CN105628821A - Rapid residual penthiopyrad quantity determination method by GC-EI-MS - Google Patents

Abstract

The invention discloses a rapid residual penthiopyrad quantity determination method by GC-EI-MS (gas chromatography-mass spectrometry with electron impact ionization). The method is mainly used for determining content of residual penthiopyrad in animal-derived food high in fat content and includes: extracting the residual penthiopyrad from a sample homogeneously by acetonitrile, subjecting an extraction solution to matrix dispersed purification by EMR (enhanced matrix removal) and extraction and concentration of a reverse extraction tube prior to GC-EI-MS detection, establishing a corrected standard working curve by a vehicle solution containing no to-be-detected pesticide, and quantifying by an external standard method. The rapid residual penthiopyrad quantity determination method by GC-EI-MS has the advantages that average recovery rate ranges from 92.7% to 93.4%, average RSD (relative standard deviation) ranges from 4.9% to 7.6%, detection limit is lower than 4.38 microgrammes/kg, and the method is simple and convenient to operate, rapid, good in impurity removal effect, high in sensitivity and characteristics, good in repeatability, accurate in qualitative and quantitative determination and capable of meeting the technical requirements of countries such as America, European Union and Japan on corresponding product safety detection so as to provide a powerful technical support for people's food safety in China and healthy development of export trade.

Description

A kind of GC-EI-MS rapid assay methods of pyrrole metsulfovax residual quantity

Technical field

The present invention relates to the GC-EI-MS rapid assay methods of a kind of pyrrole metsulfovax residual quantity, more specifically adopt gas chromatogram-electron impact ion source-mass spectrum (GC-EI-MS) qualitative, quantitative to measure the method for the pyrrole metsulfovax 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

Pyrrole metsulfovax (Penthiopyrad) is the novel acid amide fungicides of Mitsui chemical company research and development, and English general penthiopyrad by name, test code number: MTF-753, commodity are called Afetofluapro. CAS accession number: 183675-82-3, chemical name is (RS)-N-[2-(1,3-dimethylbutyl)-3-thienyl]-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide, and chemical structural formula is:

Pyrrole metsulfovax has different fungicidal spectrums from existing carboxyl acylamide antibacterial, except the same with existing carboxyl acylamide antibacterial to basidiomycetes effectively except, it is also effective to ascomycetes, imperfect fungi, existing recommended for preventing and treating gray mold and the powdery mildew that other antibacterial have resistance. The normally used effective ingredient dosage of this medicament is 100��200g/hm², it is widely used in numerous crops such as fruit tree, vegetable, lawn, control rust, sclerotiniose, gray mold, downy mildew, scab of apple and powdery mildew. Penthiopyrad in 2008 Japan obtain ratify first, for fruit tree, vegetable and ornamental plant, after again in November, 2011 Canada get permission registration. Trade mark is called 3 products of Vertisan and Fontelis (all with penthiopyrad200g/L for its active component) and Treoris (penthiopyrad100g/L+chlorothalonil (Bravo) 250g/L) and is used primarily on fruit tree, vegetable and field crops, finds that pyrrole metsulfovax has very wide application prospect in practical application.

Along with the registration of pyrrole metsulfovax, popularization and use, having formulated its maximum allowable residual quantity (MRL) in the food agricultural product such as veterinary antibiotics, Cereals and livestock products as the European Union in China's main exit market, the country such as Canadian and Australian, Canada regulation pyrrole metsulfovax MRL in the animal derived foods such as fruit and vegerable, nut, Cereals and livestock products is 0.01��30mg/kg; Australia regulation pyrrole metsulfovax MRL in the animal derived foods such as fruit and vegerable, nut, Cereals and livestock products is 0.01��5mg/kg; European Union specifies that pyrrole metsulfovax MRL in the animal derived foods such as fruit and vegerable, nut, Cereals and livestock products is 0.01��30mg/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 pyrrole metsulfovax determination of residual amount method is less, the detection method of report is mainly in vegetable and fruit pyrrole metsulfovax method for detecting residue, these detection methods all adopt Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to measure the detection method of pyrrole metsulfovax 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-EI-MS) in outfit electron impact ionization source is analyzed food Residual Pesticides in Farm Produce tool and is had great advantage, owing to electron impact ionization source mass spectrum is versatility detector, the multi-residue analysis of hundreds of kind pesticide can be realized, can be simultaneously qualitative and quantitative, moderate, therefore existing various testing agencies and enterprise are equipped with gas chromatogram-electron impact ion source-mass spectrograph (GC-EI-MS) and the pesticide residues in food agricultural product are detected, but have no the report of the GC-EI-MS detection method of pyrrole metsulfovax 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 pyrrole metsulfovax residual quantity in the dispersion purification of EMR substrate, gas chromatogram-electron impact ion source-mass spectrum (GC-EI-MS) qualitative and quantitative analysis animal derived food significant.

Summary of the invention

It is an object of the invention to provide the GC-EI-MS rapid assay methods of a kind of pyrrole metsulfovax residual quantity, be mainly used in measuring pyrrole metsulfovax 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 GC-EI-MS rapid assay methods of a kind of pyrrole metsulfovax residual quantity, 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, shift whole supernatant to saltout to the centrifuge tube equipped with anhydrous magnesium sulfate and sodium chloride, after vortex centrifugal, pipette the supernatant of certain volume, it is concentrated into after doing, constant volume is dissolved with the acetone that volume ratio is 1/1/normal hexane mixed solvent, after crossing film, treat gas chromatogram-electron impact ion source-mass spectrum (GC-EI-MS) detection.

(3) preparation of standard working solution

Same kind matrix blank sample without pyrrole metsulfovax 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 pyrrole metsulfovax 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-EI-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-EI-MS to be measured, record the chromatographic peak area of pyrrole metsulfovax in sample liquid, substitute into extraction standard working curve, obtaining pyrrole metsulfovax content in sample liquid, then the Mass Calculation of sample representated by liquid obtains pyrrole metsulfovax residual quantity in sample per sample; If pyrrole metsulfovax 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 ��m; Injector temperature 250 DEG C; Carrier gas: He, not shunt mode sample introduction, sample size: 1 �� 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: electron impact ionization, i.e. EI pattern, energy 70eV; Scan mode: Salbutamol Selected Ion Monitoring (SIM) pattern, the ion of monitoring is: 302.1,177.0,359.1,166.1.

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 pyrrole metsulfovax and detection by quantitative in conjunction with GC-EI-MS, average recovery rate is 92.7%��93.4%, average relative standard's deviation (RSD) is 4.9%��7.6%, detection limit, lower than 4.38 �� 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-EI-MS that Fig. 1 is the pyrrole metsulfovax being added in blank Carnis Sus domestica substrate selects chromatography of ions figure.

The GC-EI-MS that Fig. 2 is the Carnis Sus domestica blank sample without pyrrole metsulfovax selects chromatography of ions figure.

The pyrrole metsulfovax standard working curve that Fig. 3 is is substrate preparation with the Carnis Sus domestica blank sample without pyrrole metsulfovax.

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 98.0%, purchased from American Sigma company.

Embodiment 1: the detection of pyrrole metsulfovax 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 chromatogram-electron impact ion source-mass spectrum (GC-EI-MS) detection.

(2) preparation of standard working solution

Accurately weighing 25 �� 0.1mg standard substance in 25mL volumetric flask, dissolve with acetonitrile, constant volume obtains 1000.0 �� g/mL standard reserving solutions; Pipette 1.0mL standard reserving solution and be placed in 100mL volumetric flask, obtain 10.0 �� g/mL standard intermediate liquids with the acetone that volume ratio is 1/1/normal hexane mixed solvent constant volume; 10 �� g/mL standard solution dilutions are made into 5,2,1,0.5,0.2,0.1 �� g/mL standard solution. Carnis Sus domestica blank sample without pyrrole metsulfovax 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 �� L that 900 �� L volume ratios are 1/1, vortex mixes, and is made into 10,20,50,100,200,500 �� g/L extraction standard working solutions.

(3) gas chromatogram-electron impact ion source-mass spectrography (GC-EI-MS) measures

The standard working solution of variable concentrations gradient is injected separately into GC-EI-MS, carries out the quantitative analysis of pyrrole metsulfovax 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-EI-MS to be measured, record the chromatographic peak area of pyrrole metsulfovax in sample liquid, substitute into standard working curve, obtaining pyrrole metsulfovax content in sample liquid, then the Mass Calculation of sample representated by liquid obtains pyrrole metsulfovax 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 ��m.

Injector temperature: 250.0 DEG C, sample introduction pattern: Splitless injecting samples, sample size: 1 �� 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: electron impact ionization, i.e. EI 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: 302.1,177.0,359.1,166.1; Quota ion is 302.1.

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=3323.31X-28747.46, and correlation coefficient is 0.9993.

Recovery of standard addition and repeatability:

Carnis Sus domestica without pyrrole metsulfovax adds the pyrrole metsulfovax standard solution of 10,20 and 200 g/kg3 concentration levels of ��, 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 pyrrole metsulfovax is 92.7%��93.4%, and average relative standard's deviation (RSD) is 4.9%��7.6%, illustrates that the response rate of the inventive method is high, reproducible.

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

Detection limit:

The pyrrole metsulfovax extraction standard working solution of variable concentrations is injected GC-EI-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 pyrrole metsulfovax is limited to 4.38 �� 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 (3)

1. the GC-EI-MS rapid assay methods of a pyrrole metsulfovax residual quantity, 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, shift whole supernatant to saltout to the centrifuge tube equipped with anhydrous magnesium sulfate and sodium chloride, after vortex centrifugal, pipette the supernatant of certain volume, it is concentrated into after doing, constant volume is dissolved with the acetone that volume ratio is 1/1/normal hexane mixed solvent, after crossing film, treat gas chromatogram-electron impact ion source-mass spectrum (GC-EI-MS) detection;

(3) preparation of standard working solution

Same kind matrix blank sample without pyrrole metsulfovax 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 pyrrole metsulfovax 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-EI-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-EI-MS to be measured, record the chromatographic peak area of pyrrole metsulfovax in sample liquid, substitute into extraction standard working curve, obtaining pyrrole metsulfovax content in sample liquid, then the Mass Calculation of sample representated by liquid obtains pyrrole metsulfovax residual quantity in sample per sample; If pyrrole metsulfovax 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.

2. the GC-EI-MS rapid assay methods of a kind of pyrrole metsulfovax residual quantity 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 GC-EI-MS rapid assay methods of a kind of pyrrole metsulfovax residual quantity according to claim 1, it is characterized in that, in step (4), GC-EI-MS analysis condition is: chromatographic column: HP-5MS capillary chromatographic column, column length 30m, internal diameter 0.25mm, thickness 0.25 ��m; Injector temperature 250.0 DEG C; Carrier gas: He, not shunt mode sample introduction, sample size: 1 �� 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: electron impact ionization, i.e. EI pattern, energy 70eV; Ion source temperature 150 DEG C; Scan mode: Salbutamol Selected Ion Monitoring (SIM) pattern, the ion of monitoring is: 302.1,177.0,359.1,166.1.