CN106153800B - The HPLC-MS/MS detection methods of transgenic corns secondary metabolites - Google Patents

The HPLC-MS/MS detection methods of transgenic corns secondary metabolites Download PDF

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CN106153800B
CN106153800B CN201610460995.9A CN201610460995A CN106153800B CN 106153800 B CN106153800 B CN 106153800B CN 201610460995 A CN201610460995 A CN 201610460995A CN 106153800 B CN106153800 B CN 106153800B
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CN106153800A (en
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王俊英
王俊平
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Biotechnology Research Institute of CAAS
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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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials

Abstract

The invention discloses a kind of HPLC MS/MS detection methods of transgenic corns secondary metabolites, belong to the LC-MS detection field of corn secondary metabolites.The HPLC MS/MS detection methods of transgenic corns secondary metabolites of the present invention, include the following steps：(1) secondary metabolites in corn seed to be measured is extracted, crude extract is obtained；(2) crude extract pH is adjusted, then upper solid-phase extraction column is purified, and collects eluent；(3) HPLC MS/MS is used to carry out qualitative, quantitative determination.The present invention passes through the optimization to the extraction conditions of phenolic metabolism object, SPE condition, liquid-phase condition and Mass Spectrometry Conditions etc. in corn sample, the HPLC MS/MS detection methods of the transgenic corns secondary metabolites of foundation, sensitivity is good, there are preferable reproducibility and stability, there is no matrix effects, can be used in the detection and analysis of practical transgenic corns and phenolic metabolism object in non-transgenic corn sample.

Description

The HPLC-MS/MS detection methods of transgenic corns secondary metabolites
Technical field
The present invention relates to phenols in the detection method of transgenic corns secondary metabolites more particularly to transgenic corn seed The liquid chromatography-tandem mass of metabolin belongs to the LC-MS detection field of corn secondary metabolites.
Background technology
Phenols secondary metabolites is secondary metabolites important in plant, can help to resist various pathogens, evil The harm of worm, nematode and other plant.Phenols secondary metabolites is that crop itself is resisted in pest and disease damage approach and plays critical function A metabolite, whether the importing of external source insect resistance protein can influence the variation of crop self-defense pest and disease damage mechanism, Yi Jikang Whether degradation process of the worm albumen in crop body will produce the phenols secondary metabolites of specificity, beautiful to evaluation 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, it is used widely with its good separating effect and higher sensitivity.Establish that a kind of efficiently concentrating is pest-resistant to turn base Because of the method for secondary metabolites (flavones, phenolic acid) in corn seed, by the difference generation for analyzing transgenosis and non-transgenic corn It thanks to object, a kind of new technological means will be provided for the safety for evaluating transgenic corns.
Invention content
The technical problem to be solved by the present invention is to establish a kind of HPLC-MS/MS inspections of transgenic corns secondary metabolites Survey method, this method have many advantages, such as that sensitivity is good, good reproducibility and stability, can be used in transgenic corns and turn base with non- Because of the detection and analysis of phenolic metabolism object in corn sample.
In order to solve the above technical problems, the technical solution used in the present invention is：
The invention discloses a kind of HPLC-MS/MS detection methods of transgenic corns secondary metabolites, including following step Suddenly：(1) secondary metabolites in corn seed to be measured is extracted, crude extract is obtained；(2) crude extract pH is adjusted, then upper solid phase extraction It takes column to be purified, collects eluent；(3) HPLC-MS/MS is used to carry out qualitative, quantitative determination.
Wherein, the secondary metabolites in step (1) the extraction corn seed to be measured includes：By corn seed powder to be measured It is broken, Extraction solvent is added, is ultrasonically treated, supernatant is collected by centrifugation；Said extracted step is repeated 1-2 times, by each step institute Supernatant merge to get.The Extraction solvent is any one in methanol, ethyl alcohol, acetonitrile or ethyl acetate, preferably first Alcohol；It is furthermore preferred that the Extraction solvent is the methanol of volume fraction 50-100%, the most preferably methanol of volume fraction 70%. It is counted according to g/ml, the solid-liquid ratio of corn seed and Extraction solvent to be measured is 1：10-50, preferably 1：20；The supersound process Ultrasonic time is 0-50min, ultrasonic power 400-800W；Preferably, ultrasonic time 20min, ultrasonic power 600W.
In HPLC-MS/MS detection methods of the present invention, the liquid-phase condition of step (3) described HPLC-MS/MS includes：Color Spectrum column is Agilent ZORBAX SB-Aq chromatographic columns；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 is 0.002% to 0.01% formic acid of volume fraction-aqueous solution, and Mobile phase B is Acetonitrile；Gradient elution.Preferably, the mobile phase A is 0.002% formic acid of volume fraction-aqueous solution, and Mobile phase B is acetonitrile；Institute The step of stating gradient elution include：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 include：Ion source is that (anion scans mould in electron spray ionisation source Formula), atomization gas is 99.999% nitrogen of purity, and collision gas is 99.999% nitrogen of purity, and atomization gas pressure is 40psi, dry Temperature degree is 350 DEG C, dry gas stream amount 10L/min, capillary voltage 3500V, and detection pattern is multiple-reaction monitoring pattern (MRM)。
The HPLC-MS/MS detection methods of transgenic corns secondary metabolites of the present invention, step (2) adjust crude extract pH and are 2-6；Preferably, it is 3 to adjust crude extract pH；It is furthermore preferred that adjusting crude extract pH with formic acid；It specifically includes：By crude extract 40 DEG C rotary evaporation is redissolved to close dry, then with formic acid-aqueous solution of pH=3, and it is 3 to adjust crude extract pH.Step (2) the solid phase extraction It is Waters Oasis HLB columns to take column；During the upper solid-phase extraction column is purified, leacheate is water, and eluent is Methanol；The elution liquid nitrogen of collection is blown to after close do and is redissolved with methanol, vortex oscillation carries out LC-MS/MS inspections after centrifugation, filtering It surveys.
In HPLC-MS/MS detection methods of the present invention, step (3) the qualitative, quantitative determination includes：According to be measured Secondary metabolites is identical as the mother ion mass-to-charge ratio of secondary metabolites standard items, quota ion is identical as qualitative ion, and retains Time is identical, is determined as target secondary metabolites；Make standard curve according to the concentration of secondary metabolites standard items and peak area, it will The peak area of target secondary metabolites brings corresponding linear equation into corn seed to be measured, 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, Hyperoside, syringic acid, p-Coumaric Acid, P-hydroxybenzoic acid, sinapic acid, epicatechin, gentianic acid, trans- asafoetide Acid, caffeic acid, protocatechuic acid, (-)-times catechuic acid, high protocatechuic acid, homovanillic acid, apiolin, cinnamic acid, aromadendrin, sweet-scented osmanthus It is arbitrary in careless element, Quercetin, eriodictyol, phloretin, hesperetin, naringenin, Isorhamnetin, diosmetin or siskin isoflavonoid It is one or more.
The present invention obtains 30 a kind of optimal mass spectrum of phenolic metabolism object in corn seed by the optimization to Mass Spectrometry Conditions Condition specifically includes：
The parent ion of coumaric acid is 139.09, quota ion 95.2, and qualitative ion is 51.2；
The parent ion of gallic acid is 169.12, quota ion 125, and qualitative ion is 151；
The parent ion of vanillic acid is 167.15, quota ion 108, and qualitative ion is 91.2；
The parent ion of rutin is 609.52, quota ion 301.1, and qualitative ion is 271.2；
The parent ion of benzoic acid is 121.12, quota ion 77, and qualitative ion is 77；
The parent ion of catechin is 289.27, quota ion 245.2, and qualitative ion is 203；
The parent ion of Hyperoside is 463.4, quota ion 300, and qualitative ion is 271；
The parent ion of syringic acid is 197.17, quota ion 121.1, and qualitative ion is 153.3；
The parent ion of p-Coumaric Acid is 163.16, quota ion 119, and qualitative ion is 93.1；
The parent ion of P-hydroxybenzoic acid is 137.12, quota ion 93.0, and qualitative ion is 65.3；
The parent ion of sinapic acid is 223.21, quota ion 149, and qualitative ion is 164；
The parent ion of epicatechin is 289.27, quota ion 245.2, and qualitative ion is 203；
The parent ion of gentianic acid is 153.12, quota ion 108, and qualitative ion is 108；
The parent ion of trans-ferulaic acid is 193.18, quota ion 134.3, and qualitative ion is 134.3；
Caffeinic parent ion is 179.16, quota ion 135.1, and qualitative ion is 133.9；
The parent ion of protocatechuic acid is 153.12, quota ion 109.2, and qualitative ion is 109.2；
The parent ion of (-)-times catechuic acid is 305.27, quota ion 125, and qualitative ion is 137；
The parent ion of high protocatechuic acid is 167.15, quota ion 123, and qualitative ion is 123；
The parent ion of homovanillic acid is 181.17, quota ion 137.1, and qualitative ion is 137.1；
The parent ion of apiolin is 270.24, quota ion 117, and qualitative ion is 117；
The parent ion of cinnamic acid is 148.16, quota ion 103, and qualitative ion is 77.3；
The parent ion of aromadendrin is 288.25, quota ion 125.1, and qualitative ion is 285.9；
The parent ion of cyanidenon is 286.24, quota ion 132.8, and qualitative ion is 175.1；
The parent ion of Quercetin is 302.24, quota ion 151.1, and qualitative ion is 178.9；
The parent ion of eriodictyol is 288.25, quota ion 151.3, and qualitative ion is 135.3；
The parent ion of phloretin is 274.27, quota ion 167.4, and qualitative ion is 123.3；
The parent ion of hesperetin is 302.28, quota ion 164, and qualitative ion is 285.9；
The parent ion of naringenin is 272.25, quota ion 151, and qualitative ion is 119.1；
The parent ion of Isorhamnetin is 316.25, quota ion 300.1, and qualitative ion is 300.1；
The parent ion of diosmetin is 300.26, quota ion 284, and qualitative ion is 227；
The parent ion of siskin isoflavonoid is 270.24, quota ion 133.3, and qualitative ion is 63.3.
The liquid-phase condition of HPLC-MS/MS methods is optimized in the present invention.Chromatographic column optimum results show HILIC parents Water chromatographic column cannot good isolating target compound, will appear hangover and split phenomenon using SB-C18 chromatographic columns, and use SB-Aq chromatographic columns target compound can realize preferable separation, and peak type is symmetrically sharp, and trailing phenomenon is also obviously changed It is kind.
The optimum results of mobile phase show that Mobile phase B is the separating effect that the separating effect of acetonitrile is apparently higher than methanol, because This selects acetonitrile for Mobile phase B.The flow velocity selection result of mobile phase shows that flow velocity is the response ratio 0.3ml/ of 0.2ml/min The response of min is high, so selecting 0.2ml/min to detach flow velocity.Mobile phase acid, alkali, salt selection result show using first When aqueous acid-acetonitrile is as mobile phase, the separating effect of compound is best, and a small amount of acid is added can reach improvement peak Type reduces the purpose of hangover, therefore a small amount of volatile acid is added in selection in water phase.Further to the selection table of organic acid It is bright, in acetic acid water-acetonitrile mobile phase system, certain object gallic acids, protocatechuic acid, times catechuic acid, gentianic acid, quercitrin The response of element etc. selects formic acid water-acetonitrile mobile phase system than low in formic acid water-acetonitrile mobile phase system. The present invention further adds the formic acid of different volumes score into mobile phase, and as a result the aqueous formic acid of different volumes score is to changing Closing the separating degree of object influences less, but with the increase of aqueous formic acid concentration, removes gallic acid, gentianic acid, times catechuic acid, original Other than catechuic acid, the response of other compounds continuously decreases, and considers and selects volume fraction water-soluble for 0.002% formic acid Liquid.
If LC-MS of the present invention detection phenolic metabolism owner is detached by liquid chromatogram, by mass spectrum come into Row detection, and in the separation process of liquid chromatogram, gradient is most important link.The present invention, which optimizes gradient, to be tied Fruit shows using 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 other gradients are used, moieties cannot detach.
The present invention is also optimized corn sample extraction conditions.The selection result of Extraction solvent shows Extraction solvent For methanol when, it is ethyl alcohol, acetonitrile or acetic acid that the extraction yield of all phenolic metabolism objects, which is significantly higher than Extraction solvent, in corn sample Ethyl ester illustrates that the dissolubility of phenolic metabolism object in methyl alcohol is more preferable, therefore selects methanol as Extraction solvent.Further to extraction The optimum results of solvent ratios show methanol volume fraction be 50%-100% within the scope of, when methanol be 70% when, corn-like The extraction yield of all phenolic metabolism objects is significantly higher than the methanol of other volume fractions in product；As methanol concentration increases, extraction Rate continuously decreases.Therefore the present invention selects 70% methanol for Extraction solvent.Solid-liquid ratio optimum results show to count according to g/ml, material Liquor ratio is 1：When 20, the extraction yield highest of all phenolic metabolism objects in corn sample；With increasing for Extraction solvent, recovery rate It is gradually reduced.When ultrasonic time optimum results show that ultrasonic time is 20min, all phenolic metabolism objects carries in corn sample Acquirement rate highest；Ultrasonic time is longer, and recovery rate does not have significant change.
Phenolic metabolism object under neutral or basic conditions be in ionic condition, therefore pH value to phenolic metabolism object in Solid Phase Extraction The rate of recovery on column has a significant impact.The present invention shows in pH 2-6 ranges the optimum results of sample solution pH in Solid Phase Extraction Interior, as pH gradually increases, gallic acid, coumaric acid, the caffeinic rate of recovery continuously decrease, the rate of recovery of other compounds Decrease to some degree；As pH=3, adsorption effect of most phenolic metabolism objects on SPE columns is best, and the rate of recovery is most It is high.Therefore adjusting crude extract pH is selected to go up solid-phase extraction column again for 3.
Using HPLC-MS/MS detection methods of the present invention, a kind of concentration model of 30 polyphenols in 2.5-500 μ g/L In enclosing, linear relationship is good, and detection limit ranging from 2.5-50 μ g/L, related coefficient are all higher than 0.9991；When in a few days retaining in the daytime Between and in a few days the relative standard deviation of peak area is equal in the daytime<15%, illustrate that the basic reproducibility of the method for the present invention is good.It is deemed-to-satisfy4 The evaluation result of energy shows most phenolic metabolism objects in 10-500 μ g L-1Linear relationship is good in concentration range, and related Coefficients R2It is 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 establishes is good, there is preferable reproducibility and stability, Neng Gouyong In the analysis of corn phenolic metabolism object.
The evaluation result of matrix effect shows to obtain a kind of 30 matrix effects of phenolic metabolism object after LC-MS/MS is detected 93.17%-113.02% is should be, relative standard deviation RSD illustrates between 0.94%-7.41% 31 in corn sample Matrix effect is not present in the LC-MS/MS detections of kind phenolic metabolism object.Matrix recovery testu the result shows that, most phenol The recovery of standard addition of acid and flavones in corn sample is between 50.35%-110.69%, and relative standard deviation is in 0.51%- Between 13.91%.Illustrate that the pre-treating method of sample is good, can be used in the LC-MS/ of phenolic metabolism object in practical corn sample MS is detected.
The HPLC-MS/MS detection methods of transgenic corns secondary metabolites of the present invention can be applied to transgenic corns or The detection and analysis of phenols secondary metabolites in non-transgenic corn.Using the HPLC- of transgenic corns secondary metabolites of the present invention MS/MS detection methods are measured 30 kinds of insect-resistant transgenic corns and 14 kinds of non-transgenic corns, determine VAN (vanillic acid) For the difference metabolin of transgenic corns and non-transgenic corn, and relative amounts (1.753) of the VAN in non-transgenic corn It is higher than the relative amount (0.932) in transgenic corns.
Technical solution of the present invention compared with prior art, has the advantages that：
The present invention by extraction conditions of phenolic metabolism object in corn sample, SPE condition, liquid-phase condition and The optimization of Mass Spectrometry Conditions establishes a kind of HPLC-MS/MS detection methods of transgenic corns secondary metabolites.The present invention detects Method sensitivity is good, there is preferable reproducibility and stability, and matrix effect is not present, can be used in practical transgenosis and non-turn The detection and analysis of phenolic metabolism object in gene corn sample.The HPLC-MS/MS of transgenic corns secondary metabolites of the present invention is detected Method provides a kind of new technological means to evaluate the safety of transgenic corns by difference metabolite analysis.
Description of the drawings
Fig. 1 is the optimization of sample solution pH value；Wherein, GAL be gallic acid, COU be coumaric acid, GALLO be (-)-extremely Boheic acid, HPRO are high protocatechuic acid, PRO is protocatechuic acid, DHB is gentianic acid, HYB is P-hydroxybenzoic acid, EPI is table catechu Element, HVAN are homovanillic acid, VAN is vanillic acid, CAT is catechin, SYR is syringic acid, CAF is caffeic acid, BEN is benzene first Acid, P-COU are p-Coumaric Acid, FER is trans-ferulaic acid, RUT is rutin, HYP is Hyperoside, SIN is sinapic acid, DKF is Aromadendrin, CIN are cinnamic acid, QUE is Quercetin, ERI is eriodictyol, LUT is cyanidenon, IHN is Isorhamnetin, HES is Hesperetin, DIO are diosmetin, PHL is phloretin, NAR is naringenin, API is apiolin, GEN is siskin isoflavonoid；
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.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1, material
30 kinds of transgenic corns sample, be respectively 1420, nine jade L06, D1325 of moral list, YD238, mf222, LV1341, LD60, K60, No. 1-22 and from number from 1-21 corns, derive from Biological Technology institute, Chinese Academy of Agricultural Sciences's transgenosis Plant microbial environment supervision and inspection on safety test center.
14 kinds of non-transgenic corn sample is that force 312, high 230, Ju be rich 558, first jade 335, beautiful No. 2 expensive, carbuncle 1 respectively Number, week is No. 1 glutinous, Zheng Dan 958, it is 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.
A kind of 30 standard items (specific name is shown in Table 2) of phenolic metabolism object are purchased from Sigma Co., USA.
The HPLC-MS/MS of 1 transgenic corns secondary metabolites of embodiment is detected
1, experimental method
1.1 sample-pretreating method
1.1.1 the collection and preparation of corn sample
By 44 kinds of corn samples (wherein 30 kinds of transgenic corns sample, 14 kinds of non-transgenic corn sample), each corn After sample dries, powdered, the sieve of 60 mesh excessively is blended into pulverizer.Each corn flour is individually fitted into valve bag, mark is carried out Note, is placed under drying at room temperature environment and preserves.
1.1.2 the extraction of corn phenolic acid and flavones
1.00g (being accurate to 0.01g) corn sample accurately is weighed, is placed in 50ml tool plug centrifuge tubes, 20ml 70% is added Methanol (volume fraction), vortex 10s ultrasound (600W) 20min after mixing, in 10000r min-1, 4 DEG C of centrifugation 10min, It shifts in supernatant to 100ml round-bottomed flasks.Remaining solid residue extracts once again according still further to above-mentioned steps, will carry twice The supernatant taken merges, and is done to close in 40 DEG C of rotary evaporations, then redissolve and be settled to 5mL with formic acid-aqueous solution of pH=3, this Sample solution of the 5mL corn phenolic metabolism object crude extracts as SPE, remains to purify.
1.1.3 the purification of corn phenolic acid and flavones
Solid phase extraction column is Waters Oasis HLB columns (500mg, 6ml).Before Solid Phase Extraction loading, use first 5ml methanol and the activation of 5ml water and balance solid-phase extraction column, coutroi velocity is in 1mL/min, in the process, it should be noted that keeps solid Phase extraction column moistens.5ml sample solutions to be clean are added on SPE columns, control loading velocity ratio activation flow velocity is slow, waits for loading After outflow, the moisture in 5min removing SPE columns all is drained with 4ml water wash SPE columns, then vacuum for liquid.Last SPE columns 6ml Methanol elutes, and elution flow rate is also slow, and eluent is collected in test tube, and after eluent all outflow, it is small that vacuum drains SPE Column.It after elution liquid nitrogen is blown to close do, is redissolved with 1ml methanol, vortex oscillation 1min, 0.22 μm of organic filter membrane is crossed after centrifugation, into Row LC-MS/MS detections.
1.1.4 the measurement (HPLC-MS/MS) of corn phenolic acid and flavones
1.1.4.1 liquid-phase condition
Chromatographic column：Agilent ZORBAX SB-Aq chromatographic columns (2.1 × 150mm, 3.5 μm)；
Column temperature：25℃；
Flow velocity：0.2ml/min；
Sampling volume：1μL；
Stop acquisition time：48min；
Column presses equilibration time：14min；
Mobile phase：A：Formic acid (0.002%, volume fraction)-water；B：Acetonitrile；
Eluent gradient elution program is shown in Table 1：
1 eluent gradient elution step of table
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 degree：350℃；
Dry gas stream amount：10L/min；
Capillary voltage：3500V；
Detection pattern：Multiple-reaction monitoring pattern (MRM)；
Resolution ratio：Q1(unit)Q3(unit)；
Sweep time section and the substance of corresponding detection are as shown in table 2.
2 sweep time of table section and the substance of corresponding detection
1.1.5 LC-MS/MS is qualitative and quantitative approach
When carrying out the measurement of actual sample, it then follows the characteristics of more reaction detections (MRM) pattern that method uses, that is, pass through Retention time (RT) and the mass-to-charge ratio (m/z) of parent ion, daughter ion are come dual qualitative.If detected in sample and standard items Mother ion mass-to-charge ratio (m/z) is identical, and quota ion is identical as assisted quantitative ion (qualitative ion), and total ion current figure (TIC) The appearance time substance consistent with standard items appearance time, then it is believed that there are corresponding phenolic metabolisms in this kind of corn sample Object, thus come in corn sample phenolic acid and flavones carry out qualitative analysis.
This experiment quantitative determines actual sample using external standard method.Prepare a series of hybrid standard work of various concentrations Make liquid, and measure the peak area of each object under various concentration to draw concentration-peak area standard curve, thus comes to corn Phenolic acid and flavones 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
2.1.1.1 the selection of ion source
Ion source is the device for liquid phase molecule is atomized into gaseous state and it is made to charge, they have respective applicable model It encloses, wherein electron spray ionisation ESI and the most practical ion sources of atmospheric pressure chemical ionization APCI.
31 kinds of measured phenolic metabolism objects of this experiment belong to the substance of the easily ionizable of polarized, therefore select ESI electron sprays Source makes this 31 kinds of phenolic metabolism objects be ionized before entering mass analyzer as ion source.
2.1.1.2 the selection of positive negative mode
ESI electrospray ionization sources have two kinds of scan patterns of cation and anion.Wherein, positive ion mode is suitable for basic species The detection of matter, anion scan pattern are generally suitable for acidic sample.The structure of 31 kinds of phenolic metabolism objects of this measuring can Know there is hydroxy functional group, phenolic acid class to also have carboxyl functional group in all phenolic acid and flavones, be easy to lose a proton, fits It closes and uses anion scan pattern, therefore this experimental selection anion scan pattern is measured phenolic compound.
2.1.1.3 the selection of parent ion
Use 50% methanol as 30 a kind of single mark working solutions of phenolic metabolism object that solvent compound concentration is 100 μ g/L The chromatographic column of (standard items are purchased from Sigma Co., USA), liquid phase part is Zorbax SB-C18 1-Pack (Rapid Resolution Cartridge, 2.1 × 30mm i.d, 3.5 μm), mobile phase is water：Methanol (50:50), flow velocity 0.2ml/ Min, sample size are 1 μ L, and methanol is as blank sample and washes needle liquid.Under ESI negative ion modes, to a kind of this 30 phenolic metabolism Object carries out first mass spectrometric full scan pattern (MS2Scan) and sets mass-to-charge ratio appropriate according to the relative molecular weight of each metabolin Range finds molecular ion peak by mass spectrogram, determines the parent ion of object.Phenolic metabolism object is under ESI negative ion modes Ionization loses a proton, generates [M-H]-Molecular ion peak, if finding [M-H] in mass spectrogram-Molecular ion peak, and it is empty Without being somebody's turn to do [M-H] in white solvent-Molecular ion peak then can determine the ion [M-H]-It is the parent ion of target analytes.
2.1.1.4 the optimization of parent ion capillary outlet voltage (Fragmentor)
By taking p-Coumaric Acid (P-COU) as an example, the capillary outlet voltage of optimization parent ion m/z163.1.Scan pattern is set It is set to selection ion detection (SIM) pattern, i.e., only collects the parent ion of m/z 163.1, only allows the ion m/z for setting mass-to-charge ratio 163.1, by first level four bars (MS1), carry out the level-one scan pattern of single ion, are needed at this time to capillary outlet electricity Pressure optimizes, and keeps the ion transmission efficiency of required ion maximum, response highest.Be set separately Fragmentor be 75V, 85V, 95V, 105V compare the TIC total ion current figures of the parent ion m/z305.27 of P-COU under different capillary outlet voltages. When Fragmentor is 75V, the response highest of parent ion；When Fragmentor is more than 75V, the response of parent ion by It gradually reduces, this is because capillary outlet voltage is excessive, fragment ion can be caused to increase, parent ion is reduced.It is thus determined that P-COU Best capillary outlet voltage be 75V.
2.1.1.5 the selection of daughter ion
After the parent ion and corresponding capillary outlet voltage that each object is determined, needing, which makes parent ion enter, touches Hit pond.Mass spectrum pattern is set as daughter ion scan pattern (Product ion scan, PI), which only allows to allow defined mesh Mark parent ion enters collision cell, collides through different-energy to obtain fragment ion in collision cell, second level four bars (MS2) is to fragment Ion is scanned, and searches out the broken daughter ion of the parent ion from mass spectrogram, the highest son of selection wherein response from Son is used as quota ion, and the lower daughter ion of response is as assisted quantitative ion (qualitative ion).
2.1.1.6 the optimization of daughter ion collision energy
Obtain parent ion generation daughter ion after, need to optimize the collision energy of daughter ion, make every seed from Response highest of the son under optimum collision energy, as finally quantitative foundation.It is highest as fixed therefrom to select response Ion is measured, response is higher as assisted quantitative ion (qualitative ion).
2.1.1.7 30 a kind of Mass Spectrometry Conditions of phenolic metabolism object
A kind of 30 Mass Spectrometry Conditions of phenolic metabolism object are shown in Table 3.
The optimal Mass Spectrometry Conditions of table 30 nine kinds of phenolic acid and flavones
Note：A indicates quota ion；B indicates 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 sources, narrow diameter column is generally used in liquid phase part. This experiment has chosen three kinds of chromatographic columns and carries out method exploitations, respectively HILIC XBridgeTM Amide chromatographic columns, 2.1 × 150mm, 3.5 μm (Waters, Ireland)；Zorbax SB-C18 chromatographic columns, 2.1 × 150mm, 3.5 μm (Agilent, USA)；Zorbax SB-Aq chromatographic columns, 2.1 × 150mm, 3.5 μm (Agilent, USA).It is selected from 31 kinds of target compounds The larger compound of four kinds of polarity differences is P-hydroxybenzoic acid (HYB), coumaric acid (COU), p-coumaric acid (P-COU) respectively With trans-ferulaic acid (FER).Observe separation situation of these four compounds in different chromatographic columns.
The result shows that：(1) for HILIC hydrophilic chromatographic columns：HILIC hydrophilic Interaction Chromatography patterns, also there is referred to as reverse phase Chromatogram mode.Positive chromatography uses polar stationary phase and nonpolar liquid phase, reverse-phase chromatography to use nonpolar fixation Phase and polarity mobile phase, and HILIC hydrophilic chromatographics use the mobile phase of polar stationary phase and reverse-phase chromatography, in analytic process The middle organic phase (be typically acetonitrile) using high concentration often adds buffer salt in water phase.Reverse-phase chromatography is suitable for separating polar And low pole compound, very poor to some highly polar compounds reservations, HILIC hydrophilic Interaction Chromatographies are suitable for polar compound Separation.Different salt is added in present invention trial in water phase, observes the separation situation in these four objects.When in water phase When 10mM ammonium acetates are added, 10mM is added when 5mM ammonium acetates and 5mM ammonium formates being either added in water phase or in water phase When ammonium formate and 0.1% formic acid, either in the case of which kind of, four kinds of substances cannot all detach well.
(2) for SB-C18 chromatographic columns：SB-C18 chromatographic columns are conventional reverse-phase chromatographic columns, can be in acid mobile phase condition The lower preferable separation of realization, provides good stability and makes longest-lived, reproducibility is good.The use of pure water-acetonitrile is mobile phase, When using SB-C18 chromatographic columns as stationary phase, four kinds of substances can realize preferable separation, but since aldehydes matter contains phenol Hydroxyl is easy to be combined with stationary phase, causes trailing phenomenon.When therefore, using SB-C18 chromatographic columns, it may appear that hangover and split are existing As.
(3) SB-Aq chromatographic columns：SB-Aq chromatographic columns are best suited for increasing the acidity, alkalinity and polar compound for being difficult to detach Retention time, it is stronger than many traditional C18 liquid phase columns.There is strong reservation in the mobile phase of high-moisture, column longevity when low ph value Life length.SB-Aq columns have hydrophilic surface, make mobile phase even with 100% aqueous solution, it is possibility to have effect prevents solid Determine collapsing for phase.Highest can use the high temperature to 80 DEG C.The use of pure water-acetonitrile is mobile phase, makees using SB-Aq chromatographic columns For stationary phase when, four kinds of substances can realize preferable separation, and peak type is symmetrically sharp, trailing phenomenon also be improved significantly. So selecting SB-Aq chromatographic columns as separation chromatography column.
2.1.2.2 the flow velocity selection of mobile phase
When interface selects electron spray ESI ionization sources, Ionization Efficiency and chromatographic isolation are imitated in the selection of flow rate of mobile phase The influence of fruit is often opposite.In general, flow velocity is smaller, the efficiency of ionization is higher, but influences separating effect；Otherwise flow velocity Bigger, separating effect is better, but Ionization Efficiency is lower.So needing to take into account the two, it can both meet liquid phase part separation and want Higher electro-spray ionization efficiency can also be reached by asking, and be typically chosen flow velocity and detached in 0.2mL/min to 0.3mL/min It measures.The result shows that influence of the flow velocity of 0.2ml/min and 0.3ml/min to separating degree is not very big, but flow velocity is 0.2ml/ The response of the response ratio 0.3ml/min of min is high, so selecting 0.2ml/min to detach flow velocity.
2.1.2.3 the selection of mobile phase
In liquid chromatograph mass spectrography detection, the composition of mobile phase can be with the water phase of 100% organic phase to 100% Between change.But it is often used without 100% water phase, 100% water phase may can cause chromatography column life to reduce, and a large amount of Water phase can reduce Ionization Efficiency.Usually used mobile phase has water, methanol, acetonitrile, formic acid, second in LC-MS/MS systems Acid, ammonium formate and ammonium acetate.Mobile phase should have effumability, and easily slough, without using the buffer salt solution etc. of difficult volatilization, such as Phosphate buffer may cause pipeline and spray needle blocking etc..
In reverse-phase chromatography, mobile phase organic phase is typically chosen methanol or acetonitrile.Phenolic metabolism object is equal in methanol, acetonitrile There is good dissolubility, therefore advanced optimizes on this basis.From the results, it was seen that the separation that Mobile phase B is acetonitrile is imitated Fruit is apparently higher than the separating effect of methanol, so final choice acetonitrile is Mobile phase B.
2.1.2.4 the selection of mobile phase acid, alkali, salt
In the negative ion mode, suitable ammonium hydroxide is added in mobile phase or buffer salt (ammonium formate/ammonium acetate) perhaps can The response of compound is improved, separating degree is improved.
The total ion current figure that mobile phase A is 0.1% ammonium hydroxide is measured respectively, and mobile phase A is the total ion current of 5mM ammonium acetates Figure, mobile phase A are 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 As can be seen that when using ammonium hydroxide acetonitrile as mobile phase, the separating effect of compound is very poor, and all substances are all centered at two In a peak；When using acetic acid ammonium salt solution-acetonitrile as mobile phase, the separating effect of compound is poor, and baseline is higher；Make When using pure water acetonitrile as mobile phase, the separating effect of compound is preferable；When using aqueous formic acid-acetonitrile as mobile phase, The separating effect of compound is best, and a small amount of acid is added can reach improvement peak type, reduces the purpose of hangover.So final choosing It selects and adds a small amount of volatile acid in water phase.
Next the present invention is selected in the organic acid (formic acid/acetic acid) that mass spectrum can use.Divide in mobile phase It is 0.01% formic acid and 0.01% acetic acid not to be added to volume fraction.As a result, addition volume fraction is into mobile phase 0.01% formic acid and acetic acid, does not have significant change to separating degree.But in acetic acid water-acetonitrile mobile phase system, certain targets The response of object gallic acid, protocatechuic acid, times catechuic acid, gentianic acid, Quercetin etc. is than in formic acid water-acetonitrile mobile phase body It is low in system, so selection formic acid water-acetonitrile mobile phase system.
2.1.2.5 the optimization of Mobile Phase Additives volume fraction
The formic acid that different volumes score is added into mobile phase, can influence the pH of flow visualizing, to cause to separation The influence of degree and object response.The first of 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 μ l is added into the aqueous solution of 500ml respectively Acid, volume fraction are respectively 0.002%, 0.004%, 0.006%, 0.008%, 0.01%, and as mobile phase A, Mobile phase B is Acetonitrile, measure the mass spectrum response of 31 kinds of phenolic metabolism objects, and investigate peak shape, separating degree etc..
From the results, it was seen that the aqueous formic acid of different volumes score influences less the separating degree of compound.But with The continuous increase of aqueous formic acid concentration, removes gallic acid, gentianic acid, other than times catechuic acid, protocatechuic acid, other compounds Response continuously decrease, so consider, select volume fraction for 0.002% aqueous formic acid.
2.1.2.6 the optimization of mobile phase gradient
If LC-MS detection phenolic metabolism owner is detached by liquid chromatogram, examined by mass spectrum It surveys, and in the separation process of liquid chromatogram, gradient is most important link.When detaching various ingredients, isocratic elution It is difficult to which some wide mixtures of polarity range are separated, the advantage of gradient elution is the ratio by constantly adjusting mobile phase, The polarity of mobile phase can be changed, to be eluted in the substance of opposed polarity in different times section, reach separation Purpose.
When optimizing condition of gradient elution, a kind of 30 mixed mark solution of 500 μ g/L of polyphenols are prepared, flow velocity is 0.2mL/min, 1 μ L of sample introduction, a kind of 30 polyphenols of investigation are basically separated situation.This experiment passes through a large amount of pre- reality It tests, mainly optimizes four gradients：The total ion current figure under following four gradient (volume fraction) is measured 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 Hyperoside are equal Cannot detach, and (-)-in (a) times catechuic acid and gentianic acid are inseparable, (d) in catechin and syringic acid it is inseparable, (c) In all substances be attained by baseline separation, so finally determine gradient be (c).
2.1.2.7 the optimization of column temperature
The change of column temperature can generally influence the retention time of object, select 25 DEG C as separation temperature.
The analysis characteristic quantity of 2.2 detection methods
The 30 of 0.1,0.5,1,2.5,5,10,25,50,100,200,300,500 μ g/L of this experiment difference compound concentration It is mixed to measure various concentration for a kind of mixed standard solution of polyphenols, the 1 μ L sample introductions under the conditions of liquid chromatography mass spectrometric optimized The response for marking solution, establishes standard curve, the results are shown in Table 4.The result shows that a kind of 30 polyphenols are in 2.5-500 μ g/ Linear relationship is good in the concentration range of L, and detection limit ranging from 2.5-50 μ g/L, related coefficient are all higher than 0.9991.
With 300 μ g L-1The mixed mark 1 μ L sample introductions of solution of 17 kinds of polyphenols, in the method having had built up one day Replication six times and points of six days replications six times, under multiple-reaction monitoring pattern, calculate separately retention time and object The relative standard deviation (RSD) of peak area response, by calculating the relative standard deviation of retention time and peak area response, For the in a few days and in the daytime reproducibility of evaluation experimental method.Experimental result is as shown in table 4, the peak area of 17 kinds of polyphenols In 1.20%-5.85%, it is 3.53%-12.65%, 17 kinds of Polyphenols objects that peak area makes a variation in the daytime for the in a few days variation of response The retention time of matter in a few days makes a variation in 0.06%-1.40%, and retention time is made a variation in the daytime in 0.07%-1.39%.In the daytime in a few days Retention time and in a few days the relative standard deviation of peak area is equal in the daytime<15%, illustrate that the basic reproducibility of the method for the present invention is good.
A kind of analysis characteristic quantity of the LC-MS/MS detection methods of 4 three ten polyphenol compound of table
The optimization of 1 SPE condition of experimental example
1, the selection of solid-phase extraction column
The present invention has selected four kinds of solid phase extraction columns, be respectively Waters Oasis HLB columns (500mg, 6ml), Waters Sep-Pak C18 columns (500mg, 6ml), Agilent Bond Elut Plexa PAX (500mg, 6ml), Agela Cleanert PEP-2 columns (500mg, 6ml).Final choice Waters Oasis HLB columns are small as the Solid Phase Extraction of the present invention Column.
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 target compound and interference impurity in difference State under the conditions of pH.Phenolic metabolism object is under neutral or basic conditions ionic condition, so pH value exists to phenolic metabolism object The rate of recovery on solid phase extraction column has a significant impact, and needs to optimize loading pH value.
Compound concentration is a kind of 30 totally 5 parts of mixed samples of phenolic metabolism object of 80 μ g/L, volume 5mL, formic acid first Adjust its pH respectively to 2,3,4,5,6.HLB solid phase extraction columns are activated with 5ml methanol and 5ml water, difference loading difference pH value Sample solution, with 3ml water wash SPE pillars, vacuum is eluted after draining with 6ml methanol, and nitrogen, which is blown to, closely to be done, and finally uses 1ml methanol multiple It is molten, film is crossed, upper LC-MS/MS detects (liquid chromatography mass spectrometric condition is with embodiment 1), calculates the rate of recovery, investigates small in HLB Solid Phase Extraction The optimum pH of 30 a kind of phenolic metabolism object of target on column.The results are shown in Figure 1.
It can be seen from the figure that with the gradual increase of pH, gallic acid, coumaric acid, the caffeinic rate of recovery gradually drop It is low, the rate of recovery also decrease to some degree of other compounds.In low pH, a kind of this 30 rate of recovery of phenolic compound It is higher, this is because in acid condition, the polarity of phenolic acid declines, and is conducive to absorption of the SPE columns to polyphenols.Work as pH= When 3, adsorption effect of most phenolic metabolism objects on SPE pillars is best, rate of recovery highest.So considering, select The optimal pH of sample solution is 3.
3, the optimization of Solid Phase Extraction elution liquid proportional
In solid phase extraction procedure, the effect of leacheate be exactly wash it is miscellaneous.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, the rate of recovery of object can be influenced by not only eluting liquid proportional, if leacheate volume is excessive, Also the object being adsorbed on solid phase extraction column can be washed off, if but leacheate volume it is too small, miscellaneous effect of washing, institute is just not achieved To need to optimize leacheate volume.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 to object have good solubility, could by target compound from It is eluted on solid phase extraction column.Phenolic metabolism object has good dissolubility in methyl alcohol, so selecting methanol as washing De- liquid.The volume of eluent equally determines the eluting power of eluent.The present invention selects the most suitable eluting liquid of Solid Phase Extraction Product is 6ml.
The optimization of 2 sample extraction condition of experimental example
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, ethyl alcohol, Acetonitrile, ethyl acetate, ultrasound (600W) 20min, 10000r min-1, 4 DEG C centrifuge 10min, extract twice, then according to implementation The method purifying of 1.1.3, the phenolic metabolism object content measured is summed, total amount is denoted as in example 1, by the recovery rate for calculating total amount Select optimal Extraction solvent, the results are shown in Figure 2.
As shown in Figure 2, the extraction yield of all phenolic metabolism objects is most when the use of methanol being Extraction solvent, in corn sample Height illustrates that the dissolubility of phenolic metabolism object in methyl alcohol 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%, 80%, 90%, 100% methanol (volume fraction), ultrasound (600W) 20min, 10000r min-1, 4 DEG C of centrifugations 10min, extraction twice, then purify according to the method for 1.1.3 in embodiment 1, optimal carry are selected by calculating recovery rate Take solvent.The results are shown in Figure 3.
From the figure 3, it may be seen that when the ratio of Extraction solvent methanol is 70%, all phenolic metabolism objects extracts in corn sample Rate highest, with gradually rising for methanol concentration, recovery rate continuously decreases, this may be because the methanol of high concentration has dissolved out more Polymictic reason.So selecting methanol for 70%.
3, the optimization of solid-liquid ratio
1.00g (being accurate to 0.01g) corn sample accurately is weighed, according to solid-liquid ratio 1:10、1:20、1:30、1:40、1:50 (g/ml), 70% methanol, ultrasound (600W) 20min, 10000r min are separately added into-1, 4 DEG C centrifuge 10min, extract twice, Then it is purified according to the method for 1.1.3 in embodiment 1, optimal Extraction solvent is selected by calculating recovery rate.As a result such as Fig. 4 It is shown.
As shown in Figure 4, solid-liquid ratio 1：When 20, the extraction yield highest of all phenolic metabolism objects in corn sample.With Extraction solvent increases, and recovery rate is gradually reduced, this is because more solvent increases the time of concentration, loss amount instead It can relative increase.So selecting solid-liquid ratio for 1：20.
4, the optimization of ultrasonic time
1.00g (being accurate to 0.01g) corn sample accurately is weighed, according to feed liquid 1：20 (g/ml), are added 70% methanol (volume fraction), difference ultrasonic (600W) 0,10,20,30,40,50min, 10000r min-1, 4 DEG C of centrifugation 10min, extraction two It is secondary, it is then purified according to the method for 1.1.3 in embodiment 1, optimal Extraction solvent is selected by calculating recovery rate.As a result such as Shown in Fig. 5.
As shown in Figure 5, when ultrasonic time is 20min, the extraction yield highest of all phenolic metabolism objects in corn sample；It is super The sound time is longer, and recovery rate does not have significant change, so selecting ultrasonic time for 20min.
The evaluation of the HPLC-MS/MS detection method performances of 3 transgenic corns secondary metabolites of the present invention of experimental example
1, the range of linearity of method
To investigate the range of linearity of experimental method, 0.2 μ g L have been prepared 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-1A kind of 30 phenolic metabolism objects mix standard liquids 5mL, after the SPE condition optimized with the present invention is purified, the LC-MS/MS conditions optimized with embodiment 1 are to it It is detected, measures the peak area of object, draw peak area-concentration standard curve.With a concentration of 80 μ g L-1Phenolic metabolism Object mixed solution 5ml parallel enrichment 6 times on HLB columns, measure its peak area, calculate RSD.Correlation analysis characteristic quantity is shown in Table 5.
5 HLB of table is enriched with a kind of 30 analysis characteristic quantities of the LC-MS/MS detection methods of polyphenols
The experimental results showed that most phenolic metabolism objects are in 10-500 μ g L-1Linear relationship is good in concentration range, and Coefficient R2It is all higher than 0.9990.By the response signal of target compound be blank solvent baseline noise three times when it is dense The detection limit as method is spent, its final detection is limited to 5-100 μ g L-1.The standard deviation (RSD) of peak area is in 4.75%- Between 14.68%, illustrates that the detection method sensitivity that the present invention establishes is good, there is preferable reproducibility and stability, therefore energy It is enough in the analysis of phenolic metabolism object.
2, the evaluation of matrix effect
Matrix refers to the component other than analyte in sample, is often interfered significantly with to the analysis of analyte, and influences to divide The accuracy of result is analysed, these are influenced and interference is referred to as matrix effect.In liquid chromatography mass spectrometric detection, it should be noted that matrix is avoided to imitate It answers.There are two types of methods for the evaluation of matrix effect, are injection method and additive process after extraction after column respectively.What the present invention chose is extraction Additive process afterwards.Additive process is after blank sample is extracted purification by pre-treating method after extraction, then is added in extracting solution Determinand is detected by existing liquid chromatography mass spectrometric condition, is compared with the pure solvent of same concentration, calculates the opposite of them Ratio evaluates matrix effect.In LC-MS/MS, absolute matrix effect (%)=matrix matching standard items peak area/standard items Peak area × 100%.When absolute matrix effect is between 85%-115%, then it is assumed that matrix effect unobvious, experimental method It is feasible.
The redissolution liquid with corn sample after Solid Phase Extraction and methanol have prepared a concentration of 400 μ g L respectively-131 Kind of phenolic metabolism object mixes standard liquid, every group all do three it is parallel, after LC-MS/MS is detected, obtain a kind of 30 phenolic metabolism objects Matrix effect be 93.17%-113.02%, relative standard deviation RSD between 0.94%-7.41%, illustrate can consider Matrix effect is not present in a kind of LC-MS/MS detections of 30 phenolic metabolism object of this in corn sample, can be used in actual sample Detection.
3, matrix recovery testu
Whether it is suitable for the detection of actual sample for further confirmatory experiment method, needs to carry out matrix mark-on reclaims reality It tests.1g (being accurate to 0.01g) sample is weighed respectively, is selected basic, normal, high three mark-on levels, is added a concentration of 50 μ g L respectively-1、200μg L-1、500μg L-1A kind of 30 mixing standard liquids of phenolic metabolism object, after extracted purification, carry out LC-MS/MS It detects (with embodiment 1).Each mark-on level is done parallel three times, and the results are shown in Table 6.
A kind of 6 three ten phenolic metabolism object average recovery rate (%, RSD, n=3) in corn sample of table
As can be seen from Table 6, the recovery of standard addition of most phenolic acid and flavones in corn sample is in 50.35%- Between 110.69%, relative standard deviation is between 0.51%-13.91%.Illustrate that the pre-treating method of sample is good, Neng Gouyong The LC-MS/MS detections of phenolic metabolism object in practical corn sample.
4, the measurement of actual sample
The present invention determines 44 kinds of corn samples, wherein 30 kinds of transgenic corns, 14 kinds of non-transgenic corn altogether.It will be beautiful Rice sample is after the pre-treating method of 1.1.2 in embodiment 1 and the purification method of 1.1.3 are handled, according to the instrument of 1.1.4 Device method is measured sample.By being measured discovery to this 44 kinds of samples, tonka-bean is not contained in all corn samples This six kinds of flavones of acid, epicatechin, Quercetin, Hyperoside, phloretin, siskin isoflavonoid and Isorhamnetin.16 kinds of target phenol In acid, other than the coumaric acid be free of, other phenolic acid are nearly all present in corn seed, and content is different, but content Generally highest is p-Coumaric Acid and trans-ferulaic acid.And in 15 kinds of target flavones, other than the 6 kinds of flavones be free of, catechu Element and naringenin are all common existing in all samples, and content is also to have nothing in common with each other.
The present invention further use principal component analysis (PCA), Partial Least Squares Method (PLS-DA) and it is orthogonal partially most Small square law analysis (OPLS-DA) method determines that transgenosis group and non-transgenic group can realize separation, utilizes VIP on this basis (Variable Important Plot) value method is examined in conjunction with T and filters out the metabolin that significant changes occur.Experimental result is sieved It selects metabolin of the VIP values more than 1 and shares 6 (tables 7), be GAL, VAN, DIO, GALLO, SIN, FER respectively.
7 VIP values of table are more than 1 metabolin
With P<0.05 is the level of signifiance, determines that VAN (vanillic acid) is the difference generation of transgenic corns and non-transgenic corn Thank to object, and relative amounts (1.753) of the VAN in non-transgenic corn is higher than the relative amount in transgenic corns (0.932)。

Claims (3)

1. a kind of HPLC-MS/MS detection methods of transgenic corns secondary metabolites, which is characterized in that include the following steps：
(1) secondary metabolites in corn seed to be measured is extracted, crude extract is obtained；(2) crude extract pH is adjusted, then upper solid phase extraction It takes column to be purified, collects eluent；(3) HPLC-MS/MS is used to carry out qualitative, quantitative determination；
Wherein, the secondary metabolites in step (1) the extraction corn seed to be measured includes：Corn seed to be measured is crushed, is added Enter Extraction solvent, be ultrasonically treated, collects supernatant；The Extraction solvent is the methanol of volume fraction 70%；According to g/ml The solid-liquid ratio of meter, corn seed and Extraction solvent to be measured is 1：20；The ultrasonic time is 20min, ultrasonic power 600W；
The liquid-phase condition of step (3) described HPLC-MS/MS includes：Chromatographic column is Agilent ZORBAX SB-Aq chromatographic columns；Column Temperature is 25 DEG C；Flow velocity is 0.2ml/min；Sampling volume is 1 μ L；Mobile phase A is 0.002% formic acid of volume fraction-aqueous solution, stream Dynamic phase B is acetonitrile；Gradient elution；The step of 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 include：Ion source be electron spray ionisation source, anion scan pattern, Atomization gas is 99.999% nitrogen of purity, and collision gas is 99.999% nitrogen of purity, and atomization gas pressure is 40psi, dry temperature Degree is 350 DEG C, dry gas stream amount 10L/min, capillary voltage 3500V, and detection pattern is multiple-reaction monitoring pattern；
The secondary metabolites includes：Coumaric acid, gallic acid, vanillic acid, rutin, benzoic acid, catechin, Hyperoside, fourth Fragrant acid, p-Coumaric Acid, P-hydroxybenzoic acid, sinapic acid, epicatechin, gentianic acid, trans-ferulaic acid, caffeic acid, protocatechuic acid, (-)-times catechuic acid, high protocatechuic acid, homovanillic acid, apiolin, cinnamic acid, aromadendrin, cyanidenon, Quercetin, eriodictyol, Any one or more in phloretin, hesperetin, naringenin, Isorhamnetin, diosmetin or siskin isoflavonoid；
It is 3 that step (2), which adjusts crude extract pH,；Crude extract pH is adjusted with formic acid；
Step (2) described solid-phase extraction column is Waters Oasis HLB columns；
During step (2) the upper solid-phase extraction column is purified, leacheate is water, and eluent is methanol.
2. HPLC-MS/MS detection methods described in accordance with the claim 1, which is characterized in that step (3) qualitative, the quantitative survey Surely include：
, quota ion identical as the mother ion mass-to-charge ratio of secondary metabolites standard items and qualitative ion according to secondary metabolites to be measured It is identical, and retention time is identical, is determined as target secondary metabolites；
Make standard curve according to the concentration of secondary metabolites standard items and peak area, by target secondary metabolism in corn seed to be measured The peak area of object brings corresponding linear equation into, calculates the respective concentration of target secondary metabolites.
3. HPLC-MS/MS detection methods described in accordance with the claim 1, it is characterised in that：The mass spectrum item of the secondary metabolites Part includes：
The parent ion of coumaric acid is 139.09, quota ion 95.2, and qualitative ion is 51.2；
The parent ion of gallic acid is 169.12, quota ion 125, and qualitative ion is 151；
The parent ion of vanillic acid is 167.15, quota ion 108, and qualitative ion is 91.2；
The parent ion of rutin is 609.52, quota ion 301.1, and qualitative ion is 271.2；
The parent ion of benzoic acid is 121.12, quota ion 77, and qualitative ion is 77；
The parent ion of catechin is 289.27, quota ion 245.2, and qualitative ion is 203；
The parent ion of Hyperoside is 463.4, quota ion 300, and qualitative ion is 271；
The parent ion of syringic acid is 197.17, quota ion 121.1, and qualitative ion is 153.3；
The parent ion of p-Coumaric Acid is 163.16, quota ion 119, and qualitative ion is 93.1；
The parent ion of P-hydroxybenzoic acid is 137.12, quota ion 93.0, and qualitative ion is 65.3；
The parent ion of sinapic acid is 223.21, quota ion 149, and qualitative ion is 164；
The parent ion of epicatechin is 289.27, quota ion 245.2, and qualitative ion is 203；
The parent ion of gentianic acid is 153.12, quota ion 108, and qualitative ion is 108；
The parent ion of trans-ferulaic acid is 193.18, quota ion 134.3, and qualitative ion is 134.3；
Caffeinic parent ion is 179.16, quota ion 135.1, and qualitative ion is 133.9；
The parent ion of protocatechuic acid is 153.12, quota ion 109.2, and qualitative ion is 109.2；
The parent ion of (-)-times catechuic acid is 305.27, quota ion 125, and qualitative ion is 137；
The parent ion of high protocatechuic acid is 167.15, quota ion 123, and qualitative ion is 123；
The parent ion of homovanillic acid is 181.17, quota ion 137.1, and qualitative ion is 137.1；
The parent ion of apiolin is 270.24, quota ion 117, and qualitative ion is 117；
The parent ion of cinnamic acid is 148.16, quota ion 103, and qualitative ion is 77.3；
The parent ion of aromadendrin is 288.25, quota ion 125.1, and qualitative ion is 285.9；
The parent ion of cyanidenon is 286.24, quota ion 132.8, and qualitative ion is 175.1；
The parent ion of Quercetin is 302.24, quota ion 151.1, and qualitative ion is 178.9；
The parent ion of eriodictyol is 288.25, quota ion 151.3, and qualitative ion is 135.3；
The parent ion of phloretin is 274.27, quota ion 167.4, and qualitative ion is 123.3；
The parent ion of hesperetin is 302.28, quota ion 164, and qualitative ion is 285.9；
The parent ion of naringenin is 272.25, quota ion 151, and qualitative ion is 119.1；
The parent ion of Isorhamnetin is 316.25, quota ion 300.1, and qualitative ion is 300.1；
The parent ion of diosmetin is 300.26, quota ion 284, and qualitative ion is 227；
The parent ion of siskin isoflavonoid is 270.24, quota ion 133.3, and qualitative ion is 63.3.
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