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Abstract Imidaclothiz residue in tea was determined by high performance liquid chromatography （HPLC）， using different extractants including hexane， acetonitrile， acetone， hexane-acetonitrile （1∶1）， acetonitrile-acetone （1∶1） and hexane-ethyl acetate （1∶1）. The results indicated that all the extractants displayed excellent extraction capacities of imidaclothiz residue in tea and insignificant interference on the accuracy. Under the optimized extraction volume （20 ml）， the recovery values were in the ranges of 82.3%-100.7%， 85.8%-101.1%， 85.9%-95.2%， 82.3%-101.3%， 82.4%-100.5% and 88.6%-102.3%， respectively， with the relative standard deviations smaller than 10%. Moreover， mixed solvent showed improved extraction capacity， and the highest extraction capacity and promoted stability were approached by using hexane-ethyl acetate （1∶1） as coextractants.
Key words Imidaclothiz； HPLC； Tea； Extractant
Imidaclothiz is a kind of nitromethylene-containing nicotinic insecticide self-developed by China Agrochemical Technology Company[1]， which has very good effect in control of pests including orange caterpillar， Cruciferae vegetable aphids， rice planthoppers， greenhouse whitefly and tea leafhopper. At present， neonicotinoid insecticides acutely poison bees， and might bring unacceptable effect to survival and development of bee population[2]. The European Union has limited the application of partial neonicotinoid insecticides. Currently， imidaclothiz is determined by high performance liquid chromatography[3-6]， high performance liquid chromatography-tandem mass spectrometry HPLC-MS/MS[7-8]， and gas chromatography-tandem mass spectrometry （GC-MS/MS）[9]. During the determination， the pretreatment part often requires solid-phase extraction， and for extractants， only several single extractants have been compared[10-12]. In recent years， QuEChERS[13-14] has been widely applied in agrochemical field， but the cost is also relatively higher. In this study， the pretreatment of imidaclothiz determination in tea was subjected to optimization， during which three pure reagents， n-hexane， acetonitrile and acetone， and three mixed reagents， n-hexane-acetonitrile （volume ratio at 1∶1）， acetonitrile-acetone （1∶1） and n-hexane-ethyl acetate （1∶1）， were used as extractants， and the extraction capacities of the extractants to imidaclothiz in tea were compared from extraction time， extraction volume and extraction recovery， with an attempt to select a kind of extractant with high extraction efficiency and less impurity interference. Materials and Methods
Instruments
Ultra performance liquid chromatograph： L-30A type， Shimadzu； pressure blowing concentrator： UGC-24C series， Beijing Yousheng United Technology Co.， Ltd.； ultrasonic apparatus： KQ-300DB series， Kunshan Instrument Co.， Ltd.； electronic scale： BT125D series， Sartorius Stedim Biotech GmbH； centrifuge： LD4-2D series， Beijing Jingli Centrifuge Co.， Ltd.； vortex mixer： Vortex Genie 2 series， Shanghai P&Q Science Co.， Ltd.； ultrapure water： Ultra-pure Wster Sytem series， Hunan Kertone Water Co.， Ltd.
Experimental materials
Methanol， acetonitrile： chromatographically pure， EMerck （Germany）； n-hexane， ethyl acetate： chromatographically pure， ANPEL Laboratory Technologies （Shanghai） Inc.； acetone： chromatographically pure， Shanghai Aladdin Bio-chem Technology Co.， Ltd.； phosphoric acid： analytically pure， Sinopharm Chemical Reagent Co.， Ltd.； laboratory water： 18.25 MΩ ultrapure water， prepared by ultrapure water purifier.
Standards and preparation
Imidaclothiz standard： purity≥95.0%， ANPEL Laboratory Technologies （Shanghai） Inc..
Preparation of standard solutions： At first， 10.0 mg of standard was accurately added into a 10 ml volumetric flask， and dissolved and diluted with acetonitrile to constant volume， obtaining 1.0 mg/ml stock solution. Then， different volumes of the stock solution were pipetted and diluted with acetonitrile to 0.05， 0.10， 0.20， 0.50 and 0.80 μg/ml standard solutions.
Chromatographic conditions
Chromatographic column： Agilent C18 （5 mm×25 mm×0.45 μm）； mobile phase： methanol+ acetonitrile+0.1% phosphoric acid solution=10∶25∶65 （volume ratio）； detection wavelength： 270 nm； column temperature： 35 ℃； injection volume： 10 μl.
Sample treatment
At first， 1.00 g of sample was accurately weighed and added with 20 ml n-hexane-ethyl acetate （1∶1）. After vortex-mixing for 1 min， ultrasonic treatment was performed for 20 min and centrifuged at 4 000 r/min for 5 min. Then， 10 ml of the supernatant was accurately pipetted into a colorimetric tube， and blew to dry with nitrogen gas in a water bath at 40 ℃， and the residue was dissolved and diluted with acetonitrile to 1 ml. The solution was filtered with 0.22 μm filter membrane， obtaining filtrate for HPLC determination.
Results and Analysis
Optimization of chromatographic conditions
Selection of mobile phase
The peak shape and separation effect was compared among methanol+water （volume ratio at 25∶75）， acetonitrile+water （35∶65）， methanol+ acetonitrile+0.1% phosphorus water solution （10∶25∶65）. The results showed that the peak shape of imidaclothiz was the best with methanol+ acetonitrile+0.1% phosphorus water solution as mobile phase， and the impurity separation effect was better during sample determination. Finally， methanol+ acetonitrile+0.1% phosphorus water solution was selected as mobile phase， and the chromatogram of imidaclothiz is shown in Fig. 1. Selection of detection wavelength
The spectrum of imidaclothiz standard solution was analyzed with DAD diode array detector. It had the largest absorption at 270 nm， with less interference from impurity peaks. The peak areas at multiple wavelengths around 270 nm were compared （Table 1）， and the results showed that the peak area of imidaclothiz at 270 nm was the highest， and the spectrum of imidaclothiz standard is shown in Fig. 2.
Effect of extraction time
Multiple parts of tea samples with equal mass were added with the same standard solution， and extracted with the same volume of extractant for 5， 10， 15， 20， 25 and 30 min， respectively， and the determined recovery values are shown in Fig. 3. The results showed that when the ultrasonic time was longer than 15 min， the extraction effect was improved significantly， but more impurities were extracted over time， and the color of solution also changed very remarkably. Comprehensively considering from extraction effect and impurity interference， 20 min was selected as the optimal extraction time.
Effect of extraction volume
Multiple parts of tea blank sample were weighed and added with 1 mg/kg imidaclothiz standard solution， and 5， 10， 15， 20， 25 and 30 ml of extractant were added， respectively. The recovery results are shown in Fig. 4. It could be seen from Fig. 4 that with the increase of volume of extractant， the recovery increased faster， and reached its maximum value at 20 ml， and when the volume of extractant exceeded 20 ml， the recovery changed little with a decreasing trend. Therefore， 20 ml was selected as the extraction volume.
Sample determination
Green tea was selected as test sample for determination of sample blank and spiked sample. The samples were pre-treated according to "Effect of extraction volume" and "Sample determination"， and then determined with corresponding instrument. It could be seen from Fig. 5 that various extractants differed greatly in extraction effect and impurity interference， and among them， n-hexane-ethyl acetate （volume ratio at 1∶1） extracted the least impurities， exhibiting the most ideal extraction effect.
Recovery and precision
A certain amount of tea sample was weighed， and added with standard solutions at different concentrations， respectively， and the mixtures were prepared into spiked samples with concentrations of 0.5， 1.0， 1.5， 2.0， 3.0， 5.0 and 10.0 mg/kg， respectively.
Three parts of each sample were weighed and extracted with n-hexane， acetonitrile， acetone， n-hexane-acetonitrile （volume ratio at 1∶1）， acetonitrile-acetone （volume ratio at 1∶1）， and n-hexane-ethyl acetate （volume ratio at 1∶1）， respectively， determination was performed according to "Selection of detection wavelength"， and the recovery of each spiked sample was calculated， as shown in Table 3. It could be seen from Table 3 that the relative standard derivation of the recovery values of the three parts of sample extracted with each extractant was smaller than 10%； and the recovery values of various extractants were in the range of 80%-110%， and the average recovery obtained with n-hexane-ethyl acetate was the highest. Conclusions
In this study， the extraction capacities of n-hexane， acetonitrile， acetone， n-hexane-acetonitrile （1∶1）， acetonitrile-acetone （1∶1） and n-hexane-ethyl acetate （1∶1） to imidaclothiz in tea. Imidaclothiz was determined at 270 nm with methanol+acetonitrile+0.1% phosphoric acid as extractant， and after comparison of extraction time and extraction volume， 20 ml of extractant and 20 min of ultrasonic extraction were selected. The results showed that the six extractants all had better response to imidaclothiz， and suffered from less impurity interference， and the average recovery values were in the ranges of 82.3%-100.7%， 85.8%-101.1%， 85.9%-95.2%， 82.3%-101.3%， 82.4%-100.5% and 88.6%-102.3%， respectively. Therefore， the six extractants all had better extraction capacities to imidaclothiz， reagent combinations have better extraction capacities， and n-hexane-ethyl acetate （volume ratio at 1∶1） has stronger extraction capacity and better stability.
References
[1] WANG YK， WEN BY， WANG QL. Pesticide toxicity broad view[M]. Jinan： Shandong Technology Press， 1986： 3-4.
[2] DUAN LF， LI XB， KE CJ， et al. Risk Evaluation of implications of EUs policy on the restriction of use of neonicotinoids to domestic pesticide industry[J]. Pesticide Science and Management.， 2013， 34（9）： 15-20.
[3] HOU RY， BIAN HZ， ZHAO XX， et al. Determination of nicotinoid residues in complicated matrix vegetables by solid phase extraction and HPLC method[J]. Journal of Instrumental Analysis， 2011， 30（1）： 58-63.
[4] WANG ZM， LEI Y， LU XF. HPLC determination of imidaclothiz[J]. Journal of Nantong University： Natural Science Edition， 2005， 4（2）： 23-24.
[5] ZHANG LF. Research on residue analysis method of imidaclothiz in cabbage[J]. Journal of Hebei North University： Natural Science， 2004（2）： 60-63.
[6] HE M， JIA CH， YU PZ， et al. Residue Analysis of Imidaclothiz in Rice by HPLC[J]. Agrochemicals， 2009， 48（4）： 285-286.
[7] LIU SN， ZHAO XY， DONG XQ， et al. Determination of imidaclothiz in tea by QuEChERS cleanup and liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography， 2015， 33（11）： 1205-1209.
[8] LU XL， YE MJ， ZHOU WL. Determination of imidaclothiz residue in tea by ultra-performance liquid chromatography tandem mass spectrometry[J]. Agrochemicals， 2014（11）： 825-828.
[9] GAO ZX， WU YH， AO KH， et al. Determination of metalaxyl and imidaclothiz residues in fruits by RP-HPLC with solid-phase extraction [J]. Journal of Environment and Health， 2012， 29（3）： 258-259.
[10] ZHONG LL， HU L， LEI SR， et al. Determination of six insecticide residues in tea by HPLC-MS/MS[J]. Food Safety and Quality Detection Technology， 2016， 7（5）： 2081-2086.
[11] WU XG， CHEN XQ， XIAO HJ， et al. Determination of eight insecticide residues in tea using ultra performance liquid chromatography-electrospray tandem mass spectrometry[J]. Chinese Journal of Pesticide Science， 2013， 15（6）： 655-660.
[12] LOU ZY， TANG FB， CHEN ZM， et al. Determination of imidaclothiz residue in tea by HPLC[J]. Chinese Journal of Analysis Laboratory， 2009， 28（b05）： 76-78.
[13] SUN ZG， SHENG R， HAO JM， et al. Detected chlorpyrifos， imidacloprid， and propoxur residues in citrus by QuEChERS-high performance liquid chromatography method[J]. Food & Machinery， 2017， 33（6）： 68-72.
[14] CAO H， ZHENG JK， HUANG LY， et al. Fast determination of glyphosate and aminomethyl phosphonic acid residues in tea by QuEChERS-UPLC/MS/MS[J]. Food & Machinery， 2016， 32（4）： 84-87.
Key words Imidaclothiz； HPLC； Tea； Extractant
Imidaclothiz is a kind of nitromethylene-containing nicotinic insecticide self-developed by China Agrochemical Technology Company[1]， which has very good effect in control of pests including orange caterpillar， Cruciferae vegetable aphids， rice planthoppers， greenhouse whitefly and tea leafhopper. At present， neonicotinoid insecticides acutely poison bees， and might bring unacceptable effect to survival and development of bee population[2]. The European Union has limited the application of partial neonicotinoid insecticides. Currently， imidaclothiz is determined by high performance liquid chromatography[3-6]， high performance liquid chromatography-tandem mass spectrometry HPLC-MS/MS[7-8]， and gas chromatography-tandem mass spectrometry （GC-MS/MS）[9]. During the determination， the pretreatment part often requires solid-phase extraction， and for extractants， only several single extractants have been compared[10-12]. In recent years， QuEChERS[13-14] has been widely applied in agrochemical field， but the cost is also relatively higher. In this study， the pretreatment of imidaclothiz determination in tea was subjected to optimization， during which three pure reagents， n-hexane， acetonitrile and acetone， and three mixed reagents， n-hexane-acetonitrile （volume ratio at 1∶1）， acetonitrile-acetone （1∶1） and n-hexane-ethyl acetate （1∶1）， were used as extractants， and the extraction capacities of the extractants to imidaclothiz in tea were compared from extraction time， extraction volume and extraction recovery， with an attempt to select a kind of extractant with high extraction efficiency and less impurity interference. Materials and Methods
Instruments
Ultra performance liquid chromatograph： L-30A type， Shimadzu； pressure blowing concentrator： UGC-24C series， Beijing Yousheng United Technology Co.， Ltd.； ultrasonic apparatus： KQ-300DB series， Kunshan Instrument Co.， Ltd.； electronic scale： BT125D series， Sartorius Stedim Biotech GmbH； centrifuge： LD4-2D series， Beijing Jingli Centrifuge Co.， Ltd.； vortex mixer： Vortex Genie 2 series， Shanghai P&Q Science Co.， Ltd.； ultrapure water： Ultra-pure Wster Sytem series， Hunan Kertone Water Co.， Ltd.
Experimental materials
Methanol， acetonitrile： chromatographically pure， EMerck （Germany）； n-hexane， ethyl acetate： chromatographically pure， ANPEL Laboratory Technologies （Shanghai） Inc.； acetone： chromatographically pure， Shanghai Aladdin Bio-chem Technology Co.， Ltd.； phosphoric acid： analytically pure， Sinopharm Chemical Reagent Co.， Ltd.； laboratory water： 18.25 MΩ ultrapure water， prepared by ultrapure water purifier.
Standards and preparation
Imidaclothiz standard： purity≥95.0%， ANPEL Laboratory Technologies （Shanghai） Inc..
Preparation of standard solutions： At first， 10.0 mg of standard was accurately added into a 10 ml volumetric flask， and dissolved and diluted with acetonitrile to constant volume， obtaining 1.0 mg/ml stock solution. Then， different volumes of the stock solution were pipetted and diluted with acetonitrile to 0.05， 0.10， 0.20， 0.50 and 0.80 μg/ml standard solutions.
Chromatographic conditions
Chromatographic column： Agilent C18 （5 mm×25 mm×0.45 μm）； mobile phase： methanol+ acetonitrile+0.1% phosphoric acid solution=10∶25∶65 （volume ratio）； detection wavelength： 270 nm； column temperature： 35 ℃； injection volume： 10 μl.
Sample treatment
At first， 1.00 g of sample was accurately weighed and added with 20 ml n-hexane-ethyl acetate （1∶1）. After vortex-mixing for 1 min， ultrasonic treatment was performed for 20 min and centrifuged at 4 000 r/min for 5 min. Then， 10 ml of the supernatant was accurately pipetted into a colorimetric tube， and blew to dry with nitrogen gas in a water bath at 40 ℃， and the residue was dissolved and diluted with acetonitrile to 1 ml. The solution was filtered with 0.22 μm filter membrane， obtaining filtrate for HPLC determination.
Results and Analysis
Optimization of chromatographic conditions
Selection of mobile phase
The peak shape and separation effect was compared among methanol+water （volume ratio at 25∶75）， acetonitrile+water （35∶65）， methanol+ acetonitrile+0.1% phosphorus water solution （10∶25∶65）. The results showed that the peak shape of imidaclothiz was the best with methanol+ acetonitrile+0.1% phosphorus water solution as mobile phase， and the impurity separation effect was better during sample determination. Finally， methanol+ acetonitrile+0.1% phosphorus water solution was selected as mobile phase， and the chromatogram of imidaclothiz is shown in Fig. 1. Selection of detection wavelength
The spectrum of imidaclothiz standard solution was analyzed with DAD diode array detector. It had the largest absorption at 270 nm， with less interference from impurity peaks. The peak areas at multiple wavelengths around 270 nm were compared （Table 1）， and the results showed that the peak area of imidaclothiz at 270 nm was the highest， and the spectrum of imidaclothiz standard is shown in Fig. 2.
Effect of extraction time
Multiple parts of tea samples with equal mass were added with the same standard solution， and extracted with the same volume of extractant for 5， 10， 15， 20， 25 and 30 min， respectively， and the determined recovery values are shown in Fig. 3. The results showed that when the ultrasonic time was longer than 15 min， the extraction effect was improved significantly， but more impurities were extracted over time， and the color of solution also changed very remarkably. Comprehensively considering from extraction effect and impurity interference， 20 min was selected as the optimal extraction time.
Effect of extraction volume
Multiple parts of tea blank sample were weighed and added with 1 mg/kg imidaclothiz standard solution， and 5， 10， 15， 20， 25 and 30 ml of extractant were added， respectively. The recovery results are shown in Fig. 4. It could be seen from Fig. 4 that with the increase of volume of extractant， the recovery increased faster， and reached its maximum value at 20 ml， and when the volume of extractant exceeded 20 ml， the recovery changed little with a decreasing trend. Therefore， 20 ml was selected as the extraction volume.
Sample determination
Green tea was selected as test sample for determination of sample blank and spiked sample. The samples were pre-treated according to "Effect of extraction volume" and "Sample determination"， and then determined with corresponding instrument. It could be seen from Fig. 5 that various extractants differed greatly in extraction effect and impurity interference， and among them， n-hexane-ethyl acetate （volume ratio at 1∶1） extracted the least impurities， exhibiting the most ideal extraction effect.
Recovery and precision
A certain amount of tea sample was weighed， and added with standard solutions at different concentrations， respectively， and the mixtures were prepared into spiked samples with concentrations of 0.5， 1.0， 1.5， 2.0， 3.0， 5.0 and 10.0 mg/kg， respectively.
Three parts of each sample were weighed and extracted with n-hexane， acetonitrile， acetone， n-hexane-acetonitrile （volume ratio at 1∶1）， acetonitrile-acetone （volume ratio at 1∶1）， and n-hexane-ethyl acetate （volume ratio at 1∶1）， respectively， determination was performed according to "Selection of detection wavelength"， and the recovery of each spiked sample was calculated， as shown in Table 3. It could be seen from Table 3 that the relative standard derivation of the recovery values of the three parts of sample extracted with each extractant was smaller than 10%； and the recovery values of various extractants were in the range of 80%-110%， and the average recovery obtained with n-hexane-ethyl acetate was the highest. Conclusions
In this study， the extraction capacities of n-hexane， acetonitrile， acetone， n-hexane-acetonitrile （1∶1）， acetonitrile-acetone （1∶1） and n-hexane-ethyl acetate （1∶1） to imidaclothiz in tea. Imidaclothiz was determined at 270 nm with methanol+acetonitrile+0.1% phosphoric acid as extractant， and after comparison of extraction time and extraction volume， 20 ml of extractant and 20 min of ultrasonic extraction were selected. The results showed that the six extractants all had better response to imidaclothiz， and suffered from less impurity interference， and the average recovery values were in the ranges of 82.3%-100.7%， 85.8%-101.1%， 85.9%-95.2%， 82.3%-101.3%， 82.4%-100.5% and 88.6%-102.3%， respectively. Therefore， the six extractants all had better extraction capacities to imidaclothiz， reagent combinations have better extraction capacities， and n-hexane-ethyl acetate （volume ratio at 1∶1） has stronger extraction capacity and better stability.
References
[1] WANG YK， WEN BY， WANG QL. Pesticide toxicity broad view[M]. Jinan： Shandong Technology Press， 1986： 3-4.
[2] DUAN LF， LI XB， KE CJ， et al. Risk Evaluation of implications of EUs policy on the restriction of use of neonicotinoids to domestic pesticide industry[J]. Pesticide Science and Management.， 2013， 34（9）： 15-20.
[3] HOU RY， BIAN HZ， ZHAO XX， et al. Determination of nicotinoid residues in complicated matrix vegetables by solid phase extraction and HPLC method[J]. Journal of Instrumental Analysis， 2011， 30（1）： 58-63.
[4] WANG ZM， LEI Y， LU XF. HPLC determination of imidaclothiz[J]. Journal of Nantong University： Natural Science Edition， 2005， 4（2）： 23-24.
[5] ZHANG LF. Research on residue analysis method of imidaclothiz in cabbage[J]. Journal of Hebei North University： Natural Science， 2004（2）： 60-63.
[6] HE M， JIA CH， YU PZ， et al. Residue Analysis of Imidaclothiz in Rice by HPLC[J]. Agrochemicals， 2009， 48（4）： 285-286.
[7] LIU SN， ZHAO XY， DONG XQ， et al. Determination of imidaclothiz in tea by QuEChERS cleanup and liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography， 2015， 33（11）： 1205-1209.
[8] LU XL， YE MJ， ZHOU WL. Determination of imidaclothiz residue in tea by ultra-performance liquid chromatography tandem mass spectrometry[J]. Agrochemicals， 2014（11）： 825-828.
[9] GAO ZX， WU YH， AO KH， et al. Determination of metalaxyl and imidaclothiz residues in fruits by RP-HPLC with solid-phase extraction [J]. Journal of Environment and Health， 2012， 29（3）： 258-259.
[10] ZHONG LL， HU L， LEI SR， et al. Determination of six insecticide residues in tea by HPLC-MS/MS[J]. Food Safety and Quality Detection Technology， 2016， 7（5）： 2081-2086.
[11] WU XG， CHEN XQ， XIAO HJ， et al. Determination of eight insecticide residues in tea using ultra performance liquid chromatography-electrospray tandem mass spectrometry[J]. Chinese Journal of Pesticide Science， 2013， 15（6）： 655-660.
[12] LOU ZY， TANG FB， CHEN ZM， et al. Determination of imidaclothiz residue in tea by HPLC[J]. Chinese Journal of Analysis Laboratory， 2009， 28（b05）： 76-78.
[13] SUN ZG， SHENG R， HAO JM， et al. Detected chlorpyrifos， imidacloprid， and propoxur residues in citrus by QuEChERS-high performance liquid chromatography method[J]. Food & Machinery， 2017， 33（6）： 68-72.
[14] CAO H， ZHENG JK， HUANG LY， et al. Fast determination of glyphosate and aminomethyl phosphonic acid residues in tea by QuEChERS-UPLC/MS/MS[J]. Food & Machinery， 2016， 32（4）： 84-87.