CN109541097A - A kind of method for building up of agricultural chemical compound database and application - Google Patents
A kind of method for building up of agricultural chemical compound database and application Download PDFInfo
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Abstract
The present invention relates to Pesticides Testing technical fields, and in particular to a kind of method for building up of agricultural chemical compound database and application.The present invention is based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analyses to establish agricultural chemical compound database, the agricultural chemical compound database established using method provided by the invention, the agricultural chemical compound qualitatively screening in the case of no standard items may be implemented, and have the characteristics that high-throughput, accurate, easy and quick.
Description
Technical field
The present invention relates to Pesticides Testing technical fields, and in particular to a kind of method for building up of agricultural chemical compound database and answers
With.
Background technique
The appearance and development of pesticide, bring great convenience to our agricultural production.With explosive population growth, grain is asked
Topic highlights, and people enhance the dependence of pesticide, and usage amount increases substantially.Wherein fungicide and insecticide are using more frequency
Two numerous class pesticides, fungicide are usually used in the prevention and treatment of the Later growth or postharvest disease of crop；Insecticide due to its have low toxicity,
The features such as effect is excellent and agriculturally by the use of large area.These pesticides only rest on crop after the use of field on a small quantity
Upper doing the trick, it is most of then remain in soil or floating in air, enter water body environment by approach such as rainfall, leachings.
Enrichment and amplification of the pesticide by food chain, finally in people's cylinder accumulation, and the molecular structure of pesticide is more stable, big absolutely
Majority is difficult to be metabolized in vivo decomposition, excretion, brings irreversible injury to people's health, such as cancer, no
The diseases such as pregnant disease and endocrine disturbance are related with pollution by pesticides.
Currently used pesticide residue detection method includes Liquid Chromatography-Tandem Mass Spectrometry, high performance liquid chromatography, gas phase
Chromatography-tandem mass spectrometry and Electron-capture gas chromatography method, but these methods are usually required in the feelings for having standard items
The screening of untested compound can be realized under condition, and there is certain limitation in practical applications.
Summary of the invention
The purpose of the present invention is to provide a kind of method for building up of agricultural chemical compound database and applications, are mentioned using the present invention
The agricultural chemical compound database that the method for confession is established, can realize the accurate sieve to untested compound in the case where no standard items
It looks into, it is simple and convenient.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of method for building up of agricultural chemical compound database, comprising the following steps:
According to the title and molecular formula of agricultural chemical compound, the agriculture is established using MassHunterPCDLManager software
The initial data base of drug compound；
Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry is carried out to the standard sample solution of the agricultural chemical compound
Analysis, obtains the acquired original data of agricultural chemical compound standard sample；
Using the qualitative software of MassHunter, according to the initial data base of the agricultural chemical compound and the agricultural chemical compound
The acquired original data of standard sample, obtain the retention time of the agricultural chemical compound；When by the reservation of the agricultural chemical compound
Between input the initial data base of the agricultural chemical compound, obtain the Primary database of the agricultural chemical compound；
By the parent ion of the agricultural chemical compound, the retention time of the agricultural chemical compound and 4 different impact energies
Amount input ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry TargetedMS/MS acquisition interface, and be arranged fragment from
The karyoplasmic ratio scanning range of son, obtains the accurate molecular weight of the fragment ion；
Using the qualitative software of MassHunter, according to the Primary database of the agricultural chemical compound and the fragment ion
Accurate molecular weight obtains the accurate molecular weight of the fragment ion of the agricultural chemical compound；By the fragment of the agricultural chemical compound from
The accurate molecular weight of son inputs the Primary database of the agricultural chemical compound, obtains the second databases of the agricultural chemical compound,
Complete the foundation of the agricultural chemical compound database.
Preferably, in the standard sample solution of the agricultural chemical compound concentration of all agricultural chemical compounds independently be 10~
100μg/L。
Preferably, the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis liquid phase chromatogram condition includes:
Mobile phase: mobile phase includes mobile phase A and Mobile phase B, wherein mobile phase A is the aqueous formic acid of ammonium acetate, institute
The concentration for stating ammonium acetate in mobile phase A is 10mmol/L, and the volumn concentration of formic acid is 0.1%；The Mobile phase B is acetic acid
Formic acid-methanol aqueous solution of ammonium, the concentration of ammonium acetate is 10mmol/L in the Mobile phase B, and the volumn concentration of formic acid is
0.1~1.0%, the volumn concentration of methanol is 5~95%.
Gradient elution program: 0~5min, Mobile phase B increase to 90% by 20%；5~8min, Mobile phase B are maintained at
90%；8~10min, Mobile phase B are reduced by 90% to 20%；10~12min, Mobile phase B are maintained at 20%.
Preferably, carrying out chromatographic column used by the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry is analyzed is
Kinetex-C18 chromatographic column, specification are 2.1mm × 100mm, and packing material size is 2.6 μm；Column temperature is 35 DEG C.
Preferably, the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis Mass Spectrometry Conditions include:
Ion source is the double sources electrospray ionisation ESI of sheath stream, and scanning mode is positive ion mode, and the karyoplasmic ratio of reference ion is
121.05873 and 922.009798；Dry temperature degree is 300 DEG C, and dry gas stream speed is 10L/min；Atomization gas pressure is
207kPa；Sheath temperature degree is 300 DEG C, sheath gas 10L/min；Capillary voltage is 3100V, and fragmentation voltage is 110V, cone
Hole voltage is 35V；Full scan karyoplasmic ratio is 50~1000, sweep speed 3spectra/s.
Preferably, 4 different collision energies are 10eV, 20eV, 30eV and 40eV.
Preferably, the fragment ion karyoplasmic ratio scanning range is 50~450.
The agricultural chemical compound database obtained the present invention provides method for building up described in above-mentioned technical proposal is in screening pesticide
Application in compound.
Preferably, the described application the following steps are included:
Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis is carried out to pretreated sample to be tested, using agriculture
Primary database in drug compound database carries out level-one screening, when level-one Q-TOF score >=70, with the level-one screening
The compound obtained is target compound, Secondary Screening is carried out using the second databases in agricultural chemical compound database, when two
When grade Q-TOF score >=60, then untested compound is agricultural chemical compound that the Secondary Screening obtains in sample to be tested.
Preferably, the sample to be tested is water body example.
The present invention provides a kind of method for building up of agricultural chemical compound database, and the present invention is based on ultra performance liquid chromatographies
Agricultural chemical compound database is established in method-quadrupole time-of-flight mass spec-trometry analysis, the pesticide established using method provided by the invention
The agricultural chemical compound qualitatively screening in the case of no standard items may be implemented in compound database, and has high-throughput, accurate, letter
Just with it is quick the features such as, the agricultural chemical compound convenient for utilizing screening to go out selects corresponding standard items to carry out subsequent quantitation detection.
Detailed description of the invention
Fig. 1 is chromatographic peak comparison diagram of the Atrazine by different chromatographic columns.
Specific embodiment
The present invention provides a kind of method for building up of agricultural chemical compound database, comprising the following steps:
According to the title and molecular formula of agricultural chemical compound, the agriculture is established using MassHunterPCDL Manager software
The initial data base of drug compound；
Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry is carried out to the standard sample solution of the agricultural chemical compound
Analysis, obtains the acquired original data of agricultural chemical compound standard sample；
Using the qualitative software of MassHunter, according to the initial data base of the agricultural chemical compound and the agricultural chemical compound
The acquired original data of standard sample, obtain the retention time of the agricultural chemical compound；When by the reservation of the agricultural chemical compound
Between input the initial data base of the agricultural chemical compound, obtain the Primary database of the agricultural chemical compound；
By the parent ion of the agricultural chemical compound, the retention time of the agricultural chemical compound and 4 different impact energies
Amount inputs the acquisition method of the agricultural chemical compound, and the karyoplasmic ratio scanning range of fragment ion is arranged, obtain the fragment from
The accurate molecular weight of son；
Using the qualitative software of MassHunter, according to the Primary database of the agricultural chemical compound and the fragment ion
Accurate molecular weight obtains the accurate molecular weight of the fragment ion of the agricultural chemical compound；By the fragment of the agricultural chemical compound from
The accurate molecular weight of son inputs the Primary database of the agricultural chemical compound, obtains the second databases of the agricultural chemical compound,
Complete the foundation of the agricultural chemical compound database.
The present invention is built according to the title and molecular formula of agricultural chemical compound using MassHunter PCDL Manager software
Found the initial data base of the agricultural chemical compound.The present invention does not have the type of the agricultural chemical compound special restriction, adopts
With pesticide well known to those skilled in the art, such as insecticide and fungicide.In an embodiment of the present invention, specifically with 13 kinds
Illustrate the method for building up of agricultural chemical compound database for insecticide and 13 kinds of fungicide (specific type is listed in Table 1 below).?
In the present invention, the information of the initial data base includes the title of agricultural chemical compound, molecular formula and accurate molecular weight, by described
Initial data base can match the specific name, molecular formula and accurate molecular weight of agricultural chemical compound, be convenient for subsequent continuation
Establish Primary database.
When the present invention carries out ultra performance liquid chromatography-quadrupole rod-flight to the standard sample solution of the agricultural chemical compound
Between mass spectral analysis, obtain the acquired original data of agricultural chemical compound standard sample.In the present invention, the mark of the agricultural chemical compound
The concentration of agricultural chemical compound is independently preferably 10~100 μ g/L, more preferably 100 μ g/L in quasi- sample solution.In the present invention
In, solvent is preferably acetonitrile in the standard sample solution of the agricultural chemical compound.Mark of the present invention for the agricultural chemical compound
The manner of formulation of quasi- sample solution does not have special restriction, is prepared using mode well known to those skilled in the art.
In the present invention, ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry (UHPLC-Q-TOF/MS) analysis
Liquid phase chromatogram condition preferably include:
Mobile phase: mobile phase includes mobile phase A and Mobile phase B, wherein mobile phase A is the aqueous formic acid of ammonium acetate, institute
The concentration for stating ammonium acetate in mobile phase A is 10mmol/L, and the volumn concentration of formic acid is 0.1%；The Mobile phase B is acetic acid
Formic acid-methanol aqueous solution of ammonium, the concentration of ammonium acetate is 10mmol/L in the Mobile phase B, and the volumn concentration of formic acid is
0.1~1.0%, the volumn concentration of methanol is 5~95%.
Gradient elution program: 0~5min, Mobile phase B increase to 90% by 20%；5~8min, Mobile phase B are maintained at
90%；8~10min, Mobile phase B are reduced by 90% to 20%；10~12min, Mobile phase B are maintained at 20%.
In the present invention, the stream of mobile phase when the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis is carried out
Speed is preferably 0.3mL/min.
In the present invention, sample volume when carrying out the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis is excellent
Select 10 μ L.
In the present invention, chromatography used by the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry is analyzed is carried out
Column is preferably Kinetex-C18 chromatographic column, and specification is preferably 2.1mm × 100mm, and packing material size is preferably 2.6 μm；Column temperature is preferred
It is 35 DEG C.
In the present invention, the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis Mass Spectrometry Conditions preferably wrap
It includes:
Ion source is the double sources electrospray ionisation ESI of sheath stream, and scanning mode is positive ion mode, and the karyoplasmic ratio of reference ion is
121.05873 and 922.009798；Dry temperature degree is 300 DEG C, and dry gas stream speed is 10L/min；Atomization gas pressure is
207kPa；Sheath temperature degree is 300 DEG C, sheath gas 10L/min；Capillary voltage is 3100V, and fragmentation voltage is 110V, cone
Hole voltage is 35V；Full scan karyoplasmic ratio is 50~1000, sweep speed 3spectra/s.
In the present invention, equipment used by the ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry is analyzed is carried out
Preferably include Agilent1290series Ultra Performance Liquid Chromatography instrument (Agilent company of the U.S.)；Agilent6540 quadrupole rod-
Time of-flight mass spectrometer (Agilent company of the U.S.), equipped with double electric spray ion sources (dualJetStreamESI),
The real-time acquisition workstation of MassHunterDataAcquisition, QualitativeAnalysis off-line analysis software,
MassHunterPCDL (Personal Compound Databaseand Library) Manager people's compound database
Software and QualitativeQuantitative off-line analysis software are created with spectrogram library.
Obtain the initial data base of the agricultural chemical compound and the acquired original data of the agricultural chemical compound standard sample
Afterwards, the present invention utilizes the qualitative software of MassHunter, according to the initial data base of the agricultural chemical compound and the pesticide chemical combination
The acquired original data of object standard sample, obtain the retention time of the agricultural chemical compound；By the reservation of the agricultural chemical compound
Time inputs the initial data base of the agricultural chemical compound, obtains the Primary database of the agricultural chemical compound.In the present invention,
The information of the Primary database includes title, molecular formula, accurate molecular weight and the retention time of the agricultural chemical compound, is passed through
Accurate molecular weight and retention time can be matched with the title of the agricultural chemical compound, molecular formula, and then realize pesticide
Close the screening of object.
After obtaining the Primary database of agricultural chemical compound, the present invention is by the parent ion of the agricultural chemical compound, the pesticide
The retention time of the compound and 4 different collision energies input the collecting method of the agricultural chemical compound, and are arranged
The karyoplasmic ratio scanning range of fragment ion obtains the acquisition data of fragment ion.In the present invention, 4 different collisions
Energy is preferably 10,20,30 and 40eV；The karyoplasmic ratio scanning range of the fragment ion is preferably 50~450.In the present invention
In, preferably the pesticide is inputted in the Targeted MS/MS acquisition interface of quadrupole time-of-flight mass spec-trometry (Q-TOF/MS)
The parent ion of compound, the retention time of the agricultural chemical compound and 4 different collision energies.
After obtaining the acquisition data of fragment ion, the present invention utilizes the qualitative software of MassHunter, according to the pesticide
The Primary database of object and the accurate molecular weight of the fragment ion are closed, the accurate of the fragment ion of the agricultural chemical compound is obtained
Molecular weight；The accurate molecular weight of the fragment ion of the agricultural chemical compound is inputted to the Primary database of the agricultural chemical compound,
The second databases of the agricultural chemical compound are obtained, that is, complete the foundation of the agricultural chemical compound database.In the present invention, institute
The information for stating the second databases of agricultural chemical compound includes the title of the agricultural chemical compound, molecular formula, accurate molecular weight, reservation
The accurate molecular weight of time and fragment ion passes through retention time and accurate molecular weight, the accurate molecular weight of fragment ion
It can be matched with the title of the drug compound, molecular formula.
The agricultural chemical compound database obtained the present invention provides method for building up described in above-mentioned technical proposal is in screening pesticide
Application in compound, preferably includes following steps:
Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis is carried out to pretreated sample to be tested, using agriculture
Primary database in drug compound database carries out level-one screening, when level-one Q-TOF score >=70, with the level-one screening
The compound obtained is target compound, Secondary Screening is carried out using the second databases in agricultural chemical compound database, when two
When grade Q-TOF score >=60, then untested compound is compound that the Secondary Screening obtains in sample to be tested.
In the present invention, the sample to be tested is preferably water body example.In the present invention, the pretreated method is preferred
Using solid phase extraction, comprising the following steps:
The pH value of 100mL water body example to be measured is adjusted to 2 using hydrochloric acid, being splined on C18 solid phase extraction column, (C18 is solid
Mutually extraction pillar is successively being activated using preceding with 10mL methanol, 10mL pure water), after completion of the sample, eluted with 5mL ultrapure water
C18 solid-phase extraction column；Elution terminates, and nitrogen blows C18 solid phase extraction column to be done to close；With the mixed solvent of 10mL acetonitrile and isopropanol
(v:v=1:1) it is eluted, gained eluent is blown in 40 DEG C of nitrogen and closely does, and residue acetonitrile dissolves and be settled to 1.0mL, mistake
Film is to be measured.
In the present invention, is carried out to the pretreated sample to be tested ultra performance liquid chromatography-quadrupole rod-flight time
Liquid phase chromatogram condition and Mass Spectrometry Conditions used by mass spectral analysis are preferably established with above-mentioned technical proposal used when database
Consistent, details are not described herein.
After carrying out ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis to pretreated sample to be tested, this hair
The bright Primary database using in agricultural chemical compound database carries out level-one screening, when level-one Q-TOF score >=70, with described
The compound that level-one screening obtains is target compound, carries out second level sieve using the second databases in agricultural chemical compound database
It looks into, when second level Q-TOF score >=60, then untested compound is compound that the Secondary Screening obtains in sample to be tested.?
In the present invention, when the level-one Q-TOF score < 70, then illustrate the agricultural chemical compound in the non-database of untested compound,
Without carrying out succeeding level-two screening；When the second level Q-TOF score < 60, then illustrate the non-pesticide chemical combination of untested compound
Agricultural chemical compound in object database.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
(1) standard solution is prepared
26 kinds of standard sample of pesticide are configured to the hybrid standard stock solution that mass concentration is 1.0mg/L with acetonitrile, then
100 μ g/L are diluted to, mixed standard solution is obtained, it is spare.
(2) liquid phase chromatogram condition and Mass Spectrometry Conditions
(21) liquid phase chromatogram condition
Mobile phase: mobile phase includes mobile phase A and Mobile phase B, wherein mobile phase A is the aqueous formic acid of ammonium acetate, institute
The concentration for stating ammonium acetate in mobile phase A is 10mmol/L, and the volumn concentration of formic acid is 0.1%；The Mobile phase B is acetic acid
Formic acid-methanol aqueous solution of ammonium, the concentration of ammonium acetate is 10mmol/L in the Mobile phase B, and the volumn concentration of formic acid is
0.5%, the volumn concentration of methanol is 50%.
Gradient elution program: 0~5min, Mobile phase B increase to 90% by 20%；5~8min, Mobile phase B are maintained at
90%；8~10min, Mobile phase B are reduced by 90% to 20%；10~12min, Mobile phase B are maintained at 20%.
The flow velocity of mobile phase: 0.3mL/min.
Sample volume: 10 μ L.
Chromatographic column: Kinetex-C18 chromatographic column, specification 2.1mm × 100mm, 2.6 μm of packing material size；35 DEG C of column temperature.
(22) Mass Spectrometry Conditions
Ion source is the double sources electrospray ionisation ESI of sheath stream, and scanning mode is positive ion mode, and the karyoplasmic ratio of reference ion is
121.05873 and 922.009798；Dry temperature degree is 300 DEG C, and dry gas stream speed is 10L/min；Atomization gas pressure is
207kPa；Sheath temperature degree is 300 DEG C, sheath gas 10L/min；Capillary voltage is 3100V, and fragmentation voltage is 110V, cone
Hole voltage is 35V；Full scan karyoplasmic ratio is 50~1000, sweep speed 3spectra/s.
(3) agricultural chemical compound builds library
According to the title and molecular formula of 26 kinds of agricultural chemical compounds, institute is established using MassHunterPCDLManager software
State the initial data base of agricultural chemical compound；
According to liquid phase chromatogram condition in step (2) and Mass Spectrometry Conditions to the mixing mark of 26 kinds of agricultural chemical compounds in step (1)
Quasi- solution carries out ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis, obtains the original of agricultural chemical compound standard sample
Acquire data；
Using the qualitative software of MassHunter, according to the initial data base of the agricultural chemical compound and the agricultural chemical compound
The acquired original data of standard sample obtain the extraction chromatography of ions figure and retention time of the agricultural chemical compound；By the agriculture
The retention time of drug compound inputs the initial data base of the agricultural chemical compound, obtains the level one data of the agricultural chemical compound
Library；
By the parent ion of the agricultural chemical compound, retention time and 4 different collision energy (10eV, 20eV, 30eV
And 40eV) Primary database of the agricultural chemical compound is inputted, and fragment ion karyoplasmic ratio scanning range (50~450) is set,
Obtain the acquisition data of fragment ion；
Using the qualitative software of MassHunter, according to the Primary database of the agricultural chemical compound and the fragment ion
Data are acquired, the accurate molecular weight of the fragment ion of the agricultural chemical compound is obtained；By the fragment ion of the agricultural chemical compound
Accurate molecular weight input the Primary database of the agricultural chemical compound, obtain the second databases of the agricultural chemical compound, i.e.,
Complete the foundation of the agricultural chemical compound database.As shown in table 1.
Title, chemical formula, accurate molecular weight and the retention time of 1 26 kinds of agricultural chemical compounds of table
(4) actual sample pretreatment and screening
(41) actual sample pre-processes
16 parts of samples (respectively marked as 1-16) are taken to be used as water body example to be measured Xiamen City periphery water environment；16 parts to
Survey in practical water body example, be respectively from the populated areas such as school periphery, park, villages and small towns streamlet, take water sample compared with
It is representative.
The pH value of 100mL water body example to be measured is adjusted to 2 using hydrochloric acid, being splined on C18 solid phase extraction column, (C18 is solid
Mutually extraction pillar is successively being activated using preceding with 10mL methanol, 10mL water), after completion of the sample, eluted with 5mL ultrapure water
C18 solid-phase extraction column；Elution terminates, and nitrogen blows C18 solid phase extraction column to be done to close；With the mixed solvent of 10mL acetonitrile and isopropanol
(v:v=1:1) it is eluted, gained eluent is blown in 40 DEG C of nitrogen and closely does, and residue acetonitrile dissolves and be settled to 1.0mL, mistake
Film is to be measured.
(42) actual sample screening
According to liquid phase chromatogram condition in step (2) and Mass Spectrometry Conditions to pretreated water body example to be measured in step (41)
Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis is carried out, using the series in above-mentioned agricultural chemical compound database
Level-one screening is carried out according to library, when level-one Q-TOF score >=70, the compound obtained using the level-one screening is target chemical combination
Object carries out Secondary Screening using the second databases in agricultural chemical compound database, when second level Q-TOF score >=60, then to
Surveying untested compound in water body example is the compound that the Secondary Screening obtains.
The results show that not found in 14 parts of water body examples containing 26 kinds of pesticide chemical combination in table 1 in 16 parts of water body examples
Object, wherein 1 part is derived from detection chlopyrifos in the water body example in park, (level-one Q-TOF is scored at 94.36, and second level Q-TOF is scored at
83.47), 1 part be derived from the water body example of villages and small towns streamlet detect buttocks worm amidine (level-one Q-TOF is scored at 91.97, second level Q-TOF
It is scored at 76.24).
(5) condition optimizing
(51) selection of orifice potential
When the present invention sets mass spectrometry parameters, it is noted that orifice potential has a certain impact for the loudness value tool of object,
Therefore orifice potential is optimized in the present invention.The present invention has investigated orifice potential in 35V, 40V, 43V, 60V for 26 kinds
The influence of agricultural chemical compound.By experimental studies have found that, when orifice potential be 35V when, the response highest of object, thus this
Experimental selection orifice potential is 35V.
(52) selection of chromatographic column
The present invention has investigated Kinetex-C18 (2.1mm × 100mm, 2.6 μm), SB-C18 (2.1mm × 100mm, 3.5 μ
M), separation situation and mesh of the three kinds of different chromatographic columns of BEH (2.1mm × 100mm, 1.7 μm) for 26 kinds of agricultural chemical compounds
Mark the response of compound.It is found by Experimental comparison, separation of the Kinetex-C18 chromatographic column for agricultural chemical compound not of the same race
Effect is preferable, and peak type is good, and high (the chromatographic peak comparison that Fig. 1 passes through different chromatographic columns for Atrazine of the response of object
Figure, the corresponding SB-C18 chromatographic column of chromatographic peak 1, the corresponding Kinetex-C18 chromatographic column of chromatographic peak 2, the corresponding BEH chromatography of chromatographic peak 3
Column), therefore this experimental selection Kinetex-C18 is separation chromatography column.
(53) selection of solid phase extraction column
The present invention has investigated the enrichment purification effect of tetra- kinds of different solid phase extraction columns of C18, MAX, CN, MCX.Pass through reality
Comparison discovery is tested, C18 solid phase extraction column is best for the concentration effect of pesticide composition, the rate of recovery is higher, therefore selects C18 solid
The mutually extraction pillar that extraction pillar is tested as this.
(54) selection of eluent
The present invention investigates the type of eluant, eluent, compares acetonitrile, acetone, methanol, isopropanol, acetonitrile-acetone
(v:v=1:1), dichloromethane-ethyl acetate (v:v=1:1), acetonitrile-isopropanol (v:v=1:1) washing as eluting solvent
De- effect.It is obtained by Experimental comparison: methanol, acetone, acetonitrile-acetone (v:v=1:1), dichloromethane-ethyl acetate (v:v=
When 1:1) these four solvents are as eluant, eluent, the rate of recovery is poor, and the rate of recovery of most of pesticide is below 60%；Select polarity larger
Acetonitrile as eluting solvent when, for the pesticides such as sulfotep, nitrile bacterium azoles, diclobutrazol, diazinon, difenoconazole return
Yield is lower；It is low for the rate of recovery of the pesticides such as Rogor, malathion, sulfotep when selecting isopropanol as eluting solvent.With
Acetonitrile-isopropanol (v:v=1:1) is used as eluting solvent, the experiment proved that, under the action of the eluting solvent, 26 kinds of pesticides
Closing object can elute completely, and the rate of recovery is all larger than 80%, therefore acetonitrile-isopropanol (v:v=1:1) is selected to test as this
Eluting solvent.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method for building up of agricultural chemical compound database, comprising the following steps:
According to the title and molecular formula of agricultural chemical compound, the pesticide is established using MassHunter PCDL Manager software
The initial data base of compound；
Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis is carried out to the standard sample solution of the agricultural chemical compound,
Obtain the acquired original data of agricultural chemical compound standard sample；
Using the qualitative software of MassHunter, according to the initial data base of the agricultural chemical compound and the agricultural chemical compound standard
The acquired original data of sample, obtain the retention time of the agricultural chemical compound；The retention time of the agricultural chemical compound is defeated
The initial data base for entering the agricultural chemical compound obtains the Primary database of the agricultural chemical compound；
The parent ion of the agricultural chemical compound, the retention time of the agricultural chemical compound and 4 different collision energies are defeated
Enter ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry Targeted MS/MS acquisition interface, and fragment ion is set
Karyoplasmic ratio scanning range obtains the accurate molecular weight of the fragment ion；
Using the qualitative software of MassHunter, according to the accurate of the Primary database of the agricultural chemical compound and the fragment ion
Molecular weight obtains the accurate molecular weight of the fragment ion of the agricultural chemical compound；By the fragment ion of the agricultural chemical compound
Accurate molecular weight inputs the Primary database of the agricultural chemical compound, obtains the second databases of the agricultural chemical compound, i.e., complete
At the foundation of the agricultural chemical compound database.
2. method for building up according to claim 1, which is characterized in that institute in the standard sample solution of the agricultural chemical compound
There is the concentration of agricultural chemical compound independently to be 10~100 μ g/L.
3. method for building up according to claim 1, which is characterized in that when the ultra performance liquid chromatography-quadrupole rod-flight
Between the liquid phase chromatogram condition of mass spectral analysis include:
Mobile phase: mobile phase includes mobile phase A and Mobile phase B, wherein mobile phase A is the aqueous formic acid of ammonium acetate, the stream
The concentration of ammonium acetate is 10mmol/L in dynamic phase A, and the volumn concentration of formic acid is 0.1%；The Mobile phase B is ammonium acetate
Formic acid-methanol aqueous solution, the concentration of ammonium acetate is 10mmol/L in the Mobile phase B, the volumn concentration of formic acid is 0.1~
1.0%, the volumn concentration of methanol is 5~95%.
Gradient elution program: 0~5min, Mobile phase B increase to 90% by 20%；5~8min, Mobile phase B are maintained at 90%；8
~10min, Mobile phase B are reduced by 90% to 20%；10~12min, Mobile phase B are maintained at 20%.
4. method for building up according to claim 3, which is characterized in that carry out the ultra performance liquid chromatography-quadrupole rod-and fly
Chromatographic column used by row time mass spectrum is analyzed is Kinetex-C18 chromatographic column, and specification is 2.1mm × 100mm, and packing material size is
2.6μm；Column temperature is 35 DEG C.
5. method for building up according to claim 1, which is characterized in that when the ultra performance liquid chromatography-quadrupole rod-flight
Between the Mass Spectrometry Conditions of mass spectral analysis include:
Ion source is the double sources electrospray ionisation ESI of sheath stream, and scanning mode is positive ion mode, and the karyoplasmic ratio of reference ion is
121.05873 and 922.009798；Dry temperature degree is 300 DEG C, and dry gas stream speed is 10L/min；Atomization gas pressure is
207kPa；Sheath temperature degree is 300 DEG C, sheath gas 10L/min；Capillary voltage is 3100V, and fragmentation voltage is 110V, cone
Hole voltage is 35V；Full scan karyoplasmic ratio is 50~1000, sweep speed 3spectra/s.
6. method for building up according to claim 1, which is characterized in that 4 different collision energies be 10eV,
20eV, 30eV and 40eV.
7. method for building up according to claim 1, which is characterized in that the fragment ion karyoplasmic ratio scanning range be 50~
450。
8. the agricultural chemical compound database that any one of claim 1~7 method for building up obtains is in screening agricultural chemical compound
Application.
9. application according to claim 8, which comprises the following steps:
Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry analysis is carried out to pretreated sample to be tested, using pesticide
The Primary database closed in object database carries out level-one screening, when level-one Q-TOF score >=70, is obtained with the level-one screening
Compound be target compound, using in agricultural chemical compound database second databases carry out Secondary Screening, as second level Q-
When TOF score >=60, then untested compound is agricultural chemical compound that the Secondary Screening obtains in sample to be tested.
10. application according to claim 9, which is characterized in that the sample to be tested is water body example.
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