CN105699520A

CN105699520A - Quick measurement method for fluxapyroxad residue in animal derived foods

Info

Publication number: CN105699520A
Application number: CN201610070241.2A
Authority: CN
Inventors: 崔淑华
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-01-30
Filing date: 2016-01-30
Publication date: 2016-06-22

Abstract

The invention discloses a quick measurement method for fluxapyroxad residue in animal derived foods. The method is mainly used for measuring the content of residual fluxapyroxad in the animal derived foods with high fat content. The method comprises the following steps: homogeneously extracting residual fluxapyroxad from a sample using acetonitrile; performing EMR (enhanced matrix removal) matrix dispersion purification as well as reverse-extraction tube extraction and concentration on the extract; performing GC-NCI-MS (gas chromatography-negative chemical ionization-mass spectrometry) detection; establishing a corrected standard work curve using a blank matrix solution without the pesticide to be detected; and quantifying by an external standard method. With average recovery rate of 86.7-93.8%, average RSD (relative standard deviation) of 6.0-7.8% and detection limit lower than 2.46mug/kg, the method has the advantages of simplicity and quickness in detection, good impurity removal effect, high sensitivity, strong characteristic feature, good repeatability and qualitative and quantitative accuracies and can meet the technical requirements on the safety detection of corresponding products in countries including America, Europe and Japan and provide powerful technical support to guarantee the food safety for national people and the sound development of export trade.

Description

The rapid assay methods of fluxapyroxad residual in a kind of animal derived food

Technical field

The present invention relates to the rapid assay methods of fluxapyroxad residual in a kind of animal derived food, more specifically adopt Gas Chromatography-Negative chemical ionization source-mass spectrum (GC-NCI-MS) qualitative, quantitative to measure the method for the fluxapyroxad content of residual in the animal derived food that the fat contents such as animal muscle and goods such as Carnis Sus domestica, beef, Carnis caprae seu ovis, Carnis Gallus domesticus are high, belong to the determination techniques field of persticide residue。

Background technology

Fluxapyroxad (fluxapyroxad) is the succinate dehydrogenase inhibitors series bactericidal agent developed by BASF AG。Chemical name: 3-difluoromethyl-1-methyl-N-(3', 4', 5'-trifluoro-biphenyl-2-base)-1H-pyrazole-4-carboxamide；Molecular formula: C18H12F5N3O；Relative molecular mass: 381.3；Fusing point: 157 DEG C；Relative density: 1.42。Dissolubility (g/L, 20 DEG C) in solvent: acetone > 250, acetonitrile 168, ethyl acetate 123, methanol 53.4, toluene 20.0。The chemical structural formula of fluxapyroxad is as follows:

Fluxapyroxad has efficiently, wide spectrum, holding effect, excellent Uptake and translocation, have advantages such as preventive and therapeutic action simultaneously。It can suppress the elongation of spore-germination, spore pipe, mycelial growth and Sporulation, can effectively prevent and treat the Major Diseases of corn, Semen sojae atricolor, Semen Maydis, Brassica campestris L and special crop etc.。This product prevents and treats a series of fungal disease by blade face and seed treatment, such as the disease caused by Septoria musiva (Septoria), the pathogen of Botrytis cinerea (Botrytis), powdery mildew (Erysiphe), tail spore bacterium (Cercospora), handle rest fungus (Puccinia), rhizoctonia (Rhizoctonia), nuclear cavity bacteria (Pyrenophoraspp.) etc. on corn, Semen sojae atricolor, fruit tree and vegetable。It is particularly well-suited to bean, gray mold (Botrytiscinerea), rust, powdery mildew and the microbial disease of septoria musiva that preventing and treating is caused by alternaric bacteria (Alternariaspp.), by the microbial disease of rod method etc. on soybean rust (Phakopsora), the disease that Cotton Gossypii is caused by Rhizoctonia solani Kuhn (Rhizoctoniasolani) and Helianthi and oilseeds dish。Under all test doses, all crops are very safe。Fluxapyroxad suitability is strong, can with the composite use of other products of pyraclostrobin, triazole bactericidal agent and BASF。Research shows, fluxapyroxad has better activity than commercially available acid amide fungicides, and no matter this product is active, multi-functional, or is all that modern fungicides establishes new mark post in interior absorption and rain wash resistance etc.。BASF intends to introduce fluxapyroxad more than 50 country in the world, for 100 various crop。2011, fluxapyroxad was first in Britain's registration and listing, and the country of existing registration and listing includes Australia, the U.S., Canada, state of European Union 13, Brazilian and Chinese etc.。Company predict, this product year peak value sales volume up to 600,000,000 Euros。This is in all SDHI series bactericidal agents, the product that expected value that development company expresses is the highest。

Along with the registration of fluxapyroxad, popularization and use, its maximum allowable residual quantity (MRL) in the food agricultural product such as veterinary antibiotics, Cereals and livestock products has been formulated as countries such as the U.S. in China's main exit market, Canada, as the U.S. specifies that fluxapyroxad MRL in fruit and vegerable is 0.5～30mg/kg, in the animal derived food such as livestock products and aquatic products, MRL is 0.01mg/kg, and in nut and Cereals, MRL is 0.06～15mg/kg；Canada regulation fluxapyroxad MRL in fruit and vegerable is 0.02～2mg/kg, and in the animal derived food such as livestock products and aquatic products, MRL is 0.01～0.05mg/kg, and in nut and Cereals, MRL is 0.01～3mg/kg；If European Union, Japan and other countries regulation field use pesticide not register in this country, when not formulating corresponding residue limits standard, it is exported to the food agricultural product of its country and includes residue limits in the animal derived foods such as livestock meat and all carry out " uniform limit " of 0.01mg/L。

Present stage, the research of fluxapyroxad determination of residual amount method is less, the detection method of report is mainly in vegetable and fruit fluxapyroxad method for detecting residue, these detection methods all adopt Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to measure the detection method of fluxapyroxad residual quantity in vegetable and fruit, use LC-MS/MS to measure food Residual Pesticides in Farm Produce and have quickly, easy, sensitivity advantages of higher, but due to its price costly, a lot of testing agencies, enterprise or scientific research institutions do not configure this instrument or configuration number of units is less, when adopting LC-MS/MS detection due to different compounds, different mobile phases or chromatographic column need to be used, chromatographic column constantly changed by such needs, mobile phase also expends the long time system is balanced, this constrains the application of LC-MS/MS to a certain extent。The gaschromatographic mass spectrometry (GC-NCI-MS) in outfit negative chemical ionization source is analyzed food Residual Pesticides in Farm Produce tool and is had great advantage, Negative chemical ionization (NCI source) is referred to as mass spectrum " soft ionization source ", analyte containing electronegativity group is had high selectivity and high sensitivity, owing to its characteristic is strong, when utilizing it to carry out retention analysis, matrix interference is little, very accurately object can be carried out qualitative and quantitative analysis。Existing various testing agencies and enterprise have all purchased gas chromatograph-mass spectrometer (GC-MS), also generally all it is equipped with Negative chemical ionization (NCI), now a lot of class pesticide all contain electronegativity group, organochlorine and pyrethroid pesticide molecule and mostly contain the strong electronegative group such as-F ,-Cl ,-Br or-COO-；Organophosphorus pesticide molecule mostly contains=S ,-OR ,-P, the electronegativity group such as-O-,-Cl or-P=O；And the novel agrochemical developed in recent years contains-F group mostly, therefore, GC-NCI-MS is used can conveniently to realize the multi-residue analysis of Multiple Pesticides, compared with GC-EI-MS, better capacity of resisting disturbance, less sensitivity and better selectivity can be obtained, but have no the report of the GC-NCI-MS detection method of fluxapyroxad residual quantity in food agricultural product up to now。Additionally, it is known that, QuEChERS pretreatment technology has become sample-pretreating method most widely used in pesticide residue analysis, but QuEChERS method is primarily adapted for use in the High water cut substrate of low-fat content, such as most fruit and vegetable, when in the face of high fat content substrate limited in one's ability, it is generally required to increase normal hexane grease removal, freezing sample extracting solution goes the steps such as fat, but limited efficiency。Some company is proposed enhancement mode lipid and removes patented product-EnhancedMatrixRemoval, EMR-lipid in recent years, is mainly used in removing the lipid chaff interference with straight chain hydrocarbon structure, including free fatty, cholesterol, triglyceride, phospholipid etc.。This product has been made into matrix solid phase dispersion extractant, either directly through dispersive solid-phase extraction flow process, the lipid impurities in extracting solution can be removed after hydroactivated, the present invention applies when this pre-treating method purifies animal derived food and finds, the method is simply efficient, lipid material removal effect is good, sets up the detection method of fluxapyroxad residual quantity in the dispersion purification of EMR substrate, Gas Chromatography-Negative chemical ionization source-mass spectrum (GC-NCI-MS) qualitative and quantitative analysis animal derived food significant。

Summary of the invention

It is an object of the invention to provide the rapid assay methods of fluxapyroxad residual in a kind of animal derived food, be mainly used in measuring fluxapyroxad residual quantity in the food agricultural product that the fat contents such as animal derived food are high。

For realizing object above, the technical solution adopted in the present invention is: the rapid assay methods of fluxapyroxad residual in a kind of animal derived food, comprises the steps:

(1) extract

Weigh mixing sample in tool plug centrifuge tube, after adding suitable quantity of water recovery, be quantitatively adding acetonitrile solution homogenizing and extract, be subsequently adding sodium chloride and anhydrous magnesium sulfate, centrifugal after violent vortex 1min。

Purify

Enhancement mode lipid is removed product E MR water activate, pipette sample extracting solution and purify in pipe in the EMR activated, after vortex 1min, 7000r/min is centrifuged 5min, shifts whole supernatant and saltouts to the centrifuge tube equipped with anhydrous magnesium sulfate and sodium chloride, after vortex centrifugal, pipette the supernatant of certain volume, be concentrated into after doing, dissolve constant volume with the acetone that volume ratio is 1/1/normal hexane mixed solvent, after crossing film, treat Gas Chromatography-Negative chemical ionization source-mass spectrum (GC-NCI-MS) detection。

(3) preparation of standard working solution

The same kind matrix blank sample of not Fluxapyroxad-containsterilization is processed by above-mentioned steps (1), (2), obtain sample extraction and purify residue, adding appropriate solvent and standard solution, vortex mixes, and is configured to the fluxapyroxad series standard working solution of at least 3 concentration。

(4) measure and result calculates

The standard working solution of each Concentraton gradient in step (3) is carried out GC-NCI-MS mensuration, with the chromatographic peak area of standard working solution, its respective concentration is carried out regression analysis, obtain extraction standard working curve；Under the same conditions the sample liquid after purification in step (2) is injected GC-NCI-MS to be measured, record the chromatographic peak area of fluxapyroxad in sample liquid, substitute into extraction standard working curve, obtaining fluxapyroxad content in sample liquid, then the Mass Calculation of sample representated by liquid obtains fluxapyroxad residual quantity in sample per sample；If fluxapyroxad residual quantity exceedes the range of linearity upper limit in upper machine solution, with constant volume solvent, upper machine solution concentration need to be diluted within the range of linearity。

Step (1) if in the sample of the moisture content less such as sample animal livers, suitable quantity of water must be added before extraction and fully infiltrate。

In step (4), GC conditions is: chromatographic column: HP-5MS capillary chromatographic column, column length 30m, internal diameter 0.25mm, thickness 0.25 μm；Injector temperature 250 DEG C；Carrier gas: He, not shunt mode sample introduction, sample size: 1 μ L；Constant current mode, flow velocity 1.0mL/min；Heating schedule: initial temperature 60 DEG C keeps 2min, rises to 200 DEG C with the speed of 20 DEG C per minute, then rises to 220 DEG C with the speed of 2 DEG C per minute, then rises to 280 DEG C with the speed of 20 DEG C per minute, keeps 10min；Transmission line temperature: 280 DEG C。

In step (4), Mass Spectrometry Conditions is: ion source temperature 150 DEG C；Quadrupole rod temperature 150 DEG C；Ionization pattern: negative chemical ionization, i.e. NCI pattern, energy 70eV；Scan mode: Salbutamol Selected Ion Monitoring (SIM) pattern, the ion of monitoring is: 361.1,362.1,362.9。

When step (4) measures sample liquid and extraction standard working solution, if sample liquid Pesticides chromatographic peak retention time pesticide retention time corresponding to standard solution is consistent, and in the sample mass spectrum after background correction, selected ion all occurs, and abundance of ions is more consistent than the abundance of ions ratio with standard solution, then can determine whether sample liquid exists this pesticide；If above-mentioned two condition can not meet simultaneously, then judge without this kind of pesticide。

The beneficial effects of the present invention is:

The present invention utilizes dispersive solid-phase extraction technology, establish simplicity, quickly and the sample-pretreating method of sample mesostroma interference can be prevented effectively from, this pre-treating method is applied in Cereals, animal derived food the qualitative confirmation of fluxapyroxad and detection by quantitative in conjunction with GC-NCI-MS, average recovery rate is 86.7%～93.8%, average relative standard's deviation (RSD) is 6.0%～7.8%, detection limit, lower than 2.46 μ g/kg, has easy and simple to handle, quick, accurate, highly sensitive and reproducible advantage。The U.S., European Union, the Japan and other countries technology requirement to corresponding product safety detection can be met, for ensureing that our people's food safety and export abroad trade sound development provide strong technical support。

Accompanying drawing explanation

The GC-NCI-MS that Fig. 1 is the fluxapyroxad being added in blank Carnis Sus domestica substrate selects chromatography of ions figure。

The GC-NCI-MS that Fig. 2 is the Carnis Sus domestica blank sample of not Fluxapyroxad-containsterilization selects chromatography of ions figure。

The fluxapyroxad standard working curve that Fig. 3 is is substrate preparation with the Carnis Sus domestica blank sample of not Fluxapyroxad-containsterilization。

Detailed description of the invention

Now with following embodiment, the present invention is described, but is not restriction the scope of the present invention。

The instrument used in embodiment and reagent

T18Basic homogenizer (IKA, Germany)；CR21G III centrifuge (Hitachi, Japan)；MS3 basic model vortex mixer (IKA, Germany)；TurboVapLV pattern product automatic concentration instrument (Caliper, USA)；7890N gas chromatogram-5977C mass spectrograph (Agilent, USA)；Enhancement mode lipid is removed product (EnhancedMatrixRemoval, EMR) and is purchased from Anjelen Sci. & Tech. Inc of the U.S.。

Reagent

Acetonitrile, acetone, normal hexane (HPLC level, Merke, Germany)；Anhydrous magnesium sulfate, sodium chloride are analytical pure, all purchased from Chemical Reagent Co., Ltd., Sinopharm Group。

Standard substance: purity 99.5%, purchased from Dr.Ehrenstorfer company of Germany。

Embodiment 1: the detection of fluxapyroxad residual quantity in Carnis Sus domestica

(1) sample pre-treatments

Extract

Weigh the 5g Carnis Sus domestica sample through fully mixing in 50mL centrifuge tube, after adding 10mL water recovery 30min, accurately add 20mL acetonitrile solution, mechanical shaking extraction 20min, supersound extraction 5min, add 3g anhydrous magnesium sulfate and 2g sodium chloride, after vortex 1min, after the centrifugal 5min of 7000r/min, to be clean。

Purify

Add 4mL water in the purification pipe removing product E MR equipped with enhancement mode lipid, vortex makes it fully activate, pipette 6mL sample extracting solution and purify in pipe in the EMR activated, after vortex 1min, 7000r/min is centrifuged 5min, shift whole supernatant to saltout to the centrifuge tube equipped with anhydrous magnesium sulfate and sodium chloride, after vortex centrifugal, pipette 4mL supernatant, it is concentrated into after doing, dissolve constant volume with the acetone that volume ratio is 1/1/normal hexane mixed solvent, after crossing film, treat Gas Chromatography-Negative chemical ionization source-mass spectrum (GC-NCI-MS) detection。

(2) preparation of standard working solution

Accurately weighing 25 ± 0.1mg standard substance in 25mL volumetric flask, dissolve with methanol, constant volume obtains 1000.0 μ g/mL standard reserving solutions；Pipette 1.0mL standard reserving solution and be placed in 100mL volumetric flask, obtain 10.0 μ g/mL standard intermediate liquids with the acetone that volume ratio is 1/1/normal hexane mixed solvent constant volume；10 μ g/mL standard solution dilutions are made into 5,2,1,0.5,0.2,0.1 μ g/mL standard solution。The Carnis Sus domestica blank sample of not Fluxapyroxad-containsterilization is processed by above-mentioned pre-treatment step, obtain sample extraction and purify residue, this residue adds acetone/normal hexane mixed solvent and the 100 above-mentioned standard solution of μ L that 900 μ L volume ratios are 1/1, vortex mixes, and is made into 10,20,50,100,200,500 μ g/L extraction standard working solutions。

(3) Gas Chromatography-Negative chemical ionization source-mass spectrography (GC-NCI-MS) measures

The standard working solution of variable concentrations gradient is injected separately into GC-NCI-MS, carries out the quantitative analysis of fluxapyroxad content with external standard method, namely with the chromatographic peak area of standard working solution, its respective concentration is carried out regression analysis, obtain standard working curve；Under the same conditions sample extracting solution is injected GC-NCI-MS to be measured, record the chromatographic peak area of fluxapyroxad in sample liquid, substitute into standard working curve, obtaining fluxapyroxad content in sample liquid, then the Mass Calculation of sample representated by liquid obtains fluxapyroxad residual quantity in sample per sample。

Wherein chromatographic condition is:

Chromatographic column: HP-5MS capillary chromatographic column, column length 30m, internal diameter 0.25mm, thickness 0.25 μm。

Injector temperature: 250.0 DEG C, sample introduction pattern: Splitless injecting samples, sample size: 1 μ L。

Carrier gas: He, constant current mode, flow velocity 1.0mL/min。

Stove case heating schedule: initial temperature 60 DEG C keeps 2min, rises to 200 DEG C with the speed of 20 DEG C per minute, then rises to 220 DEG C with the speed of 2 DEG C per minute, then rises to 280 DEG C with the speed of 20 DEG C per minute, keeps 10min。

Transmission line temperature: 280 DEG C。

Wherein, mass spectrometry parameters is:

Ionization pattern: negative chemical ionization, i.e. NCI pattern, energy 70eV。

Ion source temperature: 150 DEG C；Quadrupole rod temperature 150 DEG C。

Scan mode: Salbutamol Selected Ion Monitoring (SIM) pattern；The ion of SIM monitoring is: 361.1,362.1,362.9, and quota ion is 361.1。

Qualitative Identification: at identical conditions, if sample liquid Pesticides chromatographic peak retention time pesticide retention time corresponding to standard solution is consistent, and in the sample mass spectrum after background correction, selected ion all occurs, and abundance of ions is more consistent than the abundance of ions ratio with standard solution, then can determine whether sample liquid exists this pesticide；If above-mentioned two condition can not meet simultaneously, then judge without this kind of pesticide。

Linear relationship:

With the chromatographic peak area of standard working solution, its respective concentration being carried out regression analysis, obtaining standard working curve is Y=208.32X-1299.8, and correlation coefficient is 0.9992。

Recovery of standard addition and repeatability:

The Carnis Sus domestica of not Fluxapyroxad-containsterilization adds the fluxapyroxad standard solution of 10,20 and 200 g/kg3 concentration levels of μ, add until pesticide and carry out the determination of residual amount by above-mentioned process step after 30min, mensuration concentration and pesticide theory are added concentration compare, obtain pesticide TIANZHU XINGNAO Capsul, each pitch-based sphere parallel assay 6 times, obtaining its relative standard deviation, measurement result is in Table 1。As can be seen from Table 1, in 3 mark-on levels, the average recovery rate of fluxapyroxad is 86.7%～93.8%, and average relative standard's deviation (RSD) is 6.0%～7.8%, illustrates that the response rate of the inventive method is high, reproducible。

The response rate of table 1 fluxapyroxad and repeatability (n=6)

Detection limit:

The fluxapyroxad extraction standard working solution of variable concentrations is injected GC-NCI-MS, calculating detection limit with the cycles of concentration (cycles of concentration of Carnis Sus domestica is for 1.0 times) of 3 times of signal to noise ratios of least concentration extraction standard solution chromatographic peak and sample handling processes, the detection of fluxapyroxad is limited to 2.46 μ g/kg。

Above embodiments is only that the preferred embodiment of the present invention is described; not the scope of the present invention is defined; under the premise designing spirit without departing from the present invention; various modification that technical scheme is made by this area ordinary skill technology and improvement, all should fall in the protection domain that claims of the present invention are determined。

Claims

1. the rapid assay methods of fluxapyroxad residual in an animal derived food, it is characterised in that said method comprising the steps of:

(1) extract

Weigh mixing sample in tool plug centrifuge tube, after adding suitable quantity of water, after adding the extraction of acetonitrile solution homogenizing, add sodium chloride and anhydrous magnesium sulfate, centrifugal after violent vortex 1min；

(2) purify

Enhancement mode lipid is removed product E MR water activate, pipette sample extracting solution and purify in pipe in the EMR activated, after vortex 1min, 7000r/min is centrifuged 5min, shifts whole supernatant and saltouts to the centrifuge tube equipped with anhydrous magnesium sulfate and sodium chloride, after vortex centrifugal, pipette the supernatant of certain volume, be concentrated into after doing, dissolve constant volume with the acetone that volume ratio is 1/1/normal hexane mixed solvent, after crossing film, treat Gas Chromatography-Negative chemical ionization source-mass spectrum (GC-NCI-MS) detection；

(3) preparation of standard working solution

The same kind matrix blank sample of not Fluxapyroxad-containsterilization is processed by above-mentioned steps (1), (2), obtain sample extraction and purify residue, adding appropriate solvent and standard solution, vortex mixes, and is configured to the fluxapyroxad series standard working solution of at least 3 concentration；

(4) measure and result calculates

2. the rapid assay methods of fluxapyroxad residual in a kind of animal derived food according to claim 1, it is characterised in that step (1) if in the sample of sample moisture content less, suitable quantity of water must be added before extraction and fully infiltrate。

3. the rapid assay methods of fluxapyroxad residual in a kind of animal derived food according to claim 1, it is characterized in that, in step (4), GC-NCI-MS analysis condition is: chromatographic column: HP-5MS capillary chromatographic column, column length 30m, internal diameter 0.25mm, thickness 0.25 μm；Injector temperature 250.0 DEG C；Carrier gas: He, not shunt mode sample introduction, sample size: 1 μ L；Constant current mode, flow velocity 1.0mL/min；Heating schedule: initial temperature 60 DEG C keeps 2min, rises to 200 DEG C with the speed of 20 DEG C per minute, then rises to 220 DEG C with the speed of 2 DEG C per minute, then rises to 280 DEG C with the speed of 20 DEG C per minute, keeps 10min；Transmission line temperature: 280 DEG C；Ionization pattern: negative chemical ionization, i.e. NCI pattern, energy 70eV；Ion source temperature 150 DEG C；Scan mode: Salbutamol Selected Ion Monitoring (SIM) pattern, the ion of monitoring is: 361.1,362.1,362.9。