Solid-phase extraction of fluoroquinolones from aqueous samples using a water-compatible stochiometrically imprinted polymer

Journal of Chromatography A, 1208 (2008) 62–70

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Solid-phase extraction of fluoroquinolones from aqueous samples using a water-compatible stochiometrically imprinted polymer ˜ a , Javier L. Urraca a , Börje Sellergren b , María Cruz Moreno-Bondi a,∗ Elena Benito-Pena a b

Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, E-28040 Madrid, Spain Institut für Umweltforschung, Universität Dortmund, Otto Hahn Strasse 6, D-44221 Dortmund, Germany

a r t i c l e

i n f o

Article history: Received 25 April 2008 Received in revised form 20 August 2008 Accepted 21 August 2008 Available online 4 September 2008 Keywords: Antibiotics Water-compatible imprinted polymer Solid-phase extraction Enrofloxacin

a b s t r a c t A novel and simple method for the selective cleanup and preconcentration of fluoroquinolone antibiotics in environmental water samples has been developed using molecularly imprinted polymer solid-phase extraction (MISPE). The molecularly imprinted polymer (MIP) has been prepared using enrofloxacin (ENR) as the template and a stoichiometric quantity of urea-based functional monomer to target the single oxyanionic moieties in the template molecule. The selectivity of the material for enrofloxacin, and structurally related and non-related compounds, has been evaluated using it as stationary phase in liquid chromatography. The novel polymer and the corresponding non-imprinted material (NIP) have been characterised using nitrogen adsorption–desorption isotherms and scanning electron microscopy. Various parameters affecting the extraction efficiency of the materials in the MISPE procedure were evaluated in order to achieve optimal preconcentration and to reduce non-specific interactions. The optimized MISPE/HPLC with fluorescence detection (FLD) method allows direct extraction of the antibiotics from the aqueous samples followed by a selective washing with acetonitrile/water (0.1 M 2-[4-(2-hydroxyethyl)1-piperazinyl]ethanesulfonic acid (HEPES) buffer, pH 7.5) (10/90, v/v) and elution with 2% trifluoracetic acid in methanol. Good recoveries and precision, ranging between 66 and 100% (RSD: 2–12%, n = 3) for danofloxacin, enrofloxacin, oxolinic acid and flumequine, and moderate recoveries (15–40%, RSD 4–9%, n = 3) for norfloxacin, ciprofloxacin, lomefloxacin and sarafloxacin, have been obtained for river water samples fortified with 0.50, 0.75 and 1.0 ␮g L−1 of all the antibiotics. The method detection limits ranged between 0.01 and 0.30 ␮g L−1 for all the antibiotics tested, when 100 mL water samples were processed. The results demonstrate the applicability of the optimized method for the selective extraction of fluoroquinolones in environmental water samples at the ng L−1 level. © 2008 Elsevier B.V. All rights reserved.

1. Introduction One of the most relevant topics in today’s environmental analytical chemistry is water quality control. Recent studies have shown that a multitude of drugs are present in aquatic systems [1–3]. The interest in the determination of antibiotic residues in the environment arises from the fact that they are suspected of being responsible for the appearance of antibiotic-resistant bacterial strains [4,5]. The presence of antibiotic residues in live animals and animal products is legislated (Council Directive 96/23/EC) and countries must monitor them for safety assessment. However, these antimicrobials have not been included in the list of priority and hazardous substances of the Water Framework Directive of the European Union [6]. Recent studies in Europe and North America have revealed the presence of antibacterial agents in the aquatic

∗ Corresponding author. E-mail address: [email protected] (M.C. Moreno-Bondi). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.08.109

environment at levels of ng to low ␮g L−1 [7] and the consequences of its presence are unknown. Fluoroquinolones (Fig. 1) belong to a group of antibiotics widely used in human and veterinary medicine. The mechanism of action of these drugs consists in a specific inhibition of the bacterial DNA with the subsequent blocking of bacterial multiplication [8]. Current methods of FQ analysis in biological matrices are based on liquid chromatography (LC), mainly with fluorescence [9–11], ultraviolet (UV) [12,13] or mass spectrometric (MS) detection [14,15]. Molecular imprinting is a technology to produce polymers programed to recognize a target or a class of target molecules. Firstly, a complex between a template and monomers bearing functional groups (functional monomers) is formed. The functional monomers can either be covalently linked to the template (covalent approach) or arrange themselves via non-covalent intermolecular interactions around the template molecule (non-covalent approach). The spatial assembly of these interaction sites is subsequently fixed through polymerisation in the presence of a cross-linking agent.

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Fig. 1. Molecular structures of the antibiotics tested enrofloxacin, ENR; ciprofloxacin, CIP; norfloxacin, NOR; lomefloxacin, LOM; danofloxacin, DAN; sarafloxacin, SAR; ofloxacin, OFL; flumequine, FLU; oxolinic acid, OXO; penicillin G, PEG; amoxicillin, AMX; cloxacillin, CLX and cephapirin, CEP; benzoic acid (BZ). Functional monomer 1-(4-vinylphenyl)-3-(3,5-bis(trifluoromethyl)phenyl) urea used to prepare the imprinted polymer for oxyanion recognition (1) [26].

After removal of the template, binding cavities capable of recognizing and re-binding the template molecules are obtained. The binding pockets are more or less complementary to the template regarding size, shape and arrangement of the functionalities within the cavity [16–18]. In solid-phase extraction using molecularly imprinted polymer solid-phase extraction (MISPE), the analyte (the template), or closely related compounds, will remain bound to the polymer allowing them to be subsequently eluted free of co-extractives. Thus MISPE, has been successfully employed in the determination of many analytes such as antibiotics [19], mycotoxins [20], triazines [21], endocrine disruptors [22], uracils [23], imides [24], etc. in different samples. Several important analytes are present as oxyanions. Targeting oxyanions by imprinting has previously been performed using commercially available functional monomers (e.g., vinylpyridine, N,N-diethyl-2-aminoethyl methacrylate, methacrylamide). Unfortunately, these are able to provide only weak interactions with the template molecule in solvents of low polarity [25] precluding the subsequent use of the polymers in aqueous media. Stoichiometric imprinting may here provide a solution. This is a designer variant of non-covalent imprinting where host monomers providing strong interactions with the target functional groups are used [26]. This leads to an enhanced stability of the monomer template complexes which, in turn, translates into a high-yield generation of sites of better definition in the final polymer. The higher affinity of these sites per se displayed for the target and the common use of more polar

porogens lead to polymer receptors which exhibit effective binding also in water-rich media. As a part of our ongoing efforts to develop imprinted oxyanion receptors we recently demonstrated one such example in the imprinting of penicillin G using a 1,3-diarylurea host monomer [19,27]. In this work, the characterization of a novel imprinted polymer for fluoroquinolone antibiotics using the same urea as main functional monomer has been described. The objective of this study was to optimize and characterise the extraction procedure for the analysis of enrofloxacin (ENR) and other quinolones in aqueous samples. 2. Experimental 2.1. Chemicals The urea-based functional monomer 1-(4-vinylphenyl)-3-(3,5bis(trifluoromethyl)phenyl) urea (1) was prepared as described previously [26]. Ethyleneglycol dimethacrylate (EDMA) was purchased from Sigma–Aldrich (St. Louis, MO, USA) and purified prior to use as follows: EDMA was washed sequentially with 10% NaOH (aqueous), water and then brine. After drying over MgSO4 , it was distilled under reduced pressure to give inhibitor-free monomer. Methacrylamide was obtained from fluka, ABDV was obtained from Wako (Neuss, Germany) and used as received. Enrofloxacin was supplied from Fluka (Buchs, Switzerland) and ciprofloxacin (CIP), norfloxacin (NOR), lomefloxacin (LOM), ofloxacin (OFL), flumequine (FLU), oxolinic acid (OXO), penicillin G potassium salt (PEG),

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amoxicillin anhydrous (AMX), cloxacillin salt monohydrate (CLX), cephapirin sodium salt (CEP) and benzoic acid (BZ) were supplied from Sigma–Aldrich (St. Louis, MO, USA). Sarafloxacin hydrochloride (SAR) was a gift from Fort Dodge Veterinaria (Girona, Spain). Danofloxacin (DAN) was obtained from Riedel-de-Haën (Seelze, Germany). The chemical structure of the antibiotics is shown in Fig. 1. HPLC-grade acetonitrile (ACN) and methanol (MeOH) were purchased by SDS (Peypin, France) and water was purified with a Milli-Q system (Millipore, Bedford, MA, USA). All solutions prepared for HPLC were passed through a 0.45-␮m nylon filter before use. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was supplied by Aldrich (Steinheim, Germany) and a buffer solution, pH 7.5, was prepared by dissolving 23.83 g in 1 L of milli-Q water (0.1 M). Trifluoroacetic acid (TFA) (HPLC-grade) was from Fluka (Buchs, Switzerland) and tetra-n-butyl ammonium hydrogen sulfate (TBA) was from Merck (Darmstadt, Germany). 2.2. Apparatus The pH of the buffer solutions and samples was adjusted with an Orion 710A pH/ISE meter (Beverly, MA, USA). The polymer samples were analyzed by SEM using a JEOL JSM-6400F Field Emission Gun (FEG) microscope operating at 15 kV. The samples for SEM were coated with a thin gold film before observation. The nitrogen-desorption isotherms of the synthesised polymers were measured in an ASAP 2020 instrument at 77 K (Micromeritics, Norcross, GA, USA). Before the adsorption measurements, samples were outgassed at 50 ◦ C for 2 h. The BET area of the polymer materials (SBET ) was calculated from the adsorption data using 0.162 nm2 as the molecular cross-sectional area for adsorbed nitrogen molecules. The external surface area (St ) was obtained from the so-called t-plots, obtained by Harking and Jura equation [28]. The Barret–Joyner–Halenda (BJH) method was applied to calculate the pore size distribution from the analysis of the adsorption and desorption branches of the isotherms. In the BJH analysis, the Kelvin equation was used under assumption of cylindrical pores [29,30]. Chromatographic analysis was carried out with an HP-1100 HPLC from Agilent Technologies (Palo Alto, CA, USA) equipped with a quaternary pump, an on-line degasser, an autosampler, an automatic injector and a column thermostat. A fluorescence detection (FLD) system and a diode array detection (DAD) system were used depending on the studies carried on. Chromatographic separation of the fluoroquinolones was performed on an Aqua C18 (polar endcapped; 250 mm × 4.6 mm I.D., 5 ␮m) HPLC column protected by an RP18 guard column (4.0 mm × 3.0 mm I.D., 5 ␮m), both from Phenomenex (Torrance, CA, USA). A gradient program was used with the mobile phase, combining solvent A (25 mM orthophosphoric acid adjusted to pH 3.0 with NaOH) and solvent B (acetonitrile) and solvent C (methanol) as follows: 17% B, 0% C and 1.0 mL min−1 flow rate (8 min), 17–66% B, 0–15% C and 1.0–2.0 mL min−1 flow rate (17 min), 66% B, 15% C and 2.0 mL min−1 (10 min). Column temperature was kept at 25 ◦ C. The injection volume was 10 ␮L, and all the compounds eluted within 24 min. The fluorescence excitation/emission wavelengths were programed at 280/450 nm for ENR, CIP, NOR, LOM, DAN and SAR and at 280/365 nm for OXO and FLU. The diode array detector wavelength was set at 220 nm for the ␤-lactam antibiotics PEG, AMX, CEP, CLX and BZ. Quantification was performed using external calibration peak area measurements. Linear calibration graphs were obtained in the 10–500 ␮g L−1 range for all the antibiotics (r2 > 0.999). For ␤-lactam antibiotics, the linear concentration range studied was 75–5000 ␮g L−1 (r2 > 0.999) [31].

2.3. Synthesis of imprinted and non-imprinted polymers The molecularly imprinted polymer (MIP) pre-polymerisation solution was prepared as follows: the template enrofloxacin (183.4 mg, 0.5 mmol), functional monomer 1 (187.1 mg, 0.5 mmol), methacrylamide (85 mg, 1 mmol), EDMA (3.8 mL, 20 mmol) and the free radical initiator ABDV (42.4 mg, 1% (w/w) total monomers) were dissolved in ACN (5.6 mL). After dissolution, the solution was transferred to a glass tube, cooled to 0 ◦ C and purged with N2 for 10 min. The glass tube was then sealed and polymerisation initiated thermally by placing the tube in a water bath set at 50 ◦ C. Polymerisation was allowed to proceed at this temperature for 48 h. The tube was then broken and the MIP monolith was crashed into smaller fragments. The template molecule was removed through the following sequential washing steps: MeOH (100 mL), MeOH/ water (0.1 M HCl) (90/10, v/v) (100 mL) and finally MeOH (100 mL). The MIP particles were allowed to equilibrate for ca. 24 h with each washing solution, after which the wash solution was decanted off. The washing solutions were combined and analyzed by HPLC–DAD to check template recovery which was higher than 99.9%. Thereafter, the resulting bulk polymers were grounded in the ball mill and sieved to a final size ranging between 25 and 50 ␮m. Prior to use, they were sedimented using MeOH/water (80/20, v/v) in order to remove fine particles. A non-imprinted polymer was prepared in the same way, but in the absence of the template molecule. 2.4. Chromatographic evaluation of the polymers MIP and non-imprinted material (NIP) polymers were slurry-packed in methanol into stainless steel HPLC columns (150 mm × 4.6 mm I.D.) using MeOH/water (80/20, v/v) as the pushing solvent. The polymers were first tested for their ability to retain the template ENR using mobile phases based on binary mixtures of ACN and aqueous HEPES buffer (0.1 M, pH 7.5) ranging from 100% ACN to 100% HEPES buffer. For these experiments, the following conditions were used: flow rate = 1 mL min−1 , sample volume = 20 ␮L, analyte concentration = 3 mM, detection at  = 270 nm. The flow rate was kept constant 1.0 mL min−1 throughout the study. Each elution was repeated three times. Methanol was used as void volume marker. The retention factor (k) for each analyte was calculated as k = (t − t0 )/t0 , where t and t0 are the retention times of the analyte and the void marker (methanol), respectively. The imprinting factor (IF) was calculated as IF = kMIP /kNIP , i.e., the ratio of the retention factor of each analyte in the MIP column to that in the NIP column. The selectivity of the polymer towards analogues of the template molecule was performed using (ACN/water (0.1 M HEPES, pH 7.5, 0.138/0.0027 M NaCl/KCl) (15/85, v/v)) as mobile phase that allows the elution of all the analytes within 30 min. All other conditions were as stated above except analyte concentrations, which were set at 0.5 mM because of limited solubility of some of the antibiotics tested. 2.5. Equilibrium re-binding experiment The polymer particles (10 mg) were mixed with a 2 mL ACN/water (0.1 M HEPES, pH 7.5) (10/90, v/v) solution containing different amounts of ENR (0.05–2 mM) and the mixtures were incubated for 24 h at room temperature. After incubation, the supernatant was collected and injected into the HPLC using the gradient program described in Section 2.2. The amount of bound analyte to the polymer (B) was calculated by subtracting the nonbounded amount (F), from the initial enrofloxacin concentration in the mixture. The binding experiments were carried out by triplicate.

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2.6. Optimized extraction procedure of FQs in the MIP cartridge Solid-phase extraction cartridges (Varian, Palo Alto, CA, USA) with a 3 mL volume, were packed with 150 mg of the imprinted polymer or the corresponding non-imprinted polymers. The cartridges were equilibrated with 10 mL of buffer (0.1 M HEPES, pH 7.5), and the antibiotic mixture, dissolved in buffer (0.1 M HEPES, pH 7.5), was percolated at a constant flow rate of 2.5 mL min−1 with the aid of a peristaltic pump. The cartridges were rinsed with 5 mL of ACN/water (0.1 M HEPES, pH 7.5) 10:90 (v/v), to wash out the non-specifically retained compounds. Finally, the antibiotics were eluted with 1 mL of methanol with 2% TFA. The cartridges were equilibrated with 10 mL of buffer (0.1 M HEPES, pH 7.5) before a new application. The eluates from the MISPE column were injected into the HPLC system for analysis. 2.7. Water samples analysis River water samples from Eresma River (Segovia, Spain) were collected in 2.5-L amber glass bottles, rinsed with ultrapure water, and stored at 4 ◦ C until measurement. The samples were filtered through a 0.45 ␮m filter (Whatman, Maidstone, UK) to remove suspended matter and the pH was adjusted with HEPES 0.1 M and NaOH to 7.5. The samples were fortified with the target analytes at three concentration levels, 0.5, 0.75 and 1.0 ␮g L−1 . Thereafter, they were analyzed using the MISPE cartridges and HPLC–FLD as described in Section 2.2. Non-fortified water samples were preconcentrated and spiked with the antibiotic stock solutions for calibration purposes. All the analyses have been carried out by triplicate.

Fig. 2. Retention behaviour of ENR on the MIP and the NIP for different mobile phase compositions. Sample volume, 20 ␮L; analyte concentration, 3 mM (ENR in ACN/water (0.1 M HEPES, pH 7.5 (10:90, v/v)); flow rate, 1 mL min−1 ; column dimensions, 150 mm × 4.6 mm I.D.;  = 270 nm. Methanol was used as void volume marker. Results shown are the average of three separate analyte injections.

The experimental binding data obtained with the polymers included in the study (Supplementary material, Fig. S1) displayed good adherence to the Freundlich (FI) isotherm model (Eq. (1)), which is reflected in linear log B versus log F plots according to Eq. (2): B(F) = aF m

(1)

log B = log a + m log F

(2)

3. Results and discussion 3.1. Effect of the mobile phase composition on template retention The polymers were first tested for their ability to retain the template molecule, ENR. Based on our previous experience from handling PenG MIPs, we chose to evaluate the materials using mixtures of ACN and HEPES buffer (0.1 M, pH 7.5). This pH was selected to ensure deprotonation of the carboxylic acid groups of the analytes, which promotes the formation of quadruple hydrogen bonds between the urea groups and the template carboxylate group. The results are shown in Fig. 2. In 100% acetonitrile, the retention of ENR in the imprinted and the non-imprinted polymers is low because in these conditions the template molecule is protonated and the ionic interactions between the polymer and the antibiotic are suppressed. The retention factor of the antibiotic in the MIP dramatically increases with the buffer concentration in the mobile phase. The maximum value (no elution, tMIP > 150 min) is obtained for 100% HEPES buffer (0.1 M, pH 7.5). The trend is similar for the non-imprinted polymer, but the retention factors are always significatively lower than for the MIP and almost constant for buffer concentrations over 90% in the mobile phase. Thus, moderate non-specific interactions exist but imprinting is apparent. 3.2. Determination of binding site distributions and affinities Theoretically, the efficiency of the MIP-based SPE materials should display, apart from high affinity and selectivity, appreciable binding capacities for the analyte of interest. If these requirements are fulfilled, SPE can be performed with rather small amounts of polymer, allowing the reduction, or even suppression, of nonselective adsorption effects.

where B and F are the concentrations of bound and free analyte, respectively and m is the so called heterogeneity index. The parameter m can take values from 1 to 0, increasing with decreasing heterogeneity of the material. According to Rampey et al. [32] the affinity distribution (AD) can be calculated using Eq. (2) and the experimentally derived FI fitting parameters (a and m) N(k) = 2.303 am(1 − m2 ) e2.303 log K

(3)

The ADs calculated with this equation are valid within a range of binding affinities (Kmin and Kmax ) that can be calculated from the experimental maximum and minimum free analyte concentrations (Fmin and Fmax ) and the relationship K1 = Kmin = 1/Fmax and K2 = Kmax = 1/Fmin . Table 1 summarises the fitting coefficients of the MIP and NIP, ¯ K −K , and the apparent the apparent number of binding sites, N 1 2 weighted average affinity, K¯ K1 −K2 , calculated using Eqs. (4) and (5)

Table 1 Freundlich fitting parameters, weighted average affinity (K K1 −K2 ) and number of ¯ K −K ) obtained with the experimental binding data of ENR towards the sites (N 1 2 imprinted (MIP) and non-imprinted polymer (NIP) [31] Fitting parameters

MIP

NIP

¯ K −K (␮mol g−1 ) N 1 2 a [(␮mol g−1 ) (M−1 )m ] K¯ K1 −K2 (mM−1 ) Krange (mM−1 )f m r2

15.7 ± 0.7 23.6 4.1 ± 0.3 0.70–31.2 0.65 0.99

5.1 ± 0.4 6.8 3.5 ± 0.2 0.64–17.2 0.70 0.96

Calculations are the mean of three replicate measurements with a coefficient of variation in the range of 2.5–6.1%.

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Table 2 Influence of the pH and ionic strength in the mobile phase for the retention of ENR in the imprinted and non-imprinted polymers pH

3.0

Mobile phase

kNIP

IF

A (%)

B (%)

C (%)

15 12 10

85 88 90

– – –

3.96 13.8 8.30

4.08 11.8 7.31

0.97 1.17 1.13

85 – 88 88 90 –

– 85 – 12 – 90

8.73 6.75 17.4 6.76 26.6 20.7

6.07 3.89 9.30 1.90 14.2 10.4

1.44 1.73 1.87 1.90 1.87 1.98

85 88 90

– – –

2.66 4.52 6.77

1.79 3.05 4.70

1.49 1.48 1.44

7.5

15 12 10

9.0

kMIP

15 12 10

Mobile phase composition (A/B/C) (v/v/v). A: acetonitrile; B: water (0.1 M HEPES); C: water (0.1 M HEPES, NaCl/KCl 0.138/0.0027 M). Results shown are the average of three separate analyte injections.

[32]: ¯ K −K = a(1 − m2 )(K −m − K −m ) N 1 2 1 2 K K1 −K2 =

 m m−1



(4)

 1−m

K11−m − K2

K1−m − K2−m

(5)

The values of the total number of binding sites as well as the affinity constant are higher in the imprinted polymer than in the non-imprinted polymer. The value of KMIP (K K1 −K2 ) = 4.1 mM−1 is moderately high, but typical of the stoichiometric polymers reported so far. The difference in the affinity constant between the MIP and the NIP is small (KNIP (K¯ K1 −K2 ) = 3.5 mM−1 ) but the Kmax values display larger differences. More notable is the contribution of the template molecule to the total number of binding sites for each ¯ K −K (15.7 ± 0.7 ␮mol g−1 ) is much polymer. In the case of the MIP N 1 2 ¯ K −K = 5.1 ± 0.4 mol g−1 ) leading higher than that of the NIP (N 1 2 to a differential uptake of exceeding 20 ␮mol g−1 at the highest free concentration. Thus it is clear that the template molecule displays an important role in the heterogeneity of the MIP.

3.3. Morphological analysis The SEM images revealed differences in texture between the imprinted and non-imprinted polymer (see Supplementary material, Fig. S2). Thus, the imprinted polymer exhibited a macroporous texture, which contrasts with the smoother texture of the non-imprinted polymer. These results are consistent with those of the nitrogen adsorption-desorption isotherms which provided a BET surface area of 304 ± 5 m2 g−1 and a total pore volume (Vt ) of 0.71 mL g−1 for the imprinted polymer (MIP), versus 177 ± 3 m2 g−1 and 0.33 mL g−1 , respectively, for the non-imprinted polymer (NIP). 3.4. Influence of the pH and modifier The influence of pH and ionic strength on the efficiency of enrofloxacin binding were examined with a view to improving the performance of MIP in the MISPE determination of fluoroquinolones in real water samples. The results are shown in Table 2. Tests were conducted at three different pH values, namely: 3.0, 7.5 and 9.0. At pH 3, both urea and the antibiotic were in protonated form, so they interacted only weakly. Interactions were stronger with the carboxyl group of ENR in its ionic form (pH 7.5), as reflected in an increased imprinting factor (IF). IF also increased with increasing water content in the mobile phase, which underlines the importance of the ionization state of the carboxyl group for MIP retention. The alkaline medium (pH 9.0) reduced the imprinting factor, probably through competition between the hydroxyl anions in the solvent and the antibiotic for the polymer binding sites. According to Sellergren [33], while specific binding is unaffected at buffer concentrations above 5 mM, an increased ionic strength reduces non-specific binding. Moreover, if template–monomer interactions are ionic in nature, they will be affected by the ionic strength of the sample. In order to assess this effect, we used mobile phases containing 85, 88 or 90% HEPES buffer (0.1 M, pH 7.5) in ACN in the presence of 0.138 M NaCl/0.0027 M KCl as modifier. As can be seen from Table 2, IF increased to a maximum value of 1.98 with increasing buffer concentration. We therefore chose to use a mobile phase consisting of 10:90 (v/v) acetonitrile:0.1 M HEPES buffer at pH 7.5 containing 0.138 M NaCl/0.0027 M KCl for further testing.

Fig. 3. Evaluation of the cross-selectivity of the polymers in terms of retention factors (k) towards fluoroquinolone antibiotics, penicillin antibiotics and other non-structurally related compounds (see Fig. 1). Mobile phase: ACN/ water (0.1 M HEPES, pH 7.5, 0.138/0.0027 M NaCl/KCl) (15/85, v/v), sample volume, 20 ␮L; [Analyte] = 0.5 mM; flow rate, 1 mL min−1 ; column, 150 mm × 4.6 mm;  = 220 nm. Methanol was used as void volume marker.

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3.5. Cross-selectivity The selectivity of the enrofloxacin imprinted polymer was examined by comparing the retention behaviour of other structurally related fluoroquinolones and unrelated antibiotics

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with that of the template. The mobile phase used to this end was ACN/water [0.1 M HEPES, pH 7.5, 15/85 (v/v) 0.138 NaCl/0.0027 M KCl] and allowed all analytes to be eluted within 30 min—identical cross-selectivity was obtained with ACN/water [0.1 M HEPES, pH 7.5, 10/90 (v/v)], which, however, resulted in

Fig. 4. Extraction recoveries (%) obtained on the MIP and the NIP for nine fluoroquinolones after percolation of 100 mL of water (0.1 M HEPES, pH 7.5) containing 1 ␮g L−1 of each compound using a washing step with 2.5, 5.0 and 10 mL of the solvent ACN/water (0.1 M HEPES, pH 7.5) (10/90, v/v).

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longer retention times. The results of these tests are collected in Fig. 3. The imprinted polymer exhibited a strong affinity (particularly for ENR, OXO and FLU). The template, ENR, was selectively retained by MIP (IF = 4.0). The strong retention of OXO and FLU relative to previous experiments [34–36], can be ascribed to their low pKa1 values (5.83 and 5.72 [37], respectively) relative to other quinolones, which must have facilitated their presence in carboxylate form at pH 7.5 and their interaction with the functional groups in urea as a result. Other antibiotics containing a carboxyl group but differing markedly in structure from fluoroquinolones (e.g., the ␤lactams PEG, AMX, CLX, CEP or the unrelated compound BZ) were retained by neither MIP nor NIP. This also supports the assumption that the ENR imprinted polymer contains recognition sites capable of selectively interacting with the carboxylate group in the fluoroquinolone antibiotics and accommodating the pendant substituents as a result. 3.6. MISPE procedure 3.6.1. Elution solvent selection and optimization The first step was to optimize the composition of the elution solvent. To that aim, 10 mL of samples containing 3 ␮g of ENR, dissolved in 0.1 M HEPES (pH 7.5), were percolated through the MIP/NIP cartridges and 3 mL (1 + 1 + 1 mL) of different solvents namely, methanol; methanol/acetic acid (HAc) and methanol/trifluoroacetic acid (TFA) were tested to elute the retained antibiotic. A methanolic solution of 0.05 M TBA, an ion pairing reagent proven to be useful for this purpose in combination with mixed mode polymeric sorbents [19,31] was also evaluated but double peaks were observed in the chromatogram, probably due to the zwitterionic behaviour of the ENR in this media. The best recoveries were achieved using methanol–acid mixtures. Quantitative elution of the antibiotic was achieved in 3 mL of MeOH with 5% HAc and 1 mL of MeOH with 2% TFA that was selected for further experiments. 3.6.2. Effect of the sample loading flow rate In order to evaluate the effect of the sample loading flow rate on ENR recovery, 10 mL of a solution of the antibiotic (300 ␮g L−1 in 0.1 M HEPES, pH 7.5) were loaded into the MIP/NIP cartridges at flow rates between 0.25 and 4.0 mL min−1 (Supplementary material, Fig. S3a). Recoveries close to 100% were obtained up to a flow rate of 2.5 mL min−1 , decreasing at higher rates. This behaviour can be ascribed as the decrease in the interaction time between the analyte and the polymer binding sites. Finally, a value of 2.5 mL min−1 was selected for further experiments, faster than in most MISPE methods described up to date [38]. 3.6.3. Breakthrough volume The evaluation of the breakthrough volume was carried out by percolating increasing volumes (10–250 mL) of HEPES buffer 0.1 M, pH 7.5 containing a constant amount of ENR (75 ng) through the cartridges. The results are shown in Supplementary material (Fig. S3b). Enrofloxacin recoveries did not differ significatively, at a 95% confidence limit, in the tested range. As it can be observed in the Supplementary material, Fig. S3b, the percolation of 250 mL of water allowed recoveries of 97% (RSD 3.7%, n = 3), comparable to those obtained with commercial cartridges [39,40]. Strong interactions between the MIP and ENR were thus present in this solvent allowing the preconcentration of high sample volumes with good recoveries and demonstrating the excellent performance of the synthetized material compared to other quinolone selec-

tive MIPs applied in SPE reported in the literature [19,41,42]. In order to reduce the analysis time per sample, a volume of 100 mL was selected for further optimizations leading to a 40 min/sample MISPE procedure.

3.6.4. Washing solvent selection The non-specific interactions between the antibiotics and the imprinted polymer could be minimized using a mixture of ACN/water (0.1 M HEPES, pH 7.5) (10/90, v/v), as deduced from the chromatographic experiments (Section 3.1). The use of this mixture (10% (v/v) organic solvent) also allows to wash out the less polar non-related substances present in the water samples [19] so it was selected for the washing step. To determine the optimum washing volume, 100 mL of a buffered water sample (0.1 M HEPES, pH 7.5), fortified with 1 ␮g L−1 of NOR, CIP, LOM, DAN, ENR, SAR, OXO and FLU, were loaded into the MIP/NIP cartridges, and different volumes (2.5, 5.0 and 10 mL) of the hydro-organic mixture were applied in the washing step. The antibiotics were eluted with 1 mL of MeOH with 2% TFA and the extract was analyzed by HPLC–FLD. The results are collected in Fig. 4. Higher washing volumes allowed a decrease of the non-specific binding in the non-imprinted polymer. The highest differences between the MIP and NIP were obtained using 5.0 mL of ACN/water (0.1 M HEPES, pH 7.5) (90/10, v/v). In these conditions, the extraction recoveries for all the antibiotics in the NIP ranged between 0 and 19% (RSD 4.2–9.2%, n = 3), whereas retention in the MIP ranged between 65 and 100% (n = 3) for DAN, ENR, OXO and FLU and was lower, 25–36% (n = 3) for NOR, CIP and SAR. These results are in agreement with those obtained in the HPLC selectivity studies described in Section 3.5. MISPE extracts were very clean and, practically, free from matrix interferences (Fig. 5). These results compare favourably with those reported by other authors for fluoroquinolone MISPE [34,38]. In most cases, the optimization of the MISPE procedures includes the preconcentration of high amounts of target compounds in low volumes (e.g., 2 mg L−1 , 1 mL sample volume for a MIP selective to CIP) which are far above the concentration levels usually found in environmental samples [41].

Fig. 5. Chromatograms obtained before and after preconcentration of 100 mL of river waters fortified with 0.75 ␮g L−1 of the eight fluoroquinolone antibiotics. ex/em = 280/450 nm: (1) NOR; (2) CIP; (3) LOM; (4) DAN; (5) ENR; (6) SAR. Inset: ex/em = 280/365 nm: (7) OXO; (8) FLU.

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69

Table 3 Average recoveries (R, %), relative standard deviations (RSDs, %, n = 3) and limits of detection (LOD) obtained after solid-phase extraction of 100 mL buffer HEPES 0.1 M, pH 7.5 and river water samples fortified with the fluoroquinolones at 0.5, 0.75, 1.0 ␮g L−1 concentration levels Analyte

Spiking level (␮g L−1 )

Buffer (HEPES 0.1 M, pH 7.5) Found level

NOR

CIP

LOM

DAN

ENR

SAR

OXO

FLU

(␮g L−1 )

LOD (␮g L−1 )

River water R (%)

RSD (%)

Found level

(␮g L−1 )

R (%)

RSD (%)

0.50 0.75 1.0

0.14 0.18 0.27

28 24 27

8 8 4

0.14 0.19 0.25

28 25 25

9 7 8

0.11

0.50 0.75 1.0

0.14 0.21 0.32

27 28 32

8 9 6

0.14 0.22 0.30

28 29 30

13 6 7

0.14

0.50 0.75 1.0

n.q. n.q. 0.17

– – 17

– – 9

n.q. n.q. 0.15

– – 15

– – 9

0.27

0.50 0.75 1.0

0.35 0.50 0.67

70 66 67

2 5 3

0.35 0.48 0.67

70 64 67

3 6 3

0.01

0.50 0.75 1.0

0.50 0.75 0.99

100 100 99

4 6 2

0.52 0.76 1.0

105 101 100

6 5 3

0.01

0.50 0.75 1.0

0.21 0.29 0.40

39 39 40

7 5 4

0.19 0.29 0.36

38 38 36

8 7 6

0.12

0.50 0.75 1.0

n.q. 0.54 0.69

– 71 69

– 8 10

n.q. 0.53 0.70

– 70 70

– 6 2

0.17

0.50 0.75 1.0

n.q. 0.52 0.71

– 70 71

– 7 9

n.q. 0.52 0.69

– 69 69

– 10 4

0.19

n.q., non-quantificable.

3.7. Analysis of river water samples To evaluate the applicability of the optimized MISPE procedure to real sample analysis, trueness and precision of the method were determined using fortified blank buffer and river water samples (100 mL) at three concentration levels (0.50, 0.75 and 1.00 ␮g L−1 ). The results, summarised in Table 3, show the good accuracy of the method. Errors, expressed as relative standard deviation were RSD ≤ 13% for all the concentration levels tested. Moreover, the presence of the matrix components in river samples did not affect, significatively, the preconcentration efficiency of the antibiotics onto the MIP sorbent. The limits of detection (LODs) of the MISPE method, calculated as signal-to-noise ratio of 3, were found to be in the range of 0.01–0.30 ␮g L−1 (Table 3). The values obtained are comparable to those reported [43] for the analysis of fluoroquinolones in water samples using commercial solid-phase extraction sorbents (e.g., LOD range: 0.01–0.50 ␮g L−1 using SPE-Oasis HLB [44], LOD range: 0.02–0.15 ␮g L−1 using SPE-MPC disk cartridges) demonstrating the applicability of the MIP sorbent for the preconcentration of the target antibiotics in river water. The cartridges have been reused for 80 assays without loosing their preconcentration ability. 4. Conclusions This work demonstrates the applicability of a urea-based MIP for the preconcentration of eight fluoroquinolones in environmental water samples. The optimized method is based on a MISPE procedure followed by HPLC with fluorescence detection. Using the water-compatible urea-based MIP as specific MISPE sorbent yields a suitable method to extract fluoroquinolones from environmental samples providing good recoveries and reproducibilities for ENR, DAN, FLU and OXO. The cartridges can be reused for

more than 80 assays without losing their preconcentration ability, which is promising for on-line preconcentration formats. Thus, this procedure is adequate for the analysis of the above-mentioned fluoroquinolones in environmental waters at the ng L−1 level. Acknowledgements This work has been funded by the Spanish Ministry of Science and Technology (grant CTQ2006-15610-C02), by the Madrid Community Government (ref. S-0505/AMB/0374), the European Social Fund, the European Regional Development Funds and the UCM (CCG07-UCM/AMB-2932). The authors thank M.J. Torralvo for the nitrogen adsorption-desorption studies. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2008.08.109. References [1] [2] [3] [4] [5] [6]

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