CN108562664A - A kind of method of the triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum to more pesticide residue determinations in food - Google Patents
A kind of method of the triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum to more pesticide residue determinations in food Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
Abstract
The invention belongs to field of pesticide detection, and in particular to method of the triple level four bars linear ion hydrazine mass spectrums of liquid chromatogram to more pesticide residue determinations in food.This method is characterized as that the sample size of sample is 1 μ L；Detection method is applied in food, and wherein food includes egg, leek, ginger.This method first carries out the extraction and purification of sample, then is analyzed by mass spectrometry and scans；In egg using adsorbent, extractant is made of magnesium sulfate, sodium chloride, acetonitrile；The extractant using sample in leek, ginger is formed by sodium chloride, magnesium sulfate and by acetonitrile and water composition mixture, and does not use adsorbent；This method is detected using the triple level four bars linear ion hydrazine mass spectrometry methods of liquid chromatogram and is scanned using multiple scanning of the mass spectrum pattern, and this method has matrix effect low, and the rate of recovery is high, and detection limits low advantage.
Description
Technical field
The invention belongs to field of pesticide detection, and in particular to a kind of triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum
To the method for more pesticide residue determinations in food.
Background technology
With the continuous improvement of human substance life horizontal, the food-safe requirement of people is higher and higher, food security
Also have become a hot issue being concerned.In order to improve agricultural output and quality, pesticide is in large-scale agricultural
It is used in production.But the large-scale use of pesticide also results in the appearance of Pesticide Residue.The World Health Organization is by agriculture
Medicine residual, which is defined as mankind or animal, to be the mixture of any residuals or substance caused by using pesticide and spreads out accordingly
Biology, such as degradation and converted product, reaction product and are considered toxic impurity at metabolin.
Leek is that complicated matrix largely contains because it contains pigment and sulfide in addition, ginger and garlic also contain
Sulphur compound.In order to reduce the interference of complex matrices in residue detection, extraction and method for cleaning are studied.Traditional sample
Product pre-treating method mainly has solid phase extraction, Solid-phase Microextraction, gel permeation chromatography etc..However, these methods are also deposited
It is time-consuming and laborious in disadvantage, a large amount of organic solvent is needed, being usually directed to many steps causes analyte to be lost.Since
Since Anastassiades et al. is put forward for the first time QuEChERS (quickly, simply, inexpensively, effectively, firm and safe) method, it
Have been widely used for more pesticide residue analysis in various food substrates.In order to reduce matrix effect needs in QuEChERS methods
Adsorbent adsorbing contaminant is added, but keeps the preparation process of sample complicated using adsorbent, and is had to a certain degree to instrument
Damage.Qu et al. eliminates the sulfide in leek using Microwave-assisted firing method, this is researched and proposed, and Microwave Pretreatment is usually
Before homogenizing used in leek sample, allinnase can be made to inactivate, to reduce the interference of sulfide.But this method cannot
Processing method applied to complex matrices.
Pesticide residue detection method mainly has gas-chromatography (GC), liquid chromatogram (LC), Liquid Chromatography-Tandem Mass Spectrometry (LC-
MS/MS), gas-chromatography tandem mass spectrum (GC-MS/MS) etc..In recent years, although the MRM patterns of triple level four bars mass spectrums (QqQ) exist
In most cases provide accurate and reliable analysis result, but the situation relatively high in the matrix effect of detection sample
Under, testing result can be influenced.
Lee et al. is using selectivity and the triple level four bars of the higher liquid chromatogram-mixing of sensitivity-linear ion hydrazine mass spectrum
(LC-QqLIT-MS/MS) method measures 19 kinds of triazine pesticides and its catabolite in grain sample simultaneously, with MS2It compares, MS3
Background signal can be further decreased, lower detection limit is obtained.
It is not in the prior art best to the rate of recovery of remaining pesticide ethiprole in egg or detection limit.So mesh
It is preceding accurate, detection low with reliable analysis result, matrix effect is provided to limit low, the high inspection of the rate of recovery of pesticide residue there is an urgent need for a kind of
Survey method.
Invention content
For the above-mentioned prior art the problem of, the first purpose of the invention is to provide a kind of liquid chromatograies-three
Method of the weight level four bars-linear ion hydrazine mass spectrum to more pesticide residue determinations in food.It is extracted using simple pre-treating method
Target compound, using the detection side of the triple level four bars of liquid chromatogram-mixing-linear ion hydrazine mass spectrum (LC-QqLIT-MS/MS)
Method, accumulation ability is strong, and (MS is scanned using multi-stage ms3) pattern reduction background signal, matrix effect is reduced, recycling is improved
Rate keeps sample pre-treatments simpler.
In order to solve the above technical problems, the technical scheme is that：
A kind of method of the triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum to more pesticide residue determinations in food：
The sample injection volume of this method is 1 μ L；Sample is through the triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum (LC-
) and multi-stage ms scan pattern (MS QqLIT-MS/MS3) analyzed；The liquid chromatogram is common liquid chromatogram or superelevation
Effect liquid phase chromatogram.
Second object of the present invention is to provide a kind of triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum to food
Application of the method for middle pesticide residue determination in detecting Pesticide Residues；The food includes egg, leek, ginger.
Preferably, it is ultra performance liquid chromatography that liquid chromatogram in the application of pesticide residue is detected in the food egg.
Third object of the present invention is to provide a kind of triple level four bars-of liquid chromatogram-of the application for above-mentioned egg
Linear ion hydrazine mass spectrum to the methods of more pesticide residue determinations in food, the specific steps are：
1) extraction of sample
Sample is uniformly mixed with extractant, centrifuges, takes upper solution；
2) purification of sample
Supernatant liquor is subjected to vortex mixed with adsorbent, centrifuges, upper liquid is taken to pass through 0.22 μm of nylon filter film
3) UPLC-QqLIT-MS/MS is analyzed
The filtered sample of 1 μ L is carried out through the triple level four bars of ultra performance liquid chromatography-- linear ion hydrazine mass spectrum
Detection, is analyzed using multi-stage ms scan pattern.
The sample is egg liquid.
Preferably, the extractant in step 1) is made of magnesium sulfate, sodium chloride, acetonitrile；Magnesium sulfate, sodium chloride, acetonitrile
Ratio is 2-4g：1-3g：18-22ml.
Preferably, adsorbent described in step 2) is primary secondary amine (PSA), C18, graphitic carbon black (GCB), matter
Amount is than being 75-85：75-85：8-10.
Preferably, the chromatographic column of ultra performance liquid chromatography is WatersBEH C18。
Preferably, the mobile phase A of liquid chromatogram is the aqueous solution containing 0.1% formic acid, and B is the first containing 0.1% formic acid
Alcohol.
Preferably, triple level four bars-linear ion hydrazine mass spectrum (QqLIT) uses ESI-Pattern carries out.
Preferably, multi-stage ms scan pattern is run with negative ion mode；Ion source temperature is 550 DEG C；Ion injection electricity
Pressure is -4500V.
Fourth object of the present invention is to provide a kind of liquid phase color for applications of complex matrices such as above-mentioned leek, ginger
Spectrum-triple level four bars-linear ion hydrazine mass spectrums to the methods of more pesticide residue determinations in food, the specific steps are：
1) extraction and purification of sample
Sample is uniformly mixed with extractant, centrifuges, upper solution is taken to cross 0.22 μm of filter membrane；
2) LC-QqLIT-MS/MS is analyzed
The filtered sample of 1 μ L is detected through the triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum,
It is analyzed using multi-stage ms scan pattern.
The sample is leek or ginger.
Preferably, extractant includes sodium chloride, magnesium sulfate and by acetonitrile and water composition mixture in step 1).
It is further preferred that the volume ratio of acetonitrile and water is 1:1-3:1；The mixing of sodium chloride, magnesium sulfate and acetonitrile and water
The ratio of object is 3-5g:0.8-1.2g:18-22ml.
Preferably, in step 2) sample a concentration of 10ng/mL.
Preferably, the chromatographic column Poroshell 120EC-C of the condition of liquid chromatogram (LC)18。
Preferably, the aqueous formic acid that the mobile phase A of liquid chromatogram is 0.1%；Mobile phase B is methanol.
Preferably, triple level four bars-linear ion hydrazine mass spectrum (QqLIT) uses ESI+Pattern carries out.
Preferably, multiple scanning of the mass spectrum pattern is run with positive ion mode；Ion source temperature is 550 DEG C；Ion injection electricity
Pressure is 5500V.
Beneficial effects of the present invention：
1) present invention leek, ginger application process compared with the prior art in QuEChERS methods, without using absorption
Agent shortens preparation process, improves the rate of recovery of compound, and a small amount of sample is only added during analysis reduces matrix
Effect and instrument damage rate；The mixture of acetonitrile and water is selected to improve the rate of recovery as extractant, reduce matrix effect.
2) it is of the invention in the application of egg compared with the prior art in QuEChERS methods improve compound
The rate of recovery, a small amount of sample is only added during analysis reduces matrix effect, improves the rate of recovery, reduces matrix effect
It answers.
3) present invention scans (MS using multi-stage ms3) pattern, simplify the pretreatment process of sample, reduce background letter
Number, matrix effect is reduced between -10% to 10%, reduces detection limit.
Description of the drawings
The accompanying drawings which form a part of this application are used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation do not constitute the improper restriction to the application for explaining the application.
Fig. 1 is influence of four kinds of different solvents to ethiprole and its metabolin rate of recovery；
ACE：Acetonitrile, FA：Formic acid, Me：Methanol；
Fig. 2 is influence of three kinds of different mixed adsorbents to ethiprole and its metabolite rate of recovery；
Fig. 3 is MS of the carbendazim of 1.0 μ g/kg in leek matrix3Spectrogram and MRM spectrograms；
Fig. 4 is quota ion flow graph of 4 kinds of compounds in 0.5ng/mL；
Fig. 5 is the mass spectrum optimization process of 10ng/mL ethiprole fragment ions；
The Mass Spectrometry Conditions of carbendazim optimize spectrogram when Fig. 6 is 0.05 μ g/mL；
Fig. 7 is the MS of ethiprole and its metabolite in blank egg sample3Spectrogram.
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or combination thereof.
Provided according to the standard of European SANTE 11945/2015, the rate of recovery of sample-pretreating method in 70-120%,
Matrix effect is in -20-20%.
Matrix effect (ME, %) can be calculated by the following formula：
When matrix effect is more than zero, it is referred to as matrix enhancement.When matrix effect is less than zero, it is known as matrix suppression
System.With reference to specific embodiment, the present invention is further described
Method of the embodiment 1 in the application of egg
The extraction and purification of sample
The egg sample smashed in advance is stored in -18 DEG C of refrigerator until using.Blank sample is free of target chemical combination
Object.First, 5.0g samples weighings are put into 100mL centrifuge tubes.HPLC grades of 20mL acetonitriles are added in centrifuge tube, and will be mixed
Object is closed to be ultrasonically treated 10 minutes.Then weigh 3.0g MgSO4With 2.0g NaCl and be added in mixture.It shakes 2 minutes immediately,
Then it is centrifuged 5 minutes with 5000rpm/min.Supernatant is transferred in test tube and is stored 20 minutes in -18 DEG C of refrigerator.Most
Afterwards, 10mL supernatants are transferred to containing 400mg PSA, the PP that the 15mL of 400mg C18 and 45mg GCB are intended for single use is centrifuged
Guan Zhong, vortex mixed 1 minute, and centrifuged 5 minutes with 5000rpm/min.By appropriate extracting solution by 0.22 μm of nylon filter film,
UPLC-QqLIT-MS/MS is analyzed
The condition of ultra performance liquid chromatography：The Waters that water generation company providesBEH C18Color
Column (2.1x 100mm, 1.7 μm) is composed, chromatographic isolation is carried out at 40 DEG C.Mobile phase A is the aqueous solution containing 0.1% formic acid, and B is
Methanol containing 0.1% formic acid, gradient elution program are as follows：0-1min, 20-50%B；1-2min, 50-90%B；2-4min,
90%B；4-4.1min, 90-20%B；4.1-6min, 20%B.Column temperature：40℃；Flow velocity：0.3mL/min；Sampling volume：1μL.
Quality analysis uses ESI-Source carries out.Source parameter is as follows：Curtain gas (Curtain Gas)：35.0 (arbitrary units)；
Ion source gas 1 (Ion Source Gas1) and ion source gas 2 (Ion Source Gas2)：50psi；Ion spray voltage
(Ion spray voltage)：-4500.0V；Probe temperature (Probe Temperature)：550℃；Collision gas
(Collision Gas)：It is medium.Nitrogen is used as the nebulizer gas in both of which and collision gas.
MS3Parameter：MS3Scan pattern is run with negative ion mode, and Q3 is rapidly converted into linear ion hydrazine (LIT).Mass spectrum is joined
Number is as follows：Ion source temperature：550℃；Ion injection electric：-4500.0V.For different pesticide variety carry out it is best it is female from
Son, impinging air flows, collision energy, quota ion numerical value determination.The determination method of optimal parameter is to be filled out using Dynamic ion
Quota ion is captured and is accumulated in linear ion hydrazine (LIT) by the firing time for filling time and 40msec, is finally discharged into matter
The three-level qualitatively and quantitatively ion massspectrum figure of compound is obtained in spectrum.
The method of the application of 2 leek of embodiment, ginger
The extraction and purification of sample
10.0g samples accurately are weighed, are placed in 100mL plastic centrifuge tubes, 4.0g sodium chloride, 1.0g MgSO is added4、
20mL acetonitrile/waters (75/25, v/v) centrifuge 5min in high-speed stirred 2min on homogenizer on supercentrifuge, and rotating speed is
5000r/min.It takes appropriate solution to cross 0.22 μm of filter membrane, waits for that LC-MS/MS is detected.
Liquid-phase condition：Chromatographic column Poroshell 120EC-C18(4.6 × 100mm, 2.7 μm)；Flow velocity：0.5mL/min；Column
Temperature：40℃；Sampling volume：1μL；Mobile phase A：0.1% aqueous formic acid, Mobile phase B：Methanol；Gradient elution program：0-
0.01min；25%B；0.01-1min, 75%B；1-3min, 90%B；3-11min, 90%B；11-11.1min 25%B；
11.1-14min 25%B.Dwell time is 14min.
Mass Spectrometry Conditions：Using ESI+Pattern carries out, and source parameter setting is：Curtain Gas (CUR), 35.0 is (arbitrary single
Position)；Ion source gas 1 (GS1) and ion source gas 2 (GS2), 55psi；Ion spray voltage (IS), 5500.0V；Probe temperature
It spends (TEM), 550 DEG C；Collision gas (CAD), it is medium.Nitrogen is used as spray gas and collision gas.
MS3Parameter：MS3Scan pattern is run with positive ion mode.Mass spectrometry parameters are as follows：Ion source temperature：550℃；Ion
Injection electric：5500V.Best parent ion, impinging air flows, collision energy, quota ion are carried out for different pesticide varieties
The determination of numerical value.The determination method of optimal parameter be using the firing time of Dynamic ion filling time and 40msec will it is quantitative from
Son, which captures, to be simultaneously accumulated in linear ion hydrazine (LIT), and the spectrogram that three-level qualitatively and quantitatively ion is obtained in mass spectrum is finally discharged into.
Embodiment 3
Influence of the extractant to the rate of recovery in the application of egg
Extractant selects acetonitrile, acetonitrile/formic acid (99.95/0.05, v/v), acetonitrile/formic acid (99.9/0.1, v/v) to carry out
Compare the effect for improving extraction efficiency, influence of the extractant to the rate of recovery be as shown in Figure 1, the results showed that, when using methanol
When as extractant, many impurity in matrix are extracted simultaneously.Observe solution than other extractions in centrifugal process
Agent is darker, and is layered unobvious.In order to reduce the influence of impurity in matrix, PSA (400mg), C18 (400mg) are used
Supernatant is purified with GCB (45mg).Guide, the recycling of pesticide residue are recycled according to 11945/2015 pesticide residues of European SANTE
Rate should be between 70% to 120%.By result it is found that using methanol as extractant, the rate of recovery of ethiprole is 55.7%,
The rate of recovery of his three kinds of compounds is only 70%, therefore methanol cannot act as extractant.For acetonitrile/formic acid (99.95/0.05,
V/v) and acetonitrile/formic acid (99.9/0.1, v/v), the rate of recovery is between 70.4% and 86.8%.When select acetonitrile as extractant
When, the rate of recovery is in the range of 94.4% to 101%.This may be to cause the rate of recovery relatively low because formic acid is added in acetonitrile.
Result of study shows that acetonitrile obtains the highest rate of recovery as extractant and meets 11945/2015 guides of European SANTE.
The influence of adsorbent to matrix effect in the application of egg of embodiment 4
In order to improve the accuracy of detection and reduce matrix effect, purified in pesticide residue analysis.
QuEChERS methods are used for ethiprole and its metabolite in purifying complex matrix.In this application, we have selected (A)
PSA(400mg)+GCB(45mg)；(B)PSA(400mg)+C18(400mg) and (C) PSA (400mg)+C18(400mg)+GCB
(45mg) is used as QuEChERS adsorbents.It is next pure by using mixed adsorbent set forth above in sample pretreatment process
Change supernatant.The clean-up effect of three kinds of purifying adsorbents is assessed using the rate of recovery, the results are shown in Figure 2.By comparing recycling
Rate it is found that the rate of recovery between 91.2% to 102%.Mixed adsorbent (C) has most effective clean-up effect.Mixing is inhaled
Attached dose (A), the rate of recovery range of all compounds is between 73.6% to 82.4%, for mixed adsorbent (B), rate of recovery model
It is trapped among between 76.9% to 90.1%.In view of the effect of Sample Purification on Single and concentration, therefore select mixed adsorbent (C) PSA
(400mg), C18(400mg) and GCB (45mg) are as the adsorbent during sample purification.
Matrix effect (ME) is the major defect of pesticide residue determination in LC-MS/MS, it is to selectivity, sensitivity, linearly
It is had a significant impact with accuracy.The bare substrate obtained using the above method is configured to matrix mark-on solution, matrix mark-on standard
The slope of solution is compared with the slope of solvent standard curve, and when matrix effect is when between ± 20%, we can ignore base
Mass effect.When matrix effect is between ± 20% and ± 50%, it is referred to as medium matrix effect.When matrix effect less than-
50% or higher than 50% when, be referred to as strong substrate inhibition or enhancing.
Since the ratio of lipid and protein is higher in egg, it is one that the matrix in egg, which is eliminated in sample preparation procedure,
A challenge.Present applicant proposes three kinds of mixed adsorbents to reduce matrix effect.The MS of UPLC-QqLIT-MS/MS3Pattern is used for
Detect ethiprole and its metabolin.MS3Similar detection limit not only may be implemented, and sample preparation procedure and simplification can be reduced
Chromatographic separation condition.So MS3It is the useful tool of the quantitative analysis in complex matrices.(MS is scanned with second order ms2) compare,
MS3Background signal can be further decreased and realize lower detection limit.Table 1 is summarized in different mixed adsorbent situations
Under, the matrix effect (ME) and relative standard deviation (RSDs%) of ethiprole and its metabolite.For mixed adsorbent (A),
Matrix effect value is as follows：The ethiprole of 2 μ g/kg is that the ethiprole sulfone of -23.9%, 0.2 μ g/kg is -20.7%, 0.2 μ g/kg
Ethiprole thioether be 22.7% and 0.2 the fluorine formonitrile HCN of μ g/kg be 20.6%.And other value ranges are -20% and 20%
Between.For mixed adsorbent (B), other than 0.2 μ g/kg fluorine formonitrile HCN standard items are -22.7%, other matrix effects are equal
In the range of -20% to 20%.However, mixed adsorbent (C) embodies good matrix effect, corresponding matrix effect takes
Value is in the range of -8.60% to 9.30%.By studying the matrix effect of three kinds of mixed adsorbents, corresponding experimental result
Show that when using mixed adsorbent (C) to be purified as adsorbent best purification result can be obtained.
In the case of the different mixed adsorbent of table 1, the matrix effect (ME) of ethiprole and its metabolite and relative standard
Deviation (RSDs%)
Embodiment 5
Leek, ginger application in influence of the extractant to the rate of recovery
Extractant selects acetonitrile, acetonitrile/formic acid (99.9/0.1, v/v), acetonitrile/water (75/25, v/v) and acetonitrile/water
(50/50, v/v) is compared.For acetonitrile/water (50/50, v/v), for the rate of recovery of the target agricultural chemical compound in leek
Part is more than 120%, the testing result of pesticide：Omethoate (142.8%), tebufenpyrad (140.6%), Buprofezin (163.2%)
With diflubenzuron (149.8%), for acetonitrile/water (75/25, v/v), rate of recovery 76.5-115%, be above acetonitrile and acetonitrile/
Formic acid (99.9/0.1, v/v), but the rate of recovery of acetonitrile/water (75/25, v/v) is integrally more stable, and for part agriculture
The rate of recovery of drug compound such as carbofuran, phoxim, Acetamiprid is higher than acetonitrile/water (50/50, v/v), the recycling of the pesticide of ginger
The rate of recovery of rate corresponding pesticide with leek is essentially identical.Therefore, selection acetonitrile/water (75/25, v/v) is as extractant
Extract all target compounds.
Embodiment 6
Leek, ginger application in influence of the adsorbent to the rate of recovery and matrix effect
In QuEChERS methods, in order to reduce matrix effect, need to add adsorbent adsorbing contaminant.The purification of the application
In the process, make adsorbent with inkization carbon black (GCB) to be used in reset procedure, the rate of recovery of some analytes reduces, such as more bacterium
Clever (59.6%), diflubenzuron (63.5%) and phoxim (67.6%) etc..In general, GCB cannot be selected for remain more
Analysis especially has strong preserve because it is with planar structure to the compound of planar ring structure.Some pesticides have planar junction
Structure, such as carbendazim, diflubenzuron can be absorbed by GCB.During the experiment, we chance on even if not using adsorbent, mostly
Number target compound also can ignore that matrix effect.This is potentially based on most of matrix fact soluble easily in water, most of matrix
All in water layer, target compound is in acetonitrile layer, when we use anhydrous MgSO4When going water removal, most of polarity matrix is also inhaled
It is attached.If not using adsorbent, sample preparation procedure can shorten to original one third.In this application, it is not suitable for and inhales
Attached dose, the sample of only 1 μ L is injected into LC-QqLIT-MS/MS instruments, and matrix effect is as shown in table 2 and instrument damage is equal
It is down to extremely low level.
Table 2MS3Matrix effect (n=5) of the three kinds of compounds of pattern in leek, ginger
7 multiple scanning of the mass spectrum (MS of embodiment3) with the comparison of multiple-reaction monitoring (MRM) pattern
When background signal is high or the MRM of standard is made to measure difficult strongly for interference, MS3Method is the effective of quantifying analytes
Strategy.Target compound cannot accurately be detected since there are matrix effects for the complex matrices in LC-MS/MS.At this
In the case of kind, MS3With preferably quantitative and identification sensitivity, accuracy and reproducibility.For the mite of rattling away in leek matrix
The MRM assay method mesostromas inhibition of spirit and carbendazim, the difenoconazole in ginger matrix, standard cannot be ignored.So making
Use MS3Pattern reduces substrate inhibition.As shown in table 1, pyridaben (5.3%), difenoconazole (- 7.5%) and carbendazim (-
3.2%) matrix effect is reduced to -20% to 20% range, even lower than ± 10%.In this application, using simplified
Pre-treatment processing method and MS3Pattern combines, and matrix effect is reduced in the range of -10% and 10%.With substrate inhibition
It reduces, the rate of recovery of three kinds of analytes is increased between 90.3% to 111%.MS3Scanning alternatively can be carried significantly
The detectability of compound in high complex matrices.It can be with MS by table 33Pattern has the better rate of recovery, MS compared to MRM patterns3
In the range of 90.3% to 111%, between 4.6% to 9.2%, RSDs is less than RSDs the rate of recovery of pattern target compound
European SANTE 11945/2015 recommended 20%, show that the experimental method has good preci-sion and accuracy, Neng Gouman
Foot analysis requires.
The rate of recovery and relative standard deviation (n=5) of the 3 three kinds of compounds of table in leek, ginger
a：Relative standard deviation, each concentration Parallel testing 5 times, n=5.
b：MS3The average recovery rate and relative standard deviation of mode detection.
In order to compare MS3The difference of pattern and MRM patterns in terms of the matrix effect for reducing compound, to being stirred after smashing
The carbendazim standard solvent of 1.0 μ g/kg is added in uniform leek matrix.Use the MRM and MS of LC-QqLIT-MS/MS3Pattern
Sample is analyzed respectively, the MRM spectrograms and MS of acquisition3Spectrogram is as shown in Figure 3.MRM transition m/z192.0/160.1 and m/z
192.0/132.2 background level is higher, matrix interference is stronger.When using MS3When scan pattern, the matrix interference drop in carbendazim
It is low, only m/z 192.0 → 160.1 → 132.2 and m/z 192.0 → 132.2 → 105.1, show higher selectivity and
Sensitivity, being capable of accurately qualitatively and quantitatively compound.By comparing, MS3The sound of quota ion and qualitative ion under pattern
It should be worth 20 times higher than the response under MRM pattern, and the qualitative quasi-molecular ions in MRM patterns is covered by matrix interference peak, MS3Mould
Qualitative quasi-molecular ions peak shape under formula is preferable, and response is also higher, shows higher selectivity, quantitative reliable, compared with MRM,
MS3Background signal can be further decreased and realize lower detection limit.Therefore, MS3Pattern compared to MRM patterns can base
It is applied when mass effect is very high or is difficult to detect target compound, while MS3Pattern also simplifies sample pretreatment process.
Embodiment 8 chromatography and Mass Spectrometry Conditions in the application of egg
Chromatographic condition optimizes：In order to detect the ethiprole and its metabolin in egg, chromatographic parameter is optimized.Color
It composes column and mobile phase is most important for Pesticides Testing.Research process uses water generationBEH C18Chromatography
Column analyzes 4 kinds of compounds in 6min, and packing material size is 1.7 μm, because it has good separating degree and peak shape.
The peak shape and retention time of mobile phase impact analysis object, ethiprole and its metabolin have good under neutral and acid condition
Stability.It is 0.01%, 0.05% respectively to obtain satisfied as a result, we have studied the formic acid of four kinds of various concentrations,
0.1% and 0.15%.By the research to the rate of recovery, mobile phase is finally used as using 0.1% formic acid water and methanol, it is a small amount of sour
Addition can have the function that improve separative efficiency, improve peak shape, and increase the ionizing efficiency of target compound, improve sensitive
Degree.Finally we obtain simple and excellent chromatographic condition in the case of no any buffer additive.In optimal conditions
Under, when analyzing target compound using ultra performance liquid chromatography, 4 kinds of pesticides can detect completely in 6min, and peak shape is symmetrical,
High sensitivity, quota ion flow graph of 4 kinds of compounds in 0.5ng/mL are shown in that Fig. 4, appearance time are as shown in table 5.
Mass Spectrometry Conditions optimize：In liquid chromatography-tandem mass spectrometry, the ethiprole of 10ng/mL and its metabolin are mixed into mark
Standard is injected into mass spectrograph and optimizes, and obtains best mass spectrometry parameters.These four compounds are in ESI-It is studied under pattern,
4 kinds of compounds obtain [M-H]-Peak, MS3Scanning is performed with rapid cycle time, and use is with second generation product ion m/
Narrow scan range centered on z, the scan pattern can be used for quantitative combination object.Mass Spectrometry Conditions of the ethiprole in 10ng/mL
Optimization process is shown in Fig. 5；Ethiprole (m/z 435.1) is 6 in the impinging air flows (CAD) of instrument setting, the collision energy of optimization
(CE) be 25eV when, optimize and obtain enrich highest peak in m/z 330.0, which is chosen as quota ion, use
Dynamic ion filling time and 25 milliseconds of firing time level-one fragment ion is captured as parent ion and accumulate linearly from
In sub- trap (LIT), MS3Spectrogram shows strong peak at m/z 250.0.Therefore, the qualitatively and quantitatively MS/MS/MS of ethiprole
Ion transition is respectively m/z 435.1 → 330.0 → 250.0 and 435.1 → 250.0 → 215.0.In Figure 5, highest abundance from
Son is quota ion, and second is qualitative ion.Last ethiprole and its metabolite go the mass spectrums such as cluster voltage, impact energy to join
Number is optimised, obtains best Mass Spectrometry Conditions, as shown in table 4.
The retention time and best mass spectrometry parameters of 4 ethiprole of table and its metabolin
T- retention times；* qualitative ion；aTwo level daughter ion；bThree-level daughter ion；cThe impact energy of secondary ion；dThree-level from
The impact energy of son
Embodiment 9 leek, ginger application in mass spectrographic parameter
In order to obtain maximum signal response, chromatographic parameter is optimized.Mobile phase is containing 0.1% formic acid
Ultra-pure water and methanol, the mobile phase containing formic acid can increase the ionizing efficiency of target compound, form good peak shape.It uses
Agilent EC-C18Front-end protection column protect chromatographic column, Poroshell 120EC-C18Chromatographic column carries out chromatographic isolation.
In liquid chromatography-tandem mass spectrometry, the mixed standard solution of 26 kinds of target compounds of 10ng/mL is injected into mass spectrograph and is carried out
Optimization, obtains best mass spectrometry parameters.
In MS3Under pattern, the parent ion detached first is protonation, such as carbendazim (m/z 192.0), is set in instrument
The impinging air flows (CAD) set are 6, when the collision energy (CE) of optimization is 25eV, are optimized and the highest peak of the value that meets with a response
In m/z 160.1, which is chosen as quota ion, using Dynamic ion filling time and 25 milliseconds of firing time by level-one
Fragment ion is captured and is accumulated in linear ion hydrazine (LIT), MS as parent ion3Spectrogram is shown strongly at m/z 132.2
Peak.Therefore, the qualitatively and quantitatively MS/MS/MS ion transitions of carbendazim are respectively 192.0 → 160.1 → 132.2 Hes of m/z
192.0 → 132.2 → 105.1, Mass Spectrometry Conditions optimization process of the carbendazim in 10ng/mL is shown in Fig. 6.In figure 6, highest abundance
Ion is quota ion, and second is qualitative ion.
The mixed standard solution of three kinds of target compounds of 10ng/mL, which is injected into mass spectrograph, to be optimized, and is obtained best
Mass spectrometry parameters.MS is listed in table 53The three-level mass spectrometry parameters of the three kinds of target compounds detected under pattern.
5 MS of table3The three-level mass spectrometry parameters of the three kinds of target compounds detected under pattern
aTwo level daughter ion；bThree-level daughter ion；cRetention time；dThe impact energy of secondary ion；eThe impact energy of three-level ion；*
Qualitative ion
The detection of 10 actual sample of embodiment
60 egg samples are had purchased from Chinese Supermarket, brand shop and wholesale market.Using this method to all egg samples
Product carry out ethiprole and its metabolite retention analysis.All samples are all made of the MS of UPLC-QqLIT-MS/MS3Method carries out
Analysis.Result of study shows to find ethiprole in three samples, and concentration is respectively 0.28 μ g/kg, 0.34 μ g/kg and 1.1 μ
g/kg.Maximum residue limit (MRLs) of the ethiprole in egg is different in different countries.LOQs proposed in this paper is far low
In the MRLs of global All Countries.The quantitative analysis of this method ethiprole and its metabolite suitable for egg.
It summarizes：
1) specific
Due to MS3Scanning function, UPLC-QqLIT-MS/MS provide it is very high selectivity and specificity.Pass through ratio
Compared with the MS of blank egg and matrix mark-on egg sample3Spectrogram assesses the selectivity of this method.Fig. 7 (a) shows blank chicken
The MS of ethiprole and its metabolite in egg sample3Spectrogram.Fig. 7 (b), 7 (c), 7 (d) and 7 (e) show that spiked levels are 2 μ
The ethiprole of g/kg and its MS of metabolin3Spectrogram.The result shows that not interfering with peak near the retention time of target compound
In the presence of.
2) rate of recovery and repeatability
It can be obtained from Fig. 2, the MS of UPLC-QqLIT-MS/MS methods3The rate of recovery of four kinds of target compounds of pattern exists
In the range of 90.7% to 105%, between 0.7% to 4.9%, RSDs is pushed away RSDs less than Europe SANTE 11945/2015
20% recommended shows that the experimental method has good preci-sion and accuracy, disclosure satisfy that analysis requires.
3) linear, LODs and LOQs
Application in detecting egg, on the basis of embodiment 1, by the linear of standard calibration curve assessment response,
Six concentration levels for analyzing 4 kinds of target compounds, are 0.05,0.1,0.5,1,10 and 20ng/mL respectively.Related coefficient (r2)
Linear for assessing, the results are shown in Table 6, and standard calibration curve shows good linear, the related coefficient of all compounds
(r2) it is above 0.999.
In order to assess the sensitivity of proposed method, it is thus necessary to determine that LODs and LOQs.It is calculated with 3 times of signal-to-noise ratio (S/N)
Detection limit calculates quantitative limit with 10 times of signal-to-noise ratio (S/N).Detection limit and quantitative limit are as follows：Ethiprole 0.06 μ g/kg and 0.20 μ
g/kg；Ethiprole sulfone 0.02 μ g/kg and 0.07 μ g/kg；Ethiprole thioether 0.05 μ g/kg and 0.17 μ g/kg；0.04 μ of fluorine formonitrile HCN
G/kg and 0.13 μ g/kg.These Data Summaries are in table 6.The LOQs of all analytes is below the maximum residue limit of European Union
It is worth (0.005mg/kg).Therefore, ethiprole and its metabolin of this method suitable for quantitative analysis egg.
Related coefficient, the rate of recovery, RSDs, quantitative limit and detection limit of the 64 kinds of compounds of table in egg sample
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum is to the method for more pesticide residue determinations in food, special
Sign is：The sample injection volume of this method is 1 μ L；Sample is through the triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum and multistage
Scanning of the mass spectrum pattern is analyzed；The liquid chromatogram is common liquid chromatogram or ultra performance liquid chromatography.
2. a kind of triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum is detecting Pesticide Residues method for measuring
Food egg, leek, ginger pesticide residue in application.
3. a kind of triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum is to Pesticide Residues method for measuring, feature
It is：The food is egg, the specific steps are：
1) extraction of sample
Sample is uniformly mixed with extractant, centrifuges, takes upper solution；
2) purification of sample
Supernatant liquor is subjected to vortex mixed with adsorbent, centrifuges, upper liquid is taken to pass through 0.22 μm of nylon filter film；
3) UPLC-QqLIT-MS/MS is analyzed
The filtered sample of 1 μ L is examined through the triple level four bars of ultra performance liquid chromatography-- linear ion hydrazine mass spectrum
It surveys, is analyzed using multi-stage ms scan pattern；
Preferably, the chromatographic column of ultra performance liquid chromatography is WatersBEH C18；
Preferably, triple level four bars-linear ion hydrazine mass spectrum uses ESI-Pattern carries out；
Preferably, multiple scanning of the mass spectrum pattern is run with negative ion mode；Ion source temperature is 550 DEG C；Ion injection electric be-
4500V。
4. according to the method described in claim 3, it is characterized in that：Extractant in the step 1) by magnesium sulfate, sodium chloride,
Acetonitrile forms；Magnesium sulfate, sodium chloride, acetonitrile ratio be 2-4g：1-3g：18-22ml.
5. according to the method described in claim 3, it is characterized in that：Adsorbent described in step 2) be PSA, C18 and GCB, three
The mass ratio of kind adsorbent PSA, C18 and GCB are 75-85：75-85：8-10.
6. according to the method described in claim 3, it is characterized in that：The mobile phase A of the liquid chromatogram is containing 0.1% formic acid
Aqueous solution, Mobile phase B is the methanol containing 0.1% formic acid.
7. a kind of triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum is to Pesticide Residues method for measuring, feature
It is：The food includes leek, ginger, the specific steps are：
1) extraction and purification of sample
Sample is uniformly mixed with extractant, centrifuges, upper solution is taken to cross 0.22 μm of filter membrane；
2) LC-QqLIT-MS/MS is analyzed
The filtered sample of 1 μ L is detected through the triple level four bars of liquid chromatogram-- linear ion hydrazine mass spectrum, is utilized
Multi-stage ms scan pattern is analyzed；
Preferably, in step 2) sample a concentration of 10ng/mL；
Preferably, the chromatographic column Poroshell 120EC-C of the condition of liquid chromatogram18；
Preferably, triple level four bars-linear ion hydrazine mass spectrum uses ESI+Pattern carries out；
Preferably, multi-stage ms scan pattern is run with positive ion mode；Ion source temperature is 550 DEG C；Ion injection electric is
5500V。
8. according to the method described in claim 7, it is characterized in that：Extractant includes sodium chloride, magnesium sulfate in the step 1)
And by acetonitrile and water composition mixture.
9. according to the method described in claim 7, it is characterized in that：The body of acetonitrile and water in the acetonitrile and water composition mixture
Product is than being 1:1-3:1；The ratio of the mixture of sodium chloride, magnesium sulfate and acetonitrile and water is 3-5g:0.8-1.2g:18-22ml.
10. according to the method described in claim 7, it is characterized in that：The formic acid that the mobile phase A of the liquid chromatogram is 0.1%
Aqueous solution；Mobile phase B is methanol.
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| CN114166957A (en) * | 2021-10-29 | 2022-03-11 | 湖南省食品质量监督检验研究院 | Method for detecting toxoflavin and calorimetrin in food and degradation products thereof |
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