Determination of Etoxazole Residues in Fruits and Vegetables by SPE Clean

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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20 Determination of etoxazole residues in fruits and vegetables by SPE clean-up and HPLC-DAD Farag Malhat a , Hany Badawy b , Dalia Barakat b & Ayman Saber a a Pesticide Residues and Environmental Pollution Department, Agriculture Research Center, Dokki, Giza, Egypt b Pesticides & Economic Insect Department, Faculty of Agriculture, Cairo University, Cairo, Egypt

To cite this article: Farag Malhat , Hany Badawy , Dalia Barakat & Ayman Saber (2013): Determination of etoxazole residues in fruits and vegetables by SPE clean-up and HPLC-DAD, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 48:5, 331-335 To link to this article: http://dx.doi.org/10.1080/03601234.2013.742371

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FARAG MALHAT1, HANY BADAWY2, DALIA BARAKAT2 and AYMAN SABER1 1Pesticide Residues and Environmental Pollution Department, Agriculture Research Center, Dokki, Giza Egypt 2Pesticides & Economic Insect Department, Faculty of Agriculture, Cairo University, Cairo, Egypt

A method for determination of etoxazole residues in apples, strawberries and green beans was developed and validated. The analyte was extracted with acetonitrile from foodstuff and a charcoal-celite cartridge was used for clean-up of raw extracts. Reversed phase high performance liquid chromatography with photodiode array detector (HPLC-DAD) was used for the determination and quantification of etoxazole residues in the studied samples. The calibration graphs of etoxazole in a solvent or three blank matrixes were linear within the tested intervals 0.01–2 mg L−1, with correlation coefficient of determination >0.999. The combined solid phase extraction (SPE) clean-up and the chromatographic method steps were sensitive and reliable for simultaneous determination of etoxazole residues in the studied samples. The average recoveries of etoxazole in the tested foodstuffs were between 93.4 to 102% at spiking levels of 0.01, 0.10, and 0.50 mg kg−1, with relative standard deviations ranging from 2.8 to 4.7%, in agreement with directives for method validation in residue analyses. The limit of detection (LOD) of the HPLC-DAD system was 100 pg. The limit of quantification of the entire method was 0.01 mg kg−1. Keywords: Etoxazole in food stuff, solid phase extraction, HPLC-DAD.

Introduction headaches to chronic diseases like cancer and endocrine dis- ruption, pesticide residues in food products must be closely Etoxazole (2-(2, 6-difluorophenyl)-4-[4-(1, 1-dimethyleth- monitored and strictly controlled.[5–7] In order to ensure yl)-2-ethoxyphenyl]-4, 5-dihydrooxazole), a new organo- the safety of consumable food products and supervise in- fluorine pesticide was produced by Sumitomo Chemical ternational trade, governmental authorities of each country Co. Ltd (Osaka, Japan) in 1998 and was developed world- and international organizations have established maximum wide as a new-generation acaricide for citrus, apples, grapes, residue limits (MRLs) to regulate pesticide concentration vegetables, flowers, cotton and tea.[1] Etoxazole is an alter- in food products.[8,9] native for carbamates, organochlorines and other miticides, Analytical methods that are used to monitor pesticide and works by inhibiting molting.[2,3] Main pests targeted by levels in food should be capable of measuring pesticide Downloaded by [Enstinet], [Farag Malhat] at 03:14 25 February 2013 etoxazole are tetranychid spider mites such as Panonychus residues at very low levels.[10] In addition, these methods ssp. and Tetranychus ssp. Insecticidal side effects on aphids, should be able to identify and quantity the types of pesti- green rice leafhoppers and diamondback moths have also cides found in food products.[11] Furthermore, these meth- been claimed but are of lesser importance. Etoxazole is ac- ods should be fast, robust and simple in order to minimize tive against eggs, larvae and nymphs of spider mites but the requirements for training and the time spent on sam- lacks any efficacy against male and female adults.[4] It usu- pling and maintaining equipment.[12] Although etoxazole ally exhibits high efficacy and behaves like an insect/mite has been marketed in Egypt for about 10 years, to our growth regulator, considering its effects on the different knowledge no residue data in food samples have been re- developmental stages mentioned above. ported. Studies of etoxazole focus on toxicology, mode of Since pesticides are known to cause several human health action, and efficacy.[13,14] In contrast, the papers on analyt- problems, which range from short-term sickness such as ical methods for residues of etoxazole in food stuffs seem to be rare.[15,16] The aim of this study is to develop an analytical method Address correspondence to Farag Malhat, Pesticide Residues and Environmental Pollution Department, Central Agricultural Pes- using solid phase extraction (SPE) and high performance ticide Laboratory, Agriculture Research Center, Dokki, Giza, liquid chromatography with photodiode array detector Egypt; E-mail: farag [email protected] (HPLC-DAD) to determine etoxazole residues in fruits and Received July 9, 2012. vegetables. 332 Malhat et al.

Materials and methods sodium chloride was added, the mixture was vortexed for another 2 min, and the extract was centrifuged (4000 rpm) Equipment for 10 min. An amount measuring 5 mL of the clear upper acetonitrile was transferred to a spherical flask and evapo- The HPLC system was an Agilent 1100 series (Agilent ◦ rated to dryness by using a rotary evaporator at 40 C. The Technologies, Wilmington, DE) equipped with an analyt- residue in the flask was reconstituted in a 2 mL solvent ical column (150 × 4.6 mm id, 5 µm ODS) attached to (n-hexane: acetone = 97:3, v/v). a photodiode array detector. The food processor was a The chromatographic cartridge used for clean-up was Thermomix, Vorwerk. For water purification, a Millipore- packed from bottom to top with (1) absorbent cotton, Q system was used. The vortex shaker and high-speed cen- (2) 1 cm of anhydrous sodium sulphate, (3) 1 g of charcoal- trifuge were made in Germany.The rotary evaporator was a celite chromatographic mixture (1 + 20, w/w), and (4) 1 cm Buchi. of anhydrous sodium sulphate. The packed column was pre- conditioned with a 5 ml solvent (n-hexane: acetone = 97:3, Reagents v/v). The redissolved extract was applied to the cartridge. Acetone, n-hexane, acetonitrile and methanol were sup- Etoxazole was eluted from the cartridge with a 15 mL (n- plied by Merck (Darmstadt, Germany) in HPLC grade hexane: acetone = 97:3, v/v). The elute obtained was col- quality. Anhydrous sodium sulphate and sodium chloride lected in a spherical flask and then evaporated to dryness. (made in Egypt) were baked at 130◦C for 8 h. Charcoal- The residue was redissolved with 1 mL acetonitrile, and fi- celite chromatographic mixture, one part (by weight) char- nally filtered by a 0.45 µm filter membrane for LC analysis. coal decolorizing powder, neutral, BDH, is combined with twenty parts celite 545 diatomaceous earth, mixed well and ◦ baked at 130 C over night. Prior to actual uses in a column, Chromatographic conditions it was cooled in a dessicator. Ultra pure water was prepared by the Millipore system. The neat standard of etoxazole The chromatographic conditions used for the analysis of (purity ≥98%) was supplied by Sumitomo Chemical Com- etoxazole residues were as follows: an Agilent 1100 liq- −1 uid chromatograph was used and equipped with a DAD. pany. A stock standard solution (100 mg L ) was prepared × in acetonitrile and stored at −18◦C. A hypercell ODS analytical column (150 mm 4.6 mm id, 5 µm) was used. The mobile phase was acetonitrile- + + Method development methanol-water (45 40 15, v/v/v) with a flow rate of 0.8 mL/min. The injection volume was 20 µL. Detection Samples. Apples, strawberries and green beans were pur- wavelength for etoxazole set at 240 nm. Under these con- chased from a local supermarket and those that had no ditions, the retention time of etoxazole was about 7.2 min. detectable residues were used as blank samples. Fruit and vegetable samples were comminuted by food processor and stored at −18◦C. Apple samples were also collected from an apple orchard Results and methods located at El-Hamol village, Menof province, El Menofiya governorate and treated, during June 2011, with Baroque Method validation SC (10%, etoxazole) at the recommended application rate Linearity. For the preparation of calibration curves, etox- Downloaded by [Enstinet], [Farag Malhat] at 03:14 25 February 2013 25 mL/100 L. Apple samples were randomly collected at azole standard was diluted either with pure acetonitrile or 0 (2 h after application), 1, 3, 7, 10, 12 and 15 days after with blank matrix extracts in series at 0.01, 0.05, 0.1, 0.5, 1 application. and 2 µgmL−1. A standard calibration curve of etoxazole Strawberry samples were also collected from a straw- was constructed by plotting analyte concentrations against berry field located at Qaha district, El Qaliuobiya Gover- peak areas at 240 nm. Good linear correlation between norate and treated, during March 2011, with Baroque SC the peak area and the concentration assayed (0.01–2.0 µg (10%, etoxazole) at the recommended rate of application mL−1) with correlation coefficient >0.999 for etoxazole in (25 mL/100 L). all cases, were obtained for the solvent and three foodstuffs Green bean samples were also collected from green bean (apples, strawberries and green beans). field treated, during May 2011, with Baroque SC (10%, etoxazole) at the recommended application rate 25 mL/ Accuracy. Recoveries of etoxazole were carried out in 5 100 L. replicates at 3 fortification levels (0.01, 0.10, and 0.50 mg kg−1) by spiking standard solution to 10 g of blank sam- Extraction and purification ple (Table 1). Satisfactory results were found at the three A 10 g sample was weighted into a 50 mL PTFE cen- spiking levels, with recoveries between 93.4 to 102%. The trifuge tube; 10 mL acetonitrile was added, and the mixture repeatability relative standard deviation (RSDr) values of was vortexed for 2 min. An amount measuring 5 g of the method ranged from 2.8 to 4.7%. Figure 1 shows the Analysis of etoxazole residues in produce 333

Table 1. Recovery of etoxazole residues from strawberry, apple and green bean (n = 5). Sample Spiking level, mg/kg Recovery % RSD %

Strawberry 0.01 93.4 2.9 0.10 99.6 3.7 0.50 97.4 2.8 Apple 0.01 96.8 4.6 0.10 98.4 4.7 0.50 102 3.7 Green Bean 0.01 98.6 3.6 0.10 94.8 4.7 0.50 96.2 4.4

chromatograms of the etoxazole blank and different levels of spiked samples.

Repeatability and reproducibility The repeatability of the instrument was determined by an- alyzing samples spiked at 0.5 mg kg−1 with etoxazole. The sample was injected 10 times, and the relative standard deviation (RSD) values were obtained from peak areas by LC were 2.5%. The precision of the method was determined by repeatability and reproducibility studies of the method and expressed by RSD values (Table 2). The RSDr was mea- sured by comparing the SD values of the recoveries from spiked samples analyzed the same day. The reproducibility relative standard deviation (RSDR) values, determined by the analyses of spiked samples on 3 different days by three analysts, ranged from, 4.1 to 6.2% for the spiking level shown in Table 2.

Limits of detection and quantiﬁcation The limit of detection (LOD) of LC was 100 pg and re- sulted from injecting 20 µL of standard with S/N > 3. The limit of quantiﬁcation (LOQ) of the method was 0.01 mg Downloaded by [Enstinet], [Farag Malhat] at 03:14 25 February 2013 kg−1, the S/N of which was generally >10. The matrix

Table 2. Recovery RSDr and RSDR values obtained from analyses of samples spiked with etoxazole at 0.5 mg/kg (n = 5).

Sample Analyses Day Recovery % RSDr % RSDR % Strawberry 1 98.4 5.4 4.4 2 99.8 3.5 3 100.4 4.8 Apple 1 96.8 4.8 6.2 2 99.8 8.8 3 98.0 4.7 Green Bean 1 94.8 4.5 4.1 2 94.4 3.3 Fig. 1. Chromatograms of Etoxazole (A) Standard (0.1 mg/L), 3 97.0 4.7 (B) strawberry blank, (C) strawberry spiked at 0.1 mg/kg, (D) apple blank, (E) apple spiked at 0.1 mg/kg, (F) green bean blank,

RSDr: repeatability as relative standard deviation (G) green bean spiked at 0.1 mg/kg. (Continued) RSDR: reproducibility as relative standard deviation 334 Malhat et al.

when eluted with n-hexane- acetone (97 + 3, v/v), while many interfering matrix compounds were adsorbed onto the solid phase. This indicated that charcoal-celite provides sufficient cleanup for the crude extract of apples, strawberries and green beans, and other searcher also confirmed the excellent clean up efficiency of charcoal-celite for different material extracts.[18]

Optimized wavelength for UV detection Etoxazole showed maximum UV absorption at 210 nm, but several co-extracted matrix components showed a strong absorption at this short wavelength. Further testing with matrix-matched standards indicated the perfect signal-to- noise ratio at 240 nm. Consequently, 240 nm were chosen to monitor etoxazole.

Application of the method to real samples To further demonstrate the utility and the performance of the described method, 12 fruits and vegetable samples (four apples, four strawberries and four green beans) from local markets (from Dokii, Giza, Egypt) was ana- Fig. 1. (Continued) lyzed. We also determined whether the concentrations of the etoxazole used exceeded the maximum residue limits (MRLs). Etoxazole was found in only one strawberry effect of the present method was investigated by compar- sample with concentration (0.08 mg kg−1)muchlower ing standards in solvent with matrix-matched standards than the MRL (0.2 mg kg−1) established by the European − for 5 replicates at 0.5 mg kg 1. The relative responses (re- Union. sponse matrix/response solvent) were 1.02, 0.97 and 1.07 The sample preparation technique and HPLC method- for strawberry, apple and green bean, respectively. It may ology developed in our laboratory to residue determina- be concluded that the matrix doesn’t significantly suppress tion of etoxazole in agricultural commodities were applied or enhance the response of the instrument. for analysis of apple, strawberries and green bean samples from etoxazole treated fields. In order to include samples with different concentration of the analyte, we used Optimization clean-up by SPE samples harvested at different time intervals after appli- SPE is a simple sample preparation technique based on cation from the same field plot. Each subject was treated separation by LC, where the solubility and functional with the developed technique. The results shown in Table 3

Downloaded by [Enstinet], [Farag Malhat] at 03:14 25 February 2013 group interaction of sample, solvent, and sorbent are op- demonstrated that the initial deposit of etoxazole 2 h af- timized to affect the retention and elution.[17] We investi- ter application on the apple fruits was 1.43 mg kg−1.A gated charcoal-celite for vegetable and fruit puriﬁcation in slow degradation of acaricide residues was noted from day this study. The pesticide was not retained by charcoal-celite one after application, with a reduction value of 31.5%. Its

Table 3. Residue levels of etoxazole (mg/kg ± SD∗) in green bean, apple and strawberry at different times after application. Green bean Apple Strawberry Time after application(days) Residue level (mg/kg) ± SD Residue level (mg/kg) ± SD Residue level (mg/kg) ± SD

01.13 ± 0.09 1.43 ± 0.31.04 ± 0.05 10.79 ± 0.06 0.98 ± 0.07 0.81 ± 0.2 30.45 ± .09 0.72 ± 0.03 0.62 ± 0.07 70.22 ± 0.07 0.45 ± 0.03 0.34 ± 0.07 10 0.09 ± 0.01 0.13 ± 0.05 0.15 ± 0.03 12 0.06 ± 0.01 0.08 ± 0.03 0.06 ± 0.02 15 0.01 ± 0.008 0.02 ± 0.006 0.04 ± 0.01

∗n = 3 Analysis of etoxazole residues in produce 335

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