CN110632218A - Method for detecting residual quantity of meptyldinocap and metabolites thereof in tobacco - Google Patents
Method for detecting residual quantity of meptyldinocap and metabolites thereof in tobacco Download PDFInfo
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Abstract
The invention discloses a method for detecting residual quantity of meptyldinocap and metabolites thereof in tobacco. The detection method has the advantages of simple and rapid sample pretreatment, and sensitivity, accuracy and precision all meeting the detection requirements of pesticide residue, and is suitable for simultaneous detection of residual quantity of meptyldinocap and metabolites thereof in tobacco.
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 and metabolites thereof in tobacco.
Background
The meptyldinocap is a novel powdery mildew fungicide, has good control effect on powdery mildew on crops such as pears, cucurbits, stone fruits, oranges, tobaccos, vegetables, ornamental plants and the like, and also has a certain inhibition effect on some phytophagous mites. Nitrophenyl pivoxil is a single isomer separated from 6 isomers of dinotefuran (CAS: 39300-45-3), and has been used in Europe, America and other countries for many years due to its outstanding control effect, and is registered in China in 2018. Internationally, the Maximum Residual Limit (MRL) of meptyldinocap is very strict, and the detection of the residual meptyldinocap is very important. The European Union has established the maximum residual limit of meptyldinocap (MRL, sum of meptyldinocap and metabolites) in various agricultural products, such as 0.05mg/kg in citrus, apple, pear and mango, 1 mg/kg in grape and 3 mg/kg in strawberry. Hong Kong stipulates that the MRL in grape, strawberry, melon, cucumber, pumpkin is 0.2, 0.3, 0.5, 0.7 and 0.7 mg/kg, respectively. Tobacco is an important economic crop in China, the importance of the pesticide residue problem on the safety problem of tobacco leaves is increasingly prominent, and a rapid, accurate and sensitive detection method for the residual quantity of the nitrophenyl acetate and the metabolite in the tobacco and the tobacco products is very necessary to be established for ensuring the quality safety of the tobacco leaves.
The method for detecting the residual quantity of the nitrophenyl methyl ester mainly comprises a high performance liquid chromatography method and a liquid chromatography-mass spectrometry combined method, wherein the method cannot directly measure the nitrophenyl methyl ester and the metabolite, the nitrophenyl methyl ester in a sample needs to be extracted and then hydrolyzed into corresponding phenol, and the residual quantity of the nitrophenyl methyl ester is calculated by analyzing the residual quantity of the corresponding hydrolysate phenol, so that the method is complicated and time-consuming. Therefore, it is necessary to develop a method capable of solving the above problems.
Disclosure of Invention
The invention aims to provide a method for detecting residual quantity of meptyldinocap and metabolites thereof in tobacco.
The invention aims to realize the detection method of the residual quantity of the meptyldinocap and the metabolite thereof in the tobacco, which comprises the steps of pretreatment and detection, and specifically comprises the following steps:
A. pretreatment:
1) sample treatment: accurately weighing 1g of a sample to be detected in a 50mL centrifuge tube, adding 10mL of acetonitrile, performing ultrasonic treatment and extraction, and performing primary centrifugation, shock adsorption and secondary centrifugation to prepare a sample liquid a to be detected;
2) preparing a standard solution: accurately weighing nitrobenzene bacterial ester (2, 4-DNOPC) and nitrobenzene bacterial ester metabolite (2, 4-DNOP) standard substances respectively, dissolving with methanol to a constant volume to prepare 1mg/ml nitrobenzene bacterial ester standard mother liquor b and nitrobenzene bacterial ester metabolite mother liquor c, diluting the nitrobenzene bacterial ester mother liquor b into 0.005, 0.01, 0.02, 0.05 and 0.1mg/L matrix matching standard working solutions by using a tobacco blank matrix extracting solution, diluting the nitrobenzene bacterial ester metabolite mother liquor c into 0.02, 0.05, 0.1, 0.2 and 0.5mg/L matrix matching standard working solutions, and respectively preparing matrix calibration curves of the nitrobenzene bacterial ester and the nitrobenzene bacterial ester metabolite by taking the mass concentration of the nitrobenzene bacterial ester and the nitrobenzene bacterial metabolite as an abscissa (X) and taking the peak areas of the nitrobenzene bacterial ester and the nitrobenzene bacterial metabolite as an ordinate (Y);
B. and (3) detection: and (3) detecting the sample liquid a to be detected by a gas chromatography negative chemical source tandem mass spectrometry, and measuring the residual quantity of the meptyldinocap and the meptyldinocap metabolite by combining a standard curve.
According to the method, the gas chromatography-negative chemical source tandem mass spectrometer is adopted to simultaneously determine the nitrophenyl bacteria ester and the nitrophenyl bacteria ester metabolites in the tobacco, the sample is subjected to ultrasonic extraction by adopting an organic reagent, hydrolysis is not needed, the operation steps are greatly simplified, and the time is saved.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention creatively adopts a rapid, simple and reliable extraction method for extracting the meptyldinocap and the metabolite in the tobacco sample, and adopts a high-selectivity and high-sensitivity gas chromatography negative chemical source tandem mass spectrometry to determine the residual quantity of the meptyldinocap and the metabolite in the tobacco.
2. The detection method is simple, the sample extraction steps are efficient and safe, the cost is low, the recovery rate is high, and the rapid, accurate and timely detection of the meptyldinocap and the metabolites in the tobacco is realized by optimizing the pretreatment in the detection process and controlling the chromatographic conditions in the detection.
3. The chromatographic method can simultaneously detect the meptyldinocap and the metabolite thereof, adopts a negative chemical source soft ionization and multi-reaction monitoring mode, has high selectivity and high sensitivity on the meptyldinocap and the metabolite, and is suitable for simultaneously detecting the residual quantity of the meptyldinocap and the metabolite in the tobacco.
Drawings
FIG. 1 is an MRM chromatogram of a nitrophenyl ester (2, 4-DNOPC) standard solution;
FIG. 2 is a MRM chromatogram of a nitrophenyl ester metabolite (2, 4-DNOP) standard solution;
FIG. 3 is a chromatogram of a standard solution obtained by adding meptyldinocap and meptyldinocap metabolite to a tobacco blank sample.
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 and the metabolites thereof in the tobacco comprises the steps of pretreatment and detection, and specifically comprises the following steps:
A. pretreatment:
1) sample treatment: accurately weighing 1g of a sample to be detected in a 50mL centrifuge tube, adding 10mL of acetonitrile, performing ultrasonic treatment and extraction, and performing primary centrifugation, shock adsorption and secondary centrifugation to prepare a sample liquid a to be detected;
2) preparing a standard solution: accurately weighing nitrobenzene bacterial ester (2, 4-DNOPC) and nitrobenzene bacterial ester metabolite (2, 4-DNOP) standard substances respectively, dissolving with methanol to a constant volume to prepare 1mg/ml nitrobenzene bacterial ester standard mother liquor b and nitrobenzene bacterial ester metabolite mother liquor c, diluting the nitrobenzene bacterial ester mother liquor b into 0.005, 0.01, 0.02, 0.05 and 0.1mg/L matrix matching standard working solutions by using a tobacco blank matrix extracting solution, diluting the nitrobenzene bacterial ester metabolite mother liquor c into 0.02, 0.05, 0.1, 0.2 and 0.5mg/L matrix matching standard working solutions, and respectively preparing matrix calibration curves of the nitrobenzene bacterial ester and the nitrobenzene bacterial ester metabolite by taking the mass concentration of the nitrobenzene bacterial ester and the nitrobenzene bacterial metabolite as an abscissa (X) and taking the peak areas of the nitrobenzene bacterial ester and the nitrobenzene bacterial metabolite as an ordinate (Y);
B. and (3) detection: and (3) detecting the sample liquid a to be detected by a gas chromatography negative chemical source tandem mass spectrometry, and measuring the residual quantity of the meptyldinocap and the meptyldinocap metabolite by combining a standard curve.
The ultrasonic treatment in the step 1) is ultrasonic treatment for 8 ~ 12min at the ultrasonic frequency of 60 ~ 80 KHz.
A, the centrifugation in the step 1) is performed for 2 ~ 4min at the rotating speed of 5000 ~ 8000 r/min.
The vibration adsorption in the step A1) is to purify a sample extracting solution, and specifically, 50mg of dispersion adsorbent is added into a centrifuge tube of 1ml to 1.5ml of supernatant after primary centrifugation, and the mixture is vibrated for 1 ~ 3min at the speed of 2000r/min on a vortex mixing vibration instrument, wherein the dispersion adsorbent is PSA and C18, and the particle size is 40 microns.
The second centrifugation in the step 1) is used for further purifying the extracting solution, and specifically is used for 2 ~ 4min under the condition that the rotating speed is 5000 ~ 8000 r/min.
And the chromatographic conditions in the gas chromatography negative chemical source tandem mass spectrometry in the step B are as follows: a chromatographic column: DB-5MS (30 m 0.25mm 0.25 μm); sample introduction amount: 1 mu L with the split ratio of 20: 1; a sample inlet: 250 ℃; temperature rising procedure: keeping at 115 deg.C for 1 min, heating to 280 deg.C at 7 deg.C/min, and keeping for 2 min; carrier gas: helium, 1.0 mL/min.
The analysis conditions of the mass spectrometer in the gas chromatography negative chemical source tandem mass spectrometry in the step B are as follows: negative chemical ion source (NCI) using Multiple Reaction Monitoring (MRM) mode, parent ion m/z279, daughter ion m/z 178 (CE: 12) and m/z192 (CE: 12), wherein m/z 178 is the quantitative ion.
In the step B, the detection limit of the meptyldinocap is 0.01 mg/kg, the quantification limit is 0.03 mg/kg, the detection limit of the meptyldinocap metabolite is 0.04 mg/kg, and the quantification limit is 0.1 mg/kg.
The purity of the nitrophenyl bacteria ester standard substance is 93 percent, and the purity of the nitrophenyl bacteria ester metabolite standard substance is 98.5 percent; the acetonitrile is HPLC grade.
The method for detecting the residual quantity of the meptyldinocap and the metabolites thereof in the tobacco further comprises an addition recovery experiment, wherein the addition recovery experiment adopts a tobacco blank sample for addition recovery, the adding levels of the meptyldinocap are 0.05, 0.1 and 0.5mg/kg, the adding levels of the meptyldinocap metabolites are 0.1, 0.25 and 0.5mg/kg, each adding concentration is provided with 6 parallel samples, then sample pretreatment and detection are carried out, the recovery rate of the meptyldinocap in the tobacco is 83.73-96.89%, the relative standard deviation is 3.04-4.48%, the recovery rate of the meptyldinocap metabolites in the tobacco is 82.95-96.73%, and the relative standard deviation is 3.94-5.25%.
The method for detecting the residual quantity of the meptyldinocap and the metabolites thereof in the tobacco specifically comprises the following operations:
1) preparation of a standard solution: accurately weighing the nitrophenyl bacteria ester (2, 4-DNOPC) and nitrophenyl bacteria ester metabolite (2, 4-DNOP) standard substances respectively, dissolving the nitrophenyl bacteria ester and the nitrophenyl bacteria ester metabolite in methanol to fix the volume of the mixture into 2 different brown volumetric flasks to prepare 1mg/mL single standard mother liquor, diluting the nitrophenyl bacteria ester into 0.005, 0.01, 0.02, 0.05 and 0.1mg/L matrix matching standard working solutions by using a tobacco blank matrix extracting solution, and diluting the nitrophenyl bacteria ester metabolite into 0.02, 0.05, 0.1, 0.2 and 0.5mg/L matrix matching standard working solutions.
2) Sample pretreatment: 1g of sample to be detected is accurate to 0.01 g, 10mL of acetonitrile is added into a 50mL centrifuge tube, the centrifuge tube is placed in an ultrasonic generator for 10 min, and the sample is centrifuged at 6000 r/min for 3 min. Transferring 1mL to 1.5mL of the supernatant of the sample extracting solution, adding 50mg of C18 dispersion adsorbent into a centrifuge tube, oscillating for 2min on a vortex mixing and oscillating instrument at the speed of 2000r/min, and then placing the centrifuge tube in a high-speed centrifuge for 3min at the speed of 6000 r/min. Taking the supernatant, and performing computer analysis.
3) And (3) detecting the residual quantity of the meptyldinocap: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography negative chemical source tandem mass spectrometry, and measuring the residual quantity of the meptyldinocap and the metabolite by combining the standard curve obtained in the step (1).
Further, the preparation method of the standard stock solution in the step (1) comprises the steps of accurately weighing 0.1 g of the meptyldinocap standard substance to 0.1mg, dissolving the meptyldinocap standard substance with methanol to a 100mL brown volumetric flask, and preparing the standard stock solution with the concentration of 1 mg/mL.
Further, the method for preparing the standard curve in the step (1) specifically comprises the steps of diluting the meptyldinocap into a matrix matching standard working solution of 0.005, 0.01, 0.02, 0.05 and 0.1mg/L by using a tobacco blank matrix extracting solution, diluting the meptyldinocap metabolite into a matrix matching standard working solution of 0.02, 0.05, 0.1, 0.2 and 0.5mg/L, and preparing the matrix calibration curve of the meptyldinocap and the metabolite by taking the mass concentration of the meptyldinocap and the metabolite thereof as an abscissa (X) and the peak area of the meptyldinocap and the metabolite thereof as an ordinate (Y). The standard curve of the meptyldinocap is in good linearity within the range of mass concentration of 0.005-0.1 mg/L, the standard curve of the meptyldinocap metabolite is in good linearity within the range of mass concentration of 0.02-0.5 mg/L, and the correlation coefficient r2Are both greater than 0.99.
Further, the detection method also comprises an addition recovery experiment, wherein the addition recovery experiment adopts a blank tobacco sample for addition recovery, the addition levels of the meptyldinocap are 0.05, 0.1 and 0.5mg/kg, the addition levels of the meptyldinocap metabolites are 0.1, 0.25 and 0.5mg/kg, 6 parallel samples are set for each addition concentration, then sample pretreatment and detection are carried out, the recovery rate of the meptyldinocap in the tobacco is 83.73-96.89%, the relative standard deviation is 3.04-4.48%, the recovery rate of the meptyldinocap metabolites in the tobacco is 82.95-96.73%, and the relative standard deviation is 3.94-5.25%. The method has good recovery rate and precision.
Further, the extraction in the step (2) is specifically 1g of sample to be detected, the accuracy is 0.01 g, 10mL of acetonitrile is added into a 10mL centrifuge tube, the centrifuge tube is placed in an ultrasonic generator for ultrasonic treatment for 10 min, and the centrifuge tube is centrifuged at 6000 r/min for 3 min. Transferring 1mL to 1.5mL of the supernatant of the sample extracting solution, adding 50mg of C18 dispersion adsorbent into a centrifuge tube, oscillating for 2min on a vortex mixing and oscillating instrument at the speed of 2000r/min, and then placing the centrifuge tube in a high-speed centrifuge for 3min at the speed of 6000 r/min. Taking the supernatant, and performing computer analysis.
Further, the chromatographic conditions of the gas chromatography negative chemical source tandem mass spectrometry in the step (3) are that a chromatographic column: DB-5MS (30 m × 0.25mm × 0.25 μm), sample size: 1 mu L with the split ratio of 20: 1; a sample inlet: 250 ℃; temperature rising procedure: keeping at 115 deg.C for 1 min, heating to 280 deg.C at 7 deg.C/min, and keeping for 2 min; carrier gas: helium, 1.0 mL/min.
Further, the analysis conditions of the mass spectrometer in the step (3) are as follows: negative chemical ion source (NCI) using Multiple Reaction Monitoring (MRM) mode, parent ion m/z279, daughter ion m/z 178 (CE: 12) and m/z192 (CE: 12), wherein m/z 178 is the quantitative ion.
Further, the detection limit of the meptyldinocap in the step (3) is 0.01 mg/kg, the quantification limit is 0.03 mg/kg, the detection limit of the meptyldinocap metabolite is 0.04 mg/kg, and the quantification limit is 0.1 mg/kg.
Further, the purity of the meptyldinocap 2,4-DNOPC standard is 93%, and the purity of the meptyldinocap metabolite 2,4-DNOP is 98.5%; the acetonitrile is HPLC grade; the PSA and the C18 dispersed adsorbent are both 40 μm.
The invention is further illustrated by the following specific examples:
example 1
1. Instruments and reagents: EVOQ GC-TQ gas chromatography-tandem mass spectrometer (Bruker, USA), Millipore ultra-pure water machine (Millipore silicon, USA), Eppendorf 5804 high speed centrifuge (Eppendorf, Germany), Talboys digital display type multi-tubular vortex mixer (Shanghai' an Spectroscopy instruments). Methanol, acetonitrile, HPLC grade, Fisher corporation, usa. Nitrophenyl ester (2, 4-DNOPC) standard (93% pure, available from Dr, Germany) and Nitrophenyl ester metabolite (2, 4-DNOP) standard (98.5% pure, available from 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: respectively and accurately weighing a nitrobenzene ester standard substance and a nitrobenzene ester metabolite (nitrophenolate) standard substance (accurate to 0.1 mg), dissolving the substances with methanol, fixing the volume to a 100mL brown volumetric flask, preparing a single standard stock solution of 1mg/L, storing the single standard stock solution in a refrigerator at the temperature of minus 20 ℃ in a dark place, and preparing a standard curve and carrying out quantitative detection.
2) Sample pretreatment: weighing 1g of sample to be detected, accurately measuring the sample to 0.01 g, adding 10mL of acetonitrile into a 10mL centrifuge tube, placing the centrifuge tube in an ultrasonic generator for 10 min, and centrifuging the centrifuge tube for 3min at 6000 r/min. Transferring 1mL to 1.5mL of the supernatant of the sample extracting solution, adding 50mg of C18 dispersion adsorbent into a centrifuge tube, oscillating for 2min on a vortex mixing and oscillating instrument at the speed of 2000r/min, and then placing the centrifuge tube in a high-speed centrifuge for 3min at the speed of 6000 r/min. Taking the supernatant, and performing computer analysis.
3) And (3) detecting residual amounts of meptyldinocap and metabolites: and (3) detecting the residual quantity of the meptyldinocap: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography negative chemical source tandem mass spectrometer multi-reaction monitoring mode, and measuring the residual quantity of the meptyldinocap and the metabolite thereof by combining the standard curve obtained in the step (1).
Chromatographic-mass spectrometry conditions: a chromatographic column: DB-5MS (30 m 0.25mm 0.25 μm); sample introduction amount: 1 mu L with the split ratio of 20: 1; a sample inlet: 250 ℃; temperature rising procedure: keeping at 115 deg.C for 1 min, heating to 280 deg.C at 7 deg.C/min, and keeping for 2 min; carrier gas: helium, 1.0 mL/min; negative chemical ion source (NCI), reaction gas: methane, filament current 50 muA, electron multiplier voltage 1200V, solvent delay time 6 min. And (3) monitoring mode: multiple Reaction Monitoring (MRM), parent ion m/z279, daughter ion m/z 178 (CE: 12) and m/z192 (CE: 12), where m/z 178 is the quantification ion.
Further, the detection limit of the residual amount of the meptyldinocap in the step (3) is 0.01 mg/kg, the quantification limit is 0.03 mg/kg, the detection limit of the meptyldinocap metabolite is 0.04 mg/kg, and the quantification limit is 0.1 mg/kg.
3. Results and analysis:
1) linear range and detection limit (preparation of standard curve):
diluting meptyldinocap into matrix matching standard working solution of 0.005, 0.01, 0.02, 0.05 and 0.1mg/L by using tobacco blank matrix extracting solution, diluting meptyldinocap metabolite into matrix matching standard working solution of 0.02, 0.05, 0.1, 0.2 and 0.5mg/L, and preparing matrix calibration curve of meptyldinocap and metabolite by taking mass concentration of meptyldinocap and metabolite thereof as abscissa (X) and peak area of meptyldinocap and metabolite thereof as ordinate (Y). The meptyldinocap is in good linearity within the range of 0.005-0.1 mg/L, and the curve equation isY=1.0e6 X581220, correlation coefficient r2Is 0.9967, the nitrophenyl acetate metabolite is in good linearity in the range of 0.02-0.5 mg/L, and the curve equation isY=239431X115915, correlation coefficient r2The detection limit and the quantification limit of the nifedipine are respectively determined by a signal-to-noise ratio (S/N) of 3 times and a signal-to-noise ratio (S/N) of 10 times, and the detection limit and the quantification limit of the nifedipine are respectively 0.01 mg/kg, 0.03 mg/kg, 0.04 mg/kg and 0.1 mg/kg.
2) Recovery, precision (addition recovery test):
and (4) adopting a tobacco blank sample to carry out addition recovery and precision experiment. The adding levels of the meptyldinocap are 0.05, 0.1 and 0.5mg/kg, the adding levels of the meptyldinocap metabolites are 0.1, 0.25 and 0.5mg/kg, 6 parallel samples are set for each adding concentration, and the results of sample pretreatment and detection are shown in table 1, wherein the recovery rate of the meptyldinocap in the tobacco is 83.73-96.89%, the relative standard deviation is 3.04-4.48%, the recovery rate of the meptyldinocap metabolites in the tobacco is 82.95-96.73% and the relative standard deviation is 3.94-5.25%. The method has good recovery rate and precision.
TABLE 1 recovery of Nitrophenyl bacteria esters and their metabolites in tobacco with standard addition, precision (n = 6)
3) Selection of an extraction solvent: 0.1 mg/kg of meptyldinocap and meptyldinocap metabolite are added into a tobacco blank matrix, mixed uniformly and placed for 2 hours, and the extraction efficiency of acetonitrile, methanol, ethyl acetate, n-hexane, cyclohexane, acetone and dichloromethane on target pesticides is compared respectively. The experimental result shows that the recovery rate of the acetonitrile solution as the extraction solvent is the highest, and the extraction efficiency of the methanol is the worst by using dichloromethane, normal hexane, cyclohexane, ethyl acetate and acetone, so that the acetonitrile is selected as the extraction solvent of the method.
4) Selection of purification adsorbent: respectively weighing 50mg of C18 adsorbent and PSA adsorbent in a 2.0 mL centrifuge tube, adding 0.1 mg/kg of meptyldinocap and meptyldinocap metabolite into a tobacco blank matrix, extracting according to a sample pretreatment extraction step, taking 1mL of supernatant to centrifuge tubes filled with different adsorbents, and inspecting the recovery rate influence and purification effect of the 2 adsorbents on meptyldinocap and metabolite. The experimental results show that: PSA has recovery rate of nifedipine and its metabolite lower than 55%, C18 has better purification effect on lipid, and has better recovery rate of nifedipine and metabolite, and the extraction solution is purified by selecting 50mg C18 adsorbent by comprehensively considering the impurity removal effect of two adsorbents and the adsorption condition of target compound.
5) The method examines the influence of Matrix Effect (ME), and obtains the matrix effect (matrix effect, ME) = B/A x 100%) by measuring the average response value (A, n =3) of the nitrophenyl ester and the metabolite thereof in a methanol solvent under the two concentrations of 0.01 and 0.1 mu g/mL, and obtaining the matrix effect (matrix effect, ME) = B/A x 100%, the result shows that the nitrophenyl ester and the metabolite thereof in the tobacco leaves are analyzed as matrix enhancement signals at 2 different concentrations (0.01 mu g/mL, 0.1 mu g/mL) and the matrix effect thereof is 35106% under the conditions of 0.01 mu g/mL and the substrate effect thereof is ~ 118.1% under the conditions of 0.1 mu g/mL and the substrate enhancement signals are analyzed as accurate matrix enhancement effect curves of the method by adopting a matrix effect curve of the method, wherein the matrix enhancement signals of the target compounds are enhanced by measuring the average response values (A, n =3) of the nitrophenyl ester and the metabolite thereof in the methanol solvent under the two concentrations of 0.01 and 0.1 mu g/mL.
Claims (10)
1. A method for detecting residual quantity of meptyldinocap and metabolites thereof in tobacco is characterized by comprising the steps of pretreatment and detection, and specifically comprising the following steps:
A. pretreatment:
1) sample treatment: accurately weighing 1g of a sample to be detected in a 50mL centrifuge tube, adding 10mL of acetonitrile, performing ultrasonic treatment and extraction, and performing primary centrifugation, shock adsorption and secondary centrifugation to prepare a sample liquid a to be detected;
2) preparing a standard solution: accurately weighing nitrobenzene bacterial ester (2, 4-DNOPC) and nitrobenzene bacterial ester metabolite (2, 4-DNOP) standard substances respectively, dissolving with methanol to a constant volume to prepare 1mg/ml nitrobenzene bacterial ester standard mother liquor b and nitrobenzene bacterial ester metabolite mother liquor c, diluting the nitrobenzene bacterial ester mother liquor b into 0.005, 0.01, 0.02, 0.05 and 0.1mg/L matrix matching standard working solutions by using a tobacco blank matrix extracting solution, diluting the nitrobenzene bacterial ester metabolite mother liquor c into 0.02, 0.05, 0.1, 0.2 and 0.5mg/L matrix matching standard working solutions, and respectively preparing matrix calibration curves of the nitrobenzene bacterial ester and the nitrobenzene bacterial ester metabolite by taking the mass concentration of the nitrobenzene bacterial ester and the nitrobenzene bacterial metabolite as an abscissa (X) and taking the peak areas of the nitrobenzene bacterial ester and the nitrobenzene bacterial metabolite as an ordinate (Y);
B. and (3) detection: and (3) detecting the sample liquid a to be detected by a gas chromatography negative chemical source tandem mass spectrometry, and measuring the residual quantity of the meptyldinocap and the meptyldinocap metabolite by combining a standard curve.
2. The method for detecting the residual quantity of meptyldinocap and its metabolites in tobacco as claimed in claim 1, wherein the ultrasonic treatment in step A) is ultrasonic treatment at an ultrasonic frequency of 60 ~ 80KHz for 8 ~ 12 min.
3. The method for detecting residual quantity of meptyldinocap and its metabolites in tobacco as claimed in claim 1, wherein the primary centrifugation in step a) is performed at 5000 ~ 8000r/min for 2 ~ 4 min.
4. The method for detecting residual quantity of meptyldinocap and metabolites thereof in tobacco according to claim 1, wherein the vibration adsorption in step A) is to purify a sample extract, specifically, 50mg of dispersion adsorbent is added into a centrifuge tube of 1ml to 1.5ml of supernatant after primary centrifugation, and the centrifuge tube is vibrated for 1 ~ 3min at 2000r/min speed on a vortex mixing vibrator, wherein the dispersion adsorbent is PSA and C18, and is 40 μm.
5. The method for detecting residual amount of meptyldinocap and its metabolites in tobacco according to claim 1, wherein the second centrifugation in step a) is further purifying the extract, specifically centrifugation at 5000 ~ 8000r/min for 2 ~ 4 min.
6. The method for detecting the residual quantity of meptyldinocap and metabolites thereof in tobacco according to claim 1, wherein the chromatographic conditions in the gas chromatography negative chemical source tandem mass spectrometry in step B are as follows: a chromatographic column: DB-5MS (30 m 0.25mm 0.25 μm); sample introduction amount: 1 mu L with the split ratio of 20: 1; a sample inlet: 250 ℃; temperature rising procedure: keeping at 115 deg.C for 1 min, heating to 280 deg.C at 7 deg.C/min, and keeping for 2 min; carrier gas: helium, 1.0 mL/min.
7. The method for detecting residual quantity of meptyldinocap and its metabolites in tobacco according to claim 1, wherein the analysis conditions of the mass spectrometer in the gas chromatography negative chemical source tandem mass spectrometry in step B are as follows: negative chemical ion source (NCI) using Multiple Reaction Monitoring (MRM) mode, parent ion m/z279, daughter ion m/z 178 (CE: 12) and m/z192 (CE: 12), wherein m/z 178 is the quantitative ion.
8. The method for detecting the residual quantity of the meptyldinocap and the metabolites thereof in the tobacco according to claim 1, wherein the detection limit of the meptyldinocap in the step B is 0.01 mg/kg, the quantification limit of the meptyldinocap is 0.03 mg/kg, the detection limit of the meptyldinocap metabolites is 0.04 mg/kg, and the quantification limit of the meptyldinocap metabolites is 0.1 mg/kg.
9. The method for detecting the residual quantity of the meptyldinocap and the metabolites thereof in the tobacco according to claim 1, wherein the purity of the meptyldinocap standard is 93%, and the purity of the meptyldinocap metabolite standard is 98.5%; the acetonitrile is HPLC grade.
10. The method for detecting the residual quantity of meptyldinocap and metabolites thereof in tobacco according to claim 1, further comprising an additive recovery experiment, wherein the additive recovery experiment is to perform additive recovery by using a tobacco blank sample, the addition levels of meptyldinocap are 0.05, 0.1 and 0.5mg/kg, the addition levels of meptyldinocap metabolites are 0.1, 0.25 and 0.5mg/kg, each addition concentration is set to 6 parallel samples, then sample pretreatment and detection are performed, the recovery rate of meptyldinocap in tobacco is 83.73-96.89%, the relative standard deviation is 3.04-4.48%, the recovery rate of meptyldinocap metabolites in tobacco is 82.95-96.73%, and the relative standard deviation is 3.94-5.25%.
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