CN109100448B - Method for detecting residual quantity of dipterex in tobacco - Google Patents
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Abstract
The invention discloses a method for detecting residual quantity of dipterex in tobacco. The tobacco sample is extracted by adopting an improved QuEChERS technical extraction method, C18 dispersing adsorbent is detected by adopting a gas chromatography negative chemical source mass spectrometry combined method, and the external standard method is quantitative, and the result shows that the dipterex has good linear relation and correlation coefficient (r) within the range of 0.01-0.5 mg/L2) More than 0.99, the detection limit is 0.02mg/kg, the recovery rate of the dipterex in the tobacco is 95.83-102.34%, and the relative standard deviation is 3.75-5.30%. The detection method has the advantages of simple and quick sample pretreatment, and sensitivity, accuracy and precision all meeting the detection requirements of pesticide residue, and is suitable for detecting the residual amount of the dipterex in the tobacco.
Description
Technical Field
The invention belongs to the technical field of pesticide residue detection, and particularly relates to a method for detecting the residual quantity of dipterex in tobacco.
Background
Dinocap (CAS: 39300-45-3) is a mixture of six different dinitrocrotonate isomers, is a contact type bactericide, is mainly used for preventing and treating powdery mildew in crops and fruits and vegetables, and can also be used as a non-systemic acaricide. The dinocap has high toxicity, teratogenicity and carcinogenicity, residues can be caused in crops due to improper application, and the quality and safety of tobacco leaves are seriously influenced, so that quite strict MRL values are set by various countries, the CORESTA directive residue limit (GRL) specifies that the content of the dinocap in the tobacco is 0.6 mg/kg, and the Chinese tobacco general company specifies that the Maximum Residue Limit (MRL) of the dinocap in the tobacco is 0.1 mg/kg, so that the detection of the residues is very important.
Since dipterex has thermal instability and chemical instability, is easy to hydrolyze or photolyze into corresponding phenol, and six isomers of dipterex have similar structures and are difficult to separate on a chromatogram and a mass spectrum, the residue detection of the dipterex is always a very challenging work in the field of pesticide residue detection. The detection methods reported abroad at present mainly comprise a gas chromatography-ECD method, a liquid chromatography method and a liquid chromatography-mass spectrometry combined method. The liquid chromatography or the liquid chromatography-mass spectrometry combined method has complicated and time-consuming pretreatment extraction steps, six isomers of the dinocap need to be completely hydrolyzed and converted into phenol, the hydrolysis time is long, and accurate quantification is difficult because a standard product of the dinocap hydrolyzed phenol cannot be bought; the selectivity, precision and reproducibility are poor when the gas chromatography-ECD method is used for analysis.
Based on the above problems, it is necessary to invent a method for detecting the residual amount of dipterex in tobacco.
Disclosure of Invention
The invention aims to provide a method for detecting the residual quantity of dipterex in tobacco.
The object of the invention is achieved by the following steps:
1) preparation of a standard solution: accurately weighing a dinocap standard substance, dissolving and fixing the dinocap standard substance to a brown volumetric flask to prepare a standard stock solution of 1mg/mL, storing the standard stock solution in a refrigerator at the temperature of minus 15 to minus 25 ℃ in a dark place, and diluting the standard stock solution into a series of standard working solutions for making a standard curve;
2) sample pretreatment:
extraction: weighing a sample to be detected into a centrifugal tube, adding water, oscillating until the sample is fully soaked by the water, and standing for 8-12 min; transferring 1% formic acid-acetonitrile with the same amount as water into a centrifugal tube, and oscillating at the speed of 1500-; respectively adding anhydrous magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate into a centrifugal tube, wherein the addition amount is a sample to be detected: anhydrous magnesium sulfate: sodium chloride: sodium citrate: disodium hydrogen citrate =1:2:0.5:0.5:0.25, immediately oscillating at the speed of 1500-; then centrifuging at 7000r/min of 5000-;
purifying: transferring 1mL to 1.5mL of supernatant into a centrifuge tube, adding 50-200 mg of C18 dispersion adsorbent, carrying out vortex oscillation for 2-5 min, centrifuging for 3-5 min at 5000-6000 r/min, sucking the supernatant, filtering the supernatant through a 0.22-micron organic phase filter membrane, and carrying out machine analysis;
3) and (3) detecting the residual amount of the dipterex: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography-negative chemical source-mass spectrometry combined method, and measuring the residual amount of the dipterex 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 dipterex in the tobacco sample by adopting a quick, simple and reliable extraction method, then the C18 dispersion adsorbent is added, and the residual quantity of the dipterex in the tobacco is determined by adopting a high-sensitivity gas chromatography-negative chemical source mass spectrometry combined method.
2. The gas chromatography-negative chemical source mass spectrometry combined method has high selectivity and high sensitivity on substances containing electronegative groups, the QuEChERS method is applied to extraction of the dinocap in a tobacco sample for the first time, the C18 dispersed adsorbent is added for purification, the high-sensitivity GC-NCI-MS is adopted for measuring the dinocap in the tobacco, and the treatment efficiency and the sensitivity are effectively improved.
3. The detection method is simple, the sample extraction step is efficient and safe, the cost is low, the sample purification step is simple and rapid, the purification effect and the recovery rate are high, and accurate and timely detection of the dipterex in the tobacco is realized by optimizing the pretreatment in the detection process and controlling the chromatographic condition in the detection.
4. The chromatographic method has good separation effect on dipterex isomers, adopts negative chemical source soft ionization, has high selectivity and high sensitivity on dipterex, and is suitable for accurate detection of the residual amount of dipterex in tobacco.
Drawings
FIG. 1 is a chromatogram of a standard solution of pervasive dinocap added with dinocap in a blank tobacco sample 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 amount of the dipterex in the tobacco comprises the following steps:
1) preparation of a standard solution: accurately weighing a dinocap standard substance, dissolving and fixing the dinocap standard substance to a brown volumetric flask to prepare a standard stock solution of 1mg/mL, storing the standard stock solution in a refrigerator at the temperature of minus 15 to minus 25 ℃ in a dark place, and diluting the standard stock solution into a series of standard working solutions for making a standard curve;
2) sample pretreatment:
extraction: weighing a sample to be detected into a centrifugal tube, adding water, oscillating until the sample is fully soaked by the water, and standing for 8-12 min; transferring 1% formic acid-acetonitrile with the same amount as water into a centrifugal tube, and oscillating at the speed of 1500-; respectively adding anhydrous magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate into a centrifugal tube, wherein the addition amount is a sample to be detected: anhydrous magnesium sulfate: sodium chloride: sodium citrate: disodium hydrogen citrate =1:2:0.5:0.5:0.25, immediately oscillating at the speed of 1500-; then centrifuging at 7000r/min of 5000-;
purifying: transferring 1mL to 1.5mL of supernatant into a centrifuge tube, adding 50-200 mg of C18 dispersion adsorbent, carrying out vortex oscillation for 2-5 min, centrifuging for 3-5 min at 5000-6000 r/min, sucking the supernatant, filtering the supernatant through a 0.22-micron organic phase filter membrane, and carrying out machine analysis;
3) and (3) detecting the residual amount of the dipterex: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography-negative chemical source-mass spectrometry combined method, and measuring the residual amount of the dipterex 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 0.1g of the dipterex standard substance to 0.1mg, dissolving the dipterex standard substance with methanol to a constant volume of 100mL of a brown volumetric flask, and preparing the standard stock solution of 1 mg/mL.
Further, the method for preparing the standard curve in the step (1) specifically comprises the steps of preparing a standard stock solution of the dinocap with methanol into standard working solutions of the dinocap with the mass concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L, and performing linear regression on each concentration by peak area to obtain the standard curve, wherein the standard curve is good in linearity in the range of the mass concentration of the dinocap of 0.01-0.5 mg/L, and the correlation coefficient r is2Greater than 0.99.
Further, the detection method also comprises an addition recovery experiment, wherein the addition recovery experiment adopts a tobacco blank sample for addition recovery, the standard stock solution of the dinocap is used for the addition recovery experiment, specifically, the standard recovery experiment is respectively carried out at the addition levels of 0.05, 0.1 and 0.5mg/kg 3, each addition concentration is provided with 6 parallel samples, then the sample pretreatment and detection are carried out, the recovery rate of the dinocap in the tobacco is 95.83-102.34%, and the relative standard deviation is 3.75-5.30%.
Further, the extraction in the step (2) specifically comprises weighing 2g of a sample to be detected, accurately obtaining 0.01g of the 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; transferring 10mL of 1% formic acid-acetonitrile into a centrifuge tube, and placing the centrifuge tube on a vortex mixing and oscillating instrument to oscillate for 2-5 min at the speed of 2000 r/min; 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 respectively, immediately oscillating on a vortex mixing and oscillating instrument at the speed of 2000r/min for 2-5 min, and then centrifuging at 6000r/min for 3-5 min.
Further, the purification in the step (2) is specifically to transfer the supernatant into a centrifuge tube of 1mL to 1.5mL, add 50mg of C18 dispersion adsorbent, shake with vortex for 2min, centrifuge at 6000r/min for 3min, absorb the supernatant, filter the supernatant through a 0.22 μm organic phase filter membrane, and then perform the machine analysis.
Further, the chromatographic conditions of the gas chromatography-negative chemical source mass spectrometry combined method in the step (3) are that a chromatographic column: DB-5MS (30 m 0.25mm 0.25 u m).
Further, the analysis conditions of the mass spectrometer in the step (3) are as follows: negative chemical ion source (NCI) using a Selected Ion (SIM) mode, the characteristic ions being m/z 278 and m/z 279, wherein m/z 278 is the quantitative ion.
Further, the detection limit of the residual amount of the dipterex in the step (3) is 0.02mg/kg, and the quantification limit is 0.07 mg/kg.
Further, the purity of the dipterex standard substance is 98%; the methanol, the acetonitrile and the formic acid are HPLC grades; 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 and the disodium hydrogen citrate are analytically pure.
Example 1
1. Instruments and reagents: bruker 450GC-300MS gas chromatography-tandem mass spectrometry (Bruker, USA), Millipore ultra-pure water machine (Millipore silicon, USA), Eppendorf 5804 high speed centrifuge (Eppendorf, Germany), Talboys digital display type multi-tube vortex mixer (Shanghai' an Spectroscopy instruments). Methanol, acetonitrile, formic acid, HPLC grade, Fisher, 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, analytically pure. Dimeprol standard (98% purity, supplied by Dr, Germany).
2. The method for detecting the residual amount of the dipterex in the tobacco comprises the following steps:
1) preparation of a standard solution: respectively and accurately weighing the dipterex standard substance (accurate to 0.1 mg), dissolving with methanol to a constant volume of 100mL, preparing into 1mg/L single standard stock solution, storing in a refrigerator at-20 deg.C in dark place, and making standard curve and quantitative detection.
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% formic 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 supernatant into 1-1.5 mL centrifuge tube, adding 50mg C18 dispersion adsorbent, vortex vibrating for 2min, centrifuging at 6000r/min for 3min, sucking supernatant, filtering with 0.22 μm organic phase filter membrane, and analyzing on machine.
3) And (3) detecting the residual amount of the dipterex: and (3) detecting the residual amount of the dipterex: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography-negative chemical source-mass spectrometry combined method, and measuring the residual amount of the dipterex 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 100 deg.C for 2min, heating to 280 deg.C at 10 deg.C/min, and keeping for 6 min; carrier gas: helium, 1.0mL/min, negative chemical ion source (NCI), reaction gas: methane, in Selective Ion (SIM) mode, with characteristic ions of m/z 278 and m/z 279, where m/z 278 is the quantitative ion.
3. Results and analysis:
1) linear range and detection limit (preparation of standard curve):
using methanol to prepare the dinocap into a dinocap standard working solution with the mass concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series, carrying out linear regression on the concentrations of various pesticides by peak area, and showing good linearity in the range of 0.01-0.5 mg/L, wherein the curve equation isY=2.1011e5 X-1.94e5Coefficient of correlation r20.9923, the detection limit and the quantification limit were determined by 3-fold and 10-fold signal-to-noise ratio (S/N), respectively, giving a detection limit of 0.02mg/kg and a quantification limit of 0.07mg/kg for dipterex in tobacco.
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 3 addition levels of 0.05, 0.1 and 0.5mg/kg, 6 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 result is shown in Table 1, the recovery rate of the dipterex in the tobacco is 95.83-102.34%, the relative standard deviation is 3.75-5.30%, and the recovery rate and the precision of the method are good. For example, a standard solution chromatogram of pervasive dinocap of dinotefuran is added to a blank tobacco sample in the figure 1.
TABLE 1 Standard recovery, precision of dipterex in tobacco (n = 6)
3) Selection of an extraction solvent: acetonitrile is a common pesticide residue extraction reagent, and the acetonitrile extraction solution has less interferents than other organic reagents. 0.1 mg/kg of dinocap is added into a tobacco blank substrate, mixed uniformly and placed for 2 hours, and then the extraction efficiency of acetonitrile, a 1% formic acid-acetonitrile solution and a 1% acetic acid-acetonitrile solution on target pesticides are compared respectively. The experimental result shows that the recovery rate of the 1% formic acid-acetonitrile solution as the extraction solvent is the highest (99.03%), the extraction efficiency of acetonitrile is the second (92.67%), the extraction efficiency of the 1% acetic acid-acetonitrile solution is the worst (90.52%), and 1% formic acid-acetonitrile is selected as the extraction solvent in the experiment in comprehensive consideration.
4) Selection of dispersed adsorbent:
respectively weighing 50mg, 100 mg, 150 mg and 200mg of C18 adsorbent and PSA adsorbent in a 2.0 mL centrifuge tube, adding 0.1 mg/kg of dinocap in 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 the dinocap. The experimental results show that: PSA is used as weak anion exchanger, has strong adsorption effect on polar compounds such as fatty acid, organic carbohydrate and the like, and the adsorption effect is enhanced along with the increase of the dosage of the adsorbent, so that the impurity removal is relatively clean, but the recovery rate of the dipterex is lower than 50%. The C18 has a good purifying effect on lipids and a good recovery rate on dipterex, the impurity removing effect and the recovery rate of dipterex do not change obviously with the increase of the dosage of the adsorbent, the impurity removing effect and the adsorption condition on target compounds of the two adsorbents are comprehensively considered, and 50-200 mg of C18 adsorbent is selected to purify the extracting solution.
Example 2
A method for detecting the residual quantity of dipterex in tobacco comprises the following steps:
1) preparation of a standard solution: accurately weighing 0.1g of dipterex standard substance to 0.1mg, dissolving with methanol to fix the volume to 100mL of brown volumetric flask, preparing 1mg/mL of standard stock solution, storing in a refrigerator at-15 ℃ in a dark place, and diluting the standard stock solution into a series of standard working solutions for making a standard curve;
the standard curve manufacturing method specifically comprises the steps of preparing a standard dipterex stock solution into standard dipterex working solutions with mass concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series by using methanol, carrying out linear regression on each concentration by using peak areas to obtain a standard curve, wherein the standard curve is good in linearity within the mass concentration range of the dipterex of 0.01-0.5 mg/L, and the correlation coefficient r is2Greater than 0.99;
2) sample pretreatment:
extraction: weighing a sample to be detected into a centrifugal tube, adding water, oscillating until the sample is fully soaked by the water, and standing for 8 min; transferring 1% formic acid-acetonitrile with the same amount as water into a centrifuge tube, and oscillating at 1500r/min for 5 min; respectively adding anhydrous magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate into a centrifugal tube, wherein the addition amount is a sample to be detected: anhydrous magnesium sulfate: sodium chloride: sodium citrate: disodium hydrogen citrate =1:2:0.5:0.5:0.25, immediately oscillating for 5min at the speed of 1500 r/min; then centrifuging for 5min at 5000 r/min;
purifying: transferring supernatant into a centrifuge tube of 1-1.5 mL, adding 50mg C18 dispersion adsorbent, vortex oscillating for 2min, centrifuging at 5000r/min for 5min, sucking supernatant, filtering with 0.22 μm organic phase filter membrane, and analyzing on computer;
3) and (3) detecting the residual amount of the dipterex: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography-negative chemical source-mass spectrometry combined method, and measuring the residual amount of the dipterex by combining the standard curve obtained in the step (1).
The chromatographic conditions of the gas chromatography-negative chemical source mass spectrometry combined method are that a chromatographic column: DB-5MS (30 m 0.25mm 0.25 u m). The analysis conditions of the mass spectrometer are as follows: negative chemical ion source (NCI) using a Selected Ion (SIM) mode, the characteristic ions being m/z 278 and m/z 279, wherein m/z 278 is the quantitative ion.
The purity of the dipterex standard substance is 98 percent; the methanol, the acetonitrile and the formic acid are HPLC grades; 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 and the disodium hydrogen citrate are analytically pure.
The detection method further comprises an adding and recovering experiment, wherein the adding and recovering experiment is to add and recover a tobacco blank sample, the dipterex standard stock solution is used for the adding and recovering experiment, specifically, the adding and recovering experiment is respectively carried out at the adding levels of 0.05, 0.1 and 0.5mg/kg 3, 6 parallel samples are set for each adding concentration, then, the sample pretreatment and detection are carried out, and the result proves that the recovery rate and the precision of the method are better.
Example 3
A method for detecting the residual quantity of dipterex in tobacco comprises the following steps:
1) preparation of a standard solution: accurately weighing 0.1g of dipterex standard substance to 0.1mg, dissolving with methanol to fix the volume to 100mL of brown volumetric flask, preparing 1mg/mL of standard stock solution, storing in a refrigerator at-25 ℃ in a dark place, and diluting the standard stock solution into a series of standard working solutions for making a standard curve;
the standard curve manufacturing method specifically comprises the steps of preparing a standard dipterex stock solution into standard dipterex working solutions with mass concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series by using methanol, carrying out linear regression on each concentration by using peak areas to obtain a standard curve, wherein the standard curve is good in linearity within the mass concentration range of the dipterex of 0.01-0.5 mg/L, and the correlation coefficient r is2Greater than 0.99;
2) sample pretreatment:
extraction: weighing a sample to be detected into a centrifugal tube, adding water, oscillating until the sample is fully soaked by the water, and standing for 12 min; transferring 1% formic acid-acetonitrile with the same amount as water into a centrifuge tube, and oscillating for 2min at the speed of 2500 r/min; respectively adding anhydrous magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate into a centrifugal tube, wherein the addition amount is a sample to be detected: anhydrous magnesium sulfate: sodium chloride: sodium citrate: disodium hydrogen citrate =1:2:0.5:0.5:0.25, immediately oscillating for 2min at the speed of 2500 r/min; then centrifuging at 7000r/min for 3 min;
purifying: transferring supernatant into a centrifuge tube of 1-1.5 mL, adding 200mg C18 dispersion adsorbent, vortex vibrating for 5min, centrifuging at 6000r/min for 3min, sucking supernatant, filtering with 0.22 μm organic phase filter membrane, and analyzing on machine;
3) and (3) detecting the residual amount of the dipterex: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography-negative chemical source-mass spectrometry combined method, and measuring the residual amount of the dipterex by combining the standard curve obtained in the step (1).
The chromatographic conditions of the gas chromatography-negative chemical source mass spectrometry combined method are that a chromatographic column: DB-5MS (30 m 0.25mm 0.25 u m). The analysis conditions of the mass spectrometer are as follows: negative chemical ion source (NCI) using a Selected Ion (SIM) mode, the characteristic ions being m/z 278 and m/z 279, wherein m/z 278 is the quantitative ion.
The purity of the dipterex standard substance is 98 percent; the methanol, the acetonitrile and the formic acid are HPLC grades; 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 and the disodium hydrogen citrate are analytically pure.
The detection method further comprises an adding and recovering experiment, wherein the adding and recovering experiment is to add and recover a tobacco blank sample, the dipterex standard stock solution is used for the adding and recovering experiment, specifically, the adding and recovering experiment is respectively carried out at the adding levels of 0.05, 0.1 and 0.5mg/kg 3, 6 parallel samples are set for each adding concentration, then, the sample pretreatment and detection are carried out, and the result proves that the recovery rate and the precision of the method are better.
Example 4
A method for detecting the residual quantity of dipterex in tobacco comprises the following steps:
1) preparation of a standard solution: accurately weighing 0.1g of dipterex standard substance to 0.1mg, dissolving with methanol to fix the volume to 100mL of brown volumetric flask, preparing 1mg/mL of standard stock solution, storing in a refrigerator at-20 ℃ in a dark place, and diluting the standard stock solution into a series of standard working solutions for making a standard curve;
the standard curve manufacturing method specifically comprises the steps of preparing a standard dipterex stock solution into standard dipterex working solutions with mass concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L series by using methanol, carrying out linear regression on each concentration by using peak areas to obtain a standard curve, wherein the standard curve is good in linearity within the mass concentration range of the dipterex of 0.01-0.5 mg/L, and the correlation coefficient r is2Greater than 0.99;
2) sample pretreatment:
extraction: weighing a sample to be detected into a centrifugal tube, adding water, oscillating until the sample is fully soaked by the water, and standing for 10 min; transferring 1% formic acid-acetonitrile with the same amount as water into a centrifuge tube, and oscillating at 2000r/min for 4 min; respectively adding anhydrous magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate into a centrifugal tube, wherein the addition amount is a sample to be detected: anhydrous magnesium sulfate: sodium chloride: sodium citrate: disodium hydrogen citrate =1:2:0.5:0.5:0.25, immediately oscillating for 3min at the speed of 2000 r/min; then centrifuging at 6000r/min for 4 min;
purifying: transferring the supernatant into a centrifuge tube of 1mL to 1.5mL, adding 100 mg C18 dispersion adsorbent, vortex vibrating for 3min, centrifuging at 5500 r/min for 4min, sucking the supernatant, filtering with a 0.22 μm organic phase filter membrane, and analyzing on a computer;
3) and (3) detecting the residual amount of the dipterex: and (3) detecting the sample treated in the step (2) by adopting a gas chromatography-negative chemical source-mass spectrometry combined method, and measuring the residual amount of the dipterex by combining the standard curve obtained in the step (1).
The chromatographic conditions of the gas chromatography-negative chemical source mass spectrometry combined method are that a chromatographic column: DB-5MS (30 m 0.25mm 0.25 u m). The analysis conditions of the mass spectrometer are as follows: negative chemical ion source (NCI) using a Selected Ion (SIM) mode, the characteristic ions being m/z 278 and m/z 279, wherein m/z 278 is the quantitative ion.
The purity of the dipterex standard substance is 98 percent; the methanol, the acetonitrile and the formic acid are HPLC grades; 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 and the disodium hydrogen citrate are analytically pure.
The detection method further comprises an adding and recovering experiment, wherein the adding and recovering experiment is to add and recover a tobacco blank sample, the dipterex standard stock solution is used for the adding and recovering experiment, specifically, the adding and recovering experiment is respectively carried out at the adding levels of 0.05, 0.1 and 0.5mg/kg 3, 6 parallel samples are set for each adding concentration, then, the sample pretreatment and detection are carried out, and the result proves that the recovery rate and the precision of the method are better.
Claims (7)
1. A method for detecting the residual quantity of dipterex in tobacco is characterized by comprising the following steps:
1) preparation of a standard solution: accurately weighing a dinocap standard substance, dissolving and fixing the dinocap standard substance to a brown volumetric flask to prepare a standard stock solution of 1mg/mL, storing the standard stock solution in a refrigerator at the temperature of minus 15 to minus 25 ℃ in a dark place, and diluting the standard stock solution into a series of standard working solutions for making a standard curve;
2) sample pretreatment:
extraction: weighing a sample to be detected into a centrifugal tube, adding water, oscillating until the sample is fully soaked by the water, and standing for 8-12 min; transferring 1% formic acid-acetonitrile with the same amount as water into a centrifugal tube, and oscillating at the speed of 1500-2500 r/min for 2-5 min; respectively adding anhydrous magnesium sulfate, sodium chloride, sodium citrate and disodium hydrogen citrate into a centrifugal tube, wherein the addition amount of the anhydrous magnesium sulfate, the sodium chloride, the sodium citrate and the disodium hydrogen citrate is that the ratio of the anhydrous magnesium sulfate to the sodium citrate to the disodium hydrogen citrate is =1:2:0.5:0.5:0.25, and immediately oscillating at the speed of 1500-2500 r/min for 2-5 min; then centrifuging for 3-5 min at 5000-7000 r/min;
purifying: transferring 1mL to 1.5mL of supernatant into a centrifuge tube, adding 50-200 mg of C18 dispersion adsorbent, carrying out vortex oscillation for 2-5 min, centrifuging for 3-5 min at 5000-6000 r/min, sucking the supernatant, filtering the supernatant through a 0.22-micron organic phase filter membrane, and carrying out machine analysis;
3) and (3) detecting the residual amount of the dipterex: detecting the sample treated in the step 2) by adopting a gas chromatography-negative chemical source-mass spectrometry combined method, and measuring the residual amount of the dipterex by combining the standard curve obtained in the step 1), wherein the detection limit of the residual amount of the dipterex is 0.02mg/kg, and the quantification limit is 0.07 mg/kg; the chromatographic column of the gas chromatography is DB-5MS with the size of 30m multiplied by 0.25mm multiplied by 0.25 mu m, the sample injection amount is 1 mu L, the split ratio is 20:1, the sample injection port is 250 ℃, the temperature rising program is as follows: keeping at 100 deg.C for 2min, heating to 280 deg.C at 10 deg.C/min, and keeping for 6 min; the carrier gas is helium and is 1.0 mL/min; the analysis conditions of the mass spectrometer are as follows: the negative chemical ion source NCI adopts a selective ion SIM mode, and the characteristic ions are m/z 278 and m/z 279, wherein m/z 278 is quantitative ions.
2. The method for detecting the residual amount of the dipterex in the tobacco as claimed in claim 1, wherein the preparation method of the standard stock solution in the step 1) comprises the steps of accurately weighing 0.1g of a dipterex standard substance to 0.1mg, dissolving the dipterex standard substance with methanol to a constant volume of 100mL of a brown volumetric flask, and preparing the standard stock solution with the volume of 1 mg/mL.
3. The method for detecting the residual amount of the dinocap in the tobacco as claimed in claim 1, wherein the method for preparing the standard curve in the step 1) specifically comprises the steps of preparing a standard stock solution of the dinocap with methanol to obtain standard working solutions of the dinocap with the mass concentrations of 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5mg/L, and performing linear regression on the concentrations by using peak areas to obtain the standard curve, wherein the standard curve is good in linearity within the mass concentration range of the dinocap of 0.01-0.5 mg/L, and the correlation coefficient r is2Greater than 0.99.
4. The method for detecting the residual amount of the dinocap in the tobacco as claimed in claim 1, wherein the method further comprises an addition recovery experiment, the addition recovery experiment is to perform addition recovery by using a tobacco blank sample, a standard stock solution of the dinocap is used for the addition recovery experiment, specifically, a standard addition recovery experiment is performed at the addition levels of 0.05, 0.1 and 0.5mg/kg 3 respectively, each addition concentration is provided with 6 parallel samples, then sample pretreatment and detection are performed, the recovery rate of the dinocap in the tobacco is 95.83% -102.34%, and the relative standard deviation is 3.75% -5.30%.
5. The method for detecting the residual amount of the dipterex in the tobacco as claimed in claim 1, wherein the extraction in the step 2) is specifically to weigh 2g of a sample to be detected, the sample is accurate to 0.01g, 10mL of water is added into a 50mL centrifuge tube with a cover, and the sample is shaken for 10min after being sufficiently soaked by the water; transferring 10mL of 1% formic acid-acetonitrile into a centrifuge tube, and placing the centrifuge tube on a vortex mixing and oscillating instrument to oscillate for 2-5 min at the speed of 2000 r/min; 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 respectively, immediately oscillating on a vortex mixing and oscillating instrument at the speed of 2000r/min for 2-5 min, and then centrifuging at 6000r/min for 3-5 min.
6. The method for detecting the residual amount of the dipterex in the tobacco as claimed in claim 1, wherein the purification in the step 2) is specifically to transfer the supernatant into a centrifuge tube with the volume of 1mL to 1.5mL, add 50mg of C18 dispersed adsorbent, carry out vortex oscillation for 2min, centrifuge for 3min at 6000r/min, absorb the supernatant, pass through a 0.22 μm organic phase filter membrane, and then carry out machine analysis.
7. The method for detecting the residual amount of the dinocap in the tobacco as claimed in claim 1, wherein the purity of the dinocap standard substance is 98%; methanol, acetonitrile and formic acid 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 and the disodium hydrogen citrate are analytically pure.
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