Sensitive voltammetric determination of hydroxyzine and its main metabolite cetirizine and identification of oxidation products by nuclear magnetic resonance spectroscopy

Accepted Manuscript Sensitive voltammetric determination of hydroxyzine and its main metabolite cetirizine and identification of oxidation products by nuclear magnetic resonance spectroscopy

Bruna Claudia Lourencao, Tiago Almeida Silva, Maiara da Silva Santos, Antonio Gilberto Ferreira, Orlando Fatibello-Filho PII: DOI: Reference:

S1572-6657(17)30791-9 doi:10.1016/j.jelechem.2017.11.013 JEAC 3643

To appear in:

Journal of Electroanalytical Chemistry

Received date: Revised date: Accepted date:

7 August 2017 7 October 2017 5 November 2017

Please cite this article as: Bruna Claudia Lourencao, Tiago Almeida Silva, Maiara da Silva Santos, Antonio Gilberto Ferreira, Orlando Fatibello-Filho , Sensitive voltammetric determination of hydroxyzine and its main metabolite cetirizine and identification of oxidation products by nuclear magnetic resonance spectroscopy. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jeac(2017), doi:10.1016/j.jelechem.2017.11.013

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ACCEPTED MANUSCRIPT Sensitive voltammetric determination of hydroxyzine and its main metabolite cetirizine and identification of oxidation

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products by nuclear magnetic resonance spectroscopy

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Bruna Claudia Lourencao, Tiago Almeida Silva, Maiara da Silva Santos, Antonio

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Gilberto Ferreira, Orlando Fatibello-Filho*

Department of Chemistry, Federal University of São Carlos, Rod. Washington Luís km

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235, São Carlos, P. O. Box 676, CEP: 13560-970, SP, Brazil.

*Corresponding author

Tel.: +55 16 33518098; Fax: +55 16 33518350

E-mail address: [email protected] (O. Fatibello-Filho)

ACCEPTED MANUSCRIPT Abstract The electrochemical detection and electrooxidation path of the first-generation antihistamine receptor hydroxyzine (HDZ) and its main metabolite cetirizine (CTZ) are addressed in this research. A carbon black-modified electrode to explore the electrochemical responsivity and electroanalytical detection of HDZ and CTZ was

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designed. Compared to the bare electrode, the irreversible anodic responses observed

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for HDZ and CTZ were considerably improved on the proposed carbon black-modified

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electrode, including twenty times enhancement of the anodic peak currents and the shifting of anodic peak potentials to less positive potentials. In order to identify the

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electrooxidation products resulting from the previously verified irreversible redox processes, cyclic voltammetric studies and potentiostatic electrolysis assays followed by

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products identification by nuclear resonance magnetic (NMR) spectroscopy were carried out. From the combination of electrochemistry and NMR spectroscopy data it

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was possible to propose electrooxidation reaction mechanisms for HDZ and CTZ

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molecules. By applying square-wave adsorptive anodic stripping voltammetry (SWAdASV) under optimized experimental conditions, the obtained analytical curves

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for HDZ and CTZ were linear from 2.99  10–7 to 9.81  10–6 mol L−1 and from 4.97  10–7 to 1.08  10–5 mol L−1, with limits of detection of 1.00  10–7 mol L−1and 4.00 

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10–7 mol L−1, respectively. Finally, spiked synthetic human biological fluids were analysed by the proposed SWAdASV procedures, with recovery percentages close to 100%.

Keywords: modified electrodes; mechanism elucidation; voltammetry; NMR spectroscopy.

ACCEPTED MANUSCRIPT 1. Introduction Hydroxyzine

(HDZ),

chemically

named

as

2-[2-[4-[(4-chlorophenyl)-

phenylmethyl]piperazin-1-yl]ethoxy]ethanol, is a first generation antihistamine receptor (H1), it is also presenting bronchodilator activity and analgesic effects with mild antianxiety action being beneficial in the treatment of bronchial asthma and applied in

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some cases to relax patients before surgery [1, 2]. Different concentrations of HDZ have

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been found in both the cerebrospinal fluid (CSF) and plasma after intranasal and intra-

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arterially administration [3, 4]. In the human body, it is digested in the liver to its main metabolite (45%) cetirizine (CTZ, a carboxylic acid), being mainly excreted in the urine

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almost unmodified [5].

CTZ is an active metabolite of HDZ, chemically know as 2-[2-[4-[(4-

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chlorophenyl)-phenylmethyl]piperazin-1-yl]ethoxy]acetic acid. CTZ, belongs to the second generation of antihistamines, acting in the soothing of physical symptoms and

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some other allergic disorders [6]. It is available in the market as tablets, solutions (drops

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and syrup), and compounded capsules being excreted in urine mainly as the unchanged drug [7].

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Some medical studies carried out in rats and pregnant women have presented conflicting conclusions about HDZ as a teratogenic agent, however, no definitive

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conclusion about this is provided in literature. Indeed, studies in rabbits showed that CTZ is not associated with any teratogenicity, but conclusive data on the effects of CTZ in pregnant women have not yet been published until the present time [8]. There is a recent evidence demonstrating the association of first-generation antihistamines use and increased risk of dementia, thus, the prescription of second-generation antihistamines are preferred over first-generation agents in both pregnant and non-pregnant individuals [8, 9]. Due to the absence of definitive scientific reports about the risks or side effects of

ACCEPTED MANUSCRIPT HDZ as well as its main metabolite CTZ, it is important to highlight the relevance of the rigid analytical control of both molecules in human biological fluids where they are mainly found. Different analytical methods have been reported in literature for determination of both analytes in biological fluids (urine, plasma, serum and cerebrospinal fluid). Among the main analytical proposed methods is included the

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chromatography coupled with different detectors [10-13], among others [14-17].

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However, there are few electroanalytical methods reported for HDZ or CTZ

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determination in biological fluids and pharmaceutical formulations available in the literature [18-23]. Taking into account the remarkable features of electroanalytical tools,

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such as operational simplicity, low cost instrumentation, simplified sample preparation procedure, among others, the electroanalytical methods based on use of modified

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electrodes have been widely explored in the sensing of biologically and environmentally relevant molecules [24-31]. Thus, the establishment of a sensitive electrochemical

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sensor able to quantify HDZ or CTZ compounds at biological matrices samples has

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become a prominent research focus.

One of the most important issues in the design of a high performance analytical

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electrochemical sensor is the proper selection of the working electrode material. Probably, this is the most targeted issue in electroanalysis field in the last years. In this

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sense, carbon black (CB) is a very attractive carbon-based material from the electroanalytical point of view due to its physicochemical properties such as high surface area, conductivity, chemical stability and ability to produce stable dispersions without requiring sophisticated procedures [32]. Hence, the number of papers reported in literature using CB for the preparation of electrochemical sensors and biosensors have increased in recent years [33-37]. Thus, here we describe the use of CB as electrode modifier of the glassy carbon surface to achieve an improved performance in

ACCEPTED MANUSCRIPT terms of electrode stability, sensitivity, reproductivity and analytical response with respect to the bare glassy carbon electrode (GCE). The oxidation of HDZ and CTZ under chemical and biochemical conditions have been reported in literature and its kinetics of oxidation can be monitored by chromatographic and spectroscopy techniques for possible elucidation of its oxidation

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mechanism [38-40]. However, to the best of our knowledge there are no works

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reporting the oxidation mechanism path of HDZ and CTZ promoted by electrochemical

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methods. Actually, there are some attempts to propose mechanisms based on a few electrochemical experiments, but they were not confirmed by experimental data

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obtained through structural elucidation techniques.

Considering the described, in this study we report the electrochemical behaviour

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of HDZ and CTZ molecules and the development and optimization of simple, rapid and low-cost electroanalytical methods using a CB-based electrochemical sensor for

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determination of both compounds in biological fluid samples. Particularly, an

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outstanding additional contribution was obtained from the use of NMR spectroscopy technique for the proposal of the main oxidation products of HDZ and CTZ resulting

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from the respective redox processes that occur on modified surface electrode.

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2. Experimental

2.1. Reagents, solutions and samples Standards of HDZ and CTZ with purity degree higher than 99% were purchased from Calendula Pharmacy (São Carlos, Brazil). Carbon black (CB) VULCAN® VXC72R was kindly provided by Cabot Corporation. Reagents for preparation of supporting electrolyte solutions and artificial human body fluid samples and other

ACCEPTED MANUSCRIPT reagents were of analytical grade and purchased from Sigma-Aldrich (São Paulo, Brazil). For preparation of the aqueous solutions ultrapure water supplied by a Millipore Milli-Q system (Billerica, USA) with resistivity greater than 18.0 MΩ cm was used. Stock solutions of HDZ and CTZ at 1.0 × 10−2 mol L−1 were initially prepared in methanol with final dilution in the respective supporting electrolyte solutions.

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HDZ and its main metabolite CTZ were quantified in artificial biological fluids,

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which were cerebrospinal fluid and urine. For preparation of artificial cerebrospinal

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fluid the protocol reported by Toledo et al. [41], containing the following chemical composition: 2.1 g of NaCl, 0.44 g of NaHCO3, 75 mg of KCl, 82 mg of CaCl2, 23 mg

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of glucose, 0.44 g of NaHCO3, 20 mg of urea dissolved in 250 mL of ultrapure water, was adopted, and in the case of artificial urine the protocol proposed by Laube et al.

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[42], containing the following chemical composition: 0.73 g of NaCl, 0.40 g of KCl, 0.28 g of CaCl2.2H2O, 0.56 g of Na2SO4, 0.35 g of KH2PO4, 0.25 g of NH4Cl and 6.25 g

2.2. Instrumentation

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of urea dissolved in 250 mL of ultrapure water, was adopted .

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For GCE cleaning and preparation of CB dispersions was used an ultrasonic bath UltraCleaner 1400 A from Unique Ultrasonic Systems (Indaiatuba, Brazil). The pH

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measurements were performed using an Orion Expandable Ion Analyzer (model EA940, USA), equipped with a combined glass electrode with an Ag/AgCl (3.0 mol L1 KCl) external reference electrode. A μAutolab Type III (EcoChemie, Netherlands) potentiostat/galvanostat controlled by GPES 4.9 software was employed for the electrochemical measurements. The electrochemical measurements were conducted in a three-electrode configuration glass cell, being the working electrode, the modified

ACCEPTED MANUSCRIPT electrode (CB-GCE), the counter electrode a Pt foil and the reference electrode an Ag/AgCl (3.0 mol L1 KCl). All NMR measurements were carried out using a Bruker Avance III NMR spectrometer (14.1 Tesla / 600.23 MHz for ¹H) equipped with a TCI 5 mm triple resonance (1H/13C/15N) z-field gradient cryo-probe. The ¹H spectra were acquired using

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the presaturation pulse sequences and processed by applying a Fourier transform to

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65536 points and with a line broadening factor of 0.3 Hz. For the two-dimensional

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spectra, the data was processed by applying a Fourier transform with 4096 points in F2 (SIF2) and 1024 points in F1 (SIF1), using the sine squared apodization function

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(QSINE). The chemical shift scale of all spectra was referenced against the methyl signal of the TMSP-d4 (3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt)

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employing an external capillary. The values of ¹³C chemical shifts were obtained from

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HSCQ (Heteronuclear Single Quantum Coherence) experiments.

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2.3. Preparation of carbon black based sensor The carbon black based sensor was designed using as substrate a GCE (Ø = 3.0

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mm or Ø = 5.0 mm for electrolysis). Prior to modification process, the GCE surface was carefully polished to a mirror finish with 0.05 μm alumina slurries and rinsed

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thoroughly with ultrapure water. Following, it was sonicated in isopropyl alcohol for about 1 min. The steps taken for construction of the CB based sensor are outlined in Scheme 1. Initially, CB dispersion was prepared from the mixture of 1.0 mL of dimethylformamide (DMF) and 1.0 mg of CB. This mixture was subjected to sonication for 30 min, after which a stable black dispersion of CB was obtained. DMF was selected for CB dispersion preparation because homogeneous and very-well adhered films on GCE have been obtained from the casting of carbon nanomaterials dispersed in this

ACCEPTED MANUSCRIPT solvent [32]. Next, 6 μL of CB dispersion was then cast on the GCE surface and the solvent evaporated at room temperature overnight to finally obtain the modified electrode, which was simply referred to as CB-GCE. The modified CB-GCE was then applied in the development of voltammetric procedures, described in the next section.

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2.4. Analytical procedure

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Initially, the voltammetric response of HDZ and CTZ molecules was accessed

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by cyclic voltammetry. In this study, the cyclic voltammetric assays were carried out using the bare GCE and modified CB-GCE, in order to verify the improvements from

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the GCE modification with nanostructured carbon black. Following, the experimental conditions for HDZ and CTZ determination were systematically optimized, including

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analysis of the supporting electrolyte, pre-concentration potential (Epc) and time (tpc), and technical parameters of the square-wave adsorptive anodic stripping voltammetry

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(SWAdASV) technique, i.e., frequency (f), amplitude (a) and, potential increment

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(ΔEs). Under the optimized experimental conditions, the analytical curves for HDZ and CTZ were individually constructed from the addition of small aliquots of HDZ or CTZ

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stock solutions in the electrochemical cell containing 10 mL of supporting electrolyte. From the anodic peak currents (Ip) recorded at each analyte concentration level, the

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analytical curves were constructed (Ip vs. [concentration]). From these, the analytical parameters for each analyte were obtained, being these: correlation coefficient, linear concentration range and analytical sensitivity. The limits of detection (LOD) were experimentally determined using the 3 × (signal/noise) relationship [43]. The precision of the proposed sensor was evaluated from intra- and inter-day repeatability studies. Finally, artificial biologic fluid samples were spiked with a known concentration of

ACCEPTED MANUSCRIPT HDZ or CTZ and directly analysed by the proposed voltammetric procedure. The results of sample analysis were reported as recovery percentages.

3. Results and Discussion

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3.1. Morphological characterization of the CB film

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From images of scanning electron microscopy (SEM) recorded for GCE

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modified with CB in a previously published work by our research group, it was possible to notice that the deposited material consisted of multiple grains/nanoparticles of

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amorphous carbon with sizes of ca. 25 nm (Please, consult Fig. 3 of Ref. [32]). Moreover, in terms of electrochemical performance, the porous surface morphology

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obtained from the GCE surface modification with CB film provided a larger electroactive surface area and faster heterogeneous electron transfer. For more detailed

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additional information about the morphological, structural and electrochemical

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characterization of different types of CB nanomaterial we suggest that the reader

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consults our recent previous publications [32, 37, 44].

3.2. Electrochemical behaviour of HDZ and CTZ

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Cyclic voltammograms recorded using GCE and CB-GCE in the absence and presence of HDZ or CTZ at pH 4.0 are shown in Figs. 1 (a) and (b), respectively. On the modified CB-GCE a very sharp oxidation process was verified during the anodic potential scanning in both cases, located at +0.97 V (HDZ) and +1.07 V (CTZ) vs. Ag/AgCl (3.0 mol L−1 KCl), respectively. After the potential scanning direction inversion, non-equivalent reduction process was observed, demonstrating the irreversible behaviour of the HDZ and CTZ oxidation processes. On the other hand,

ACCEPTED MANUSCRIPT using the bare GCE, only a small variation of the anodic current at around +1.2 V (HDZ) and +1.3 V (CTZ) vs. Ag/AgCl (3.0 mol L−1 KCl) was verified in the presence of the analytes, without the occurrence of a well-defined anodic peak. Thus, the incorporation of carbon black on the GCE surface improved significantly the voltammetric response of HDZ and CTZ, with an increment of approximately twenty

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times in the anodic peak current for HDZ and CTZ. Moreover, the anodic peak

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potentials were shifted to less negative potentials in nearly 200 mV, showing the

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apparent “electrocatalytic activity” of the nanostructured carbon black film. As demonstrated in the further sections, the electrochemical responses of HDZ and CTZ

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were adsorption-controlled processes. In this way, part of the electrocatalytic effect verified on the CB-based electrode can be credited to the thin layer effects originated

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from the porous structure of the CB film, as well discussed by Compton and collaborators using porous electrodes based on carbon nanotubes [45-47]. This

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remarkable enhancement of the GCE electrochemical performance associated with the

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very low cost of carbon black, places this material as a carbon nanomaterial ideal for the

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development of electrochemical sensors and biosensors.

3.3. Effect of pH

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The influence of phosphate buffer solution pH used as supporting electrolyte was evaluated in the pH range from 2.0 to 10.3. The cyclic voltammograms recorded for HDZ and CTZ through different pHs are shown in Fig. 2. As can be seen, in both cases, the peak potentials (Ep) shifted to less positive values with the increase of pH, showing that protons are involved in the respective oxidation reactions. Besides that, it is also possible to note from Figs. 2 (a) and (b) that for pH higher than 4.0, both molecules presented two anodic peaks, which became more separated as the pH value increased.

ACCEPTED MANUSCRIPT Considering the variation of the peak potential for the first peak (insets (i) of Figs 2. (a) and (b)), in the case of HDZ, two linear relationships were obtained, one in the pH range from 2.0 to 7.0 and another one in the interval from 7.0 to 10.3, in accordance with the linear regression equations (1) and (2). On the other hand, in the case of CTZ three linear relationships were observed, in the pH ranges from 2.0 to 4.0, from 4.0 to

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8.0 and finally from 8.0 to 10.3, as represented by the linear regression equations (3),

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(4) and (5), respectively.

HDZ:

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(pH range: 2.0 - 7.0): Ep (V) = 1.188 V – 0.055 (V pH−1) pH (r = 0.999) (1)

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(pH range: 7.0 - 10.3): Ep (V) = 0.816 V – 0.004 (V pH−1) pH (r = 0.913)

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(2)

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CTZ:

(pH range: 2.0 - 4.0): Ep (V) = 1.230 V – 0.039 pH (V pH−1) (r = 0.998)

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(3)

(4)

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(pH range: 4.0 - 8.0): Ep (V) = 1.309 V – 0.060 pH (V pH−1) (r = 0.996)

(pH range: 8.0 - 10.3): Ep (V) = 0.977 V – 0.017 pH (V pH−1) (r = 0.966) (5)

Electrochemical processes involving weak acids or bases typically have variations of potential versus pH showing inflection points where pH = pKa ou pKb. Then, using previously found data and the intersections of the Ep versus pH curves, the

ACCEPTED MANUSCRIPT respective conditional pKa values (pKa*) could be estimated for both molecules. According to literature, for HDZ the related pKa* values are pKa,1* = 3.00 (attributed to the nitrogen of amine nearest to the aromatic rings) and pKa,2* = 7.00 (amine) (in 0.04 mol L−1 BR buffer solution) and for CTZ pKa,1* = 2.24 (attributed to the nitrogen of amine nearest to the aromatic rings), pKa,2* = 3.47 (carboxylic acid group) and pKa,3* =

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7.90 (amine) (using ionic strength = 0.05 mol L−1) [19, 48]. The estimated pKa* values

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in the employed experimental conditions were pKa,2* = 7.29 for HDZ and pKa,2* = 3.95

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and pKa,3* = 7.75 for CTZ, respectively. For both molecules, the pKa,1* could not be estimated, since these values are close to the first studied pH (2.0). The obtained

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experimental values of pKa* were very close to those values previously reported in literature for both molecules. Moreover, from the linear equations (1), (3) and (4) it is

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clear that slopes close to the theoretical Nernst slope of –0.0592 V pH−1 were observed for HDZ and CTZ, which suggested that the same number of protons and electrons are

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involved in the respective electrooxidation reactions.

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Analysing the insets (ii) of Figs. 2 (a) and (b), it is also possible to observe that the magnitude of the peak current varied with variations of pH value for both cases.

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Besides, for HDZ the maximum current value was reached at pH = 6.0 and for CTZ at pH = 3.0 and 4.0. Considering all the obtained results, in both cases, the supporting

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electrolyte solution chosen for further studies was the 0.2 mol L−1 phosphate buffer solution at pH = 4.0, once only one and well-defined oxidation peak was obtained.

3.4. Effect of pre-concentration potential and time The effect of pre-concentration potential on the oxidation peak currents was evaluated in the range from −0.5 V to 0.7 V, with a constant pre-concentration time of 120 s. In both cases, the oxidation peak current decreased from the application of pre-

ACCEPTED MANUSCRIPT concentration potential more positive than −0.3 V, and remained practically constant in more negative values (data not shown). In our recent publication [36], the potential of zero charge (PZC), which plays an important role in the extension of adsorption onto the electrode surface, has been determined from electrochemical impedance spectroscopy (EIS) measurements for the CB-GCE electrode. From this strategy, the

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PZC of CB-GCE was determined as being −0.3 V. Looking at the pKa values of HDZ

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and CTZ provided in the previous section, it is possible to conclude that at pH 4.0 the

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most part of HDZ and CTZ molecules are in their respective neutral forms (zero charge). It is known that adsorption of neutral species is more effective at potentials

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relatively near the PZC [49]. This effect happens because the adsorption of a neutral molecule requires the displacement of water molecules from the surface, and when the

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interface is polarized, the water is tightly bound and its displacement by a less dipolar substance is energetically unfavourable [49]. Based on this explanation, it is very clear

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why the pre-concentration potential of −0.3 V (PZC) provided the highest peak current

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signals.

In addition, the effect of pre-concentration time was also evaluated in the range

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from 30 to 180 s for HDZ and CTZ. The anodic peak currents increased up to 120 s, and for longer times remained practically constant, probably due to the working electrode

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saturation by the analyte molecules (data not shown). Thus, a pre-concentration time of 120 s was selected for further studies.

3.5. HDZ and CTZ electrooxidation reactions: cyclic voltammetric studies, potentiostatic electrolysis and, products identification by NMR spectroscopy

3.5.1. Effect of scan rate

ACCEPTED MANUSCRIPT The effect of scan rate on the electrochemical response of both analytes at the CBGCE was carried out by cyclic voltammetry in the potential scan rates range of 10 mV s−1 to 400 mV s −1. From the cyclic voltammograms of Figs. 3 (a) and (b), it is possible to note that for both analytes there was a shift in the anodic peak potentials with increasing scan rate potentials. In the case of HDZ, a linear relationship (r = 0.995) was

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obtained between the logarithm of the oxidation peak current (log Ip) and the logarithm

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of the scan rate (log v), with a slope of 0.70 (inset (i) of Fig. 3 (a)). This value indicates

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that the HDZ oxidation process on the CB-GCE surface could be controlled by both processes, diffusion or adsorption of the species, since the obtained slope value was

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between 0.5 (diffusion-controlled process) and 1.0 (adsorption-controlled process). Thus, the linearity between the anodic peak current (Ip) and the scan rate potential (v)

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and between the anodic peak current (Ip) and the square root of the scan rate (v1/2) was also evaluated. A linear dependence of Ip versus v was obtained (inset (ii) of Fig 3 (a)

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and linear regression equation (6)), indicating that the electrooxidation of HDZ was

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governed by an adsorption process. Similar conclusions were obtained in the CTZ case, with a slope for the linear log Ip versus log v plot (r = 0.999) of 0.83 (inset (i) of Fig. 3

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(b)) and a linear relationship between Ip and v (inset (ii) of Fig. 3 (b)) according to the linear regression equation (7), therefore, showing that the electrooxidation of CTZ was

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an adsorption-controlled process. Other authors have observed the same behaviour for HDZ and CTZ in previously published studies [19, 23].

HDZ: Ip (A) = 2.26 × 10−5 A + 5.72 × 10−4 (A V−1 s) v (V s−1) (r = 0.997)

(6)

CTZ: Ip (A) = 1.15 × 10−5 A + 6.55 × 10−4 (A V−1 s) v (V s−1) (r = 0.998)

(7)

3.5.2. Potentiostatic electrolysis and NMR spectroscopy investigations

ACCEPTED MANUSCRIPT To investigate a possible mechanism for HDZ and CTZ oxidations on the CBGCE electrode, NMR spectroscopy was used to identify the HDZ and CTZ oxidation products (HDZox and CTZox) and the following strategy was carried out. The NMR measurements were performed ex situ, because the magnetoeletrolysis effect present in in situ measurements could interfere in the kinetics and/or mechanism of oxidation

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reaction [50]. Firstly, a 1.0 × 10−3 mol L−1 HDZ solution was subjected to potentiostatic

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electrolysis by the chronoamperometric application of an electrode potential of +1.4 V for 5 h; then, after this time the final solution was freeze-dried for 24h and the resulting

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residue was dissolved in 600 L of D2O. The ¹H NMR spectrum of this resulting

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solution was compared to the ¹H NMR spectrum of HDZ in the buffer solution, before the potentiostatic electrolysis experiment. Both spectra were very similar, which

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evidenced that no expressive oxidation of HDZ occurred (see Fig. 4). An evidence for this may be observed from the charge (Q = 1.1 C) obtained in the electrolysis,

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suggesting that the adsorption of HDZ oxidation products lead to an electrode fouling.

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Despite the low expressive oxidation of HDZ it was possible to observe some signals of minor compounds and from them suggest two possible oxidation products

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(HDZox1 and HDZox2). The singlets at 6.00 and 7.85 ppm (H-a and H-b, respectively) shown in the Fig. 4 (b) indicate the bond breakage between N and C-g in the HDZ

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molecule and formation of the oxidation products illustrated in the Fig. 4. This assumption is based in the ¹H and ¹³C chemical shifts, supported by the correlations observed in the 2D experiments, which are shown in the supplementary material (Fig. S1 (a)). The HDZox1 has been previously reported in literature [39] as a degradation product of HDZ and in this study, it was evidenced by the chemical shifts of H-a and Ca. Additionally, in the correlation map obtained from HMCB (Heteronuclear Multiple

ACCEPTED MANUSCRIPT Bond Correlation) experiment, correlations between the H-a and the carbons atoms of aromatic rings were observed, but there were no correlations with any carbon atoms of the nitrogen ring. For HDZox2, similar information was obtained from the NMR experiments. The deshielding chemical shifts of H-b and C-b indicate that a double bond was formed between C-b and N and, the lack of correlations between H-b with the

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nearest carbon atom of aromatic rings, in addition to the correlation between the H-b

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and carbon atom of the nitrogen ring in the HMBC experiment corroborate with our

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assumptions.

According to literature [19], the oxidation reaction of HDZ occurs in the alcohol

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function of the aliphatic chain, transforming it into an aldehyde. However, in the present work the formation of this supposed product was not observed. If this compound had

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been formed, some characteristic NMR signal would be observed, for example singlets around 4.50 and 9.00 ppm in the ¹H NMR spectrum, corresponding to hydrogen atoms

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of alpha-methylene group and hydrogen of aldehyde functional group, respectively;

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and/or some long-range heteronuclear correlation with the carbonyl carbon around 200 ppm.

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Similarly, the potentiostatic electrolysis of CTZ was evaluated, but applying an electrode potential of +1.3 V, and the NMR measurements were carried out also in the

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same conditions. The ¹H NMR spectra of CTZ before and after the potentiostatic electrolysis experiment were compared and just a slight ¹H chemical shifts variation of major signals was observed, further, both spectra have similar profiles (Fig. S2). This observation suggests that the majority of the solution compound obtained after electrolysis was CTZ, evidencing that the oxidation reaction was inefficient (charge of 0.98 C), similarly to the outcomes obtained for HDZ, possibly for the same reason. CTZ chemical shifts are more sensible to slight changes of pH, concentrations and/or

ACCEPTED MANUSCRIPT temperature than HDZ, since at pH = 4 CTZ is in its zwitterionic form, while the neutral HDZ form has no electric charges at this pH [51]. The formation of a CTZ dimer by electro polymerization is described in the literature as a suggestion of CTZ oxidation product [22], nevertheless the signal correspondent to the hydrogen atom that should be eliminated in the polymerization (

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= 4.71 ppm) was observed in the ¹H NMR spectrum. This hydrogen atom is bonded

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exactly to the carbon atom that would be involved in the suggested reaction and show

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long-range correlations with other carbons atoms of CTZ.

In the ¹H NMR spectrum of CTZ after electrolysis, a great number of small

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signals evidencing the formation of minor oxidation products, was also noted. Among these signals, the singlets at 5.84 and 7.71 and their correlations in the HSQC and

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HMBC experiments, suggested the same bond breakage described for HDZ (see Fig. S1 (b)). The oxidation products (CTZox1 and CTZox2, highlighting that CTZox1 = HDZox1)

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are shown in the Fig. S2. Finally, considering the results obtained by ¹H NMR

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measurements, a mechanism for HDZ and CTZ oxidation is proposed (Fig. 5), in which two protons and two electrons are involved. This result is in agreement with

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electroanalytical data.

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3.6. Optimization of SWAdASV parameters and analytical curve The SWAdASV parameters, square-wave frequency (f), pulse amplitude (a), and scan increment (ΔEs) were optimized for 5.0 × 10−6 mol L−1 HDZ or CTZ solution in 0.2 mol L−1 phosphate buffer (pH 4.0). The studied ranges and selected values are shown in Table S1. The values were selected taking into account baseline stability, repeatability and magnitude of analytical signal onto the CB-GCE for HDZ and CTZ determination.

ACCEPTED MANUSCRIPT Under optimized experimental conditions, SWAdAS voltammograms were obtained after successive additions of HDZ or CTZ stock solutions in the electrochemical cell containing previously supporting electrolyte solution and the respective current signal was measured using the CB-CGE electrode for the different HDZ or CTZ concentrations. In Fig. 6 (a) are provided the SWAdAS voltammograms

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obtained for HDZ molecule in the concentration range of 2.99 × 10−7 to 9.81 × 10−6 mol

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L−1 and, similarly, the SWAdAS voltammograms obtained for CTZ in the concentration

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range of 4.97 × 10−7 to 1.08 × 10−5 mol L−1 are shown in Fig. 6 (b). The respective analytical curves were then constructed for both analytes (see insets of Figs. 6 (a) and

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(b)) and their calibration equations were:

(8)

CTZ: Ip (µA) = −0.731 µA + 7.31 (µA mol–1 L) [CTZ] (mol L–1) (r = 0.999)

(9)

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HDZ: Ip (µA) = −1.97 µA + 5.74 (µA mol–1 L) [HDZ] (mol L–1) (r = 0.999)

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The LOD were calculated by the 3 × (signal/noise) relationship (as described in Section 2.4), and the following values were determined, respectively: 1.0 × 10−7 mol L–1

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for HDZ and 4.0 × 10−7 mol L−1 for CTZ. According to literature, previous works were reported on the individual electroanalytical determination of HDZ or CTZ as well as the

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simultaneous determination of CTZ and other analytes of interest. Table 1 comparatively lists the analytical parameters obtained from these works with those attained in the present work using SWAdASV and the CB-GCE as electrochemical sensor. In the case of HDZ, improved analytical parameters were reported in two works, such as linear range and LOD. However, in both cases, the authors described problems with adsorption onto the electrode surface and thus, an electrode-cleaning step for regeneration of the electrode surface after recording each voltammogram was necessary.

ACCEPTED MANUSCRIPT In the case of CTZ the linear range was similar or higher than the obtained in previous works and the LOD was also similar or improved. The proposed voltammetric method measurement precision was evaluated from intra- and inter-day repeatability tests. Basically, these tests consisted in monitoring the stability of the anodic peak current signals recorded for HDZ and CTZ on the proposed

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CB-GCE through successive SWAdASV measurements performed in the same day (n =

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10, intra-day repeatability) or different days (n = 3, inter-day repeatability). For intra-

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day repeatability study, it was used the same electrode and, for intra-day repeatability study it was employed fresh modified electrodes prepared in different days. In both

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cases, the repeatabilities were tested at two different HDZ and CTZ concentration levels. The relative standard deviations (RSD) calculated for the registered analytical

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signals are organized in Table 2. From the results shown in the Table 2, it was possible to conclude that the developed voltammetric methods presented excellent measurement

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precision, with RSD values lower than 10%. Moreover, the proposed sensor does not

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require sophisticated procedures to be prepared, and it provides reproducible analytical signals without appreciated adsorption surface or any fouling by products from the

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respective redox reactions.

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3.7. Analysis of HDZ and CTZ in human biological fluid samples The real applicability of the optimized and developed novel voltammetric procedures was investigated from the determination of HDZ and CTZ species in spiked artificial cerebrospinal fluid and urine samples. These samples were directly analysed by the proposed procedures, and the experimental recovered concentrations were determined by the interpolation of the analytical signals in the respective analytical curves (equations (8) and (9)). The predicted recovery percentage values are listed in

ACCEPTED MANUSCRIPT Table 3. As can be seen, for the analysis of HDZ spiked samples, the recovery percentages ranged from 99 to 101% while in the case of CTZ spiked samples ranged from 96 to 108%. This set of recovery percentages proved the accuracy of the proposed voltammetric procedures based on the use of the CB-based sensor.

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4. Conclusions

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We reported that the incorporation of a CB film on the GCE surface produced a

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modified electrode with remarkable “electrocatalytic activity” toward the oxidations of HDZ and CTZ. Exploring the designed CB-GCE, the electrochemical behaviour of

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HDZ and CTZ compounds was explored via different cyclic voltammetric studies and potentiostatic electrolysis assays followed by products identification by NMR

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spectroscopy. From this set of electrochemistry and NMR spectroscopy results, the possible electrooxidation mechanisms for HDZ and CTZ were proposed. SWAdASV

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methods were optimized and developed using the proposed modified electrode, and

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linear ranges and LODs at micromolar levels were achieved in both cases. The applicability of the developed SWAdASV has been proved from the analysis of

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synthetic urine and human serum fluids.

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Acknowledgments

The financial support from the following Brazilian funding agencies is gratefully acknowledged: CNPq (process no. 444150/2014-5), CAPES, and São Paulo Research Foundation – FAPESP (process no. 2014/03019-7).

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ACCEPTED MANUSCRIPT Scheme Captions

Scheme 1. Steps for preparation of the carbon black (CB) based sensor.

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Figure Captions

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Fig. 1. Cyclic voltammograms recorded in a 0.2 mol L−1 phosphate buffer solution (pH

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= 4.0) containing (a) 5.0 × 10−5 mol L−1 HDZ or (b) 5.0 × 10−5 mol L−1 CTZ using a GCE and the proposed CB-GCE. Scan rate = 50 mV s −1. Pre-concentration conditions:

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Epc = −0.3 V and tpc = 120 s.

Fig. 2. Cyclic voltammograms for 0.2 mol L−1 phosphate buffer solutions at pH ranging

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from 2.0 to 10.3 containing (a) 5.0 × 10−5 mol L−1 HDZ or (b) 5.0 × 10−5 mol L−1 CTZ

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using the proposed CB-GCE. Scan rate = 50 mV s −1. Pre-concentration conditions: Epc = −0.3 V and tpc = 120 s. Insets: (i) Plots of Ep versus pH and (ii) Plots of Ip versus pH.

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Fig. 3. Cyclic voltammograms for 0.2 mol L−1 phosphate buffer solution (pH = 4.0)

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containing (a) 5.0 × 10−5 mol L−1 HDZ or (b) 5.0 × 10−5 mol L−1 CTZ using the proposed CB-GCE at different scan rates (1: 10 mV s

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to 13: 400 mV s

−1

). Pre-

concentration conditions: Epc = −0.3 V and tpc = 120 s. Insets: (i) Plots of log Ip versus log v and (ii) Plots of Ip versus v.

Fig. 4. ¹H NMR spectra of: (a) HDZ in buffer solution before the electrolysis with assignments of the HDZ signals; (b) solution obtained after electrolysis with expansion of region where the oxidation products signals appear and assignments of them.

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Fig. 5. Possible electrooxidation reactions proposed for (a) HDZ and (b) CTZ.

Fig. 6. (a) SWAdAS voltammograms recorded for 0.2 mol L−1 phosphate buffer solution (pH = 4.0) containing different concentration of HDZ: (1) 0.0 (blank solution);

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(2) 2.99 × 10−7; (3) 4.98 × 10−7; (4) 6.95 × 10−7; (5) 9.90 × 10−7; (6) 2.96 × 10−6; (7) 4.93 × 10−6; (8) 6.89 × 10−6 and (9) 9.81 × 10−6 mol L−1. Experimental parameters: f =

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20 Hz, a = 90 mV and ΔEs = 10 mV. Pre-concentration conditions: Epc = −0.3 V and tpc

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= 120 s. (b) SWAdAS voltammograms recorded for 0.2 mol L−1 phosphate buffer solution (pH = 4.0) containing different concentration of CTZ: (1) 0.0 (blank solution);

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(2) 4.97 × 10−7; (3) 6.95 × 10−7; (4) 9.90 × 10−7; (5) 2.97 × 10−6; (6) 4.93 × 10−6; (7)

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6.89 × 10−6; and (8) 1.08 × 10−5 mol L−1. Experimental parameters: f = 30 Hz, a = 80

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mV and ΔEs = 10 mV. Pre-concentration conditions: Epc = −0.3 V and tpc = 120 s.

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Table 1. Comparison of the analytical parameters obtained in the electroanalytical determination of HDZ and CTZ using different techniques and electrode materials Electrode Technique Analytical parameter Reference −1 Linear range (mol L ) LOD (mol L−1) HDZ CTZ HDZ CTZ BDD SWV 5.0  10−7– 2.0 --[21] 4.3  --–7 −5 10  10 a −7 MWCNTAdsDPV ----5.86 [22] 1.9  10 – −4 PtNPs  1.93  10 10–8 MWNTs/GC CVb --[20] 5.0  10–8 – 5.0  --–9 –5 10 2.5  10 –5 GCE DPV ----- 4.5  [18] 2.0  10 – –6 –4 10 1.0  10 MWCNT/GC CV ----7.07 [23] 5.0  10–7 – –5  1.0  10 10–8 GCE SWAdASc --[19] 5.0  10–8 – 1.5  --–8 –6 10 4.0  10 CB-GCE SWAdAS 2.99  10–7 – 4.97  10–7 – 1.00 4.00 This work  10–  9.81  10–6 1.08  10–5 7 10–7

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BDD: boron-doped diamond; MWCNT-PtNPs: carbon paste electrode modified with multiwalled carbon nanotube-platinum nanoparticles; MWNTs/GC and MWCNT/GC: multi-walled carbon nanotubesmodified glassy carbon electrode; aDPV used after 90 s of potentiostatic accumulation of CTZ; bCV used after 180 s of potentiostatic accumulation of HDZ at open circuit under stirring, the authors reported problems with adsorption onto the electrode surface; cSWV used after 180 s of potentiostatic accumulation of HDZ, after recording each voltammogram the CGE was cleaned for re-generation of the electrode surface.

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Table 2. Results obtained for the intra- and inter-day repeatability studies Analyte

Intra-day repeatability

Inter-day repeatability

(RSD %)*

(RSD %)**

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9.78

4.71

5.66

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HDZ (µmol L−1)

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5.55 1.97

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Table 3. Results obtained in the determination of HDZ or CTZ in spiked artificial cerebrospinal fluid and urine samples by the here-proposed voltammetric methods using a CB-GCE sensor. Concentration (µmol L−1)

HDZ

Found*

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1.00 ± 0.05

4.9

4.86 ± 0.07

fluid Urine

CTZ

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Cerebrospinal

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Recovery (%)**

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0.99

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5.3 ± 0.3

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Graphical abstract

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Carbon black (CB) material was used to develop the proposed modified electrode (ME)

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Hydroxyzine (HDZ) and cetirizine (CET) were determined by voltammetry with

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a ME Similar or lower LODs were attained in comparison with all electroanalytical

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The HDZ and CET electrooxidation products were identified by NMR

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spectroscopy.

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methods