Baseline-Corrected Second-Order Derivative Electroanalysis

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Baseline-Corrected Second-Order Derivative Electroanalysis Combined With Ultrasound-Assisted Liquid–Liquid Microextraction: Simultaneous Quantification of Fluoroquinolones at Low Levels Luiz Henrique de Oliveira, and Magno Aparecido Gonçalves Trindade Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01379 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 4, 2016

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Analytical Chemistry

Baseline-Corrected Second-Order Derivative Electroanalysis Combined With Ultrasound-Assisted Liquid–Liquid Microextraction: Simultaneous Quantification of Fluoroquinolones at Low Levels Luiz Henrique de Oliveira, Magno Aparecido Gonçalves Trindade* Faculdade de Ciências Exatas e Tecnologia, Universidade Federal da Grande Dourados, Rodovia Dourados-Itahum, km 12. Dourados-MS, 79804-970, Brazil. * E-mail: [email protected]; Fax: +55 67 3410-2072 ABSTRACT: A baseline-corrected second-order derivative procedure and a miniaturized sample preparation based on low-density solvent and ultrasound-assisted liquid–liquid microextraction (LDS-UA-LLME) was combined to provide the simultaneous electroanalysis of three fluoroquinolones (FQ) as emerging contaminants (ECs). The enhanced mathematical processing provided the best separation with an accurate measurement of the overlapping peaks during the simultaneous electro-oxidation of target FQs that were directly dropped on the surface of carbon nanofiber-modified screen-printed electrodes. The adapted LDS-UA-LLME protocol was the key step involved in the sample preparation, which pre-concentrate target analytes from diluted tap water samples with an enrichment factor of around 80 times, allowing their quantification at trace levels. This combined feature demonstrated the unique application of an electroanalytical technique for the simultaneous electroanalysis of three FQs in spiked tap water samples, with recovery values remarkably close to 100%.

Fluoroquinolones (FQ) are a class of modern and efficient synthetic antibiotics that are commonly administered in humans and animals. FQs are used by veterinaries for disease treatment, control, and prevention in animals, but many times it is inappropriately utilized in therapeutic dosages to maintain the health and to promote the growth of livestock.1-4 As a result, FQs are commonly used worldwide, and in the last decade, reports of the environment and food contaminated with unchanged antimicrobial agents and/or their active metabolites have been documented.5-7 Discharge from industrial sources, domestic activities and antibiotics excreted by treated animals are responsible for the introduction and accumulation of FQs in aquatic environment, and this created a new challenge for drinking and wastewater treatment systems.8-14 The presence of broad-spectrum antibacterial agents in tap and drinking waters as well as foodstuffs (even at low concentrations), is seriously damaging human health and is responsible for the proliferation of antibacterial drug resistance.15,16 Studies developed in countries such as India,5 Brazil,2,17 China,18-21 Switzerland,22 Japan,23 Estonia,24 Canada,25 and United States 26,27 have reported the presence of considerable levels of unchanged FQs or their active metabolites in wastewater effluents or aquatic environments, which affected general and drinking water quality. Considering the impact of these chemical compounds on human health, the development of sensitive and selective analytical methods, especially for the quantification of FQs residues in drinking water, is very important and highly desirable in meeting existing environmental challenges. Regardless of the analytical technique used, the final result inevitably depends on the sample preparation steps, between them the clean-up and the extraction processes of the target analyte, capital stages to achieve selective enrichment of diluted samples.15,28-29 Ideally, the analytical technique used should be able to detect target analytes at residual levels (as low as ng

L-1), and be able to simultaneously detect multi-class compounds.15,28-29 FQs in aquatic environmental samples represent a great challenge in analytical method development owing to notable low analyte concentration, matrix effect interferences from concomitant elements and, possibly the most important feature, the need for sample volume adjustment to enrich and achieve the required detection limit. 16,30-32 To overcome these challenges, new strategies for sample preparation/preconcentration are required and/or the existing ones must be improved, in order to meet the aforementioned requirements and to reduce the associated costs, time, solvent consumption (and toxicity), and possible interferences. All these can be potentially achieved in a miniaturized extraction system.31,33-35 Thus, to accomplish selective extractions of the analytes, attaining high enrichment factors, in this study was developed a liquid-liquid microextraction (LLME) processed with ultrasound and utilizing low-density solvents. Despite a considerable number of researches aimed effectively determining FQs in pharmaceutical and biological samples, to our best knowledge, there has not been presented a study specifically focusing on the analysis of FQs as emerging contaminants (ECs) in tap and drinking water samples using electroanalytical techniques. Here, we demonstrate for the first time the advantages of the association between miniaturized extraction method using low-density solvent and ultrasoundassisted liquid-liquid microextraction (LDS-UA-LLME) and electroanalytical techniques. In this work, the LDS-UA-LLME provide the adjustment in sample volume as well as preconcentrate target analytes in tap water samples while the baseline-corrected second-order derivative method allows to process the voltammetric data during the simultaneous electroanalysis of FQs (chemical structure in Figure S1, Supporting Information) at residual concentrations.

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EXPERIMENTAL SECTION Instrumentation and electrochemical system. Voltammetric experiments were performed using a PGSTAT 204 potentiostat/galvanostat (Metrohm Autolab®, Utrecht, The Netherlands), controlled by the Nova 1.11 software. Specific boxed connector obtained from DropSens® (model DSC) was used as the interface between the screen-printed electrodes (SPEs) and the potentiostat, based on a suitable configuration to work with microvolumes. Working electrodes composed by different materials were tested and they include screen-printed carbon electrodes (SPCE), graphene-modified SPEs (GPH/SPE), carbon nanofiber-modified SPEs (CNF/SPE), and carbon nanotube SPEs (CNT/SPE), all of which were purchased from Drop-Sens® (Oviedo, Spain). All measurements and/or adjustments of pH were performed using a combined glass electrode (Hanna®, model HI 1131B, Texas, United States) connected to a digital pH-meter (Hanna®, model HI 3221, Texas, United States). The ultrapure water (R ≥ 18.2 MΩ cm) was obtained with a Milli-Q Plus system (Millipore®, São Paulo, Brazil). An ultrasonicator (Bransonic®, model 1800, New York, United States) was used for chemical dissolution and homogenization of the prepared working solutions, as well as to assist the sample preparation in LDS-UA-LLME process. Extractions were performed using conical tubes measuring 15 and/or 50 mL (Falcon®, Curitiba, PR, Brazil), a vortex mixer (Fisatom®, model 774, São Paulo, Brazil), and a centrifuge (Hettich®, EBA 200, Tuttlingen, Germany). When necessary, a laboratory oven (Ethik® technology, São Paulo, Brazil) was used to evaporate the solvent during the extraction process. Chemicals, solutions, and samples. The FQs: levofloxacin (LEVO), norfloxacin (NOR) and danofloxacin (DANO) were purchased from Sigma Aldrich® (São Paulo, Brazil) and used as received. The standard stock solutions were prepared at the concentration of 1.0 mmol L-1 by dissolving the respective FQs in 1.0% methanol (Vetec®, Rio de Janeiro, Brazil) and 0.5% of acetic acid (Vetec®, Rio de Janeiro, Brazil) and further diluted with ultrapure water. The working solutions, in the concentration range 0.008 to 10.0 µmol L-1, were prepared daily by diluting the stock standard solutions. For the LDSAU-LLME procedure, samples were prepared by adding aliquots of the stock solutions into an Eppendorf tube, followed by dropping the sample onto the electrode surface for electrochemical measurements. The surfactants tested include sodium dodecyl sulfate (SDS), dioctyl sodium sulfosuccinate (DSS), cetyltrimethylammonium bromide (CTAB), tetraethylammonium chloride (TEAC), tetra-n-butylammonium bromide (TBAB), and Triton X-100 (TX-100), all of which were purchased from Sigma-Aldrich® (São Paulo, Brazil) and prepared via serial dilutions to give solution concentrations in the range of 0-350 µmol L-1 using ultrapure water (R ≥ 18.2 MΩ cm). All other reagents and solvents used were of analytical grade and purchased from Sigma-Aldrich® (São Paulo, Brazil) and used as received. The buffer Britton-Robinson (BR) was used as the supporting electrolyte solution and the composition was prepared by a mixture of acetic acid, phosphoric acid, and boric acid (all purchased from Sigma-Aldrich®, São Paulo, Brazil) at a concentration of 0.04 mol L-1. The required pH values were adjusted using hydrochloric acid or sodium hydroxide (Vetec®, Rio de Janeiro, Brazil) at a concentration of 1.0 mol L-1. Tap water samples were collected from the water supply network of the Laboratory of Analytical Chemistry at the Federal University of Grande Dourados (Dourados, MS, Brazil). All

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water samples were stored in dark glass bottles, conserved at 4.0 °C, and analyzed without any pre-treatment or filtration. Prior to use, all selected water samples were certified to be free from the target FQs. Sample preparation, LDS-UA-LLME procedure, and electrochemical analysis. Before the samples preparations and electrochemical analysis, all the bottles and glassware used were immersed in a 10% nitric acid (Vetec®, Rio de Janeiro, Brazil) solution for 12 hours to clean them thoroughly and subsequently, washed in ultrapure water thrice. Six tap water samples were spiked with LEVO, NOR, and DANO at concentrations levels of 3.0, 2.6, and 2.9 as well as 30, 26, and 29 µg L-1, respectively, followed by an adjustment of the pH value to 10 with sodium hydroxide and manual or vortex shaking for 5 min. The matrix effects were tested by the additionrecovery experiments carried out using the target FQ-spiked samples. To carry out the LDS-UA-LLME procedure, 12 and/or 35 mL of target-spiked samples were placed into conical tubes and pH-adjusted to 2.0, 7.0, and 10 with acetic acid or ammonium hydroxide (both from Vetec®, Rio de Janeiro, Brazil). Subsequently, NaCl (in the concentration range of 5.0 to 25% (m/V)) was added to the samples and the tubes were shaken by a vortex mixer until the solid dissolved completely. The extractor solvent, acetone (Vetec®, Rio de Janeiro, Brazil), was rapidly added into the conical tubes containing the tap water samples using a micropipette, with volume varying between 12.5 and 25% (v/v). The samples were shaken using the vortex mixer, followed by ultrasonication at 60 °C, both varied time in 5 and 15, which first allowed dispersion and then a separation between the aqueous phase and the low density organic solvent, as indicated by the blurred color on the surface of the aqueous phase. In this step, conical tube was sealed to prevent loss of the organic phase. After this step, the mixture was centrifuged for 5 min at 3000 rpm and the top organic layer (acetone) was then collected with a microsyringe (Hamilton, São Paulo, Brazil) into an Eppendorf tube. The enriched residue from the organic phase collected was concentrated in a laboratory oven at 60 °C. After drying, the residue was redissolved in 150 µL of the supporting electrolyte solution, BR buffer (pH 5.0), and an appropriate amount of surfactant was added, followed by ultrasonication for an extra 1.0 min to achieve homogenization. The voltammetric measurements were performed using 30 µL of target-enriched solution (with an appropriate electrolytic medium) and directly dropped onto the SPE surface, using boxed connector for SPE. The standard addition method was used to check the additionrecovery measurements and to calculate the enrichment factor (EF). The EF was calculated using the following equation: EF = Ccol./Co, which Ccol. and Co are the concentration of analyte in the collected organic phase and the initial concentration of analyte in the spiked tap water samples, respectively. Unless otherwise indicated, all the measurements were performed in triplicates and the standard deviation were also calculated. After each electrochemical measurement, the SPE surface was cleaned with isopropyl alcohol (Vetec®, Rio de Janeiro, Brazil), rinsed with ultrapure water, and dried at room temperature. The SPE was then cycled via reverse scanning, in the potential range between 0.4 and -1.0 V vs. Ag at a scan rate of 10 mV s-1 (2 cycles), in the supporting electrolyte solution composed of the B-R buffer. This approach enabled the single use of CNF/SPE for at least 40 times without losing precision.


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Baseline-corrected second-order derivative process. After registering the original DP voltammograms, the signal transformation was performed using the Originlab® (version 9.0) or Nova 1.11 (Metrohm Autolab®) software to obtain an improved peak separation of the target FQs during simultaneous detection. However, it was preferably used the Nova 1.11 by automating all the mathematical process, inserting the firstorder derivative mathematical function linked directly in the current and potential, following of the smoothed (factor 2) according to the Savitsky-Golay algorithm. For better definition of the FQs peaks, the derivative function was set again, which approaches provided more accurate measurements as well as the intensification of all target peaks. Data treatment using a factorial design. The extraction time was valued through of the variables vortex, ultrasound-assisted and centrifugation in terms of recovery values of LEVO, NOR and DANO. Data treatment was carried out using a complete factorial design (23), taking into consideration the low (-) and high (+) points for all factors and interactions that are available in the Pareto chart. In addition, the Doehlert matrix was also applied, with a central point, and both methods were employed at 95% confidence level. The statistical treatment was performed using the StatSoft Statistica® 10.0 software package (Statsoft, Tulsa, U.S.).

behavior of LEVO, NOR, and DANO during their electrooxidation onto SPCE, CNT/SPE, GPH/SPE, and CNF/SPE, respectively. On the bare SPCE (Figure 1, curve a) as well as the CNT/SPE (Figure 1, curve b), broad and undistinguishable peaks were observed and the current intensity could not be accurately measured. However, on the GPH/SPE (Figure 1, curve c) and CNF/SPE (Figure 1, curve d), three peaks (though not well defined) could be clearly distinguished, corresponding to the electro-oxidation of the target FQs. Therefore, based on the preliminary study, regardless of the properties of the SPE surface used as working electrode, it is somewhat difficult to simultaneously detect the individual FQs from a mixture owing to the impossibility of accurately differentiating the three target peaks of LEVO (Ep = 0.75 V), NOR (Ep = 0.82 V), and DANO (Ep = 0.90 V) (Figure S2, Supporting Information). Among these, the CNF/SPE-based working electrode (Figure 1, curve d) was chosen because even though the three peaks were only observed at partially distinguishable potential values. CNF/SPE provided enhanced electrochemical responses that can be attributed to their superior electrocatalytic activity and this is ideal to facilitate a further studies aiming at exploring the analytical performance as well as the peak separation efficiency of the electrode.

RESULTS AND DISCUSSION Voltammetric study. Most FQ derivatives are structurally similar and generally, have a different substituent on the piperazinyl moiety at the 7-position. With respect to the thermodynamics factor, the peak potential (Ep) of the modified piperazinyl group for the corresponding redox processes are nearly similar, not sufficiently different to provide peak separation when two or more FQs are simultaneously electro-oxidized. Although the mechanism for the electro-oxidation of some FQs on carbon electrodes has not been fully elucidated yet, previous studies suggest that the oxidation step occurs at the piperazinyl group via an irreversible electrochemical process.36-38 This implies that during a simultaneous electrochemical detection process, this electroactive group can cause overlapping or broad voltammetric peaks owing to the energetically similar process, hence making it difficult to distinguish accurately the peaks during electroanalysis. In a previous work, Bilibio and coworkers39 have shown that the electrooxidation of NOR and LEVO, i.e., FQs that contain the piperazinyl group at the 7-position (Figure S1, Supporting Information), on a bare glassy carbon electrode occurs at very close potential values, 0.97 and 1.04 V vs. Ag/AgCl(3 mol L-1), respectively. The broad voltammetric peaks hindered the simultaneous determination, even at best working conditions, because the positions of the target peaks were undistinguishable and peak current could not be accurately measured.39 It was also shown that the mathematical processing procedure based on deconvolution allowed a satisfactory peak separation and offered clear advantages in treating the voltammetric data during the simultaneous electroanalysis of the target FQs. The data acquisition process was simple and was not costly and time-consuming.39 Taking the aforementioned factors into consideration, the performances of different unmodified and modified SPEs were tested, in order to identify an electrode array that can maximize the output signal of electrocatalytic activities, providing better detectability with clear voltammetric peaks during the simultaneous detection of oxidized target FQs. Figure 1 shows the DP voltammograms obtained, comparing the voltammetric

Figure 1. DPV registered for LEVO, NOR and DANO (18 µmol L-1) on the SPEs. Parameter: 0.04 mol L-1 of BR buffer (pH 5.0) as supporting electrolyte. Parameters: Step potential (∆Es) = 4.0 mV, pulse amplitude (Esw) = 30 mV and scan rate (υ) = 10 mV/s.

Enhanced voltammetric responses via mathematical processing. Considering the poor ability of DPV technique to distinguish the peaks obtained during the simultaneous detection of target FQs on the CNF/SPE (Figure 1), the next challenge was to overcome this limited selectivity; aiming at the accurate measurement of the partially distinguishable FQs peaks. The overlapping peaks observed in the electrooxidation of structurally similar FQs is a common occurrence.39 This aspect was clearly demonstrated in the voltammetric studies even after optimizing working conditions that included various instrumental and experimental parameters (Figures S3 and Table S1, Supporting Information), different surfactants and their respective ideal concentrations (TX-100, Figure S4, Supporting Information), and different responses from the electrode surface. Heretofore, no methods provided an avenue for FQs to be simultaneously determined, especially at residual levels (i.e., as low as µg L-1). Thus, derivative transformation was introduced to separate the overlapping voltammetric peaks.



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To perform the signal transformation and to separate the illdefined peaks, we first evaluated different mathematical processing based on the first or second-derivative voltammograms (Figure 2), followed by choosing the type of baseline modes that have been previously reported in typical peak current measurement (Figure 3).40-43 Figure 2 displays in detail the comparison of original, first and second-order derivative DP voltammograms, recorded during simultaneous electroanalysis of target FQs onto CNF/SPE. Based on the original voltammogram (Figure 2I), it is clear that even though there is a discrimination between the LEVO (Ep = 0.71 V) and NOR (Ep = 0.81 V) peaks, they are still partially overlapping and the shorter distance between the neighboring peaks undertakes the accurate measurement. With respect to DANO (Ep = 0.89 V), its peaks was not completely resolved and it was practically superimposed on a broad NOR wave (at Ep = 0.81 V), resulting in an undetectable peak current intensity. However, once the original voltammogram was mathematically-processed by first-order (Figure 2II) and second-order (Figure 2III) derivate, the three FQs peaks were clearly distinguished. Hence, this means that both mathematical processing provide noticeable peaks separations followed by its amplification without background interferences.

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current intensification less complicated for any practical electrochemical applications. Hence, the peak processing applied herein can be used as an effective approach to improve analytes peaks resolution as well as its intensification during simultaneous multi-residual analysis, fulfilling the requirements of a sensing protocol. However, taking advantage of target signal transformation, it is also observed that the baseline-corrected second-order derivative method (Figure 3IV) lead to a more accurate measurement to obtain the peak current intensity when compared with baseline-corrected mode (Figure 3II). Thus, it was chosen the combined baselinecorrected second-order derivative associated with the peakpeak baseline mode selection strategy to measure accurately the peak current intensity for all detected FQs.

Figure 3. DP voltammogram showing: first-order (I) and baseline-corrected first-order derivative (II), second-order (III) and baseline-corrected second-order derivative (IV). (III) Also showing the conventional baseline adjustment for a second-order DP voltammogram, in which (a) front-peak horizontal baseline, (b) peak-peak baseline and (c) rear-peak horizontal baseline. Conditions as in Figure 2.

Figure 2. DP voltammogram registered for LEVO, NOR and DANO (18 µmol L-1) on the CNF/SPE showing: (I) only baselinecorrection, (II) after first-order and (III) after second-order derivatives processing. Conditions: 0.04 mol L-1 of BR buffer (pH 5.0) containing TX-100 (100 µmol L-1) as supporting electrolyte. ∆Es = 4.0 mV, Esw = 30 mV and υ = 10 mV s-1.

The selection of the baseline type in the first or second-order derivative model is essential in enabling an accurate measurement of the peak current intensity. 41 Figure 3 (I-IV) illustrates in detail typical derivatives voltammograms and the different baseline modes involved in the measurements of peak current intensity. Indeed, the peak current can be measured using any of the three modes specified for baseline adjustments in Figure 3 (III). However, it is worth emphasizing that these approaches are time-consuming and require a greater level of operator precision to measure accurately the peak current intensity. From Figure 3 (II and IV) is demonstrated that when the baseline-corrected derivative mode is associated with the peakpeak baseline mode selection, the measurement of the individual FQ peak current intensity can be accurately explored to generate reliable analytical quantifications. In both case (Figure 3, II and IV), the voltammetric curves behave consistently with the Gaussian model during mathematical processing, which makes the peak identification and

Analytical curve and electroanalytical performance via LDS-UA-LLME. Using the optimized experimental and instrumental conditions (Table S1, Supporting Information), the quantitative parameters of the newly developed method were evaluated via an analytical curve that was obtained in the concentration range of 4.8 to 31 µmol L−1 (Figure S5, Supporting Information). Clearly, in the absence of the LDS-UALLME pre-concentration step, the linear range was satisfactory for all the FQs studied with a correlation coefficient (r) greater than 0.998 (n = 7); however, the detection limit, ranging between 1.32 and 2.53 µmol L−1 (Table S2, Supporting Information) did not meet with the requirements of the residual analyte determination. As previously mentioned, the drawback of electroanalytical techniques in bulk analysis is their inability, in certain cases, to support multi-analyte residual determination. In addition, the measurement of voltammetric peaks can be strongly affected by broad background waves and/or interference peaks that overlap the required analyte peaks, difficulting their discrimination during trace analysis.40,44 In such scenarios, the signal transformation eliminates background interference with improved peak identification, specifically for the ill-defined DANO peak. For the residual analyte determination, sample preparation is a crucial step to reduce the matrix effect. The adjustment in the sample volume during the pre-concentration step is also vital in ensuring an accurate lower limit of quantification.15,31 For instance, two previous studies have proposed


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avoiding the use of conventional bulky analytical instruments but advocated a miniaturized sample preparation protocol to remove the matrix effect and to pre-concentrate the target analyte, in order to reach the desired quantitation limit. Both methods used in situ ionic liquid-assisted dispersive liquid– liquid microextraction, followed by an ultrasound-assisted microvolume back-extraction to enable the detection of 2,4,6trinitrotoluene and mercury using SPCEs45 and gold nanostructured SPCE,46 respectively. The miniaturized protocol and the electrochemical transducers allowed the determination of target analytes in aqueous samples at low levels. Here, the proposed analytical strategy involves the use of a powerful voltammetric peak processing combined with a preconcentration step via LDS-UA-LLME, to afford a low quantitation limit. The LDS-UA-LLME protocol was tested to evaluate the resulting analytical performance and a comparison against the direct detection of target FQs using electrochemical pre-concentration was also established (Figure 4). Both pre-concentration steps were performed by spiking the samples with target FQs (at 30 µg L-1) that were prepared in a BR buffer solution using tap water as solvent, and analyzed under the previously optimized conditions as in the Table 1. Table 1. Optimized conditions used for the preconcentration step shown in Figure 4. Electroanalytical Electrolyte [BR buffer] Buffer pH [TX-100] ∆Es (mV) Esw (mV) υ (mV s-1)

BR buffer 0.04 5.0 100 4.0 25 10

LDS-UA-LLME Sample vol. (mL) Sample pH [NaCl] (%) Acetone vol. (mL) Vortex shaking (min.) Ultrasound time (min.) Centrifugation time (min.)

12 10 25 2.5 10 5 5

[BR buffer] in mol L-1; [TX-100] in µmol L-1; vol.: volume.

In the absence of mathematical processing (Figure 4, curves C and D), undefined peak currents can be observed in the original DP voltammograms, showing that the simultaneous determination of the three FQs is unfeasible. However, the advantage of the LDS-UA-LLME protocol, used in combination with the baseline-corrected second-order derivative signal transformation, is clearly proven as the peak currents are almost four-fold (LEVO and NOR) and fifteen-fold (DANO) (Figure 4D, inset) higher than those obtained with direct detection using electrochemical pre-concentration (Figure 4C, inset). Hence, the proposed LDS-UA-LLME approach (Figure 4D) maximized the peak current and may be suitably adapted for practical applications in electroanalysis, giving a high detectable peaks that can be translated into quantitative information (even for DANO) during the simultaneous determination of tap water samples at trace levels. In Figure 4C, it is noteworthy that the CNF/SPE when subjected to electrochemical pre-concentration produces poor voltammetric peaks, which may not be related to the poor FQs accumulation itself. As well-know, the electrochemical preconcentration has a disadvantage during the analysis of polar organic compounds as the generated products can passivate the electrode surface via fouling and thus, hinder the electron transfer process.39,44 Owing to the surface roughness of the CNF/SPE, the removal of surface fouling between each measurement is difficult, which in turn impedes the ability to reestablish the electrode-surface-activity. In agreement with these statements, we have found that the peak current regis-

tered was markedly lowered after successive potential scanning or during continuous measurements, confirming the existence of electrode-surface-fouling. Also, we have observed that the adsorption complication affected the CNF/SPE performance even when cleaning the surface with organic solvent and further electrochemical pretreatment before recording the DP voltammograms.

Figure 4. Conventional DP voltammograms and (inset) baselinecorrected second-order derivative method registered for LEVO, NOR and DANO at 8.3×10-8 mol L-1. (A) Blank: BR buffer solution prepared in tap water sample, (B) Direct detection at tap water sample without any pre-concentration step, (C) detection using electrochemical pre-concentration at 0.4 V for 400 s and (D) pre-concentration via LDS-UA-LLME. Conditions as in Table 1.

Optimization of the LDS-UA-LLME method. The LLME applications are well-established and there is not much novelty regarding the experimental parameters that are to be evaluated to achieve the highest extraction efficiency.47-52 Commonly, to obtain a satisfactory enrichment factor with high extraction efficiency for multi-residual analytes, the type and volume of extractor solvent, ionic strength by salt addition, sample pH, temperature, extraction and centrifugation time, as well as the ultrasonication and/or vortex agitation time are important parameters to be optimized. 47-50 Thus, the LDS-UA-LLME method was chosen after evaluating the many adaptations of specific LLME procedures featured in previously published work papers.49-52 We have attempted to reduce the number of measurements and the main adjustment made was in the elimination of the dispersive solvent by choosing less toxic solvents, as well as to make the procedure faster, cheaper, safe, and reliable. Briefly, the main steps for the protocol include pre-concentrating the target FQs in a smaller volume to obtain high enrichment efficiencies, and establishing a working condition that is easily adaptable to the electrochemical system where the collected drop was directly deposited onto the CNF/SPE surface (droplet-based). To optimize the relevant parameters (Table S3-S5, Supporting Information), the recovery values obtained via addition-recovery test were used to evaluate the extraction effectiveness of FQs from spiked tap water samples. Initially, the recovery of target FQs was studied together with the formation of the organic phase by varying the concentration of acetone and the ionic strength (based on NaCl amount), both of which varied between 4.0% and 25.0% (Figure 5). The increased recoveries of the multi-residue extraction that were observed between 15% and 25% of NaCl and acetone could be explained by the “salting-out” effect. Such an effect has max-



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imum extraction efficiency at 25% of the mixture, organic solvent and NaCl, with recovery values between 67% and 80% for the target FQs. While the highest extraction efficiencies could be achieved at 25% (m/v) of NaCl, amounts greater than 25% loaded a large amount of salt into the organic phase, which affected the performance of the electrochemical measurement. The salt concentration is the key factor in fine-tuning the LDS-UA-LLME protocol since an adjustment in the surfactant concentration is part of the strategy that prevents interference from matrix constituents, and it protects the electrode surface from the adsorption of electro-generated products that are strongly dependent on NaCl amount. High values in acetone percentage, not provide significant increase in the recovery values beyond contribute to the time consumption during the solvent evaporation process. Thus, with 25% of NaCl (mass-based) and acetone (volume-based), all FQs were suitably extracted and this condition was used in subsequent experiments.

Figure 5. Recoveries of target FQs in spiked tap water samples performed to optimize the extraction efficiency via LDS-UALLME based on the volume of extractor solvent and ionic strength by salt addition. LDS-UA-LLME conditions: sample volume = 12 mL; sample pH = 10; vortex shaking time = 15 min; ultrasound-assisted time = 5 min and centrifugation time = 5 min.

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The combined parameters, such as vortex, ultrasonication, and centrifugation time, may have a decisive influence on the enrichment factor (EF)32,47-50 beyond the reduced number of experiments, and thus allow the LDS-UA-LLME protocol to be developed in a fast and economic way. The results obtained according to a 23 factorial design are shown in Table S4 (Supporting Information) and the careful combination of these three specific parameters was performed by a Pareto chart (Figure S6, Supporting Information). The analysis of this chart indicated that the time of centrifuge extraction was not significant. However, significant effects were observed with an upper level vortex, as consequence of the longer interaction time between the aqueous and organic phases, facilitating a more efficient target FQs migration into the organic phase. The ultrasonication time shows a slight negative influence, confirming literature reports53-54 that a higher exposure time under ultrasonic irradiation degrades certain FQs. Nonetheless, the ultrasonication time used in this study (5.0 min) promoted a fast transfer of the target FQs into the organic phase and did not cause any degradation. The main variable effects were applied using the Doehlert matrix and the results obtained (Table S5, Supporting Information) were evaluated by the analysis of variance (ANOVA). The F-test (Table S7, Supporting Information) indicated that the quadratic regression model is significant, being that lack of fit of the Fcalculated < Ftabeled (LEVO = 7.17, NOR = 2.75, and DANO = 1.78 < 18.51). In summary, the maximum extraction efficiency is observed on the response surfaces (Figure 6), which show the combined parameters and their influence on the EF, as measured by the recovery value. The highest recovery values for LEVO (A), NOR (B), and DANO (C) could be achieved with 25% (3.0 mL) acetone, 25% NaCl, 15 min of vortex time, and 5 min of ultrasonication time. Hence, using these parameters, an EF of around 80 times was obtained for the newly developed LDS-UA-LLME procedure, which is satisfactory for an analytical method that is capable of obtaining acceptable quantitative information during simultaneous determinations at trace levels.

Figure 6. Response surface for LEVO (A) NOR (B) and DANO (C) obtained to factors vortex and ultrasound. Samples analysis. To establish the accuracy and the reliability of the proposed method, the recovery tests were evaluated using addition-recovery experiments carried out in tap water samples contaminated with known amounts of target FQs (Table 2) and the pre-concentration step via LDS-UA-LLME was performed as described in the experimental section. Figure 7 illustrates the DP baseline-corrected voltammograms

recorded after: FQs pre-concentration via LDS-UA-LLME (curve a), and the successive addition of the target FQs, i.e., directly dropped onto the CNF/SPE surface (curves b-e) to achieve the quantitation via the standard-addition method curve. According to the results, there is no significant matrix effect that compromises the accurate determination of the target FQs, once the proper linearity for all standard addition


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curves showed a correlation coefficient close to 0.99 (Table S7, Supporting Information). Similarly, the detection limits for every FQ is not significant affected when compared to the standard analytical curve (Figure S5 and Table S2, Supporting Information). It is noteworthy that, even in the presence of tap water sample, the overlapping DANO peak can be quantified accurately and this meets the initial goal of simultaneously determining three FQs that have partially overlapping peaks in a reliable manner using both an improved sample preparation method and mathematical processing. Table 2 summarizes the recovery data for tap water samples (pH-adjusted at three different values) contaminated with two different FQs concentration levels. In tap water samples spiked with 30, 26, and 29 µg L-1 of LEVO, NOR, and DANO, respectively, the best recovery values ranged between 94.3% and 98.8% when the pH was adjusted to 10. At pH 7.0, the recovery values decreased and fell in the range of 69% to 96%. As expected, at low pH values, no significant recovery values were obtained owing to the fact that the FQs are present in the protonated form and are not attracted to the organic phase even at high ionic strength. In general, the analytical precisions, expressed as coefficient of variation (CV), are lower than 14% and are in agreement with the expected values when the Horwitz equation55 is used, suggesting that the newly developed method can be reliably used to quantify the target FQs in the water samples collected. Fixing the pH 10 to the spiked sample with 30, 26 and 29 mg L-1 of LEVO, NOR e DANO, respectively, and using the optimum conditions the LDS-UA-LLME protocol was performed to obtain the repeatability (measured peak current with the same CNF/SPE) and the reproducibility (measured peak current with five CNF/SPE). After triplicate measurements, the average RSD were: LEVO = 5.7%, NOR = 2.5% and DANO = 0.3% for the repeatability and LEVO = 8.9% NOR = 9.0% and DANO = 5.1% for the reproducibility, revealing an acceptable repeatability and reproducibility to perform the electroanalysis of target FQs using the reported approach.

Table 2. Recovery assay to assess the accuracy and precision of the proposed method for quantitative determination of LEVO, NOR and DANO in tap water samples. FQs

pH

Added (µg L-1)

Found [a] (µg L-1)

Recovery (%)

RSD (%)

LEVO

2.0 7.0 10 2.0 7.0 10 2.0 7.0 10

30

10.4 24.7 29.6 14.4 23.0 24.5 16.0 20.0 28.0

34.7 82.3 98.8 55.3 88.0 94.3 55.0 69.0 96.5

5.8 6.9 8.4 4.2 7.8 5.0 6.4 12.7 13.9

LEVO

10

3.00

2.60

86.8

11.0

NOR

10

2.60

2.10

80.8

11.9

DANO

10

2.90

2.23

77.0

10.5

NOR

DANO

26

29

[a]

Average of four determinations, RSD = relative standard deviation. The values: 3.0, 2.6 and 2.9 µg L-1 as well as 30, 26 and 29 µg L-1 represents, respectively, 8.3×10-9 mol L-1 and 8.3×10-8 mol L-1 of LEVO, NOR and DANO.

Tap water samples containing 3.0, 2.6, and 2.9 µg L-1 of the target FQs were also tested to assay the recovery test (Table 2). Once the pH value of the samples was fixed at 10, the matrix effect and the adjustment of the sample volume to preconcentrate the target analytes were tested using the aforementioned experimental conditions. No matrix effect was observed during the DP voltammetry; however, as can be seen in the Table 2, the recovery values were lower than 90%. The recovery values (ranging from 77% to 86.8%) may be related to the trace analyses, which can result in a loss of analytes during the sample preparation step. Nevertheless, if the trace-level concentration is taken into consideration, the recovery values are satisfactory and corroborate the feasibility of the newly developed method in simultaneously determining three FQ species that contain partially overlapping peaks.

CONCLUSION

Figure 7. DP voltammograms for: (a) 30 µL of tap water sample containing 30, 26 e 29 mg L-1 of LEVO, NOR and DANO, (b-d) successive additions (30 µL) of FQs (0.10 mmol L-1). Inset: Standard addition curve for the addition-recovery measurements. Conditions for LDS-UA-LLME: sample volume = 12 mL; sample pH = 10; NaCl concentration = 25%; acetone volume = 2.5 mL; vortex shaking time = 15 min; centrifugation time = 5 min and ultrasound-assisted time = 5 min. Others condition as in Table S1.

The combined electroanalytical approaches were proven successful when used for the simultaneous electro-oxidation of three FQs as ECs that were directly dropped on the CNF/SPE surface. Firstly, an accurate definition of voltammetric peaks could be achieved (i.e., separating three overlapping FQs peaks) using an improved mathematical processing via baseline-corrected second-order derivative approach and secondly, an adjustment in the sample volume using the LDS-UALLME protocol provided a high enrichment factor that enabled the quantitation of target analytes at trace levels. Both strategies reduced the background interferences and gave the best definition of the overlapping peaks, thus making the developed method useful, not only for the trace analysis of tap water samples, but also more complex samples such as wastewater effluents and groundwater.

SUPPORTING INFORMATION Additional information as noted in the text include chemical structures of FQs, Figures and Tables to provides further Information of the analytical performance and samples analysis as well as to describe the optimization of the LDS-UA-LLME method.



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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ; Fax: +55 67 3410-2072 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support provided by the FUNDECT/MS and Brazilian funding agency CNPq (contract: 23/200.258/2014 and 23/200.680/2012). L.H.O. is especially grateful for the scholarship awarded via CAPES program.

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< for TOC only >


Baseline-Corrected Second-Order Derivative Electroanalysis

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