CN106153800A - The HPLC MS/MS detection method of transgenic corns secondary metabolites - Google Patents
The HPLC MS/MS detection method of transgenic corns secondary metabolites Download PDFInfo
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Abstract
The invention discloses the HPLC MS/MS detection method of a kind of transgenic corns secondary metabolites, belong to the LC-MS detection field of Semen Maydis secondary metabolites.The HPLC MS/MS detection method of transgenic corns secondary metabolites of the present invention, comprises the following steps: (1) extracts the secondary metabolites in corn seed to be measured, obtains crude extract；(2) regulation crude extract pH, then goes up solid-phase extraction column and purifies, and collects eluent；(3) HPLC MS/MS is used to carry out qualitative, quantitative determination.The present invention is by the optimization of the extraction conditions of phenolic metabolism thing, Solid-Phase Extraction condition, liquid-phase condition and Mass Spectrometry Conditions etc. in corn sample, the HPLC MS/MS detection method of the transgenic corns secondary metabolites set up, sensitivity is good, there are preferable repeatability and stability, there is not matrix effect, it is possible to be used for detection and the analysis of phenolic metabolism thing in actual transgenic corns and non-transgenic corn sample.
Description
Technical field
The present invention relates to the detection method of transgenic corns secondary metabolites, particularly relate to phenols in transgenic corn seed
The liquid chromatography-tandem mass of metabolite, belongs to the LC-MS detection field of Semen Maydis secondary metabolites.
Background technology
Phenols secondary metabolites is secondary metabolites important in milpa, can help to resist various pathogen, evil
The harm of worm, nematicide and other plant.Phenols secondary metabolites is that crop self resists performance critical function in pest and disease damage approach
A metabolite, the importing of external source insect resistance protein whether can affect crop self-defense pest and disease damage mechanism change, Yi Jikang
Whether worm albumen degradation process in crop body can produce specific phenols secondary metabolites, beautiful to evaluating insect-resistant transgenic
The biological safety of rice is significant.
The modern analytical techniques such as LC-MS, LC-MS/MS, GC-MS and CE-MS have been applied to the analysis of plant metabolites
In research, the separating effect good with it and higher sensitivity are used widely.Set up that a kind of efficiently concentrating is pest-resistant turns base
Because of the method for secondary metabolites (flavone, phenolic acid) in corn seed, by analyzing the difference generation of transgenic and non-transgenic corn
Thank to thing, by the safety a kind of new technological means of offer for evaluating transgenic corns.
Summary of the invention
The technical problem to be solved is to set up the HPLC-MS/MS inspection of a kind of transgenic corns secondary metabolites
Survey method, the advantage such as the method has that sensitivity is good, repeatability and good stability, it is possible to turn base with non-for transgenic corns
Because of the detection analysis of phenolic metabolism thing in corn sample.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
The invention discloses the HPLC-MS/MS detection method of a kind of transgenic corns secondary metabolites, including following step
Rapid: (1) extracts the secondary metabolites in corn seed to be measured, obtains crude extract；(2) regulation crude extract pH, then goes up solid phase extraction
Take post to purify, collect eluent；(3) HPLC-MS/MS is used to carry out qualitative, quantitative determination.
Wherein, the secondary metabolites in step (1) described extraction corn seed to be measured includes: by corn seed powder to be measured
Broken, add Extraction solvent, carry out supersound process, centrifugal collection supernatant；Repeat said extracted step 1-2 time, by each step institute
Obtain supernatant to merge, to obtain final product.Described Extraction solvent is any one in methanol, ethanol, acetonitrile or ethyl acetate, preferably first
Alcohol；It is furthermore preferred that described Extraction solvent is the methanol of volume fraction 50-100%, the most preferably methanol of volume fraction 70%.
Counting according to g/ml, corn seed to be measured is 1:10-50, preferably 1:20 with the solid-liquid ratio of Extraction solvent；Described supersound process
Ultrasonic time is 0-50min, and ultrasonic power is 400-800W；Preferably, ultrasonic time is 20min, and ultrasonic power is 600W.
In HPLC-MS/MS detection method of the present invention, the liquid-phase condition of step (3) described HPLC-MS/MS includes: color
Spectrum post is Agilent ZORBAX SB-Aq chromatographic column；Column temperature is 25 DEG C；Flow velocity is 0.2ml/min-0.3mL/min, is preferably
0.2ml/min；Sampling volume is 1 μ L；Mobile phase A is volume fraction 0.002% to 0.01% formic acid-aqueous solution, and Mobile phase B is
Acetonitrile；Gradient elution.Preferably, described mobile phase A is volume fraction 0.002% formic acid-aqueous solution, and Mobile phase B is acetonitrile；Institute
The step stating gradient elution includes: according to volume fraction, 0min, 0%B；1min, 10%B；25min, 20%B；33min,
30%B；34min, 40%B；38min, 40%B；39min, 70%B；41min, 70%B；43min, 0%B.
The Mass Spectrometry Conditions of step (3) described HPLC-MS/MS includes: ion source is electron spray ionisation source (anion scanning mould
Formula), atomization gas is purity 99.999% nitrogen, and collision gas is purity 99.999% nitrogen, and atomization gas pressure is 40psi, is dried
Temperature is 350 DEG C, and dry gas stream amount is 10L/min, and capillary voltage is 3500V, and detection pattern is multiple-reaction monitoring pattern
(MRM)。
The HPLC-MS/MS detection method of transgenic corns secondary metabolites of the present invention, step (2) regulation crude extract pH is
2-6；Preferably, regulation crude extract pH is 3；It is furthermore preferred that regulate crude extract pH with formic acid；Specifically include: by crude extract 40
DEG C rotary evaporation is near dry, then redissolves with the formic acid of pH=3-aqueous solution, and regulating crude extract pH is 3.Step (2) described solid phase extracts
Taking post is Waters Oasis HLB post；During described upper solid-phase extraction column purifies, leacheate is water, and eluent is
Methanol；The eluting liquid nitrogen of collection is blown near dry after redissolve with methanol, vortex oscillation, centrifugal, filter after, carry out LC-MS/MS inspection
Survey.
In HPLC-MS/MS detection method of the present invention, step (3) described qualitative, quantitative determination include: according to be measured
Secondary metabolites is identical with the parent ion mass-to-charge ratio of secondary metabolites standard substance, quota ion is identical with qualitative ion, and retains
Time is identical, is defined as target secondary metabolites；Concentration according to secondary metabolites standard substance and peak area make standard curve, will
In corn seed to be measured, the peak area of target secondary metabolites brings corresponding linear equation into, calculates the phase of target secondary metabolites
Answer concentration.
Secondary metabolites of the present invention includes but not limited to: coumaric acid, gallic acid, vanillic acid, rutin, benzoic acid,
Catechin, hyperin, syringic acid, P-coumaric acid, P-hydroxybenzoic acid, sinapic acid, epicatechin, gentisic acid, trans Resina Ferulae
Acid, caffeic acid, protocatechuic acid, (-)-times catechuic acid, high protocatechuic acid, 4-hydroxy-3-methoxy-.alpha.-toluic acid., apigenin, cinnamic acid, aromadendrin, sweet-scented osmanthus
Any in grass element, Quercetin, eriodictyol, phloretin, hesperetin, naringenin, isorhamnetin, diosmetin or genistein
One or more.
The present invention, by the optimization to Mass Spectrometry Conditions, obtains the optimum mass spectrum of 31 kinds of phenolic metabolism things in corn seed
Condition, specifically includes:
The parent ion of coumaric acid is 139.09, and quota ion is 95.2, and qualitative ion is 51.2；
The parent ion of gallic acid is 169.12, and quota ion is 125, and qualitative ion is 151；
The parent ion of vanillic acid is 167.15, and quota ion is 108, and qualitative ion is 91.2；
The parent ion of rutin is 609.52, and quota ion is 301.1, and qualitative ion is 271.2；
Benzoic parent ion is 121.12, and quota ion is 77, and qualitative ion is 77；
The parent ion of catechin is 289.27, and quota ion is 245.2, and qualitative ion is 203；
The parent ion of hyperin is 463.4, and quota ion is 300, and qualitative ion is 271；
The parent ion of syringic acid is 197.17, and quota ion is 121.1, and qualitative ion is 153.3；
The parent ion of P-coumaric acid is 163.16, and quota ion is 119, and qualitative ion is 93.1；
The parent ion of P-hydroxybenzoic acid is 137.12, and quota ion is 93.0, and qualitative ion is 65.3；
The parent ion of sinapic acid is 223.21, and quota ion is 149, and qualitative ion is 164；
The parent ion of epicatechin is 289.27, and quota ion is 245.2, and qualitative ion is 203；
The parent ion of gentisic acid is 153.12, and quota ion is 108, and qualitative ion is 108；
The parent ion of trans-ferulaic acid is 193.18, and quota ion is 134.3, and qualitative ion is 134.3；
Caffeinic parent ion is 179.16, and quota ion is 135.1, and qualitative ion is 133.9；
The parent ion of protocatechuic acid is 153.12, and quota ion is 109.2, and qualitative ion is 109.2；
The parent ion of (-)-times catechuic acid is 305.27, and quota ion is 125, and qualitative ion is 137；
The parent ion of high protocatechuic acid is 167.15, and quota ion is 123, and qualitative ion is 123；
The parent ion of 4-hydroxy-3-methoxy-.alpha.-toluic acid. is 181.17, and quota ion is 137.1, and qualitative ion is 137.1；
The parent ion of apigenin is 270.24, and quota ion is 117, and qualitative ion is 117；
The parent ion of cinnamic acid is 148.16, and quota ion is 103, and qualitative ion is 77.3；
The parent ion of aromadendrin is 288.25, and quota ion is 125.1, and qualitative ion is 285.9；
The parent ion of luteolin is 286.24, and quota ion is 132.8, and qualitative ion is 175.1；
The parent ion of Quercetin is 302.24, and quota ion is 151.1, and qualitative ion is 178.9；
The parent ion of eriodictyol is 288.25, and quota ion is 151.3, and qualitative ion is 135.3；
The parent ion of phloretin is 274.27, and quota ion is 167.4, and qualitative ion is 123.3；
The parent ion of hesperetin is 302.28, and quota ion is 164, and qualitative ion is 285.9；
The parent ion of naringenin is 272.25, and quota ion is 151, and qualitative ion is 119.1；
The parent ion of isorhamnetin is 316.25, and quota ion is 300.1, and qualitative ion is 300.1；
The parent ion of diosmetin is 300.26, and quota ion is 284, and qualitative ion is 227；
The parent ion of genistein is 270.24, and quota ion is 133.3, and qualitative ion is 63.3.
The liquid-phase condition of HPLC-MS/MS method is optimized by the present invention.Chromatographic column optimum results shows, HILIC parent
Water chromatographic column can not good isolating target compound, use SB-C18 chromatographic column to there will be hangover and split phenomenon, and use
SB-Aq chromatographic column target compound can realize preferably separation, and peak type is symmetrical sharp-pointed, and conditions of streaking is the most substantially changed
Kind.
The optimum results of flowing phase shows, Mobile phase B is the separating effect separating effect apparently higher than methanol of acetonitrile, because of
This selects acetonitrile to be Mobile phase B.The flow velocity of flowing phase selects result to show, flow velocity is that the response value of 0.2ml/min compares 0.3ml/
The response value of min is high, so selecting 0.2ml/min for separating flow velocity.Flowing acid mutually, alkali, the selection result of salt show, use first
Aqueous acid-acetonitrile is as flowing phase time, and the separating effect of compound is best, and adds a small amount of acid and can reach to improve peak
Type, reduces the purpose of hangover, therefore selects to add a small amount of volatile acid in aqueous phase.Selection table to organic acid further
Bright, in acetic acid water-acetonitrile mobile phase system, some object gallic acid, protocatechuic acid, times catechuic acid, gentisic acid, Cortex querci dentatae
Low than in formic acid water-acetonitrile mobile phase system of the response value of element etc., therefore selects formic acid water-acetonitrile mobile phase system.
The present invention adds the formic acid of different volumes mark further in flowing mutually, and the aqueous formic acid of result different volumes mark is to change
The separating degree impact of compound is little, but along with the increase of aqueous formic acid concentration, except gallic acid, and gentisic acid, times catechuic acid, former
Beyond catechuic acid, the response value of other compounds is gradually lowered, and considers the formic acid that selective body fraction is 0.002% water-soluble
Liquid.
If LC-MS of the present invention detection phenolic metabolism owner separated by liquid chromatograph, entered by mass spectrum
Row detection, and in the separation process of liquid chromatograph, gradient is most important link.Gradient optimization is tied by the present invention
Fruit shows, uses gradient: according to volume fraction, 0min, 0%B；1min, 10%B；25min, 20%B；33min,
30%B；34min, 40%B；38min, 40%B；39min, 70%B；41min, 70%B；43min, 0%B, all substances can
Reach baseline separation；And using other gradient, moieties can not separate.
Corn sample extraction conditions is also optimized by the present invention.The selection result of Extraction solvent shows, Extraction solvent
During for methanol, in corn sample, the extraction yield of all phenolic metabolism things is significantly higher than Extraction solvent is ethanol, acetonitrile or acetic acid
Ethyl ester, illustrates that phenolic metabolism thing dissolubility in methanol is more preferable, therefore selects methanol as Extraction solvent.Further to extraction
The optimum results of solvent ratios shows, in the range of methanol volume fraction is 50%-100%, when methanol is 70%, and corn-like
In product, the extraction yield of all phenolic metabolism things is significantly higher than the methanol of other volume fractions；Along with methanol concentration raises, extract
Rate is gradually lowered.Therefore the present invention selects 70% methanol to be Extraction solvent.Solid-liquid ratio optimum results shows, counts according to g/ml, material
When liquor ratio is 1:20, in corn sample, the extraction yield of all phenolic metabolism things is the highest；Along with increasing of Extraction solvent, extraction ratio
It is gradually reduced.Ultrasonic time optimum results shows, when ultrasonic time is 20min, in corn sample, all phenolic metabolism things carries
Acquirement rate is the highest；Ultrasonic time is the longest, and extraction ratio does not has significant change.
Phenolic metabolism thing under neutral or basic conditions in ionic condition, therefore pH value to phenolic metabolism thing in Solid-Phase Extraction
The response rate on post has a significant impact.The optimum results of sample solution pH in Solid-Phase Extraction is shown by the present invention, in pH 2-6 scope
In, along with pH is gradually increased, gallic acid, coumaric acid, the caffeinic response rate are gradually lowered, and the response rate of other compounds is also
Decrease to some degree；As pH=3, overwhelming majority phenolic metabolism thing adsorption effects on SPE post are best, and the response rate is
High.Therefore selecting regulation crude extract pH is 3 to go up solid-phase extraction column again.
Applying HPLC-MS/MS detection method of the present invention, 31 kinds of polyphenols are at the concentration model of 2.5-500 μ g/L
In enclosing, linear relationship is good, and detection limit scope is 2.5-50 μ g/L, and correlation coefficient is all higher than 0.9991；When retaining the most in the daytime
Between and the most in the daytime peak area relative standard deviation all < 15%, illustrate that the basic repeatability of the inventive method is good.Deemed-to-satisfy4
The evaluation result of energy shows, most phenolic metabolism things are at 10-500 μ g L-1Concentration range internal linear relation is good, and relevant
Coefficients R2Being all higher than 0.9990, detection is limited to 5-100 μ g L-1, the standard deviation (RSD) of peak area 4.75%-14.68% it
Between, illustrate that the HPLC-MS/MS detection method sensitivity that the present invention sets up is good, have preferable repeatability and stability, it is possible to use
Analysis in Semen Maydis phenolic metabolism thing.
The evaluation result of matrix effect shows, obtains the substrate effect of 31 kinds of phenolic metabolism things after LC-MS/MS detects
Should be 93.17%-113.02%, relative standard deviation RSD, between 0.94%-7.41%, illustrates in corn sample 31
There is not matrix effect in the LC-MS/MS detection planting phenolic metabolism thing.Substrate recovery testu result shows, most phenol
Acid and flavone recovery of standard addition in corn sample are between 50.35%-110.69%, and relative standard deviation is at 0.51%-
Between 13.91%.Illustrate that the pre-treating method of sample is good, it is possible to the LC-MS/ of phenolic metabolism thing in actual corn sample
MS detects.
The HPLC-MS/MS detection method of transgenic corns secondary metabolites of the present invention can be applied to transgenic corns or
The detection analysis of phenols secondary metabolites in non-transgenic corn.Apply the HPLC-of transgenic corns secondary metabolites of the present invention
30 kinds of insect-resistant transgenic Semen Maydiss and 14 kinds of non-transgenic corn are measured, determine VAN (vanillic acid) by MS/MS detection method
For the difference metabolite of transgenic corns and non-transgenic corn, and the relative amount (1.753) that VAN is in non-transgenic corn
The relative amount (0.932) being higher than in transgenic corns.
Technical solution of the present invention compared with prior art, has the advantages that
The present invention by the extraction conditions of phenolic metabolism thing in corn sample, Solid-Phase Extraction condition, liquid-phase condition and
The optimization of Mass Spectrometry Conditions, establishes the HPLC-MS/MS detection method of a kind of transgenic corns secondary metabolites.The present invention detects
Method sensitivity is good, has preferable repeatability and stability, there is not matrix effect, it is possible to for actual transgenic and non-turn
The detection analysis of phenolic metabolism thing in gene corn sample.The HPLC-MS/MS detection of transgenic corns secondary metabolites of the present invention
Method, for evaluating the safety of transgenic corns, it is provided that a kind of new technological means by difference metabolite analysis.
Accompanying drawing explanation
Fig. 1 is the optimization of sample solution pH value；Wherein, GAL be gallic acid, COU be coumaric acid, GALLO for (-)-extremely
Boheic acid, HPRO be high protocatechuic acid, PRO be protocatechuic acid, DHB be gentisic acid, HYB be P-hydroxybenzoic acid, EPI be table catechu
Element, HVAN be 4-hydroxy-3-methoxy-.alpha.-toluic acid., VAN be vanillic acid, CAT be catechin, SYR be syringic acid, CAF be caffeic acid, BEN be benzene first
Acid, P-COU be P-coumaric acid, FER be trans-ferulaic acid, RUT be rutin, HYP be hyperin, SIN be that sinapic acid, DKF are
Aromadendrin, CIN be cinnamic acid, QUE be Quercetin, ERI be eriodictyol, LUT be luteolin, IHN be that isorhamnetin, HES are
Hesperetin, DIO be diosmetin, PHL be phloretin, NAR be naringenin, API be apigenin, GEN be genistein；
Fig. 2 is the optimization of sample extraction solvent；
Fig. 3 is the optimization of sample extraction solvent ratios；
Fig. 4 is the optimization of sample extraction solid-liquid ratio；
Fig. 5 is the optimization of sample extraction ultrasonic time.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or replaces, but these amendments or replacement each fall within protection scope of the present invention.
1, material
30 kinds of transgenic corns sample, be respectively the beautiful L06 of moral list 1420, nine, D1325, YD238, mf222, LV1341,
LD60, K60, No. 1-22 and from numbering from 1-21 Semen Maydis, derive from Biological Technology institute, Chinese Academy of Agricultural Sciences's transgenic
Plant microbial environment supervision and inspection on safety test center.
Non-transgenic corn sample 14 kinds, is force 312, jade 335 high by 230, rich by 558, first, expensive beautiful No. 2, carbuncle 1 respectively
Number, glutinous No. 1 of week, Zheng Dan 958, grand flat 206, Su Yu 20, calabash shell serving as a dipper jade 16, breathe out and make every effort to overcome popcorn, Autumn Gold popcorn, green valley head
Popcorn, purchased from oneself farm.
The standard substance (specific name is shown in Table 2) of 31 kinds of phenolic metabolism things, purchased from Sigma Co., USA.
The HPLC-MS/MS detection of embodiment 1 transgenic corns secondary metabolites
1, experimental technique
1.1 sample-pretreating method
1.1.1 the collection of corn sample and preparation
By 44 kinds of corn samples (wherein 30 kinds of transgenic corns sample, non-transgenic corn sample 14 kinds), every kind of Semen Maydis
After sample dries, blend with pulverizer powdered, cross 60 mesh sieves.Every kind of Semen Maydis powder is individually loaded in valve bag, carries out mark
Note, is placed under drying at room temperature environment preservation.
1.1.2 Semen Maydis phenolic acid and the extraction of flavone
Accurately weigh 1.00g (being accurate to 0.01g) corn sample, be placed in 50ml tool plug centrifuge tube, add 20ml 70%
Methanol (volume fraction), ultrasonic (600W) 20min after vortex 10s mix homogeneously, at 10000r min-1, 4 DEG C of centrifugal 10min,
Transfer supernatant is in 100ml round-bottomed flask.Remaining solid residue extracts once again according still further to above-mentioned steps, carries twice
The supernatant taken merges, and at 40 DEG C of rotary evaporations near dry, then redissolves with the formic acid-aqueous solution of pH=3 and is settled to 5mL, this
5mL Semen Maydis phenolic metabolism thing crude extract, as the sample solution of SPE, remains to purify.
1.1.3 Semen Maydis phenolic acid and the purification of flavone
Solid phase extraction column is Waters Oasis HLB post (500mg, 6ml).Before Solid-Phase Extraction loading, first use
5ml methanol and 5ml water activate and balance solid-phase extraction column, and coutroi velocity is at 1mL/min, in the process, it should be noted that keep solid
Phase extraction column moistens.5ml sample solution to be clean is joined on SPE post, controls loading velocity ratio activation flow velocity slow, treat loading
After liquid all flows out, with 4ml water wash SPE post, then vacuum is drained 5min and is removed the moisture in SPE post.Last SPE post 6ml
Methanol-eluted fractions, elution flow rate is also slow, is collected in test tube by eluent, and after eluent all flows out, it is little that vacuum drains SPE
Post.After eluting liquid nitrogen is blown near doing, redissolve with 1ml methanol, vortex oscillation 1min, cross the 0.22 organic filter membrane of μm after being centrifuged, enter
Row LC-MS/MS detects.
1.1.4 the mensuration (HPLC-MS/MS) of Semen Maydis phenolic acid and flavone
1.1.4.1 liquid-phase condition
Chromatographic column: Agilent ZORBAX SB-Aq chromatographic column (2.1 × 150mm, 3.5 μm)；
Column temperature: 25 DEG C；
Flow velocity: 0.2ml/min；
Sampling volume: 1 μ L；
Stop acquisition time: 48min；
Post pressure balanced time: 14min；
Flowing phase: A: formic acid (0.002%, volume fraction)-water；B: acetonitrile；
Eluent gradient elution program is shown in Table 1:
Table 1 eluent gradient elution step
1.1.4.2 Mass Spectrometry Conditions
Ion source: electron spray ionisation source (ESI)；
Atomization gas: nitrogen (99.999%)；
Collision gas: nitrogen (99.999%)；
Atomization gas pressure: 40psi；
Dry temperature: 350 DEG C；
Dry gas stream amount: 10L/min；
Capillary voltage: 3500V；
Detection pattern: multiple-reaction monitoring pattern (MRM)；
Resolution: Q1 (unit) Q3 (unit)；
Sweep time section and the material of corresponding detection as shown in table 2.
Table 2 section sweep time and the material of corresponding detection
1.1.5 LC-MS/MS is qualitative and quantitative approach
When carrying out the mensuration of actual sample, it then follows the feature of many reaction detection (MRM) pattern that method uses, i.e. pass through
The mass-to-charge ratio (m/z) of retention time (RT) and parent ion, daughter ion is come dual qualitative.If sample detects and standard substance
Parent ion mass-to-charge ratio (m/z) is identical, and quota ion is identical with assisted quantitative ion (qualitative ion), and total ion current figure (TIC)
The appearance time material consistent with standard substance appearance time, then it is believed that this kind of corn sample exists corresponding phenolic metabolism
Thing, thus comes the phenolic acid in corn sample and flavone are carried out qualitative analysis.
This experiment uses external standard method to quantitative determine actual sample.Prepare the hybrid standard work of a series of variable concentrations
Make liquid, and measure the peak area of every kind of object under variable concentrations and, to draw concentration-peak area standard curve, thus come Semen Maydis
Phenolic acid and flavone in sample carry out quantitative analysis.
2, experimental result
The optimization of 2.1 LC-MS/MS conditions
2.1.1 the optimization of Mass Spectrometry Conditions
The most ionogenic selection
Ion source is used to liquid phase molecule is atomized into gaseous state and makes its charged device, and they have respective applicable model
Enclose, wherein electron spray ionisation ESI and the most practical ion source of Atmosphere Pressure Chemical Ionization (APCI) APCI.
31 kinds of phenolic metabolism things measured by this experiment all belong to the material of the easily ionizable of polarized, therefore select ESI electron spray
Source, as ion source, makes the ionizing before entering mass analyzer of these 31 kinds of phenolic metabolism things.
2.1.1.2 the selection of positive negative mode
ESI electrospray ionization source has cation and two kinds of scan patterns of anion.Wherein, positive ion mode is suitable for basic species
The detection of matter, anion scan pattern is generally suitable for acidic sample.The structure of 31 kinds of phenolic metabolism things of this measuring can
Knowing all there is hydroxy functional group in all of phenolic acid and flavone, phenolic acids also has carboxyl functional group, easily loses a proton, suitable
Close and use anion scan pattern, therefore phenolic compound is measured by this experimental selection anion scan pattern.
2.1.1.3 the selection of parent ion
It is the list mark working solution of 31 kinds of phenolic metabolism things of 100 μ g/L as solvent compound concentration with 50% methanol
(standard substance are purchased from Sigma Co., USA), the chromatographic column of liquid phase part is Zorbax SB-C18 1-Pack (Rapid
Resolution Cartridge, 2.1 × 30mm i.d, 3.5 μm), flowing is water mutually: methanol (50:50), flow velocity is 0.2ml/
Min, sample size is 1 μ L, and methanol is as blank sample and washes pin liquid.Under ESI negative ion mode, to these 31 kinds of phenolic metabolisms
Thing carries out first mass spectrometric full scan pattern (MS2Scan), according to the relative molecular weight of every kind of metabolite, sets suitable mass-to-charge ratio
Scope, finds molecular ion peak by mass spectrum, determines the parent ion of object.Phenolic metabolism thing is under ESI negative ion mode
Ionization loses a proton, produces [M-H]-Molecular ion peak, if find [M-H] in mass spectrum-Molecular ion peak, and empty
White solvent is not somebody's turn to do [M-H]-Molecular ion peak, then can determine that this ion [M-H]-It it is the parent ion of target analytes.
2.1.1.4 the optimization of parent ion capillary outlet voltage (Fragmentor)
As a example by P-coumaric acid (P-COU), optimize the capillary outlet voltage of parent ion m/z163.1.Scan pattern is set
It is set to select ion detection (SIM) pattern, the most only collects the parent ion of m/z 163.1, only allow to set the ion m/z of mass-to-charge ratio
163.1 pass through first level Four bar (MS1), carry out the one-level scan pattern of single ion, now need capillary outlet electricity
Pressure is optimized, and the ion transmission efficiency making desired ion is maximum, and response value is the highest.Set respectively Fragmentor as 75V,
85V, 95V, 105V, contrast the TIC total ion current figure of the parent ion m/z305.27 of P-COU under different capillary outlet voltages.
When Fragmentor is 75V, the response value of parent ion is the highest；When Fragmentor more than 75V time, the response value of parent ion by
Gradually reducing, this is because capillary outlet voltage is excessive, fragment ion can be caused to increase, parent ion reduces.It is thus determined that P-COU
Optimal capillary outlet voltage be 75V.
2.1.1.5 the selection of daughter ion
After the capillary outlet voltage of the parent ion and correspondence that determine every kind of object, need to make parent ion enter and touch
Hit pond.Setting mass spectrum pattern as daughter ion scan pattern (Product ion scan, PI), this pattern only allows to allow the mesh of regulation
Mark parent ion enters collision cell, obtains fragment ion at collision cell through different-energy collision, and second level Four bar (MS2) is to fragment
Ion is scanned, search out from mass spectrum this parent ion broken after daughter ion, select son that wherein response value is the highest from
Son is as quota ion, and the relatively low daughter ion of response value is as assisted quantitative ion (qualitative ion).
2.1.1.6 the optimization of daughter ion collision energy
Obtain parent ion produce daughter ion after, need the collision energy of daughter ion is optimized, make every seed from
Son response value under optimum collision energy is the highest, as the most quantitative foundation.Therefrom select the highest conduct of response value fixed
Amount ion, response value higher as assisted quantitative ion (qualitative ion).
2.1.1.7 the Mass Spectrometry Conditions of 31 kinds of phenolic metabolism things
The Mass Spectrometry Conditions of 31 kinds of phenolic metabolism things is shown in Table 3.
30 nine kinds of phenolic acid of table and the optimum spectral condition of flavone
Note: a represents quota ion；B represents qualitative ion.
2.1.2 the optimization of liquid-phase condition
2.1.2.1 the selection of chromatographic column
When liquid chromatography-mass spectrometry uses electron spray ESI ion source, generally use narrow footpath post at liquid phase part.
This experiment have chosen three kinds of chromatographic columns and carries out method exploitation, respectively HILIC XBridgeTM Amide chromatographic column, 2.1 ×
150mm, 3.5 μm (Waters, Ireland)；Zorbax SB-C18 chromatographic column, 2.1 × 150mm, 3.5 μm (Agilent,
USA)；Zorbax SB-Aq chromatographic column, 2.1 × 150mm, 3.5 μm (Agilent, USA).Select from 31 kinds of target compounds
The compound that four kinds of polarity differences are bigger, is P-hydroxybenzoic acid (HYB), coumaric acid (COU), p-coumaric acid (P-COU) respectively
With trans-ferulaic acid (FER).Observe these four compound separation situation in different chromatographic columns.
Result shows: (1) is for HILIC hydrophilic chromatographic post: HILIC hydrophilic Interaction Chromatography pattern, also has the most anti-phase
Chromatogram mode.Forward chromatograph uses polar stationary phase and nonpolar liquid phase, and reversed phase chromatography uses nonpolar fixing
Flow phase mutually with polarity, and HILIC hydrophilic chromatographic uses the flowing phase of polar stationary phase and reversed phase chromatography, in the process of analysis
The organic facies (typically acetonitrile) of middle use high concentration, often adds buffer salt in aqueous phase.Reversed phase chromatography is applicable to separating polar
And low pole compound, to some highly polar compounds retain very poor, HILIC hydrophilic Interaction Chromatography be applicable to polar compound
Separation.The present invention attempts adding different salt in aqueous phase, observes the separation situation at these four object.When in aqueous phase
When adding 10mM ammonium acetate, or when adding 5mM ammonium acetate and 5mM ammonium formate in aqueous phase, or in aqueous phase, add 10mM
When ammonium formate and 0.1% formic acid, either in the case of which kind of, four kinds of materials all can not well separate.
(2) for SB-C18 chromatographic column: SB-C18 chromatographic column is conventional reversed phase chromatographic column, can be in acidity flowing phase condition
Lower realization preferably separates, it is provided that good stability also makes longest-lived, and repeatability is good.Using pure water-acetonitrile is flowing phase,
Using SB-C18 chromatographic column can realize preferably separation as fixing phase time, four kinds of materials, but owing to aldehydes matter contains phenol
Hydroxyl, easily combines with fixing, causes conditions of streaking.Therefore, when using SB-C18 chromatographic column, it may appear that hangover and split are existing
As.
(3) SB-Aq chromatographic column: SB-Aq chromatographic column is best suited for increasing acidity, alkalescence and the polar compound being difficult to separate
Retention time, stronger than the traditional C18 liquid phase posts of many.Strong reservation, post longevity during low ph value is had in the flowing mutually of high-moisture
Life is long.SB-Aq post has hydrophilic surface, and the aqueous solution even with 100% makees flowing phase, it is also possible to effectively prevent solid
Determine subsiding of phase.The highest high temperature that can use 80 DEG C.Use pure water-acetonitrile for flowing phase, make using SB-Aq chromatographic column
For fixing phase time, four kinds of materials can realize preferably separation, and peak type is symmetrical sharp-pointed, conditions of streaking also be improved significantly.
So selecting SB-Aq chromatographic column as separation chromatography post.
2.1.2.2 the flow velocity of flowing phase selects
When interface selects electron spray ESI ionization source, Ionization Efficiency and chromatographic isolation are imitated by the selection of flow rate of mobile phase
The impact of fruit is the most contrary.Generally, flow velocity is the least, and the efficiency of ionizing is the highest, but affects separating effect；Otherwise flow velocity
The biggest, separating effect is the best, but Ionization Efficiency is the lowest.So needing to take into account both, both can meet liquid phase part separation and having wanted
Seek the electro-spray ionization efficiency that also can reach higher, be typically chosen flow velocity and separate when 0.2mL/min to 0.3mL/min
Measure.Result shows, the flow velocity of 0.2ml/min and 0.3ml/min is not very big on the impact of separating degree, but flow velocity is 0.2ml/
The response value of the min response value than 0.3ml/min is high, so selecting 0.2ml/min for separating flow velocity.
The selection of the phase that 2.1.2.3 flows
In liquid chromatograph mass spectrography detects, the composition of flowing phase can be with the organic facies aqueous phase to 100% of 100%
Between change.But being often used without 100% aqueous phase, 100% aqueous phase can may result in the minimizing of chromatographic column life-span, and substantial amounts of
Aqueous phase can reduce Ionization Efficiency.In LC-MS/MS system, normally used flowing has water, methanol, acetonitrile, formic acid, second mutually
Acid, ammonium formate and ammonium acetate.Flowing has effumability accordingly, and easily sloughs, and does not use the buffer salt solution etc. of difficult volatilization, as
Phosphate buffer, is likely to result in pipeline and spray needle blocking etc..
In reversed phase chromatography, flowing phase organic facies is typically chosen methanol or acetonitrile.Phenolic metabolism thing is equal in methanol, acetonitrile
There is good dissolubility, therefore do optimization further on this basis.From the results, it was seen that the separation effect that Mobile phase B is acetonitrile
Fruit is apparently higher than the separating effect of methanol, so finally selecting acetonitrile is Mobile phase B.
2.1.2.4 flow the selection of acid, alkali, salt mutually
In the negative ion mode, in flowing mutually, add appropriate ammonia or buffer salt (ammonium formate/ammonium acetate) perhaps can
Improve the response value of compound, improve separating degree.
Measuring the total ion current figure that mobile phase A is 0.1% ammonia respectively, mobile phase A is the total ion current of 5mM ammonium acetate
Figure, mobile phase A is the total ion current figure of pure water, and the total ion current figure that mobile phase A is 0.02% aqueous formic acid.By result
It can be seen that use ammonia acetonitrile as flowing phase time, the separating effect of compound is very poor, and all of material is all centered at two
In individual peak；Using ammonium acetate saline solution-acetonitrile as flowing phase time, the separating effect of compound is poor, and baseline is higher；Make
With pure water acetonitrile as flowing phase time, the separating effect of compound is preferable；Use aqueous formic acid-acetonitrile as flowing phase time,
The separating effect of compound is best, and adds a small amount of acid and can reach to improve peak type, reduces the purpose of hangover.So finally selecting
Select in aqueous phase, add a small amount of volatile acid.
Next the present invention is selected in the organic acid (formic acid/acetic acid) that mass spectrum can use.Flowing mutually divides
Not with the addition of formic acid and the acetic acid of 0.01% that volume fraction is 0.01%.As a result, adding volume fraction in flowing mutually is
The formic acid of 0.01% and acetic acid, do not have significant change to separating degree.But in acetic acid water-acetonitrile mobile phase system, some target
The response value ratio of thing gallic acid, protocatechuic acid, times catechuic acid, gentisic acid, Quercetin etc. is at formic acid water-acetonitrile mobile phase body
Low in system, so selecting formic acid water-acetonitrile mobile phase system.
2.1.2.5 the optimization of Mobile Phase Additives volume fraction
In flowing mutually, add the formic acid of different volumes mark, the pH of flow visualizing can be affected, thus cause separation
Degree and the impact of object response value.10 μ l, 20 μ l, 30 μ l, 40 μ l, the first of 50 μ l is added respectively in the aqueous solution of 500ml
Acid, volume fraction is respectively 0.002%, 0.004%, 0.006%, 0.008%, 0.01%, and as mobile phase A, Mobile phase B is
Acetonitrile, measures the mass spectrum response value of 31 kinds of phenolic metabolism things, and investigates peak shape, separating degree etc..
From the results, it was seen that the separating degree of compound is affected little by the aqueous formic acid of different volumes mark.But with
The continuous increase of aqueous formic acid concentration, except gallic acid, gentisic acid, beyond times catechuic acid, protocatechuic acid, other compounds
Response value be gradually lowered, so considering, selective body fraction is the aqueous formic acid of 0.002%.
2.1.2.6 the optimization of flowing phase gradient
If LC-MS detection phenolic metabolism owner separated by liquid chromatograph, examined by mass spectrum
Survey, and in the separation process of liquid chromatograph, gradient is most important link.When separating various ingredients, isocratic elution
Being difficult to by some wide for polarity scope mixture separately, the advantage of gradient elution is the ratio by constantly adjusting flowing phase,
The polarity of flowing phase can be changed, thus the material of opposed polarity was eluted within the different time periods, reach separation
Purpose.
When optimizing condition of gradient elution, the 500 μ g/L mixed mark solution of 31 kinds of polyphenols of preparation, flow velocity is
0.2mL/min, sample introduction 1 μ L, that investigates 31 kinds of polyphenols is basically separated situation.This experiment is by substantial amounts of pre-reality
Test, mainly optimize four gradients: measure the total ion current figure under following four gradient (volume fraction) respectively.
(a): 0min5%B；15min, 20%B；25min, 30%B；35min, 40%B；40min, 70%B；42min,
70%B；44min, 0%B；
(b): 0min, 0%B, 15min, 20%B, 30min, 30%B；35min, 40%B；40min, 80%B；42min,
80%B, 44min, 0%B；
(c): 0min, 0%B；1min, 10%B；25min, 20%B；33min, 30%B；34min, 40%B；38min,
40%B；39min, 70%B；41min, 70%B；43min, 0%B；
(d): 0min, 0%B；10min, 15%B；25min, 30%B；35min, 50%B；40min, 70%B；42min,
70%B；44min, 0%B.
From the results, it was seen that in (a), (b), (d) gradient, ferulic acid, sinapic acid, rutin and hyperin are equal
Can not separate, and in (a) (-)-times catechuic acid and gentisic acid inseparable, catechin and syringic acid in (d) are inseparable, (c)
In all substances be attained by baseline separation, so finally determining that gradient is (c).
2.1.2.7 the optimization of column temperature
The change of column temperature typically can affect the retention time of object, selects 25 DEG C as separation temperature.
The analysis characteristic quantity of 2.2 detection methods
The 30 of this experiment compound concentration 0.1,0.5,1,2.5,5,10,25,50,100,200,300,500 μ g/L respectively
A kind of mixed standard solution of polyphenols, 1 μ L sample introduction under the conditions of the liquid chromatography mass spectrometric optimized, measure variable concentrations and mix
The response value of mark solution, Criterion curve, the results are shown in Table 4.Result shows, 31 kinds of polyphenols are at 2.5-500 μ g/
The concentration range internal linear relation of L is good, and detection limit scope is 2.5-50 μ g/L, and correlation coefficient is all higher than 0.9991.
With 300 μ g L-117 kinds of polyphenols mixed mark solution 1 μ L sample introduction, by the method having built up in one day
Replication six times and points of six days replications six times, under multiple-reaction monitoring pattern, calculate retention time and object respectively
The relative standard deviation (RSD) of peak area response, by calculating retention time and the relative standard deviation of peak area response,
In a few days and in the daytime repeatability for evaluation experimental method.Experimental result is as shown in table 4, the peak area of 17 kinds of polyphenols
The in a few days variation of response value is at 1.20%-5.85%, and peak area makes a variation in the daytime as 3.53%-12.65%, 17 kinds of Polyphenols things
The retention time of matter in a few days makes a variation at 0.06%-1.40%, and retention time makes a variation in the daytime at 0.07%-1.39%.The most in the daytime
The relative standard deviation of retention time and the most in the daytime peak area all < 15%, illustrate that the basic repeatability of the inventive method is good.
The analysis characteristic quantity of the LC-MS/MS detection method of 4 three ten one kinds of polyphenol compounds of table
The optimization of experimental example 1 Solid-Phase Extraction condition
1, the selection of solid-phase extraction column
The present invention have selected four kinds of solid phase extraction columns, be respectively Waters Oasis HLB post (500mg, 6ml),
Waters Sep-Pak C18 post (500mg, 6ml), Agilent Bond Elut Plexa PAX (500mg, 6ml), Agela
Cleanert PEP-2 post (500mg, 6ml).Final selection Waters Oasis HLB post is little as the Solid-Phase Extraction of the present invention
Post.
2, the optimization of Solid-Phase Extraction sample solution pH
When using solid phase extraction method purification of target compound, it is necessary to consider that target compound and interference impurity are in difference
State under the conditions of pH.Phenolic metabolism thing is under neutral or basic conditions in ionic condition, so phenolic metabolism thing is existed by pH value
The response rate on solid phase extraction column has a significant impact, and needs to be optimized loading pH value.
First compound concentration is the mixed sample totally 5 parts of 31 kinds of phenolic metabolism things of 80 μ g/L, volume 5mL, formic acid
Adjust its pH respectively to 2,3,4,5,6.HLB solid phase extraction column is activated with 5ml methanol and 5ml water, difference loading difference pH value
Sample solution, with 3ml water wash SPE pillar, vacuum uses 6ml methanol-eluted fractions after draining, and nitrogen is blown near dry, finally multiple with 1ml methanol
Molten, cross film, upper LC-MS/MS detects (liquid chromatography mass spectrometric condition is with embodiment 1), calculates the response rate, investigates little in HLB Solid-Phase Extraction
The optimum pH of 31 kinds of phenolic metabolism things of target on post.Result is as shown in Figure 1.
It can be seen that being gradually increased along with pH, gallic acid, coumaric acid, the caffeinic response rate gradually drop
Low, the also decrease to some degree of the response rate of other compounds.When low pH, the response rate of these 31 kinds of phenolic compounds
Higher, this is because in acid condition, the polarity of phenolic acid declines, the absorption to polyphenols of the beneficially SPE post.Work as pH=
When 3, overwhelming majority phenolic metabolism thing adsorption effect on SPE pillar is best, and the response rate is the highest.So considering, select
The optimum pH of sample solution is 3.
3, the optimization of Solid-Phase Extraction leacheate ratio
In solid phase extraction procedure, the effect of leacheate is washed miscellaneous exactly.The Solid-Phase Extraction leacheate that the present invention selects is water.
4, the optimization of Solid-Phase Extraction leacheate volume
In solid phase extraction procedure, not only leacheate affects the response rate of object than regular meeting, if leacheate volume is excessive,
Also absorption object on solid phase extraction column can be washed off, if but leacheate volume is too small, just do not reach miscellaneous effect of washing, institute
With needs, leacheate volume is optimized.The Solid-Phase Extraction leacheate volume that the present invention selects is 4ml.
5, the optimization of Solid-Phase Extraction effluent volume
In solid phase extraction procedure, eluent must have good solubility to object, could by target compound from
Elute on solid phase extraction column.Phenolic metabolism thing all has good dissolubility in methanol, so selecting methanol as washing
De-liquid.The volume of eluent determines the eluting power of eluent equally.The present invention selects the suitableeest eluting liquid of Solid-Phase Extraction
Amass as 6ml.
The optimization of experimental example 2 sample extraction condition
1, Extraction solvent selects
Accurately weigh 1.00g (being accurate to 0.01g) corn sample, solid-liquid ratio 1:20, be separately added into 20ml methanol, ethanol,
Acetonitrile, ethyl acetate, ultrasonic (600W) 20min, 10000r min-1, 4 DEG C of centrifugal 10min, extract twice, then according to implement
The method purification of 1.1.3 in example 1, the phenolic metabolism thing content summation that will record, it is designated as total amount, by calculating the extraction ratio of total amount
Selecting the Extraction solvent of optimum, result is as shown in Figure 2.
As shown in Figure 2, using methanol when being Extraction solvent, in corn sample, the extraction yield of all phenolic metabolism things is
Height, illustrates that phenolic metabolism thing dissolubility in methanol is more preferable.So selecting methanol as Extraction solvent.
2, the optimization of Extraction solvent ratio
Accurately weigh 1.00g (being accurate to 0.01g) corn sample, solid-liquid ratio 1:20, be separately added into 20ml 50%, 60%,
70%, the methanol (volume fraction) of 80%, 90%, 100%, ultrasonic (600W) 20min, 10000r min-1, 4 DEG C are centrifuged
10min, extracts twice, then according to the method purification of 1.1.3 in embodiment 1, selects carrying of optimum by calculating extraction ratio
Take solvent.Result is as shown in Figure 3.
During from the figure 3, it may be seen that the ratio of Extraction solvent methanol is 70%, in corn sample, all phenolic metabolism things extracts
Rate is the highest, along with gradually rising of methanol concentration, extraction ratio is gradually lowered, and this is possibly due to the methanol dissolution of high concentration more
Polymictic reason.So selecting methanol is 70%.
3, the optimization of solid-liquid ratio
Accurately weigh 1.00g (being accurate to 0.01g) corn sample, according to solid-liquid ratio 1:10,1:20,1:30,1:40,1:50
(g/ml), it is separately added into the methanol of 70%, ultrasonic (600W) 20min, 10000r min-1, 4 DEG C of centrifugal 10min, extract twice,
Then according to the method purification of 1.1.3 in embodiment 1, select the Extraction solvent of optimum by calculating extraction ratio.Result such as Fig. 4
Shown in.
As shown in Figure 4, when solid-liquid ratio is 1:20, in corn sample, the extraction yield of all phenolic metabolism things is the highest.Along with
Increasing of Extraction solvent, extraction ratio is gradually reduced, this is because more solvent adds the time of concentration on the contrary, loss amount is also
Can relatively increase.So selecting solid-liquid ratio is 1:20.
4, the optimization of ultrasonic time
Accurately weigh 1.00g (being accurate to 0.01g) corn sample, according to feed liquid 1:20 (g/ml), add the methanol of 70%
(volume fraction), respectively ultrasonic (600W) 0,10,20,30,40,50min, 10000r min-1, 4 DEG C of centrifugal 10min, extract two
Secondary, then according to the method purification of 1.1.3 in embodiment 1, select the Extraction solvent of optimum by calculating extraction ratio.Result is such as
Shown in Fig. 5.
As shown in Figure 5, when ultrasonic time is 20min, in corn sample, the extraction yield of all phenolic metabolism things is the highest；Super
The sound time is the longest, and extraction ratio does not has significant change, so selecting ultrasonic time is 20min.
The evaluation of the HPLC-MS/MS detection method performance of experimental example 3 transgenic corns of the present invention secondary metabolites
1, the range of linearity of method
For investigating the range of linearity of experimental technique, prepare 0.2 μ g L respectively-1、1μg L-1、2μg L-1、5μg L-1、10μ
g L-1、20μg L-1、40μg L-1、60μg L-1、80μg L-1、100μg L-131 kinds of phenolic metabolism things mixing mark liquid
5mL, after the Solid-Phase Extraction condition optimized by the present invention purifies, the LC-MS/MS condition optimized by embodiment 1 is to it
Detect, measure the peak area of object, draw peak area-concentration standard curve.It is 80 μ g L by concentration-1Phenolic metabolism
Thing mixed solution 5ml is parallel enrichment 6 times on HLB post, measure its peak area, calculate RSD.Correlation analysis characteristic quantity is shown in Table 5.
The analysis characteristic quantity of the LC-MS/MS detection method of table 5 HLB 31 kinds of polyphenols of enrichment
Test result indicate that, most phenolic metabolism things are at 10-500 μ g L-1Concentration range internal linear relation is good, and
Coefficient R2It is all higher than 0.9990.Dense by during three times of baseline noise that response signal is blank solvent of target compound
Spending the detection limit as method, finally its detection is limited to 5-100 μ g L-1.The standard deviation (RSD) of peak area is at 4.75%-
Between 14.68%, illustrate that the detection method sensitivity that the present invention sets up is good, have preferable repeatability and stability, therefore can
It is enough in the analysis of phenolic metabolism thing.
2, the evaluation of matrix effect
Substrate refers to that the component beyond analyte in sample, the often analysis to analyte interfere significantly with, and impact point
The accuracy of analysis result, these impacts and interference are referred to as matrix effect.In liquid chromatography mass spectrometric detects, it should be noted that avoid substrate to imitate
Should.The evaluation of matrix effect has two kinds of methods, is injection and additive process after extracting after post respectively.What the present invention chose is to extract
Rear additive process.After extraction additive process be blank sample is carried out extraction and cleaning by pre-treating method after, then in extracting solution add
Determinand, is detected by existing liquid chromatography mass spectrometric condition, with as the neat solvent of concentration compare, calculate the relative of them
Ratio evaluates matrix effect.In LC-MS/MS, absolute base mass effect (%)=matrix matching standard substance peak area/standard substance
Peak area × 100%.When absolute base mass effect is between 85%-115%, then it is assumed that matrix effect is inconspicuous, experimental technique
Feasible.
Having prepared concentration with corn sample redissolution liquid after Solid-Phase Extraction and methanol respectively is 400 μ g L-131
Kind of phenolic metabolism thing mixing mark liquid, often organize all do three parallel, after LC-MS/MS detects, obtain 31 kinds of phenolic metabolism things
Matrix effect be 93.17%-113.02%, relative standard deviation RSD between 0.94%-7.41%, illustrate it is believed that
In corn sample there is not matrix effect in the LC-MS/MS detection of these 31 kinds of phenolic metabolism things, it is possible to for actual sample
Detection.
3, substrate recovery testu
Whether it is applicable to the detection of actual sample for further confirmatory experiment method, needs to carry out substrate mark-on and reclaim real
Test.Weighing 1g (being accurate to 0.01g) sample respectively, select basic, normal, high three mark-on levels, adding concentration respectively is 50 μ g L-1、200μg L-1、500μg L-1The mixing mark liquid of 31 kinds of phenolic metabolism things, after extracted purification, carry out LC-MS/MS
Detection (with embodiment 1).Each mark-on level do three times parallel, result is as shown in table 6.
Table 6 three ten one kinds of phenolic metabolism things average recovery rate (%, RSD, n=3) in corn sample
As can be seen from Table 6, most phenolic acid and flavone recovery of standard addition in corn sample are at 50.35%-
Between 110.69%, relative standard deviation is between 0.51%-13.91%.Illustrate that the pre-treating method of sample is good, it is possible to use
The LC-MS/MS detection of phenolic metabolism thing in actual corn sample.
4, the mensuration of actual sample
The present invention determines altogether 44 kinds of corn samples, wherein transgenic corns 30 kinds, non-transgenic corn 14 kinds.By jade
Rice sample is after the pre-treating method of 1.1.2 in embodiment 1 and the purification method of 1.1.3 process, according to the instrument of 1.1.4
Sample is measured by device method.By these 44 kinds of samples being measured discovery, all corn samples all do not contain tonkabean
These six kinds of flavone of acid, epicatechin, Quercetin, hyperin, phloretin, genistein and isorhamnetin.16 kinds of target phenol
In acid, in addition to the coumaric acid all not contained, other phenolic acid are nearly all present in corn seed, and content is each variant, but content
The highest is P-coumaric acid and trans-ferulaic acid.And in 15 kinds of target flavone, in addition to the 6 kinds of flavone all not contained, catechu
Element and naringenin are all common in all samples to be existed, and content is also had nothing in common with each other.
The present invention use further principal component analysis (PCA), Partial Least Squares Method (PLS-DA) and orthogonal partially
Young waiter in a wineshop or an inn's squares analysis (OPLS-DA) method determines that transgenic group and non-transgenic group are capable of separating, and utilizes VIP on this basis
(Variable Important Plot) value method, selects the metabolite that notable change occurs in conjunction with T test sieve.Experimental result is sieved
Select the VIP value metabolite more than 1 and have 6 (tables 7), be GAL, VAN, DIO, GALLO, SIN, FER respectively.
The table 7 VIP value metabolite more than 1
With P < 0.05 as significant level, determine the difference generation that VAN (vanillic acid) is transgenic corns and non-transgenic corn
Thank thing, and the relative amount that the relative amount (1.753) that VAN is in non-transgenic corn is higher than in transgenic corns
(0.932)。
Claims (10)
1. the HPLC-MS/MS detection method of a transgenic corns secondary metabolites, it is characterised in that comprise the following steps:
(1) extract the secondary metabolites in corn seed to be measured, obtain crude extract；(2) regulation crude extract pH, then goes up solid phase extraction
Take post to purify, collect eluent；(3) HPLC-MS/MS is used to carry out qualitative, quantitative determination.
2. according to the HPLC-MS/MS detection method described in claim 1, it is characterised in that step (1) described extraction jade to be measured
Secondary metabolites in rice seed includes: pulverized by corn seed to be measured, adds Extraction solvent, carries out supersound process, in collection
Clear liquid.
3. according to the HPLC-MS/MS detection method described in claim 2, it is characterised in that: described Extraction solvent is methanol, second
Any one in alcohol, acetonitrile or ethyl acetate, preferably methanol；It is furthermore preferred that described Extraction solvent is volume fraction 50-
The methanol of 100%, the most preferably methanol of volume fraction 70%.
4. according to the HPLC-MS/MS detection method described in claim 2, it is characterised in that: count according to g/ml, maize seed to be measured
Son is 1:10-50, preferably 1:20 with the solid-liquid ratio of Extraction solvent；The ultrasonic time of described supersound process is 0-50min, ultrasonic
Power is 400-800W；Preferably, ultrasonic time is 20min, and ultrasonic power is 600W.
5. according to the HPLC-MS/MS detection method described in claim 1, it is characterised in that step (3) described HPLC-MS/MS
Liquid-phase condition include: chromatographic column is Agilent ZORBAX SB-Aq chromatographic column；Column temperature is 25 DEG C；Flow velocity is 0.2ml/min-
0.3mL/min, preferably 0.2ml/min；Sampling volume is 1 μ L；Mobile phase A be volume fraction 0.002% to 0.01% formic acid-
Aqueous solution, Mobile phase B is acetonitrile；Gradient elution.
6. according to the HPLC-MS/MS detection method described in claim 5, it is characterised in that: described mobile phase A is volume fraction
0.002% formic acid-aqueous solution, Mobile phase B is acetonitrile；
The step of described gradient elution includes: according to volume fraction, 0min, 0%B；1min, 10%B；25min, 20%B；
33min, 30%B；34min, 40%B；38min, 40%B；39min, 70%B；41min, 70%B；43min, 0%B.
7. according to the HPLC-MS/MS detection method described in claim 1, it is characterised in that step (3) described HPLC-MS/MS
Mass Spectrometry Conditions include: ion source is electron spray ionisation source, anion scan pattern, and atomization gas is purity 99.999% nitrogen,
Collision gas is purity 99.999% nitrogen, and atomization gas pressure is 40psi, and dry temperature is 350 DEG C, and dry gas stream amount is 10L/
Min, capillary voltage is 3500V, and detection pattern is multiple-reaction monitoring pattern.
8. according to the HPLC-MS/MS detection method described in claim 1, it is characterised in that step (2) regulation crude extract pH is
2-6；Preferably, regulation crude extract pH is 3；It is furthermore preferred that regulate crude extract pH with formic acid；
Step (2) described solid-phase extraction column is Waters Oasis HLB post；
During the described upper solid-phase extraction column of step (2) purifies, leacheate is water, and eluent is methanol.
9. according to the HPLC-MS/MS detection method described in claim 1, it is characterised in that step (3) described qualitative, quantitatively survey
Surely include:
, quota ion identical with the parent ion mass-to-charge ratio of secondary metabolites standard substance according to secondary metabolites to be measured and qualitative ion
Identical, and retention time is identical, is defined as target secondary metabolites；
Concentration according to secondary metabolites standard substance and peak area make standard curve, by target secondary metabolism in corn seed to be measured
The peak area of thing brings corresponding linear equation into, calculates the respective concentration of target secondary metabolites.
10. according to the HPLC-MS/MS detection method described in claim 1, it is characterised in that: described secondary metabolites includes: fragrant
Bean acid, gallic acid, vanillic acid, rutin, benzoic acid, catechin, hyperin, syringic acid, P-coumaric acid, para hydroxybenzene first
Acid, sinapic acid, epicatechin, gentisic acid, trans-ferulaic acid, caffeic acid, protocatechuic acid, (-)-times catechuic acid, high protocatechuic acid,
4-hydroxy-3-methoxy-.alpha.-toluic acid., apigenin, cinnamic acid, aromadendrin, luteolin, Quercetin, eriodictyol, phloretin, hesperetin, naringenin, different
Any one or more in rhamnetin, diosmetin or genistein；
The Mass Spectrometry Conditions of described secondary metabolites includes:
The parent ion of coumaric acid is 139.09, and quota ion is 95.2, and qualitative ion is 51.2；
The parent ion of gallic acid is 169.12, and quota ion is 125, and qualitative ion is 151；
The parent ion of vanillic acid is 167.15, and quota ion is 108, and qualitative ion is 91.2；
The parent ion of rutin is 609.52, and quota ion is 301.1, and qualitative ion is 271.2；
Benzoic parent ion is 121.12, and quota ion is 77, and qualitative ion is 77；
The parent ion of catechin is 289.27, and quota ion is 245.2, and qualitative ion is 203；
The parent ion of hyperin is 463.4, and quota ion is 300, and qualitative ion is 271；
The parent ion of syringic acid is 197.17, and quota ion is 121.1, and qualitative ion is 153.3；
The parent ion of P-coumaric acid is 163.16, and quota ion is 119, and qualitative ion is 93.1；
The parent ion of P-hydroxybenzoic acid is 137.12, and quota ion is 93.0, and qualitative ion is 65.3；
The parent ion of sinapic acid is 223.21, and quota ion is 149, and qualitative ion is 164；
The parent ion of epicatechin is 289.27, and quota ion is 245.2, and qualitative ion is 203；
The parent ion of gentisic acid is 153.12, and quota ion is 108, and qualitative ion is 108；
The parent ion of trans-ferulaic acid is 193.18, and quota ion is 134.3, and qualitative ion is 134.3；
Caffeinic parent ion is 179.16, and quota ion is 135.1, and qualitative ion is 133.9；
The parent ion of protocatechuic acid is 153.12, and quota ion is 109.2, and qualitative ion is 109.2；
The parent ion of (-)-times catechuic acid is 305.27, and quota ion is 125, and qualitative ion is 137；
The parent ion of high protocatechuic acid is 167.15, and quota ion is 123, and qualitative ion is 123；
The parent ion of 4-hydroxy-3-methoxy-.alpha.-toluic acid. is 181.17, and quota ion is 137.1, and qualitative ion is 137.1；
The parent ion of apigenin is 270.24, and quota ion is 117, and qualitative ion is 117；
The parent ion of cinnamic acid is 148.16, and quota ion is 103, and qualitative ion is 77.3；
The parent ion of aromadendrin is 288.25, and quota ion is 125.1, and qualitative ion is 285.9；
The parent ion of luteolin is 286.24, and quota ion is 132.8, and qualitative ion is 175.1；
The parent ion of Quercetin is 302.24, and quota ion is 151.1, and qualitative ion is 178.9；
The parent ion of eriodictyol is 288.25, and quota ion is 151.3, and qualitative ion is 135.3；
The parent ion of phloretin is 274.27, and quota ion is 167.4, and qualitative ion is 123.3；
The parent ion of hesperetin is 302.28, and quota ion is 164, and qualitative ion is 285.9；
The parent ion of naringenin is 272.25, and quota ion is 151, and qualitative ion is 119.1；
The parent ion of isorhamnetin is 316.25, and quota ion is 300.1, and qualitative ion is 300.1；
The parent ion of diosmetin is 300.26, and quota ion is 284, and qualitative ion is 227；
The parent ion of genistein is 270.24, and quota ion is 133.3, and qualitative ion is 63.3.
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