ORIGINAL PAPER

Determination of eight pesticides of varying polarity in surfacewaters using solid phase extraction with multiwalled carbonnanotubes and liquid chromatography-linear ion trap massspectrometry

Soraya Dahane & María Dolores Gil García &

Ana Uclés Moreno & María Martínez Galera &

María del Mar Socías Viciana & Aicha Derdour

Received: 18 March 2014 /Accepted: 13 May 2014# Springer-Verlag Wien 2014

Abstract We describe a MWCNT-based method for thesolid-phase extraction of eight pesticides from environmentalwater samples. The analytes are extracted from 100 mL sam-ples at pH 5.0 (containing 5 mmol L−1 of KCl) by passing thesolution through a column filled with 20 mg of multiwalledcarbon nanotubes. Following elution, the pesticides were de-termined by LC and electrospray ionization hybrid quadru-pole linear ion trap MS. Two selected reaction monitoringtransitions were monitored per compound, the most intenseone being used for quantification and the second one forconfirmation. In addition, an information-dependent acquisi-tion experiment was performed for unequivocal confirmationof positive findings. Matrix effect was not found in real watersand therefore the quantitation was carried out with calibration

graphs built with solvent based standards. Except forcymoxanil, the detection and quantitation limits in surfacewaters are in the range from 0.3 to 9.5 ng L−1 and 1.6 to45.2 ng L−1, respectively. Recoveries from spiked ultrapurewater are ~100 %, except for the most polar pesticidesmethomyl and cymoxanil. The same behavior is found forreal water samples (except for phosalone). The relative stan-dard deviation is <10 % in all cases.

Keywords Pesticides . Solid-phase extraction .Multiwalledcarbon nanotubes . Liquid-chromatography-massspectrometry

Introduction

Since removal of organo chlorine insecticides from use, or-ganophosphate insecticides have become the most widelyused insecticides available today. More than forty of themare currently registered for use and all run the risk of acuteand subacute toxicity. All of them apparently share a commonmechanism of cholinesterase inhibition by disrupting the en-zyme that regulates acetylcholine, a neurotransmitter, and cancause similar symptoms. Therefore, exposure to one or mul-tiple organophosphates can lead to serious additive toxicity[1].

N-methyl carbamate insecticides are widely used and theyshare with organophosphates the capacity to inhibit cholines-terase enzymes and, therefore, they share similar symptom-atology during acute and chronic exposures [2].

Fungicides also have wide use in agriculture and, althoughthe acute toxicity of fungicides to humans is generally con-sidered to be low, they must also be controlled in water

Electronic supplementary material The online version of this article(doi:10.1007/s00604-014-1290-x) contains supplementary material,which is available to authorized users.

M. D. Gil García :A. Uclés Moreno :M. Martínez Galera (*) :M. M. Socías VicianaDepartment of Chemistry and Physics, Area of Analytical Chemistry,University of Almería, E-04120 La Cañada de San Urbano, Almería,Spaine-mail: mmartine@ual.es

M. D. Gil García :A. Uclés Moreno :M. Martínez Galera :M. M. Socías VicianaCentro de Investigación en Biotecnología Agroalimentaria, BITAL,La Cañada de San Urbano, E-04120 Almería, Spain

M. D. Gil García :A. Uclés Moreno :M. Martínez Galera :M. M. Socías VicianaCampus de Excelencia Internacional Agroalimentario CeiA3, LaCañada de San Urbano, Almería, Spain

S. Dahane :A. DerdourDepartment of Chemistry, University of Oran, Oran, Algeria

Microchim ActaDOI 10.1007/s00604-014-1290-x

sources, following the European Union Directive on drinkingwater quality (98/83/EC) [3].

Before determining contaminants in water samples, apreliminary sample pre-concentration and clean step mustbe carried out, the most popular technique being solidphase extraction (SPE). In environmental analysis, par-ticularly in the isolation and pre-concentration of pesti-cides and their transformation products from aqueoussamples, the use of hydrophobic non-selective sorbents,such as C18-bonded silica and styrene-divinylbenzeneco-polymers has been considered the best option [4].However, new advances have been developed, relatedto research on novel sorbent materials for SPE. Thus, FAugusto et al. [5] published a recent review devoted tonew materials and trends in sorbents for SPE and KPyrzynska [6] has reviewed the use of nanomaterials(metallic, silica and carbon-based) in sample preparation.

Carbon nanotubes (CNTs) are among the most usednanomaterials for SPE [6, 7]. They interact strongly withorganic molecules via non-covalent forces such as hydropho-bic, electrostatic and van der Waals interactions, hydrogenbonding and π stacking and these interactions, along withtheirs hollow and layered structures which involve largesurface-to-volume ratios, makes them good candidates foruse as sorbents, specially multi wallet carbon nanotubes(MWCNTs) [6].

CNTs have been applied for the extraction of both organicand inorganic compounds. As for the first ones, CNTs havebeen widely used for extraction of pesticides, as can be seenfrom the information gathered by LM Ravelo-Pérez et al. [7],K Pyrzynska [8] and BT Zhang et al. [9]. Thus, pesticides withdifferent chemical properties were pre-concentrated from wa-ter samples [10–16], soils [17, 18], fruit juices [19], virginolive oils [20] or vegetables and fruits [21].

In response to environmental problems, due to the bioac-cumulation and global transport of volatile apolar pesticides,the modern ones are more polar, thermo-labile and less vola-tile compounds [22]. Therefore, the analytical approaches todetermine pesticides have been adapted to these propertiesand, even though gas chromatography (GC) with mass spec-trometry (MS) detection is still used [23, 24], the coupling ofliquid chromatography (LC) with mass spectrometry (MS) viaatmospheric pressure ionization (API) is the best choice todetermine current pesticides.

Although LC-tandem MS (LC-MS2) techniques, such astriple quadrupole (QqQ) and ion trap (IT) are routinely used inenvironmental analysis, more recent approaches in LC-MS2

such as linear ion trap (LIT), new generation QqQs and hybridinstruments such as quadrupole-time of flight (Qq-TOF) andQ-linear trap (Qq-LIT), which offers advantages such as highscanning speed, accurate mass measurement (Qq-TOF) andincreased sensitivity (LIT and new-generation QqQ), are be-ing increasingly used.

The hybrid quadrupole-linear ion trap (Qq-LIT) is a QqQin which the third quadrupole can be operated as either aquadrupole or an ion trap. In this way, the same analyzer canbe run in two different modes, retaining the classical QqQscan functions, such as selected reaction monitoring (SRM),product ion, neutral loss and precursor ion, while providingaccess to sensitive IT experiments. This allows very powerfulscan runs which can be combined in one single experimentwhen performing information-dependent data acquisition(IDA). The QqLIT enables extraction of nearly an order ofmagnitude more ions into the linear ion trap compared withthe conventional 3D ion traps, when both are operated at thesame ion density. This is because the LIT, in contrast to the 3Dion trap, omits the quadrupolar electric field in the axialdirection which means that fewer ions are lost during theprocess of filling and emptying the trap [25]. As a conse-quence, it enables higher sensitivity for product-ion scan andoffers some extra scans possibilities such as enhanced fullscan (EMS), enhanced product ion (EPI) and MS3 [26].

However, in spite of these advances, determination andcorrect quantification of a large number of pesticides of dif-ferent chemical classes at low concentrations and/or in com-plex matrices still remains a challenge. The main goal of thepresent paper was to investigate the potential of MWCNTs assorbents for extracting eight pesticides of different polarityincluding organophosphate insecticides (methidation,parathion-methyl, malathion, phosalone and diazinon), fungi-cides (cymoxanil and penconazole) and one carbamate(methomyl), from environmental water samples as well as toexploit the improvements in sensitivity and specificity provid-ed by the LC-hybrid MSQqLIT detection for quantitating andconfirming the above mentioned pesticides.

Experimental

Reagents

Pesticide standards of methomyl (MET), cymoxanil (CYM),methidathion (MET), parathion-methyl (PMT), malathion(MAT), penconazole (PEN), phosalone (PHO) and diazinon(DIA) with >99 % purity, were purchased from Riedel-deHaën (Seelze, Germany, www.riedeldehaen.com). Chemicalstructures and principal physicochemical properties ofselected pesticides are included in the ElectronicSupplementary Material (ESM) (See Table S1).

Individual stock standard solutions of the target analytes(400 mg L−1) were prepared by weighing and dissolving thecorresponding compounds in acetonitrile and stored in thedark at −20 °C, being stable for at least 3 months. Workingstandard solutions were prepared daily by appropriate dilutionof individual stock standard solutions in acetonitrile-ultrapurewater (10:90, v/v).

S. Dahane et al.

Real water sample collection

Two types of surface waters (river and natural dam) were usedto evaluate the ability of MWCNTs as sorbent for pre-concentration of the eight pesticides in environmental waters.Two water samples were collected from rivers located inAlmería (Spain), one sample from Nacimiento River (sampleNCR) and the other from Andarax River (sample AXR).Nacimiento River flows through a scarcely populated areaand discharges into the Andarax River, which flows througha populated area. The sample NCR was picked up in theNacimiento River at the top of the stream, whereas the sampleAXR was picked up in the Andarax River near a wastewatertreatment plant which discharges its effluent in the riverflowing. In addition, two water samples were collected fromtwo natural dams (samples BND and GND), both located inAlmería (Spain). The sample BND was picked up in a damwhose water comes from the Andarax River and is used toirrigate crops, whereas the sample GND was picked from adam whose water comes from the rainfall and is used fordrinking by animals living in the area.

All real water samples were collected using 1 L amberbottles, with Teflon lined caps, which were previously rinsedwith the sample water on site, and were immediately carried tothe laboratory. Both, river and natural dam water sampleswere filtered through 0.45 μm cellulose filters and stored at4ºC in refrigerator.

Solid-phase extraction

The selected pesticides were extracted from environmentalwater samples by SPE using MWCNTs as sorbent. A BüchiVac V-500 (Flawil, Switzerland, www.buchi.com) vacuumpump, connected to an extraction manifold of Waters(Milford, MA, USA, www.waters.com) was used in thisprocess.

MWCNTs with an average external diameter (O.D.) of 6–13 nm, length (L) of 2.5–20 μm and 98 % carbon basic wereacquired from Sigma–Aldrich (Madrid, Spain, www.sigmaaldrich.com/spain.html). The SPE cartridges packedwith 20 mg of MWCNTs sorbent were prepared in thelaboratory using 1 mL polypropylene body syringes and twopolyethylene frits, both from Supelco (Madrid, Spain, www.sigmaaldrich.com/spain.html), to retain inside the sorbentmaterial.

To activate the sorbent, the MWCNT-SPE cartridgewas firstly conditioned with 5 mL of a mixtureacetone:n-hexane (50:50 v/v) and 5 mL of ultrapure wateradjusted at pH 5.0±0.1 at a flow rate of 1 mL min−1.Then, 100 mL of water sample, previously filtered, wasadjusted to pH 5.0±0.1 and both potassium chloride0.005 mol L−1 and 1 % methanol (organic modifier) wereadded to the sample prior pre-concentration.

The water sample was pre-concentrated onto the MWCNTcartridge at a flow rate of 1 mL min−1 and the sorbent wasdried by successively passing air through it for 5 min and thenN2 for another 5 min. The retained analytes were eluted with3 mL of a mixture acetone:n-hexano (50:50, v/v) and theeluate was evaporated to dryness using a N2 stream. Further-more, the obtained residue was dissolved in 1 mL ofacetonitrile:water (10:90, v/v) and finally, 10μL of the filteredextract were injected.

LC-QqLIT-MS/MS procedure

The chromatographic separation of the pesticides wasachieved in an Agilent 1200 LC (Agilent Technologies, CA,USA www.agilent.com) provided with a binary pump. Theanalytical column was a ZORBAX Eclipse XDB C8(150 mm×4.6 mm, 5 μm particle size) from Agilent. TheLC mobile phase consisted of a mixture of acetonitrile(solvent A) and LC-grade water containing 0.1 % formic acid(solvent B). The elution gradient program was as follows:initially 0.5 min of 10 % A, 9 min linear gradient to 100 %A, 5min of 100%A, 0.1 min to return to initial conditions andfinally initial conditions were reached in 10min. The flow rateof the mobile phase was set constant at 0.4 mL min−1 duringthe whole process and the injection volume was 10 μL.

The LC was connected to a hybrid triple quadrupole-linearion trap (QqLIT) mass spectrometer 5500 QTRAP® (ABSciex Instruments, CA, USA, www.absciex.com) with anelectrospray interface (ESI) which was operated in positiveionization mode (PI). The Turbo Ion Spray source settingswere: Ion Spray Voltage (IS) 5,500 V, Source Temperature(TEM) 500 °C, Curtain Gas (CUR) 30 psi, Collision Gas(CAD) Medium, both Ion Source Gas 1 (GS1) and Ion SourceGas 2 (GS2) 50 psi. Nitrogen was used as nebulizer gas andcollision gas.

For the SRM mode, the optimization was performed bydirect injection of individual standard solutions of each pesti-cide in methanol at 0.1 mg L−1. The operational MS/MSparameters for each pesticide such as declustering potential(DP), entrance potential (EP) for precursor ions, collisionenergy (CE) and collision cell exit potential (CXP) for productions, for each pesticide, are included in ESM (See Table S2).

In the IDA method, only the main SRM transition for eachsubstance is recorded and, therefore, 8 transitions were mon-itored, all of them in positive ion mode. This SRM experimentwas recorded by using the ScheduledMRMoption, setting thetarget scan time at 1 s with a MRM detection window of 40s.Q1 was set at low resolution and Q3 at unit. Regarding theIDA criteria, the intensity threshold was set to 500 cps andwithout exclusion after dynamic background subtraction ofthe survey scan. EPI scans were recorded at DP=100 Vat onecollision energy value CE=25 V, but using the collisionenergy spread (CES=5 V) and the LIT scanning from 80 to

Determination of pesticides in environmental waters using SPE with MWCNTs and LC-QqLIT

450 amu at a scan rate of 10,000 amu.s−1. The dynamic fill-time option was selected on the ion trap and EPI scans weremonitored in each SRM-IDA-EPI cycle, being the completeSRM-IDA-EPI cycle time of 1.58 s.

The ABSciex Analyst software was used for data acquisi-tion and processing.

Results and discussions

Optimization of LC-QqLIT-MS/MS method

The optimization of the chromatographic separation of theeight pesticides was performed by modification of a previous-ly published method [27].

For all the pesticides, positive electrospray ionization (PI)mode showed higher response and, thus the protonated mol-ecule [M + H+] was selected as the precursor ion. In order tocomply with the EU requirements the two most intense tran-sitions (SRM1 and SRM2) were selected for each pesticide[28] (See Table S2).

The SRM1was used for quantitation purposes, whereas thetwo SRM1 and SRMwere used for identification. In addition,in order to avoid overestimations or false positive findings inquantitative analysis, other criteria were used for identifica-tion: (i) the pesticide tR in the real sample must be within ±2%the pesticide tR in the standards and (ii) the relative abun-dances of the two selected SRM transitions (SRM2/SRM1) inthe real samples must be between±20 % of the SRM2/SRM1ratios in the analytical standards [29].

When working with standards, the abundances of the twoselected fragments (SRM1 and SRM2) were similar for mostanalytes, with SRM2/SRM1 between 0.84 and 0.70, exceptfor cymoxanil and phosalone with SRM2/SRM1 <0.3. Inthese cases, the SRM2 transition cannot be used for identifi-cation at low concentration levels, a drawback that can beovercome by the IDA approach, as EPI spectra can be obtain-ed at very high sensitivity levels providing confirmation in-formation at low concentrations [30]. Thus, working in theIDA mode provided further structural information for confir-mation of cymoxanil and phosalone, as well as extra confir-mation for the rest of the analytes.

The IDA approach used combined the SRM acquisitionmode with an enhanced product ion scan (EPI). EPI spectrawere acquired using the collision energy spread (CES) withCE=25 V and CES of 5 V, in such a way that the trap wasautomatically filled with fragment ions obtained at three dif-ferent energies (CE-CES, CE, CE + CES) [29], which were20, 25 and 30 V, and enough fragmentation was obtained forall pesticides (See Figure S1, ESM).

The EPI spectra generated for each pesticide were matchedagainst a mass spectral library, the good agreement between

the reference spectrum of the library and the spectrum obtain-ed in the samples being given by the fit, reverse fit (RevFit)and purity values provided by the software. The confirmationcriteria for identification of the target compounds were a fitvalue or purity higher than 70 %.

Optimization of the SPE procedure

Selection of type and volume of eluent solvent

CNTs have shown high adsorption capacity for organic com-pounds, the complete elution of the retained analytes from theCNTsorbent being very difficult in some cases [7, 31]. There-fore, in order to choose the best elution solvent, preliminarystudies were carried out using methanol, acetonitrile, acetone,n-hexane, dichloromethane and ethyl acetate. In all theseexperiments, 10 mL of ultrapure water, spiked with 20 ng ofthe pesticides, were passed through the SPE cartridge contain-ing 20 mg of MWCNTs sorbent, previously conditioned with5 mL of the selected elution solvent followed by 5 mL ofultrapure water. The elution of the retained analytes wasperformed with 2 mL of the elution solvent.

It was found that all pure solvents assayed gave poorelution efficiency, except acetone that showed recoverieshigher than 60 % for most compounds except for phosalone,whose recovery was lower than 30 %. This behaviour can bedue to the low polarity of this pesticide (log Kow=4.01), whichremains retained onto the sorbent. With the aim of improvingthe elution of phosalone, a mixture acetone:n-hexane (50:50,v/v) was assayed and a recovery of 70 %was achieved for thispesticide and also for the most polar methomyl. Therefore,acetone and n-hexane (50:50, v/v) was chosen as the elutionsolvent in the following experiments (Fig. 1a).

Next, the elution volume of the selected solvent (acetone:n-hexane 50:50, v/v) was studied between 2 and 7 mL. Theobtained results (Fig. 1b) showed that a volume of 3 mL ofacetone/n-hexane (50:50 v/v) allowed the complete elution ofall pesticides from the MWCNTs sorbent, with recoveries of80 % for methomyl and phosalone and about 100 % for theother ones.

Optimization of sample pH, organic modifier and ionicstrength

The pH of the water sample exerts an important effect on theSPE retention because it defines the molecular state of thetarget compounds (ionic or neutral state) and therefore, theiradsorption onto the MWCNTs sorbent.

Most selected pesticides do not show acidic-basic proper-ties, except penconazole, diazinon and cymoxanil with pKavalues of 1.5, 2.6 and 9.3, respectively. Therefore, the effect ofsample pH was evaluated in the range 3.0–9.0. Extreme pH

S. Dahane et al.

values were not considered due to the possible hydrolysis ofsome pesticides under strong acidic or basic conditions.

Experiments were performed using 50mL of ultrapure watercontaining 20 ng of each pesticide, adjusted at different pHvalues with hydrochloric acid 0.1 mol L−1 or sodium hydroxide0.1 mol L−1. In general, recoveries were similar between pH 3and 7, except for methomyl which showed better recoveries atpH 5 and, therefore, in the light of these results (Fig. 2a), pH 5was considered the optimum value for the water sample.

The influence of the salt content in the water sample on theextraction efficiency, was also investigated. The addition ofsalt to the water sample usually improves the retention ofanalytes into SPE sorbents due to the salting out effect [32].The increase in the pesticide extraction can be explained bythe engagement of water molecules in the hydration spheresaround the ionic salt and hence in the reduction of the waterconcentration available to dissolve the analytes [33]. Howev-er, at higher salt concentration the interaction between the saltions and the analytes predominates, thus reducing their capac-ity to be retained in the sorbent [33].

The effect of the salt content in the water sample wasexamined by analyzing ultrapure water samples containingdifferent concentrations of KCl (0–0.2 mol L−1). The extractionefficiencies increased or remained constant for most pesticidesup to concentration of 0.005 mol L−1 KCl and higher concen-trations of salt in the water sample decreased the retention formost of them (Fig. 2b). Thus, in all cases, 0.005 mol L−1 KClwas added to the water sample before SPE extraction.

Finally, the addition of organic modifier was evaluated inorder to improve pesticide recoveries. With this aim, 50 mL ofultrapure water samples spiked with 20 ng of each pesticide(adjusted at pH 5.0±0.1 and containing 0.005 mol L−1 KCl)were modified by the addition of different amounts of meth-anol (0–3 % v/v) and 1 % was selected as a compromisebetween the optimal conditions for the most polar pesticidesand the less polar ones (Fig. 2c).

MWCNTs amounts and flow rate

The effect of the amount of sorbent on the pesticide retentioninto SPE cartridge was investigated using 20 and 50 mg of

MWCNTs. Higher amounts of sorbent were not checkedbecause the small particle size of MWCNTs we used resultsin compactness of the sorbent, which difficult the pass of thewater sample through it, in addition to the volume of organicsolvent required for elution increases significantly [31]. 20mgof MWCNTs were selected as a compromise as the bestconditions for methomyl (50 mg with recovery 91 %) werethe worse ones for phosalone (50 mg with recovery 0 %).

On the other hand, in a previous work we found that thesmall particle size of MWCNTs reduces the sample flow ratethrough the SPE-sorbent and this fact leads to a significantlyincreasing of the pre-concentration time [31]. The abovementioned study showed that flow rates higher than1 mL min−1 led to low recoveries and, therefore, in this casethe flow rate was restricted to this value.

Sample loading volume (Breakthrough)

The breakthrough volume was estimated by analyzing differ-ent volumes of ultrapure water, all of them containing 20 ng ofeach pesticide. The retention of the analytes in the sorbentremained constant up to a volume of 500 mL (Fig. 2d), exceptfor methomyl, which eluted completely when more than250 mL of water were passed through the cartridge. Although250 mL could be considered as the breakthrough volume, dueto the high sensitivity of the LC-QqLIT method and with theaim of reducing the time spent in this step, 100 mL of waterwere chosen for further studies.

Method validation

Detection and quantitation limits

The instrumental detection (IDL) and quantitation (IQL)limits were estimated by direct injection of mixtures of pesti-cide standards in solvent (acetonitrile:ultrapure water, 1:9,v/v) at low concentrations. The IDLs were determined as thelowest pesticide concentration whose SRM2 transitionshowed a S/N=3 whereas the IQLs were determined as thelowest pesticide concentration whose SRM1 transitionshowed a S/N=10 (See Table S3, ESM). The IDLs achieved

Fig. 1 Optimization of SPEparameters: a) sample pH, b) saltcontent (KCl), c) percentage oforganic modifier (Methanol) and(d) water sample volume(breakthrough) on the retentioninto MWCNTs cartridges of: (1)Methomyl, (2) Cymoxanil, (3)Methidathion, (4) Malathion, (5)Penconazole, (6) Diazinon, (7)Parathion-methyl and (8)Phosalone

Determination of pesticides in environmental waters using SPE with MWCNTs and LC-QqLIT

for all pesticides were ranged from 0.02 to 0.25μg L−1 and theIQL were ranged from 0.07 to 0.93 μg L−1.

In addition, detection (LOD) and quantitation (LOQ) limitswere calculated using the criterion described above but ana-lyzing SPE blank extracts of four different environmentalwater samples (two river and two dump waters) spiked atlow concentrations of pesticides (See Table S3, ESM). TheLOD and LOQ were similar for all environmental waters butslightly higher than in solvent for most pesticides (LODbetween 0.03 and 0.95 μg L−1 and LOQ between 0.16 and4.52 μg L−1), except for cymoxanil whose LOD and LOQcalculated in real waters were significantly higher than insolvent.

Taking into account that the pre-concentration factorachieved by SPE-MWCNTs was 100, the LODs and LOQsof the method in the water samples were 121.0 and541.4 ng L−1 for cymoxanil and between 0.3 and 9.5 ng L−1

and between 1.6 and 45.2 ng L−1, respectively for the otherpesticides.

The SPE-MWCNT method allowed the quantitation of theselected pesticides in all types of environmental waters atconcentration levels lower than the ones established by theEU, except for cymoxanil, as can be deduced from theircorresponding LOQ values.

Linearity and matrix effect

The linearity in the response was studied by using standardsolutions of the target analytes prepared in solvent, rangingfrom the IQL of each analyte up to 200 μg L−1. The responsewas linear with the concentration between the corresponding

IQL and 10 μg L−1 for diazinon, 50 μg L−1 for cymoxanil,malathion and methomyl, 100 μg L−1 for methidathion,parathion-methyl and phosalone and 200 μg L−1 forpenconazole, with R2 >0.99 (See Table S4, ESM).

On the other hand, the effect of the co-eluting residualmatrix components present in the real water samples wasevaluated by comparing the slopes of calibration graphs ob-tained using standards prepared in solvent and blank extractsof environmental water (river and dumpwaters) by means of at-test [34]. Significant differences in the slopes of calibrationgraphs were not found for any pesticide and we stated thatmatrix effect was absent for all pesticides in the four environ-mental waters assayed. Therefore, quantitation of pesticides inreal water samples was performed using external calibrationcurves with standards prepared in solvent.

Accuracy and precision studies

The recovery of the pesticides was evaluated by spiking, intriplicate, ultrapure and environmental waters at two concen-tration levels (0.05 and 0.10 μg L−1 for diazinon and 0.25 and0.50 μg L−1 for the other pesticides). Recoveries were about100 %, except for methomyl and cymoxanil (Table 1), whoselow recoveries can be explained by their high water solubility(See Table S1, ESM), being only partially retained.

The precision of the overall method was calculated as therelative standard deviation (RSD%) of three replicate samplesgiving satisfactory results (Table 1), with values ranging from3.0 to 9.3 %.

To demonstrate the applicability of SPE-MWCNT-LC-QqLIT-MS/MS method, the two river waters and the two

Fig. 2 Optimization of SPEparameters: a) sample pH, b) saltcontent (KCL), c) percentage oforganic modifier (Methanol, (2)Cymoxanil, (3) Methidathion, (4)Malathion, (5) Penconazole, (6)Diazinon, (7) Parathion-methyland (8) Phosalone

S. Dahane et al.

dump waters, spiked at the same concentration levels thatultrapure water, were analyzed in triplicate.

Firstly, retention times of the eight pesticides in the fourreal water samples were within ±2 % of the corresponding tRin the standards and the relative abundances of the two select-ed transitions were within ±20% of the two SRM ratios in theanalytical standards (See Table S2, ESM). In addition, the EPIspectra acquired during the analysis of real water sampleswere identified by the spectral library with a match qualityof 70 %.

As for recoveries, the target pesticides kept in real watersthe same behaviour as in ultrapure water but, a significantdecrease in the recoveries of the less polar pesticide phosalonewas found in three of the four real water samples, which canbe due to the matrix components existing in these watersamples, according to the sampling sites. Humic acids(HAs), with 26.3 % of aromatic carbon (more than fulvicacids), can interact with electron-rich sites of MWCNTs sur-faces via π-π interactions [35] and compete with organicmolecules by direct site competition and pore blockage onMWCNTs. In addition, due to its high aromatic carbon per-centage, HAs can interact with the more hydrophobic com-pounds, which would reduce their adsorption toMWCNTs. Inthe light of these results, we propose this last mechanism toexplain the low recoveries obtained for phosalone in the morecomplex environmental water.

Although it seems a contradiction between the low recov-eries obtained for phosalone and the absence of matrix effect,this behaviour can be explained because most organic matterwas removed in the SPE procedure (by interaction withelectron-rich sites of MWCNTs surfaces via π-π interactions)and, therefore, it was absent in the blank extracts when thematrix matched calibration graphs were built. However, whenthe pesticides were added to the real water samples, they werepreferably adsorbed onto the MWCNTs whereas the organicmatter present in these samples interacted with the most apolar

pesticide phosalone reducing its adsorption toMWCNTs. Thisdemonstrates that comparing the slopes of calibration graphsbuilt in solvent and in blank extract of real samples is notalways a safe procedure to check the effect of matrix compo-nents and the standard addition method should be used whenthere is a risk for any analyte. In this way, a deep study aboutthe physico-chemical properties of analytes should be alwaysundertaken as the first step when developing an analyticalmethod.

Comparative study of the MWCNTs-LC-QqLIT method

According to the literature, the pre-concentration of the targetpesticides in aqueous samples was carried out with differentconventional SPE sorbents, the most frequently used beingOasis HLB although another sorbents such as Chroma bond oreven Sep-Pak C18 cartridges in tandem have been used (SeeTable S5, ESM). As can be seen the LODs obtained with ourmethod (using 20 mg of MWCNTs) were in the same order orlower than those obtained using the above mentionedsorbents.

The recoveries were also in the same order, except for themost polar pesticides methomyl and cymoxanil. In fact, ourprevious studies showed recoveries near 90 % for these twopesticides when using 50 mg of sorbent, but phosalone wasnot recoveried at all (0 % recovery). Therefore, a compromisebetween both situations was chosen.

As for methods using MWCNTs, the high sensitivity pro-vided by the QqLIT detection allowed to use less quantity ofsorbent, unlike the methods using this sorbent and less sensi-tive detection, such as UV (300 mg) or MS (100 mg). Also,the highly sensitive QqLIT detection allowed working withlow sample volumes, thus allowing low pre-concentrationfactors (100 in our work v.s. 250–1,000 in the other cases).An additional advantage provided by the QqLIT detection is

Table 1 Mean recovery (%) and RSD (%) obtained in the pre-concentration of pesticides in ultrapure and environmental waters, using MWCNTs assorbent

Pesticides Ultrapure water Sample NCR Sample ANX Sample BNT Sample GUA

0.25(μg L−1)

0.50(μg L−1)

0.25(μg L−1)

0.50(μg L−1)

0.25(μg L−1)

0.50(μg L−1)

0.25(μg L−1)

0.50(μg L−1)

0.25(μg L−1)

0.50(μg L−1)

Methomyl 51 (7) 54 (6) 43 (5) 50 (4) 49 (6) 52 (6) 60 (5) 53 (6) 56 (7) 58 (7)

Cymoxanil 80 (6) 59 (4) 86 (10) 57 (7) 79 (8) 71 (8) 71 (7) 68 (5) 74 (6) 68 (4)

Methidathion 100 (5) 95 (5) 98 (8) 98 (6) 84 (7) 86 (5) 93 (4) 86 (4) 84 (6) 90 (5)

Malathion 110 (5) 122 (6) 86 (8) 97 (6) 77 (6) 107 (4) 89 (5) 107 (4) 109 (6) 76 (3)

Penconazole 100 (4) 86 (4) 66 (9) 69 (7) 87 (6) 102 (4) 86 (7) 73 (5) 87 (6) 91 (6)

Diazinona 106 (3) 121 (4) 91 (6) 58 (4) 111 (3) 96 (4) 94 (7) 70 (5) 71 (5) 72 (5)

Parathion-methyl 89 (6) 81 (5) 81 (8) 83 (6) 79 (6) 72 (5) 95 (4) 79 (4) 84 (6) 72 (4)

Phosalone 115 (5) 81 (3) 98 (7) 89 (4) 65 (8) 64 (8) 57 (6) 57 (4) 58 (7) 60 (7)

a 0.05 and 0.1 μg L−1 RSD, relative standard deviation (n=3) between parentheses

Determination of pesticides in environmental waters using SPE with MWCNTs and LC-QqLIT

the absence of matrix effect, which is present in the methodsusing LC-MS.

However, in addition to the analytical performance, it isalso interesting to take into account the cost of the adsorbent.In this sense, the price of one Oasis HLB cartridge was about7.5 euros, whereas the price of one MWCNTs cartridge usedin this work was 4.9 euros, including the polypropylenesyringe and frits, which involves a cost reduction of 30 %.

Moreover, MWCNTs cartridges used to pre-concentratesolvent or clean water samples can be re-used up to ten times,after washing with 5 mL methanol, whereas conventionalcartridges are usually discarded.

Conclusions

In the present paper, SPE cartridges packed in the labo-ratory with 20 mg of MWCNTs were successfully usedto pre-concentrate eight pesticides in surface water sam-ples. High efficiency was obtained except for the mostpolar ones methomyl and cymoxanyl, whose recoverieswere lowered to get acceptable recoveries for the mostapolar phosalone. We can conclude that differences inthe polarity of the analytes are not compatible with goodrecoveries using MWCNTs. The pesticides were analyzedby liquid chromatography coupled to a hybrid triplequadrupole-linear ion trap-mass spectrometer at tracelevels working in SRM and IDA modes.

The developed methodology was applied to the anal-ysis of two river waters and two natural dam watersamples, spiked with the target pesticides, and the twocriteria to avoid overestimations (tR and SRM2/SRM1 inreal samples) were accomplished. The IDA mode provid-ed additional EPI spectra at low concentration levels,thus allowing reliable identification at the quantitationlevel for those pesticides with SRM2/SRM1 <0.3(cymoxanyl and phosalone) and providing additionalconfirmation for the other ones. The interaction of HAswith the more hydrophobic compound (phosalone) re-duced its adsorption to MWCNTs.

Comparing the slopes of calibration graphs is not always asafe procedure to checkmatrix effect and the standard additionmethod should be used when there is risk for any analyte,which could be predicted from its physico-chemicalproperties.

Acknowledgments The authors grateful to Agencia Española deCooperación Internacional (AECI, Acción Integrada A1/035959/11) forfinancial support. Soraya Dahane thanks to Agencia Española deCooperación Internacional for a research fellowship associated to theAcción Integrada A1/035959/2011.

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Determination of pesticides in environmental waters using SPE with MWCNTs and LC-QqLIT