CN107941939A - A kind of method that organic rice and non-organic rice are distinguished using metabonomic technology - Google Patents
A kind of method that organic rice and non-organic rice are distinguished using metabonomic technology Download PDFInfo
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
A kind of method that organic rice and non-organic rice are distinguished using metabonomic technology, belongs to grain quality detection technique field.The method is as follows：After organic rice sample and non-organic rice sample are carried out pre-treatment respectively, separation and the measure to the chemical composition in the sample after pre-treatment are realized using ultra performance liquid chromatography series connection level Four bar flight time high resolution mass spectrum method, then the initial data of two kinds of samples to obtaining pre-processes, finally organic rice and non-organic rice are distinguished using the orthogonal partial least squares discriminant analysis model of Multielement statistical analysis method, and the factor having a great influence to discrimination is obtained using S curve figure, by increasing income, these materials are identified in online database massbank.Analysis method using the present invention can effectively distinguish organic rice and non-organic rice, can intuitively differentiate its classification situation, without carrying out associated verification and analysis, can with it is concluded that, testing result is accurately and reliably.
Description
Technical field
The invention belongs to grain quality detection technique field, and in particular to one kind is distinguished organic big using metabonomic technology
The method of rice and non-organic rice.
Background technology
Rice is one of world's Three major grain crops, and the population in the whole world about 1/3 is using rice as staple food.The production of rice
Amount and consumption figure are always most in cereal crops for a long time, and substantial majority Rice Cropping person and consumer exist
Asia.The living standard and health of the raising of rice quality and these regional people are all inseparable.Rice is that China is exhausted
The main staple food of most people, while be also the main exported product in China.With the continuous improvement of people's living standards, to big
The requirement of meter Pin Zhi is also higher and higher, and organic rice has gradually come into the dining table of consumer.
Organic rice refers to come from organic agriculture production system, according to international organic agriculture production requirement and corresponding mark
Quasi- production and processing, and pass through the rice product of independent Organic Food Attestation agency qualification.Due to organic paddy rice plantation and
The whole process of growth does not allow using pesticide, chemical fertilizer, growth regulator of any chemical synthesis etc., it is easier to is subject to disease pest
The influence of the factors such as crop smothering, therefore organic rice product has the characteristics that low output, polished rice rate are low, of high cost.China has at present
The amount of consumption of machine rice is increased with annual 30%~50% speed, it was predicted that in coming 10 years, China's organic rice increases every year
Long rate will be up to 20%~30%.Also with problems while organic rice industry is fast-developing, many manufacturers are " with secondary
Substitute the bad for the good, mix the spurious with the genuine " behavior, greatly upset the normal development in organic rice product market.Therefore, a kind of differentiate is established
The method of organic rice has very strong Practical significance.
Metabolism group is the important branch in systems biology, is mainly to endogenous and exogeneous small molecule metabolite
Carry out a science that is qualitative and quantitatively detecting.Metabolism group more can comprehensively study plant complexity metabolic process and its production
Thing, therefore received significant attention in recent years in plant research field.Plant Metabolome develops into analysis Secondary Metabolism of Plant
Affiliation between network structure, rate-limiting step, parsing cellular activity process and looking for plant etc. provides possibility.
According to the difference of research purpose, metabolism group can be divided into non-targeted metabolism group and targeting metabolism group.It is non-
It is a kind of metabonomic analysis of no deviation to target metabolism group, and it is comprehensive to carry out system mainly for organism endogenous metabolism thing
Analysis；It is a kind of analysis of orientation to target metabolism group, the analysis carried out mainly for specific a certain metabolite.Mesh
Before, non-targeted metabonomic analysis makes it in active substance of plant difference due to its Difference to secondary metabolite
It is widely used in terms of the otherness discriminating of analysis, metabolic mechanism and associated metabolic network, especially plant variety and the place of production.
The existing detection project to organic rice is only confined in several common indexs, can not be to organic rice quality
Make comprehensive and effectively judge, and artificial determination methods are had a great influence by various factors, and it is too weak to convince power.
The content of the invention
It is comprehensive and effective the purpose of the present invention is to solve that can not be made using existing detection means to rice quality
A kind of the problem of judgement, there is provided method that organic rice and non-organic rice are distinguished using metabonomic technology.
To achieve the above object, the technical solution that the present invention takes is as follows：
A kind of method that organic rice and non-organic rice are distinguished using metabonomic technology, the method are as follows：
After organic rice sample and non-organic rice sample are respectively adopted organic solvent progress pre-treatment, using ultra high efficiency
Liquid chromatography tandem level Four bar-flight time high resolution mass spectrum method is realized to the organic rice sample after pre-treatment and non-organic
The separation of chemical composition in rice sample and measure, then to the super of obtained organic rice sample and non-organic rice sample
High performance liquid chromatography series connection level Four bar-flight time high resolution mass spectrum initial data is pre-processed, finally using multivariate statistics
The orthogonal offset minimum binary of analysis method-discriminant analysis model distinguishes organic rice and non-organic rice, and is obtained using S curve figure
The factor having a great influence to discrimination, by increasing income, these materials are identified in online database massbank.
It is of the invention to be relative to the beneficial effect of the prior art：
(1) metabonomic technology combination ultra performance liquid chromatography series connection level Four bar-flight time high resolution mass spectrum is applied
Technology analyzes secondary metabolite in rice sample, and it is high to convince power.Non-targeted metabonomic technology can be to the whole of metabolin
Body situation is studied, more can reflected sample overall condition, meanwhile, can applied statistics gain knowledge find otherness metabolin,
Technical support is provided for further research.
(2) analysis method using the present invention can effectively distinguish organic rice and non-organic rice, as a result with OPLS-DA
The form displaying of shot chart, and otherness material (potential source biomolecule marker) is identified using online database of increasing income.Energy
It is enough intuitively to differentiate its classification situation, without carrying out associated verification and analysis, can with it is concluded that, testing result is accurately and reliably.
(3) rice sample pre-treating method simple and fast, method require testing staff's operating technology after establishing relatively low.
Whole pretreatment process, in order to ensure the metabolin information of acquisition rice sample as much as possible, extracts reagent is methanol
Test sample is dissolved with the mixed solvent of water, machine testing, pre-treatment step can be gone up by centrifuging organic filter membrane after ultrasound
Simply, it is easily operated.By experimental verification, unsuitable pre-treating method cannot extract the endogenous of rice sample to greatest extent
Property metabolite, it will cause to be not easy the testing result distinguished so that testing result is inaccurate.
(4) streamline operation：Traditional detection needs to configure standard sample, draws standard curve, while need to more
Kind chemical substance is measured, and process is cumbersome, time-consuming and laborious.It is measured using non-targeted metabolism group method, pre-treatment
Process is simple, easy to operate, and it is also more convenient to operate the computer, and can carry out batch processing, time saving and energy saving, with a high credibility.
(5) have for ultra performance liquid chromatography series connection level Four bar-flight time high resolution mass spectrum application metabolism group differentiation
The analysis method of machine rice and non-organic rice, for the present invention according to the characteristics of rice sample, optimal screening, which obtains one group, makes score
Analysis sample obtains ultra performance liquid chromatography condition and level Four bar-flight time high-resolution of optimal separation effect and detection result
Mass spectrographic process conditions, by verification experimental verification, by the process conditions, can on OPLS-DA shot charts by organic rice with
Non-organic rice is effectively distinguished.
(6) on the basis of OPLS-DA models, the factor having a great influence to separation is filtered out using S curve figure, and utilize
These materials are identified in database.The material identified can be as the discriminatory analysis that target substance is organic rice from now on
Lay the first stone.
(7) present invention uses level Four bar-flight time high resolution mass spectrum, resolution ratio is higher, with other detection methods
Compare, more accurate and more materials can be obtained, this has significant advantage in data analysis.
Brief description of the drawings
Fig. 1 is organic rice and non-organic rice sample OPLS-DA scatterplot shot charts；
Fig. 2 is the S curve figure of OPLS-DA models.
Embodiment
Technical scheme is further described with reference to the accompanying drawings and examples, but is not limited thereto,
It is every to technical solution of the present invention technical scheme is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention,
It should all cover in protection scope of the present invention.
Embodiment one：What present embodiment was recorded is a kind of using metabonomic technology differentiation organic rice and non-
The method of organic rice, the method are as follows：
After organic rice sample and non-organic rice sample are respectively adopted organic solvent progress pre-treatment, using ultra high efficiency
Liquid chromatography tandem level Four bar-flight time high resolution mass spectrum method is realized to the organic rice sample after pre-treatment and non-organic
The separation of chemical composition in rice sample and measure, then to the super of obtained organic rice sample and non-organic rice sample
High performance liquid chromatography series connection level Four bar-flight time high resolution mass spectrum (UHPLC-Q-TOF MS) initial data is pre-processed,
Finally organic rice and non-is distinguished using orthogonal offset minimum binary-discriminant analysis (OPLS-DA) model of Multielement statistical analysis method
Organic rice, and the factor (potential source biomolecule marker) having a great influence to discrimination is obtained using S curve figure (S-plot), by opening
Source online database massbank (http://www.massbank.jp/) these materials are identified.
Embodiment two：One kind described in embodiment one using metabonomic technology distinguish organic rice and
The method of non-organic rice, the organic rice sample and non-organic rice sample kind are in the rice fragrance of a flower, imperial round-grained rice, loose round-grained rice
One or more.
Embodiment three：One kind described in embodiment one using metabonomic technology distinguish organic rice and
The method of non-organic rice, the pre-treatment are specially：Organic rice sample and non-organic rice sample are crushed, cross 1mm
Aperture sieve, then the organic rice sample after sieving and non-organic rice sample are mixed with organic solvent, ultrasound, centrifugation, crossing has
Machine filter film, that is, complete pre-treatment, the sample for the detection that obtains being available on the machine.
Embodiment four：One kind described in embodiment three using metabonomic technology distinguish organic rice and
The method of non-organic rice, the organic rice sample and the adding proportion of non-organic rice sample and organic solvent are
1g：(3~10) mL, preferably 1g：5mL, it is ensured that metabolin extraction effect is preferable in sample；The organic solvent is methanol
Aqueous solution, volume fraction are 60~85%, are most preferably 70%.
Embodiment five：One kind described in embodiment three using metabonomic technology distinguish organic rice and
The method of non-organic rice, ultrasonic time are 10~40min, are preferably 30min；The actual conditions of centrifugation is：Under the conditions of 4 DEG C
8000~12000rpm centrifuges 15~30min, preferably 10000rpm centrifugations 20min.
Embodiment six：One kind described in embodiment three using metabonomic technology distinguish organic rice and
The method of non-organic rice, organic filter sizes are 0.20~0.25 μm, are preferably 0.22 μm.
Embodiment seven：One kind described in embodiment one using metabonomic technology distinguish organic rice and
The method of non-organic rice, the ultra performance liquid chromatography condition are：Using octadecyl silane column (C18 columns)； A
Phase：Aqueous formic acid, B phases：Formic acid acetonitrile solution, gradient elution flow：0~1.5min, 15%B；1.5~5.0min, 15-
55%B；5.0~17.0min, 55~70%B；17.0~20.0min, 70-90%B；20.0~21.0min, 90-15%B.
Embodiment eight：One kind described in embodiment seven using metabonomic technology distinguish organic rice and
The method of non-organic rice, the volume fraction of formic acid is 0.1% in the aqueous formic acid；In the formic acid acetonitrile solution
The volume fraction of formic acid is 0.1%；Flow velocity 0.3mL/min, 36 DEG C of column temperature；5 μ L of sample size.
Embodiment nine：One kind described in embodiment one using metabonomic technology distinguish organic rice and
The method of non-organic rice, level Four bar-flight time high resolution mass spectrum select Aglient 6540UHD accurate-
Mass QTOF spectrometer, Mass Spectrometry Conditions are：Double ESI sources, positive ion mode, 325 DEG C of dry gas temperature, flow velocity
9L/min；Nebulizer pressure 45psi；Capillary voltage 4000V；Sampling spiroid voltage, 140V；Extract taper voltage, 65V；Scanning
Scope, m/z：50~2000；Scan pattern：Full Scan (full scan)；It is m/z with reference to ion：301.998139 and
1033.988109。
Embodiment ten：One kind described in embodiment one using metabonomic technology distinguish organic rice and
The method of non-organic rice, the UHPLC-Q-TOF MS initial data to obtained organic rice sample and non-organic rice sample
Carry out turning lattice using MS Convert softwares, then pre-processed with XCMS softwares, which refers to total ion chromatogram
The extraction of chromatographic peak in initial data, peak alignment, go noise treatment, obtain the retention time at each peak, peak height, peak area and
Mass-to-charge ratio data, then distinguishes result by the form of Multivariate OPLS-DA shot charts and is shown, and bent by S
Line chart filters out the material (potential marker) having a great influence to identification result, using online database massbank to potential
Marker is differentiated.
Term is explained：Multielement statistical analysis method is built upon a kind of processing multivariate statistics in multivariate statistics distributed basis
The general name of data method, is the important branch with abundant theoretical result and numerous application processes in statistics.It is common more
First statistical analysis technique mainly includes：It is multiple regression analysis, cluster analysis, discriminant analysis, principal component analysis, factorial analysis, right
It should analyze, canonical correlation analysis etc..The present invention mainly uses orthogonal offset minimum binary-techniques of discriminant analysis (Orthogonal
Partial least squares-discriminant analysis, OPLS-DA).
Instrument and equipment：
Aglient 1290UHPLC system, German agilent company；
Acquity BEH C18column (1.7 μm of 2.1id × 150mm, particle size), U.S. Waters are public
Department；
Aglient 6540UHD accurate-mass QTOF spectrometer, German Aglient companies；
3K15 laboratories high speed desktop refrigerated centrifuge, German Sigma companies；
Milli-Q pure water meters, Millipore companies of the U.S.；
MX-S type vortex instruments, Dragon Laboratory Instruments (Beijing) Co., Ltd.；
KQ-700DE type numerical control supersonic instruments, Kunshan Ultrasonic Instruments Co., Ltd.；
Material and reagent：
Organic rice (through Zhong Lv China Organic Food Attestation center certification), is obtained by each rice production company；
Non-organic rice, is obtained by various regions peasant household；
Ultra-pure water (18.2M Ω cm), Milli-Q pure water meters obtain；
Acetonitrile (chromatographically pure), German Merck companies；
Methanol (chromatographically pure), Sigma Co., USA；
Anhydrous formic acid (chromatographically pure), German Merck companies.
The principle of analysis method of the present invention is：Different planting patterns can cause the difference of rice endogenous metabolism material,
These differences directly affects the quality of rice product, can separate the chemical substance in rice using UHPLC, uses mass spectrum
Technology is detected, and can obtain the liquid phase and mass spectrometric data of rice, then using metabonomic analysis technology, to the number of gained
Analyzed according to using OPLS-DA technologies, so as to intuitively distinguish organic and non-organic rice sample.
High performance liquid chromatography is a kind of using little particle filler chromatographic column (particle diameter is less than 2 μm) and extra high voltage system (pressure
More than 105kPa) emerging liquid chromatography technology, the separating degree and detection sensitivity of chromatographic peak can be significantly improved, while contract significantly
Short analytical cycle, separation and high pass quantity research suitable for micro complex mixture.Meanwhile Aglient 6540UHD
Accurate-mass QTOF spectrometer have the characteristics that superelevation separating degree, ultraspeed, hypersensitivity, can be with
Chemical substance in rice is more accurately detected, makes result more reliable, more there is conviction power.
Specifically include following steps：
After organic rice sample and non-organic rice sample are respectively adopted organic solvent progress pre-treatment, using ultra high efficiency
Liquid chromatography tandem level Four bar-flight time high resolution mass spectrum method realizes the separation to the chemical composition of the sample after pre-treatment
With measure, then the UHPLC-Q-TOF MS initial data of obtained rice sample is carried out turning lattice and pretreatment, finally should
Organic rice sample is distinguished with orthogonal offset minimum binary-discriminant analysis (OPLS-DA) model of Multielement statistical analysis method and non-is had
Machine rice sample.
For the rice sample of different sources, planting environment is also subject to the shadows such as height above sea level, longitude and latitude, illumination, temperature, humidity
Ring, so even if being all organic rice, the metabolome in rice sample between different sources is into will not complete phase with content
Together, therefore between them certain difference can be presented, but pass through the experimental verification of the present invention, this has no effect on final detection knot
Fruit, can effectively be distinguished.In certain embodiments of the present invention, the rice sample selection rice fragrance of a flower, loose round-grained rice, imperial round-grained rice
In one or more.
In whole pretreatment process, in order to ensure the metabolin information of acquisition sample as much as possible, extracts reagent choosing
It is that the mixed solvent of first alcohol and water dissolves test sample, machine testing can be gone up by centrifuging organic filter membrane after ultrasound,
Pre-treatment step is simple, easily operated.In a preferred embodiment of the invention, in the methanol aqueous solution methanol volume fraction
For 70%.The present invention has also carried out the research of other extracts reagents, such as ethanol water, acetone soln and aqueous isopropanol,
But obtained effect is unsatisfactory, the metabolin in rice sample can not be comprehensively extracted.
In some preferred embodiments of the present invention, sample pretreatment process includes：Rice sample is crushed, sieve (1mm
Aperture), mixed with organic solvent, ultrasound, centrifugation, excessively organic filter membrane, the sample for the detection that obtains being available on the machine.Wherein, ultrasonic time
For 10~40min, preferably 30min；Centrifugal condition：8000~12000rpm centrifuges 15~30min under the conditions of 4 DEG C, preferably
10000rpm centrifuges 20min；Organic filter sizes are 0.20~0.25 μm, are preferably 0.22 μm；To ensure metabolin in sample
Extraction effect is preferable, and the adding proportion of sample and organic solvent is 1g：(3~10) mL, preferably 1g：5mL.Wherein, the rice
The adding proportion of sample and organic solvent is more crucial, and test sample fully can be dissolved, so as to obtain more sample generations
Thank to thing information so that final detection result is more accurate.
The separation of chromatography and the collection of mass spectrometric data are carried out at the same time, in order to make each component be separated and reflected
It is fixed, it is necessary to select suitable chromatography and mass spectral analysis condition.
Present invention the characteristics of being directed to rice sample component, investigated mobile phase in ultra performance liquid chromatography, gradient elution
Influence of the conditions such as flow, column temperature and sample size to separative efficiency and analyze speed, final optimization pass, which screens to obtain one group, causes sample
Product obtain the ultra performance liquid chromatography condition of optimal separation effect.
In the preferred embodiment of the invention, ultra performance liquid chromatography condition is：Using octadecyl silane column
(C18 columns)；A phases：Aqueous formic acid, B phases：Formic acid acetonitrile, gradient elution flow：0-1.5min, 15%B；1.5-5.0min
15-55%B；5.0-17.0min, 55-70%B；17.0-20.0min 70-90%B；20.0-21.0min 90-15%B.First
The volume fraction of formic acid is 0.1% in aqueous acid, and the volume fraction of formic acid is 0.1% in formic acid acetonitrile solution；Flow velocity 0.3
ML/min, 36 DEG C of column temperature；5 μ L of sample size.
Rice sample is the sample of a complicated component, and the gradient elution program that present invention screening obtains can be preferably right
The component of complex material carries out strong separation in rice sample, differentiates that organic rice and non-organic rice carry out base to be follow-up
Plinth.The present invention additionally uses other gradient elution programs, finds inappropriate gradient elution program, can not make each component
Efficiently separated, and then can not effectively distinguish organic and non-organic rice.
The present invention is directed to the component feature of rice sample, to improve the atomization of compound and ionization situation, improves sensitivity,
By being investigated to conditions such as resolution ratio, gas flow rate, spray voltages, final optimization pass, which screens to obtain one group, causes detection result
Accurate level Four bar-flight time high resolution mass spectrum condition.
In the preferred embodiment of the invention, level Four bar-flight time high resolution mass spectrum selects Aglient 6540UHD
Accurate-mass Q-TOF spectrometer, Mass Spectrometry Conditions are：Double ESI sources, positive ion mode, dry gas temperature
325 DEG C, flow velocity 9L/min；Nebulizer pressure 45psi；Capillary voltage 4000V；Sampling spiroid voltage, 140V；Extract taper electricity
Pressure, 65V；Scanning range, m/z：50~2000；Scan pattern：Full Scan (full scan)；It is m/z with reference to ion：
301.998139 and 1033.988109.
, can be in OPLS-DA shot charts by the ultra performance liquid chromatography and mass spectrographic process conditions by verification experimental verification
On organic rice sample and non-organic rice sample are effectively distinguished.
The qualitative and quantitative information of many endogenous compounds can be measured using metabonomic technology.These information are defeated
Many signal peaks are shown as on the spectrogram gone out, different retention times are shown as on chromatographic mass spectrometry figure and chromatographic peak occur.
For treatment effect and convenience, in the preferred embodiment of the invention, to obtained rice sample and non-have
The UHPLC-Q-TOF MS initial data of machine rice sample carries out turning lattice using MS Convert softwares, then is carried out with XCMS softwares
Pretreatment, the pretreatment refer to extraction to the chromatographic peak in total ion chromatogram initial data, peak alignment, remove noise etc.
Reason, obtains retention time, peak height, peak area and the mass-to-charge ratio data at each peak；Then Multielement statistical analysis method OPLS- is passed through
The form of DA shot charts is shown to distinguishing result.
Wherein, MS Convert softwares be Proteo Wizard companies develop a kind of data conversion format software, XCMS
It is a kind of software of general procedure LC-MS initial data of Scripps Center exploitations.
Differentiating method is：In OPLS-DA shot charts, organic rice sample spot and non-organic rice sample point are belonged to
Region be compared, if both sample belongs to two regions being clearly separated, then both differences of explanation are obvious.
Embodiment 1：
A kind of method that organic rice and non-organic rice are distinguished using metabonomic technology, is comprised the following steps：
(1) sample pre-treatments
Rice sample is crushed, sieves in (1mm apertures), weighs 600mg samples in 5mL centrifuge tubes, adds 3mL extractions
Solvent (70% methanol solution (v/v)), is vortexed and mixes dissolving, ultrasonic 30min, 10000rpm centrifugations 20min under the conditions of 4 DEG C, so
Supernatant is crossed into 0.22 μm of organic filter membrane, upper machine afterwards, applied sample amount is 5 μ L.
(2) Aglient 1290UHPLC Cascade System Aglient 6540UHD accurate-mass Q-TOF are applied
Spectrometer instruments realize the separation and measure to chemical composition in sample.
(1) liquid chromatogram parameter
Chromatographic column：Acquity BEH C18column (1.7 μm of 2.1id × 150mm, particle size)
(Acquity, Waters, Milford, MA, USA).
Liquid phase：A phases, 0.1% aqueous formic acid, B phases：0.1% formic acid acetonitrile；Flow velocity：0.3mL/min；36 DEG C of column temperature.
| Time/min | A/% | B/% |
| 0 | 85 | 15 |
| 1.5 | 85 | 15 |
| 5.0 | 45 | 55 |
| 17.0 | 30 | 70 |
| 20.0 | 10 | 90 |
| 21.0 | 85 | 15 |
(2) mass spectrometry parameters
Positive ion mode
| Ionization pattern | Double ESI sources |
| Scan pattern | FullScan |
| Dry gas temperature | 325℃ |
| Flow velocity | 9L/min |
| Nebulizer pressure | 45psi |
| Capillary voltage | 4000V |
| Sampling spiroid voltage | 140V |
| Extract taper voltage | 65V |
| Scanning range m/z | 50~2000 |
| With reference to ion m/z | 301.998139 and 1033.988109 |
(3) data processing and multi-variate statistical analysis
The UHPLC-Q-TOF MS initial data of rice sample to obtaining uses MS Convert into the row format transformation of ownership, and
Pre-processed with XCMS software kits, which refers to the extraction to the chromatographic peak in total ionic chromatographic initial data, peak
Alignment, goes noise etc. to manage, and obtains the retention time at each peak, peak height, goes noise etc. to manage, when obtaining the reservation at each peak
Between, peak height, peak area and mass-to-charge ratio data, then pass through the Multielement statistical analysis method OPLS-DA scores of SIMCA-P softwares
The form of figure is shown identification result.
OPLS-DA is a kind of method of inspection for having supervision, is a kind of regression modeling method of multivariate response to more independents variable.
OPLS-DA is the extension of PLS-DA, i.e., first uses Orthogonal Signal Correction Analyze technology, by X matrix information decomposition into and not phase related to Y
Two category informations closed, then filter out the information unrelated with classification, relevant information is concentrated mainly on first prediction component
In, compared with PLS-DA models, OPLS-DA can better discriminate between sample group difference, and the validity for improving model is conciliate
Analysis ability, makes analysis result become simple and be easy to explain, it differentiates that effect visualization is more obvious.
(4) achievements exhibition of application：
Rice sample is 20 organic rice samples, and 20 non-organic rice samples, carry out according to the flow of (1)~(3)
Operation, obtains the OPLS-DA scatterplot shot charts of organic rice and non-organic rice.
Aggregation and dispersion degree from each sample of OPLS-DA scatterplot it can be seen from the figure thats, each point represent a sample
Product, wherein O1-O20 represent 20 organic rice samples (round dot), and C1-C20 represents 20 non-organic rice samples (square).Such as
Shown in Fig. 1, in the left-half (X-axis bears semiaxis) of scatter diagram, non-organic rice is concentrated mainly on scattered organic rice sample distribution
The right half part (X-axis positive axis) of point diagram.This illustrates organic rice and non-organic rice in the component of secondary metabolite and contains
There is certain difference in amount, while illustrate that OPLS-DA models can be well by organic rice sample and non-organic rice sample
Distinguish.
According to the high reliability (correlation) and high-magnitude degree (covariance) of data, some are to OPLS-DA category of model shadows
Ring the larger factor to be chosen by manual (apart from the point of main body farther out), we mark referred to as potentiality biology
Know thing (potential biomarker).As shown in Figure 2, totally 30 potential source biomolecule markers are screened out, wherein being overexpressed
The factor (the S curve figure upper right corner) 15, the low expression factor (the S curve figure lower left corner) 15.
Cut-off does not have the mirror that authoritative criterion or normative reference can be applied to non-targeted metabolism group compound to current
In settled.In the present embodiment, we utilize online PostgreSQL database Massbank (http://www.massbank.jp/) it is right
30 potential source biomolecule markers of above-mentioned most statistical significance carry out material discriminating, and the results are shown in Table 1.Wherein 8 chemical combination
Thing, we illustrates the higher compound name of possibility, 12 compounds therein, we annotate molecular formula.Histidinol
(Histidinol), malvin (Malvin), rosin spirit (Pinoresinol), lagochiline (Lagochiline), 4-
Methyl umbelliferone (4-Methylumbelliferyl glucuronide), Coumarin 106 (Coumarin 106), N α-benzene first
Acyl-L-arginine (N α-Benzoyl-L-arginine) and eight kinds of materials of hydrocinchonine (Hydrocinchonine) can by regarding
Make the significant material of discriminating organic rice and non-organic rice.
The identification of 1 potential source biomolecule marker of table
In table 1,
1. determined by Metlin and MassBank databases
2. it is based on peak ion
3. identify rank：1. the compound being identified, the annotation compound of 2. presumptions, the characteristic compounds of 3. presumptions,
4. unknown compound
4.NM- is without matching.
Claims (10)
- A kind of 1. method that organic rice and non-organic rice are distinguished using metabonomic technology, it is characterised in that：The method It is as follows：After organic rice sample and non-organic rice sample are respectively adopted organic solvent progress pre-treatment, using ultra high efficiency liquid phase Chromatographic tandem level Four bar-flight time high resolution mass spectrum method is realized to the organic rice sample after pre-treatment and non-organic rice The separation of chemical composition in sample and measure, then the organic rice sample and the ultra high efficiency of non-organic rice sample to obtaining Liquid chromatography tandem level Four bar-flight time high resolution mass spectrum initial data is pre-processed, finally using multi-variate statistical analysis The orthogonal offset minimum binary of method-discriminant analysis model distinguishes organic rice and non-organic rice, and is obtained using S curve figure to distinguishing The factor not having a great influence, by increasing income, these materials are identified in online database massbank.
- 2. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 1, It is characterized in that：The organic rice sample and non-organic rice sample kind are one kind in the rice fragrance of a flower, imperial round-grained rice, loose round-grained rice It is or a variety of.
- 3. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 1, It is characterized in that：The pre-treatment is specially：Organic rice sample and non-organic rice sample are crushed, cross 1mm aperture sieves, The organic rice sample after sieving and non-organic rice sample are mixed with organic solvent again, ultrasound, centrifugation, excessively organic filter membrane, Pre-treatment is completed, the sample for the detection that obtains being available on the machine.
- 4. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 3, It is characterized in that：The organic rice sample and the adding proportion of non-organic rice sample and organic solvent is 1g：(3~ 10)mL；The organic solvent is methanol aqueous solution, and volume fraction is 60~85%.
- 5. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 3, It is characterized in that：Ultrasonic time is 10~40min；The actual conditions of centrifugation is：8000~12000rpm centrifugations 15 under the conditions of 4 DEG C ~30min.
- 6. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 3, It is characterized in that：Organic filter sizes are 0.20~0.25 μm.
- 7. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 1, It is characterized in that：The ultra performance liquid chromatography condition is：Using octadecyl silane column；A phases：Aqueous formic acid, B Phase：Formic acid acetonitrile solution, gradient elution flow：0~1.5min, 15%B；1.5~5.0min, 15-55%B；5.0~ 17.0min, 55~70%B；17.0~20.0min, 70-90%B；20.0~21.0min, 90-15%B.
- 8. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 7, It is characterized in that：The volume fraction of formic acid is 0.1% in the aqueous formic acid；Formic acid in the formic acid acetonitrile solution Volume fraction is 0.1%；Flow velocity 0.3mL/min, 36 DEG C of column temperature；5 μ L of sample size.
- 9. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 1, It is characterized in that：The level Four bar-flight time high resolution mass spectrum selects 6540 UHD accurate-mass of Aglient QTOF spectrometer, Mass Spectrometry Conditions are：Double ESI sources, positive ion mode, 325 DEG C of dry gas temperature, flow velocity 9L/min； Nebulizer pressure 45psi；Capillary voltage 4000V；Sampling spiroid voltage, 140V；Extract taper voltage, 65V；Scanning range, m/ z：50~2000；Scan pattern：Full Scan；It is m/z with reference to ion：301.998139 and 1033.988109.
- 10. a kind of method that organic rice and non-organic rice are distinguished using metabonomic technology according to claim 1, It is characterized in that：MS is used to obtained organic rice sample and the UHPLC-Q-TOF MS initial data of non-organic rice sample Convert softwares carry out turning lattice, then are pre-processed with XCMS softwares, which refers to total ion chromatogram initial data In chromatographic peak extraction, peak alignment, go noise treatment, obtain retention time, peak height, peak area and the mass-to-charge ratio number at each peak According to then distinguishing result by the forms of Multivariate OPLS-DA shot charts and be shown, and screened by S curve figure Go out the material having a great influence to identification result, potential marker is differentiated using online database massbank.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110007017A (en) * | 2019-03-04 | 2019-07-12 | 山东省中医药研究院 | It is a kind of to identify mulberry leaf whether through white detection method |
| CN110007017B (en) * | 2019-03-04 | 2022-08-23 | 山东省中医药研究院 | Detection method for identifying frosting of mulberry leaves |
| CN110887893A (en) * | 2019-11-08 | 2020-03-17 | 山东省分析测试中心 | MALDI-MS-based method for rapidly identifying fritillaria species |
| CN113419000A (en) * | 2021-06-16 | 2021-09-21 | 中国中医科学院中药研究所 | Method for identifying panax notoginseng with 25 heads and less than 80 heads based on non-targeted metabonomics |
| CN113419000B (en) * | 2021-06-16 | 2022-11-29 | 中国中医科学院中药研究所 | Method for identifying panax notoginseng with 25 heads and less than 80 heads based on non-targeted metabonomics |
| CN113311076A (en) * | 2021-07-02 | 2021-08-27 | 上海应用技术大学 | Method for rapidly distinguishing different varieties of rice based on aldehyde compounds |
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