Analytica Chimica Acta 559 (2006) 186–194

Behaviour of triazine herbicides and their hydroxylated and dealkylatedmetabolites on a propazine-imprinted polymer

Comparative study in organic and aqueous media

R. Carabias-Martınez∗, E. Rodrıguez-Gonzalo, E. Herrero-HernandezDepartamento de Quımica Analıtica, Nutricion y Bromatologıa, Facultad de Quımica, Universidad de Salamanca, 37008 Salamanca, Spain

Received 2 September 2005; received in revised form 25 November 2005; accepted 30 November 2005Available online 17 January 2006

Abstract

A molecularly imprinted polymer (MIP) obtained by precipitation polymerization with methacrylic acid as functional monomer and propazineas template was tested as a sorbent for the solid phase extraction of chloro- and methylthio-triazines and some of their dealkylated and hydroxylatedmetabolites. In order to confirm the interaction between the functional monomer and the template,1H NMR (CD2Cl2) analyses were made. Itw ds with thef

media andd ained aftero ot retainedo

nteractions,t IP througn©

K

1

itcilwiwl0

iouslysis,

alky-am-theloro-zine

instep.

reat-suchples.narybeenTheseibody

0d

as observed that the hydrogen and/or nitrogen of the amino group of the propazine were involved in the formation of hydrogen bonunctional monomer.

The possibilities offered by the propazine-MIP for the extraction of triazine and derivatives were studied in organic and aqueousifferent types of behaviour were observed. From organic medium, selective extractions of chloro- and methylthio-triazines were obtptimization of the extraction process, with recoveries higher than 75% in both cases. However, the hydroxylated metabolites were nn the propazine-MIP in organic medium.In aqueous medium, the chloro-triazines and the dealkylated chloro-metabolites were retained on the propazine-MIP by specific i

he recoveries obtained being a function of their polarity. Regarding the hydroxylated metabolites, these are retained on the propazine-Mhon-specific hydrogen-bridge interactions. The methylthio-triazines undergo poor molecular recognition in this medium.2005 Elsevier B.V. All rights reserved.

eywords: Molecularly imprinted polymers; Precipitation polymerization; Triazine herbicides; Dealkylated and hydroxylated metabolites

. Introduction

Triazine herbicides are some of the more commonly usedn Europe as selective pre- and post-emergence herbicides forhe control of broadleaf and grassy weeds in many agriculturalrops. Their prolonged use involves the risk of their retentionn crops and soils from which in turn, due to washing andeaching processes, these substances pass to surface and groundaters[1,2]. In previous work[3,4] evaluating these herbicides

n an agricultural zone close to the City of Salamanca (Spain),e observed that surface and ground water samples contained

evels of atrazine, terbutryne and deethylatrazine (DEA) above.1�g L−1.

∗ Corresponding author. Fax: +923 294 574.E-mail address: rcm@usal.es (R. Carabias-Martınez).

In water, parent triazine herbicides are subject to varbiotic and abiotic degradation processes such as photooxidation, hydrolysis and biodegradation, leading to the delation of amine groups, dechlorination, hydroxylation, deination, etc.[5,6]. For example, with respect to atrazinemain degradation products in water are the dealkylated chmetabolites: deethylatrazine (DEA) and deisopropylatra(DIA) [7,8].

Analytical methods for the determination of pollutantsenvironmental samples usually require a preconcentrationSolid-phase extraction (SPE) is a powerful tool for the pretment of environmental waters and other kind of matrices,as biological fluids, plant tissue culture and forensic samThe main problem associated with SPE with ordinary statiophases is its low selectivity. More selective sorbents havedeveloped, based on molecular recognition mechanisms.include immunosorbents that use selective antigen–antinteractions and molecularly imprinted polymers (MIPs).

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.11.081

R. Carabias-Martınez et al. / Analytica Chimica Acta 559 (2006) 186–194 187

Different authors have employed MIPs to extract triazinesfrom different matrices such as water[9–11], vegetable extracts[12], beef liver [13], urine, apple extract[14] and soil [15]and also an atrazine sensor based on MIP modified electrodes[16] has been developed. Most of these works attempt to studythe retention of parent compounds (chloro- and methylthio-triazine herbicides) using MIPs obtained by bulk polymeriza-tion. Although in some cases dealkylated chloro-metaboliteshave also been included[11,17], few works have addressedthe behaviour of hydroxylated metabolites. Only the works ofChapuis et al.[15,18] included hydroxyterbutylazine amongthe target analytes for retention studies on a terbutylazine-MIPobtained by bulk polymerization. As far as we are aware, thereare no reports that have addressed the retention behaviour ofN-dealkylated hydroxy-metabolites.

The aim of this work was to check the possibilities ofMIPs prepared by precipitation polymerization for the molec-ular recognition of different triazinic derivatives (chloro- andmethylthio-triazines and some of their hydroxylated, dealky-lated and dealkylated hydroxy-metabolites) when the samplepassing through the sorbent is organic or aqueous. The mecha-nism of the retention in both media was studied and the resultswere compared with those reported by other authors with MIPsobtained by bulk polymerization.

The selectivity of the MIP for the specific retention of thesemetabolites in organic and aqueous solutions was assesseda hlor-s

2

2

DrE t fur-tO l-s ASR -1( 30-

55-2] andlenacil (Len) 3-cyclohexyl-1,5,6,7-tetrahydrocyclo-pentapyrimidine-2,4(3H)-dione, CAS RN [2164-08-1].

Stock solutions of each herbicide and metabolite wereprepared in acetonitrile at 500�g mL−1 except in the caseof the hydroxylated metabolites which were prepared inacetonitrile–0.1 M hydrochloric acid (80:20, v/v) at a concen-tration of 200�g mL−1.

Methacrylic acid (MAA), ethylene glycol dimethacrylate(EGDMA) and 2,2′-Azobis(2-methyl-propionitrile) (AIBN)were obtained from Acros Organics (Geel, Belgium). Theorganic solvents, toluene, dichloromethane, acetonitrile andmethanol were of HPLC grade (Merck) and were used asreceived. Ultra-high quality UHQ water (purity 18.2 M� cm at25◦C) was obtained with an Elgastat UHQ water purificationsystem (Elga Ltd., Bucks, UK). All other chemicals were ofanalytical reagent grade.

2.2. Synthesis and characterization of the imprintedpolymer

The procedure followed to obtain the MIP by precipitationwas as follows: the template, propazine (1 mmol) and the func-tional monomer, MAA (4 mmol), were added to a glass tubeand after 5 min EGDMA (20 mmol), toluene (30 mL) and AIBN(0.2 mmol) were added. The solution was degassed with nitro-g r thisa poly-m t3 witha ed byS tureu zedb

re-p latet

byn sure-m on aG ss,G

TG lites

HHllHl

lllCH3CH3

he eq ents.

gainst non-triazine herbicides in frequent use: lenacil, culfuron, isoproturon and linuron.

. Experimental

.1. Chemicals

The herbicides and the metabolites were obtained fromhrenstorfer (Augsburg, Germany) and were used withou

her purification. The triazine compounds are listed inTable 1.ther herbicides were:chlorsulfuron (Cls) 1-(2-chloropheny

ulfonyl)-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea CN [64902-72-3]; isoproturon (Ipn) 3-(4-isopropylphenyl),1-dimethylurea CAS RN [34123-59-6];linuron (Lin) 3-3,4-dichlorophenyl)-1-methoxy-1-methylurea CAS RN [3

able 1eneral structure and some properties of triazine herbicides and metabo

Compounds R1

Deisopropylhydroxyatrazine (DIHA) –ODeethylhydroxyatrazine (DEHA) –ODeisopropylatrazine (DIA) –CDeethylatrazine (DEA) –CHydroxyterbutylazine (HT) –OSimazine (Smz) –CAtrazine (Atz) –CPropazine (Ppz) –CTerbutylazine (Tbz) –CPrometryn (Pmn) –STerbutryn (Tbn) –S

a logKow: n-octanol–water partition coefficients, defined as the ratio of t

.

en for 5 min and the tubes were closed and sealed undetmosphere. Microspheres were obtained by precipitationerization in a water bath at 60◦C for 10 h with stirring a50 rpm. The resulting microspheres were washed twicecetone to remove the fine particles. Template was removoxhlet extraction with a methanol:acetic acid (9:1, v/v) mixntil no template was found in the washing solution (analyy HPLC-DAD UV).

Blank polymers or non-imprinted polymers (NIP) were pared in the same way but without the addition of the temp

o the polymerization mixture.Characterization of the imprinted polymer was made

itrogen sorption and scanning electron microscopy meaents. Nitrogen sorption measurements were performedemini 2375 (Micromeritics Instrument Corporation, NorcroA) at the Department of Inorganic Chemistry (University

considered in this work

R2 R3 logKowa pKa

–NH2 –NH–CH2–CH3 −0.1 4.65–NH–CH(CH3)2 –NH2 0.2 4.57–4.75–NH2 –NH–CH2–CH3 1.15 1.30–1.58–NH–CH(CH3)2 –NH2 1.52 1.30–1.65–NH–C(CH3)3 –NH–CH2–CH3 – 5.2–NH–CH2–CH3 –NH–CH2–CH3 2.18 1.65

–NH–CH(CH3)2 –NH–CH2–CH3 2.5 1.68–NH–CH(CH3)2 –NH–CH(CH3)2 2.93 1.85–NH–C(CH3)3 –NH–CH2–CH3 3.21 2.00

–NH–CH(CH3)2 –NH–CH(CH3)2 3.51 4.05–NH–C(CH3)3 –NH–CH2–CH3 3.65 4.38

uilibrium concentrations of a dissolved substance in two immiscible solv

188 R. Carabias-Martınez et al. / Analytica Chimica Acta 559 (2006) 186–194

of Salamanca). The sample (ca. 80–100 mg) was previouslydegassed in flowing nitrogen at 100◦C for 12 h in a Flow-Prep 060 (Micromeritics) in order to remove physisorbed water.The specific surface area was evaluated using the BET method,and specific pore volume and the average pore diameter wereassessed according to the BJH theory. The physical character-istics of the propazine-MIP material are as follows: specificsurface area, 57.73 m2/g; specific pore volume, 0.063 cm3/g;average pore diameter, 3.78 nm. The polymer particles had aregular spherical morphology, with an average particle diameterof ∼1�m as determined by scanning electron microscopy usinga Zeiss DSM 949 (Zeiss, Oberkochen, Germany) device at theServicio de Microscopıa Electronica (University of Salamanca).

2.3. Procedure for preconcentrating triazines in organicmedium

For the experiments on extraction, 200 mg of polymer weredry-packed in empty solid-phase extraction cartridges of 3 mLand conditioned with 5.0 mL of a mixture acetonitrile:acetic acid(9:1, v/v) and 5.0 mL of toluene. The MIP columns were thenloaded with 2.0 mL of a mixture of triazines and metabolites ata concentration of 200�g L−1 in toluene. After an exhaustivedrying step, 10.0 mL of dichloromethane was percolated throughthe sorbent in order to eliminate non-specific interactions. Thetarget analytes were eluted from the cartridge with 10 mL of ana singa ithd lvea d ba

2m

tionw samp thes e of2 aftew vacu mLo per-f in thp

2

riesc any)e er, aa wac datac ndt as2p V

detector was set at 210 and 245 nm. UV spectra were recordedin the 190–400 nm range.

A gradient acetonitrile (solvent A)–5 mM phosphate bufferpH 7.2 (solvent B) was used: from 5 to 45% of solvent A in15 min, from 45 to 50% of solvent A in 15 min and returned toinitial conditions in 2 min, with 3 min for column equilibration.The flow rate was 1 mL min−1 and the volume injected was100�L. The analytical column was thermostated at 25◦C.

The elution order (retention time in brackets) was as follows:DIHA (7.32 min), DEHA (8.46 min), DIA (11.20 min), DEA(13.38 min), HT (14.91 min), simazine (17.15 min), atrazine(19.73 min), propazine (23.64 min), terbutylazine (25.41 min),prometryn (29.43 min) and terbutryn (30.87 min).

2.6. 1H NMR study

Sample was prepared with a fixed concentration of propazine(58.0 mmol L−1) in CD2Cl2 and varying concentrations of MAA(from 0 to 246 mmol L−1). 1H NMR spectra were recorded atroom temperature on a Bruker Avance DRX 400 MHz NMRspectrometer (Bruker, Karlsruhe, Germany).

3. Results and discussion

A MIP with propazine as template has been developedand its use as sorbent for the solid-phase extraction of tri-a beens thet ub-g d ter-b yn),h tedc yla-t iso-p ine,D rtiesa

er-i elys dings ent-i thea

3

platea g tod mersb thesei

erea ft rdingt ine.

gt n

cetonitrile–acetic acid (9:1, v/v) mixture and analysed un HPLC-DAD UV system. Adjustable-volume pipettors wisposable tips were used to load the sample, washing sond eluent into the cartridge; percolation was accomplishepplying a vacuum of−15 mmHg.

.4. Procedure for preconcentrating triazines in aqueousedium

A procedure similar to that describe in the previous secas used for the extraction of triazines from aqueousles, with the difference that in the final conditioning stepolvent employed was water instead of toluene. A volum.0 mL of aqueous sample was passed through the MIP,hich an exhaustive drying step was implemented (under aum of −15 mmHg for 30 min). For the washing step, 5f dichloromethane were employed. Analyte elution was

ormed under the same conditions as those describedrevious section.

.5. Chromatographic conditions

HPLC-DAD UV was performed on an HP 1100 Sehromatograph from Hewlett Packard (Waldbronn, Germquipped with a quaternary pump, a membrane degassutosampler and a diode-array UV detector. The systemontrolled by a HP ChemStation, which also performedollection from the diode array UV detector (DAD UV) ahe quantitative measurements. The analytical column w50 mm× 4.0 mm i.d. Spherisorb S5 ODS2 packed with 5�marticles (Waters, Milford, MA, USA). The diode-array U

nty

-

r-

e

ns

a

zine herbicides and some of their major metabolites hastudied. The analytes included in this study belong toriazine family and can be classified in the following sroups: chloro-triazines (simazine, atrazine, propazine anutylazine), methylthio-triazines (prometryn and terbutrydroxy-metabolites (hydroxyterbutylazine, HT) dealkylahloro-metabolites (deisopropylatrazine, DIA and deethrazine, DEA) and dealkylated hydroxy-metabolites (deropylhydroxyatrazine, DIHA and deethylhydroxyatrazEHA). Their chemical structures and some of their propere shown inTable 1.

The propazine-MIP was obtained by precipitation polymzation; this way of obtaining the desired product is relativimple and has the advantage of not requiring a polymer grintep, which could alter some of the polymer’s cavities, prevng such sites from becoming available for later binding tonalytes.

.1. 1H NMR study

Study of the intermolecular interaction between the temnd functional monomer prior to polymerization is interestinetermine the recognition mechanism of the imprinted polyecause the recognition of imprinted polymers is based on

nteractions.Here the interactions between MAA and the template w

ssessed experimentally using1H NMR analysis. The effect ohe MAA on the propazine spectrum was evaluated by recohe spectra in the presence of different ratios of MAA/propaz

Upon plotting the chemical shifts (δ) that occur upon varyinhe concentration of MAA in CD2Cl2 (Fig. 1(a)), it may be see

R. Carabias-Martınez et al. / Analytica Chimica Acta 559 (2006) 186–194 189

Fig. 1. 1H NMR study in CD2Cl2. (a) Chemical shift,δ, of propazine hydrogens against the concentration of the functional monomer; (b) chemical shift,δ, ofhydrogen H3 of propazine against the functional monomer/template ratio.

that the only hydrogen that undergoes a shift corresponds to theamine hydrogen. This suggests that amine hydrogen would beinvolved in the formation of hydrogen bridges with the func-tional monomer. When the shift is plotted against the functionalmonomer/template ratio, it is seen that the stoichiometric ratiobetween them is equal to unity (Fig. 1(b)). Bearing in mindmethacrylic acid is also a hydrogen donor, it is likely that thenitrogen atom of the triazine ring will be involved in hydrogenbonding as a hydrogen acceptor. The fact that the stoichiometryobserved was 1:1 suggests that one molecule of MAA wouldbind to one molecule of propazine through the formation of twohydrogen bonds.

3.2. Study of the retention process in organic medium

Initially, we studied the ability of the MIPs obtained byprecipitation polymerization to specifically recognise triazinesand metabolites from different organic media (toluene anddichloromethane). This was evaluated working in parallelwith cartridges filled with imprinted polymer (propazine-MIP)and blank or non-imprinted polymer (NIP), both obtained asdescribed in the experimental section. The loading step wasaccomplished using a standard mixture of herbicides in tolueneor dichloromethane spiked with an approximate concentrationof a 200�g L−1 of each of the triazines and metabolites studied.I anal ntedu vente anda anal

rougt inedf grem tm esiso

For samples in toluene, different types of behaviour wereobserved: (a) the chloro- and methylthio-triazines were specifi-cally retained on the MIP, (b) the dealkylated chloro-metabolites– DEA and DIA – were retained in both the imprinted and thenon-imprinted polymer and (c) the hydroxylated metabolites –HT, DIHA and DEHA – were not retained neither on the MIPnor on the NIP.

Accordingly, the results obtained indicate that toluene is asuitable organic medium for the specific retention of triazines.They also suggest that dichloromethane can be used as a goodwashing solvent for eliminating non-specific interactions.

3.2.1. Influence of the amount of sorbent usedTo check the influence of this in the recovery values, a series

of empty SPE cartridges were filled with different amounts ofpolymer. After the cartridges had been conditioned, 2 mL oftoluene with 200�g L−1 of each analyte were passed throughthem. For most of the triazines, the recoveries obtained were notsignificantly better when amounts of sorbent above 200 mg wereused, whereas for the dealkylated chloro-metabolites DIA andDEA recovery continued to increase with the increase in polymermass. In the case of the hydroxylated metabolites DIHA, DEHAand HT, the recoveries obtained were lower than 10% in all cases.

Taking into account the physical characteristics observed forthe imprinted polymer (see Section2.2), the difficulty in passingt se inp g ofp

3ecific

i con-s sol-v sev luesa e lat-t t that

n order to prevent non-specific interactions between theytes and the MIP, a cartridge-washing step was implemesing, initially, 10 mL of toluene because this was the solmployed to obtain the polymer. A mixture of acetonitrilecetic acid (9:1, v/v) was used to elute specifically retained

ytes.The recoveries obtained when the sample was passed th

he MIP dissolved in toluene were higher than those obtaor samples in dichloromethane. These results are in good aent with those reported in literature[11,18] in which the besedium for molecular recognition is that used for the synthf the MIP.

-

-

h

e-

he sample through the system increases with the increaolymer mass. Accordingly, it is recommended that 200 molymer be used (Fig. 2).

.2.2. Influence of the washing solventThe washing solvents assayed here to remove non-sp

nteractions were selected as a function of their dielectrictant (�) and the hydrogen bond parameter (HBP). Theseents were toluene, dichloromethane and acetonitrile who�alues are 2.5, 9.1 and 36.2, respectively, while their HBP vare, respectively, 3.6, 2.2 and 7.0. Taking into account thes

er values, acetonitrile would be expected to be the solven

190 R. Carabias-Martınez et al. / Analytica Chimica Acta 559 (2006) 186–194

Fig. 2. Influence of polymer mass on the recoveries.Sample: 2 mL of toluene spiked with 200�g L−1 of each herbicide (n = 2). The MIP was washed with 10 mLof toluene.

removes the greatest number of hydrogen bridges and hencewould suppress the greatest number of specific interactions inthe washing step; dichloromethane lies at the opposite end of thisrange. The results obtained (Fig. 3) on using these washing sol-vents on the MIP and in the blank (NIP) are consistent with theHBP values of the solvents. When the sample was percolated intothe MIP and washing step was carried out with dichloromethaneor toluene, higher recovery values were obtained than when it

Fodb

was carried out with acetonitrile (Fig. 3(a)). In contrast, whenthe same experiment was performed using a NIP as sorbent anddichloromethane as washing solvent, the recoveries obtainedwere always lower than those achieved when washing was car-ried out with toluene (Fig. 3(b)) or acetonitrile (Fig. 3(a)). Thus,dichloromethane is the solvent of choice for the most efficientremoval of non-specific interactions (Fig. 3(c)).

To determine the selectivity of the imprinted polymeragainst other analytes in organic medium and under the opti-mum conditions found up to this point, four widely usedherbicides were employed: chlorsulfuron (logKow =−0.99),lenacil (logKow = 2.31), isoproturon (logKow = 2.50) and lin-uron (logKow = 3.0). All have chemical characteristics differentfrom those of the template and they cover a broad range ofpolarities.Table 2shows the recoveries obtained for chloro-and methylthio-triazines and the dealkylated chloro-metabolitesDIA and DEA. As may be seen, none of the other herbicidesincluded to study selectivity was retained in any case. Fromthe results obtained it may be inferred that, when the sam-ple is passed through the cartridge in toluene and is washedwith dichloromethane, the propazine-MIP selectively retains thechloro- and methylthio-triazines, with similar recovery valuesfor both, ranging between 89 and 77%.

The behaviour of the methylthio-triazines on the propazine-MIP obtained by precipitation observed here differs from that

TR

DDS

ig. 3. Influence of washing solvent in the recoveries of the analytes fromrganic medium. Washing solvent: (a) acetonitrile, (b) toluene and (c)ichloromethane.Sample: 2 mL of toluene spiked with 200�g L−1 of each her-icide (n = 3).

APTPTCLIL

S fd

able 2ecoveries of some triazines and non-related herbicides

Recoveries (%)± S.D.a

MIP NIP

IA 96 ± 11 66± 5EA 93 ± 7 43 ± 6imazine 87± 10 5 ± 0.7trazine 79± 8 –ropazine 83± 9 –erbutylazine 77± 10 –rometryn 89± 7 –erbutryn 84± 9 –hlorsulfuron 2± 0.3 –enacil – –

soproturon – –inuron – –

ample: 2 mL of toluene spiked with 200�g L−1. Washing solvent: 10 mL oichloromethane. (–) Not detected.a S.D., standard deviations forn = 3.

R. Carabias-Martınez et al. / Analytica Chimica Acta 559 (2006) 186–194 191

reported by other authors[17] for imprinted polymers obtainedby bulk polymerization, in which the –SCH3 group of themethylthio-triazines seems to exert a steric effect that hin-ders their specific retention on the polymer. This difference inbehaviour seems to be related to the way in which the polymerwas obtained.

Regarding the dealkylated chloro-metabolites DIA and DEA,recoveries higher than 93% were obtained but it was observedthat these compounds were retained by specific and non-specificinteractions. In contrast, the hydroxylated metabolites DIHA,DEHA and HT were not retained on the MIP. Similar resultshave been reported by other authors[18] using an imprintedpolymer obtained by bulk polymerization.

3.3. Study of the process of retention in aqueous medium

With a view to being able to use the MIP for the pretreat-ment of environmental waters contaminated by herbicides, thebehaviour of this imprinted polymer in the preconcentration oftriazines from aqueous samples was studied.

The behaviour of different chloro- and methylthio-triazinesin aqueous medium using MIPs obtained by bulk polymerizationhas been described[10,11], but few authors have addressed thejoint behaviour of the hydroxylated metabolites of these com-pounds[18].

solt yings ithmt polym hoseo ted

F aque-o hane.S

polymer (Fig. 4(a)), suggesting that the interactions occurringon the MIP were not specific.

Bearing in mind the results obtained previously, the abilityof dichloromethane as a washing solvent to disrupt non-specificinteractions was studied when triazines were passed throughthe cartridges in aqueous medium. The recoveries obtained(Fig. 4(b)) showed that by using dichloromethane as the wash-ing solvent all the triazines were removed from the NIP with theexception of the hydroxylated metabolites, regardless of whetherthey were dealkylated or not. Thus, under the experimentalconditions employed three different types of behaviour wereobserved: (a) the chloro-triazines and the dealkylated chloro-metabolites – DIA and DEA – were retained by specific inter-actions; (b) the hydroxylated metabolites – DIHA, DEHA andHT – were retained by non-specific interactions on the poly-meric matrix; (c) the methylthio-triazines were not extracted,instead they were washed out of the cartridge by the action ofdichloromethane. The retention of the hydroxylated metabolitesby the NIP seems to be related to the negative charge generatedby the hydroxyl groups, which may act as donors and acceptorsof hydrogen bridges with the molecules of the NIP[15].

3.3.1. Effect of the drying stepThe retention of the analytes on a sorbent from an aque-

ous medium may be strongly affected by the presence of waterembedded in the sorbent after passage of the sample. Accord-i tiono d bya s

Fwith 200�g L−1 of each herbicide (n = 3). Washing solvent: 10 mL ofdichloromethane. Elution with 10 mL of a mixture acetonitrile:acetic acid (9:1,v/v).

The propazine-MIP was exposed to a standard aqueousion containing the eleven herbicides. After an exhaustive drtep, the cartridge was washed with toluene and eluted wixture of acetonitrile–acetic acid (9:1, v/v) (Fig. 4(a)). When

he same experiment was carried out using a non-imprinteder (NIP) as sorbent, the recoveries were very similar to tbtained when extraction was performed with the imprin

ig. 4. Influence of washing solvent in the recoveries of the analytes fromus medium. Washing solvent: 10 mL of (a) toluene and (b) dichlorometample: 2 mL of water spiked with 200�g L−1 of each herbicide (n = 3).

u-

a

-

ngly, we next studied how the drying time affected the retenf the analytes in both the MIP and NIP. The sorbent was driepplying a vacuum of−15 mmHg.Fig. 5shows the recoverie

ig. 5. Effect of MIP and NIP drying time.Sample: 2 mL of water spiked

192 R. Carabias-Martınez et al. / Analytica Chimica Acta 559 (2006) 186–194

Table 3Influence of the volume of dichlorometane used in the washing step

Percentage of analyte removed in each fractiona

First fraction,2 mL

Secondfraction, 3 mL

Third fraction,5 mL

Fourthfraction 5 mL

Fifth fraction,5 mL

Totalwashed

Triazines and metabolitesDIHA – – – – – –DEHA – – – – – –DIA – – – – – –DEA – – – – – –HT – – – – – –Simazine – – – 23 14 37Atrazine – – 10 24 16 50Propazine 18 22 13 16 11 80Terbutylazine 25 21 11 15 8 80Prometryn 39 27 15 7 5 93Terbutryn 54 26 10 3 – 93

Other herbicidesChlorsulfuron 63 25 – – – 88Lenacil 62 24 – – – 86Isoproturon 88 – – – – 88Linuron 96 – – – – 96

Sample: 2 mL of water spiked with 200�g L−1 of each herbicide.a Mean value of two replicates.

obtained on using 3 or 30 min for sorbent drying. As the dryingtime increased so did the recoveries of all the analytes, this effectbeing more pronounced in the case of the chloro-triazines. Whenthe experiments were carried out with a NIP, the effect was lessmarked and a slight decrease in the recoveries of the hydroxy-lated metabolites was observed upon increasing the drying timeand the low retention of the chloro-triazines was eliminated.

3.3.2. Effect of volume of washing solvent on MIPselectivity

The selectivity of the imprinted polymer for specificallyextracting triazines from aqueous solutions was assessed againstthe above non-triazine herbicides—lenacil, chlorsulfuron, iso-proturon and linuron. To accomplish this, the MIP was washedsuccessively with increasing volumes of dichloromethane andthe fractions thus obtained were analysed chromatographically(Table 3). It was observed that the non-triazine herbicideswere eluted efficiently (>80%) in the first two fractions ofdichloromethane. In contrast, in the case of the triazine ana-lytes the order of elution in the washing step was inverselyrelated to their hydrophobicity (Table 1). Thus, the methylthio-triazines prometryn and terbutryn were removed at 39% and54%, respectively, in the first dichloromethane fraction andwere almost completely washed from the MIP with 10 mL ofdichloromethane.

Chlorsulfuron, with a logK value of −0.99, lower thant n aw reaD

hat at dn d on

the polymer through hydrophobic interactions and, on the other,it generates an organic medium in which the specific inter-actions are developed that facilitate the retention of triazineson the MIP. The competition between these two effects hasunfavourable results for the more apolar triazines and the largerones, the methylthio-triazines, which elute in the organic wash-ing medium before developing specific interactions with theMIP.

These results led us to employ a compromise solution inwhich, using a washing step with 5 mL of dichloromethane,the chloro-triazines and the dealkylated chloro-metabolites caninteract in a specific way with the MIP, recoveries rangingbetween 38% for terbutylazine and 96% for DIA being obtained.In the case of the methylthio-triazines, the recovery values werelower than 12% since these were the most apolar compounds.For the hydroxylated metabolites, DIHA, DEHA and HT, recov-eries higher than 92% were obtained, although for these analytesno specific recognition by the MIP was observed and their reten-tion must be due non-specific hydrophilic interactions (hydrogenbridges) with the polymeric matrix, which are not eliminated bydichloromethane.

The results indicate that when the sample is passed throughthe cartridge in aqueous medium the analytes are retainedby non-selective interactions whereas later washing withdichloromethane generates an organic medium in which non-selective interactions are converted into that are specific for thec cov-e t ber ad, tot dieda withd

owhat of DIA (logKow = 1.15), was eluted from the sorbent wheashing volume of 2 mL of dichloromethane was used, wheIA was not washed out even with 20 mL.These results show that washing with dichloromethane

wo effects: on one hand, the solvent washes analytes thot belong to the triazine family because they are retaine

s

so

hloro-triazines and dealkylated chloro-metabolites. The rery values obtained for the methylthio-triazines should noelated to a greater retention of these analytes but, instehe fact that they are the most apolar of the triazines stund are therefore removed more easily in the washing stepichloromethane.

R. Carabias-Martınez et al. / Analytica Chimica Acta 559 (2006) 186–194 193

Table 4Behaviour of triazine herbicides and related metabolites on the propazine-MIP

Analytes Organicmedium

Aqueousmedium

MIP NIP MIP NIP

Chloro-triazines:Simazine +++ − ++ −AtrazazinePropazineTerbutylazine

Methylthio-triazines:Prometryn +++ − − −Terbutryn

Hydroxy-metabolites:Hydroxyterbutylazine (HT) − − +++ +++

Dealkylated chloro-metabolites:Deisopropylatrazine (DIA) +++ ++ +++ −Deethylatrazine (DEA)

Dealkylated hydroxy-metabolites:Deisopropylhydroxyatrazine (DIHA) − − +++ +++Deethylhydroxyatrazine (DEHA)

Recoveries: +++, >80%; 80% > ++ > 50%;− <20%.

3.4. Behaviour in organic medium versus aqueous medium

The possibilities of using the propazine-MIP as a selectivesorbent for triazines and metabolites depend on the extractiomedium and on the type of analyte being studied. A summaryof the behaviour of the MIP under the conditions employed inthe present work can be found inTable 4.

3.4.1. Organic mediumIn organic medium the propazine-MIP prepared by precipi-

tation polymerization exhibited highly selective binding for thechloro- and methylthio-triazines, with recoveries higher than75% in both cases.

The dealkylated chloro-metabolites – DIA and DEA – areretained by a combination of specific and non-specific interactions, although these latter can be taken advantage of to carrout extraction since they were not eliminated in the washingstep. However, in this medium it was not possible to extract thehydroxy-derivatives (DIHA, DEHA and HT) since these do notinteract with the MIP in a specific way and their non-specificinteractions with the polymeric matrix are weak.

3.4.2. Aqueous mediumIn aqueous medium, choice of the volume of the washing sol-

vent is more critical than in organic medium. This is because, onone hand, the medium eliminates (washes out) the non-specifih tablem

lites( A)i andt thed tesb

such that its logKow value would be expected to lie betweenthese values. However, the behaviour of HT in both the MIP andNIP was similar to that of compounds of greater polarity, suchas DIHA (logKow =−0.1) and DEHA (logKow = 0.2).

The propazine-MIP exhibited selective binding for chloro-triazines and dealkylated chloro-metabolites. It can also be usedin the extraction of the hydroxy- and dealkylated hydroxy-derivatives of the triazines since these compounds were retainedin a non-specific way by hydrogen bridge interactions that werenot eliminated in the step involving washing with dichloro-methane.

4. Conclusions

The propazine-MIP prepared by precipitation polymeriza-tion showed some advantages over MIPs obtained by bulkpolymerization. It exhibited highly selective binding for tri-azines, dealkylated chloro-metabolites, dealkylated hydroxy-metabolites and hydroxy-metabolites and thus, it can be usedto preconcentrate these analytes. However, different types ofbehaviour were observed as a function of the sample mediumand of the type of analyte being studied. Accordingly, depend-ing on the type of triazine to be preconcentrated, the appropriatemedium should organic or aqueous. Additionally, it may be con-cluded that the behaviour of the hydroxy-metabolites (HT) andd nlyg e thet

pro-t

A

U-2 tad cialF

R

tec-

tion,

a,

.02)

30

723

ry,

cel

[ togr.

ydrophobic interactions, and, on the other, it provides a suiedium for molecular recognition of the target analytes.In this medium, the behaviour of the hydroxy-metabo

HT) and dealkylated hydroxy-metabolites (DEHA and DIHs mainly governed by the presence of the hydroxyl grouphese three compounds show similar behaviour despiteifferences in polarity. Thus, chromatographically HT eluetween DEA (logKow = 1.52) and simazine (logKow = 2.18),

n

-y

c

ir

ealkylated hydroxy-metabolites (DEHA and DIHA) is maioverned by the presence of the hydroxyl group and henc

hree metabolites show similar behaviour.Other non-related herbicides (lenacil, chlorsulfuron, iso

uron, linuron) were not specifically retained.

cknowledgements

This work was supported by the MCYT-DGI (Project BQ002-02314) and by the Consejerıa de Cultura y Turismo, June Castilla y Leon, and the European Union (European Sound, Project SA044/02).

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