Extraction and Preconcentration of Some Triazole Pesticidesin Grape Juice by Salting Out HomogeneousLiquid–Liquid Extraction in a Narrow-Bore Tube Priorto Their Determination by Gas Chromatography–FlameIonization Detection

Mir Ali Farajzadeh & Saheleh Sheykhizadeh &

Parisa Khorram

Received: 28 July 2013 /Accepted: 2 October 2013# Springer Science+Business Media New York 2013

Abstract A fast and simple extraction and preconcentrationmethod for some triazole pesticides has been developed using ahomogeneous liquid–liquid extraction method performed in anarrow-bore tube. The extraction is based on phase separationof a water-miscible organic solvent from aqueous solution inthe presence of a salting out agent. In this work, the homoge-neous solution of water and acetonitrile (water-solubleextraction solvent) was broken by addition of 30 %, w /v,sodium chloride (salting out agent). After sonication, a smallvolume of acetonitrile was collected on top of the tube and theextracted analytes in the collected phase were determined bygas chromatography–flame ionization detection. The effect ofvarious experimental parameters including kind and volume ofthe water-soluble organic solvent, amount of salt, length anddiameter of tube, and pH of sample solution was investigated.Under the optimum conditions, calibration graphs were linearover the range of 3–5,000 μg L−1. Relative standard deviationswere less than 5.4 % for six repeated determinations (C =100 μg L−1). Furthermore, the limits of detection (S /N =3)and quantification (S /N =10) were obtained in the ranges of0.60–4.8 and 1.9–16 μg L−1, respectively. This method is verysimple and rapid, requiring less than 10 min for sample prep-aration. It has been successfully utilized for the analysis oftriazole pesticides in the grape juice samples.

Keywords Gas chromatography . Grape juice .

Homogeneous extraction . Sample preparation .

Triazole pesticide

Introduction

The use of pesticides to control pests is clearly inevitable.Triazole pesticides are one of the major classes of the pesticidesthat are greatly used in a variety of vegetables, fruits, and graincrops (Kahle et al. 2008). Besides their antifungal activity, thesecompounds are also discussed as a group that disturbs endocrineactivity in human (Carbone et al. 2007). The determination ofresidue levels of pesticides is necessary for food safety moni-toring and controlling goal. Pesticides are usually determined bygas chromatography (GC), liquid chromatography, or capillaryelectrophoresis, depending on their polarity, volatility, and ther-mal stability (Picó et al. 2007). However, when their concentra-tions are low, an enrichment step is usually needed beforeanalysis. Sample preparation is one of the most important stepsin a chemical analysis. The aim of this challenging and criticalstep is to prepurify, concentrate, and transfer the analyte into aform that is compatible with the analytical system (Ulrich2000). The preconcentration techniques, which are commonlyapplied to monitor pesticides in different samples, are liquid–liquid extraction (LLE) (Fernandez-Alba et al. 1998; Navarroet al. 2000) and solid-phase extraction (SPE) (Zhou et al. 2007;Montes et al. 2009). These techniques are time consuming,expensive, and, especially relating to LLE, hazardous to healthdue to the high volume of potentially toxic solvents used (Xiaoet al. 2006). Recent investigations have focused on the devel-opment of methods that reduce the analysis time, the cost, and

M. A. Farajzadeh (*) : S. Sheykhizadeh : P. KhorramDepartment of Analytical Chemistry, Faculty of Chemistry,University of Tabriz, Tabriz, Irane-mail: mafarajzadeh@yahoo.com

M. A. Farajzadehe-mail: mafarajzadeh@tabrizu.ac.ir

Food Anal. MethodsDOI 10.1007/s12161-013-9737-y

the solvent consumption. Solid-phase microextraction is a sam-ple preparation technique, introduced by Arthur and Pawliszyn(1990), that has been applied to determine a wide variety oforganic compounds from numerous types of samples (Jinnoet al. 2001; Zeng et al. 2001; Millan et al. 2003; Bianchi et al.2008). Miniaturization of LLE technique can be achieved by adrastic reduction of the extractant phase volume. Based on thisprinciple, three new methodologies, i.e., single-dropmicroextraction, hollow fiber liquid-phase microextraction,and dispersive liquid–liquid microextraction (DLLME), havebeen introduced (Jeannot and Cantwell 1997; Pedersen-Bjergaard and Rasmussen 1999; Rezaee et al. 2006). Thesemethods are cheap, and there is considerable freedom to selectsuitable solvents for extraction of different analytes. On theother hand, homogeneous liquid–liquid extraction (HLLE) is asimple and powerful preconcentration method that reduces theextraction time, cost, solvent consumption, and exposure to theorganic solvent (Ebrahimzadeh et al. 2007). An HLLE methodcan be defined as “using the phase separation phenomenon in ahomogeneous solution and simultaneously extracting the targetsolutes into the separated phase” (Igarashi and Yottuyanagi1992). In HLLE, the initial status is a homogeneous solution,namely the contact area between the aqueous phase and organicphase is extremely large and the equilibrium state is obtainedquickly; therefore, no vigorous mechanical shaking is necessary(Ghiasvand et al. 2005; Rezaee et al. 2006). In HLLE technique,phase separation can be based on salting-out phenomenon (withaddition of a salt or an auxiliary solvent), temperature, pH, orion-pair formation (Anthemidis and Ioannou 2009). HLLEbased on salting out (SHLLE) utilizes the salt-induced phaseseparation phenomenon, whereby the water-soluble organicsolvent is separated from the homogeneous solution and simul-taneously the target solutes are extracted into the separatedorganic phase when a salting-out reagent is added. Recently,HLLE has been utilized for the extraction of some organic andinorganic analytes (Rodila et al. 2006; Zhang et al. 2009; Liuet al. 2010; Zhao et al. 2012). Many polar organic solvents,which can be homogenized with water, such as acetonitrile,isopropanol, methanol, ethanol, acetone, and dimethyl sulfoxidecan be used as an extractant (Anthemidis and Ioannou 2009).The water-soluble organic solvents used by this extractiontechnique are usually non-halogenated and non-aromatic sol-vents and therefore thought to be more environmentally friendlythan solvents such as benzene, chloroform, and so on (Cai et al.2007). HLLE and SHLLE are different fromDLLME. DLLMEis a miniaturized sample pretreatment preconcentration tech-nique based on ternary component solvent system. But inHLLE and SHLLE, the homogeneous mixture of extractionsolvent and aqueous phase is produced prior to separation oftwo phases by adding either an auxiliary solvent or a salt. On theother hand, in DLLME a suspension is accomplished instantlyby adding rapidly themixture of disperser and extraction solventinto the aqueous solution and no homogeneous form is

appeared. Different methodologies such as coupling stir barsorptive extraction-dispersive liquid–liquid microextraction(Farajzadeh et al. 2010), air-assisted liquid–liquid microex-traction (Farajzadeh and Khoshmaram 2013), dynamicsingle-drop microextraction (Farajzadeh et al. 2011), disper-sive liquid–liquid microextraction (Farajzadeh et al. 2012a),and combination of solid-phase extraction–hollow fiber(Farajzadeh et al. 2012b) have been applied for the extractionof triazole pesticides from grape juice.

The aim of this work is to use the SHLLE method in a newmode performed in a narrow-bore tube before determinationof triazole pesticides in the grape juice samples by gas chro-matography–flame ionization detection (GC–FID). Longlength of the narrow-bore tube increases the contact areabetween salt and homogeneous solution, and an effectivephase separation results along with good extraction efficiency.Also, because of narrow diameter of the tube, the collection ofthe small volume of organic phase is feasible. In this study, thephase separation occurs by the addition of NaCl to a homo-geneous solution of acetonitrile and water. Then analytes areextracted into the produced fine droplets of extraction solvent(acetonitrile) which are collected on the surface of aqueousphase after a short time. The collected organic phase volume isat microliter level, and it is expected that the higher enrich-ment factors (EFs) are obtainable. The effect of various ex-perimental parameters was studied, and the performance ofpresented method for the analysis of real samples was tested.

Experimental

Reagents and Solutions

The studied pesticides were penconazole, hexaconazole,diniconazole, tebuconazole, triticonazole, and difenconazole.All pesticides had a purity of >98 % and were provided byGYAH Corporation (Karadj, Iran). Acetonitrile, methanol, ac-etone, and tetrahydrofuran (THF) as tested extraction solventswere obtained from Merck (Darmstadt, Germany). Also, sodi-um chloride, sodium hydroxide, and hydrochloric acid werepurchased from Merck. De-ionized water (Ghazi Company,Tabriz, Iran) was used for the preparation of standard solutionsand dilution of real samples. A stock solution of the studiedcompounds was prepared by dissolving six pesticides in aceto-nitrile to obtain a 1,000 mg L−1 solution (of each pesticide) andstored in a refrigerator at 4 °C. A standard solution containing200 mg L−1 of each pesticide in acetonitrile was prepared anddaily injected into the separation system (three times) so that thesystem (GC–FID) quality could be evaluated (repeatability andresponse of detector for daily peaks areas were considered).The obtained peaks areas were used in calculation of EFs anddetermination of the collected extraction solvent volume afterextraction as it has been explained in the related section.

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Real Samples

Three packed grape juice samples (Moneh, Mikhak, andSunich) and a fresh grape sample were purchased from localmarkets (Tabriz, Iran). Moneh, Mikhak, and Sunich sampleswere diluted with de-ionized water at ratios of 1:1, 1:1, and 1:5,respectively. In the case of fresh grape, 50 g was squeezedwithin a mortar and the supernatant was transferred into a testtube and centrifuged at 4,000 rpm for 5min. Then, it was dilutedwith de-ionized water at a ratio of 1:7. To determine dilutionratio of samples, they diluted at different ratios (1:1, 1:2, 1:3,…). Initially the analytes′ contents of the diluted samples wereevaluated by performing the proposedmethod. Then, the dilutedsamples along with de-ionized water spiked with analytes at asame concentration and proposed method was carried out onthem. The obtained analytical signals for both solutions werecompared. The dilution ratio in which the analytical signals arecomparable (<30 % differences) was selected as a suitable one.It is noted that analytes′ signals of unspiked samples weresubtracted in the cases of spiked samples. Also, it is noted thatdilution ratio were hold at low level due to obtaining relativelylow detection limits for themethod. pH of all samples was in therange of 6–8.

Apparatus

Separation and monitoring of the selected triazole pesticideswas carried out using a gas chromatograph (2014, Shimadzu,Japan) equipped with an FID. Helium (99.999 %, Gulf Cryo,United Arab Emirates) was used as the carrier gas at a constantlinear velocity of 30 cm s−1. Separation was carried out on a PT-5 capillary column (5 % diphenyl-95%dimethyl siloxane,30 m×0.25 mm i.d., and film thickness of 0.25 μm) (Supelco,Bellefonte, PA, USA). A splitless/split injector which was heldat a constant temperature of 290 °C with a sampling time of1 min was used. Samples were analyzed using the followingoven temperature programming: initial temperature 100 °C(held for 2 min), increased by 20 °C min−1 to 200 °C, thenincreased by 8 °Cmin−1 to 290 °C, and held at 290 °C for 6min.The FID temperature was maintained at 290 °C. Hydrogen gaswas generated with a hydrogen generator (OPGU-1500S,Shimadzu, Japan) for FID at a flow rate of 30 mL min−1. Theflow rate of air for FID was 300 mL min−1. Flow rate of makeup gas (helium) was 30 mL min−1. Gas chromatography–massspectrometry (GC–MS) analysis was carried out by a GC-MS-QP2010 plus (Shimadzu, Kyoto, Japan). The separation wascarried out on a ZebronTM ZB-5MS capillary column (20 m×0.18 mm i.d. and film thickness 0.18 μm) [poly (5 %-diphenyl-95 %-dimethylsiloxane)] (Phenomenex, Torrance, CA, USA).Data acquisition was performed in full scan mode from m /z 50to 350. Direct coupling capillary column to MS at 300 °C wasused. Ionization was done with an electron impact (temperature250 °C) mode at 70 ev, and the produced ions was separated by

a quadrupole mass analyzer. Helium was used as the carrier gasat a linear velocity of 40 cm s−1. The oven temperature pro-gramming was the same as GC–FID analysis mentioned above.A D-7200 centrifuge from Hettich (Kirchlengern, Germany)was used for preparation of fresh grape juice for extraction.An ultrasonic bath (Zyklusmed, Germany) was used.

HLLE Procedure

The set-up included a narrow-bore glass tube (100 cm×5 mmi.d.). One end of the tube was closedwith a septum. Initially, theinner wall of the tube was cleaned by filling it with 3M sodiumhydroxide solution. After 30 min, it was rinsed thoroughly withde-ionized water. This prevents the fine droplets of extractionsolvent to adhere to the inner wall of the tube during extraction.The prepared homogenous solution (18 mL) containing aceto-nitrile (3.1mL) as extraction solvent (17.3%, v /v) in the dilutedsample solution or standard solution (14.9 mL) of the targetanalytes was filled into the tube using a 20-mL syringe. Then,5.4 g of NaCl was added to the solution from top of the tube. Itis noted that NaCl was grained manually in a mortar beforeusing. During addition of NaCl to the solution, many finedroplets of acetonitrile were formed. The lower end of tubewas placed into an ultrasonic bath and after sonication for5 min, almost all of the fine droplets reached the top of thetube and floated on the surface of aqueous phase as a separatelayer due to lower density of acetonitrile compared to its ofwater. During this step, the analytes were extracted into the finedroplets. It is noted that the volume of collected acetonitrile was17±3 μL. Finally, a portion of the gathered phase was easilyremoved by a capillary tube (glass capillary tube, 100 mmlength and 1.5 mm o.d., Electrothermal, Denmark) and 1 μLof it was injected into the GC–FID or GC–MS for analysis. Theextraction procedure is shown as a schematic in Fig. 1.

Calculation of EF

EF is defined as the ratio between the analyte concentration inthe collected phase (C coll) and the initial concentration ofanalyte (Co) in aqueous sample.

EF ¼ Ccoll=Co ð1Þ

C coll was obtained from comparison of analytes peaksareas in two cases: direct injection of standard solution(200 mg L−1 of each pesticide) and injection of the collectedphase after performing the proposed method.

Results and Discussion

In order to obtain the optimum SHLLE conditions for thedetermination of triazole pesticides in aqueous samples,

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different experimental parameters including kind and volume ofthe water-soluble organic solvent, amount of salt, length anddiameter of tube, and pH of sample solution on the performanceof SHLLE should be optimized.

Water-Miscible Organic Solvent Nature

The selection of an appropriate extraction solvent is verycritical for an SHLLE procedure. In this method, organicsolvent is selected based on its miscibility with aqueous phase,the ability of two-phase system formation after adding a phaseseparation agent, density lower than water, extraction capabil-ity of the interested compounds, and less toxicity. Based onthese facts, acetonitrile (density, 0.786 g mL−1), acetone (den-sity, 0.791 g mL−1), methanol (density, 0.792 g mL−1), andTHF (density, 0.889 g mL−1) were chosen and tested as theextraction solvent and the phase separation was performed inthe tube with dimensions 70 cm×5 mm i.d. with a capacity of12 mL. The experimental results revealed that among thetested solvents, acetonitrile and THF can be quickly separatedfrom aqueous phase, while other solvents cannot form thetwo-phase system after addition of 30%,w /v, NaCl as a phaseseparator reagent. Due to high volatility of THF and its adhe-sion to the inner wall of the tube after sonication, low repeat-able results were obtained in this case. Thus, acetonitrile wasselected as the extraction solvent for the subsequent experi-ments. Also, the experiments showed that phase separationcan be achieved at less than 20 %, v /v, acetonitrile in water.

Study of Acetonitrile Volume

The volume of extraction solvent can affect the repeatability ofresults and extraction efficiency. Extraction solvent volume was

studied in the range of 17.0–19.0 %, v /v. The volume of thegathered phase (17, 35, 90, 150, and 390 μL for 17.3, 17.7 18.0,18.4, and 19.0 % (v/v), respectively) and amounts of the ex-tracted analytes increased by increasing the volume of acetoni-trile, but dilution caused the analytical signals to be decreased athigh volumes of extractant. On the other hand, at less than17.3 %, v /v, acetonitrile the phase separation was not accom-plished or the boundary between acetonitrile and the aqueousphase was unclear. Therefore, the removal of organic phase bythe capillary tubewas difficult. Thus, as shown in Fig. 2, 17.3%,v/v, acetonitrile was selected as a suitable volume in this study.

The Influence of Sodium Chloride Amount on the PhaseSeparation

Sodium chloride is an efficient and common salt to inducephase separation of acetonitrile from aqueous phase. InSHLLE, salt concentration has a great influence on the phaseseparation. In most cases, high concentration of a salt wouldenhance the phase separation and increase the extraction per-formance of analytes (Majors 2009; Anastassiades et al. 2003).Here, the amount of sodium chloride was studied in the rangeof 3.0–4.8 g, and the experimental results are shown in Fig. 3.The analytical signals increased by increasing amount of NaClup to 3.6 g and then decreased due to dilution effect withincreasing the collected phase volume. Consequently, 3.6 gNaCl was selected for the subsequent experiments. It is notedthat at lower than 3.0 g NaCl, phase separation did not occur.

Effect of pH

In all extraction methods, pH is an important parameter for theanalytes which are acidic or basic. Also, the hydrolysis of

Fig. 1 Scheme of salting-out homogeneous liquid–liquid extraction pro-cedure. (a) The narrow-bore tube filled with the mixture of acetonitrileand aqueous solution of analytes, (b) formation of cloudy solution after

addition of sodium chloride, and (c) removal of a portion of extractant(acetonitrile) by a capillary tube after ultrasonication in order to inject intothe separation system

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analytes in very acidic or alkaline medium is possible bywhich the extraction efficiency can be disturbed in thosesolutions. Additionally, pH has another role in HLLE becauseit may be effective in phase separation step. Therefore, in thisstudy, the effect of pH on the extraction efficiency of theanalytes was examined in the range of 2–12 adjusted by HClor NaOH 1 M solutions. As shown in Fig. 4, the higheranalytical signals were obtained at pHs 4–8. At pH valueshigher than 8 and lower than 4, the analytical signals for thetested pesticides were decreased. Decreasing in extractionefficiency of the target analytes can be attributed to the hy-drolysis of pesticides in highly acidic or alkaline pHs. On thebasis of these results, pH values in the range of 4–8 can beused for the further analysis. It should be noted that pH of allsamples used in this study was within the range of 6–8 andhence pH adjustment was not required.

Optimization of Sample Volume

For this purpose, the SHLLE procedure was performed inthree tubes having different volumes (12, 18, and 43 mL):two tubes with different lengths (70 and 100 cm) and aconstant i.d. (5 mm) and one tube with 100 cm length andi.d. of 7 mm. The results (Fig. 5) showed that by increasing thevolume of aqueous phase, the analytical signals were alsoincreased. But in the case of the tube with 7 mm i.d., removalof the collected organic phase from the surface of aqueousphase was difficult. Also, because of less sample and extrac-tion solvent consumption, the tube with dimensions 100 cm×5 mm i.d. with a capacity of 18 mL was selected for thefollowing experiments.

0

500000

1000000

1500000

2000000

2500000

3000000

2 4 6 8 10 12

Pea

k ar

ea

pH

PenconazoleHexaconazoleDiniconazoleTebuconazoleTriticonazoleDifenconazole

Fig. 4 Effect of sample solution pH on the extraction efficiency. Extrac-tion conditions: the same as Fig. 3, except 3.6 g NaCl was used. The errorbars indicate the maximum and minimum of three determinations

0

1000000

2000000

3000000

4000000

5000000

6000000

12 (70) 18 (100) 43 (100)

Pea

k ar

ea

Sample volume, mL (Tube length, cm)

PenconazoleHexaconazoleDiniconazoleTebuconazoleTriticonazoleDifenconazole

Fig. 5 Effect of sample volume on extraction efficiency. Extractionconditions: the same as Fig. 4. The i.d. of the first two tubes was 5 mm,and the last one was 7 mm. In order to remain NaCl concentrationconstant, its amount was 3.6, 5.4, and 12.9 g, respectively. The errorbars indicate the maximum and minimum of three determinations

Fig. 3 Optimization of salt amount. Extraction conditions: as the same asFig. 2, except 17.3 % (v /v) acetonitrile was used. The error bars indicatethe maximum and minimum of three determinations

Fig. 2 Optimization of extraction solvent volume on the extractionefficiency. Extraction conditions: aqueous sample volume, 12 mL (nar-row-bore tube dimensions, 70 cm×5 mm i.d.); salt amount, 3 g; andanalytes concentrations, 2 mg L−1 of each pesticide. The error barsindicate the maximum and minimum of three determinations

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Table 1 Quantitative features of the proposed method for the selected pesticides

Analyte LRa (μg L−1) rb LODc (μg L−1) LOQd (μg L−1) RSD%e

EF ± SDf

Penconazole 6–5,000 0.9994 1.3 4.5 3.0 318±17

Hexaconazole 6–5,000 0.9994 1.2 3.9 2.9 305±6

Diniconazole 3–5,000 0.9989 0.60 1.9 2.2 351±18

Tebuconazole 12–5,000 0.9984 1.4 4.5 5.4 319±30

Triticonazole 24–5,000 0.9989 4.0 13 2.1 261±9

Difenconazole 24–5,000 0.9989 4.8 16 2.2 389±16

a Linear rangeb Correlation coefficientc Limit of detection, S /N =3d Limit of quantification, S /N =10e Relative standard deviation (100 μg L−1 , n =6)fMean enrichment factor ± standard deviation, n =3

Table 2 Study of matrix effect in samples spiked at different concentrations

Analyte Mean relative recoverya (%) ± standard deviation (n =3)

Packed grape juice

Moneh Mikhak Sunich Fresh grape juice

All samples were spiked with each analyte at a concentration of 250 μg L−1.

Penconazole 83±4 72±2 85±10 77±6

Hexaconazole 85±7 75±3 81±8 75±3

Diniconazole 80±5 70±7 79±7 75±6

Tebuconazole 89±3 80±7 77±5 76±7

Triticonazole 72±7 71±2 76±6 80±9

Difenconazole 70±6 75±3 71±4 73±7

All samples were spiked with each analyte at a concentration of 500 μg L−1.

Penconazole 94±3 78±1 81±3 85±2

Hexaconazole 90±2 74±2 83±8 83±2

Diniconazole 89±5 77±2 86±6 84±3

Tebuconazole 88±3 79±4 99±5 84±9

Triticonazole 78±6 109±3 92±7 82±6

Difenconazole 86±7 80±5 90±9 79±5

All samples were spiked with each analyte at a concentration of 1,000 μg L−1.

Penconazole 95±1 97±3 96±2 91±4

Hexaconazole 93±3 92±4 84±4 87±2

Diniconazole 91±2 90±2 86±4 83±3

Tebuconazole 89±6 97±3 83±3 85±4

Triticonazole 88±7 106±6 85±4 88±5

Difenconazole 90±5 88±5 92±2 86±6

In all cases, peak areas were obtained after performing the proposed method and injection of the enriched analytes into GC-FIDa Relative recoveries were calculated from the following equation: RR% ¼ A1−As

A2� 100

RR%=Relative recovery

A1=Peak area of analyte in spiked samples

As=Peak area of analyte in unspiked samples

A2=Peak area of analyte in spiked de-ionized water

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Determination of the Gathered Organic Phase VolumeAfter Extraction

Determination of organic phase volume gathered on the surfaceof aqueous sample after extraction was performed based on thephase separation of an analyte-free blank solution (18 mLsolution containing 17.3 %, v /v, acetonitrile in water in which5.4 g NaCl was added). Then, 5 μL (Vadded) of 200 mg L−1

standard solution of each tested pesticide in acetonitrile wasadded to the gathered organic phase on top of the tube and 1μLof it injected into the separation system. In order to calculate thevolume of extraction solvent (Vcoll) after extraction, the follow-ing equation was used:

V coll ¼ Aext=Adirð Þ � V added½ �−V added ð2Þ

where A dir is the peak area of each pesticide after directinjection of 1 μL of their standard solution (200 mg L−1 inacetonitrile) into GC, and A ext is the peak area of each pesti-cide in the obtained chromatogram after addition of theirstandard solution (5 μL) into the collected phase on top ofthe tube (obtained from blank). The calculated gathered phasevolume after extraction was 17±3 μL (n =3) based on Eq. 2.

Evaluation of the Method Performance

To assess the analytical characteristics of the method, somequantitative parameters including linear range of calibrationgraphs (LR), correlation coefficients (r), limits of detection(LOD), limits of quantification (LOQ), relative standard devi-ations (RSD), and EFs were investigated. Table 1 summarizessome analytical characteristics of the optimized method. Asgiven in this table, LRs are in the ranges of 6–5,000 μg L−1

for penconazole and hexaconazole, 3–5,000 μg L−1 fordiniconazole, 12–5,000 μg L−1 for tebuconazole, and24–5,000 μg L−1 for difenconazole and triticonazole withcorrelation coefficients in the range of 0.9984–0.9994. LODsand LOQs for the tested pesticides are in the ranges of 0.60–4.8and 1.9–16 μg L−1, respectively. Also, the repeatability studywas performed at a concentration of 100 μg L−1 of eachpesticide and the RSD percentages were obtained in the rangeof 2.1 – 5.4 % for six repeated determinations. High EFsranging from 261 to 389 were obtained. Wide linear ranges,low LODs and LOQs, and high EFs are the main advantages ofthe proposed method.

Real Sample Analysis

Three packed grape juice samples from different pro-ducers and one fresh grape sample were bought fromlocal markets (Tabriz, Iran), and the proposed methodwas performed on them. Initially, these samples werespiked with the pesticides standard solutions at differentconcentrations, diluted, and then mixed with acetonitrileto assess their matrix effects. Due to strong matrixeffects and low recovery values in samples, all of themwere diluted with de-ionized water at different ratiosbefore applying the method on them (as described in“Experimental”). By this action, matrix effects in thementioned samples reduced considerably. The relativerecoveries, obtained in comparison with de-ionized wa-ter spiked at the same levels, are summarized inTable 2. Typical GC–FID chromatograms of Monehgrape juice (before and after spiking at 500 μg L−1 ofeach pesticide) after performing the proposed method onthem are shown in Fig. 6. As it can be seen from the

Fig. 6 GC-FID chromatogramsof (a) unspiked Moneh grapejuice and (b) spiked Moneh grapejuice with 500 μg L−1 of eachpesticide. In both cases, thepreconcentration technique underthe optimized conditions wasperformed on them and 1 μL ofthe collected organic phase wasinjected into GC. Peaksidentification: 1 penconazole,2 hexaconazole, 3 diniconazole,4 tebuconazole, 5 triticonazole,and 6 difenconazole

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chromatogram, one peak is observed in the retentiontime belonging to penconazole in Moneh sample.Other samples were free of the target analytes. In orderto verify the presence of the mentioned pesticide inMoneh sample, it was injected into GC–MS system.As shown in Fig. 7, the presence of penconazole wasverified in Moneh sample. Its concentration was

calculated using GC–FID data and was 63±8 (n =3)μg kg−1. It is noted that European Commission (ECCommission Regulation 149/2008) has proposedpenconazole 50 μg kg−1 as maximum limit residue(MLR) for penconazole in grape. By considering thissubject, penconazole was found higher than MLR inMoneh sample

Table 3 Comparison of the proposed method with the other methods used in triazole pesticides determination

Method LODa LRb RSDc (%) Extractiontime (min)

EFd Sample Reference

DSDME–GC–FIDe 2–112 μg L−1 10–5,000 μg L−1 2.9–4.5 17 141–214 Grape juice Farajzadeh et al. (2011)

DLLME–SFO–HPLC–DADf

0.06–0.08 μg L−1 0.5–200 μg L−1 4.6–5.7 >9 190–450 Environmentalwater

Wang et al. (2011)

SPE–HF–GC–FIDg 0.6–4.5 μg L−1 2–15 μg L−1 4.5–8.7 60 870–950 Grape juice Farajzadeh et al. (2012b)

SPE–GC–MSh 3–5 ng g−1 10–200 ng g−1 – >30 – Crops Balinova et al. (2007)

SPE–GC–ECDi 0.5–4.0 ng g−1 5–200 ng g−1 – >30 – Crops Balinova et al. (2007)

SHLLE–GC–FID 0.6–4.8 μg L−1 3–5,000 μg L−1 2.1–5.4 10 261–389 Grape juice This method

a Limit of detectionb Linear rangec Relative standard deviationd Enrichment factore Dynamic single drop microextraction–gas chromatography–flame ionization detectorf Dispersive liquid–liquid microextraction–solidification of floating organic droplet–high performance liquid chromatography–diode array detectorg Solid-phase extraction–hollow fiber–gas chromatography–flame ionization detectionh Solid-phase extraction–gas chromatography–mass spectrometryi Solid-phase extraction–gas chromatography–electron capture detector

Fig. 7 Total ions currentchromatogram of Moneh sample(a), mass spectra of penconazole(b) and scan 5934, retention time12.888 min (c)

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Comparison of the Proposed Method with Others

Table 3 summarizes LOD, LR, RSD, extraction time,and EF of the presented method and other methods forextraction and determination of the selected pesticides.The proposed method has LODs comparable or lowerthan those of the other methods in which GC–FIDsystem was used (DSDME–GC–FID, SPE–HF–FID). Itshould be noted that in some of the techniques men-tioned, two preconcentration steps or a high sensitivedetection system such as MS or electron capture detec-tor were used which are inherently more sensitive thanFID. In comparison with the other methods, the presentmethod has wide LRs and a short extraction time. RSDpercentage of the method is better than or comparablewith those of other mentioned methods. EFs of themethod are better than those of the other mentionedmethods (except SPE–HF–GC–FID). These results re-veal that the proposed method is rapid, sensitive, effi-cient, and easy to use and it can be utilized for thepesticide residues preconcentration from aqueoussamples.

Conclusion

This paper describes an SHLLE method performed in anarrow-bore tube for the extraction/preconcentration of sometriazole pesticides in the aqueous samples prior to GC–FIDdetermination. This method is based on the phase separationof 17.3 % (v /v ) acetonitrile in water containing target analytesin the presence of 30 %, w /v, NaCl in a narrow-bore tube(dimensions, 100 cm×5 mm i.d). This method applied aceto-nitrile, a green chemistry solvent, as an extractant instead ofchlorinated or aromatic solvents used in most extractivemethods. Compared to the other conventional sample prepa-ration methods, it offers advantages such as a relatively shortanalysis time and high EFs (ranging from 261 to 389). Thedeveloped method is considered to be economical, facile, andsuitable for preconcentration, separation, and determination oftriazole pesticides at micrograms-per-liter levels in aqueoussamples such as grape juice.

Acknowledgments The authors thank the Research Council ofUniversity of Tabriz for financial support and Gyah Corporation (Karaj,Iran) for preparing standard triazole fungicides.

Conflict of Interest Mir Ali Farajzadeh has received research grantsfrom University of Tabriz. Saheleh Sheykhizadeh declares that she has noconflict of interest. Parisa Khorram declares that she has no conflict ofinterest. This article does not contain any studies with human or animalsubjects.

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