Microchemical Journal 112 (2014) 119–126

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Microchemical Journal

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Determination of nineteen pesticides residues (organophosphates,organochlorine, pyrethroids, carbamate, thiocarbamate and strobilurin)in coconut water by SDME/GC–MS

Jeancarlo Pereira. dos Anjos, Jailson B. de Andrade ⁎Universidade Federal da Bahia, Instituto de Química, 40170-290 Salvador, BA, BrazilCentro Interdisciplinar de Energia e Ambiente— CIEnAm, Universidade Federal da Bahia, Ondina 40170-290 Salvador, BA, BrazilINCT de Energia e Ambiente, UFBA, 40170-290 Salvador, BA, Brazil

⁎ Corresponding author. Tel./fax: + 55 71 32836821.E-mail address: jailsong@ufba.br (J.B. de Andrade).

0026-265X/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.microc.2013.10.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 August 2013Accepted 1 October 2013Available online 11 October 2013

Keywords:PesticidesCoconut waterSDMEGC–MS

Coconut water is a natural isotonic drink and a rich source of sugars, salts, vitamins, minerals and amino acids,and can be served as a beverage to quench thirst. Palm trees are often attacked by insects and/or pests, thereforereducing their productivity. In order to enhance coconut production, pesticides are often used. Thus, the mainobjective of this study is to propose a simple and efficient method for the determination of pesticide residues,from different chemical classes, in samples of industrialized and natural coconut water, using single-dropmicroextraction (SDME), followed by gas chromatography coupled to mass spectrometry (GC–MS). Theextraction step using SDME was optimized and it was found that the best experimental conditions for10 mL of samples were obtained using toluene as an extraction solvent; stirring time of 30 min at 200 rpm;drop volume of 1.0 μL; and acidification with HCl without salt addition. The chromatographic method wasvalidated and good values were found for the figures of merit, with LOD ranging between 0.1 and 0.88 μg L−1,and LOQ between 1.21 and 6.69 μg L−1. The method was successfully applied to real samples of natural andindustrialized coconut water, and the pesticides sulfotep, demeton-O, dimethoate, disulfoton, fenitrothion andmalathion were determined at concentrations ranging from bLOQ to 12.1 μg L−1. The proposed methodologypresents high sensitivity and the capability for detecting and quantifying low levels of pesticides in coconutwater samples.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Coconut water is a natural isotonic drink and a rich source of sugars,salts, vitamins, minerals and amino acids, and can be served directlyas a beverage to quench thirst. It is a clear, colorless and sweet liquid,presenting a slightly acidic flavor, with pH ranging from 4.2 to 6.0. It ismarketed in its natural form or after processing, in which it is subjectedto various sterilization steps and addition of preservatives [1–3].

Palm trees are often attacked by various insects and/or pests, there-fore reducing their productivity. In order to enhance coconut produc-tion, pesticides are often used in various forms, either by spraying onthe leaves or by injection through the stem and root systems. Pesticideinjection in the trunk of palm trees began in the mid-1950s and isstill being continued in several countries of South Asia. Pesticides likemonocrotophos, carbofuran and its metabolite 3-hydroxycarbofuranwere found in samples subjected to spraying or injection on trunkor leaves. The presence of residues of these compounds in the coco-nut water may be a risk to consumers' health, due to their potentialtoxicity [3–5].

ights reserved.

Nowadays, pesticides from different chemical classes are frequentlyused for the control of pests in palm crops, especially carbamates(carbofuran), pyrethroids (bifenthrin), organophosphates (dimethoate,malathion, methyl parathion and trichlorfon), among others [6].Despite the intensive use of pesticides, the Brazilian government hasnot established a maximum limit for pesticide residues in coconutwater [7].

Several analyticalmethods have beenproposed for the simultaneousdetermination of multiresidues of pesticides in aqueous matrices. Thedevelopment of such methods is often difficult, since the compoundshave different degrees of polarity, solubility and volatility, as well asdifferent octanol–water partition coefficients (Table 1), which maketheir extraction and analysis difficult [8]. Therefore, the routinemethodsfor the determination of pesticide residues in the environment and infood usually require many steps of sample preparation before instru-mental analysis, such as extraction, clean-up and pre-concentration[9]. In the specific case of coconut water, the fact that it is a complexmedium should be taken into account, considering both its physicaland chemical properties. Thus, even with the recent analytical in-strumentation advances, there is little information available in theopen literature about analytical methodologies that have identifiedand quantified pesticides in coconut water. Until now, it was only

Table 1Physicochemical properties of the pesticides selected for study [10].

Pesticide Molecular formula Chemical group log Kow — pH 7; 20 °C Water solubility — 20 °C (mg L−1)

Carbofuran C12H15NO3 Carbamate 1.8 3.22 × 102

Molinate C9H17NOS Thiocarbamate 2.86 1.10 × 103

Sulfotep C8H20O5P2S2 Organophosphate 3.99 10Dimethoate C5H12NO3PS2 Organophosphate 0.704 3.98 × 104

Demeton-o C6H15O3PS2 Organophosphate 0.47 330Diazinon C12H21N2O3PS Organophosphate 3.69 60Disulfoton C8H19O2PS3 Organophosphate 3.95 25Methyl parathion C8H10NO5PS Organophosphate 3 55Fenitrothion C8H10NO5PS Organophosphate 3.32 19Malathion C10H19O6PS2 Organophosphate 2.75 1.48 × 102

Fenthion C10H15O3PS2 Organophosphate 4.84 4.2Dursban C9H11Cl3NO3PS Organophosphate 4.7 1.05Parathion C10H14NO5PS Organophosphate 3.83 12.4Endosulfan C9H6Cl6O3S Organochlorine 4.75 3.20 × 10−1

Ethion C9H22O4P2S4 Organophosphate 5.07 2Bifenthrin C23H22ClF3O2 Pyrethroid 6.6 10−3

Permethrin i C21H20Cl2O3 Pyrethroid 0.2 6.1Permethrin ii C21H20Cl2O3 Pyrethroid 0.2 6.1Azoxystrobin C22H17N3O5 Strobilurin 2.5 6.7

120 J.P. dos Anjos, J.B. de Andrade / Microchemical Journal 112 (2014) 119–126

the quantification of malathion that was found. This was possibledue to the use of several procedures for sample clean-up andpreconcentration [3].

Recent studies have focused on the development of analyticalmethods which are economic, miniaturized and with a low impacton the environment. In this context, the single-drop microextraction(SDME) technique has emerged as a simple, virtually free of solvents,fast and cheap sample preparation procedure. This technique is basedon the principle of distribution of analytes between a microdrop of or-ganic solvent, on the tip of a microsyringe needle, and the aqueousphase (sample). The microdrop is exposed to an aqueous sample inwhich the analyte is extracted into the drop. After extraction, themicrodrop is retracted into the microsyringe and injected into instru-ments for liquid or gas chromatography for analysis [9,11]. This tech-nique has been successfully used in the determination of differentclasses of substances, such as phenolic compounds [12], sulfur com-pounds [13], metals [14,15], amines [16], as well as for the extractionof pesticides [9,17–20] in different matrices.

Therefore, the aim of this study was to develop and validate an effi-cient method for the determination of pesticide residues, from differentchemical classes, in samples of industrialized and natural coconutwater, using single-drop microextraction (SDME), followed by gaschromatography coupled to mass spectrometry (GC–MS), and thenapply the developed method to real samples of coconut water.

2. Experimental

2.1. Standards, reagents and samples

The certified standards employed for pesticide analysis werecarbofuran,molinate, sulfotep, demeton-O, dimethoate, diazinon, disul-foton, methyl parathion, fenitrothion, malathion, fenthion, dursban,parathion, endosulfan, ethion, bifenthrin, permethrin and azoxystrobin,all acquired from AccuStandard.

The toluene used was acquired fromMerck; methanol, cyclohexaneand isooctane (HPLC grade) were obtained from JT Baker. Fuminghydrochloric acid, phosphoric acid (Merck) and sodium chloride(99.6%) (JT Baker) were also used.

Samples of industrialized coconut water (from 3 different brands,among which 5 different batches were evaluated) were acquired in acommercial establishment in the city of Salvador, Bahia, Brazil, and sam-ples of natural coconut water were acquired in open markets.

2.2. Preparation of standards and samples

Stock solutions were prepared at concentrations of 100 mg L−1

(demeton-O, diazinon, disulfoton, ethion, malathion, methyl parathionand parathion), 500 mg L−1 (bifenthrin, azoxystrobin, dursban and di-methoate), 600 mg L−1 (carbofuran) and 1000 mg L−1 (permethrin,molinate, sulfotep, fenitrothion and fenthion), solubilized in methanol,except for endosulfan (500 mg L−1), which was solubilized in ethylacetate; the solutions were kept under refrigeration.

Themethod used to quantify the compoundswas external standard-ization. For the construction of calibration curves, dilutions of an inter-mediate solution were performed in ultrapure water, containing amixture of all standards at the same concentration (1.0 mg L−1), andthis mixture was obtained by diluting the previously prepared stocksolutions in methanol.

Before the SDME procedure, 15-mL aliquots of the samples werecentrifuged (3000 g) for 5 min in order to remove coconut particlessuspended in the beverage, since they harmed the stability of themicrodrop at the end of the syringe needle.

2.3. Single-drop microextraction (SDME)

For the SDME procedure, a 10-μL Hamilton microsyringe was usedto measure and introduce the microdrop of the extraction solvent(toluene) in a glass vial containing the extraction solution or the sample,a magnetic stir bar and a silicone septum. Before each extraction, thesyringe was rinsed 6 times with methanol, then 3 times with toluene.The plunger was then placed in position zero and 2 μL of toluene wasdrawn into the syringe. Then, the microsyringe needle was insertedthrough the septum and directly immersed in the extraction solutionor sample (10 mL) under stirring (200 g). The microsyringe plungerwas depressed (up to the position 1 μL — drop volume) to expose thetoluene drop, so that the analyte transfer occurred from the aqueousphase to the organic drop. After the extraction time (30 min), the dropwas drawn into the microsyringe, removing the needle out of theglass vial, and immediately injected into a gas chromatography systemcoupled to a mass spectrometer (GC–MS) for later analysis.

2.4. Chromatographic analyses

The pesticide analyses were performed using a Shimadzu gaschromatograph coupled to a mass spectrometer (GCMS-2010 Plus)equipped with a split/splitless injector, operating in the splitless

Table 2Experimental parameters selected for the analysis of pesticides by GC–MS.

Segments (min) Analyte Ion quantification (m/z) Ion confirmation (m/z) tR (min)

0.00–6.00 Carbofuran 164 149 5.736.01–8.20 Molinate 126 55 6.97

Sulfotep 322 97 7.66Demeton-o 88 60 7.94Dimethoate 87 125 7.66

8.21–8.70 Diazinon 137 304 8.35Disulfoton 88 60 8.45methyl parathion 109 125 8.45

8.71–10.00 Fenitrothion 109 125 8.97Malathion 93 125 9.37Fenthion 278 125 9.53Dursban 97 197 9.59Parathion 291 109 9.59

10.01–11.20 Endosulfan 241 339 10.7311.21–12.00 Ethion 231 384 11.5712.01–14.00 Bifenthrin 181 105 13.0214.01–15.50 Permethrin I 183 163 14.55

Permethrin II 183 163 14.6615.51–20.00 Azoxystrobin 344 388 17.21

Fig. 1. Chromatogram of the standard solution of pesticides (10 μg L−1) (A) and a coconut water sample (B) after optimization of the extraction parameters by SDME. Identification of thepeaks: 1— carbofuran; 2—molinate; 3— sulfotep; 4— dimethoate; 5— demeton-O; 6— diazinon; 7— disulfoton; 8—methyl parathion; 9— fenitrothion; 10—malathion; 11— fenthion;12— dursban; 13— parathion; 14— endosulfan; 15— ethion; 16— bifenthrin; 17 — permethrin I; 18— permethrin II; and 19 — azoxystrobin.

121J.P. dos Anjos, J.B. de Andrade / Microchemical Journal 112 (2014) 119–126

Fig. 3. Areas obtained for extraction time optimization (solvent: toluene; drop volume:1,0 μL). Standard solution (50 μg L−1): 1 — carbofuran; 2 — molinate; 3 — sulfotep;4— demeton-O; 5— dimethoate; 6— diazinon; 7— disulfoton; 8—methyl parathion;9 — fenitrothion; 10 — malathion; 11 — fenthion; 12 — dursban; 13 — parathion;14 — endosulfan; 15 — ethion; 16 — bifenthrin; 17 — permethrin I; 18 — permethrin II;and 19— azoxystrobin.

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mode at a temperature of 300 °C during the chromatographic run,with a purge time of 0.75 min. Pesticide separations were performedusing a Restek Rtx®-1MS capillary column (30 m × 0.25 mm ID;0.25 μm film thickness). The carrier gas used was helium (99.999%purity) under a 1.0 mL min−1

flow. The oven temperature programwas 60 °C (1.0 min); 200 °C at 25 °C min−1; 280 °C at 10 °C min−1;300 °C at 5 °C min−1and hold time at 300 °C for 1.40 min, withtotal run time of 20 min. For the mass detector, transfer line temper-ature and ion source at 300 °C were used, as well as ionization byelectron impact at 70 eV.

The optimization of the retention times was performed in SCANmode, using a standard solution of pesticides at a concentration of10 mg L−1. The quantification of pesticides in the samples of coconutwater was performed in SIM (Selected Ion Monitoring) mode, andtwo specific ions were chosen for each analyte: the first ion was usedfor quantitation and the second for confirmation (Table 2). For per-methrin, which presents stereoisomerism, two peaks were detected,corresponding to the cis (Z) and trans (E) isomers.

Fig. 1 shows the chromatographic profile of the standard solution ofpesticides at a concentration of 10 μg L−1 with the identification of therespective compounds, according to the time segments listed in Table 2.

2.5. Validation of the chromatographic method

In order to ensure the analytical quality of the results, procedureswere performed to validate the method, in which the following param-eters were evaluated: selectivity, linearity, precision, limit of detection,limit of quantification and accuracy [21,22].

3. Results and discussion

3.1. Optimization of experimental conditions for SDME

The choice of a suitable solvent is very important for the obtention ofa good selectivity and better extraction efficiency of the analytes. Thus,the selected solvent should present low solubility in water, low toxicityand good stability of the formed drop and, above all, be able to extracteffectively the analytes [23].

In a previous work [19,24] multivariate experimental design wasapplied for the optimization of experimental conditions for SDME inthe analysis of organophosphate pesticides and pyrethroid in waterevaluating, among others, the following variables: solvent, extractiontime and drop volume. Based on the previous finding it was possibleto use in this work univariate experimental design, for evaluation:

Fig. 2. Areas obtained for solvent optimization (extraction time: 30 min; drop volume:1.0 μL). Standard solution (50 μg L−1): 1 — carbofuran; 2 — molinate; 3 — sulfotep;4— demeton-O; 5— dimethoate; 6— diazinon; 7— disulfoton; 8—methyl parathion;9 — fenitrothion; 10 — malathion; 11 — fenthion; 12 — dursban; 13 — parathion;14 — endosulfan; 15 — ethion; 16 — bifenthrin; 17 — permethrin I; 18 — permethrin II;and 19— azoxystrobin.

solvent (toluene, cyclohexane, isooctane); extraction time (10, 20, 30and 40min) and drop volume (0.5, 0.8 and 1.0 μL).

In Fig. 2 it can be observed that all solvents studied presented goodperformance in the pesticide extraction. Hence, toluene was selecteddue to their low toxicity, when compared to the other solvents testedand, for their compatibility with the analysis of pesticides by gaschromatography.

Usually, when SDME method is applied the extraction efficiency in-creases with the time. Meanwhile, the selected time must be sufficientto permit the microdrop to extract properly the target analytes, butnot too long in order to prevent microdroplet dissolution [23]. In Fig. 3it can be observed that, the extraction equilibrium was reached at30 min, for most part of the pesticides. After this time the microdropdissolution was enhanced making the microdrop size control difficult.Hence, the extraction time of 30 min was chosen.

In general, the use of a greater drop volume results in an increase inthe response of the analytical instrument. However, very voluminousdrops may be difficult to handle and are less reliable. Furthermore,there may be a fall in the diffusion process of the analytes because ofthe increase in the drop volume, so there will be the need of a longertime to reach equilibrium [23]. Therefore, the chosen drop volumewas 1.0 μL, for showing a good drop stability, as well as a better

Fig. 4. Areas obtained for drop volume optimization (solvent: toluene; extraction time:30 min). Standard solution (50 μg L−1): 1 — carbofuran; 2 — molinate; 3 — sulfotep;4— demeton-O; 5— dimethoate; 6— diazinon; 7— disulfoton; 8—methyl parathion;9 — fenitrothion; 10 — malathion; 11 — fenthion; 12 — dursban; 13 — parathion;14 — endosulfan; 15 — ethion; 16 — bifenthrin; 17 — permethrin I; 18 — permethrin II;and 19— azoxystrobin.

Table 3Figures of merit obtained for the studied pesticides.

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extraction efficiency of pesticides, in order to ensure a good reproduc-ibility and allow the use of rapid stirring (Fig. 4).

Further tests were performed in order to obtain a better response foreach pesticide analyzed. In this way, those evaluated were the solutionvolume (1, 5 and 10 mL), stirring speed (without stirring, 100, 200 and300 rpm), acidification of the extraction medium (without acid, withH3PO4 and with HCl), and the salting-out effect (without salt and with10% NaCl). For the tests performed with different solution volumes,although the areas obtained were very close for the volumes of 5 and10mL, the use of 10 mL of solution was chosen, since this volumeproved to be better placed in the vial when stirring was performedwith a magnetic stir bar submerged in the solution.

It was also observed that stirring was a factor that determined asignificant increase in the peak area of pesticides. Better extractionefficiency was obtained when using rapid stirring, due to the fact thatstirring enables a continuous exposure of the extraction surface bythe sample. It was possible to observe higher extraction efficienciesof pesticides for the speeds 200 and 300 rpm. However, 200 rpm wasthe chosen speed since, in this speed, a smaller solution disturbancewould occur, when compared to 300 rpm, and, consequently, greatermicrodrop stability at the tip of the microsyringe needle. For speedsgreater than 300 rpm, it was found that the drop detached very easily,preventing its collection to be subsequently injected into the chromato-graphic system.

Ito et al. [25] suggested thatmolecules of certain pesticides are oftenmore stable in acidic aqueous medium, when compared to the basicmedium, as in the case of fenitrothion. In this way, the effect of the acid-ification of themedium in the extraction efficiency of the pesticideswasstudied using non-oxidant acids: hydrochloric acid and phosphoric acid.It can be observed in Fig. 5 that, with the use of both acids, there is asignificant positive linear correlation between the data obtained forthe extraction without the use of acid and the extraction acidifying themedium. Correlation coefficients (R) of 0.9924 were obtained for theuse of hydrochloric acid and 0.9593 for the use of phosphoric acid,revealing that the acidification of the medium is an important factorfor the extraction of pesticides using SDME. Furthermore, it was ob-served that the slope of the straight line obtained for the correlationusing hydrochloric acid (1.6023)was higher, compared to that obtainedby using phosphoric acid (0.8216), demonstrating a greater increase inextraction efficiency when hydrochloric acid was used. In this context,acidification of themedium for the extraction of pesticideswas adopted,and hydrochloric acid was chosen, due to its higher response tothe analysis of pesticides after the extraction process, especiallyto organophosphorus pesticides, such as disulfoton, fenitrothion,fenthion and dursban.

The salting-out effect is related to the connection between waterand salt, thus reducing the hydration of molecules in aqueous solution

Fig. 5. Graph for the correlation between the responses obtained for the extraction ofpesticides without the use of acid and acidifying the medium.

(solvation) [26]. In this context, salts such as sodium chloride improvethe structuring of the aqueous phase and, consequently, thewater bind-ing energy, due to their strong interaction with its dipoles, promoting achange in the partition equilibrium of neutral organic solutes towardsnon-aqueous phases, facilitating the extraction of analytes containedin aqueous phases [27]. On the other hand, Kin and Huat [23] showedthat the addition of salt caused a slight reduction in the extraction effi-ciency for most of the studied pesticides and was more pronounced forless polar compounds. However, in this study, no significant influencewas observed for the extraction of pesticides when salt was added tothe standard solution, because the areas obtained between the solutionswith and without the addition of salt were very close. Therefore, in thisstudy, no salt was added to the standard solution for the extractionof pesticides.

In the presentwork, the optimization of the experimental conditionsfor SDME extraction showed that the best parameters for the analysis ofthe studied pesticideswere: solvent (toluene), extraction time (30 min);drop volume (1.0 μL); solution volume (10 mL); stirring rate (200 rpm);with acidification of the medium with HCl and without salt addition,since it allows a better extraction of analytes from the aqueous medium.

3.2. Validation of the method

3.2.1. SelectivityUnder the chromatographic conditions employed, it was observed

that the samples showed no interfering substances in the retentiontimes of the pesticides analyzed, which was verified by the comparisonof the chromatogram of a sample without the addition of the pesticidestandard and of the same sample fortified with the standards of thecompounds analyzed at a concentration of 10 μg L−1 for each analyte,thus confirming the selectivity of the analytical method.

3.2.2. LinearityBy constructing calibration curves, coefficients of determination (R2)

ranging from 0.9254 (malathion) to 0.9996 (ethion) (Table 3) were ob-tained, showing the strong linear correlation between the concentrationof the compounds analyzed and the peak areas, enabling the quantifica-tion of pesticides in coconut water.

3.2.3. Limits of detection and quantificationThe limits of detection and quantification were obtained using the

parameters of the calibration curves constructed and calculated bytheir mathematical relationships: LOD= 3SD/m and LOQ= 10SD/m(where SD= estimative of the standard deviation of the regression

Pesticide Linear range (μg L−1) r2 LODa (μg L−1) LOQa (μg L−1)

Carbofuran 1.0–25.0 0.9911 0.88 2.94Molinate 0.5–15.0 0.9997 0.38 1.28Sulfotep 0.5–15.0 0.9922 0.10 2.51Demeton-O 0.5–15.0 0.9955 0.25 3.26Dimethoate 0.25–15.0 0.9927 0.25 6.11Diazinon 0.5–15.0 0.9957 0.10 2.53Disulfoton 0.5–15.0 0.9974 0.10 2.37Methyl parathion 0.5–15.0 0.9914 0.50 6.65Fenitrothion 0.5–15.0 0.9922 0.50 3.47Malathion 1.0–25.0 0.9254 0.73 2.42Fenthion 0.5–15.0 0.9951 0.10 2.96Dursban 0.5–15.0 0.9968 0.25 2.48Parathion 0.5–15.0 0.9913 0.25 6.69Endosulfan 0.25–10.0 0.9984 0.10 1.99Ethion 0.5–15.0 0.9996 0.10 2.73Bifenthrin 0.5–25.0 0.9885 0.36 1.21Permethrin I 0.5–15.0 0.9916 0.25 1.81Permethrin II 0.5–25.0 0.9981 0.10 2.42Azoxystrobin 0.5–15.0 0.9925 0.25 2.67

a LOD= limit of detection; LOQ= limit of quantification.

Table 4Peak area averages (n = 5) and relative standard deviation (RSD) obtained for each pesticide in the evaluation of method repeatability.

Compound 0.5 μg L−1 1 μg L−1 5 μg L−1 10 μg L−1

Area RSD (%) Area RSD (%) Area RSD (%) Area RSD (%)

Carbofuran – – – – 4340.0 25.0 14975.6 13.3Molinate – – 31566.6 11.3 196889.2 12.6 378529.0 6.10Sulfotep – – 8016.2 7.95 43669.6 16.7 76874.0 14.5Demeton-O – – 15714.6 16.3 122354.5 12.3 211692.8 14.5Dimethoate 833.0 10.3 3311.3 4.32 24165.2 9.61 35506.6 7.11Diazinon – – 36523.8 10.5 207875.6 18.2 324939.2 13.2Disulfoton – – 99546.5 3.09 817022.3 37.9 1179516.8 4.99Methyl parathion – – 3765.3 5.89 32950.8 19.1 52343.8 8.68Fenitrothion – – – – 104945.0 21.1 384215.0 10.4Malathion – – 23396.8 15.6 35880.75 13.6 91883.4 18.5Fenthion – – 65840.5 9.91 244304.2 13.5 724325.8 12.2Dursban – – 61876.7 9.70 257517.2 9.56 737409.2 11.4Parathion – – 3193.2 9.87 13300.2 17.2 38411.4 8.63Endosulfan 1261.6 14.5 6586.7 7.46 25121.4 8.28 69107.6 10.1Ethion – – 31874.5 7.71 90983.6 14.6 292495.2 15.4Bifenthrin – – 10324.3 7.16 23774.7 17.9 47672.6 7.53Permethrin I – – 3476.3 6.78 8942.8 16.9 21943.8 12.5Permethrin II – – 8677.0 3.95 23752.5 19.3 62438.8 14.0Azoxystrobin – – 8775.8 12.3 16148.4 19.9 39810.2 21.4

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line and m= angular coefficient of the calibration line) [22]. The limitsof detection and quantification found for the pesticides ranged from 0.1to 0.88 μg L−1 and from 1.21 to 6.69 μg L−1, respectively (Table 3).

3.2.4. PrecisionThe precision of the analytical method was evaluated in relation to

the levels of repeatability, estimating the relative standard deviation(RSD) for each compound analyzed from successive measurements(Table 4). The peak areas obtained from the injection of standard solu-tions of pesticides in three concentration levels (1, 5 and 10 μg L−1)were evaluated. However, for the compounds dimethoate and endosul-fan, the concentration of 0.5 μg L−1 was also measured, since it wasthe lowest concentration relative to the respective calibration curves.The method showed a satisfactory precision for most of the pesticidesanalyzed in coconutwater, despite the complexity of the type of analyteextraction used in this study. For the pesticides carbofuran andfenitrothion, considerable repeatability values were not obtainedfor the lowest levels of concentration and, therefore, they werenot presented.

Table 5Recovery of phenolic compounds in samples of coconut water.a

Compound Concentration in thesample (μg L−1)

Added concentration (μg L−1)

0.5 1 5 10

Recovery (%)

Carbofuran ND NQ 110 99.3Molinate ND 37.1 78.2 72.6Sulfotep ND 133 90.5 104Dimethoate ND 160 93.4 106Demeton-O bLOD 80.0 117 92.2Diazinon ND 142 119 102Disulfoton bLOQ 55.3 75.7 83.2Methyl parathion ND 114 128 102Fenitrothion 5.51 72.6 133 110Malathion 3.57 95.4 86.4 95.9Fenthion ND 121 89.2 102Dursban ND 110 55.9 77.4Parathion ND 118 82.6 84.2Endosulfan ND 28.3 28.3 30.4Ethion ND 88.3 52.7 57.0Bifenthrin ND NQ 65.6 82.1Permethrin I ND 27.9 40.7 49.6Permethrin II ND 54.0 40.5 40.6Azoxystrobin ND 160 143 100

a ND= not detected; NQ= not quantified; bLOD= lower than the limit of detection;bLOQ= lower than the limit of quantification.

3.2.5. AccuracyThe accuracy of the analytical method was evaluated by recovery

experiments (Table 5). It is possible to observe that, in general, themethod showed a good recovery for compounds whose average valueswere included in the acceptable limits (between 50% and 120%, due tothe complexity of the extraction of analytes using SDME as theextraction/preconcentration technique).

It is important to note that for some compounds, such as permethrinI and permethrin II, low recovery values were obtained (39.4% and45.0%, respectively), since they are compoundswhich present relativelylow partition coefficients (Table 1), presenting thus a greater affinity forthe aqueous phase. Therefore, it is possible to notice a greater difficultyof migration of these analytes from the aqueous phase (sample) to theorganic phase (drop).

For the compound endosulfan, despite having a relatively high par-tition coefficient compared to other analytes, it was observed thatthere was an abnormality in its behavior during the extraction process,since the recovery value obtainedwas low (29.0%) and it should presenta greater affinity for the organic phase and thus a better extraction. Thedifficulty in the extraction of this compound is probably related to itsinteraction with other substances present in coconut water. This factcan be explained by the higher polarity of the molecules of organo-chlorine compounds, compared to other classes of compounds suchas organophosphates, which promote a greater affinity for aqueousphases than for organic phases, thus hampering the extraction ofsuch substances.

3.3. Application of the method to real samples of coconut water

After obtaining the optimum conditions for the extraction/preconcentration of the pesticides and the validation of the method,it was applied in the evaluation of these analytes in samples of indus-trialized and natural coconut water. Table 6 shows the concentra-tions of pesticides found in samples of coconut water.

By the results obtained, it is possible to observe that the pesticidesfound in the samples of coconut water were sulfotep, demeton-O,dimethoate, disulfoton, fenitrothion andmalathion. Among the analytesfound in the analyzed samples, the concentrations ranged from bLOQ to12.1 μg L−1 (dimethoate). It is noteworthy that, among the pesticidesfound in the analyzed samples, the insecticides and acaricidesmost commonly used to control the action of pests in palm cropsare dimethoate and malathion [6], which showed the highest con-centrations in samples of coconut water, especially in the samplesof industrial origin.

Table 6Concentrations of pesticides (μg L−1) found for samples of industrialized and natural coconut water.

Pesticide Concentration (μg L−1)a

Brand 1 Brand 2 Brand 3 Natural coconut

Lot 1 Lot 2 Lot 3 Lot 4 Lot 5 Lot 1 Lot 2 Lot 3 Lot 4 Lot 5 Lot 1 Lot 2 Lot 3 Lot 4 Lot 5 1 2 3 4 5

Carbofuran ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDMolinate ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDSulfotep ND ND ND ND ND ND ND bLOQ ND ND ND ND ND ND ND ND ND ND ND NDDimethoate bLOQ ND ND 12.1

±7.48ND ND bLOQ ND ND ND ND bLOQ ND ND ND ND ND ND ND ND

Demeton-O bLOQ bLOQ bLQ bLOQ bLOQ ND bLOQ bLOQ bLOQ bLOQ bLOQ bLOQ bLOQ ND bLOQ ND ND ND ND NDDiazinon ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDDisulfoton ND ND ND ND bLOQ ND bLOQ ND ND ND ND ND ND ND ND ND ND ND ND NDMethyl parathion ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDFenitrothion bLOQ bLOQ bLOQ bLOQ 5.51

±1.37bLOQ bLOQ bLOQ bLOQ bLOQ bLOQ bLOQ bLOQ ND bLOQ ND ND bLOQ bLOQ bLOQ

Malathion 3.41±0.27

4.23±0.82

3.28±0.06

3.70±0.58

3.57±0.70

ND 3.31±0.18

3.52±0.90

ND ND 5.57±2.43

5.11±0.24

3.65±0.80

ND ND ND ND 3.14±0.13

ND 3.33±0.30

Fenthion ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDDursban ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDParathion ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDEndosulfan ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDEthion ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDBifenthrin ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDPermethrin I ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDPermethrin II ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND NDAzoxystrobin ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

a Mean ± standard deviation; ND= not detected; bLOQ= below limit of quantification.

125J.P. dos Anjos, J.B. de Andrade / Microchemical Journal 112 (2014) 119–126

Studies performed by Deme et al. [3] using solid phase extraction(SPE) followed by LC–MS/MS analysis showed that, among the pesti-cides analyzed in coconut water, only malathion was found in 23% ofthe samples, at concentrations ranging from 24 to 45 ng L−1. The au-thors comment that the detection of this analyte in the samples waspossible only after the execution of different preconcentration steps ofthe samples. In this context, the method proposed in this study hasthe advantage of simplicity and execution of only one step for theextraction and preconcentration of the samples.

Other studies reported in the literature using different instrumentalmethods and techniques of extraction/preconcentration were appliedin the analysis of pesticides in coconutwater. Techniques such asmatrixsolid-phase dispersion (MSPD) followed by analysis by liquid chroma-tography with ultraviolet detection or gas chromatography coupled tomass spectrometry showed that it was not possible to detect pesticideresidues in samples of coconut water, probably because they did notreach detection levels compatible for this type of analysis [7,28].

Because there is no specific legislation for the presence of pesticidesin coconut water, the results obtained in this study were compared tothe drinking water standards for chemicals that present health risk.Brazilian law establishes maximum limits for two pesticides used inthis study, endosulfan (20 μg L−1) and permethrin (20 μg L−1) [29],while international law establishes a maximum allowable limit of40 mg L−1 for the pesticide carbofuran in drinking water [30].Thus, the results obtained for these pesticides in coconut waterwere below the limits set in relation to Brazilian and internationallegislation for drinking water, since they were not detected in theanalyzed samples.

4. Conclusions

After obtaining the optimal experimental conditions for SDME,the developed method was successfully applied to real samples ofcoconut water, being fast and efficient. The method enabled theidentification and quantification of pesticides from different chemi-cal classes in the samples analyzed, and the presence of the pesti-cides sulfotep, demeton-O, dimethoate, disulfoton, fenitrothion andmalathion was verified. It was found that the highest concentrationsof pesticides were observed for samples of industrialized coconut

water, since there is great concern on the part of the industry tohave a good performance in its production.

Acknowledgments

The authors wish to thank Brazilian agencies who have fundedthis work: CAPES, CNPq, FAPESB, PRONEX and INCT (Energy andEnvironmental).

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