD energy is set as 50 and loses one
Fragment ion after individual hydroxyl and alanine group, karyoplasmic ratio is 116.03441, Fig. 8 A and Fig. 8 B.P-Coumaric Acid is only in HCD
Energy just obtains 4 fragment ion such as Fig. 9 A, Fig. 9 B, Fig. 9 C and Fig. 9 D when being set as 50, karyoplasmic ratio is respectively 120.05233,
109.06503rd, 95.04947,73.02895, it is to lose the fragment ion after a carboxyl, molecular ion at double bond to break respectively
Fragment ion, phenolic groups fragment ion, acrylic acid groups fragment ion after splitting.Figure 10 A, Figure 10 B cinnamic acids are set in HCD
Two fragment ions are obtained when 50, are that fragment ion karyoplasmic ratio is 91.05464, matter after being broken at acrylic acid side chain double bond respectively
Fragment ion of the core than 104.05780 is to fall the fragment ion after a carboxyl；Figure 10 C are cinnamic acid when HCD is set as 150
The fragment ion of acquisition, exact mass number is 79.05458, is the fragment ion of benzene radicals.See Figure 11 Chrysins in HCD energy
Amount is set as obtaining the fragment ion that karyoplasmic ratio is 147.04364 when 50, is that Chrysin molecular ion loses a phenyl ring and two
Fragment ion after individual hydroxyl.Tyrosine obtains two and a fragment ion when HCD energy is set as 50 and 150 such as respectively
Figure 12 A and Figure 12 B, the fragment ion that karyoplasmic ratio is 136.07545 is to fall the fragment ion after a carboxyl, and 119.04916 are
Fall the fragment ion after a carboxyl and amino, 95.04958 be phenol group fragment ion.
The significant metabolin fragment ion information table of the chaste honey of table 2
The present embodiment, which is mainly, to be analyzed the metabolin difference of chaste 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 chaste honey and the adulterated chaste honey of syrup, it is characterized in that, comprise the following steps：
By true chaste honey sample and organic solvent is respectively adopted with the adulterated chaste 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 chaste honey sample and chaste honey sample to be measured
Pretreatment, finally using Multielement statistical analysis method principal component analysis（PCA）Model distinguishes true chaste honey and the adulterated twigs of the chaste tree 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 0.22 μ
m；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 acetic acid
The concentration of aqueous ammonium is 10 mM, and 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；Scan pattern：Full scan.
6. analysis method as claimed in claim 1, it is characterized in that：By obtained chaste honey sample and the adulterated chaste 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 chaste honey for being different from syrup, it is characterized in that, comprise the following steps：
Chaste honey sample and syrup are respectively adopted organic solvent to carry out after pre-treatment, using any described super in described 1 ~ 6
High performance liquid chromatography-quadrupole rod-track trap high resolution mass spectrum method realizes point to the chemical composition in the sample after pre-treatment
From with measure, then the UHPLC-MS initial data for obtaining chaste honey sample and syrup is pre-processed, finally using polynary
Statistical analysis technique principal component model carry out difference screening compound, it is determined that with appraisal mark metabolin；
It is preferred that, when obtaining load diagram, by setting Ratio ＞ 20 or ＜ 0.5, P value ＜ 0.01, to screen potential mark
Property 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 chaste 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 chaste honey sample.
9. the significant metabolin of the chaste 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：Phenylalanine, leucine, pantothenic acid, p-Coumaric Acid, Chinese cassia tree
Acid, Chrysin, tyrosine.
10. the significant metabolin of the chaste honey for being different from rice syrup described in claim 9 is differentiating the adulterated chaste tree of rice syrup
Application in bar honey.
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| CN112285180B (en) * | 2020-10-26 | 2022-11-04 | 杭州电子科技大学 | Electrochemical analysis method for identifying raw sugar |
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