CN105823832A - LC-Q-TOF/MS detection technology for 544 pesticide residues in kernel fruit - Google Patents
LC-Q-TOF/MS detection technology for 544 pesticide residues in kernel fruit Download PDFInfo
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Abstract
The invention provides an LC-Q-TOF/MS detection technology for 544 pesticide residues in kernel fruit. In a TOF/MS mode, the LC-Q-TOF/MS detection technology separately determines the retention time of each pesticide standard under designated gas chromatography-mass spectrography conditions, determines the ionization form and chemical formula of a compound under the condition of an ESI source and acquires the accurate mass number of the parent ion of each compound so as to form a TOF/MS database. In a Q-TOF/MS mode, the LC-Q-TOF/MS detection technology separately acquires the fragment ion mass spectrum of each pesticide standard under the condition of three to five different collision energies and introduces acquired information into PCDL software so as to form a Q-TOF/MS database. Through comparison of the retention time, mass spectrometry information and tandem mass spectrometry information of a kernel fruit sample, whether the sample contains pesticide residues or not can be determined; and compounds with high primary scores are subjected to secondary confirmation, and if secondary scores are high, it is confirmed that related pesticide residues are detected. The LC-Q-TOF/MS detection technology provided by the invention has the advantages of fastness, high throughput, high accuracy, high reliability and the like, and can accurately screen pesticides in the kernel fruit.
Description
Technical field
The present invention relates to food hygiene, agricultural product security detection field, particularly to one in the pip fruit 544
Plant the method for detecting of pesticide residues.
Background technology
Common as far back as World Health Organization (WHO) (WHO) in 1976, food and agricultural organization (FAO) and United Nations Environment Programme (UNEP)
Establish global environment detecting system/food item (Global Environment Monitoring System, GEMS/
Food), it is intended to grasp member state's food pollution situation, understand food contaminant intake, protect health, give a impetus to trade and send out
Exhibition.Now, countries in the world all rise to food safety the strategic position of national security.Pesticide Residue is food safety mark
One of accurate, also it is international trade access threshold.Meanwhile, the requirement of pesticide residues presenting kind and gets more and more, limitation is more come
The strictest development trend, the Pesticide Residue threshold that namely international trade is set up is more and more higher.European Union formulates now
162248 MRL standards of 839 kinds of pesticide, the U.S. has formulated 39147 MRL standards of 351 kinds of pesticide, and Japan formulates
51600 multinomial MRL standards of 579 kinds of pesticide, China has issued 3650 MRL standards of 381 kinds of pesticide for 2014.At present, state
Uniform limit limitation commonly used on border is 10 μ g/kg.Therefore, the quick agriculture of high flux is all called in food safety and international trade
Medicine residue detection technology, this provides opportunities and challenges the most also to vast pesticide residue analysis worker.Current numerous
In Analytical Techniques of Pesticide Residues, hydrolysis and condensation is to realize the optimized analysis means that the many residuals of high flux quickly detect.
Pesticide residue analysis is many with gas chromatogram, liquid chromatograph, gas chromatography-mass spectrum and liquid phase chromatogram-mass spectrometry combination at present
It is main by technology.These detection techniques all carry out qualitative firstly the need of standard sample of pesticide comparison.Such as, the inspection to 100 kinds of pesticide
Survey is accomplished by preparing corresponding 100 kinds of standard sample of pesticide comparison, and the pesticide outside these 100 kinds all can be missed.At agricultural and veterinary chemicals
In the real work of results from residue tests room, most laboratorys are all without laying in hundreds of standard sample of pesticide, and its reason is pesticide
Standard substance are the most expensive, and effect duration only has 2,3 years, needs overlapping investment.The standing standard sample of pesticide of common laboratory
Only tens kinds, the pesticide species of its daily monitoring is the most just only limited to these tens kinds, thereby results in food safety monitoring leak.
Summary of the invention
The present invention, on the basis of have rated the world and commonly use the mass spectrum behavior characteristics of 1000 Multiple Pesticides, sets up innovatively
544 kinds of pesticide TOF/MS and Q-TOF/MS accurate mass data base；Establish innovatively with high-resolution accurate mass number, with
The 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 mass
The LC-Q-TOF/MS technical method that 544 kinds of pesticide are detected and confirmed by data base；Develop matched innovatively
Fruit and vegetable Pesticide Residues high flux sample preparation technology.Achieve have only to sample prepare, single injected sampling detection, just
To the rapid detection of 500 Multiple Pesticides in fruit and vegetable, and the pesticide detected can be carried out Q-TOF/MS data base
Confirmation, revolutionizes original qualitative model with compound standard thing as reference, is that one need not reference material comparison, soon
Speed, high flux, accurately and reliably Detecting Pesticide new technique.
One, novelty of the present invention is:
1, establish 544 kinds of pesticide accurate masses innovatively and can reach the accurate mass data base of 0.0001m/z, including
TOF/MS data base and Q-TOF/MS data base.
2, TOF/MS data base comprise the English name of 544 kinds of pesticide, chemical molecular formula, retention time, accurate mass number,
The full scan mass spectrum information of Isotopic Distribution, isotope abundance and parent ion and fragment ion, contains much information, the most entirely
Face.
3, the full scan mass spectrum information of every kind of pesticide fragment ion in Q-TOF/MS data base, is by eight differences
Under collision energy, full scan mass spectrum is preferred, and under informative four the different collision energies of ion obtained, fragment ion is swept entirely
Tracing.
4, establish innovatively with compound mass spectrums such as high-resolution accurate mass number, Isotopic Distribution and isotope abundances
Information is criterion of identification, according to TOF/MS and Q-TOF/MS accurate mass data base, 544 kinds of pesticide is detected and is confirmed
LC-Q-TOF/MS technical method, revolutionize original qualitative model with compound standard thing as reference, be that one is not required to
Reference material is wanted to compare, quick, high flux, accurately and reliably Detecting Pesticide new technique.
5, the residual detection techniques method that the present invention sets up can be according to the retention time of target compound, accurate mass
The information such as number, Isotopic Distribution and isotope abundance, by comparing with the corresponding informance retrieval of compound in TOF/MS data base
Right, provide the matching degree score value of target compound.Score value according to target compound, it is achieved the qualitative detecting to pesticide.
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 ion
Figure information, by with the full scan mass spectrum information retrieval comparison of compound fragment ion in Q-TOF/MS data base, provide mesh
The matching degree score value of mark compound, it is achieved the qualitative confirmation to target compound.Compare traditional detection qualitative confirmation method,
Owing to have employed full scan mass spectrum information comparison, there is higher qualitative accuracy.
7, the Residual Pesticides in Farm Produce high flux sample preparation technology supporting with remaining detection techniques method is developed.Real
Showed have only to sample prepare, twice sample detection, it is possible to the rapid detection of 500 Multiple Pesticides in agricultural product and really
Card.
8, LC-Q-TOF/MS main detection techniques index: sweep limits 50-1600m/z；Measure accurate mass up to
0.0001m/z；Within Mass accuracy can be controlled in the 10ppm of molecular mass；Scanning speed is per second 4 in 50-1600m/z scope
Secondary.LC-Q-TOF/MS main detection techniques feature: sample is once prepared, can complete 544 kinds of pesticide detecting, qualitative in 1 hour
With confirmation；More than 80% pesticide confirmation point reaches more than 10；In the 544 kinds of pesticide detected at the same time, 327 kinds of pesticide detecting spirits
Sensitivity is less than the micro-g kg of uniform limit 10, accounting 64.1%, substantially meets the requirement of the horizontal examination of various countries' pesticide residues MRL.
Two, the technical solution used in the present invention:
1, accurate mass data base is set up
The foundation of 1.1 TOF/MS data bases
Under TOF/MS pattern, measure every kind of pesticide standard thing retention time under specifying chromatograph Mass Spectrometry Conditions respectively,
Determine the ionized form (+H ,+NH under this compound ESI source4,+Na) and chemical formula, obtain the essence of every kind of compound parent ion
Really mass number, isotopic peak distribution and abundance ratio.By the title of 544 kinds of pesticide, retention time, molecular formula, accurate mass number, with
The information such as position element peak distribution and abundance ratio import in data base's CSV software document, form TOF/MS data base.
The foundation of 1.2 Q-TOF/MS data bases
Agilent MassHunter data acquisition interface sequentially input in TOF/MS data base the mother of 544 kinds of pesticide from
Son, under Q-TOF/MS pattern, to 544 kinds of pesticide respectively under 8 collision energies, carries out fragment ion full scan mass spectrum and adopts
Collection.Under informative 3-5 the different collision energies of ion, fragment ion full scan mass spectrum importing data base is soft preferably wherein
In part file, and it is associated with corresponding pesticide information, builds up Q-TOF/MS data base.
2, set up with compound information such as high-resolution accurate mass number, Isotopic Distribution and isotope abundances as criterion of identification
, 544 kinds of pesticide LC-Q-TOF/MS detectings and confirmation technical method, technical step includes:
Prepared by 2.1 samples: pip fruit sample extracts through 1% acetate acetonitrile, Carbon/NH2Column purification, acetonitrile+first
Benzene (3+1, v/v) eluting residual pesticide, concentrated, dissolve and filter after make and treat sample measuring liquid；
Described pip fruit includes Fructus Mali pumilae, pears, Folium Eriobotryae and Fructus Crataegi；
2.2 chromatographic mass spectrometry detection: use be furnished with reversed phase chromatographic column (ZORBAX SB-C18column of 2.1mm ×
100mm, 3.5 μm) liquid chromatograph separate the pesticide treating in sample measuring liquid, be furnished with the LC-Q-TOF-MS detection of electric spray ion source；
The 2.3 qualitative method for detecting of TOF/MS data base: call in the qualitative software of Agilent MassHunter and set up
TOF/MS data base, according to TOF/MS data base to target compound retrieval by header, search argument retention time deviation limits
For ± 0.5min, accurate mass deviation is defined to ± 10ppm, ionized form selection+H ,+NH4,+Na pattern, software can foundation
The measured value of the key elements such as every kind of Compound Retention time, accurate mass and the deviation of theoretical value, Yi Jitong in TOF/MS data base
Position element distribution and four parameters of isotope ratio, by scientifically setting each key element weight in matching degree score calculates
(wherein the weight of accurate mass number deviation is the highest), provides retrieval matching score value, retrieves the compound that matching score value is higher,
For doubtful detection compound, according to the method accuracy of TOF/MS data base qualitative detecting Residual Pesticides in Farm Produce, reach
More than 90%；
2.4 Q-TOF/MS data base qualitative confirmation method: under Targeted MS/MS drainage pattern, input doubtful inspection
Go out the parent ion of compound, retention time and optimal collision energy, treat sample measuring liquid and again detect, will detection mass spectrum and Q-
The full scan mass spectrum of compound fragment ion in TOF/MS data base, mates, according to characteristic ion under the conditions of mirror image contrasts
Being distributed the most consistent with spectrum storehouse, the ratio of secondary fragment accurate mass number deviation and fragment ion provides score, its matching score
It is worth higher, i.e. confirms to detect this target compound.This confirmation method uses full scan mass spectrum coupling confirmation, makes more than 80% agriculture
Medicine confirmation point can reach more than 10, and ratio 4 confirmation point requirements of standard have higher qualitative accuracy.
Three, the technology contents of the present invention:
The present invention provides the accurate mass data base of two kinds of 544 kinds of pesticide and one for detecting in fruit and vegetable 544
Plant pesticide residues detection techniques.
1, two kinds of accurate mass data bases that the present invention provides are TOF/MS data base and Q-TOF/MS data base.
1.1 TOF/MS database technology contents include: the chemical formula of 544 kinds of pesticide, accurate mass number, retention time with
And the information such as pesticide English name.In TOF/MS data base, 3 kinds of compounding techniques content example are as follows:
1.2 Q-TOF/MS database technology contents include: 544 kinds of pesticide fragment ion information under 4 collision energies
Mass spectrum.
2, the invention provides 544 kinds of pesticide LC-Q-TOF/MS detectings and confirmation technical method in a kind of fruit and vegetable.Detect
Survey and confirmation technical method content includes the following:
Prepared by 2.1 samples
2.1.1 pip fruit sample takes edible part chopping, mixing, seals, indicates labelling.
2.1.2 weigh 10g pip fruit sample (being accurate to 0.01g), in 80mL centrifuge tube, add 40mL 1% vinegar
Acid acetonitrile, with high-speed homogenization machine 13500r/min, homogenate is extracted 1min, is added 1g sodium chloride, 4g anhydrous magnesium sulfate, vibration
5min, under 4200r/min, centrifugal 5min, takes supernatant 20mL, and in 40 DEG C of water-baths, rotary evaporation is concentrated into about 1mL, treats clean
Change.
2.1.3 in Carbon/NH2 post, add about 2cm height anhydrous sodium sulfate.First with 4mL acetonitrile+toluene (3+1, v/v)
Drip washing SPE post, and discard effluent, when liquid level arrives the top of sodium sulfate, rapidly sample concentration liquid is transferred to decontaminating column
On, under connect new Cor Gigeriae Galli bottle graft receive.The most every time with 2mL acetonitrile+toluene (3+1, v/v) washing sample liquid bottle three times, and cleaning mixture is moved
Enter in SPE post.Post connects 50mL liquid reservoir, then with 25mL acetonitrile+toluene (3+1, v/v) eluting pesticide, is incorporated in Cor Gigeriae Galli
In Ping, and in 40 DEG C of water-baths spin concentration to about 0.5mL.
2.1.4 concentrated solution is placed under nitrogen and dries up, add acetonitrile+0.1% formic acid water (2+8, the v/v) mixing of 2mL, warp
Constant volume after 0.2 μm membrane filtration, obtains testing sample solution a.
2.2 LC-Q-TOF/MS detections
2.2.1 liquid chromatograph separates: testing sample solution a is by being furnished with reversed phase chromatographic column (ZORBAX SB-C18column
Of2.1mm × 100mm, 3.5 μm) liquid chromatographic system separate；Mobile phase A is ammonium acetate-0.1% formic acid-water of 5mM；Stream
Dynamic phase B is acetonitrile；Liquid chromatograph 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, run 4min afterwards；Flow velocity is 0.4mL/
min；Column temperature: 40 DEG C；Sample size: 10 μ L.
2.2.2 Mass Spectrometer Method: Agilent 6530LC-Q-TOF/MS is at electron spray ionisation positive ion mode (ESI+)；Hair
Tubule voltage: 4000V；Dry temperature: 325 DEG C；Dry gas stream amount 10L/min, sheath gas flow velocity 11L/min, sheath gas temperature
Degree is 325 DEG C；Atomization gas pressure 40psi, taper hole voltage 60V, fragmentation voltage 140V.Full scan karyoplasmic ratio scope is 50-
1600m/z, and use internal standard reference solution that instrument quality precision is carried out real time correction.By Agilent MassHunter
Workstation Software (version is B.05.00) is to mass spectrometric data collection and process.
2.3 the qualitative detecting of TOF/MS data base
In the qualitative software of Agilent MassHunter, call the TOF/MS data base set up, search argument is set and protects
Staying time deviation is ± 0.5min, and accurate mass deviation is ± 10ppm, ionized form selection+H ,+NH4,+Na pattern；Foundation
TOF/MS data base carries out retrieval by header to the sample solution mass spectrometric data of above-mentioned collection, goes out every kind of compound by computed in software
Retrieval matching score value, the compound of retrieval matching score value ＞ 70, for doubtful detection compound.
The 2.4 qualitative confirmations of Q-TOF/MS data base
Under Targeted MS/MS drainage pattern, inputting the parent ion of doubtful detection compound, retention time is with optimal
Collision energy, testing sample solution a is detected again, will in detection mass spectrum and Q-TOF/MS data base compound fragment from
The full scan mass spectrum of son, coupling confirmation under the conditions of mirror image contrasts.Its matching score value ＞ 70, i.e. confirms to detect this targeted
Compound.
The present invention is had the beneficial effect that compared to existing technology
1, the present invention is with compound information such as high-resolution accurate mass number, Isotopic Distribution and isotope abundances for identifying mark
Standard, 500 Multiple Pesticides detectings of foundation and confirmation technical method, revolutionize original determining with compound standard thing as reference
Sexual norm, is that one need not reference material comparison, quick, high flux, accurately and reliably Detecting Pesticide new technique；
2, the present invention is on the premise of being not required to reference material comparison, it is achieved that the qualitative detecting of 500 Multiple Pesticides residuals is with true
Card, can be greatly saved the cost buying reference material, the most more environmentally-friendly, safety；
3, the present invention can complete 500 Multiple Pesticides detectings, qualitative and confirmation in 1 hour, compared with traditional detection method
Relatively, work efficiency hundreds times can be improved；
4, the present invention can accomplish 500 Multiple Pesticides in pip fruit sample are remained disposable extraction and cleaning, once
Sample detection, compared with traditional detection method, can save testing cost, improve work efficiency hundreds times；
5, in 544 kinds of pesticide that the present invention detects simultaneously, there are 327 kinds of pesticide detecting sensitivity less than 10 micro-g kg, account for
Ratio 64.1%, substantially meets the requirement of the horizontal examination of various countries' pesticide residues MRL；
6, the present invention is on the premise of being not required to the comparison of pesticide standard thing, according to accurate mass data base to target compound
Qualitative and confirmation, make more than 80% pesticide confirmation point reach more than 10, whole pesticide meet European Union to compound 4 confirm point
Requirement, qualitative results is more accurately and reliably；
7, the pip fruit sample Pesticides that the present invention can test includes the class in following material or a few class: organic
Phosphorus pesticide, carbamate chemicals for agriculture, Ion pairing, sulfonylurea pesticide, nicotinoids pesticide, cinerins agriculture
Medicine and other kinds of pesticide and metabolite；
8, the detection techniques index that the present invention sets up: sweep limits 50-1600m/z；Measure accurate mass up to
0.0001m/z；Within Mass accuracy can be controlled in the 10ppm of molecular mass；Scanning speed is per second 4 in 50-600m/z scope
Secondary；
9, accurate mass data base and the one of two kinds of 544 kinds of pesticide that the present invention provides is used for detecting in fruit and vegetable
544 kinds of pesticide residues detection techniques are promoted at 7 pesticide residue analysis laboratorys and apply, through to 30 provinces and cities
The detecting checking of more than 500 commercially available fruit and vegetable sample, achieves important residual detecting data.
Accompanying drawing explanation
Fig. 1 Fructus Mali pumilae sample full scan pattern TOF shot chart；
Two grades of acquisition method exemplary plot of doubtful pesticide in Fig. 2 Fructus Mali pumilae sample；
Fig. 3 Fructus Mali pumilae sample detects two grades of TOF/MS of pesticide and confirms shot chart；
Fig. 4 pears sample full scan pattern TOF shot chart；
Two grades of acquisition method exemplary plot of doubtful pesticide in Fig. 5 pears sample；
Fig. 6 pears sample detects two grades of TOF/MS of pesticide and confirms shot chart；
Fig. 7 Fructus Crataegi sample full scan pattern TOF shot chart；
Two grades of acquisition method exemplary plot of doubtful pesticide in Fig. 8 Fructus Crataegi sample；
Fig. 9 Fructus Crataegi sample detects two grades of TOF/MS of pesticide and confirms shot chart.
Detailed description of the invention
Embodiment 1
544 kinds of pesticide (being shown in Table 1) LC-Q-TOF/MS detectings and confirmation technology implementation in pip fruit (as a example by Fructus Mali pumilae)
Example, comprises the steps:
1, the concrete steps of Sample Pretreatment Technique:
1.1 Fructus Mali pumilae samples take edible part chopping, mixing, seal, indicate labelling；
1.2 weigh 10g Fructus Mali pumilae sample (being accurate to 0.01g), in 80mL centrifuge tube, add 40mL1% acetate acetonitrile, use
High-speed homogenization machine 13500r/min, homogenate extraction 1min, add 1g sodium chloride, 4g anhydrous magnesium sulfate, and vibrate 5min, at 4200r/
Under min, centrifugal 5min, takes supernatant 20mL, and in 40 DEG C of water-baths, rotary evaporation is concentrated into about 1mL, to be clean.
1.3 at Carbon/NH2Post adds about 2cm height anhydrous sodium sulfate.First drench with 4mL acetonitrile+toluene (3+1, v/v)
Wash SPE post, and discard effluent, when liquid level arrives the top of sodium sulfate, rapidly sample concentration liquid is transferred in SPE post,
Under connect new Cor Gigeriae Galli bottle graft receive.The most every time with 2mL acetonitrile+toluene (3+1, v/v) washing sample liquid bottle three times, and cleaning mixture is moved into
In SPE post.Post connects 50mL liquid reservoir, with 25mL acetonitrile+toluene (3+1, v/v) eluting pesticide and related chemicals, closes
And in Cor Gigeriae Galli bottle, and in 40 DEG C of water-baths spin concentration to about 0.5mL.
Concentrated solution is placed under nitrogen and to dry up by 1.4, adds acetonitrile+water (2+8, v/v) mixing of 2mL, through 0.2 μm filter membrane mistake
Constant volume after filter, obtains testing sample solution.
2, LC-Q-TOF/MS operating condition
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, run 4min afterwards；Flow velocity is 0.4mL/min；Column temperature: 40 DEG C；Sample size: 10 μ L.
Mass Spectrometry Conditions: the mass spectrometric capillary voltage of Agilent 6530LC-Q-TOF/MS: 4000V；Dry temperature:
325℃；Dry gas stream amount 10L/min, sheath gas flow velocity 11L/min, sheath gas temperature is 325 DEG C；Atomization gas pressure 40psi,
Taper hole voltage 60V, fragmentation voltage 140V.Full scan karyoplasmic ratio scope is 50-1600m/z, and uses internal standard reference solution to instrument
Device Mass accuracy carries out real time correction.By Agilent MassHunter Workstation Software (version
B.05.00) mass spectrum testing result gathered and process.
3 pip fruits (as a example by Fructus Mali pumilae) sample Pesticide Residues is detected
3.1 measure sample solution under full scan pattern, detecting result are compared with TOF data storehouse, draw 1 grade
TOF score, is shown in Fig. 1.
3.2 meet the compound of requirement for score, set up two grades of acquisition methods in software, refer to Fig. 2.
3.3, under MS/MS pattern, rerun sample solution, it is thus achieved that sample fragment ion full scan data, by it with two
In level mass spectrum picture library, fragment ion information is compared, and obtains two grades of QTOF scores, sees Fig. 3.
In 4 somewhere Fructus Mali pumilae samples, LC-Q-TOF/MS detects result
Gathering 19, somewhere commercially available Fructus Mali pumilae sample, application LC-Q-TOF/MS technology carries out 544 kinds of pesticide residues detectings,
LC-Q-TOF/MS technology detection 8 kinds of pesticide residues, altogether 28 frequencys, relate to 19, sample, and concrete outcome is shown in Table 2.
1 544 kinds of pesticide LC-Q-TOF/MS detection parameters tables of table
In the Fructus Mali pumilae sample of table 2 somewhere, LC-Q-TOF/MS detects result
Embodiment 2
In pip fruit (as a example by pears), 544 kinds of pesticide (being shown in Table 1) LC-Q-TOF/MS detectings and confirmation technology implementation are real
Example.
Sample pre-treatments step, LC-Q-TOF/MS operating condition are all with reference to the treatment conditions in embodiment 1.
Pip fruit (as a example by pears) sample Pesticide Residues is detected
Under full scan pattern, measure sample solution, detecting result is compared with TOF data storehouse, show that 1 grade of TOF obtains
Point, see Fig. 4；Score is met to the compound of requirement, set up two grades of acquisition methods in software, refer to Fig. 5；At MS/MS mould
Under formula, rerun sample solution, it is thus achieved that sample fragment ion full scan data, by itself and fragment ion in second order ms picture library
Information is compared, and obtains two grades of QTOF scores, sees Fig. 6.
In the pears sample of somewhere, LC-Q-TOF/MS detects result: gather 19, somewhere commercially available pears sample, applies LC-Q-
TOF/MS technology carries out 544 kinds of pesticide residues detectings, LC-Q-TOF/MS technology detection 11 kinds of pesticide residues, altogether 26 frequencys, relates to
And 19, sample, concrete outcome is shown in Table 3.
In the pears sample of table 3 somewhere, LC-Q-TOF/MS detects result
Embodiment 3
544 kinds of pesticide (being shown in Table 1) LC-Q-TOF/MS detectings and confirmation technology in pip fruit (as a example by Fructus Crataegi) sample
Embodiment, comprises the steps:
Sample pre-treatments step, LC-Q-TOF/MS operating condition are all with reference to the treatment conditions in embodiment 1.
Pip fruit (as a example by Fructus Crataegi) sample Pesticide Residues is detected
Under full scan pattern, measure sample solution, detecting result is compared with TOF data storehouse, show that 1 grade of TOF obtains
Point, see Fig. 7；Score is met to the compound of requirement, set up two grades of acquisition methods in software, refer to Fig. 8；At MS/MS mould
Under formula, rerun sample solution, it is thus achieved that sample fragment ion full scan data, by itself and fragment ion in second order ms picture library
Information is compared, and obtains two grades of QTOF scores, sees Fig. 9.
In the Fructus Crataegi sample of somewhere, LC-Q-TOF/MS detects result: gather 8, somewhere commercially available Fructus Crataegi sample, applies LC-
Q-TOF/MS technology carries out 544 kinds of pesticide residues detectings, and LC-Q-TOF/MS technology detects 14 kinds of pesticide residues, altogether 32 frequencys,
Relating to 8, sample, concrete outcome is shown in Table 4.
In the Fructus Crataegi sample of table 4 somewhere, LC-Q-TOF/MS detects result
Claims (10)
1. one kind is used for detecting the method for detecting of 544 kinds of pesticide residues in pip fruit, it is characterised in that: by setting up 544
Plant TOF/MS data base and the Q-TOF/MS data base of pesticide, by the chromatographic mass spectrometry information of target compound and TOF/MS data base
Information retrieval with Q-TOF/MS data base is mated, it is achieved that compare without reference material, simultaneously to more than in agricultural product 500 kinds
Pesticide carries out qualitative detecting and confirmation；544 kinds of described pesticide are the pesticide of numbered 1-544 in table 1.
2. a TOF/MS data base as claimed in claim 1, it is characterised in that: build data base by the following method:
Under TOF/MS pattern, measure every kind of pesticide standard thing retention time under specifying chromatograph Mass Spectrometry Conditions respectively, determine this chemical combination
Ionized form under thing ESI source, selection+H ,+NH4,+Na, and chemical formula, obtain the accurate mass of every kind of compound parent ion
Number, isotopic peak distribution and abundance ratio, by the title of 544 kinds of pesticide, retention time, molecular formula, accurate mass number, isotopic peak
Distribution and abundance ratio information import in data base's CSV software document, form TOF/MS data base.
3. a Q-TOF/MS data base as claimed in claim 1, it is characterised in that: build data base by the following method:
The parent ion of 544 kinds of pesticide in data acquisition interface sequentially inputs TOF/MS data base as stated in claim 2, at Q-
Under TOF/MS pattern, to 544 kinds of pesticide respectively under 8 collision energies, carry out fragment ion full scan mass spectrum collection, preferably
Under informative 3-5 the different collision energies of its intermediate ion, fragment ion full scan mass spectrum imports database software file
In, and be associated with corresponding pesticide information, build up Q-TOF/MS data base.
Detection techniques method the most according to claim 1, it is characterised in that: the detecting for pesticide residues need not pesticide
Reference material compares.
Method for detecting the most according to claim 1, it is characterised in that described method includes following procedure:
A prepared by () sample: pip fruit sample extracts through acetate acetonitrile homogenate, after being dehydrated and be centrifuged, concentrating, then warp
Carbon/NH2Column purification, acetonitrile+toluene eluting residual pesticide, concentrated, filter after make and treat sample measuring liquid a；
B () chromatographic mass spectrometry detects: use the liquid chromatograph being furnished with reversed phase chromatographic column, under special color spectral condition, separates and treats test sample
Pesticide in liquid a；Using the LC-Q-TOF/MS-MS being furnished with electric spray ion source, under specific Mass Spectrometry Conditions, detection pesticide is residual
Stay；
(c) TOF/MS data base's examination: call the TOF/MS data as claimed in claim 2 set up in qualitative software
Storehouse, limits target compound retrieval by header in mass spectrum testing result, search argument retention time deviation according to TOF/MS data base
Being set to ± 0.5min, accurate mass deviation is defined to ± 10ppm, ionized form selection+H ,+NH4,+Na pattern, according to every kind
The measured value of each key element of compound and the deviation of theoretical value in TOF/MS data base, provide retrieval matching score value, retrieval coupling
The compound of score value ＞ 70, for doubtful detection compound；
D () Q-TOF/MS data base confirms: under Targeted MS/MS drainage pattern, input the mother of doubtful detection compound from
Son, retention time and optimal collision energy, treat sample measuring liquid a and again detect, and will detect spectrogram with as claimed in claim 3
Spectrogram in Q-TOF/MS data base, coupling confirmation under the conditions of mirror image contrasts, its matching score value ＞ 70, i.e. confirm to detect this mesh
Mark compound.
Method for detecting the most according to claim 5, it is characterised in that: in step (a), described pip fruit includes Herba Marsileae Quadrifoliae
Really, pears, Folium Eriobotryae and Fructus Crataegi.
Method for detecting the most according to claim 5, it is characterised in that: in step (a), described Carbon/NH2Column purification walks
Rapid as follows: at Carbon/NH2Post adds anhydrous sodium sulfate, first with acetonitrile+toluene mixture liquid drip washing SPE post, and discards outflow
Liquid, when liquid level arrive sodium sulfate top time, rapidly sample concentration liquid is transferred on decontaminating column, under connect new Cor Gigeriae Galli bottle graft receive；
Acetonitrile+toluene mixture liquid washing sample liquid bottle three times again, and cleaning mixture is moved in SPE post；Again with acetonitrile+toluene mixture liquid eluting
Pesticide, merges eluent in Cor Gigeriae Galli bottle.
Method for detecting the most according to claim 5, it is characterised in that: in step (a), described concentrated, filter after make
Treat sample measuring liquid a refer to claim 7 obtained by eluent, spin concentration in 40 DEG C of water-baths, and concentrated solution is placed in nitrogen
Under dry up, add the mixing of acetonitrile+formic acid water, constant volume after membrane filtration, obtain testing sample solution a.
Method for detecting the most according to claim 5, it is characterised 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, runs 4min afterwards；Flow velocity is 0.4mL/min；Column temperature: 40 DEG C；Sample size: 10 μ L.
Method for detecting the most according to claim 5, it is characterised in that: in step (b), described specific Mass Spectrometry Conditions refers to:
The mass spectrometric capillary voltage of Agilent 6530LC-Q-TOF/MS: 4000V；Dry temperature: 325 DEG C；Dry gas stream amount
10L/min, sheath gas flow velocity 11L/min, sheath gas temperature is 325 DEG C；Atomization gas pressure 40psi, taper hole voltage 60V, fragmentation
Voltage 140V, full scan karyoplasmic ratio scope is 50-1600m/z, and uses internal standard reference solution that instrument quality precision is carried out reality
Shi Jiaozheng, is gathered and process mass spectrum testing result by Agilent MassHunter Workstation Software.
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| CN109884200A (en) * | 2019-02-22 | 2019-06-14 | 嘉兴迈维代谢生物科技有限公司 | A kind of quick exploitation and the method for establishing standard items LC-MS database |
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