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Journal of Chromatography A, 1147 (2007) 135–143

Analysis of carbamate and phenylurea pesticide residues in fruit juices bysolid-phase microextraction and liquid chromatography–mass spectrometry

Gianni Sagratini a,∗, Jordi Manes b, Dario Giardina a, Pietro Damiani c, Yolanda Pico b

a Dipartimento di Scienze Chimiche, Facolta di Farmacia, Universita di Camerino, via S.Agostino 1, 62032 Camerino, Italyb Laboratori de Bromatologia i Toxicologia, Facultat de Farmacia, Universitat de Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjassot, Valencia, Spain

c Istituto di Chimica Bromatologica, Facolta di Farmacia, Universita di Perugia, via S.Costanzo, 06100 Perugia, Italy

Received 22 December 2006; received in revised form 14 February 2007; accepted 19 February 2007Available online 24 February 2007

bstract

A new analysis method to detect carbamates and phenylurea pesticide residues in fruit juices was developed using solid-phase microextractionSPME) coupled with liquid chromatography–single quadrupole mass spectrometry (LC/MS) and liquid chromatography–quadrupole ion trapass spectrometry (LC/QIT-MS). The pesticide residues present in watery matrices as fruit juices were extracted using three types of fibers: 50-m Carbowax/templated resin (CW/TPR), 60-�m poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) and 85-�m polyacrylate. The differentxtraction conditions were evaluated choosing as the best parameters 90 min (time), 20 ◦C (temperature) and 1 ml (volume). After extraction, theesorption (in a static mode) was performed in the specific interface chamber SPME/HPLC, previously filled with 70% methanol and 30% water.he best recoveries, evaluated at two fortification levels (0.2 and 0.5 mg kg−1) in fruit juices, were obtained using PDMS/DVB and CW/TPRbers, and ranged from 25 to 82% (monolinuron, diuron and diethofencarb), with relative standard deviations (RSDs) from 1 to 17%. All the

imits of quantification (LOQs) were in the range of 0.005–0.05 �g ml−1 and, in any case, equal to, or lower than, maximum residue limits (MRLs)stablished by Italian and Spanish legislations. The mass spectrometry analyses were carried out using an electrospray ionization (ESI) source

perating in the positive mode both for single quadrupole and for QIT mass analysers, operating in selected ion monitoring (SIM) and in multipleeaction monitoring (MRM) modes, respectively. The proposed new method can be applied to the determination of selected pesticides in realamples of fruit juices.

2007 Elsevier B.V. All rights reserved.

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eywords: Food analysis; Fruit juices; Solid-phase microextraction; Liquid chr

. Introduction

The evaluation of pesticide residues in food is nowadays ariority objective to ensure food quality and to protect consumersgainst possible health risks. As the residues of pesticides arerequently found in citrus fruit and grapes, they may easily beransferred into relative juices and become a worrying risk foronsumer health.

In order to obtain a practical and rapid method for determin-ng pesticide residues in food complex matrices, several sample

reparation methods were developed, including liquid–liquidxtraction (LLE) [1], supercritical fluid extraction (SFE) [2],olid-phase extraction (SPE) [3] and solid-phase microextrac-

∗ Corresponding author. Tel.: +39 0737402344; fax: +39 0737637345.E-mail address: gianni.sagratini@unicam.it (G. Sagratini).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.02.066

ography–mass spectrometry; Pesticide residues

ion (SPME) [4]. However, in LLE and SPE, big disadvantagesre the large quantities of solvent utilized, the multiple opera-ion steps needed, the preconcentration of the extract requiredrior to analysis and the interfering compounds that are moreikely to be coextracted. SPME, introduced in 1990 by Arthurnd Pawliszyn [5], is an extraction technique using a fused silicaber externally coated with an appropriate stationary phase. Itonstitutes a convenient alternative extraction method becauset integrates sampling, extraction, concentration and samplentroduction into a single step without the use of solvents. Its usually combined with GC, GC/MS and HPLC, HPLC/MSor determining a wide variety of compounds, including pesti-ides, agrochemicals and other contaminants in food samples

4,6–8].

Nowadays, liquid chromatography coupled with masspectrometry (LC/MS) is one of the most powerful techniquesor the analysis of pesticides in fruit and vegetables [9,10].

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n particular, it has been shown that, in combination withandem mass spectrometry (ion trap or triple quadrupole), LCs a very sensitive technique to reveal pesticide residues inruits [11]. Some methods using the SPME technique and ahromatographic routine with MS detection or flame thermionicetection (FTD) have been developed to discover and quantifyesticide residues in watery matrices, as organophosphorus inruit juices by SPME/GC/FTD/MS [12–14], some carbamatesnd phenylureas in water and wine by SPME/LC/MS [15], andolar fungicides in fruit by SPME/LC/MS [4].

The aim of the present work was to develop a new analyticalethod for determination of various carbamate and phenylurea

esticides in different types of fruit juices using SPME/LC/MSnd SPME/LC/MS/MS that, till to this day, are not documentedn literature. The study of the MS–MS transitions of the analysedesticides in the proposed method meets the requirements ofonfirmatory criteria, established by the European Union [16].

In particular, we investigated two different classes of commonesticides: (a) carbamates such as carbosulfan, benfuracarb, car-ofuran, pirimicarb (insecticides) and diethofencarb (fungicide)nd (b) phenylurea such as diuron, monuron and monolinuronherbicides).

Due to their high solubility in water [17], carbamate insec-icides, mainly used in agriculture, are distributed in aqueousood such as fruit and related derivatives. Phenylurea herbicidesre widely used in pre- and post-emergency situations in cotton,ruit or growing cereal [18]. Diethofencarb is a very effectiveungicide for controlling various fungal species, such as Botry-is spp., Cercospora spp. and Venturia spp., that are resistant toenzimidazole fungicides [19].

. Experimental

.1. Materials and standards

Carbamates (carbosulfan, benfuracarb, carbofuran, pirim-carb, diethofencarb) and phenylurea (diuron, monuron and

onolinuron) were supplied by Riedel-de Haen (Seelze, Ger-any). Individual stock solutions were prepared by dissolving

00 mg of each compound in 100 ml of methanol and were storedn glass-stopper bottles at 4 ◦C. Standard working solutions atarious concentrations were daily prepared by appropriate dilu-ion of aliquots of the stock solutions in methanol.

HPLC-grade methanol was supplied by Merck (Darmstadt,ermany), and sodium chloride (99.5%) by Panreac (Barcelona,pain). Deionized water (<8 M� cm resistivity) was obtainedrom the Milli-Q SP Reagent Water System (Millipore, Bedford,

A, USA). All the solvents and solutions were filtered through a.45-�m cellulose filter from Scharlau (Barcelona, Spain) beforese.

Fruit juices were centrifuged in 10-ml tubes using an Eppen-orf 5804 Centrifuge (Hamburg, Germany) and were filteredhrough a 25-mm/0.45-�m nylon filter purchased from Analisis

inicos (Tomelloso, Spain).

The manual fiber holders for SPME and the SPME/HPLCnterface were purchased from Supelco (Bellefonte, PA, USA)s were the three types of tested fibers: 50-�m Carbowax/

PceO

r. A 1147 (2007) 135–143

emplated resin (CW/TPR), 60-�m poly(dimethylsiloxane)/ivinylbenzene (PDMS/DVB) and 85-�m polyacrylate. Theew fibers were conditioned in methanol with stirring for 30 min,nd the used ones were cleaned in methanol with stirring for0 min before new extraction. The SPME/HPLC interface, usedor injection of analytes into the column, consists of a six-portnjection valve and a desorption chamber replacing the injectionoop of the HPLC system [4].

.2. Sample preparation

All the fruit juices (orange, apple, cherry and strawberry),roduced by different companies, were purchased from localarkets of the Valencia community. Fruit juice samples were

tored at room temperature before use. Once opened, they weretored in the specific food containers at 4 ◦C and analysed withindays.A 7-ml aliquot of fresh juice was centrifuged at 4000 r.p.m.

or 15 min, and then the supernatant liquid portion was filteredhrough a 0.45-�m nylon filter. Before extraction, 0.5 ml of theuice sample was diluted with 0.5 ml of water in a 1.5-ml screwap vial containing 0.3 g of NaCl and a small magnetic stirrerar.

Extraction recoveries were determined by spiking 5–10 �l ofroperly diluted fresh juices with standard pesticide mixtures atwo concentration levels – 0.2 and 0.5 mg kg−1.

.3. SPME procedure

The SPME fiber was directly immersed in the sample solu-ion with continuous stirring at 1000 r.p.m. for different timeeriods (30–120 min). To achieve the best extraction conditions,valuating parameters such as time, temperature and sample vol-me, the desorption was, at first, carried out introducing the fibernto a 1.5-ml screw cap vial that contains 1 ml of methanol inontinuous magnetic stirring (1000 r.p.m.) for 15 min.

After this, the desorption was performed in a static mode:he fiber was placed into the desorption chamber, which hadeen filled with methanol/water (70:30 v/v), for 15 min usinghe same procedure as that reported in a previous work [4].

.4. LC/MS and LC/MS/MS analysis

Separation was achieved on a Luna C18 analytical column150 mm × 4.6 mm I.D., 5 �m) preceded by a C18 securityuard cartridge (4 mm × 2 mm I.D.), both from PhenomenexCheshire, UK). The mobile phase was methanol–water at a flowate of 0.5 ml min−1. The solvent composition varied from 80%ethanol for the first 5 min to 95% methanol from 5 to 30 min.he separation conditions were the same for the two LC/MSpparatuses used: the single quadrupole and the quadrupole ionrap.

Liquid chromatography was performed using a Hewlett-

ackard (Palo Alto, CA, USA) HP-1100 Series LC/MSD systemonsisting of an autosampler, a binary solvent pump and a MSDquipped with an ESI interface in positive ionization (PI) mode.ptimization of the LC/MS conditions was carried out by vary-

G. Sagratini et al. / J. Chromatogr. A 1147 (2007) 135–143 137

Table 1Structure and molecular and fragment ions obtained by LC/ESI-MS

Compound (MW) Structure Time (min) SIM ion (m/z) Main ions (m/z)(relative abundance)

Confirmatory ions(m/z)

Carbofuran (221.25) 5.75 222.1 222.1 [M + H]+ (100) 165.1[M–CONCH3]+165.1 [M–CONCH3]+

(99)

Monuron (198.65) 6.06 199.0 199.0 [M + H]+ (100)221.0 [M + Na]+ (88)

Pirimicarb (238.29) 6.74 239.4 239.4 [M + H]+ (100)261.1 [M + Na]+ (64)

Monolinuron (214.65) 6.76 215.0 215.0 [M + H]+ (100)

237.0 [M + Na]+ (100)

Diuron (233.09) 7.80 233.0 233.0 [M + H]+ (100)255.0 [M + Na]+ (86)

Diethofencarb (267.32) 7.93 268.1 268.1 [M + H]+ (89) 226.1[M–C(CH3)2]+226.1 [M–C(CH3)2]+

(100)

Benfuracarb (410.53) 13.44 411.0 411.0 [M + H]+ (81) 190[M–C9H19N2O2S]+190.0

[M–C9H19N2O2S]+

(100)

Carbosulfan (380.54) 19.12 381.2 381.2 [M + H]+ (100)

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38 G. Sagratini et al. / J. Chro

ng them in flow injection analysis (FIA) of the analytes (20 �lf a 50-�g ml−1 individual standard solution). The optimizedarameters of the interface were: vaporizer temperature, 325 ◦C;ebulizer gas (nitrogen) pressure, 10 psi; drying gas (nitrogen)ow rate, 7 ml min−1; temperature, 250 ◦C; capillary voltage,000 V; fragmentator, 90 V; gain, 3. Time scheduled conditionsor monitoring pesticides are reported in Table 1.

The MS/MS study was performed using an Esquire 3000quipped with an ESI source operating in positive ionizationode. The mass spectrometer was tuned for each compound,

ptimizing the ionization source parameters, voltages of theenses and trap conditions in the ExpertTune mode of thealtonic Esquire Control software, whilst infusing a stan-ard solution (10 �g ml−1) via a syringe pump at a flow ratef 4 �l min−1, which was mixed with the mobile phase at.5 ml min−1 by means of a T-piece. Operating conditions ofhe source were the same.

The mass spectrometer was run in full scan and MRM modes.ositive ions were detected using the standard scan at normalesolution (scan speed 10 300 m/z/s; peak with 0.6 FWHM/m/z).he trap parameters were set in ion charge control (ICC) using

olling averaging set at 2 with a target of 20 000, and max-mum accumulation time of 50 ms at m/z range from 50 to00 u. To detect pesticides, the following time program wassed: transitions for carbofuran, monuron, pirimicarb, monolin-ron, diuron and diethofencarb were monitored from 0 to 10 min,hereas those for benfuracarb and carbosulfan were monitored

rom 10 to 15 min and from 15 to 30 min, respectively. Theragments, fragmentation and time conditions are reported inable 2.

. Results and discussion

.1. Liquid chromatography–mass spectrometry

Chemical structures and molecular weights of the studiedompounds together with the main ions obtained by LC/ESI-S operating in positive polarity, with their relative abundances

nd tentative assignation, are shown in Table 1.For each pesticide, the ions monitored in selected ion mon-

toring (SIM) (Table 1) were the base peak of the mass

pectrum, corresponding to the protonated molecule [M + H]+.or monuron, pirimicarb, monolinuron and diuron, the secondost abundant fragment was the sodium adduct [M + Na]+. For

arbofuran, we observed, in agreement with what is reported in

dptr

able 2ransitions and conditions used by quadrupole ion trap (QIT)

ompound Transition Cut-off

arbofuran 221.9 → 164.9 70onuron 198.0 –

irimicarb 238.9 → 182.0 80onolinuron 214.8 → 147.9 70iuron 232.8 → 159.0 80iethofencarb 267.9 → 225.9 90enfuracarb 411.0 → 251.8 100arbosulfan 381.2 → 159.9 120

r. A 1147 (2007) 135–143

he literature [20], the loss of an isocyanate fragment (−57 amu)nd the consequent formation of a second most abundant frag-ent with m/z 165.1. For diethofencarb, we recorded the loss

f an isopropyl group (−42 amu) which generated the principalragment ion with m/z 226.1, while in the case of benfuracarbhe principal ion with m/z 190 was obtained by loss of the m/z21 fragment from the [M + H]+ at m/z 411. For carbosulfan, webserved only the presence of the protonated form.

The base peak of the mass spectrum was subjected to the firsttage of MS/MS for all studied compounds. The MS2 analysisf tested pesticides with the ESI interface in positive polarityrovided ions for all of them except for monuron (see Table 2).he product ions monitoring the transitions of carbofuran andiethofencarb by QIT (m/z 164.9 and m/z 225.9, respectively)isplayed the same abundant ions observed in mass spectra bySI-MS in single quadrupole (m/z 165.1 and m/z 226.1, respec-

ively). On the contrary, for benfuracarb the MS/MS transition ofolecular ion (m/z 411) produced an ion at m/z 251.8, which was

ifferent from the most abundant fragment (m/z 190) observedn the single quadrupole.

.2. Optimization of SPME conditions

To improve the reproducibility and quantification of SPME,areful control and monitoring of some extraction parametersre required, e.g., salt addition, time, temperature and volume13]. In this work, we studied and optimized all four variables,y testing first the CW/TPR fiber in orange juice.

The salt addition was the first studied parameter because itan affect the recovery performance [4]. It was found that theddition of 30% of NaCl to the fruit juice improved the percentf pesticide recovery as result of reduced solubility and a con-emporary increase of fiber performance, according to previouseported experiments [4]. In consequence of these results, allollowing studies in fruit juice samples were performed afterddition of 30% of NaCl.

To study the influence of temperature in the extraction pro-ess of the fiber, the vial containing 1 ml of the spiked fruituice was placed in a water bath, and then the right temperatureas set using an electric vertex. After 15 min equilibration of

he vial temperature with the external bath, the fiber was intro-

uced in the vial through the cap septum and the extractionerformed for 90 min. The following five different tempera-ures were tested: 20, 30, 40, 50 and 60 ◦C. The results areeported in Fig. 1. For most pesticides, 20 ◦C proved to be the

Amplitude Width Time program (min)

1.50 4.0 0–10– 4.0 0–101.80 4.0 0–101.80 4.0 0–102.50 4.0 0–101.00 4.0 0–100.80 4.0 10–151.00 4.0 15–30

G. Sagratini et al. / J. Chromatogr. A 1147 (2007) 135–143 139

Fig. 1. Effect of temperature on extraction capacity of the 50-�m Car-bowax/templated resin fiber (CW/TPR), evaluated in orange juice spiked at0t

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Fig. 2. Effect of time on extraction capacity of the 50-�m Carbowax/templatedresin fiber (CW/TPR), evaluated in orange juice spiked at 0.5 mg kg−1 with amixture of pesticides. All experiments were performed in triplicate with RSDvalues ranging from 5 to 15%.

TP

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P

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.5 mg kg−1 with a mixture of pesticides. All experiments were performed inriplicate with RSD values ranging from 3 to 12%.

est extraction temperature, except diethofencarb, which dis-layed a different behaviour with a maximum recovery at 40 ◦C.onolinuron, diuron and diethofencarb went from 20 to 60 ◦C,

hereby displaying a greater drop in recovery than the other pes-icides. Pirimicarb showed the least ability for extraction withhe CW/TPR fiber in the whole range of temperatures tested. Inhese experiments, significant modifications of the chromato-raphic peak structure were observed at 50–60 ◦C for someesticides.

To investigate the time required for reaching an equilib-ium between the stationary phase (fiber), sample solution (fruituice, 1 ml) and the studied analytes, four different extractionimes, i.e., 30, 60, 90 and 120 min, were tested at 20 ◦C. Theesults, reported in Fig. 2, indicated that the best recoveriesere obtained with 90 min of equilibration time for all pesti-

ides, except for carbofuran and diethofencarb, for which the

est equilibrium conditions were 60 and 120 min, respectively.lso in this experiment, the fiber displayed the lowest ability to

xtract the pesticide pirimicarb at all tested times.

Fig. 3. Effect of volume on extraction capacity of the 50-�m Car-bowax/templated resin fiber (CW/TPR), evaluated in orange juice spiked at0.5 mg kg−1 with a mixture of pesticides. All experiments were performed intriplicate with RSD values ranging from 2 to 10%.

able 3ercent recovery and repeatabilitya of the method using PDMS/DVB fiber in water and fruit juices spiked at two fortification levels

esticide % Recovery

ompound Concentration (mg kg−1) Water Orange Strawberry and cherry Apple

arbofuran 0.5 34 37 41 440.2 36 39 40 46

onuron 0.5 25 19 19 180.2 27 20 22 21

irimicarb 0.5 42 3 2 20.2 40 2 2 3

onolinuron 0.5 52 55 55 500.2 50 54 59 53

iuron 0.5 45 44 48 430.2 47 42 49 45

iethofencarb 0.5 59 55 82 630.2 60 51 81 62

enfuracarb 0.5 12 0 5 00.2 15 1 4 0

arbosulfan 0.5 8 8 11 90.2 5 6 13 8

a All the extractions were performed in triplicate with % RSD values ranging from 1 to 17%.

140 G. Sagratini et al. / J. Chromatogr. A 1147 (2007) 135–143

Table 4Percent recovery and repeatabilitya of the method using CW/TPR fiber in water and fruit juices spiked at two fortification levels

Pesticide % Recovery

Compound Concentration (mg kg−1) Water Orange Strawberry and cherry Apple

Carbofuran 0.5 15 16 27 200.2 17 19 28 18

Monuron 0.5 34 18 24 180.2 37 16 28 16

Pirimicarb 0.5 13 1 2 10.2 11 2 2 3

Monolinuron 0.5 38 35 44 250.2 33 34 44 26

Diuron 0.5 65 53 68 390.2 67 56 70 41

Diethofencarb 0.5 79 37 73 500.2 75 34 75 53

Benfuracarb 0.5 13 0 0 00.2 10 0 0 0

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a All the extractions were performed in triplicate with % RSD values ranging

Other experiments were conducted at 20 ◦C and 90 min ofxtraction to select the right solution volume to be left in contactith the fiber to ensure the best extraction. The results obtainedy testing 1, 3 and 5 ml of fruit juice samples are reported in his-

ograms in Fig. 3. The diagram clearly shows that most pesticidesave the best recovery with 1 ml of juice volume. In particular,onolinuron, diethofencarb and diuron showed the greatest dif-

erence in percent recovery between 1 ml and the other tested

(tsp

able 5ntra- and inter-day precision and accuracy for the tested pesticides from orange juortification levels

ompound Amount added(mg kg−1)

Intra-day

Amount detecteda

(mg kg−1)Accuracy (%

arbofuran 0.5 0.48 960.2 0.21 105

onuron 0.5 0.49 980.2 0.19 95

irimicarb 0.5 0.52 1040.2 0.2 100

onolinuron 0.5 0.5 1000.2 0.21 105

iuron 0.5 0.51 1020.2 0.2 100

iethofencarb 0.5 0.49 980.2 0.19 95

enfuracarb 0.5 0.52 1040.2 0.19 95

arbosulfan 0.5 0.51 1020.2 0.2 100

a The values are means of three replicate determinations.

9 11 912 13 7

1 to 16%.

olumes. On the contrary, carbosulfan, and especially pirimi-arb, displayed very low percent recoveries with no differenceetween volumes.

To optimize the extraction, the best conditions of temperature

20 ◦C), time (90 min) and volume (1 ml) were applied to choosehe best performing SPME fibers among the three differentelected types: Carbowax/templated resin (CW/TPR), 60-�moly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) and 85-

ice (blank sample) by SPME/LC/MS using CW/TPR fiber evaluated at two

Inter-day

) RSD (%) Amount detecteda

(mg kg−1)Accuracy (%) RSD (%)

9 0.5 100 37 0.19 95 10

4 0.49 98 73 0.21 105 12

6 0.51 102 210 0.21 105 5

5 0.48 96 1414 0.19 95 9

12 0.5 100 67 0.21 105 4

8 0.48 96 82 0.2 100 15

15 0.53 106 105 0.21 105 11

4 0.5 102 511 0.19 95 4

G. Sagratini et al. / J. Chromatogr. A 1147 (2007) 135–143 141

F e quas R fibt onoli

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ig. 4. Representative determinative LC/MS chromatograms obtained by singlpiked with 50 ng of a standard mixture of pesticides and extracted with CW/TPime, the list of pesticides is: (1) carbofuran, (2) monuron, (3) pirimicarb, (4) m

m polyacrylate. During the experiments with the apple, orange,herry and strawberry fruit juices spiked with the tested pesti-ides at two levels of concentrations, the best performances werebtained using the CW/TPR and the PDMS/DVB fibers.

tcr

able 6RLs for the tested pesticides in Spanish and Italian legislation, and limits of detecti

ompound MRLsa Spanish legislation (mg kg−1) MRLsa I

Orange Strawberryand cherry

Apple Orange

arbofuran 0.3 0.1 0.1 0.3onuronb – – – –

irimicarb 0.5 0.5 0.5 0.2onolinuron 0.05 0.05 0.05 0.05iuron 0.3 0.1 0.2 0.05iethofencarb 0.05 0.5 0.05 1c

enfuracarb 0.05 0.05 0.05 0.05arbosulfan 0.05 0.05 0.05 0.05

a MRLs, referred to fruits, are available on Internet sites indicated in Refs. [20] andb In Spanish and Italian legislation monuron was forbidden by regulation (CE) N. 2c Sole available datum, referred to grapes.

drupole of (A) 10 ng of standard mixture of tested pesticides, (B) orange juiceer and (C) orange juice extracted with CW/TPR fiber. With regard to retentionnuron, (5) diuron, (6) diethofencarb, (7) benfuracarb and (8) carbosulfan.

From the results reported in Tables 3 and 4, it can be observedhat the PDMS/DVB fiber showed good recovery, that in someases exceeded 50%, in all three juices and in water, used aseference. The pesticides displaying the highest recoveries were

on and quantification of the method

talian legislation (mg kg−1) LOD(mg kg−1)

LOQ (mg kg−1)

Strawberryand cherry

Apple

0.1 0.1 0.01 0.03– – 0.005 0.020.2 0.2 0.001 0.0050.05 0.05 0.01 0.040.05 0.05 0.005 0.021c 1c 0.01 0.050.05 0.05 0.01 0.050.05 0.05 0.01 0.03

[21].076/2002.

1 matogr. A 1147 (2007) 135–143

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42 G. Sagratini et al. / J. Chro

arbofuran, monolinuron, diuron and diethofencarb. On the con-rary, pirimicarb and benfuracarb showed the least recovery withalues of around zero. This kind of fiber exhibited a very goodecovery reproducibility showing values very similar in all threeypes of fruit juices.

With regard to the more polar CW/TPR fiber, good extractionapacity was observed for monolinuron, diuron and diethofen-arb, a lower capacity for carbofuran and monuron, and noapacity for pirimicarb and benfuracarb. In this case, signifi-antly different recovery values were observed for monolinuron,iuron and diethofencarb in the three tested juices, indicating thenfluence of the different fruit matrices.

The results obtained with polyacrylate fiber (data not shown)ndicated that, especially in the cherry and strawberry fruituices, the recoveries were comparable with those obtained usinghe PDMS/DVB and CW/TPR fibers, whereas in the other juices

30–40% loss of recovery, as in the cases of monuron andarbofuran, was observed.

The results also showed that compounds such as pirimicarbnd benfuracarb were extracted with difficulty by any kind ofber used, probably due to their high solubility in water or

n watery matrices, as already known for pirimicarb (2.7 g/l)21].

The centrifugation and filtration steps before the SPMExtraction process are very important in this kind of analysis.n fact, as already reported [14], the suspended matter presentn fruit juice can interfere with the extraction process of SPMEber, decreasing the percent recovery values. We demonstrated

hat with samples fortified after centrifugation, an increase inecovery, greater than 35%, was generally observed, confirminghe reported interference. With regard to this, the lower values ofome pesticides in the apple and orange juices, compared withherry and strawberry, could be explained by their high pec-ine content that could interfere, as the suspended matter, in thePME extraction process.

To improve the recovery, the effect of dilution with water onhe fortified juice was studied [12]: from 10- to 50-fold dilutionhe recoveries became very similar to those from water; however,cceptable recovery values, indicated by significant LOD data,ere achieved with a 1:1 juice–water dilution. Consequently, we

hose a 1:1 sample dilution to increase the instrumental method’sensitivity.

.3. Method validation

Calibration curves of the analysed compounds were con-tructed at five different concentrations (0.05, 0.1, 0.5, 1 and�g ml−1), injecting 20 �l of standard solutions in the LC/MSnd LC-QIT/MS in the MRM mode. Three replications for eachoncentration were performed and the relative standard devi-tions (RSDs) ranged from 1 to 5% for run-to-run precision,nd from 1 to 6% for day-to-day precision. All the calibrationurves of the analysed pesticides showed a correlation coeffi-

ient greater than 0.996.

The intra- and inter-day precision and accuracy for the orangeuice using CW/TPR fiber and LC/MS are presented in Table 5.he intra-day precision was less than 15% and the accuracy

wan

ber and (C) orange juice extracted with CW/TPR fiber. With regard to retentionime, the list of pesticides is: (1) carbofuran, (2) monuron, (3) pirimicarb, (4)

onolinuron, (5) diuron, (6) diethofencarb, (7) benfuracarb and (8) carbosulfan.

anged from 95 to 105% for the tested concentrations, while thenter-day precision was less than 15% and the accuracy was inhe range 95 to 106%. The response factor test (e.g., relativetandard deviation of the average of the response factors <15%)onfirmed that the linearity of the obtained analytical responseas adequate.

In Table 6 the MRLs of tested pesticides are reported together

ith the limits of detection (LODs) and the LOQ values. LODnd LOQ were estimated on the basis of 3:1 and 10:1 signal-to-oise ratios obtained with standards containing the compounds

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[21] The Merck Index, Merck & Co., Whitehouse Station, NJ, 2001.

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f interest at low-concentration levels. The method sensitivityas confirmed by the obtained LOQs that were of the samerder of magnitude (diethofencarb, benfuracarb) as, or lowerhan, the MRLs established by Spanish and Italian laws refer-ing to fruit [22,23]. Fig. 4 shows, for the tested pesticides, theC/MS chromatograms obtained by single quadrupole injec-

ion (A) of 10 ng of standard mixture, (B) an orange juicepiked with 50 ng of pesticides standard mixture and extractedith CW/TPR fiber and (C) an orange juice extracted withW/TPR fiber. Pesticide numbers were assigned according to

heir retention time on chromatographic analysis. Fig. 5 showshe LC/MS/MS chromatograms obtained by ion-trap injectionA) of 10 ng of standard mixture, (B) an orange juice spikedith 50 ng of pesticides standard mixture and extracted withW/TPR fiber and (C) an orange juice extracted with CW/TPRber.

. Conclusions

A new method to extract and detect carbamate and pheny-urea pesticide residues in fruit juice was developed. The appliedPME technique allowed us to work with practically no sam-le treatment from extraction to HPLC analysis. The besterformances were achieved using PDMS/DVB and CW/TPRbers under the following extraction conditions: 90 min (time),0 ◦C (temperature), 1 ml (volume). The SPME/HPLC inter-ace proved to be a valid method to connect the solid-phaseicroextraction to the liquid chromatography system, allowing

he SPME to be used not only for molecules in the gaseous phaseut also in the liquid system. LC/MS, and especially LC/MS/MS,llowed pesticide residues to be detected in food matrices whileorking with a high level of sensitivity. In addition, the resultseriving from both instrumental methods proved similar with

espect to the recoveries obtained using different fibers and thenalysed fruit juices. This method can be applied for analysingeal samples of fruit juices for the screening of the reportedesticides.

[

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r. A 1147 (2007) 135–143 143

cknowledgements

This work was financially supported by the Spanish Min-stry of Science and Technology and the European Regionalevelopment Funds (ERDF) (Project AGL2003-01407).

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