CN107422054B - method for detecting residual quantity of meptyldinocap in tobacco - Google Patents
method for detecting residual quantity of meptyldinocap in tobacco Download PDFInfo
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- CN107422054B CN107422054B CN201710604136.7A CN201710604136A CN107422054B CN 107422054 B CN107422054 B CN 107422054B CN 201710604136 A CN201710604136 A CN 201710604136A CN 107422054 B CN107422054 B CN 107422054B
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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
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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
- 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
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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
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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
- 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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Abstract
the invention discloses a method for detecting the residual quantity of nitrophenyl ester in tobacco, which is characterized in that the residual quantity of the nitrophenyl ester in the tobacco is detected by detecting a hydrolysate 2,4-dinitro-6- (1-methylheptyl) phenyl crotonate,2,4-DNOPC, corresponding to the nitrophenyl ester (2,4-dinitro-6- (1-methylheptyl) phenol (2,4-dinitro-6- (1-methylheptyl) phenyl, 2, 4-DNOP), a tobacco sample is extracted by adopting an improved method, sample introduction analysis is carried out after heating and ultrasonic hydrolysis under an alkaline condition, detection is carried out by adopting an ultra-performance liquid chromatography-tandem negative ion multiple reaction monitoring mode, an external standard method is adopted for quantification precision, the result shows that the linear relation of the nitrophenyl ester in a range of 0.001-0.5mg/L is good, a correlation coefficient (r 2) is more than 0.999, the detection limit is 3 mu g/kg, the relative sensitivity of the nitrophenyl ester in the tobacco is 32%, the relative sensitivity of the nitrobenzene in the tobacco is 100.52%, and the detection is suitable for simple pretreatment method for detecting the residual quantity of the tobacco, and the detection accuracy of the detection of the nitrobenzene residue in the tobacco is 3% detection method, and the detection method is suitable for detecting the detection of pesticides.
Description
Technical Field
The invention belongs to the technical field of pesticide residue detection, and particularly relates to a method for detecting residual quantity of meptyldinocap in tobacco.
Background
Nitrophenyl baconate (2,4-dinitro-6- (1-methylheptyl) phenyl crotonate,2,4-DNOPC) and CAS (131-72-6) are novel powdery mildew bactericides and are mainly used for preventing and treating powdery mildew of crops and fruit trees, and have the function of killing mite eggs, so that the meptyldinonyl crotonate is more and more widely used at present. Nitrophenyl pivoxil is a single isomer isolated from 6 isomers of the bactericidal (acaricidal) agent dinocap (dinocap, CAS: 39300-45-3), developed by the United states of America, Yinong, and registered in Europe, America, China, etc., and has improved toxicity relative to dinocap, which has a long service life, but still belongs to a highly toxic pesticide, and the European Union has established the maximum residual limit of nitrophenyl pivoxil (MRL, sum of 2,4-DNOP and OPC 2, 4-DNOP) in various agricultural products, such as 0.05mg/kg of MRL in citrus, apple, pear and mango, and 1mg/kg of MRL in grape.
The detection methods reported abroad at present comprise a high performance liquid chromatography, a gas chromatography-ECD method, a gas chromatography-mass spectrometry combined method and a liquid chromatography-mass spectrometry combined method, and the detection methods have thermal instability and chemical instability and are easy to hydrolyze or photolyze into corresponding phenol, so that the selectivity, precision and reproducibility are poor during analysis by using a Gas Chromatography (GC) or a gas chromatography-mass spectrometry (GC-MS), the LC method has the defects of poor sensitivity and selectivity and poor matrix interference resistance, and the LC-MS/MS has the advantages of high sensitivity and selectivity. The method mainly comprises the steps of adopting an LC method or an LC-MS/MS method to calculate the residual quantity of a parent body by analyzing the residual quantity of phenol which is a hydrolysate corresponding to the meptyldinocap, and adopting an organic solvent to extract in the currently reported method, adding a sodium hydroxide solution to hydrolyze the obtained product into corresponding phenol after liquid-liquid distribution.
Disclosure of Invention
the invention aims to provide a method for detecting the residual quantity of meptyldinocap in tobacco.
The object of the invention is achieved by the following steps:
1) preparation of a standard solution: accurately weighing 2,4-DNOP standard substance to 0.1mg, dissolving with methanol to fix volume to 100mL brown volumetric flask, preparing into 1mg/L single standard stock solution, storing in refrigerator at-20 deg.C in dark place, and making standard curve and quantitative detection;
2) The method comprises the steps of sample pretreatment, namely weighing 2 g-5 g of a sample to be detected, accurately obtaining the sample to be detected to 0.01g, adding 10 mL-20 mL of water into a 50mL centrifuge tube with a cover, oscillating until the sample is fully soaked by the water, standing for 10min, transferring 10mL of acetic acid-acetonitrile mixed solution into the centrifuge tube, placing the centrifuge tube on a vortex mixing oscillator, oscillating for 2-5 min at the speed of 2000r/min, freezing and storing the centrifuge tube at-18 ℃ for 10min, taking out, adding 4g of anhydrous magnesium sulfate, 1g of sodium chloride, 1g of sodium citrate and 0.5g of disodium hydrogen citrate into the centrifuge tube, immediately oscillating for 2-5 min at the speed of 2000r/min on the vortex mixing oscillator, then centrifuging for 3-5 min at the speed of 6000r/min, transferring 1mL of sample extraction supernatant into a 1.5mL centrifuge tube, adding 10-20 mu L of alkaline solution, placing the centrifuge tube on the vortex mixing oscillator for 2-5 min at the speed of 2000r/min, placing the centrifuge tube at the ultrasonic wave 40 8560 ℃ for 40min, filtering through a 0.22 m filter membrane, and carrying out sample analysis on a sample to;
3) And (3) detecting the residual quantity of the meptyldinocap: and (3) detecting the sample treated in the step (2) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry anion multi-reaction monitoring mode, and measuring the residual amount of the meptyldinocap by combining the standard curve obtained in the step (1).
Compared with the prior art, the invention has the beneficial effects that:
1. The method is creatively used for extracting the 2,4-DNOPC in the tobacco sample by adopting a quick, simple and reliable extraction method, then alkaline solution (ammonia water) is added to be heated and hydrolyzed into the 2,4-DNOP in ultrasonic waves, and the residual quantity of the 2,4-DNOP in the tobacco is measured by using UPLC-MS/MS with higher efficiency, rapidness and high sensitivity to calculate the parent compound 2, 4-DNOPC.
2. The detection method can complete the detection of the sample within 3min, is simple and rapid, has short analysis time and high sensitivity, accuracy and precision, and is suitable for rapid detection of the residual quantity of the meptyldinocap in the tobacco.
3. The detection method is simple, the pretreatment of the sample is efficient and safe, the cost is low, and the accurate and timely detection of the meptyldinocap in the tobacco is realized by optimizing the pretreatment in the detection process and controlling the chromatographic conditions in the detection.
drawings
FIG. 1 is a MRM chromatogram of a tobacco blank sample in example 1 of the present invention with 0.1. mu.g/g 2,4-DNOPC added thereto;
FIG. 2 is a graph showing the effect of different extraction solvents on the extraction of meptyldinocap in example 1 of the present invention;
FIG. 3 is a graph comparing the hydrolysis efficiency of meptyldinocap at different temperatures and different ultrasonic times in example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following examples and the accompanying drawings, but the present invention is not limited thereto in any way, and any modifications or alterations based on the teaching of the present invention are within the scope of the present invention.
The method for detecting the residual quantity of the meptyldinocap in the tobacco comprises the following steps:
1) Preparation of a standard solution: accurately weighing 2,4-DNOP standard substance to 0.1mg, dissolving with methanol to fix volume to 100mL brown volumetric flask, preparing into 1mg/L single standard stock solution, storing in refrigerator at-20 deg.C in dark place, and making standard curve and quantitative detection;
2) The method comprises the steps of sample pretreatment, namely weighing 2 g-5 g of a sample to be detected, accurately obtaining the sample to be detected to 0.01g, adding 10 mL-20 mL of water into a 50mL centrifuge tube with a cover, oscillating until the sample is fully soaked by the water, standing for 10min, transferring 10mL of acetic acid-acetonitrile mixed solution into the centrifuge tube, placing the centrifuge tube on a vortex mixing oscillator, oscillating for 2-5 min at the speed of 2000r/min, freezing and storing the centrifuge tube at-18 ℃ for 10min, taking out, adding 4g of anhydrous magnesium sulfate, 1g of sodium chloride, 1g of sodium citrate and 0.5g of disodium hydrogen citrate into the centrifuge tube, immediately oscillating for 2-5 min at the speed of 2000r/min on the vortex mixing oscillator, then centrifuging for 3-5 min at the speed of 6000r/min, transferring 1mL of sample extraction supernatant into a 1.5mL centrifuge tube, adding 10-20 mu L of alkaline solution, placing the centrifuge tube on the vortex mixing oscillator for 2-5 min at the speed of 2000r/min, placing the centrifuge tube at the ultrasonic wave 40 8560 ℃ for 40min, filtering through a 0.22 m filter membrane, and carrying out sample analysis on a sample to;
3) And (3) detecting the residual quantity of the meptyldinocap: and (3) detecting the sample treated in the step (2) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry anion multi-reaction monitoring mode, and measuring the residual amount of the meptyldinocap by combining the standard curve obtained in the step (1).
The step (1) also comprises the preparation of a 2,4-DNOPC standard solution, specifically, accurately weighing a 2,4-DNOPC standard substance to be accurate to 0.1mg, dissolving with methanol to fix the volume to a 100mL brown volumetric flask, preparing a 1mg/L single standard stock solution, storing in a refrigerator at-20 ℃ in a dark place, and performing an addition recovery test; the adding recovery test specifically comprises the steps of performing standard addition recovery tests at 0.01, 0.05 and 0.1mg/kg of 3 adding levels respectively, setting 5 parallel samples for each adding concentration, and then performing sample pretreatment and detection, wherein the recovery rate of the 2,4-DNOPC in tobacco is 92.37-100.52%, and the relative standard deviation is 2.43-3.30%.
The standard curve preparation in the step (1) is specifically to prepare 2,4-DNOP matrix matching standard working solution with the series of mass concentrations of 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L from tobacco blank matrix extracting solution, and perform linear regression on each concentration by peak area to obtain a standard curve, wherein the standard curve is good in linearity in the range of the mass concentration of the 2,4-DNOP being 0.001-0.5mg/L, and the correlation coefficient r 2 is greater than 0.999.
The acetic acid-acetonitrile mixed solution in the step (2) is a mixed solution of 1% acetic acid-99% acetonitrile.
The alkaline solution in the step (2) is 25% ammonia water.
The ultrasonic wave in the step (2) is ultrasonic at 40 ℃ for 60 min.
The chromatographic conditions of the ultra-high performance liquid chromatography in the step (3) are that a chromatographic column: 5mm × 2.1mm, 1.7 μm ACQUITY UPLC BEH C18; column temperature: 25 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: methanol-water solution containing 0.2mmol/L ammonium acetate (50 +50, V/V) or acetonitrile water containing 0.2mmol/L ammonium acetate (50 +50, V/V), flow rate: 0.4mL/min, time: 3 min.
The analysis conditions of the mass spectrum in the step (3) are as follows: an ion source: ESI, scan mode: negative ion mode, air curtain airflow rate: 25 psi; flow rate of atomizing gas: 55 psi; flow rate of auxiliary gas: 55 psi; ion source temperature: 500 ℃; spraying voltage: 4500V, declustering voltage: -100V; in the multi-reaction monitoring mode, the parent ion is m/z 295.1, the daughter ion is m/z134.1, CE: -50V and m/z 163.0, CE: -50V, wherein m/z134.1 is the quantitative ion.
the detection limit of the residual quantity of the meptyldinocap in the step (3) is 3 mug/kg, and the quantification limit is 10 mug/kg.
The purity of the 2,4-DNOP standard substance is 98.5 percent, and the purity of the 2,4-DNOPC standard substance is 93 percent; the methanol and the acetonitrile are HPLC grade; the anhydrous magnesium sulfate is analytically pure, is burned for 4 hours at 650 ℃ before use, and is stored in a dryer for later use; the sodium chloride, the sodium citrate, the disodium hydrogen citrate, the sodium hydroxide and the ammonia water are all analytically pure.
Example 1
1. Instruments and reagents: ultra high performance liquid chromatography (Waters, usa), AB Sciex QTRAP 5500 mass spectrometry (AB Sciex, usa), Millipore silica personal ultra pure water machine (Millipore, usa), Eppendorf 5804 high speed centrifuge (Eppendorf, germany), Talboys digital display multitubular vortex mixer (shanghai spectral science instruments). Methanol, acetonitrile, HPLC grade, Fisher corporation, usa. Anhydrous magnesium sulfate, analytically pure. It should be burned at 650 deg.C for 4 hr before use, and stored in a desiccator for use. Sodium chloride, sodium citrate, disodium hydrogen citrate, sodium hydroxide, ammonia water and analytically pure. 2,4-DNOP and 2,4-DNOPC standards (98.5% and 93% purity, respectively, as supplied by Dr, Germany).
2. The detection method of the residual amount of the meptyldinocap in the tobacco comprises the following steps:
1) preparation of a standard solution: accurately weighing 2,4-DNOPC and 2,4-DNOP standard substances (accurate to 0.1 mg) respectively, dissolving with methanol to fix the volume to 100mL of brown volumetric flask, preparing 1mg/L single standard stock solution, storing in a refrigerator at-20 ℃ in a dark place, using the 2,4-DNOPC standard solution (namely the standard stock solution) for adding and recycling tests, and using the 2,4-DNOP standard solution (namely the standard stock solution) for preparing a standard curve and quantitatively detecting.
2) Sample pretreatment: weighing about 2g of sample, accurately obtaining 0.01g of sample, adding 10mL of water into a 50mL centrifuge tube with a cover, oscillating until the sample is fully soaked by the water, and standing for 10 min. 10mL of 1% acetic acid-acetonitrile was transferred to a centrifuge tube and placed on a vortex mixing shaker at 2000r/min for 2 min. Freezing and storing the centrifugal tube in a freezer (-18 deg.C) for 10min, and taking out. 4g of anhydrous magnesium sulfate, 1g of sodium chloride, 1g of sodium citrate and 0.5g of disodium hydrogen citrate are respectively added into a centrifugal tube, immediately shaken on a vortex mixing and shaking instrument for 2min at the speed of 2000r/min to prevent the anhydrous magnesium sulfate from reacting with water to cause local overheating and caking, and then centrifuged at 6000r/min for 3 min. Transferring 1mL of sample extraction supernatant into a 1.5mL centrifuge tube, adding 20 μ L of 25% ammonia water, oscillating for 2min at a speed of 2000r/min on a vortex mixing and oscillating instrument, placing the sample in an ultrasonic wave at 40 ℃ for 60min, and filtering the sample to a sample injection bottle through a 0.22 μm organic phase filter membrane for analysis.
3) and (3) detecting the residual quantity of the meptyldinocap: and (3) detecting the sample treated in the step (2) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry anion multi-reaction monitoring mode, and measuring the residual amount of the meptyldinocap by combining the standard curve obtained in the step (1).
Chromatographic-mass spectrometric analysis conditions of chromatographic column ACQUITY UPLC BEH C18 (5 mm. times.2.1 mm, 1.7 μm), column temperature 25 ℃, sample introduction 2 μ L, mobile phase methanol-water solution (50 +50, V/V) containing 0.2mmol/L ammonium acetate, flow rate 0.4mL/min, time 3min, ion source ESI, scanning mode negative ion mode, gas curtain flow rate 25psi, atomizing gas flow rate 55psi, auxiliary gas flow rate 55psi, ion source temperature 500 ℃, spraying voltage-0V, declustering voltage-100V, multi-reaction monitoring (MRM) mode with parent ion m/z 295.1, daughter ion m/z134.1 (CE: -50V) and m/z 163.0(CE: -50V), where m/z134.1 is quantitative ion.
3. results and analysis:
1) linear range and detection limit (preparation of standard curve):
Preparing 2,4-DNOP matrix matching standard working solution with 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series of mass concentrations by using tobacco blank matrix extracting solution, performing linear regression on the concentrations of various pesticides by using peak areas, and determining the detection limit and the quantitative limit of the pesticide within the range of 0.001-0.5mg/L, wherein the curve equation is Y =3.85e +007 X +5.71e +004, the correlation coefficient r 2 is 0.9994, and the detection limit and the quantitative limit are determined by using 3-time and 10-time signal-to-noise ratios (S/N) respectively, so that the detection limit of the 2,4-DNOPC in the tobacco is 3 mu g/kg, and the conversion calculation formula of the concentration of the quantitative limit of 10 mu g/kg. of the nitrobenzoate and the corresponding phenol concentration is as follows:
2) Recovery, precision and matrix effect (addition recovery test):
and (4) adopting a tobacco blank sample to carry out addition recovery and precision experiment. The standard addition recovery experiments are respectively carried out at 0.01, 0.05 and 0.1mg/kg of 3 addition levels, 5 parallel samples are arranged at each addition concentration, and the sample treatment and measurement are carried out according to the experimental method, so that the recovery rate of 2,4-DNOPC in tobacco is 92.37-100.52%, the Relative Standard Deviation (RSD) is 2.43-3.30%, and the recovery rate and the precision of the method are better. FIG. 1 shows the MRM chromatogram of tobacco blank sample with 0.1. mu.g/g 2,4-DNOPC added.
Since the tobacco leaf matrix is very complex, the influence of the Matrix Effect (ME) was examined. The Matrix Effect (Matrix Effect, ME) = B/a × 100%) is obtained by measuring an average response value (a, n =3) of 2,4-DNOP in methanol and an average response value (B, n =3) of a Matrix matching standard solution obtained by pretreatment according to 2.3, and experimental results show that the Matrix has a strong inhibitory Effect on the response of 2,4-DNOP, and thus the Matrix Effect is compensated by performing calibration and quantification by using a Matrix matching standard solution external standard method.
TABLE 12 spiked recovery, precision (n = 5) and matrix effect of 4-DNOPC in tobacco
3) Selection of an extraction solvent: adding 10 mu g/kg of 2,4-DNOPC into a tobacco blank substrate, uniformly mixing, standing for 2h, adding 10mL of water, oscillating for 2min, and standing for 10min, wherein the extraction efficiencies of methanol, a 1% formic acid-methanol solution, a 1% acetic acid-methanol solution, 2% ammonia water-methanol, acetonitrile, a 1% formic acid-acetonitrile solution, 1% acetic acid-acetonitrile and a 2% ammonia water-acetonitrile solution on target pesticides are respectively compared. The experimental result shows that the extraction effect of the methanol-water system is poorer than that of the acetonitrile-water system, and mainly shows that when methanol, acidic methanol and alkaline methanol are used as extraction reagents, the peak shape of 2,4-DNOP is poorer, and the methanol and the water are likely to be mutually soluble, so that a co-extract is added, the emulsification phenomenon is more serious, and the impurity interference is large; in the acetonitrile-water solution system, the acetonitrile extracting solution has less interferents, and the addition of the salt can promote the layering of acetonitrile and water and reduce the interference of water-soluble substances. In an acetonitrile-water system, the recovery rate of the most extracted solvent of 1% acetic acid-acetonitrile solution is highest (100.07%), the extraction efficiency of 2% ammonia water is second (89.74%), the extraction efficiency of the most extracted solvent of 1% formic acid-acetonitrile solution is worst (19.59%), the peak shape is poor, the acidity of formic acid is probably strong, the extraction of the nitrophenyl acetate is not favorable, the acidity of acetic acid is weaker than that of formic acid, the extraction efficiency of the nitrophenyl acetate is high, and the 1% acetic acid-acetonitrile solution is selected as the extraction solvent of the experiment in comprehensive consideration. FIG. 2 is a graph showing the effect of different extraction solvents on the extraction of meptyldinocap.
4) Optimizing hydrolysis conditions:
(1) selection of hydrolysis reagents: the ester can be completely hydrolyzed by heating under the alkaline condition, the experiment compares the hydrolysis effects of 2mol/L sodium hydroxide solution, 5mol/L sodium hydroxide solution and 25% ammonia water solution, and the experiment finds that the reagent with the best hydrolysis efficiency is 25% ammonia water, then 2mol/L sodium hydroxide solution, and the 5mol/L sodium hydroxide solution has the lowest hydrolysis efficiency. Therefore, 25% ammonia was selected as the hydrolysis reagent for this experiment.
(2) Selection of hydrolysis time: compared with hydrolysis effects of 40 ℃, 50 ℃ and 60 ℃ ultrasonic waves for 10, 20, 30, 40, 50, 60, 70 and 80min respectively, experiments show that the recovery rate of 40 ℃ hydrolysis for 60min is the highest, the ultrasonic time is continuously prolonged, the recovery rate change is small, the recovery rates of 50 ℃ and 60 ℃ hydrolysis for 40min are the highest, but the recovery rates are lower than the recovery rate of 40 ℃ hydrolysis for 60min, so 40 ℃ ultrasonic waves for 60min are selected for hydrolysis in the experiment.
FIG. 3 is a graph comparing the hydrolysis efficiency of meptyldinocap at different temperatures and different ultrasonic times.
5) Selection of mobile phase system:
In this experiment, the influence of methanol water (50 +50, V/V), methanol water (50 +50, V/V) containing 0.1% (V/V) formic acid, methanol water (50 +50, V/V) and acetonitrile water (50 +50, V/V) containing 0.2mmol/L ammonium acetate, acetonitrile water (50 +50, V/V) containing 0.1% (V/V) formic acid, acetonitrile water (50 +50, 8655) containing 0.2mmol/L ammonium acetate, respectively, on the chromatographic peak shape and response value was examined with 0.01 mg/L2, 4-DNOP sample, the influence of the flow of DNphase on the 2, 4-OP peak shape was greater for the 2, 4-OP sample, and the influence of the flow of the DNOP sample on the chromatographic peak shape and response value was found to be greater for the DNphase in the system with the highest possible difference between methanol water (50 +50, 3635), acetonitrile water (50 +50, V/V) containing 0.1% (V/V) formic acid, the sample, the influence of the acetonitrile water (50 +50, 3650, V/V) containing 0.1% ammonium acetate, the difference between the peaks of the methanol water (50 +50, 3625, 25, respectively.
6) selection of chromatographic column: as the column for detecting agricultural chemicals, a T3 column and a C18 column were mostly used, and for this purpose, ACQUITY UPLC BEH C18 (100 mm. times.2.1 mm, 1.7 μm), ACQUITY UPLCBEH C18 (150 mm. times.2.1 mm, 1.7 μm), ACQUITY UPLC BEH T3 (100 mm. times.2.1 mm, 1.7 μm), ACQUITY UPLC BEH T3 (150 mm. times.2.1 mm, 1.7 μm), and 2,4-DNOP produced by Waters were compared in experiments, and as the column for this study, ACQUITY LC UPBEH T3 (50 mm. times.2.1 mm, 1.7 μm) having an earlier peak time and a sharper peak shape was finally selected.
The invention establishes a detection method for determining the residual amount of the meptyldinocap in tobacco by detecting 2,4-DNOP corresponding to 2,4-DNOPC, a tobacco sample adopts a quick, simple and reliable extraction method, an extracting solution is heated and hydrolyzed under an alkaline condition, UPLC-MS/MS analysis with high sensitivity and high selectivity is used, and the detection of the sample can be completed within 3 min.
example 2
A method for detecting residual quantity of meptyldinocap in tobacco comprises the following steps:
1) Preparation of a standard solution: accurately weighing 2,4-DNOP standard substance to 0.1mg, dissolving with methanol to fix volume to 100mL brown volumetric flask, preparing into 1mg/L single standard stock solution, storing in refrigerator at-20 deg.C in dark place, and making standard curve and quantitative detection;
The standard curve is specifically prepared by preparing 2,4-DNOP matrix matching standard working solution with 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series of mass concentrations by using tobacco blank matrix extracting solution, carrying out linear regression on each concentration by using peak area to obtain a standard curve, wherein the standard curve is good in linearity in the range of the mass concentration of the 2,4-DNOP being 0.001-0.5mg/L, and the correlation coefficient r 2 is more than 0.999;
Preparing a 2,4-DNOPC standard solution: accurately weighing 2,4-DNOPC standard substance to 0.1mg, dissolving with methanol to fix volume to 100mL brown volumetric flask, preparing into 1mg/L single standard stock solution, storing in refrigerator at-20 deg.C in dark place for addition recovery test; the adding recovery test specifically comprises the steps of carrying out a labeling recovery test at addition levels of 0.01, 0.05 and 0.1mg/kg of 3 respectively, setting 5 parallel samples for each addition concentration, and then carrying out sample pretreatment and detection, wherein the recovery rate of the 2,4-DNOPC in tobacco is 92.37%, and the relative standard deviation is 2.43%.
2) sample pretreatment: weighing 2g of a sample to be detected, accurately measuring the sample to 0.01g, adding 10mL of water into a 50mL centrifuge tube with a cover, oscillating until the sample is fully soaked by the water, and standing for 10 min; transferring 10mL of 1% acetic acid-acetonitrile (volume ratio is 1: 99) into a centrifuge tube, and placing the centrifuge tube on a vortex mixing and oscillating instrument to oscillate for 2min at the speed of 2000 r/min; freezing and storing the centrifugal tube at-18 deg.C for 10min, and taking out; respectively adding 4g of anhydrous magnesium sulfate, 1g of sodium chloride, 1g of sodium citrate and 0.5g of disodium hydrogen citrate into a centrifugal tube, immediately oscillating for 2min on a vortex mixing and oscillating instrument at the speed of 2000r/min, and then centrifuging for 3min at 6000 r/min; then transferring 1mL of sample extraction supernatant into a 1.5mL centrifuge tube, adding 10 μ L of 25% ammonia water, oscillating for 2min on a vortex mixing oscillator at a speed of 2000r/min, placing the sample in an ultrasonic wave at 40 ℃ for 40min, filtering the sample through a 0.22 μm organic phase filter membrane to a sample injection bottle for analysis;
3) and (3) detecting the residual quantity of the meptyldinocap: and (3) detecting the sample treated in the step (2) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry anion multi-reaction monitoring mode, and measuring the residual amount of the meptyldinocap by combining the standard curve obtained in the step (1).
The chromatographic conditions of the ultra-high performance liquid chromatography are that a chromatographic column: 5mm × 2.1mm, 1.7 μm ACQUITY UPLC BEH C18; column temperature: 25 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: methanol-water solution containing 0.2mmol/L ammonium acetate (50 +50, V/V) or acetonitrile water containing 0.2mmol/L ammonium acetate (50 +50, V/V), flow rate: 0.4mL/min, time: 3 min.
The analysis conditions of the mass spectrum are as follows: an ion source: ESI, scan mode: negative ion mode, air curtain airflow rate: 25 psi; flow rate of atomizing gas: 55 psi; flow rate of auxiliary gas: 55 psi; ion source temperature: 500 ℃; spraying voltage: 4500V, declustering voltage: -100V; in the multi-reaction monitoring mode, the parent ion is m/z 295.1, the daughter ion is m/z134.1, CE: -50V and m/z 163.0, CE: -50V, wherein m/z134.1 is the quantitative ion. The detection limit of the residual amount of the meptyldinocap is 3 mug/kg, and the quantification limit is 10 mug/kg.
example 3
A method for detecting residual quantity of meptyldinocap in tobacco comprises the following steps:
1) Preparation of a standard solution: accurately weighing 2,4-DNOP standard substance to 0.1mg, dissolving with methanol to fix volume to 100mL brown volumetric flask, preparing into 1mg/L single standard stock solution, storing in refrigerator at-20 deg.C in dark place, and making standard curve and quantitative detection;
the standard curve is specifically prepared by preparing 2,4-DNOP matrix matching standard working solution with 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series of mass concentrations by using tobacco blank matrix extracting solution, carrying out linear regression on each concentration by using peak area to obtain a standard curve, wherein the standard curve is good in linearity in the range of the mass concentration of the 2,4-DNOP being 0.001-0.5mg/L, and the correlation coefficient r 2 is more than 0.999;
The step (1) also comprises the preparation of a 2,4-DNOPC standard solution, specifically, accurately weighing a 2,4-DNOPC standard substance to be accurate to 0.1mg, dissolving with methanol to fix the volume to a 100mL brown volumetric flask, preparing a 1mg/L single standard stock solution, storing in a refrigerator at-20 ℃ in a dark place, and performing an addition recovery test; the adding recovery test specifically comprises the steps of respectively carrying out standard adding recovery tests at 0.01, 0.05 and 0.1mg/kg of 3 adding levels, setting 5 parallel samples for each adding concentration, then carrying out sample pretreatment and detection, wherein the recovery rate of the 2,4-DNOPC in tobacco is 100.52%, and the relative standard deviation is 3.30%.
2) sample pretreatment: weighing 5g of a sample to be detected, accurately measuring the sample to 0.01g, adding 20mL of water into a 50mL centrifuge tube with a cover, oscillating until the sample is fully soaked by the water, and standing for 10 min; transferring 10mL of 1% acetic acid-acetonitrile (volume ratio is 1: 99) into a centrifuge tube, and placing the centrifuge tube on a vortex mixing and oscillating instrument to oscillate for 5min at the speed of 2000 r/min; freezing and storing the centrifugal tube at-18 deg.C for 10min, and taking out; respectively adding 4g of anhydrous magnesium sulfate, 1g of sodium chloride, 1g of sodium citrate and 0.5g of disodium hydrogen citrate into a centrifugal tube, immediately oscillating for 5min on a vortex mixing and oscillating instrument at the speed of 2000r/min, and then centrifuging for 5min at 6000 r/min; then transferring 1mL of sample extraction supernatant into a 1.5mL centrifuge tube, adding 20 μ L of 25% ammonia water, oscillating for 5min on a vortex mixing oscillator at a speed of 2000r/min, placing the sample in an ultrasonic wave at 60 ℃ for ultrasonic 60min, and filtering the sample to a sample injection bottle through a 0.22 μm organic phase filter membrane for analysis;
3) And (3) detecting the residual quantity of the meptyldinocap: and (3) detecting the sample treated in the step (2) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry anion multi-reaction monitoring mode, and measuring the residual amount of the meptyldinocap by combining the standard curve obtained in the step (1).
The chromatographic conditions of the ultra-high performance liquid chromatography are that a chromatographic column: 5mm × 2.1mm, 1.7 μm ACQUITY UPLC BEH C18; column temperature: 25 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: methanol-water solution containing 0.2mmol/L ammonium acetate (50 +50, V/V) or acetonitrile water containing 0.2mmol/L ammonium acetate (50 +50, V/V), flow rate: 0.4mL/min, time: 3 min.
the analysis conditions of the mass spectrum are as follows: an ion source: ESI, scan mode: negative ion mode, air curtain airflow rate: 25 psi; flow rate of atomizing gas: 55 psi; flow rate of auxiliary gas: 55 psi; ion source temperature: 500 ℃; spraying voltage: 4500V, declustering voltage: -100V; in the multi-reaction monitoring mode, the parent ion is m/z 295.1, the daughter ion is m/z134.1, CE: -50V and m/z 163.0, CE: -50V, wherein m/z134.1 is the quantitative ion. The detection limit of the residual amount of the meptyldinocap is 3 mug/kg, and the quantification limit is 10 mug/kg.
Claims (9)
1. A detection method for residual quantity of meptyldinocap in tobacco is characterized by comprising the following steps:
1) Preparation of a standard solution: accurately weighing 2,4-DNOP standard substance to 0.1mg, dissolving with methanol to fix volume to 100mL brown volumetric flask, preparing into 1mg/L single standard stock solution, storing in refrigerator at-20 deg.C in dark place, and making standard curve and quantitative detection;
2) Pre-treatment of a sample, namely weighing 2g ~ g of a sample to be detected, accurately measuring the sample to 0.01g, adding 10mL ~ mL of water into a 50mL centrifuge tube with a cover, oscillating until the sample is fully soaked by the water, standing for 10min, transferring 10mL of acetic acid-acetonitrile mixed solution into the centrifuge tube, placing the centrifuge tube on a vortex mixing oscillator, oscillating for 2 3875 min at the speed of 2000r/min, freezing and storing the centrifuge tube at-18 ℃ for 10min, taking out, adding 4g of anhydrous magnesium sulfate, 1g of sodium chloride, 1g of sodium citrate and 0.5g of disodium hydrogen citrate into the centrifuge tube, immediately oscillating for 2 ~ min at the speed of 2000r/min on the vortex mixing oscillator, then centrifuging for 3 ~ min at 6000r/min, transferring 1mL of sample extraction supernatant into 1.5mL centrifuge tube, adding 10 ~ μ L of alkaline solution, placing the sample extraction supernatant into the centrifuge tube at the speed of 2000r/min, oscillating for 2 ~ min on the vortex mixing oscillator, placing the centrifuge tube at the ultrasonic wave of ~ ℃, performing ultrasonic analysis at the temperature of 3640 ℃ for 0.78 min, and filtering the sample to a filter bottle for organic phase analysis;
3) And (3) detecting the residual quantity of the meptyldinocap: detecting the sample treated in the step 2) by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry anion multi-reaction monitoring mode, and measuring the residual amount of the meptyldinocap by combining the standard curve obtained in the step 1); the chromatographic conditions of the ultra-high performance liquid chromatography are that a chromatographic column: 5mm × 2.1mm, 1.7 μm ACQUITY UPLC BEH C18; column temperature: 25 ℃; sample introduction amount: 2 mu L of the solution; mobile phase: methanol-water solution containing 0.2mmol/L ammonium acetate or acetonitrile water containing 0.2mmol/L ammonium acetate, flow rate: 0.4mL/min, time: 3 min; the methanol-water solution is V/V50 +50, and the acetonitrile water is V/V50 + 50.
2. The detection method according to claim 1, wherein the step 1) further comprises preparation of a 2,4-DNOPC standard solution, specifically comprises accurately weighing the 2,4-DNOPC standard, accurately weighing the 2,4-DNOPC standard to 0.1mg, dissolving the volume to 100mL of brown volumetric flask with methanol, preparing a 1mg/L single standard stock solution, storing the stock solution in a refrigerator at-20 ℃ in a dark place for performing an addition recovery test, wherein the addition recovery test specifically comprises performing an addition recovery test at 0.01, 0.05 and 0.1mg/kg of 3 addition levels, setting 5 parallel samples for each addition concentration, and performing sample pretreatment and detection, wherein the recovery rate of the 2,4-DNOPC in tobacco is 92.37% ~ 100.52%, and the relative standard deviation is 2.43% ~ 3.30.30%.
3. The detection method according to claim 1, wherein the preparing of the standard curve in step 1) is to prepare a 2,4-DNOP matrix matching standard working solution with 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series of mass concentrations from the tobacco blank matrix extract, and perform linear regression on each concentration by peak area to obtain a standard curve, wherein the standard curve is linear in the range of 0.001-0.5mg/L of the mass concentration of 2,4-DNOP, and the correlation coefficient r 2 is greater than 0.999.
4. The detection method according to claim 1, wherein the acetic acid-acetonitrile mixed solution in the step 2) is a mixed solution of 1% acetic acid-99% acetonitrile.
5. The detection method according to claim 1, wherein the alkaline solution in step 2) is 25% ammonia water.
6. The detection method according to claim 1, wherein the ultrasonic wave in the step 2) is ultrasonic at 40 ℃ for 60 min.
7. the detection method according to claim 1, wherein the analysis conditions of the mass spectrum in step 3) are: an ion source: ESI, scan mode: negative ion mode, air curtain airflow rate: 25 psi; flow rate of atomizing gas: 55 psi; flow rate of auxiliary gas: 55 psi; ion source temperature: 500 ℃; spraying voltage: 4500V, declustering voltage: -100V; in the multi-reaction monitoring mode, the parent ion is m/z 295.1, the daughter ion is m/z134.1, CE: -50V and m/z 163.0, CE: -50V, wherein m/z134.1 is the quantitative ion.
8. The detection method according to claim 1, wherein the detection limit of the residual quantity of meptyldinocap in step 3) is 3 μ g/kg, and the quantification limit is 10 μ g/kg.
9. The detection method according to claim 1, wherein the purity of the 2,4-DNOP standard is 98.5%, and the purity of the 2,4-DNOPC standard is 93%; the methanol and the acetonitrile are HPLC grade; the anhydrous magnesium sulfate is analytically pure, is burned for 4 hours at 650 ℃ before use, and is stored in a dryer for later use; the sodium chloride, the sodium citrate, the disodium hydrogen citrate, the sodium hydroxide and the ammonia water are all analytically pure.
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