CN105606749A - LC-Q-TOF/MS detection technology for 544 types of pesticide residues in brassica vegetables - Google Patents
LC-Q-TOF/MS detection technology for 544 types of pesticide residues in brassica vegetables Download PDFInfo
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Abstract
The invention relates to an LC-Q-TOF/MS detection technology for 544 types of pesticide residues in brassica vegetables. The detection technology comprises the following steps: when LC-Q-TOF/MS is at a TOF/MS mode, respectively determining the retention time of each pesticide reference substance under the condition of a specified chromatography-mass spectrometry, determining an ionic form and a chemical formula of a compound under an ESI source to obtain an accurate mass number of parent ions of each compound, and forming a TOF/MS database; when the LC-Q-TOF/MS is at a Q-TOF/MS mode, respectively acquiring a fragmental ion mass spectrum of each pesticide reference substance under 3 to 5 different collision energies, and importing the acquired information into PCDL software to form a Q-TOF/MS database; and comparing the information of the retention time, a primary mass spectrum and a secondary mass spectrum in a brassica vegetable sample, determining whether the sample contains pesticide residues or not, secondarily verifying the compound with relatively higher score at the primary level, and confirming the detection of the relevant pesticide residues if the score at the secondary level is relatively higher. The LC-Q-TOF/MS detection technology has the advantages of high speed, high flux, high precision and high reliability and can accurately screen the pesticide in the brassica vegetables.
Description
Technical field
The present invention relates to food hygiene, agricultural product security detection field, particularly one belongs to class vegetables for rapeThe method for detecting of 544 kinds of residues of pesticides.
Background technology
Common as far back as the World Health Organization (WHO), food and agricultural organization (FAO) and United Nations Environment Programme (UNEP) in 1976Global environment detection system/food item (GlobalEnvironmentMonitoringSystem, GEMS/ are set upFood), be intended to grasp member state's food pollution situation, understand food contaminant intake, protection health, gives a impetus to trade and sends outExhibition. Now, countries in the world all rise to food security the strategic position of national security. Pesticide Residue is food security markOne of accurate, be also international trade access threshold. Meanwhile, the requirement of residues of pesticides is presented to kind more and more, limit the quantity more nextStricter development trend, the Pesticide Residue threshold that namely international trade is set up is more and more higher. European Union formulates now162248 MRL standards of 839 kinds of agricultural chemicals, the U.S. has formulated 39147 MRL standards of 351 kinds of agricultural chemicals, Japan has formulated51600 multinomial MRL standards of 579 kinds of agricultural chemicals, China has issued 3650 MRL standards of 381 kinds of agricultural chemicals for 2014. At present, stateThe uniform limit limitation generally adopting on border is 10 μ g/kg. Therefore, the quick agriculture of high flux is all called in food security and international tradeMedicine residue detection technology, this provides opportunities and challenges also to undoubtedly vast pesticide residue analysis worker. Current numerousIn Analytical Techniques of Pesticide Residues, hydrolysis and condensation is the optimized analysis means that realize the how residual fast detecting of high flux.
Pesticide residue analysis is mainly with gas-chromatography, liquid chromatogram, gas chromatography-mass spectrum and liquid phase chromatogram-mass spectrometry combination at presentBe main by technology. First these detection techniques all need standard sample of pesticide contrast to carry out qualitative. For example, the inspection to 100 kinds of agricultural chemicalsSurvey and just need to prepare corresponding 100 kinds of standard sample of pesticide and contrast, and agricultural chemicals outside these 100 kinds all can be missed. At agricultural and veterinary chemicalsIn the real work of results from residue tests chamber, hundreds of standard sample of pesticide can not laid in most laboratories, and its reason is agricultural chemicalsStandard items are not only expensive, and the term of validity only has 2,3 years, need overlapping investment. The standing standard sample of pesticide of common laboratoryOnly have tens kinds, the pesticide species of its daily monitoring is also just only limited to these tens kinds, causes thus food safety monitoring leak.
Inventor team is through concentrating on studies for many years, researched and developed two kinds of 544 kinds of agricultural chemicals accurate mass database andRape belongs to a residual detection techniques for 544 kinds of agricultural chemicals in class vegetables, has realized and has not needed standard control, can belong to class to rapeMore than 500 kinds residues of pesticides in vegetables, rapid detection detects simultaneously, has met Agricultural Products Pesticide Residues high flux quickThe urgent need detecting.
Summary of the invention
The present invention, on the basis of mass spectrum behavioural characteristic of having evaluated conventional 1000 Multiple Pesticides in the world, sets up innovatively544 kinds of agricultural chemicals TOF/MS and Q-TOF/MS accurate mass database; Set up innovatively with high-resolution accurate mass number, sameThe compound Information in Mass Spectras such as position element distribution and isotope abundance are criterion of identification, according to TOF/MS and Q-TOF/MS accurate massThe LC-Q-TOF/MS technical method that database is detected and confirmed 544 kinds of agricultural chemicals; Develop innovatively supporting with itRape belongs to class Pesticide Residues in Vegetables high flux sample preparation technology. Realize and only needed a sample preparation, single injected sampling inspectionSurvey, just can belong to rape the rapid detection of 500 Multiple Pesticides in class vegetables, and can carry out Q-TOF/ to the agricultural chemicals detectingMS database confirmation, has thoroughly changed original qualitative model taking compound reference material as reference, is that one does not need reference materialContrast, fast, high flux, Detecting Pesticide new technology accurately and reliably.
One, novelty of the present invention is:
1, the accurate mass database of having set up innovatively 544 kinds of agricultural chemicals accurate masses and can reach 0.0001m/z, comprisesTOF/MS database and Q-TOF/MS database.
English name that 2, TOF/MS database comprises 544 kinds of agricultural chemicals, chemical molecular formula, retention time, accurate mass number,The full scan mass spectrogram information of isotope distribution, isotope abundance and parent ion and fragment ion, contains much information, accurately completeFace.
3, the full scan mass spectrogram information of every kind of agricultural chemicals fragment ion in Q-TOF/MS database is by eight differencesUnder collision energy, full scan mass spectrogram is preferred, and under informative four the different collision energies of ion that obtain, fragment ion is swept entirelyTracing.
4, set up innovatively with compound mass spectrums such as high-resolution accurate mass number, isotope distribution and isotope abundancesInformation is criterion of identification, according to TOF/MS and Q-TOF/MS accurate mass database, 544 kinds of agricultural chemicals is detected and is confirmedLC-Q-TOF/MS technical method, thoroughly changed original qualitative model taking compound reference material as reference, be that one does not needWant reference material contrast, fast, high flux, Detecting Pesticide new technology accurately and reliably.
5, the residual detection techniques method that the present invention sets up can be according to the retention time of target compound, accurate massNumber, isotope distribute and the information such as isotope abundance, by with TOF/MS database in the corresponding informance retrieval ratio of compoundRight, the matching degree that provides target compound obtains score value. According to the score value that obtains of target compound, realize the qualitative detecting to agricultural chemicals.
6, the residual detection techniques method that the present invention sets up can be according to the full scan mass spectrum of target compound fragment ionFigure information, by with Q-TOF/MS database in the full scan mass spectrogram information retrieval of compound fragment ion compare, provide orderThe matching degree of mark compound obtains score value, realizes the qualitative confirmation to target compound. Compare the qualitative confirmation method of traditional detection,Owing to having adopted the comparison of full scan mass spectrogram information, there is the higher qualitative degree of accuracy.
7, developed the Residual Pesticides in Farm Produce high flux sample preparation technology supporting with residual detection techniques method. RealShow an only sample preparation of needs, twice sample detection, just can be to the rapid detection of 500 Multiple Pesticides in agricultural product and trueCard.
8, the main detection techniques index of LC-Q-TOF/MS: sweep limits 50-1600m/z; The accurate mass of measuring can reach0.0001m/z; Quality precision can be controlled in the 10ppm of molecular mass; Sweep speed is per second 4 in 50-1600m/z scopeInferior. The main detection techniques feature of LC-Q-TOF/MS: sample is once prepared can complete 544 kinds of agricultural chemicals detectings, qualitative in 1 hourWith confirmation; 80% above agricultural chemicals confirmation point reaches more than 10; In 544 kinds of agricultural chemicals of detecting, 327 kinds of agricultural chemicals detectings are clever at the same timeSensitivity lower than uniform limit 10 micrograms/kilogram, accounting 64.1%, meets the requirement of the horizontal examination of various countries residues of pesticides MRL substantially.
Two, the technical solution used in the present invention:
1, set up accurate mass database
The foundation of 1.1TOF/MS database
Under TOF/MS pattern, measure respectively every kind of pesticide standard thing in the retention time of specifying under chromatogram mass spectrum condition,Determine ionized form (+H ,+NH under this compd E SI source4,+Na) and chemical formula, obtain the essence of every kind of compound parent ionReally mass number, isotopic peak distributes and abundance ratio. By the title of 544 kinds of agricultural chemicals, retention time, molecular formula, accurate mass number, sameThe information such as position plain peak distribution and abundance ratio import in database CSV software document, form TOF/MS database.
The foundation of 1.2Q-TOF/MS database
In TOF/MS database is inputted at AgilentMassHunter data acquisition interface successively the mother of 544 kinds of agricultural chemicals fromSon, under Q-TOF/MS pattern,, carries out fragment ion full scan mass spectrogram and adopts respectively under 8 collision energies 544 kinds of agricultural chemicalsCollection. Preferably under the informative 3-5 of its intermediate ion different collision energies, fragment ion full scan mass spectrogram importing database is softIn part file, and be associated with corresponding agricultural chemicals information, build up Q-TOF/MS database.
2, set up taking compound information such as high-resolution accurate mass number, isotope distribution and isotope abundances as criterion of identification, 544 kinds of agricultural chemicals LC-Q-TOF/MS detect and confirmation technical method, and technical step comprises:
2.1 sample preparations: rape belongs to class vegetable sample extracts through 1% acetate acetonitrile, Carbon/NH2Column purification, acetonitrile+Toluene (3+1, v/v) wash-out remains of pesticide is made and is treated sample measuring liquid after concentrating, dissolve and filtering;
Described rape belongs to class vegetables and comprises tender flower stalk, wild cabbage, broccoli, cabbage mustard, purple wild cabbage and cauliflower;
2.2 chromatogram Mass Spectrometer Method: employing is furnished with reverse-phase chromatographic column, and (3.5 μ m) for ZORBAXSB-C18,2.1mm × 100mmLiquid chromatogram separate and treat agricultural chemicals in sample measuring liquid, the LC-Q-TOF-MS that is furnished with electric spray ion source detects;
The qualitative method for detecting of 2.3TOF/MS database: call and set up in the qualitative software of AgilentMassHunterTOF/MS database, according to TOF/MS database to target compound retrieval by header, search argument retention time deviation limitFor ± 0.5min, be defined as ± 10ppm of accurate mass deviation, ionized form selection+H ,+NH4,+Na pattern, software can foundationThe deviation of theoretical value in the measured value of the key elements such as every kind of compound retention time, accurate mass and TOF/MS database, Yi JitongPosition element distributes and four parameters of isotope ratio, by scientifically setting the weight of each key element in matching degree score is calculated(wherein accurate mass count the weight of deviation the highest), provides retrieval matching score value, the higher compound of retrieval matching score value,For the doubtful compound that detects, according to the method degree of accuracy of the qualitative detecting Residual Pesticides in Farm Produce of TOF/MS database, reachMore than 90%;
The qualitative confirmation method of 2.4Q-TOF/MS database: under TargetedMS/MS drainage pattern, input doubtful detectingThe parent ion of compound, retention time and best collision energy, treat sample measuring liquid and again detect, and will detect mass spectrogram and Q-The full scan mass spectrogram of compound fragment ion in TOF/MS database mates, according to characteristic ion under mirror image contrast conditionWhether distribute consistent with spectrum storehouse, the ratio that secondary fragment accurate mass is counted deviation and fragment ion provides score, its matching scoreBe worth highlyer, confirm to detect this target compound. This confirmation method adopts full scan mass spectrogram coupling confirmation, makes 80% above agricultureMedicine confirmation point can reach more than 10, than 4 confirmation points requirement of standard, has the higher qualitative degree of accuracy.
Three, technology contents of the present invention:
The accurate mass database and the one that the invention provides two kinds of 544 kinds of agricultural chemicals belong in class vegetables for detection of rape544 kinds of residues of pesticides detection techniques.
1, two kinds of accurate mass databases provided by the invention are TOF/MS database and Q-TOF/MS database.
1.1TOF/MS database technology content comprises: the chemical formula of 544 kinds of agricultural chemicals, accurate mass number, retention time andThe information such as agricultural chemicals English name. In TOF/MS database, 3 kinds of compound technology contents examples are as follows:
1.2Q-TOF/MS database technology content comprises: 544 kinds of agricultural chemicals fragment ion information under 4 collision energiesMass spectrogram. Q-TOF/MS database technology content example as shown in Figure 1.
2, the invention provides a kind of rape and belong in class vegetables 544 kinds of agricultural chemicals LC-Q-TOF/MS detectings and confirmation technical sideMethod. Detecting and confirmation technical method content comprise as follows:
2.1 sample preparation
2.1.1 rape belongs to class vegetable sample and gets edible part chopping, mixes, and sealing, indicates mark.
2.1.2 take 10g rape and belong to class vegetable sample (being accurate to 0.01g), in 80mL centrifuge tube, add 40mL1%Acetate acetonitrile, with high-speed homogenization machine 13500r/min, 1min is extracted in homogenate, adds 1g sodium chloride, 4g anhydrous magnesium sulfate, vibration5min, centrifugal 5min under 4200r/min, gets supernatant 20mL, and in 40 DEG C of water-baths, rotary evaporation is concentrated into about 1mL, treats cleanChange.
2.1.3 in Carbon/NH2 post, add the high anhydrous sodium sulfate of about 2cm. First use 4mL acetonitrile+toluene (3+1, v/v)Drip washing SPE post, and discard efflux, in the time that liquid level arrives the top of sodium sulphate, rapidly sample concentration liquid is transferred to decontaminating columnAbove, under, connecing new heart bottle graft receives. Use again 2mL acetonitrile+toluene (3+1, v/v) washing sample liquid bottle three times at every turn, and cleaning solution is movedEnter in SPE post. On post, connect 50mL liquid reservoir, then use 25mL acetonitrile+toluene (3+1, v/v) wash-out agricultural chemicals, be incorporated in heartIn bottle, and rotation is concentrated into about 0.5mL in 40 DEG C of water-baths.
2.1.4 concentrate is placed under nitrogen and is dried up, add the acetonitrile+0.1% formic acid water (2+8, v/v) of 2mL to mix, warpConstant volume after 0.2 μ m membrane filtration, obtains testing sample solution a.
2.2LC-Q-TOF/MS detect
2.2.1 liquid chromatogram separates: testing sample solution a is by being furnished with reverse-phase chromatographic column (ZORBAXSB-C18,2.1mm× 100mm, 3.5 μ liquid chromatographic system m) separates; Mobile phase A is ammonium acetate-0.1% formic acid-water of 5mM; Mobile phase B isAcetonitrile; Liquid chromatogram gradient elution program is: 0min:1%B, and 3min:30%B, 6min:40%B, 9min:40%B, 15min:60%B, 19min:90%B, 23min:90%B, 23.01min:1%B, rear operation 4min; Flow velocity is 0.4mL/min; Column temperature:40 DEG C; Sample size: 10 μ L.
2.2.2 Mass Spectrometer Method: Agilent6530LC-Q-TOF/MS is at electron spray ionisation positive ion mode (ESI+); HairTubule voltage: 4000V; Dry gas temperature: 325 DEG C; Dry gas flow 10L/min, sheath gas flow velocity 11L/min, sheath gas temperatureDegree is 325 DEG C; Atomization gas pressure 40psi, taper hole voltage 60V, cracked voltage 140V. Full scan karyoplasmic ratio scope is 50-1600m/z, and in adopting, mark reference solution carries out real time correction to instrument quality precision. Pass through AgilentMassHunterWorkstationSoftware (versionB.05.00) is to mass spectrum data acquisition and processing.
The qualitative detecting of 2.3TOF/MS database
In the qualitative software of AgilentMassHunter, call the TOF/MS database of having set up, search argument is set and protectsStay time deviation to be ± 0.5min, accurate mass deviation is ± 10ppm, ionized form selection+H ,+NH4,+Na pattern; FoundationTOF/MS database carries out retrieval by header to the sample solution mass spectrometric data of above-mentioned collection, calculates every kind of compound by softwareRetrieval matching score value, the compound of retrieval matching score value > 70, is the doubtful compound that detects.
The qualitative confirmation of 2.4Q-TOF/MS database
Under TargetedMS/MS drainage pattern, input the doubtful parent ion that detects compound, retention time and the bestCollision energy, to testing sample solution, a detects again, will detect in mass spectrogram and Q-TOF/MS database compound fragment fromThe full scan mass spectrogram of son, coupling confirmation under mirror image contrast condition. Its matching score value > 70, confirms to detect this targetedCompound.
The beneficial effect that the present invention had is compared to existing technology:
1, the present invention is taking compound information such as high-resolution accurate mass number, isotope distribution and isotope abundances as identification markStandard, 500 Multiple Pesticides detectings and the confirmation technical method of foundation, thoroughly changed original determining taking compound reference material as referenceSexual norm, is that one does not need reference material contrast, fast, high flux, Detecting Pesticide new technology accurately and reliably;
2, the present invention is under the prerequisite that is not needing reference material contrast, realized the residual qualitative detecting of 500 Multiple Pesticides and reallyCard, can save the cost of buying reference material, also environmental protection more, safety greatly;
3, the present invention can complete 500 Multiple Pesticides detectings, qualitative and confirmation in 1 hour, compared with traditional detection method, can increase work efficiency hundreds of times;
4, the present invention can accomplish rape to belong to the residual disposable extraction and cleaning of 500 Multiple Pesticides in class vegetable sample, oneInferior sample detection, compares with traditional detection method, can save testing cost, increase work efficiency hundreds of times;
5, in 544 kinds of agricultural chemicals that the present invention detects simultaneously, have 327 kinds of agricultural chemicals detecting sensitivity lower than 10 micrograms/kilogram, account forThan 64.1%, substantially meet the requirement of the horizontal examination of various countries residues of pesticides MRL;
6, the present invention is under the prerequisite that is not needing pesticide standard thing contrast, according to accurate mass database to target compoundQualitative and confirmation, reaches more than 10 80% above agricultural chemicals confirmation point, and all agricultural chemicals meet European Union 4 of compounds are confirmed to pointsRequirement, qualitative results is more accurately and reliably;
7, the rape that the present invention can test belongs to class vegetable sample Pesticides and comprises a class or a few class in following material: haveThe Phosphorus agricultural chemicals of machine, carbamate chemicals for agriculture, benzimidazole agricultural chemicals, sulfonylurea pesticide, nicotinoids agricultural chemicals, cinerinsThe agricultural chemicals of agricultural chemicals and other types and metabolin;
8, the detection techniques index that the present invention sets up: sweep limits 50-1600m/z; The accurate mass of measuring can reach0.0001m/z; Quality precision can be controlled in the 10ppm of molecular mass; Sweep speed is per second 4 in 50-600m/z scopeInferior;
9, the accurate mass database of provided by the invention two kinds of 544 kinds of agricultural chemicals and one belong to class vegetables for detection of rapeIn dish, 544 kinds of residues of pesticides detection techniques are promoted and apply in 7 pesticide residue analysis laboratories, and process is to 30 provincesThe detecting checking that the commercially available rape in more than 500, city belongs to class vegetable sample, has obtained important residual detecting data.
Brief description of the drawings
Fig. 1 broccoli sample full scan pattern TOF shot chart;
Doubtful agricultural chemicals secondary acquisition method exemplary plot in Fig. 2 broccoli sample;
In Fig. 3 broccoli sample, detect agricultural chemicals secondary Q-TOF/MS confirmation shot chart;
Fig. 4 tender flower stalk sample full scan pattern TOF shot chart;
Doubtful agricultural chemicals secondary acquisition method exemplary plot in Fig. 5 tender flower stalk sample;
In Fig. 6 tender flower stalk sample, detect agricultural chemicals secondary Q-TOF/MS confirmation shot chart;
Fig. 7 wild cabbage sample full scan pattern TOF shot chart;
Doubtful agricultural chemicals secondary acquisition method exemplary plot in Fig. 8 wild cabbage sample;
In Fig. 9 wild cabbage sample, detect agricultural chemicals secondary Q-TOF/MS confirmation shot chart.
Detailed description of the invention
Embodiment 1
544 kinds of agricultural chemicals (in table 1) LC-Q-TOF/MS detecting and confirmation technology in Btassica class vegetables (taking broccoli as example)Embodiment, comprises the steps:
1, the concrete steps of Sample Pretreatment Technique:
1.1 broccoli samples are got edible part chopping, mix, and sealing, indicates mark;
1.2 take 10g broccoli sample (being accurate to 0.01g), in 80mL centrifuge tube, add 40mL1% acetate acetonitrile,With high-speed homogenization machine 13500r/min, 1min is extracted in homogenate, adds 1g sodium chloride, 4g anhydrous magnesium sulfate, and vibration 5min,Centrifugal 5min under 4200r/min, gets supernatant 20mL, and in 40 DEG C of water-baths, rotary evaporation is concentrated into about 1mL, to be clean.
1.3 at Carbon/NH2In post, add the high anhydrous sodium sulfate of about 2cm. First use 4mL acetonitrile+toluene (3+1, v/v) to drenchWash SPE post, and discard efflux, in the time that liquid level arrives the top of sodium sulphate, rapidly sample concentration liquid is transferred in SPE post,Under connect new heart bottle graft and receive. Use again 2mL acetonitrile+toluene (3+1, v/v) washing sample liquid bottle three times at every turn, and cleaning solution is moved intoIn SPE post. On post, connect 50mL liquid reservoir, with 25mL acetonitrile+toluene (3+1, v/v) wash-out agricultural chemicals and related chemicals, closeAnd in heart bottle, and rotation is concentrated into about 0.5mL in 40 DEG C of water-baths.
1.4 are placed in concentrate under nitrogen and dry up, and add acetonitrile+water (2+8, v/v) of 2mL to mix, through 0.2 μ m filter membrane mistakeConstant volume after filter, obtains testing sample solution.
2, LC-Q-TOF/MS operating condition
Chromatographic condition: ammonium acetate-0.1% formic acid-water that liquid chromatogram mobile phase A is 5mM; Mobile phase B is acetonitrile; GradientElution program is: 0min:1%B, 3min:30%B, 6min:40%B, 9min:40%B, 15min:60%B, 19min:90%B, 23min:90%B, 23.01min:1%B, rear operation 4min; Flow velocity is 0.4mL/min; Column temperature: 40 DEG C; Sample size: 10 μ L.
The mass spectrometric capillary voltage of mass spectrum condition: Agilent6530LC-Q-TOF/MS: 4000V; Dry gas temperature:325 DEG C; Dry gas flow 10L/min, sheath gas flow velocity 11L/min, sheath gas temperature is 325 DEG C; Atomization gas pressure 40psi,Taper hole voltage 60V, cracked voltage 140V. Full scan karyoplasmic ratio scope is 50-1600m/z, and in adopting, marks reference solution to instrumentDevice quality precision is carried out real time correction. By AgilentMassHunterWorkstationSoftware (versionB.05.00) mass spectrum testing result is gathered and processed.
3 Btassica class vegetables (taking broccoli as example) Pesticide Residues detectings
3.1 under full scan pattern working sample solution, detecting result and TOF database are compared, draw 1 gradeTOF score, is shown in Fig. 1.
3.2 compounds that meet the demands for score are set up secondary acquisition method in software, refer to Fig. 2.
3.3 under MS/MS pattern, reruns sample solution, obtains sample fragment ion full scan data, by itself and twoIn level mass spectrum picture library, fragment ion information is compared, and obtains secondary QTOF score, sees Fig. 3.
LC-Q-TOF/MS detecting result in 4 certain provincial capital's broccoli sample
Gather 19, the commercially available broccoli sample of certain provincial capital, application LC-Q-TOF/MS technology is carried out 544 kinds of residues of pesticidesDetecting, LC-Q-TOF/MS technology detects 5 kinds of residues of pesticides, amounts to 9 frequencys, relates to 6, sample, and concrete outcome is in table 2.
Table 1544 kind of agricultural chemicals LC-Q-TOF/MS detection parameters table
LC-Q-TOF/MS detecting result in the broccoli sample of table 2 somewhere
Embodiment 2
In Btassica class vegetables (taking tender flower stalk as example), 544 kinds of agricultural chemicals (in table 1) LC-Q-TOF/MS detecting and confirmation technology are realExecute example.
Sample pre-treatments step, LC-Q-TOF/MS operating condition are all with reference to the treatment conditions in embodiment 1.
Working sample solution under full scan pattern, compares detecting result and TOF database, show that 1 grade of TOF obtainsPoint, see Fig. 4; The compound meeting the demands for score is set up secondary acquisition method in software, refers to Fig. 5; At MS/MS mouldUnder formula, rerun sample solution, obtain sample fragment ion full scan data, by fragment ion in itself and second order ms picture libraryInformation is compared, and obtains secondary QTOF score, sees Fig. 6.
LC-Q-TOF/MS detecting result in certain provincial capital's tender flower stalk sample: gather the commercially available tender flower stalk sample 19 of certain provincial capitalIndividual, application LC-Q-TOF/MS technology is carried out 544 kinds of residues of pesticides detectings, and LC-Q-TOF/MS technology detects 20 kinds of residues of pesticides,Amount to 70 frequencys, relate to 19, sample, concrete outcome is in table 3.
LC-Q-TOF/MS detecting result in the tender flower stalk sample of table 3 somewhere
Embodiment 3
In Btassica class vegetables (taking wild cabbage as example), 544 kinds of agricultural chemicals (in table 1) LC-Q-TOF/MS detecting and confirmation technology are realExecute example, comprise the steps:
Sample pre-treatments step, LC-Q-TOF/MS operating condition are all with reference to the treatment conditions in embodiment 1.
Working sample solution under full scan pattern, compares detecting result and TOF database, show that 1 grade of TOF obtainsPoint, see Fig. 7; The compound meeting the demands for score is set up secondary acquisition method in software, refers to Fig. 8; At MS/MS mouldUnder formula, rerun sample solution, obtain sample fragment ion full scan data, by fragment ion in itself and second order ms picture libraryInformation is compared, and obtains secondary QTOF score, sees Fig. 9.
LC-Q-TOF/MS detecting result in certain provincial capital's wild cabbage sample: gather the commercially available wild cabbage sample 19 of certain provincial capitalIndividual, application LC-Q-TOF/MS technology is carried out 544 kinds of residues of pesticides detectings, and LC-Q-TOF/MS technology detects 7 kinds of residues of pesticides, altogetherCount 19 frequencys, relate to 12, sample, concrete outcome is in table 4.
LC-Q-TOF/MS detecting result in the wild cabbage sample of table 4 somewhere
Claims (10)
1. a method for detecting that belongs to 544 kinds of residues of pesticides in class vegetables for detection of rape, is characterized in that: by setting upThe TOF/MS database of 544 kinds of agricultural chemicals and Q-TOF/MS database, by the chromatogram Information in Mass Spectra of target compound and TOF/MS numberAccording to the information retrieval coupling of storehouse and Q-TOF/MS database, realize without reference material and having compared, simultaneously in agricultural product 500 kindsAbove agricultural chemicals carries out qualitative detecting and confirmation; 544 kinds of described agricultural chemicals are the agricultural chemicals that is numbered 1-544 in table 1.
2. a TOF/MS database as claimed in claim 1, is characterized in that: build by the following method database:Under TOF/MS pattern, measure respectively every kind of pesticide standard thing in the retention time of specifying under chromatogram mass spectrum condition, determine this chemical combinationIonized form under thing ESI source, selection+H ,+NH4,+Na, and chemical formula, obtain the accurate mass of every kind of compound parent ionNumber, isotopic peak distributes and abundance ratio, by the title of 544 kinds of agricultural chemicals, retention time, molecular formula, accurate mass number, isotopic peakDistribution and abundance ratio information import in database csv file, form TOF/MS database.
3. a Q-TOF/MS database as claimed in claim 1, is characterized in that: build by the following method database:Input successively described in claim 2 parent ion of 544 kinds of agricultural chemicals in TOF/MS database at data acquisition interface, at Q-Under TOF/MS pattern, 544 kinds of agricultural chemicals respectively under 8 collision energies, are carried out to the collection of fragment ion full scan mass spectrogram, preferablyUnder the informative 3-5 of its intermediate ion different collision energies, fragment ion full scan mass spectrogram imports database software fileIn, and be associated with corresponding agricultural chemicals information, build up Q-TOF/MS database.
4. method for detecting according to claim 1, is characterized in that: the detecting for residues of pesticides does not need pesticide standardThing compares.
5. method for detecting according to claim 1, is characterized in that, described method comprises following program:
(a) sample preparation: rape belongs to class vegetable sample extracts through acetate acetonitrile homogenate, through dehydration and centrifugal, concentrate after, then warpCarbon/NH2Column purification, acetonitrile+toluene wash-out remains of pesticide is made and is treated sample measuring liquid a after concentrating, filtering;
(b) chromatogram Mass Spectrometer Method: adopt the liquid chromatogram of being furnished with reverse-phase chromatographic column, under special color spectral condition, separate and treat test sampleAgricultural chemicals in liquid a; Employing is furnished with the LC-Q-TOF/MS-MS of electric spray ion source, under specific mass spectrum condition, detects agricultural chemicals;
(c) TOF/MS database examination: call the TOF/MS data as claimed in claim 2 of having set up in qualitative softwareStorehouse, according to TOF/MS database, to target compound retrieval by header in mass spectrum testing result, search argument retention time deviation is limitBe decided to be ± 0.5min be defined as ± 10ppm of accurate mass deviation, ionized form selection+H ,+NH4,+Na pattern, according to every kindThe deviation of theoretical value in the measured value of the each key element of compound and TOF/MS database, provides retrieval matching score value, retrieval couplingThe compound that obtains score value > 70 is the doubtful compound that detects;
(d) Q-TOF/MS database confirmation: under TargetedMS/MS drainage pattern, input the doubtful mother who detects compound fromSon, retention time and best collision energy, treat sample measuring liquid a and again detect, and will detect spectrogram and as claimed in claim 3Spectrogram in Q-TOF/MS database, coupling confirmation under mirror image contrast condition, its matching score value > 70, confirms to detect this orderMark compound.
6. method for detecting according to claim 5, is characterized in that: in step (a), described rape belongs to class vegetables and comprises dishA kind of sedge, wild cabbage, broccoli, cabbage mustard, purple wild cabbage and cauliflower.
7. method for detecting according to claim 5, is characterized in that: in step (a), and described Carbon/NH2Column purification stepRapid as follows: at Carbon/NH2In post, add anhydrous sodium sulfate, first use acetonitrile+toluene mixture liquid drip washing SPE post, and discard outflowLiquid, in the time that liquid level arrives the top of sodium sulphate, is transferred to sample concentration liquid on decontaminating column rapidly, under connect new heart bottle graft and receive;Acetonitrile again+toluene mixture liquid washing sample liquid bottle three times, and cleaning solution is moved in SPE post; Use again acetonitrile+toluene mixture liquid wash-outAgricultural chemicals, merges eluent in heart bottle.
8. method for detecting according to claim 5, is characterized in that: in step (a), described through concentrated, make after filteringTreat that sample measuring liquid a refers to the eluent that claim 7 obtains, in 40 DEG C of water-baths, rotation concentrates, and concentrate is placed in to nitrogenUnder dry up, add acetonitrile+formic acid water to mix, constant volume after membrane filtration, obtains testing sample solution a.
9. method for detecting according to claim 5, is characterized in that: in step (b), described special color spectral condition refers to:Liquid chromatogram mobile phase A is ammonium acetate-0.1% formic acid-water of 5mM; Mobile phase B is acetonitrile; Gradient elution program is: 0min:1％B，3min：30％B，6min：40％B，9min：40％B，15min：60％B，19min：90％B，23min：90％B，23.01min:1%B, rear operation 4min; Flow velocity is 0.4mL/min; Column temperature: 40 DEG C; Sample size: 10 μ L.
10. method for detecting according to claim 5, is characterized in that: in step (b), described specific mass spectrum condition refers to:The mass spectrometric capillary voltage of Agilent6530LC-Q-TOF/MS: 4000V; Dry gas temperature: 325 DEG C; Dry gas flow10L/min, sheath gas flow velocity 11L/min, sheath gas temperature is 325 DEG C; Atomization gas pressure 40psi, taper hole voltage 60V, crackedVoltage 140V, full scan karyoplasmic ratio scope is 50-1600m/z, and in adopting, mark reference solution carries out reality to instrument quality precisionShi Jiaozheng, is gathered and processes mass spectrum testing result by AgilentMassHunterWorkstationSoftware.
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