CN105548435A - LC-Q-TOF/MS (liquid chromatography-quadrupole-time offlight/ mass spectrometer) detection technology for detecting 544 kinds of pesticide residues in leguminous vegetables - Google Patents
LC-Q-TOF/MS (liquid chromatography-quadrupole-time offlight/ mass spectrometer) detection technology for detecting 544 kinds of pesticide residues in leguminous vegetables Download PDFInfo
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Abstract
The invention discloses an LC-Q-TOF/MS (liquid chromatography-quadrupole-time offlight/ mass spectrometer) detection technology for detecting 544 kinds of pesticide residues in leguminous vegetables. In a TOF/MS mode, the LC-Q-TOF/MS is used for detecting the retention time of each pesticide standard substance under specified conditions of chromatogram and mass spectrum, determining the ionized form of the compound under an ESI (Electrospray Ionization) source and the chemical formula thereof, and obtaining the precise mass number of the parent ions of each compound, respectively, so as to form a TOF/MS database. In a Q-TOF/MS mode, the LC-Q-TOF/MS detection technology is used for acquiring fragment ion mass spectrum of each pesticide standard substance under 3-5 collision energies respectively and importing the acquired information into PCDL (Personal Compound Database and Library) software to form a Q-TOF/MS database. The leguminous vegetable samples are compared in the aspects of the retention time, the primary mass spectrum and the second mass spectrum to determine whether the sample contains the pesticide residue; and the compound having high scores in the primary comparison is subjected to secondary confirmation, if the sample has high scores in the secondary comparison, the sample is determined to have the pesticide residue. The technology has the advantages of rapidness, high throughout, high precision and high reliability, and can accurately detect the pesticide in leguminous vegetables.
Description
Technical field
The present invention relates to food hygiene, agricultural product security detection field, particularly a kind of method for detecting for 544 kinds of residues of pesticides in legume vegetable.
Background technology
Global environment detection system/food item (GlobalEnvironmentMonitoringSystem is jointly established as far back as the World Health Organization (WHO) (WHO), food and agricultural organization (FAO) and United Nations Environment Programme (UNEP) (UNEP) in 1976; GEMS/Food); be intended to grasp member state's food pollution situation; understand food contaminant intake; protection health, give a impetus to trade development.Now, countries in the world all rise to food security the strategic position of national security.Pesticide Residue is one of food security standard, is also international trade access threshold.Meanwhile, present kind get more and more to the requirement of residues of pesticides, more and more stricter development trend of limiting the quantity, the Pesticide Residue threshold that namely international trade is set up is more and more higher.Present European Union has formulated 162248 MRL standards of 839 kinds of agricultural chemicals, and the U.S. has formulated 39147 MRL standards of 351 kinds of agricultural chemicals, and Japan has formulated 51600 multinomial MRL standards of 579 kinds of agricultural chemicals, and China has issued 3650 MRL standards of 381 kinds of agricultural chemicals for 2014.At present, the uniform limit limitation generally adopted in the world is 10 μ g/kg.Therefore, the quick the Detection Technologies of Pesticide Residues of high flux is all called in food security and international trade, and this provides opportunities and challenges also to undoubtedly vast pesticide residue analysis worker.In current numerous Analytical Techniques of Pesticide Residues, hydrolysis and condensation realizes the many residual optimized analysis means detected fast of high flux.
Current pesticide residue analysis is main mainly with gas chromatography, liquid chromatography, gas chromatography-mass spectrum and LC-MS-MS.All first these detection techniques need standard sample of pesticide contrast to carry out qualitative.Such as, just need to prepare corresponding 100 kinds of standard sample of pesticide contrast to the detection of 100 kinds of agricultural chemicals, and the agricultural chemicals outside these 100 kinds all can be missed.In the real work of agricultural and veterinary chemicals results from residue tests room, hundreds of standard sample of pesticide all can not be laid in most laboratory, and its reason is that standard sample of pesticide is not only expensive, and the term of validity only has 2,3 years, needs overlapping investment.The standing standard sample of pesticide of common laboratory only has tens kinds, and the pesticide species of its daily monitoring is also just only limited to these tens kinds, causes food safety monitoring leak thus.
The present inventor team is through concentrating on studies for many years, have developed the residual detection techniques of 544 kinds of agricultural chemicals in the accurate mass database of two kinds of 544 kinds of agricultural chemicals and a kind of legume vegetable, achieve do not need standard control, can to more than 500 kinds residues of pesticides in legume vegetable, rapid detection detects simultaneously, meets the urgent need that Agricultural Products Pesticide Residues high flux detects fast.
Summary of the invention
The present invention commonly uses on the basis of mass spectrum behavioural characteristic of 1000 Multiple Pesticides have rated the world, establishes 544 kinds of agricultural chemicals TOF/MS and Q-TOF/MS accurate mass database innovatively; What to establish with high-resolution accurate mass number, isotope distribution and the compound Information in Mass Spectra such as isotopic abundance be innovatively criterion of identification, the LC-Q-TOF/MS technical method of 544 kinds of agricultural chemicals being detected and being confirmed according to TOF/MS and Q-TOF/MS accurate mass database; Develop legume vegetable Pesticide Residues high flux sample preparation technology supporting with it innovatively.Achieve and only need a sample preparation, single injected sampling detection, just can to the rapid detection of 500 Multiple Pesticides in legume vegetable, and Q-TOF/MS database confirmation can be carried out to the agricultural chemicals detected, revolutionize original qualitative model being reference with compound standard thing, that one does not need reference material to contrast, quick, high flux, accurately and reliably Detecting Pesticide new technology.
One, novelty of the present invention is:
1, establish the accurate mass database that 544 kinds of agricultural chemicals accurate masses can reach 0.0001m/z innovatively, comprise TOF/MS database and Q-TOF/MS database.
2, TOF/MS database comprises the full scan mass spectrogram information of the English name of 544 kinds of agricultural chemicals, chemical molecular formula, retention time, accurate mass number, isotope distribution, isotopic abundance and parent ion and fragmention, contains much information, accurately comprehensively.
3, the full scan mass spectrogram information of often kind of agricultural chemicals fragmention in Q-TOF/MS database is by preferred to full scan mass spectrogram under eight different collision energies, fragmention full scan figure under informative four the different collision energies of the ion obtained.
What 4, to establish with high-resolution accurate mass number, isotope distribution and the compound Information in Mass Spectra such as isotopic abundance be innovatively criterion of identification, according to the LC-Q-TOF/MS technical method that TOF/MS and Q-TOF/MS accurate mass database is detected 544 kinds of agricultural chemicals and confirmed, revolutionize original qualitative model being reference with compound standard thing, that one does not need reference material to contrast, quick, high flux, accurately and reliably Detecting Pesticide new technology.
5, the residual detection techniques method that the present invention sets up can according to information such as the retention time of target compound, accurate mass number, isotope distribution and isotopic abundances, by retrieving comparison with the corresponding informance of compound in TOF/MS database, provide the matching degree score value of target compound.According to the score value of target compound, realize the qualitative detecting to agricultural chemicals.
6, the residual detection techniques method that the present invention sets up can according to the full scan mass spectrogram information of target compound fragmention, by the full scan mass spectrogram information retrieval comparison with compound fragmention in Q-TOF/MS database, provide the matching degree score value of target compound, realize the qualitative confirmation to target compound.Compare the qualitative confirmation method of traditional detection, owing to have employed the comparison of full scan mass spectrogram information, there is higher qualitative accuracy.
7, the Residual Pesticides in Farm Produce high flux sample preparation technology supporting with residual detection techniques method is developed.Achieve and only need a sample preparation, twice sample detection, just can to the rapid detection of 500 Multiple Pesticides in agricultural product and confirmation.
8, the main detection techniques index of LC-Q-TOF/MS: sweep limit 50-1600m/z; The accurate mass measured can reach 0.0001m/z; Within Mass accuracy can be controlled in the 10ppm of molecular mass; Sweep velocity is 4 times per second in 50-1600m/z scope.The main detection techniques feature of LC-Q-TOF/MS: sample is once prepared, can complete 544 kinds of agricultural chemicals detecting, qualitative with confirmation in 1 hour; More than 80% agricultural chemicals confirmation point reaches more than 10; In the 544 kinds of agricultural chemicals detected at the same time, 327 kinds of agricultural chemicals detecting sensitivity lower than uniform limit 10 microgram/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, accurate mass database is set up
The foundation of 1.1TOF/MS database
Under TOF/MS pattern, measuring often kind of pesticide standard thing respectively specifying the retention time under chromatogram Mass Spectrometry Conditions, determining ionized form (+the H ,+NH under this compd E SI source
4,+Na) and chemical formula, obtain the accurate mass number of often kind of compound parent ion, isotopic peak distribution and abundance ratio.The information such as the title of 544 kinds of agricultural chemicals, retention time, molecular formula, accurate mass number, isotopic peak distribution and abundance ratio are imported in database CSV software document, forms TOF/MS database.
The foundation of 1.2Q-TOF/MS database
Input the parent ion of 544 kinds of agricultural chemicals in TOF/MS database at AgilentMassHunter data acquisition interface successively, under Q-TOF/MS pattern, to 544 kinds of agricultural chemicals respectively under 8 collision energies, carry out the collection of fragmention full scan mass spectrogram.Preferably under the informative 3-5 of its intermediate ion different collision energy, fragmention full scan mass spectrogram imports in database software file, and is associated with corresponding agricultural chemicals information, builds up Q-TOF/MS database.
What 2, to set up with high-resolution accurate mass number, isotope distribution and the compound information such as isotopic abundance be 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: legume vegetable sample extracts through 1% acetate acetonitrile, Carbon/NH
2column purification, acetonitrile+toluene (3+1, v/v) wash-out remains of pesticide, makes and treats sample measuring liquid after concentrating, dissolve and filtering;
Described legume vegetable comprises fresh kidney beans, cowpea, Kidney bean, pea, sword bean, edible podded pea and Fresh edible soybean.
2.2 chromatographic mass spectrometry detect: adopt the liquid chromatography of being furnished with reverse-phase chromatographic column (ZORBAXSB-C18,2.1mm × 100mm, 3.5 μm) to be separated the agricultural chemicals treated in sample measuring liquid, the LC-Q-TOF-MS being furnished with electric spray ion source detects.
The qualitative method for detecting of 2.3TOF/MS database: call the TOF/MS database set up in the qualitative software of AgilentMassHunter, according to TOF/MS database to target compound retrieval by header, search argument retention time deviation is defined as ± 0.5min, accurate mass deviation is defined as ± 10ppm, ionized form selection+H ,+NH
4+ Na pattern, software can according to the deviation of theoretical value in the measured value of the key elements such as often kind of Compound Retention time, accurate mass and TOF/MS database, and isotope distributes and isotope ratio four parameters, by scientifically setting the weight (wherein the weight of accurate mass number deviation the highest) of each key element in matching degree score calculates, provide retrieval matching score value, the compound that retrieval matching score value is higher, compound is detected for doubtful, according to the method accuracy of the qualitative detecting Residual Pesticides in Farm Produce of TOF/MS database, reach more than 90%.
The qualitative confirmation method of 2.4Q-TOF/MS database: under TargetedMS/MS drainage pattern, input the doubtful parent ion detecting compound, retention time and best collision energy, treat sample measuring liquid again to detect, the full scan mass spectrogram of compound fragmention in mass spectrogram and Q-TOF/MS database will be detected, mate under mirror image contrast condition, whether consistent with spectrum storehouse according to characteristic ion distribution, the ratio of secondary fragment accurate mass number deviation and fragmention provides score, its matching score value is higher, namely confirms to detect this target compound.This confirmation method adopts full scan mass spectrogram coupling confirmation, makes more than 80% agricultural chemicals confirmation point can reach more than 10, than 4 confirmation points requirement of standard, has higher qualitative accuracy.
Three, technology contents of the present invention:
The invention provides the accurate mass database of two kinds of 544 kinds of agricultural chemicals and one for detecting 544 kinds of residues of pesticides detection techniques in legume vegetable.
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, the information such as retention time and agricultural chemicals English name.In TOF/MS database, 3 kinds of compounding techniques content example are as follows:
1.2Q-TOF/MS database technology content comprises: the fragmention information mass spectrogram of 544 kinds of agricultural chemicals under 4 collision energies.Q-TOF/MS database technology content example as shown in Figure 1.
2, the invention provides 544 kinds of agricultural chemicals LC-Q-TOF/MS in a kind of legume vegetable to detect and confirmation technical method.Detecting and confirmation technical method content comprise as follows:
2.1 sample preparation
2.1.1 edible part chopping got by legume vegetable sample, mixing, and sealing, indicates mark.
2.1.2 10g legume vegetable sample (being accurate to 0.01g) is taken, in 80mL centrifuge tube, add 40mL1% acetate acetonitrile, with high-speed homogenization machine 13500r/min, 1min is extracted in homogenate, add 1g sodium chloride, 4g anhydrous magnesium sulfate, vibration 5min, centrifugal 5min under 4200r/min, get supernatant 20mL, in 40 DEG C of water-baths, rotary evaporation is concentrated into about 1mL, to be clean.
2.1.3 in Carbon/NH2 post, add about 2cm height anhydrous sodium sulfate.First use 4mL acetonitrile+toluene (3+1, v/v) drip washing SPE post, and discard efflux, when liquid level arrives the top of sodium sulphate, rapidly sample concentration liquid is transferred on decontaminating column, under connect new heart bottle graft and receive.Use 2mL acetonitrile+toluene (3+1, v/v) to wash sample liquid bottle three times more at every turn, and cleansing solution is moved in SPE post.Post connects 50mL liquid reservoir, then uses 25mL acetonitrile+toluene (3+1, v/v) wash-out agricultural chemicals, be incorporated in heart bottle, and in 40 DEG C of water-baths spin concentration to about 0.5mL.
2.1.4 dry up under concentrate being placed in nitrogen, add acetonitrile+0.1% formic acid water (2+8, the v/v) mixing of 2mL, constant volume after 0.2 μm of membrane filtration, obtains testing sample solution a.
2.2LC-Q-TOF/MS detect
2.2.1 liquid chromatography is separated: testing sample solution a is separated by the liquid chromatographic system being furnished with reverse-phase chromatographic column (ZORBAXSB-C18,2.1mm × 100mm, 3.5 μm); Mobile phase A is ammonium acetate-0.1% formic acid-water of 5mM; Mobile phase B is acetonitrile; Liquid chromatography 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.
2.2.2 Mass Spectrometer Method: Agilent6530LC-Q-TOF/MS is at electron spray ionisation positive ion mode (ESI+); Capillary voltage: 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, mark reference solution carries out real time correction to instrument quality precision.By AgilentMassHunterWorkstationSoftware (versionB.05.00) to mass spectrometric data Acquire and process.
The qualitative detecting of 2.3TOF/MS database
In the qualitative software of AgilentMassHunter, call the TOF/MS database set up, arranging search argument retention time deviation is ± 0.5min, and accurate mass deviation is ± 10ppm, ionized form selection+H ,+NH
4,+Na pattern; Carry out retrieval by header according to the sample solution mass spectrometric data of TOF/MS database to above-mentioned collection, calculate often kind of compound retrieval matching score value by software, the compound of retrieval matching score value > 70, detects compound for doubtful.
The qualitative confirmation of 2.4Q-TOF/MS database
Under TargetedMS/MS drainage pattern, input the doubtful parent ion detecting compound, retention time and best collision energy, testing sample solution a is detected again, the full scan mass spectrogram of compound fragmention in mass spectrogram and Q-TOF/MS database will be detected, coupling confirmation under mirror image contrast condition.Its matching score value > 70, namely confirms to detect this target compound.
The beneficial effect that the present invention is had compared to existing technology is:
1, the present invention with high-resolution accurate mass number, isotope distribution and the compound information such as isotopic abundance for criterion of identification, the 500 Multiple Pesticides detectings of setting up and confirmation technical method, revolutionize original qualitative model being reference with compound standard thing, that one does not need reference material to contrast, quick, high flux, accurately and reliably Detecting Pesticide new technology;
2, the present invention is under the prerequisite not needing reference material to contrast, and achieves the residual qualitative detecting of 500 Multiple Pesticides and confirmation, greatly can save the cost buying reference material, also environmental protection more, safety;
3, the present invention can complete 500 Multiple Pesticides detectings, qualitative with confirmation in 1 hour, compared with traditional detection method, can increase work efficiency hundreds of times;
4, the present invention can accomplish to remain disposable extraction and cleaning to 500 Multiple Pesticides in legume vegetable sample, and single injected sampling detects, and compared 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, accounting 64.1%, meets the requirement of the horizontal examination of various countries residues of pesticides MRL substantially;
6, the present invention is under the prerequisite not needing pesticide standard thing to contrast, according to accurate mass database to the qualitative of target compound and confirmation, make more than 80% agricultural chemicals confirmation point reach more than 10, whole agricultural chemicals meets the requirement of European Union to compound 4 confirmation point, and qualitative results more accurately and reliably;
7, the legume vegetable sample Pesticides that the present invention can test comprises a class in following material or a few class: organophosphorus insecticide, carbamate chemicals for agriculture, Ion pairing, sulfonylurea pesticide, nicotinoids agricultural chemicals, the agricultural chemicals of cinerins agricultural chemicals and other types and metabolin;
8, the detection techniques index of the present invention's foundation: sweep limit 50-1600m/z; The accurate mass measured can reach 0.0001m/z; Within Mass accuracy can be controlled in the 10ppm of molecular mass; Sweep velocity is 4 times per second in 50-600m/z scope;
9, the accurate mass database of provided by the invention two kinds of 544 kinds of agricultural chemicals and one are promoted and apply in 7 pesticide residue analysis laboratories for detecting 544 kinds of residues of pesticides detection techniques in legume vegetable, through the detecting checking to 30 provinces and cities' more than 500 commercially available legume vegetable samples, achieve important residual detecting data.
Accompanying drawing explanation
Fig. 1 fresh kidney beans sample full scan pattern TOF shot chart;
Doubtful agricultural chemicals secondary acquisition method exemplary plot in Fig. 2 fresh kidney beans sample;
Detect agricultural chemicals secondary Q-TOF/MS in Fig. 3 fresh kidney beans sample and confirm shot chart.
Fig. 4 Kidney bean sample full scan pattern TOF shot chart;
Doubtful agricultural chemicals secondary acquisition method exemplary plot in Fig. 5 Kidney bean sample;
Detect agricultural chemicals secondary Q-TOF/MS in Fig. 6 Kidney bean sample and confirm shot chart.
Fig. 7 cowpea sample full scan pattern TOF shot chart;
Doubtful agricultural chemicals secondary acquisition method exemplary plot in Fig. 8 cowpea sample;
Detect agricultural chemicals secondary Q-TOF/MS in Fig. 9 cowpea sample and confirm shot chart.
Embodiment
Embodiment 1
In legume vegetable (for fresh kidney beans), 544 kinds of agricultural chemicals (see table 1) LC-Q-TOF/MS detect and confirmation technology embodiment, comprise the steps:
1, the concrete steps of Sample Pretreatment Technique:
Edible part chopping got by 1.1 fresh kidney beans samples, mixing, and sealing, indicates mark;
1.2 take 10g fresh kidney beans 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, add 1g sodium chloride, 4g anhydrous magnesium sulfate, vibration 5min, centrifugal 5min under 4200r/min, get supernatant 20mL, in 40 DEG C of water-baths, rotary evaporation is concentrated into about 1mL, to be clean.
1.3 at Carbon/NH
2about 2cm height anhydrous sodium sulfate is added in post.First use 4mL acetonitrile+toluene (3+1, v/v) drip washing SPE post, and discard efflux, when 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 2mL acetonitrile+toluene (3+1, v/v) to wash sample liquid bottle three times more at every turn, and cleansing solution is moved in SPE post.Post connects 50mL liquid reservoir, with 25mL acetonitrile+toluene (3+1, v/v) wash-out agricultural chemicals and related chemicals, is incorporated in heart bottle, and in 40 DEG C of water-baths spin concentration to about 0.5mL.
1.4 concentrate is placed in nitrogen under dry up, add acetonitrile+water (2+8, v/v) mixing of 2mL, constant volume after 0.2 μm of membrane filtration, obtains testing sample solution.
2, LC-Q-TOF/MS operating conditions
Chromatographic condition: 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.
The mass spectrometric capillary voltage of Mass Spectrometry Conditions: 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, mark reference solution carries out real time correction to instrument quality precision.By AgilentMassHunterWorkstationSoftware (versionB.05.00) to mass spectrum testing result Acquire and process.
3 legume vegetables (for fresh kidney beans) sample Pesticide Residues is detected
3.1 under full scan pattern working sample solution, detecting result and TOF data storehouse are compared, draw 1 grade of TOF score, see Fig. 1.
3.2 compounds met the demands for score, set up secondary acquisition method in software, refer to Fig. 2.
3.3, under MS/MS pattern, rerun sample solution, obtain sample fragmention full scan data, fragmention information in itself and second order ms picture library are compared, obtain secondary QTOF score, see Fig. 3.
In 4 certain provincial capital's fresh kidney beans sample, LC-Q-TOF/MS detects result
Gather 13, the commercially available fresh kidney beans sample of certain provincial capital, application LC-Q-TOF/MS technology carries out 544 kinds of residues of pesticides detectings, and LC-Q-TOF/MS technology detects 9 kinds of residues of pesticides, and amount to 16 frequencys, relate to 9, sample, concrete outcome is in table 2.
Table 1544 kind of agricultural chemicals LC-Q-TOF/MS detection parameters table
In the fresh kidney beans sample of table 2 somewhere, LC-Q-TOF/MS detects result
Embodiment 2
In legume vegetable (for Kidney bean), 544 kinds of agricultural chemicals (see table 1) LC-Q-TOF/MS detect and confirmation technology embodiment.
Sample pre-treatments step, LC-Q-TOF/MS operating conditions are all with reference to the treatment conditions in embodiment 1.
Working sample solution under full scan pattern, compares detecting result and TOF data storehouse, draws 1 grade of TOF score, sees Fig. 4; For the compound that score meets the demands, set up secondary acquisition method in software, refer to Fig. 5; Under MS/MS pattern, rerun sample solution, obtain sample fragmention full scan data, fragmention information in itself and second order ms picture library is compared, obtains secondary QTOF score, see Fig. 6.
In certain provincial capital's Kidney bean sample, LC-Q-TOF/MS detects result: gather 7, the commercially available Kidney bean sample of certain provincial capital, application LC-Q-TOF/MS technology carries out 544 kinds of residues of pesticides detectings, LC-Q-TOF/MS technology detects 15 kinds of residues of pesticides, amount to 29 frequencys, relate to 7, sample, concrete outcome is in table 3.
In the Kidney bean sample of table 3 somewhere, LC-Q-TOF/MS detects result
Embodiment 3
In legume vegetable (for cowpea), 544 kinds of agricultural chemicals (see table 1) LC-Q-TOF/MS detect and confirmation technology embodiment.
Sample pre-treatments step, LC-Q-TOF/MS operating conditions are all with reference to the treatment conditions in embodiment 1.
Working sample solution under full scan pattern, compares detecting result and TOF data storehouse, draws 1 grade of TOF score, sees Fig. 7; For the compound that score meets the demands, set up secondary acquisition method in software, refer to Fig. 8; Under MS/MS pattern, rerun sample solution, obtain sample fragmention full scan data, fragmention information in itself and second order ms picture library is compared, obtains secondary QTOF score, see Fig. 9.
In certain provincial capital's cowpea sample, LC-Q-TOF/MS detects result: gather 6, the commercially available cowpea sample of certain provincial capital, application LC-Q-TOF/MS technology carries out 544 kinds of residues of pesticides detectings, LC-Q-TOF/MS technology detects 9 kinds of residues of pesticides, amount to 19 frequencys, relate to 5, sample, concrete outcome is in table 4.
In the cowpea sample of table 4 somewhere, LC-Q-TOF/MS detects result
Claims (10)
1. one kind for detecting the method for detecting of 544 kinds of residues of pesticides in legume vegetable, it is characterized in that: by setting up TOF/MS database and the Q-TOF/MS database of 544 kinds of agricultural chemicals, the information retrieval of the chromatographic mass spectrometry information of target compound with TOF/MS database and Q-TOF/MS database is mated, achieve and compare without the need to reference material, qualitative detecting and confirmation are carried out to more than 500 kinds agricultural chemicals in agricultural product simultaneously; 544 kinds of described agricultural chemicals are the agricultural chemicals being numbered 1-544 in table 1.
2. a TOF/MS database as claimed in claim 1, it is characterized in that: build database by the following method: under TOF/MS pattern, measure often kind of pesticide standard thing respectively and specify the retention time under chromatogram Mass Spectrometry Conditions, determine the ionized form under this compd E SI source, selection+H ,+NH
4+ Na, and chemical formula, obtain the accurate mass number of often kind of compound parent ion, isotopic peak distribution and abundance ratio, the title of 544 kinds of agricultural chemicals, retention time, molecular formula, accurate mass number, isotopic peak distribution and abundance ratio information are imported in database csv file, forms TOF/MS database.
3. a Q-TOF/MS database as claimed in claim 1, it is characterized in that: build database by the following method: the parent ion of 544 kinds of agricultural chemicals in data acquisition interface inputs TOF/MS database as stated in claim 2 successively, under Q-TOF/MS pattern, to 544 kinds of agricultural chemicals respectively under 8 collision energies, carry out the collection of fragmention full scan mass spectrogram, preferably under the informative 3-5 of its intermediate ion different collision energy, fragmention full scan mass spectrogram imports in database software file, 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 standard thing to compare.
5. method for detecting according to claim 1, is characterized in that, described method comprises following program:
A () sample preparation: legume vegetable sample extracts through acetate acetonitrile homogenate, after dewatering and be centrifugal, concentrated, then through Carbon/NH
2column purification, acetonitrile+toluene wash-out remains of pesticide, makes and treats sample measuring liquid a after concentrating, filtering;
B () chromatographic mass spectrometry detects: adopt the liquid chromatography of being furnished with reverse-phase chromatographic column, under special color spectral condition, is separated the agricultural chemicals treated in sample measuring liquid a; Adopt the LC-Q-TOF/MS-MS being furnished with electric spray ion source, under specific Mass Spectrometry Conditions, detect agricultural chemicals;
(c) TOF/MS database examination: call the TOF/MS database as claimed in claim 2 set up in qualitative software, according to TOF/MS database to target compound retrieval by header in mass spectrum testing result, search argument retention time deviation is defined as ± 0.5min, accurate mass deviation is defined as ± 10ppm, ionized form selection+H ,+NH
4,+Na pattern, according to the deviation of theoretical value in measured value and the TOF/MS database of often kind of each key element of compound, provides retrieval matching score value, retrieves the compound of matching score value > 70, detect compound for doubtful;
D () Q-TOF/MS database is confirmed: under TargetedMS/MS drainage pattern, input the doubtful parent ion detecting compound, retention time and best collision energy, treat sample measuring liquid a again to detect, spectrogram in spectrogram and Q-TOF/MS database as claimed in claim 3 will be detected, coupling confirmation under mirror image contrast condition, its matching score value > 70, namely confirms to detect this target compound.
6. method for detecting according to claim 5, is characterized in that: in step (a), described legume vegetable comprises fresh kidney beans, cowpea, Kidney bean, pea, sword bean, edible podded pea and Fresh edible soybean.
7. method for detecting according to claim 5, is characterized in that: in step (a), described Carbon/NH
2column purification step is as follows: at Carbon/NH
2add anhydrous sodium sulfate in post, first use acetonitrile+toluene mixture liquid drip washing SPE post, and discard efflux, when liquid level arrives the top of sodium sulphate, rapidly sample concentration liquid is transferred on decontaminating column, under connect new heart bottle graft and receive; Acetonitrile again+toluene mixture liquid washing sample liquid bottle three times, and cleansing solution is moved in SPE post; Use acetonitrile+toluene mixture liquid wash-out agricultural chemicals again, merge eluent in heart bottle.
8. method for detecting according to claim 5, it is characterized in that: in step (a), described making after concentrating, filtering treats that sample measuring liquid a refers to the eluent that claim 7 obtains, spin concentration in 40 DEG C of water-baths, and dry up under concentrate being placed in nitrogen, add acetonitrile+formic acid water mixing, 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), and 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), and described specific Mass Spectrometry Conditions refers to: the mass spectrometric capillary voltage of 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 mark reference solution carries out real time correction to instrument quality precision, by AgilentMassHunterWorkstationSoftware to mass spectrum testing result Acquire and process in adopting.
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