ORIGINAL PAPER

Pesticide residue analysis in cereal-based baby foodsusing multi-walled carbon nanotubes dispersive solid-phaseextraction

Miguel Ángel González-Curbelo &

María Asensio-Ramos & Antonio V. Herrera-Herrera &

Javier Hernández-Borges

Received: 7 February 2012 /Revised: 25 April 2012 /Accepted: 4 May 2012 /Published online: 24 May 2012# Springer-Verlag 2012

Abstract In the present study, a new analytical method hasbeen developed for the simultaneous quantification of 15organophosphorus pesticides, including some of theirmetabolites, (disulfoton-sulfoxide, ethoprophos, cadusafos,dimethoate, terbufos, disulfoton, chlorpyrifos-methyl,malaoxon, fenitrothion, pirimiphos-methyl, malathion,chlorpyrifos, terbufos-sulfone, disulfoton-sulfone and fen-sulfothion) in three different types of commercial cereal-based baby foods. Dispersive solid-phase extraction (dSPE)with multi-walled carbon nanotubes (MWCNTs) was usedtogether with gas chromatography with nitrogen phosphorusdetection. Most favorable conditions involved a previousultrasound-assisted extraction of the sample with acetoni-trile containing formic acid. After evaporation of the extractand redissolution in water, a dSPE procedure was carried outwith MWCNTs. The whole method was validated in termsof repeatability, linearity, precision and accuracy and matrixeffect was also evaluated. Absolute recoveries were in therange 64–105 % with relative standard deviation valuesbelow 7.6 %. Limits of quantification achieved ranged from0.31 to 5.50 μg/kg, which were lower than the EuropeanUnion maximum residue limits for pesticide residues incereal-based baby foods.

Keywords Pesticides . Multi-walled carbon nanotubes .

Dispersive solid-phase extraction .Cereal-based baby foods .

Gas chromatography–nitrogen phosphorus detection

Introduction

In the last decades, special concern has arisen regardingfoods devoted to infants (children under the age of12 months) and young children (from 1 to 3 years old) asa result of their higher food intake than adults, expressed ona per kilogram body weight basis. Because of their impor-tant role in the first years of life, special care is being takenconcerning the presence of harmful contaminants or resi-dues like, for example, polycyclic aromatic hydrocarbons[1, 2], mycotoxins [3, 4] or pesticides [5, 6]. In this last case,and despite the fact that significant efforts are being made toavoid the appearance of pesticide residues in such products,the truth is that monitoring programs should always bedeveloped to ensure their safe consumption and that newanalytical methods are also highly welcome, especially forthe analysis of samples of such importance at the requiredmaximum residue limits (MRLs).

One of the most important achievements of the EuropeanUnion (EU) regarding food safety, has probably been theestablishment of specific and strict legislation like the one ofCommission Directive 2006/125/EC of 5 December 2006on processed cereal-based foods and baby foods for infantsand young children. In that directive, it is clearly indicatedthat no detectable levels of pesticide should appear in babyfoods, which means no more than 0.01 mg/kg. The Direc-tive also banned the use of certain very toxic pesticides (i.e.disulfoton, fensulfothion, etc.) in the production of babyfoods. In that case, if such pesticide residues do not exceed

Electronic supplementary material The online version of this article(doi:10.1007/s00216-012-6103-7) contains supplementary material,which is available to authorized users.

M. Á. González-Curbelo :M. Asensio-Ramos :A. V. Herrera-Herrera : J. Hernández-Borges (*)Departamento de Química Analítica, Nutrición y Bromatología,Facultad de Química, Universidad de La Laguna (ULL),Avenida Astrofísico Francisco Sánchez, s/no. 38206,La Laguna, Tenerife, Spaine-mail: jhborges@ull.es

Anal Bioanal Chem (2012) 404:183–196DOI 10.1007/s00216-012-6103-7

0.003 mg/kg they are considered not to have been used. TheDirective also established specific and MRLs lower than0.01 mg/kg (i.e., 0.006 or 0.008 mg/kg) for certain pesti-cides (i.e., cadusafos, ethoprofos, etc.).

Apart from that, and concerning only the analysis ofcereal samples, the EU has also developed in the last deca-des several programs for the monitoring of pesticide resi-dues in products of plant origin, in which residues ofchlorpyrifos, chlorpyrifos-methyl, dimethoate, malathionand pirimifos-methyl among others were found in cerealsamples [7]. As a result, it is interesting to include suchpesticides in forthcoming monitoring programs as well as inany analytical method devoted to the analysis of suchsamples.

Up to now, and concerning published works, the analysisof pesticides in cereal-based baby foods has only beendeveloped in very few works [5, 6, 8, 9] in which differentmodifications of the QuEChERS approach were used. Lean-dro et al. [8], for example, determined 12 priority pesticidesand transformation products by gas chromatography (GC)–tandem mass spectrometry (MS/MS) in different babyfoods, some of them containing individual cereals mixedwith fruits. The method, although slightly changed, was alsoapplied in a later work of the same authors [5] for thedetermination of 16 pesticides by high-performance liquidchromatography and ultra-performance liquid chromatogra-phy (UPLC)-MS/MS in similar samples and also for theanalysis of 52 pesticides by UPLC-MS/MS in oranges,potatoes and cereal-based baby foods [6]. On the other hand,very recently González-Curbelo et al. [9] optimized QuECh-ERS conditions by means of an experimental design for theextraction of a group of 15 pesticides and metabolites fromtoasted maize and wheat flours. For comparison purposes,the method was also applied to the extraction of one babycereal sample. Apart from these works, in which only onetype of a single baby cereal have been analyzed in twooccasions, and to the best of our knowledge, no furtherstudy has been developed regarding pesticide analysis ofthese complex and, at the same time, important samplesfrom a food safety point of view.

Since their first report in 1991, carbon nanotubes (CNTs)have attracted great interest of the scientific community. Inthe Analytical Chemistry field they are currently beingstudied/applied as stationary phases in gas chromatography[10, 11], liquid chromatography (LC) [10, 11] or capillaryelectrochromatography [10], pseudostationary phases incapillary electrophoresis (CE) [11], as matrix-assisted laserdesorption/ionization matrices [12], in the fabrication offiltration membranes [11], as part of electrochemical detec-tors [13] and also as sorbents for solid-phase extraction(SPE) [11, 14, 15] or microextraction [11, 15, 16] of bothorganic and inorganic analytes. Regarding their applicationin the SPE field, they have been employed in a good number

of occasions as pesticide extraction sorbents [15] mainlyfrom water samples for which they have demonstrated toprovide high recovery values. However, their applications inother fields are still very scarce and only in very few works,have they been extracted from food matrices [17–19]. Thisis the case of the extraction of a single pesticide (methyl-parathion) from garlic and its later determination by square-wave voltammetry [17], eight organophosphorus pesticidesand a thiadiazine from fruit juices determined by GC-nitrogen phosphorus detection (NPD) [18] and eight multi-class pesticides from olive oil previous to their GC-MSanalysis [19]. As a result, new CNTs-SPE applications inthe food analysis field are highly welcome, especially due tothe complexity of these samples.

All these previous works in the pesticide food analysisfield involved the application of the SPE procedure in theconventional mode using laboratory-made CNTs-SPE car-tridges, since this type of cartridges are not still commer-cialized. One of the variations of the procedure whichclearly reduces extraction time and simplifies the wholeprocedure is called dispersive SPE (dSPE). This approachinvolves the extraction of the analytes in the bulk solution(which defines how the mode is called) and not in a column,without previous conditioning and/or initially locating thematerial onto the cartridge as it happens in traditional SPE.Subsequently, a SPE tube is only used to carry out theelution step. Besides, extraction time is highly reduced sincethe sample does not have to be passed slowly through theextraction column. As a result, the whole extraction proce-dure is highly simplified and can be performed more quick-ly. dSPE has been mainly used for clean-up purposes byadding a suitable sorbent to the organic matrix solutioncontaining the analytes and potential interferences, remov-ing the last of them, rather than for the performance of theextraction. Even so, some works have shown the use of thedSPE mode with good results using different types of sorb-ents [20–22] and, as far as we know, very few [23–27] haveused multi-walled carbon nanotubes (MWCNTs) for dSPE.Two of these works have been developed by our group. Inone of them, 9 pesticides were analyzed in water samples bynano-LC [23] and in the other, 11 quinolone antibiotics weredetermined in environmental waters by CE [24] providingpromising results. Moreover, only in one occasion haveMWCNTs been used as sorbents in dSPE for the analysisof foods, in particular estrogens in milk [26].

Therefore, the aim of this work is to develop an analyticalmethod based on the use of MWCNTs of 6–13 nm o.d. and2.5–20 μm length as SPE materials for the simultaneousdetermination of 15 pesticides (some of them EU prioritypesticides in baby foods, i.e., terbufos, disulfoton, fensulfo-thion, cadusafos and ethoprofos) in cereal-based baby foods.The method includes the pesticides most frequently found inEU monitoring programs in cereal samples. In this case an

184 M.Á. González-Curbelo et al.

ultrasound-assisted extraction (USE) together with a dSPEapproach of the baby food aqueous extract was applied inorder to simplify the overall procedure, in combination withGC-NPD analysis. A strong intralaboratory validation pro-cedure was carried out in order to demonstrate the feasibilityof the procedure for the analysis of cereal-based baby foodsof different composition. To the best of our knowledge, thisis the first work in the literature regarding the application ofMWCNTs as dSPE materials for the extraction of pesticidesfrom foods and, in particular, from baby foods, also allow-ing their determination at the low levels required by thecurrent EU legislation.

Materials and methods

Chemicals

Pesticide analytical standards of disulfoton-sulfoxide (O,O-diethyl S-(2-(ethylsulfinyl)ethyl) phosphorodithioate), etho-prophos (O-ethyl S,S-dipropyl phosphorodithioate), cadusa-fos (S ,S-di-sec-butyl O-ethyl phosphorodithioate),dimethoate (2-dimethoxyphosphinothioylthio-N-methylace-tamide), terbufos (S-tert-butylthiomethyl O,O-diethyl phos-phorodithioate), disulfoton (O,O-diethyl S-2-ethylthioethylphosphorodithioate), chlorpyrifos-methyl (O,O-dimethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), malaoxon(diethyl 2-(dimethoxyphosphorylsulfanyl)butanedioate),fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphoro-thioate), pirimiphos-methyl (O-2-diethylamino-6-methyl-pyrimidin-4-yl O,O-dimethyl phosphorothioate), malathion(diethyl (dimethoxyphosphinothioylthio)succinate), chlor-pyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphor-othioate), terbufos-sulfone (S-{(1,1-dimethylethyl)sulfonylmethyl} O,O-diethyl ester), disulfoton-sulfone (O,O-diethyl S-(2-ethsulfonylethyl)phosphorodithioate) andfensulfothion (O,O-diethyl O-4-methylsulfinylphenyl phos-phorothioate) were obtained from Riedel-de-Haën (Sigma-Aldrich Chemie, Madrid, Spain). Purity of the pesticidestandards was higher than 92.6 % and they were usedwithout further purification. Individual stock solutions ofeach analyte were prepared in cyclohexane and stored inthe darkness at 4 °C. A composite stock standard solution ofall pesticides was prepared by combination and dilutionwith cyclohexane. Working solutions were daily preparedby dilution of these mixtures with the same solvent.

Distilled water was deionized by using a Milli-Q systemfrom Millipore (Bedford, MA, USA). Dichloromethane(DCM) stabilized with 50 ppm of amylene was from Schar-lau Chemie S.A. (Barcelona, Spain) and acetonitrile (ACN),cyclohexane, methanol (MeOH) and acetone were fromMerck (Darmstadt, Germany). Formic acid (98 %) and

diethyl ether stabilized with 6 ppm of butylhydroxytoluenewere from Panreac (Barcelona, Spain).

MWCNTs with o.d. of 6–13 nm and 2.5–20 μm lengthwere purchased from Sigma-Aldrich Chemie (Madrid,Spain). Empty glass SPE tubes of 6 mL volume (7.5 cmlength, 1.5 cm o.d., 1.2 cm i.d.) and polytetrafluoroethylene(PTFE) frits (20 μm porosity, 11.5 mm diameter, 3 mmwidth) were from Supelco (Madrid, Spain).

Cereal-based baby food samples

Three different types of commercial cereal-based babyfoods were obtained from local supermarkets of Tenerife,Canary Islands, Spain. Baby food 1 (five-cereal sample) wascomposed of a mixture of wheat, barley, rye, maize and oat.Baby food 2 (eight-cereal sample) was composed of wheat,barley, rye, maize, rice, oat, sorghum and millet, while Babyfood 3 (five-cereal sample) was composed of wheat, barley,rye, maize and rice. Sample homogenization was not foundnecessary because of their dusty nature. Fat amount in eachsample was determined by Soxhlet extraction with diethylether for 8 h, using 5 g of previously dried samples for 1 h at120 °C. This procedure was carried out in duplicate. Fatspercentages were 1.56±0.05 % for Baby food 1, 0.82±0.07 % for Baby food 2 and 0.50±0.11 % for Baby food 3.

Apparatus and software

The samples were analyzed by GC using a Varian 3800(Walnut Creek, CA, USA) GC-NPD system, equipped witha Varian Combipal Autosampler and a Equity™-5 fusedsilica capillary column (30 m×0.25 mm, 0.25 μm film),poly(5 % diphenyl/95 % dimethylsiloxane), from Supelco(Bellefonte, PA, USA) with the Varian Star ChromatographyWorkstation v.6.41 Software. Chromatographic conditionswere taken from a previous work of our group [9]. Thecolumn was initially maintained for 1 min at 50 °C, andthen the temperature was increased to 160 °C at a rate of10 °C/min, held for 5 min, then increased to 190 °C at a rateof 1.5 °C/min and finally increased to 280 °C at a rate of16 °C/min and held for 8 min. Total run time was 50.6 min.Nitrogen was employed as the carrier gas (1.0 mL/min) andalso as make-up (30 mL/min). Hydrogen flow was kept at4 mL/min. Two microliters of a standard or sample solutionwere injected in the split/splitless mode at 280 °C and theNPD was maintained at 320 °C. The StatGraphics PlusSoftware Version 5.1 (StatPoint Technologies, Inc., Warren-ton, VA, USA) was used for data processing.

Sample extraction and dSPE procedure

A 1.5 g portion of sample was accurately weighed in a50 mL centrifuge tube and a mixture of the analytes was

Pesticide residue analysis in cereal-based baby foods 185

added (for matrix-matched calibration, they were addedafter the performance of the USE-dSPE method). Then,20 mL of ACN containing 1.25 % (v/v) formic acid wereadded and the sample was vigorously shaken by hand for2 min. Afterwards, the mixture was sonicated for 5 min in anUltrasons ultrasonic bath from Selecta (Barcelona, Spain),working at 50/60 Hz and 100 W. Then, centrifugation wascarried out for 8 min at 4,000 rpm (3,000×g) in a 5702centrifuge from Eppendorf (Hamburg, Germany). After that,the supernatant phase was collected after filtration (0.45 μmPET filter, Chromafil® Xtra PET-45/25 Macherey-NagelGmbH & Co. KG, Düren, Germany) and the sample wassubjected to the same extraction process again. The twosupernatant phases were joined together and evaporated todryness at 40 °C and 205 mbar using a Rotavapor R-200equiped with a V-800 vacuum controller and a vacuumpump V-500, all from Büchi Labortechnik (Flawil, Switzer-land). The dry residue was redissolved in 50 mL of water,adjusted to pH 6 with 0.1 M NaOH, filtrated (0.45 μm PETfilter, Chromafil®) and located into a volumetric flask con-taining 80 mg of MWCNTs. The flask was shaken for 2 minand transferred into a glass SPE tube containing two PTFEfrits, using a SPE vacuum extraction manifold from Waters(Milford, MA, USA), where the solution was filtered, re-moving the water and leaving the stationary phase on thefrit. A new PTFE frit was introduced onto the column andthe cartridge was dried under vacuum for 20 min at−20 mmHg. The retained pesticides were eluted with30 mL of DCM, coupling a PTFE 0.20 μm filter (Chroma-fil® O-20/25 from Macherey-Nagel GmbH & Co. KG)under the glass tube. The organic solvent was then evapo-rated to dryness at 40 °C and 700 mbar using a rotavapor.The residue was dissolved in 1 mL of cyclohexane, a verysmall amount of anhydrous Na2SO4 was added and thesupernatant was finally filtered through PTFE filters (Chro-mafil® O-20/25) and injected in the GC system.

Results and discussion

Optimization of the dSPE procedure

SPE conditions including elution solvent type and volume,pH, amount of MWCNTs and extraction time were initiallyoptimized in order to achieve the highest extraction efficien-cy in terms of absolute recovery values. For this purpose,SPE optimization was carried out in the dispersive modewith 50 mL of spiked Milli-Q water for a better understand-ing of extraction influencing parameters. All experimentswere carried out comparing the results obtained in each casewith the results achieved through the standards dissolved inthe respective final extracts after the dSPE procedure.

Elution solvent selection

Preliminary experiments were developed by adding 40 mg ofMWCNTs to 50 mL of spiked Milli-Q water (concentration2.27–16.18 μg/L) at pH 7.0. Water samples were shaken withthe CNTs for 15 min (extraction/contact time) in an automaticshaker and then introduced in an empty glass SPE columncontaining two PTFE frit as described in the “Sample extrac-tion and dSPE procedure” section. Initially, 20 mL of fourorganic solvents of different polarity i.e., MeOH, ACN, ace-tone and DCM were used. As shown in Fig. 1, the use ofDCM provided the highest recovery values (in the range 78–98 %) for most of the pesticides. On the contrary, the use ofMeOH and ACN provided mean recovery values in the range17–86 % and 23–90 %, respectively, allowing only a partialelution of the pesticides. Regarding the use of acetone, al-though recovery values were also acceptable, between 67 and103%, but slightly lower than using DCM, repeatability of theextraction was not found appropriate as has also been reportedin previous occasions [23, 28]. As a result, DCMwas selectedfor subsequent experiments.

Effect of pH

The pH exerts an important effect on SPE recoveries since itdefines the molecular state of the target compounds (ionic orneutral state) and therefore, their adsorption onto MWCNTssurface. On the other hand, extreme pH values are notfrequently suitable since the possible hydrolysis of someof the pesticides under strong acidic or basic conditionscan happen. Although the selected pesticides do not havepKa values, in this work, the effect of pH was evaluated inthe range 4.0–9.0 by adding appropriate volumes of 0.1 MNaOH or 0.1 M HCl solutions under the same previousconditions: 50 mL of water, 40 mg MWCNTs and 15 minextraction time. Experimental results showed that recoveryvalues for most pesticides were lower at pH≤5 or pH≥8. Infact, the lowest recoveries between 39–99 % and 58–100 %were found at pH 4.0 and 9.0, respectively. In contrast,satisfactory recoveries were obtained in the range ofpH 6.0–7.0, between 81–102 % for the first value and 78–98 % for the second. According to this, pH 6.0 was selected.(See Electronic Supplementary Material Figure S1).

Elution solvent volume

The volume of the elution solvent is another importantparameter that must be considered since it should be suffi-cient to elute the pesticides adsorbed onto MWCNTs. Forthis purpose, DCM volume was investigated from 10 to40 mL. As shown in Fig. 2, for most of the pesticidesrecoveries increased significantly when 20 mL were used,i.e., 81–102 %. Further increase in the elution volume did

186 M.Á. González-Curbelo et al.

0

20

40

60

80

100

120

Rec

ove

ry (

%)

Pesticide

MeOH

ACN

Acetone

DCM

Fig. 1 Effect of the type of elution solvent on the recoveries of the 15pesticides under study from Milli-Q water of the dSPE procedure.Extraction conditions, 50 mL spiked Milli-Q water sample at pH 7.0

(concentration 2.27–16.18 μg/L); 40 mg of MWCNTs; 15 min dSPEextraction time and elution volume 20 mL

40

50

60

70

80

90

100

110

10 20 30 40

Rec

ove

ry (

%)

Volume (mL)

Disulfoton sulfoxideEthoprophosCadusafosDimethoateTerbufosDisulfotonChlorpyrifos-methyl

60

70

80

90

100

110

10 20 30 40

Rec

ove

ry (

%)

Volume (mL)

MalaoxonFenitrothionPirimiphos-methylMalathionChlorpyrifosTerbufos sulfoneDisulfoton sulfoneFensulfothion

Fig. 2 Effect of DCM volumeon the recoveries of the selectedpesticides from Milli-Q water ofthe dSPE procedure. Extractionconditions, 50 mL spikedMilli-Q water sample atpH 6.0 (concentration 2.27–16.18 μg/L); 40 mg MWCNTs;15 min dSPE extraction time

Pesticide residue analysis in cereal-based baby foods 187

not provide any significant improvement. Thus, 20 mL ofDCM was selected for the following studies.

Cereal baby food extraction conditions

Up to now, elution solvent type and volume as well as the pHhave been satisfactory optimized using spiked Milli-Q water.In order to extend the developed methodology to the quanti-tative determination of this group of 15 pesticides to cereal-based baby foods, a previous solvent extraction should becarried out. Initial experiments were developed using 1.5 gof a spiked cereal-based baby food (concentration 76–539μg/kg). This sample was composed of a mixture of wheat,barley, rye, maize, and oat, defined as Baby food 1 in thesection “Cereal-based baby food samples”, which was the onewith the highest fat content (1.56±0.05 %). Once more, ex-traction recoveries were calculated by comparison with thestandards dissolved in the final matrix extract. Non-spikedsamples were also initially analyzed to check the absence ofthese pesticides or matrix interferences that could difficult thecorrect quantification of the target analytes. No coelutingpeaks were found in any of the analyzed samples.

Before the dSPE procedure, an USE with 20 mL of ACNwas carried out since it has proven to be a good pesticideextraction solvent for different types of cereal grains andflours [29, 30] as well as from different baby foods

including those cereal-based [5, 6] and also because of thelow amount of coextracted fats that may interfere in the laterGC analysis [31, 32]. Under these conditions, unsatisfactoryrecoveries in the range 27–79 % were obtained. Recoveryvalues were found particularly lower (between 27 and 48 %)for terbufos, disulfoton, chlorpyrifos and chlorpyrifos-methyl. These results suggested the need of optimizing theamount of MWCNTs (which was increased up to 125 mg),since matrix components may block active sites of thestationary phase, and also the initial composition of theextraction solvent as well as the extraction time, whichwas initially kept constant for 15 min. An increase in theamount of CNTs may also require an increase in the elutionsolvent (which was increased up to 30 mL).

In this sense, recovery values increased when increasingthe sorbent amount up to 80 mg for most pesticides with30 mL of elution solvent, obtaining recoveries in the range59–99 %, except, once more, for terbufos (47 %), disulfoton(35 %), chlorpyrifos (40 %) and chlorpyrifos-methyl (50 %).When the sorbent amount was enlarged up to 125 mg, therecoveries did not change. An increase in the elution volumedid not also improve these results. Therefore, and in order touse the lowest sorbent amount as possible, 80 mg ofMWCNTs was selected with 30 mL of elution solvent.

The following approach involved several changes on theextraction of the cereal samples with ACN. Several

0

20

40

60

80

100

120

Rec

ove

ry (

%)

Pesticide

20 mL30 mL2x20 mL20 mL with formic acid30 mL with formic acid2x20 mL with formic acid

Fig. 3 Effect of different extraction conditions on the recoveries of the15 pesticides under study from cereal-based Baby food 1 after theMWCNTs-dSPE-GC-NPD procedure. Conditions, 1.5 g spiked Babyfood 1 (concentration 76–539 μg/kg) with variable volumes of ACN;

after evaporation, the extract was reconstituted in 50 mL of Milli-Qwater, pH adjusted to 6.0 and extracted with 80 mg MWCNTs andelution with 30 mL DCM

188 M.Á. González-Curbelo et al.

Table 1 Calibration data of the selected group of pesticides from standards prepared in cyclohexane and in the final extract of each baby food afterthe MWCNTs-dSPE-GC-NPD method and assessment of the matrix effect

Peak Pesticide Matrix Calibration data (n06) Matrix effect

Range of concentrationtested (mg/L)

Slope Intercept R2

1 Disulfoton-sulfoxide Standard 0.004–0.89 3.62·105±0.18·105 2.83·103±8.88·103 0.999 –

Baby food 1 0.004–0.89 5.95·105±0.25·105 −6.67·103±1.35·103 0.999 Yes

Baby food 2 0.004–0.89 5.07·105±0.95·104 3.88·103±5.19·103 0.999 Yes

Baby food 3 0.004–0.89 6.01·105±0.30·105 8.21·103±1.64·103 0.999 Yes

2 Ethoprophos Standard 0.004–0.86 6.11·105±0.25·105 1.25·104±1.21·104 0.999 –

Baby food 1 0.004–0.86 9.35·105±0.72·105 −8.36·103±3.77·103 0.998 Yes

Baby food 2 0.004–0.86 8.18·105±0.10·105 1.09·104±5.41·103 0.999 Yes

Baby food 3 0.004–0.86 9.62·105±0.34·105 1.62·104±1.80·103 0.999 Yes

3 Cadusafos Standard 0.004–0.84 4.58·105±0.14·105 5.80·103±6.52·103 0.999 –

Baby food 1 0.004–0.86 6.97·105±0.53·105 −3.56·103±2.70·103 0.998 Yes

Baby food 2 0.004–0.86 6.08·105±0.09·105 4.78·103±5.58·103 0.999 Yes

Baby food 3 0.004–0.86 6.96·105±0.24·105 5.66·103±1.22·103 0.999 Yes

4 Dimethoate Standard 0.016–3.10 3.63·105±0.15·105 1.72·104±2.61·104 0.999 –

Baby food 1 0.016–3.10 5.53·105±0.26·105 −5.33·103±4.91·103 0.999 Yes

Baby food 2 0.016–3.10 4.67·105±0.29·105 2.11·104±5.50·103 0.999 Yes

Baby food 3 0.016–3.10 6.06·105±0.45·105 3.72·104±8.54·103 0.998 Yes

5 Terbufos Standard 0.006–1.10 1.05·106±0.36·105 3.68·104±2.24·104 0.999 –

Baby food 1 0.006–1.10 1.47·106±0.60·105 −3.14·103±4.07·103 0.999 Yes

Baby food 2 0.006–1.10 1.28·106±0.76·105 4.68·104±3.72·103 0.999 Yes

Baby food 3 0.006–1.10 1.48·106±0.10·106 5.68·104±6.91·103 0.999 Yes

6 Disulfoton Standard 0.002–0.45 8.07·105±0.24·105 5.00·103±6.06·103 0.999 –

Baby food 1 0.002–0.45 1.15·106±1.00·106 −3.09·103±2.76·103 0.997 Yes

Baby food 2 0.002–0.45 1.04·106±0.19·105 1.39·103±5.28·103 0.999 Yes

Baby food 3 0.002–0.45 1.19·106±0.25·105 3.55·103±7.06·103 0.999 Yes

7 Chlorpyrifos-methyl Standard 0.004–0.76 3.75·105±0.93·104 4.18·103±3.96·103 0.999 –

Baby food 1 0.004–0.76 5.22·105±0.25·105 −4.89·103±1.14·103 0.999 Yes

Baby food 2 0.004–0.76 5.01·105±0.24·105 2.53·103±1.11·103 0.999 Yes

Baby food 3 0.004–0.76 6.10·105±0.19·105 4.01·103±8.64·103 0.999 Yes

8 Malaoxon Standard 0.016–3.24 1.18·105±0.52·104 −4.63·103±1.04·104 0.999 –

Baby food 1 0.016–3.24 1.71·105±0.13·105 −1.41·103±2.50·103 0.998 Yes

Baby food 2 0.016–3.24 1.57·105±0.86·105 6.16·102±1.70·103 0.999 Yes

Baby food 3 0.016–3.24 2.11·105±0.11·105 4.12·103±2.10·103 0.999 Yes

9 Fenitrothion Standard 0.007–1.49 2.40·105±0.16·104 1.41·103±1.35·103 0.999 –

Baby food 1 0.007–1.49 3.43·105±0.23·105 −3.49·103±2.10·103 0.998 Yes

Baby food 2 0.007–1.49 3.04·105±0.14·105 2.21·103±1.31·103 0.999 Yes

Baby food 3 0.007–1.49 3.94·105±0.16·105 2.81·103±1.49·103 0.999 Yes

10 Pirimiphos-methyl Standard 0.004–0.76 4.88·105±0.25·105 1.10·104±1.06·104 0.999 –

Baby food 1 0.004–0.76 6.74·105±0.26·105 1.72·103±1.20·103 0.999 Yes

Baby food 2 0.004–0.76 6.65·105±0.16·105 4.39·103±7.44·103 0.999 Yes

Baby food 3 0.004–0.76 7.76·105±0.26·105 9.35·103±1.13·104 0.999 Yes

11 Malathion Standard 0.005–0.93 3.40·105±0.10·105 4.91·103±5.40·103 0.999 –

Baby food 1 0.005–0.93 4.76·105±0.31·105 −4.59·103±1.76·103 0.998 Yes

Baby food 2 0.005–0.93 4.31·105±0.25·105 1.82·103±1.44·103 0.999 Yes

Baby food 3 0.005–0.93 5.31·105±0.12·105 5.30·103±6.94·103 0.999 Yes

Pesticide residue analysis in cereal-based baby foods 189

experiments which included the increase of the amount ofsolvent, as well as the development of consecutive extractionsand the addition of different amounts of formic acid, which hasalso proven to provide good results to improve the extractionof planar pesticides from cereals [9, 33], were studied. Figure 3shows the most representative results of these experiments. Ascan be seen, when a double extraction with ACN was carriedout, recovery values increased for all pesticides, however,these recovery values were still low for terbufos, disulfoton,chlorpyrifos and chlorpyrifos-methyl. On the other hand, aslight increase in recovery values was also found for somepesticides when 20 or 30 mL of 1.25 % (v/v) formic acid inACNwere used. Only when a double extraction with 20 mL of1.25 % (v/v) formic acid in ACN was used for sample extrac-tion, the highest and highly acceptable recoveries wereachieved: 62–102 %. Such conditions were therefore selected.

Following, and with the aim of decreasing the method’sLOD, the sample amount was increased up to 2.5 g. When2.0 and 2.5 g were used, recovery values were lower, below50 % for many of the pesticides. For the improvement of therecovery values, higher amounts of extraction solvent oreven superior amounts of MWCNTs or elution solvent(DCM) would probably be needed. However, since as it willbe described later, the limits of detection (LODs) and limitsof quantification (LOQs) of the whole method achievedwere enough to ensure the determination of these pesticidesat the required EU MRLs in these complex samples, it wasnot found necessary to develop this study. On the other

hand, when 1.0 g of baby food was analyzed, similar recov-ery values like the ones obtained with 1.5 g were obtained.No differences were found between both of them, not evenfrom a chromatographic point of view in terms of interferingcompounds or dirtier chromatograms.

Finally, extraction time was also evaluated between 2 and15 min. No decrease in the recoveries was observed for any ofthe pesticides under study when changing the contact timeand, as a result, extraction time was not a critical parameter.Subsequent extractions were therefore performed for 2 min.

Method validation

To validate the USE-MWCNTs-dSPE-GC-NPD methodol-ogy and taking into account the matrix effect, calibration,recovery as well as precision and accuracy studies werecarried out for three baby food samples (see “Cereal-basedbaby food samples” section for details). For this purpose,the absence of the pesticides in the samples under study aswell as any chromatographic interference was initiallychecked.

Linearity study and matrix effect evaluation

Calibration linearity was verified based on injections of thestandard solutions prepared in cyclohexane and also in thefinal extract of each baby food sample after the proposedmethodology (also finally dissolved in cyclohexane). In all

Table 1 (continued)

Peak Pesticide Matrix Calibration data (n06) Matrix effect

Range of concentrationtested (mg/L)

Slope Intercept R2

12 Chlorpyrifos Standard 0.004–0.83 1.23·106±0.85·105 4.21·104±3.96·104 0.999 –

Baby food 1 0.004–0.83 1.64·106±0.55·105 1.75·104±2.82·104 0.999 Yes

Baby food 2 0.004–0.83 1.57·106±0.44·105 2.85·104±2.22·104 0.999 Yes

Baby food 3 0.004–0.83 1.79·106±0.99·105 4.14·104±5.04·103 0.999 Yes

13 Terbufos-sulfone Standard 0.005–1.05 4.05·105±0.22·105 1.21·104±1.27·104 0.999 –

Baby food 1 0.005–1.05 5.22·105±0.24·105 5.36·103±1.56·103 0.999 Yes

Baby food 2 0.005–1.05 5.66·105±0.31·105 9.47·102±2.00·103 0.999 Yes

Baby food 3 0.005–1.05 6.10·105±0.17·105 1.02·104±1.07·103 0.999 Yes

14 Disulfoton-sulfone Standard 0.005–1.04 4.78·105±0.25·105 1.28·104±1.45·104 0.999 –

Baby food 1 0.005–1.04 6.09·105±0.30·105 1.15·104±1.88·103 0.999 Yes

Baby food 2 0.005–1.04 5.99·105±0.14·105 1.45·104±1.17·103 0.999 Yes

Baby food 3 0.005–1.04 7.14·105±0.33·105 2.03·104±2.08·103 0.999 Yes

15 Fensulfothion Standard 0.005–1.03 4.11·105±0.23·105 5.52·103±1.29·104 0.999 –

Baby food 1 0.005–1.03 5.25·105±0.35·105 1.46·104±2.20·103 0.998 Yes

Baby food 2 0.005–1.03 5.21·105±0.18·105 1.40·104±1.17·103 0.999 Yes

Baby food 3 0.005–1.03 6.14·105±0.14·105 1.67·104±2.20·103 0.999 Yes

R2 determination coefficient

190 M.Á. González-Curbelo et al.

cases, triplicate injections of six different concentrationslevels (n06) were considered (See Table 1). Determinationcoefficients (R2) were higher than 0.997 for all compounds.

One of the commonly used methods to compensate GCmatrix effects is the development of a matrix-matched cal-ibration which involves the construction of the calibrationcurve of the pesticides dissolved in the final matrix extract[34]. In this sense, statistical comparison between the cali-bration in cyclohexane and in each type of baby food wascarried out using a statistical program (Statgraphics Plus)that calculates F and p values comparing the slopes and theintercepts of the calibration curves for each analyte. Statis-tical differences between slopes, intercepts or both of themwere found for all pesticides because p values were below orequal to 0.1 and thus, calibration with the baby food matrixwas found necessary.

From the calibration data of each of the 15 pesticides stud-ied, instrumental/chromatographic LODs and LOQs, definedas the concentration of the standards of the analytes dissolved incyclohexane that yielded a S/N ratio of three and ten,

respectively, were estimated. Instrumental LODs were in theranges 0.17–1.55 μg/L and instrumental LOQs in the range0.55–5.17 μg/L which were experimentally checked.

Recovery, precision and accuracy of the method

The repeatability of the method was initially studied by theexamination of a recovery study which was performed byanalyzing each baby food sample spiked at two concentra-tion levels in quintuplicate (n05) and injecting each of themin triplicate in the GC-NPD system. Figure 4 shows thechromatograms of the spiked and non-spiked Baby food 1,after the application of the MWCNTs-dSPE-GC-NPD meth-od. As can be observed, the sample was free of the selectedpesticides and no chromatographic interferences that over-lapped with the selected pesticides were found. Mean re-covery values were calculated from a comparison of thepesticide peak areas with the ones obtained for matrix-matched standards. Table 2 shows the absolute recoveriesand relative standard deviation (RSD) values obtained for

10 20 30 40 Time (min)500

Baby food 1

mV

olts

4

1

2

3

5

6 7

8 910

11

12

13

14

15

0

25

50

75

100

0

25

50

75

100

10 20 30 40 Time (min)500

Fig. 4 GC-NPD chromatogramof the spiked and non-spikedBaby food 1 sample after theapplication of the proposedMWCNTs-dSPE-GC-NPDmethod. Peak identification andconcentration: (1) disulfoton-sulfoxide (444 μg/kg); (2)ethoprofos (428 μg/kg); (3)cadusafos (419 μg/kg); (4) di-methoate (1,551 μg/kg); (5)terbufos (552 μg/kg); (6) disul-foton (227 μg/kg); (7)chlorpyrifos-methyl (380 μg/kg); (8) malaoxon (1618 μg/kg); (9) fenitrothion (744 μg/kg); (10) pirimiphos-methyl(378 μg/kg); (11) malathion(467 μg/kg); (12) chlorpyrifos(417 μg/kg); (13) terbufos-sulfone (524 μg/kg); (14)disulfoton-sulfone (520 μg/kg);(15) fensulfothion (513 μg/kg).See the section “Apparatus andsoftware” for other conditions

Pesticide residue analysis in cereal-based baby foods 191

Tab

le2

Meanrecoveries

(n05),R

SDvalues

(betweenparenthesis),add

edconcentration(betweenparenthesis)andLODsof

theselected

pesticides

indifferentbaby

food

cerealsamples

afterthe

MWCNTs-dS

PE-G

C-N

PD

metho

d

Pesticide

Cereal-basedBabyfood

1Cereal-basedBabyfood

2Cereal-basedBabyfood

3

Meanrecovery

(RSD%) n05

(add

edμg

/kg)

LODmethod(μg/kg

)Meanrecovery

(RSD%) n05

(add

edμg

/kg)

LODmethod(μg/kg

)Meanrecovery

(RSD%) n05

(add

edμg

/kg)

LODmethod(μg/kg

)

Level

1Level

2Level

1Level

2Level

1Level

2

Disulfoton-sulfox

ide

96(7)(44)

105(2)(444

)0.12

97(2)(44)

102(5)(444

)0.12

96(5)(44)

101(6)(444

)0.09

Ethop

roph

os85

(5)(43)

98(4)(428

)0.18

82(6)(43)

91(3)(428

)0.19

88(3)(43)

83(3)(428

)0.17

Cadusafos

85(4)(42)

96(4)(419

)0.38

88(7)(42)

92(4)(419

)0.39

90(4)(42)

86(5)(419

)0.33

Dim

etho

ate

72(5)(155

)72

(3)(155

1)0.46

76(3)(155

)71

(3)(155

1)0.54

73(4)(155

)70

(5)(155

1)0.33

Terbu

fos

64(3)(55)

64(4)(552

)0.21

67(4)(55)

69(5)(552

)0.22

74(4)(55)

67(2)(552

)0.18

Disulfoton

65(4)(23)

64(4)(227

)0.33

66(3)(23)

68(4)(227

)0.34

70(3)(23)

66(5)(227

)0.26

Chlorpy

rifos-methy

l74

(4)(38)

70(2)(380

)0.54

73(4)(38)

70(5)(380

)0.60

78(4)(38)

71(4)(380

)0.49

Malaoxo

n74

(4)(162

)76

(3)(161

8)1.38

73(5)(162

)74

(3)(161

8)1.65

72(4)(162

)75

(5)(161

8)1.32

Fenitrothion

83(5)(74)

91(3)(744

)0.61

83(6)(74)

88(4)(744

)0.67

85(3)(74)

86(4)(744

)0.52

Pirim

ipho

s-methy

l72

(4)(38)

77(4)(378

)0.38

74(3)(38)

74(2)(378

)0.38

84(5)(38)

76(3)(378

)0.33

Malathion

80(4)(47)

90(2)(467

)0.46

79(1)(47)

80(4)(467

)0.49

87(4)(47)

78(4)(467

)0.44

Chlorpy

rifos

73(7)(42)

74(3)(417

)0.23

72(4)(42)

71(2)(417

)0.27

76(5)(42)

76(3)(417

)0.20

Terbu

fos-sulfon

e86

(3)(52)

98(4)(524

)0.32

89(4)(52)

89(3)(524

)0.33

94(3)(52)

87(1)(524

)0.28

Disulfoton-sulfon

e84

(6)(52)

93(3)(520

)0.17

86(3)(52)

83(4)(520

)0.18

98(5)(52)

80(6)(520

)0.16

Fensulfothion

90(4)(51)

102(3)(513

)0.13

91(8)(51)

96(2)(513

)0.13

102(3)(51)

90(4)(513

)0.12

192 M.Á. González-Curbelo et al.

the spiked samples at each concentration level. For allcompounds, average recoveries were satisfactory, rangingfrom 64 to 105 % for Baby food 1, from 66 to 102 % forBaby food 2 and from 66 to 102 % for Baby food 3, withRSD values lower than 7.6 % in all cases. Table 2 alsoshows the determined LODs of the proposed method foreach matrix, which were defined as the lowest concentrationof the standards in the samples giving a S/N ratio of three. ForBaby food 1, LODs ranged between 0.12 and 1.38 μg/kg,0.12 and 1.65 μg/kg for Baby food 2 and 0.09 and 1.32 μg/kgfor Baby food 3. These LODs were also experimentallychecked by analyzing spiked samples at the LOD level andthe subsequent calculation of the S/N. Concerning LOQsvalues, they ranged between 0.40 and 4.59 μg/kg for Babyfood 1, 0.40 and 5.50 μg/kg for Baby food 2 and 0.31 and4.40 μg/kg for Baby food 3, calculated from a S/N ratio of ten.All these results revealed that the developed method is highlyrepeatable and sensitive, as well as very selective. Besides,these LOQs are below MRLs specified in the EU Baby FoodDirective 2003/13/EC for these pesticides [35].

In addition, recoveries, LODs and LOQs obtained arecomparable to those previously obtained in the literaturefor similar pesticides and matrices [6] or even lower [9].As an example, and as previously indicated, Leandro et al.[6] developed a QuEChERS-UPLC-MS/MS method to de-termine 52 pesticides in potato, orange and cereal-basedbaby foods, obtaining recoveries between 73 and 114 %for cereal-based baby foods. Regarding also baby foods,but containing both cereals and fruits or other ingredients,the LODs achieved in this work were also comparable tothose obtained in previous methods [36–38] (see Table 3).

To evaluate the precision and accuracy of the method,pesticides-free baby food samples were spiked at two dif-ferent concentration levels. Five consecutive extractions(n05) were carried out at each level in order to comparethe concentration found with the spiked one. Table 4 showsthe results of this study, in which a Student’s t test revealedthat there were no statistical significant differences betweenthe spiked and the obtained values since experimental tvalues (t≤2.78) were below or equal to the tabulated one(t402.78 for n05 and α00.05). Finally, accuracy percen-tages ranged between 92 and 107 %, which shows the highrobustness of the method developed.

Conclusions

In this work, a new dSPE method using MWCNTs has beenproposed for the first time for the extraction of 15 pesticidesresidues from cereal-based baby foods aqueous extracts.Optimization of extraction and dSPE parameters as well asmatrix-matched calibration, recovery, precision and accura-cy studies were carried out for three different types of cereal T

able

3Com

parisonof

thismetho

dwith

previous

ones

dealingwith

theanalysisof

OPPsin

baby

food

sby

othertechniqu

esandmetho

ds

Analytes

Typ

eof

baby

food

Sam

pletreatm

ent

Separationtechniques

LODs

Reference

50multiclass

pesticides

includ

ing10

OPPs(m

ethamidop

hos,acephate,om

etho

ate,

oxydem

eton-m

ethy

l,demeton

-S-m

ethy

lsulfon

e,dimetho

ate,ph

oratesulfox

ide,

phoratesulfone,azinph

os-m

ethy

landph

orate)

Cereal-based

QuE

ChE

RS

UPL

C-M

S/MS

LCL:0.005mg/kg

[6]

15OPPs(disulfoton-sulfox

ide,etho

prop

hos,cadu

safos,dimetho

ate,terbufos,disulfoton

,chlorpyrifos-m

ethy

l,malaoxo

n,fenitrothion

,pirimipho

s-methy

l,malathion

,chlorpyrifos,

terbufos-sulfone,disulfoton

-sulfone

andfensulfothion)

Cereal-based

QuE

ChE

RS

GC-N

PD

0.07

–34.8

μg/kg

[9]

109multiclass

pesticides

includ

ing29

OPPs(m

ethamidopho

s,dichlorvos,mevinph

os,

acephate,heptenop

hos,om

etho

ate,mon

ocrotoph

os,dimetho

ate,diazinon

,ph

osph

amidon

I+II,chlorpyrifos-m

ethy

l,parathion-methy

l,pirimipho

s-methy

l,fenitrothion

,malathion

,chlorpyrifos,fenthion

,parathion,

chlorfenvinp

hosI+

II,qu

inalph

os,methidathion,

ethion

,triazophos,phosmet,ph

osalon

e,azinph

os-m

ethy

landazinph

os-ethyl)

App

le-based

QuE

ChE

RSor

EtOAc-GPCmethod

GC-TOF-M

SLCL:2.5–

25μg

/kg

[36]

5OPPs(dim

ethoate,chlorpyrifos,methidathion,

phosaloneanddiazinon

)Fruit-based

EtOACandaceton

epartition

metho

dsGC-N

PD

0.001–

0.1mg/kg

[37]

44multiclass

pesticides

includ

ing12

OPPs(dichlorvo

s,methamidop

hos,mevinph

os,

acephate,om

etho

ate,dimetho

ate,diazinon

,pirimipho

s-methy

l,chlorpyrifos,ethion

,azinphos-m

ethylandcoum

apho

s)

App

le-based

QuE

ChE

RS

GC-M

SLCL:2

.5–200μg

/kg

[38]

15OPPs(disulfoton-sulfox

ide,etho

prop

hos,cadu

safos,dimetho

ate,terbufos,disulfoton

,chlorpyrifos-m

ethy

l,malaoxo

n,fenitrothion

,pirimipho

s-methy

l,malathion

,chlorpyrifos,

terbufos-sulfone,disulfoton

-sulfone

andfensulfothion)

Cereal-based

dSPEwith

MWCNTs

GC-N

PD

0.09

–1.65

μg/kg

Thiswork

LCLlowestcalib

ratio

nlevel

Pesticide residue analysis in cereal-based baby foods 193

Table 4 Results of assays to check the precision and accuracy of the proposed method for the selected pesticides in each baby food sample

Pesticide Spiked level (μg/kg) Sample Found (μg/kg)a Accuracy (%) texpb

Disulfoton-sulfoxide 30 Baby food 1 29±5 99 0.20

Baby food 2 30±6 102 0.84

Baby food 3 32±4 107 1.64

296 Baby food 1 284±1 96 2.64

Baby food 2 304±1 103 0.72

Baby food 3 309±1 104 1.39

Ethoprophos 29 Baby food 1 30±2 105 1.85

Baby food 2 29±6 101 0.15

Baby food 3 27±3 94 1.94

286 Baby food 1 276±1 97 1.75

Baby food 2 289±1 101 0.83

Baby food 3 295±1 103 1.72

Cadusafos 28 Baby food 1 28±3 100 0.10

Baby food 2 27±3 97 2.70

Baby food 3 26±6 95 1.52

279 Baby food 1 288±1 103 1.04

Baby food 2 274±1 98 1.01

Baby food 3 280±1 100 0.16

Dimethoate 103 Baby food 1 97±3 94 2.45

Baby food 2 97±4 94 2.05

Baby food 3 96±4 93 1.83

1034 Baby food 1 1069±1 103 2.12

Baby food 2 973±1 94 2.36

Baby food 3 1024±1 99 0.43

Terbufos 37 Baby food 1 36±4 98 1.24

Baby food 2 36±6 99 0.51

Baby food 3 34±3 93 2.75

368 Baby food 1 369±1 100 0.50

Baby food 2 349±3 95 2.52

Baby food 3 353±1 96 0.99

Disulfoton 15 Baby food 1 14±4 96 1.24

Baby food 2 15±5 99 0.38

Baby food 3 14±5 95 1.62

151 Baby food 1 143±2 95 2.05

Baby food 2 151±2 100 0.11

Baby food 3 146±2 97 2.30

Chlorpyrifos-methyl 25 Baby food 1 26±5 104 1.90

Baby food 2 25±9 99 0.49

Baby food 3 25±6 100 0.47

253 Baby food 1 247±2 98 0.94

Baby food 2 237±3 94 2.47

Baby food 3 235±3 93 2.15

Malaoxon 108 Baby food 1 103±4 96 1.89

Baby food 2 107±4 99 0.15

Baby food 3 102±7 94 1.42

1079 Baby food 1 1036±1 96 1.44

Baby food 2 1017±2 94 2.28

Baby food 3 1037±2 96 2.14

194 M.Á. González-Curbelo et al.

baby foods. Mean recovery values ranged between 64 and105 % (RSDs <7.6 %) for all types of samples and for the 15

studied analytes. LOQs, which ranged between 0.31 and5.50 μg/kg were below the EU MRLs of these pesticides

Table 4 (continued)

Pesticide Spiked level (μg/kg) Sample Found (μg/kg)a Accuracy (%) texpb

Fenitrothion 50 Baby food 1 53±3 107 2.76

Baby food 2 52±5 104 1.25

Baby food 3 48±3 97 0.99

496 Baby food 1 457±1 92 2.72

Baby food 2 499±1 101 0.42

Baby food 3 477±1 96 1.59

Pirimiphos-methyl 25 Baby food 1 26±3 103 1.19

Baby food 2 27±5 106 2.27

Baby food 3 26±6 101 0.47

252 Baby food 1 247±1 98 1.05

Baby food 2 251±2 100 0.22

Baby food 3 243±2 96 1.10

Malathion 31 Baby food 1 32±4 102 0.80

Baby food 2 33±4 105 2.04

Baby food 3 31±6 101 0.30

311 Baby food 1 298±1 96 2.40

Baby food 2 313±1 101 0.36

Baby food 3 315±2 101 0.67

Chlorpyrifos 28 Baby food 1 27±3 99 0.77

Baby food 2 29±5 104 2.08

Baby food 3 28±6 102 1.01

278 Baby food 1 268±1 96 1.09

Baby food 2 295±2 106 2.55

Baby food 3 264±2 95 2.72

Terbufos-sulfone 35 Baby food 1 35±4 101 0.76

Baby food 2 34±5 98 1.35

Baby food 3 33±8 95 1.20

349 Baby food 1 331±1 95 1.77

Baby food 2 339±1 97 0.94

Baby food 3 352±1 101 0.38

Disulfoton-sulfone 35 Baby food 1 36±5 105 1.37

Baby food 2 32±8 93 2.78

Baby food 3 34±5 97 1.31

347 Baby food 1 341±1 98 0.47

Baby food 2 356±2 103 1.32

Baby food 3 349±2 101 0.42

Fensulfothion 34 Baby food 1 35±4 102 0.55

Baby food 2 32±9 93 2.64

Baby food 3 34±6 98 0.58

342 Baby food 1 330±1 96 2.23

Baby food 2 355±1 104 2.19

Baby food 3 360±2 105 1.94

a Average value±standard deviation of five determinations (95 % confidence level)b Experimental t value (ttab02.78, α00.05)

Pesticide residue analysis in cereal-based baby foods 195

in cereal-based baby foods. The method involved the use oflow amounts of MWCNTs, only 80 mg, which is consider-ably lower than the amounts frequently used in SPE withconventional sorbents. Besides, the application of the dSPEmode considerably reduces extraction time and simplifiesthe extraction compared to conventional SPE, since it is notcarried out in the column but in the bulk solution. This workrepresents the first application of CNTs-dSPE for the extrac-tion of pesticides in food analysis.

Acknowledgments M.A.G.C., M.A.R. and A.V.H.H. wish to thankthe Spanish Ministry of Education for the FPU grant at the Universityof La Laguna. J.H.B. thanks the Spanish Ministry of Science andInnovation for the Ramón y Cajal contract at the University of LaLaguna. This work has been supported by the Spanish Ministry ofScience and Innovation (project AGL2009-07884).

References

1. Ciecierska M, Obiedziński MW (2010) Food Control 21:1166–11722. Rey-Salgueiro L, Martínez-Carballo E, García-Falcón MS, González-

Barreiro C, Simal-Gándara J (2009) Food Chem 115:814–8193. Brera C, Debegnach F, De Santis B, Pannunzi E, Berdini C,

Prantera E, Gregori E, Miraglia M (2011) Talanta 83:1442–14464. D’Arco G, Fernández-Franzón M, Font G, Damiani P, Mañes J

(2008) J Chromatogr A 1209:188–1945. Leandro CC, Hancock P, Fussell RJ, Keely BJ (2006) J Chroma-

togr A 1103:94–1016. Leandro CC, Hancock P, Fussell RJ, Keely BJ (2007) J Chroma-

togr A 1144:161–1697. Monitoring of pesticide residues in products of plant origin in the

EU, Norway, Iceland and Liechtenstein. http://ec.europa.eu/food/fvo/specialreports/pesticides_index_en.htm, Accessed 1 Feb. 2012

8. Leandro CC, Fussell RJ, Keely BJ (2005) J Chromatogr A1085:207–212

9. González-Curbelo MA, Hernández-Borges J, Borges-Miquel TM,Rodríguez-Delgado MA (2012) Food Addit Contam 29:104–116

10. Duan AH, Xie SM, Yuan LM (2011) Trac-Trend Anal Chem30:484–491

11. Valcárcel M, Cárdenas S, Simonet BM, Moliner-Martínez Y,Lucena R (2008) Trac-Trend Anal Chem 27:34–43

12. Li XS, Wu JH, Xu LD, Zhao Q, Luo YB, Yuan BF, Feng YQ(2011) Chem Commun 47:9816–9818

13. Jacobs CB, Peairs MJ, Venton BJ (2010) Anal Chim Acta662:105–127

14. Ma J, Xiao R, Li J, Yu J, Zhang Y (2010) Chen L 1217:5462–5469

15. Ravelo-Pérez LM, Herrera-Herrera AV, Hernández-Borges J,Rodríguez-Delgado MA (2010) J Chromatogr A 1217:2618–2641

16. Pyrzynska K (2011) Chemosphere 83:1407–141317. Du D, Wang M, Zhang J, Cai J, Tu H, Zhang A (2008) Electro-

chem Commun 10:85–8918. Ravelo-Pérez LM, Hernández-Borges J, Rodríguez-Delgado MA

(2008) J Chromatogr A 1211:33–4219. López-Feria S, Cárdenas S, Valcárcel M (2009) J Chromatogr A

1216:7346–735020. Tsai WH, Huang TC, Huang JJ, Hsue YH, Chuang HY (2009) J

Chromatogr A 1216:2263–226921. Lu Q, Chen X, Nie L, Jiang H, Chen L, Hu Q, Du S, Zhang Z

(2010) Talanta 81:959–96622. Peng Y, Xie Y, Luo J, Nie L, Chen Y, Chen L, Du S, Zhang Z

(2010) Anal Chim Acta 674:190–20023. Asensio-Ramos M, D’Orazio G, Hernández-Borges J, Rocco A,

Fanali S (2011) Anal Bioanal Chem 400:1113–112324. Herrera-Herrera AV, Ravelo-Pérez LM, Hernández-Borges J,

Afonso MM, Palenzuela J, Rodríguez-Delgado MA (2011) J Chro-matogr A 1218:5352–5361

25. Morales-Cid G, Fakete A, Simonet BM, Lehmann R, Cárdenas S,Zhang X, Valcárcel M, Schmitt-Kopplin P (2010) Anal Chem82:2743–2752

26. Ding J, Gao Q, Li XS, Huang W, Shi ZG, Feng YQ (2011) J SepSci 34:2498–2504

27. Pardasani D, Kanaujia PK, Purohit AK, Shrivastava AR, DubeyDK (2011) Talanta 86:248–255

28. Asensio-Ramos M, Hernández-Borges J, Borges-Miquel TM,Rodríguez-Delgado MA (2009) Anal Chim Acta 647:167–176

29. Lee SJ, Park HJ, Kim W, Jin JS, El-Aty AMA, Shim JH, Shin SC(2009) Biomed Chromatogr 23:434–442

30. Liu X, Xu J, Li Y, Dong F, Li J, Song W, Zheng Y (2011) AnalBioanal Chem 399:2539–2547

31. Chung SWC, Chen BLS (2011) J Chromatogr A 1218:5555–556732. Gilbert-López B, García-Reyes JF, Molina-Díaz A (2009) Talanta

79:109–12833. Koesukwiwat U, Sanguankaew K, Leepipatpiboon N (2008) Anal

Chim Acta 626:10–2034. Poole CF (2007) J Chromatogr A 1158:241–25035. Commission Directive 2003/13/EC of 10 February 2003 amending

Directive 96/5/EC on processed cereal-based foods and baby foodsfor infants and young children, Off. J. Eur. Commun. L41 (2003)

36. Căjka T, Hajšlová J, Lacina O, Maštovská K, Lehotay SJ (2008) JChromatogr A 1186:281–294

37. Georgakopoulos P, Mylona A, Athanasopoulos P, Drosinos EH,Skandamis PN (2009) Food Chem 115:1164–1169

38. Căjka T, Maštovská K, Lehotay SJ, Hajšlová J (2005) J Sep Sci28:1048–1060

196 M.Á. González-Curbelo et al.