CN105548444A - GC-MS/MS (gas chromatography-tandem mass spectrometry) determining method of aniline pyrazolone residual quantity - Google Patents
GC-MS/MS (gas chromatography-tandem mass spectrometry) determining method of aniline pyrazolone residual quantity Download PDFInfo
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- 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
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- 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
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- G—PHYSICS
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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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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
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- G01N2030/062—Preparation extracting sample from raw material
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
Abstract
The invention discloses a GC-MS/MS (gas chromatography-tandem mass spectrometry) determining method of aniline pyrazolone residual quantity. The method is a method mainly used for determining the residual aniline pyrazolone content in agricultural products of complicated matrix food such as cereal grains and animal derived food. Acetonitrile or an acetonitrile solution containing one-percent acetic acid is used for performing homogeneous extraction of the residual aniline pyrazolone in samples; after C<18>/PSA (primary secondary amine) solid phase extraction column purification and concentration, GC-MS/MS detection is performed; a corrected standard work curve is built by using a blank matrix solution not containing pesticide to be detected; an external reference method is used for quantification. The GC-MS/MS determining method has the advantages that the average recovery rate is 88.8 percent to 97.8 percent; the average RSD (relative standard deviation) is 6.3 percent to 8.2 percent; the detection limit is lower than 0.42mug/kg; the operation is simple, convenient and fast; the impurity removal effect is good; the sensitivity is high; the characteristic performance is high; the repeatability is good; the qualitative and quantitative analysis is accurate. The technical requirements of countries such as America, European Union and Japan on corresponding product safety detection can be met; powerful technical support is provided for guaranteeing the food safety of Chinese people and healthy development of export abroad trade.
Description
Technical field
The present invention relates to a kind of GC-MS/MS assay method of amine Phenopyrazone residual quantity, be more particularly the method adopting gas chromatography tandem mass spectrum (GC-MS/MS) qualitative, quantitative to measure amine Phenopyrazone content residual in the animals and plants derived food of the complex matrices such as animal muscle and goods such as Cereals, pork, beef, mutton, chicken, belong to the determination techniques field of persticide residue.
Background technology
Amine Phenopyrazone (Fenpyrazamine) is the heterocyclic fungicide based on pyridine of SUMITOMO CHEMICAL KCC research and development, by suppressing the pathogen of the mechanism of action of ergosterol biosynthesis pathway, demonstrate the inhibiting effect to mycelial growth and spore germination and pollen tube growth.No. CAS is: 473798-59-3, and molecular formula is C
17h
21n
3o
2s, molecular weight is 331.43, and the chemical structural formula of amine Phenopyrazone is as follows:
Amine benzene pyrrole bacterium ketone is the novel germicide of Sumitomo Chemical stand-alone development, there is novel chemical constitution, can infiltrate rapidly does in object, to the fungi energy fast onset drug effect infected, is mainly used in controlling gray mold, sclerotiniose and the chain sclerotinia sclerotiorum in grape, carpolities, vegetable crop.In addition, amine benzene pyrrole bacterium ketone, to mammal high safety, can be degraded rapidly in the environment, can apply before various crop is gathered.Amine benzene pyrrole bacterium ketone is released in Korea S at first, after the former medicine of amine benzene pyrrole bacterium ketone obtains first whole world registration, Sumitomo Chemical releases its germifuge new product containing the former medicine of amine benzene pyrrole bacterium ketone in Korea S again, and sell with Botrycide trade (brand) name, this product is the suspending agent type of 30%, for fruits and vegetables crop.2009, Sumitomo Chemical was in applying for the vegetable crops such as the tomato of European Union's registration for greenhouse production, eggplant, capsicum and cucurbit, and also have the Field information of grape, but only obtained the interim registration of 3 years by a definite date at that time in Poland, commodity are called Prolectu.In June, 2012, European Union's food and animal health standing committee have voted its application for registration through, amine benzene pyrrole bacterium ketone has obtained European Union's approval registration, and will formally list the registered effective constituent list under European Union's agricultural chemicals 1107/2009 regulation in 1 day January in 2013, the term of validity is 10 years.Amine benzene pyrrole bacterium ketone, after European Union obtains registration, will be used for grape and some chamber crops, as tomato, eggplant, capsicum and cucurbit etc.Amine benzene pyrrole bacterium ketone has excellent prevention and control ability to botrytis, and Sumitomo Chemical will further in 2012/13 year to Italy and this product of Chilean marketing, and its global commodity are called Prolectus.Now register in the U.S. and a lot of Asian countries, there is very wide application prospect.
Along with the registration of amine Phenopyrazone, popularization and use, the country such as European Union, the U.S., Canada as China's main exit market has formulated its maximum maximum permission quantity (MRL) in the food agricultural product such as veterinary antibiotics, Cereals and livestock products, and European Union formulates the maximum residue limit standard of germifuge amine benzene pyrrole bacterium ketone in various crop.This standard comes into force on May 7th, 2012.The maximum residue limit of amine benzene pyrrole bacterium ketone in potato, capsicum, eggplant is 3mg/kg; Maximum residue limit in cucurbit, cucumber, cuke, Courgettes is 0.7mg/kg; Residue limits in the fruit such as oranges and tangerines, grape fruit, lemon and various dry fruit is the strictest, is 0.01mg/kg.The U.S. specifies that the maximum residue limit of amine Phenopyrazone in almond is 0.02mg/kg; Maximum residue limit in almond shell is 1.5mg/kg; Maximum residue limit in American pistachios is 0.02mg/kg; Maximum residue limit in lettuce heads is 1.5mg/kg; Maximum residue limit in leaf lettuce is 2mg/kg; Maximum residue limit in Cranberry subgroup 13-07A is 5mg/kg; Maximum residue limit in bush fruit class subgroup 13-07B is 5mg/kg; In small-sized fruit 13-07F subgroup of overgrowing, the maximum residue limit of (except Kiwi berry) is 3mg/kg; Maximum residue limit in grape juice is 4mg/kg; Maximum residue limit in short youngberry class subgroup 13-07G is 3mg/kg; Maximum residue limit in ginseng is 0.7mg/kg.The MRL of Canada's regulation amine Phenopyrazone in grape juice, romaine lettuce, lettuce, ginseng, almond nut, American pistachios is respectively 4.0mg/kg, 2.0mg/kg, 1.5mg/kg, 0.7mg/kg and 0.02mg/kg; When European Union, Japan and other countries specify the residue limits standard not formulating corresponding agricultural chemicals, the food agricultural product being exported to its country comprise " uniform limit " that residue limits in the animal derived foods such as livestock meat all carries out 0.01mg/L.
Present stage, less to the research of amine Phenopyrazone determination of residual amount method, the detection method of report is mainly amine Phenopyrazone method for detecting residue in vegetables and fruit, these detection methods all adopt Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to measure the detection method of amine Phenopyrazone residual quantity in vegetables and fruit, using LC-MS/MS to measure food Residual Pesticides in Farm Produce has fast, easy, sensitivity advantages of higher, but due to its price costly, a lot of testing agency, enterprise or scientific research institutions do not configure this instrument or configuration number of units is less, during due to different compounds employing LC-MS/MS detection, different mobile phases or chromatographic column need be used, such needs constantly change chromatographic column, mobile phase also expends the long time and balances system, this constrains the application of LC-MS/MS to a certain extent.Using gas chromatography tandem mass spectrum (GC-MS/MS) to analyze food Residual Pesticides in Farm Produce has fast, easy, highly sensitive, the advantages such as selectivity is strong, the multi-residue analysis of hundreds of kind agricultural chemicals can be realized, but have no the report of the GC-MS/MS detection method of amine Phenopyrazone residual quantity in food agricultural product up to now, due to Cereals, the food agricultural product matrix more complicated such as animal derived food, the good sample-pretreating method of clean-up effect must be set up and instrumental conditions could meet testing requirement, therefore, the detection method setting up amine Phenopyrazone residual quantity in gas chromatography tandem mass spectrum (GC-MS/MS) qualitative and quantitative analysis Cereals and animal derived food is significant.
Summary of the invention
The object of this invention is to provide a kind of GC-MS/MS assay method of amine Phenopyrazone residual quantity, be mainly used in measuring amine Phenopyrazone residual quantity in the complex matrices such as Cereals, animal derived food food agricultural product.
For realizing above object, the technical solution adopted in the present invention is: a kind of GC-MS/MS assay method of amine Phenopyrazone residual quantity, comprises the steps:
(1) extract
Take mixing sample in tool plug centrifuge tube, after adding suitable quantity of water recovery, quantitatively add acetonitrile or extract containing the acetonitrile solution homogeneous of 1% acetic acid or oscillating ultrasonic, then adding the one in sodium chloride or sodium acetate and anhydrous magnesium sulfate, centrifugal after violent vortex 1min.
(2) purify
Pipette certain volume sample extracting solution, be concentrated into about 1mL, through C
18/ PSA Solid-Phase Extraction column purification, acetonitrile, collects eluent, get elution fractions be concentrated into dry after, dissolve constant volume with acetone/normal hexane mixed solvent that volume ratio is 1/1, after crossing film, treat that gas chromatography tandem mass spectrum (GC-MS/MS) detects.
(3) preparation of standard working solution
When same kind matrix blank sample not containing amine Phenopyrazone is processed by above-mentioned steps (1), (2), obtain sample extraction purification residue, add appropriate solvent and standard solution, vortex mixes, and is mixed with the amine Phenopyrazone series standard working fluid of at least 3 concentration.
(4) gas chromatography tandem mass spectrometry (GC-MS/MS) measures
The standard working solution of each concentration gradient in step (3) is carried out GC-MS/MS mensuration, with the chromatographic peak area of standard working solution, regretional analysis is carried out to its respective concentration, obtain standard working curve; Under the same conditions the sample liquid after purification in step (2) is injected GC-MS/MS to measure, record the chromatographic peak area of amine Phenopyrazone in sample liquid, substitute into standard working curve, obtain amine Phenopyrazone content in sample liquid, then the Mass Calculation of sample representated by liquid obtains amine Phenopyrazone residual quantity in sample per sample.If amine Phenopyrazone residual quantity exceedes the range of linearity upper limit in upper machine solution, with constant volume solvent, upper machine solution concentration need be diluted within the range of linearity.
Step (1), if the sample of middle sample moisture content less, must add suitable quantity of water and fully infiltrate before extraction.
Add sodium chloride when adopting acetonitrile to extract in step (1) to saltout, add sodium acetate when adopting the acetonitrile solution containing 1% acetic acid to extract and saltout.
Step carries out C in (2)
18/ PSA Solid phase extraction, during acetonitrile, elution volume is 6 ~ 8mL.
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 1.2mL/min; Heating schedule: initial temperature 60 DEG C keeps 2min, rises to 200 DEG C, then rises to 220 DEG C with the speed of 2 DEG C per minute, then rise to 280 DEG C with the speed of 20 DEG C per minute with the speed of 20 DEG C per minute, keeps 10min; Transmission line temperature: 290 DEG C.
In step (4), Mass Spectrometry Conditions is: ionization pattern: electron impact ionization (EI, 70eV); Ion source temperature 280 DEG C; Collision gas: argon gas; Multiple-reaction monitoring scan mode MRM, monitoring parameter is:
When measuring sample liquid and extraction standard working solution in step (4), if sample liquid Pesticides chromatographic peak retention time agricultural chemicals retention time corresponding to standard solution is consistent, and in the sample mass spectrogram after background correction, selected ion all occurs, and abundance of ions than with the abundance of ions of standard solution than consistent, then can judge to exist in sample liquid this agricultural chemicals; If above-mentioned two conditions can not meet simultaneously, then judge not containing this kind of agricultural chemicals.
Beneficial effect of the present invention is:
The present invention utilizes dispersive solid-phase extraction technology, establish sample-pretreating method that is easy, that also can effectively avoid sample mesostroma to disturb fast, this pre-treating method to be applied in Cereals, animal derived food the qualitative confirmation of amine Phenopyrazone in conjunction with GC-MS/MS and quantitatively to detect, average recovery rate is 88.8% ~ 97.8%, average relative standard's deviation (RSD) is 6.3% ~ 8.2%, detection limit, lower than 0.42 μ g/kg, has easy and simple to handle, quick, accurate, highly sensitive and reproducible advantage.The U.S., European Union, Japan and other countries being met to the technical requirement of corresponding product safety detection, providing strong technical support for ensureing that our people's food security and export abroad trade develop in a healthy way.
Accompanying drawing explanation
Fig. 1 is the GC-MS/MS Selective ion mode chromatogram of the amine Phenopyrazone mark liquid be added in blank pork matrix.
Fig. 2 is not containing the GC-MS/MS Selective ion mode chromatogram of the pork blank sample of amine Phenopyrazone.
The amine Phenopyrazone standard working curve that Fig. 3 is is substrate preparation with the pork blank sample not containing amine Phenopyrazone.
Embodiment
Now with following embodiment, the present invention is described, but is not limit the scope of the invention.
The instrument used in embodiment and reagent
T18Basic homogenizer (IKA, Germany); CR21G III hydro-extractor (Hitachi, Japan); MS3 basic model vortex mixer (IKA, Germany); TurboVapLV type sample automatic concentration instrument (Caliper, USA); 7890N gas chromatography-5977C mass spectrometer (Agilent, USA); C
18/ PSA solid-phase extraction column (6mL, 500mg/500mg) is purchased from Tianjin Bonaaijieer Technology Co., Ltd.
Reagent
Acetonitrile, acetone, normal hexane (HPLC level, Merke, Germany); Acetic acid (HPLC level, CNW, Germany); Anhydrous magnesium sulfate, sodium chloride and sodium acetate are pure for analyzing, all purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
Standard substance: purity 98.0%, purchased from Japan and Guang Chun agro-industry Co., Ltd..
Embodiment 1: the detection of amine Phenopyrazone residual quantity in wheat
(1) sample pre-treatments
Extract
Take 5g wheat samples (grinding to form flour) through fully mixing in 50mL centrifuge tube, after adding 20mL water recovery 30min, accurately add the acetonitrile solution of 20mL containing 1% acetic acid, mechanical shaking extraction 20min, ultrasonic extraction 5min, add 3g anhydrous magnesium sulfate and 2g sodium acetate, after vortex 1min, the centrifugal 5min of 7000r/min.After centrifugal, get 8mL acetonitrile extract and revolve steaming or nitrogen blows near dry in 40 DEG C, after adding 1mL acetonitrile vortex, to be clean.
Purification
With 5mL acetonitrile prewashing C
18/ PSA solid-phase extraction column, when liquid level arrives the top of adsorbent, said extracted solution is proceeded in post, with 2mL acetonitrile wash test tube, and cleansing solution is moved in SPE post, when solution reaches adsorbent top, add on 4mL acetonitrile to pillar and carry out wash-out, eluent all receives in quantitative test tube, nitrogen dry up rear volume ratio be 1/1 acetone/normal hexane mixed solvent be settled to 1mL, after crossing 0.22 μm of filter membrane, treat that GC-MS/MS measures.
(2) preparation of standard working solution
Accurately take 25 ± 0.1mg standard items in 25mL volumetric flask, dissolve with acetonitrile, 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 acetone/normal hexane mixed solvent constant volume that volume ratio is 1/1; 10 μ g/mL standard solution dilutions are made into 5,2,1,0.5,0.2,0.1 μ g/mL standard solution.Wheat blank sample not containing amine Phenopyrazone is pressed above-mentioned pre-treatment step process, obtain sample extraction purification residue, acetone/normal hexane mixed solvent and the above-mentioned standard solution of 100 μ L that 900 μ L volume ratios are 1/1 is added in this residue, vortex mixes, and is made into 10,20,50,100,200,500 μ g/L extraction standard working solutions.
(3) gas chromatography tandem mass spectrometry (GC-MS/MS) measures
The standard working solution of variable concentrations gradient is injected GC-MS/MS respectively, carries out the quantitative test of amine Phenopyrazone content with external standard method, namely with the chromatographic peak area of standard working solution, regretional analysis is carried out to its respective concentration, obtain standard working curve; Under the same conditions sample extracting solution is injected GC-MS/MS to measure, record the chromatographic peak area of amine Phenopyrazone in sample liquid, substitute into standard working curve, obtain amine Phenopyrazone content in sample liquid, then the Mass Calculation of sample representated by liquid obtains amine Phenopyrazone 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 1.2mL/min.
Stove case heating schedule: initial temperature 60 DEG C keeps 2min, rises to 200 DEG C, then rises to 220 DEG C with the speed of 2 DEG C per minute, then rise to 280 DEG C with the speed of 20 DEG C per minute with the speed of 20 DEG C per minute, keeps 10min.
Transmission line temperature: 290 DEG C.
Wherein, mass spectrometry parameters is:
Ionization pattern: electron impact ionization, i.e. EI pattern, energy 70eV.
Ion source temperature: 280 DEG C; Collision gas: argon gas.
Scan mode: multiple-reaction monitoring (SRM) scan pattern; MRM detected parameters is in table 1.
Table 1: the MRM detected parameters of embodiment 1
*for quota ion pair.
Qualitative Identification: at identical conditions, if sample liquid Pesticides chromatographic peak retention time agricultural chemicals retention time corresponding to standard solution is consistent, and in the sample mass spectrogram after background correction, selected ion all occurs, and abundance of ions than with the abundance of ions of standard solution than consistent, then can judge to exist in sample liquid this agricultural chemicals; If above-mentioned two conditions can not meet simultaneously, then judge not containing this kind of agricultural chemicals.
With the chromatographic peak area of standard working solution, regretional analysis is carried out to its respective concentration, obtain standard working curve as table 2.
The standard working curve of amine Phenopyrazone in table 2 wheat bare substrate
| Title | Retention time (min) | Regression equation | Related coefficient |
| Amine Phenopyrazone | 26.64 | Y＝9325.5X-76963 | 0.9995 |
Recovery of standard addition and repeatability:
In the wheat not containing amine Phenopyrazone, add the amine Phenopyrazone standard solution of 10, a 20 and 200 μ g/kg3 concentration level, add after 30min until agricultural chemicals and carry out the determination of residual amount by above-mentioned treatment step.Mensuration concentration and agricultural chemicals theory are added concentration compare, obtain agricultural chemicals TIANZHU XINGNAO Capsul, each Pitch-based sphere replicate determination 6 times, obtain its relative standard deviation, measurement result is in table 3.As can be seen from Table 3, in 3 mark-on levels, the average recovery rate of amine Phenopyrazone is 88.8% ~ 96.9%, and average relative standard's deviation (RSD) is 6.3% ~ 8.2%, illustrates that the recovery of the inventive method is higher, reproducible.
The recovery of table 3 amine Phenopyrazone and repeatability (n=6)
Detection limit:
The amine Phenopyrazone extraction standard working solution of variable concentrations is injected GC-MS/MS, calculate detection limit with the cycles of concentration (cycles of concentration of wheat is 2.0 times) of 3 times of signal to noise ratio (S/N ratio)s of least concentration extraction standard solution chromatographic peak and sample handling processes, detecting of amine Phenopyrazone is limited to 0.42 μ g/kg.
Embodiment 2: the detection of amine Phenopyrazone residual quantity in pork
(1) sample pre-treatments
Extract
Take 5g pork sample through fully mixing in 50mL centrifuge tube, add the mixing of 5mL water, place 30min, accurately add 20mL acetonitrile, homogeneous extracts 2min, adds 3g anhydrous magnesium sulfate and 2g sodium chloride, after vortex 1min, and the centrifugal 5min of 7000r/min.After centrifugal, get 8mL acetonitrile extract in 40 DEG C revolve steaming or nitrogen blow to about 1mL, to be clean.
Purification
With 5mL acetonitrile prewashing C
18/ PSA solid-phase extraction column, when liquid level arrives the top of adsorbent, said extracted solution is proceeded in post, with 2mL acetonitrile wash test tube, and cleansing solution is moved in SPE post, when solution reaches adsorbent top, add on 4mL acetonitrile to pillar and carry out wash-out, eluent all receives in quantitative test tube, nitrogen dry up rear volume ratio be 1/1 acetone/normal hexane mixed solvent be settled to 1mL, after crossing 0.22 μm of filter membrane, treat that GC-MS/MS measures.
(2) preparation of standard working solution
Acetone/normal hexane the mixed solvent being 1/1 by 1000 μ g/mL standard solution volume ratios is diluted to 10 μ g/mL standard intermediate liquids, and 10 μ g/mL standard solution dilutions are made into 5,2,1,0.5,0.2,0.1 μ g/mL standard solution.Pork blank sample not containing amine Phenopyrazone is pressed above-mentioned pre-treatment step process, obtain sample extraction purification residue, acetone/normal hexane mixed solvent and the above-mentioned standard solution of 100 μ L that 900 μ L volume ratios are 1/1 is added in this residue, vortex mixes, and is made into 10,20,50,100,200,500 μ g/L extraction standard working solutions.
(3) gas chromatography tandem mass spectrometry (GC-MS/MS) measures
Operation steps, chromatogram are consistent with the mensuration of amine Phenopyrazone in above-mentioned wheat samples with Mass Spectrometry Conditions.
Qualitative Identification: consistent with the mensuration of amine Phenopyrazone in above-mentioned wheat samples.
Linear relationship:
Carry out regretional analysis with the chromatographic peak area of standard working solution to its respective concentration, obtaining standard working curve is Y=10222X-82664, and related coefficient is 0.9997.
Recovery of standard addition and repeatability:
The amine Phenopyrazone standard solution of 10, a 20 and 200 μ g/kg3 concentration level is added in the pork not containing amine Phenopyrazone, add after 30min until agricultural chemicals and carry out the determination of residual amount by above-mentioned treatment step, mensuration concentration and agricultural chemicals theory are added concentration compare, obtain agricultural chemicals TIANZHU XINGNAO Capsul, each Pitch-based sphere replicate determination 6 times, obtain its relative standard deviation, measurement result is in table 4.As can be seen from Table 4, in 3 mark-on levels, the average recovery rate of amine Phenopyrazone is 89.6% ~ 97.8%, and average relative standard's deviation (RSD) is 7.1% ~ 8.0%, illustrates that the recovery of the inventive method is high, reproducible.
The recovery of table 4 amine Phenopyrazone and repeatability (n=6)
Detection limit:
The amine Phenopyrazone extraction standard working solution of variable concentrations is injected GC-MS/MS, calculate detection limit with the cycles of concentration (cycles of concentration of pork is 2.0 times) of 3 times of signal to noise ratio (S/N ratio)s of least concentration extraction standard solution chromatographic peak and sample handling processes, detecting of amine Phenopyrazone is limited to 0.35 μ g/kg.
Above embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various modification that the common engineering in this area is made technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determine.
Claims (5)
1. a GC-MS/MS assay method for amine Phenopyrazone residual quantity, is characterized in that, said method comprising the steps of:
(1) extract
Take mixing sample in tool plug centrifuge tube, after adding suitable quantity of water, add acetonitrile or after extracting containing the acetonitrile solution homogeneous of 1% acetic acid or oscillating ultrasonic, add the one in sodium chloride or sodium acetate and anhydrous magnesium sulfate, centrifugal after violent vortex 1min;
(2) purify
Pipette certain volume sample extracting solution, be concentrated into about 1mL, through C
18/ PSA Solid-Phase Extraction column purification, acetonitrile, collects eluent, get elution fractions be concentrated into dry after, dissolve constant volume with acetone/normal hexane mixed solvent that volume ratio is 1/1, after crossing film, treat that gas chromatography tandem mass spectrum (GC-MS/MS) detects;
(3) preparation of standard working solution
Same kind matrix blank sample not containing amine Phenopyrazone is processed by above-mentioned steps (1), (2), obtain sample extraction purification residue, add appropriate solvent and standard solution, vortex mixes, and is mixed with the amine Phenopyrazone series standard working fluid of at least 3 concentration;
(4) mensuration and result calculate
The standard working solution of each concentration gradient in step (3) is carried out GC-MS/MS mensuration, with the chromatographic peak area of standard working solution, regretional analysis is carried out to its respective concentration, obtain extraction standard working curve; Under the same conditions the sample liquid after purification in step (2) is injected GC-MS/MS to measure, record the chromatographic peak area of amine Phenopyrazone in sample liquid, substitute into extraction standard working curve, obtain amine Phenopyrazone content in sample liquid, then the Mass Calculation of sample representated by liquid obtains amine Phenopyrazone residual quantity in sample per sample; If amine Phenopyrazone residual quantity exceedes the range of linearity upper limit in upper machine solution, with constant volume solvent, upper machine solution concentration need be diluted within the range of linearity.
2. the GC-MS/MS assay method of a kind of amine Phenopyrazone residual quantity according to claim 1, is characterized in that, step (1), if the sample of middle sample moisture content less, must add suitable quantity of water and fully infiltrate before extraction.
3. the GC-MS/MS assay method of a kind of amine Phenopyrazone residual quantity according to claim 1, it is characterized in that, sodium chloride need be added when adopting acetonitrile to extract in step (1) to saltout, sodium acetate need be added when adopting the acetonitrile solution containing 1% acetic acid to extract and saltout.
4. the GC-MS/MS assay method of a kind of amine Phenopyrazone residual quantity according to claim 1, it is characterized in that, step carries out C in (2)
18/ PSA Solid phase extraction, during acetonitrile, elution volume is 6 ~ 8mL.
5. the GC-MS/MS assay method of a kind of amine Phenopyrazone residual quantity according to claim 1, it is characterized in that, in step (4), GC-MS/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 1.2mL/min; Heating schedule: initial temperature 60 DEG C keeps 2min, rises to 200 DEG C, then rises to 220 DEG C with the speed of 2 DEG C per minute, then rise to 280 DEG C with the speed of 20 DEG C per minute with the speed of 20 DEG C per minute, keeps 10min; Transmission line temperature: 290 DEG C; Ionization pattern: electron impact ionization (EI, 70eV); Ion source temperature 280 DEG C; Collision gas: argon gas; Multiple-reaction monitoring scan mode MRM, monitoring parameter is:
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