Method for detecting imidacloprid residues in tobacco

Abstract

The invention discloses a method for detecting imidacloprid residues in tobacco, which comprises the steps of pretreatment, extraction, purification and analysis, and is characterized in that a tobacco sample is dried, crushed and sieved after surface dust is brushed off, and the tobacco sample is dissolved by acetonitrile in an extraction stage to obtain a supernatant liquid. And air-drying the supernatant by using nitrogen, dissolving the supernatant by using acetonitrile, passing through an amino column, air-drying again, and fixing the volume by using acetonitrile to finish purification. The purified liquid is subjected to high performance liquid chromatography to detect the imidacloprid in the sample. According to the method for detecting imidacloprid residues in tobacco disclosed by the invention, few types of organic solvents are used in the process of extracting, purifying and analyzing imidacloprid, so that the detection cost is saved, the working intensity of analysts is reduced, and meanwhile, the pollution of the organic solvents to the environment is reduced; meanwhile, tests prove that the method disclosed by the invention is high in extraction rate of pesticide residue imidacloprid and good in data reproducibility, and the detection accuracy is improved.

Description

Technical Field

[0001] The present application relates to the technical field of pesticide residue detection, and in particular to a method for detecting imidacloprid pesticide residues in tobacco. Background Art

[0002] Imidacloprid is a highly effective broad-spectrum insecticide that selectively inhibits the nicotinic acid acetylcholinesterase receptors in the insect nervous system, thereby hindering the normal conduction of the insect central nervous system, causing the insect to paralyze and die. It has a significant efficacy, so it has always ranked first in the use of insecticides worldwide. Although imidacloprid has a good insecticidal effect, it also has certain chronic toxicological effects on humans and mammals. Imidacloprid at a concentration of 0.05 mg / kg can damage the genetic material DNA of human lymphocytes. With the continuous increase in the use of imidacloprid pesticides in tobacco cultivation in recent years, its residue has always received widespread attention. At the same time, tobacco is a widely used consumer product. Therefore, in order to protect the health of consumers, it is very important to invent a method for quickly and effectively extracting and detecting the residual amount of imidacloprid pesticide residues in tobacco. Summary of the invention

[0003] The invention discloses a method for detecting imidacloprid pesticide residue in tobacco quickly and effectively with low cost.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for detecting imidacloprid residues in tobacco, comprising:

[0006] Pretreatment: pretreatment of tobacco samples to obtain pretreatment products;

[0007] Extraction: vortex the pretreated product with water and acetonitrile, then add sodium chloride, let stand and centrifuge to obtain the extract;

[0008] Purification: The extracted product is purified by acetonitrile to obtain the solution to be analyzed;

[0009] Analysis: The imidacloprid peak area of ​​the liquid to be analyzed was obtained by high performance liquid chromatography using the external standard method peak area percentage calculation.

[0010] Furthermore, the preprocessing specifically includes:

[0011] Randomly select some tobacco samples;

[0012] Brushing away impurities on the surface of the tobacco sample, and extracting and discarding the main veins of the tobacco leaves in the tobacco sample;

[0013] The tobacco sample is dried, crushed and sieved to obtain a pretreated product.

[0014] Furthermore, the drying temperature is less than or equal to 40°C.

[0015] Further, the extracting includes:

[0016] Weigh a certain mass of the pre-treated product;

[0017] Add the pretreated product, water, and acetonitrile into a centrifuge tube;

[0018] After vortexing the centrifuge tube, add sodium chloride into the centrifuge tube;

[0019] The centrifuge tube was allowed to stand and then centrifuged to allow a lower layer of sediment and extract to appear in the centrifuge tube.

[0020] Further, the mass of the pre-treated product is weighed to be: 2 to 5 g;

[0021] The volume of the acetonitrile is: 15 to 20 mL;

[0022] The volume ratio of the water to the acetonitrile is 1:2 to 1:3.

[0023] Furthermore, the mass of the sodium chloride is 1 to 1.5 g.

[0024] Further, the purification comprises:

[0025] Extract a certain volume of extract;

[0026] The extract was blown dry by nitrogen, and the remaining solid was dissolved with acetonitrile-ethyl acetate solution;

[0027] The dissolved solution was passed through an acetonitrile pre-eluted amino column and the effluent was collected;

[0028] The effluent was blown dry with nitrogen, dissolved and fixed to volume with acetonitrile, and passed through an organic filter membrane to obtain the analysis solution.

[0029] Further, the extraction volume of the extract is 3 to 5 mL;

[0030] The acetonitrile-ethyl acetate solution is 5 to 8 ml, and the volume ratio of acetonitrile to ethyl acetate is 3:1 to 1:1;

[0031] The effluent was dried with nitrogen, and the dried product was dissolved in acetonitrile and fixed to 1 mL.

[0032] Furthermore, the organic filter membrane is a 0.45 μm organic filter membrane.

[0033] Further, a high performance liquid chromatograph equipped with an ultraviolet detector and a fluorescence detector is selected;

[0034] Adopt 4.6mm×250mm MP C 18 Reversed phase chromatography columns;

[0035] Detection wavelength 270nm; flow rate 0.5 to 1mL / min; column temperature 25 to 30℃; analysis solution injection volume 10 to 20μL;

[0036] Gradient elution was used, with mobile phase A being methanol and mobile phase B being water, and the running time being 30 to 40 min; the gradient elution program was:

[0037] A:B = 10:90, maintain for 7 to 10 minutes;

[0038] A: B = 60:40, maintain for 13 to 15 minutes;

[0039] A:B=90:10, maintain for 10 to 15 minutes.

[0040] The beneficial effects of the present invention are:

[0041] The tobacco samples are extracted by vortex oscillation. The instrument is easy to operate and the vortex extraction time is short, which improves the efficiency of analysis and detection, shortens the extraction operation time, is conducive to batch and rapid processing of tobacco samples, and is suitable for tobacco detection operations with large sample volumes.

[0042] Less organic solvents are used in the extraction, purification and analysis of imidacloprid, which saves testing costs, reduces the workload of analysts, and reduces the pollution of organic solvents to the environment.

[0043] The method of the present invention has a high extraction rate for the pesticide residue imidacloprid, good data reproducibility, and improves the accuracy of detection;

[0044] The experimental process has relatively low requirements on experimental conditions, and general analysis and detection laboratories can meet the extraction and detection conditions, so the present invention has the characteristics of universal application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the liquid chromatogram of the 5 mg / L imidacloprid standard sample solution;

[0046] Figure 2 It is the liquid chromatogram of the sample extract measurement;

[0047] Figure 3 yes Figure 2 Liquid chromatogram of homologous samples spiked with 100 mg / L. DETAILED DESCRIPTION

[0048] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0049] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0050] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0051] The method for detecting imidacloprid residues in tobacco disclosed by the present invention comprises the following steps:

[0052] Pretreatment: pretreatment of tobacco samples to obtain pretreatment products;

[0053] Extraction: vortex the pretreated product with water and acetonitrile, then add sodium chloride, let stand and centrifuge to obtain the extract;

[0054] Purification: The extracted product is purified by acetonitrile to obtain the solution to be analyzed;

[0055] Analysis: The solution to be analyzed was blown dry and dissolved in acetonitrile. The peak area of ​​imidacloprid was obtained by high performance liquid chromatography using the external standard method peak area percentage calculation.

[0056] In one feasible embodiment of the present invention, the water is distilled water. In other feasible embodiments of the present invention, the water may be deionized water or pure water.

[0057] The preprocessing process includes:

[0058] Randomly select some tobacco samples, brush off impurities on the surface of the tobacco samples, extract and discard the main veins of tobacco leaves in the tobacco samples; dry the tobacco samples, crush and sieve them to obtain pretreated products.

[0059] In this embodiment, the impurities on the surface of the tobacco sample are preferably brushed off with a soft-bristled brush, the purpose of which is to brush off the fine soil and sand on the surface of the tobacco leaves without damaging the surface of the tobacco leaves, so as to avoid damaging the surface of the tobacco leaves and reducing the amount of imidacloprid residue. The main vein of the tobacco leaves will occupy the volume of the tobacco sample after drying in subsequent tests, and the main vein of the tobacco is often removed in the subsequent processing of tobacco products, so the main vein of the tobacco leaves is extracted to reduce interference with the detection.

[0060] After brushing off impurities and extracting the main vein of the tobacco, the tobacco sample is dried at a temperature not higher than 40° C., and the dried product is crushed and sieved to obtain a pretreated product.

[0061] The extraction comprises:

[0062] Weigh a certain mass of the pre-treated product;

[0063] Add the pretreated product, water and acetonitrile into a centrifuge tube; wherein the volume ratio of water to acetonitrile is 1:2 to 1:3;

[0064] After vortexing the centrifuge tube, add sodium chloride into the centrifuge tube;

[0065] The centrifuge tube was allowed to stand and then centrifuged to allow a lower layer of sediment and extract to appear in the centrifuge tube.

[0066] In a feasible embodiment of the present invention, 1-1.5g of pre-treated product, 5-10ml of distilled water, and 15-20ml of acetonitrile are placed in a centrifuge tube, and the centrifuge tube cover is tightened. The centrifuge tube is vortexed for 2-4min to fully mix the materials in the centrifuge tube, and 1-1.5g of sodium chloride is added after shaking and shaken again. After the centrifuge tube is left to stand for 5-10min, it is centrifuged for 5-10min at 3000-4000r / min. After the above operation, a lower precipitate and an upper clear liquid are produced in the centrifuge tube, wherein the upper clear liquid is an extract, and the extract is made from dried tobacco leaves, and part of the organic matter and imidacloprid in the tobacco leaves are dissolved in an acetonitrile-water solution.

[0067] The purification comprises:

[0068] Extract a certain volume of extract;

[0069] The extract was blown dry by nitrogen, and the remaining solid was dissolved with acetonitrile-ethyl acetate solution;

[0070] The dissolved solution was passed through an acetonitrile pre-eluted amino column and the effluent was collected;

[0071] The effluent was blown dry with nitrogen, dissolved and fixed to volume with acetonitrile, and passed through an organic filter membrane to obtain the analysis solution.

[0072] In a feasible embodiment of the present invention, the volume of the extract obtained is 3 to 5 mL. After the extract is dried with nitrogen, the remaining solid is dissolved with a solution having an acetonitrile:ethyl acetate volume ratio ranging from 3:1 to 1:1, and the volume of the solution used is 5 to 8 mL.

[0073] Take the amino column and elute it with 5 mL of acetonitrile. When the amino column is almost dry, transfer the above solution to the amino column, collect the effluent, and blow dry it with nitrogen. Collect the product after drying, dissolve it with acetonitrile and make it to 1 mL. After making it to volume, filter the liquid through a 0.45 μm organic filter membrane to obtain the analysis solution.

[0074] After the above extraction and purification, the analysis method is as follows:

[0075] Select a high performance liquid chromatograph equipped with an ultraviolet detector and a fluorescence detector;

[0076] 4.6mm×250mm MP C 18 Reversed phase chromatography columns;

[0077] Detection wavelength 270nm; flow rate 0.5 to 1mL / min; column temperature 25 to 30℃; analysis solution injection volume 10 to 20μL;

[0078] Gradient elution was used, with mobile phase A being methanol and mobile phase B being water, and the running time being 30 to 40 min; the gradient elution program was:

[0079] A:B = 10:90, maintain for 7 to 10 minutes;

[0080] A: B = 60:40, maintain for 13 to 15 minutes;

[0081] A:B=90:10, maintain for 10 to 15 minutes.

[0082] The present invention detects imidacloprid in the analysis liquid by the following method:

[0083] A series of imidacloprid standard sample solutions with concentrations of 1.5, 2.5, 5, and 6 mg / L were prepared, and the samples were analyzed by high performance liquid chromatography. The measurement conditions were the same as those in the above embodiment. Specifically, the flow rate was 0.5 mL / min, the column temperature was 25°C, the injection volume was 20 μL, the mobile phase A was methanol, B was water, the running time was 30 min, and the elution program was: A: B = 10:90, maintained for 7 min; A: B = 60:40, maintained for 13 min; A: B = 90:10, maintained for 10 min. The sample concentration was calculated by the external standard method using the peak area percentage, and the liquid chromatogram of the 5 mg / L imidacloprid standard solution was obtained as shown in the figure below: Figure 1 As shown, by measuring the imidacloprid standard curve of the series of standard solutions, Area=203.3827*c-17710, wherein Area is the peak area of ​​imidacloprid, c is the imidacloprid concentration, and the correlation coefficient r=0.99998, the standard curve has good linearity and can meet the analysis needs.

[0084] Tobacco samples were randomly selected as test objects, and the samples and spiked samples were measured three times in parallel according to the following treatment, and the spiked recovery rate was calculated. Among them, the spiked samples were the same as the samples except that 0.5 mL of 100 mg / L imidacloprid standard solution was added after weighing. In addition to the spiked samples and samples, a blank control test was also performed.

[0085] Processing method:

[0086] Randomly select a portion of tobacco samples, use a soft brush to brush off the fine soil and gravel on the tobacco leaves; remove the main veins, dry in a 40℃ oven, take out the dried product, crush and sieve it, and store the sieved powder in a dry and clean wide-mouth bottle;

[0087] Take 2.5 g of dried tobacco sample and place it in a 50 mL centrifuge tube with a lid, add 10 mL of distilled water and shake well, then add 20 mL of acetonitrile to the centrifuge tube, tighten the centrifuge tube lid, vortex and shake for 2 minutes, then add 1 g of sodium chloride and shake well, let it stand for 5 minutes, and then centrifuge it at 4000 r / min for 10 minutes.

[0088] Take 4 mL of the supernatant in the centrifuge tube and blow it dry under nitrogen. Dissolve the dry product with 5 mL of acetonitrile:ethyl acetate (volume ratio 3:1); take 5 mL of acetonitrile to elute the amino column. When the amino column is almost dry, transfer the dissolved solution to the amino column, collect the effluent, blow it dry with nitrogen again, dissolve the dry product with acetonitrile and make it to 1 mL. After passing through a 0.45 μm organic filter membrane, transfer it to a 1.5 mL brown injection bottle for HPLC analysis.

[0089] The preparation method of spiked samples was the same as the above treatment, with the only difference being that 2.5 g of dried tobacco sample was placed in a 50 mL centrifuge tube with a cap, 10 mL of distilled water was added and shaken, and 0.5 mL of 100 mg / L imidacloprid standard solution was additionally added, and the subsequent treatment was the same as the above treatment method.

[0090] After the HPLC analysis method and conditions of the standard sample solution as described above, the liquid chromatograms of the measured sample and the spiked sample were as follows: Figure 2 and Figure 3 As shown, the spike recovery rate was 85.51% and the standard deviation was 3.567, indicating that the analytical data of this method is accurate and can be used as a reference.

[0091] The tobacco samples are extracted by vortex oscillation. The instrument is easy to operate and the vortex extraction time is short, which improves the efficiency of analysis and detection, shortens the extraction operation time, is conducive to batch and rapid processing of tobacco samples, and is suitable for tobacco detection operations with large sample volumes.

[0092] The present invention uses fewer organic solvents in the pretreatment, extraction and purification and subsequent detection processes, and only uses three organic solvents, namely, acetonitrile, ethyl acetate and methanol, thereby saving detection costs, reducing the work intensity and operating costs of analysts, and reducing the pollution of the environment by organic solvents.

[0093] The method of the present invention has a high extraction rate for the pesticide residue imidacloprid, good data reproducibility, and improves the accuracy of detection;

[0094] The experimental process has relatively low requirements on experimental conditions, and general analysis and detection laboratories can meet the extraction and detection conditions, so the detection method disclosed in the present invention can be generally applicable.

[0095] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for detecting imidacloprid residues in tobacco, characterized in that: include: Pretreatment: pretreatment of tobacco samples to obtain pretreatment products; Extraction: vortex the pretreated product with water and acetonitrile, then add sodium chloride, let stand and centrifuge to obtain the extract; Purification: The extracted product is purified by acetonitrile to obtain the solution to be analyzed; Analysis: The imidacloprid peak area of ​​the liquid to be analyzed was obtained by high performance liquid chromatography using the external standard method peak area percentage calculation.

2. The method for detecting imidacloprid residues in tobacco according to claim 1, characterized in that: The pre-processing specifically includes: Randomly select some tobacco samples; Brushing away impurities on the surface of the tobacco sample, and extracting and discarding the main veins of the tobacco leaves in the tobacco sample; The tobacco sample is dried, crushed and sieved to obtain a pretreated product.

3. The method for detecting imidacloprid residues in tobacco according to claim 2, characterized in that: The drying temperature is less than or equal to 40°C.

4. The method for detecting imidacloprid residues in tobacco according to claim 1, characterized in that: The extraction comprises: Weigh a certain mass of the pre-treated product; Add the pretreated product, water, and acetonitrile into a centrifuge tube; After vortexing the centrifuge tube, add sodium chloride into the centrifuge tube; The centrifuge tube was allowed to stand and then centrifuged to allow a lower layer of sediment and extract to appear in the centrifuge tube.

5. The method for detecting imidacloprid residues in tobacco according to claim 4, characterized in that: Weigh the mass of the pre-treated product: 2 to 5 g; The volume of the acetonitrile is: 15 to 20 mL; The volume ratio of the water to the acetonitrile is 1:2 to 1:

3.

6. The method for detecting imidacloprid residues in tobacco according to claim 4, characterized in that: The mass of the sodium chloride is 1 to 1.5 g.

7. The method for detecting imidacloprid residues in tobacco according to claim 1, characterized in that: The purification comprises: Extract a certain volume of extract; The extract was blown dry by nitrogen, and the remaining solid was dissolved with acetonitrile-ethyl acetate solution; The dissolved solution was passed through an acetonitrile pre-eluted amino column and the effluent was collected; The effluent was blown dry with nitrogen, dissolved and fixed to volume with acetonitrile, and passed through an organic filter membrane to obtain the analysis solution.

8. The method for detecting imidacloprid residues in tobacco according to claim 7, characterized in that: The extraction volume of the extract is 3 to 5 mL; The acetonitrile-ethyl acetate solution is 5 to 8 ml, and the volume ratio of acetonitrile to ethyl acetate is 3:1 to 1:1; The effluent was dried with nitrogen, and the dried product was dissolved in acetonitrile and fixed to 1 mL.

9. The method for detecting imidacloprid residues in tobacco according to claim 7, characterized in that: The organic filter membrane is a 0.45 μm organic filter membrane.

10. The method for detecting imidacloprid residues in tobacco according to claim 1, characterized in that: Select a high performance liquid chromatograph equipped with an ultraviolet detector and a fluorescence detector; Adopt 4.6mm×250mm MP C 18 Reversed phase chromatography columns; Detection wavelength 270nm; flow rate 0.5 to 1mL / min; column temperature 25 to 30℃; analysis solution injection volume 10 to 20μL; Gradient elution was used, with mobile phase A being methanol and mobile phase B being water, and the running time being 30 to 40 min; the gradient elution program was: A:B = 10:90, maintain for 7 to 10 minutes; A: B = 60:40, maintain for 13 to 15 minutes; A:B=90:10, maintain for 10 to 15 minutes.

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