Empleo De Nuevos (Nano)Materiales Para El Desarrollo De Metodologías Analíticas En Los Campos Alimentarios, Farmacéuticos Y Bioanalíticos

EMPLEO DE NUEVOS (NANO)MATERIALES PARA EL DESARROLLO DE METODOLOGÍAS ANALÍTICAS EN LOS CAMPOS ALIMENTARIOS, FARMACÉUTICOS Y BIOANALÍTICOS

USE OF NEW (NANO)MATERIALS FOR THE DEVELOPMENT OF ANALYTICAL METHODOLOGIES IN FOOD, PHARMACEUTICAL AND BIOANALYTICAL FIELDS

Khaled Ali Murtada

Departament of Analytical Chemistry and Food Technology

Faculty of Sciences and Chemistry Technology

University of Castilla-La Mancha

Ciudad Real, 2019

EMPLEO DE NUEVOS (NANO)MATERIALES PARA EL DESARROLLO DE METODOLOGÍAS ANALÍTICAS EN LOS CAMPOS ALIMENTARIOS, FARMACÉUTICOS Y BIOANALÍTICOS

USE OF NEW (NANO)MATERIALS FOR THE DEVELOPMENT OF ANALYTICAL METHODOLOGIES IN FOOD, PHARMACEUTICAL AND BIOANALYTICAL FIELDS

Supervised by

Signed. Dr. Ángel Ríos Castro Signed. Dr. Mohammed Zougagh Zariouh

Full professor Associate professor

Department of Analytical Chemistry and Department of Analytical Chemistry and Food Technology, Faculty of Chemical Food Technology, Faculty of Pharmacy Science and Technology

University of Castilla-La Mancha, Spain University of Castilla-La Mancha, Spain

Thesis submitted to obtain the degree of International Doctorate in Chemistry

Signed. Khaled Ali Murtada

Master´s Degree in Chemistry

Department of Analytical Chemistry and Food Technology

Dr. Gregorio Castañeda Peñalvo, Associate Professor and Secretary of the Department of Analytical Chemistry and Food Technology, University of Castilla-La Mancha.

CERTIFY THAT:

This research work entitled “USE OF NEW (NANO)MATERIALS FOR THE DEVELOPMENT OF ANALYTICAL METHODOLOGIES IN FOOD, PHARMACEUTICAL AND BIOANALYTICAL FIELDS” constitutes the Doctoral Thesis presented by Mr. Khaled Ali Murtada to obtain the degree of International Doctorate in Chemistry. The Thesis has been developed at the Department of Analytical Chemistry and Food Technology, fulfilling all the academic requirements, under the supervision of Prof. Ángel Ríos Castro and Dr. Mohammed Zougagh Zariouh.

And for the record, I hereby issue, and sign this present certificate in Ciudad Real on 26 March, 2019.

Signed and approved by:

Rosa C. Rodríguez Martín-Doimeadiós Gregorio Castañeda Peñalvo

Head of the Department Secretary of the Department

Mención Doctorado Internacional

Programa de Doctorado en Química por la UCLM, verificado por R.D. 99/2011.

Mediante la defensa de esta Memoria de Tesis Doctoral se pretende optar a la obtención de la Mención de “Doctorado Internacional”, habida cuenta de que se reúnen los requisitos que establece el R.D. 99/2011, de 28 de Enero de 2011:

1. Cuenta con los informes favorables de dos doctores pertenecientes a instituciones de Enseñanza Superior de países distintos a España. 2. Uno de los miembros del tribunal que ha de evaluar la Tesis pertenece a un centro de Enseñanza Superior de otro país distinto a España. 3. Parte de la Tesis Doctoral se ha redactado y presentado en una lengua habitual para la comunicación científica de su campo de conocimiento, distinta a cualquiera de las lenguas oficiales de España. 4. El doctorando ha realizado una estancia de tres meses en la Escuela Nacional de Ciencias Aplicadas de la Universidad IBN ZOHR de Agadir (Marruecos), que ha contribuido a su formación y ha permitido desarrollar parte del trabajo experimental de esta Memoria.

Index / Índice

Index / Índice ...... IX Dedication / Dedicación ...... I Acknowledgements / Agradecimientos ...... II Abstract / Resumen ...... III Abbreviations / Acrónimos...... V Aim / Objetivo ...... 1 1. INTRODUCTION ...... 3 1.1. Analytical Chemistry and Analytical Science ...... 4 1.2. New (nano)materials as analytical tools in non-chromatographic separation techniques: treatment/separation of analytes in food, pharmaceutical and bioanalytical samples ...... 10 1.2.1. Types of new (nano)materials used in separation by extraction ...... 11 1.2.2. Types of extraction techniques involving (nano)materials ...... 21 1.3. New nanomaterials as tools for analytical detection of residues in food, pharmaceutical and bioanalytical samples ...... 26 References ...... 32 2. MATERIALS, EQUIPMENT AND METHODS ...... 41 2.1. Standards, reagents and samples ...... 42 2.2. Equipment ...... 47 2.3. Methods ...... 56 2.3.1. Synthesis, decoration and functionalization of nanoparticles ...... 56 2.3.2. General Procedures used in the extraction/separation of analytes...... 61 References ...... 66 3. RESULTS AND DISCUSSION ...... 67 3.1. Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation ...... 68 3.1.1. Strategies for Antidepressants Extraction from Biological Species Using Nanotechnology: A Review ...... 70 3.1.2. Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites and separation by capillary electrophoresis ...... 117 3.1.3. Magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites for the extraction and LC-MS determination of catecholamines and related compounds in red deer urine and hair ...... 142

Index / Índice

3.1.4. A simple poly(styrene-co-divinylbenzene)-coated glass blood spot method for monitoring of seven antidepressants using capillary liquid chromatography-mass spectrometry ...... 166 3.2. Nanoparticles in electrochemical sensors for food and pharmaceutical samples monitoring ...... 188

3.2.1. Development of an Aluminium Doped TiO2 Nanoparticles-modified Screen Printed Carbon Electrode for Electrochemical Sensing of Vanillin in Food Samples ...... 191 3.2.2. A Sensitive Electrochemical Sensor Based on Aluminium Doped Copper Selenide Nanoparticles-modified Screen Printed Carbon Electrode for Determination of L-tyrosine in Pharmaceutical Samples ...... 209 3.2.3. Decoration of Graphene oxide with Copper Selenide in Supercritical Carbon Dioxide Medium as a Novel Approach for Electrochemical Sensing of Eugenol in Various Samples ...... 230 4. Conclusions / Conclusiones ...... 253 5. Scientific Self-assessment / Autoevaluación científica ...... 257 Annexes / Anexos ...... 261 Annexe I. Figure captions / Índice de figuras ...... 262 Annexe II. Table captions / Índice de tablas ...... 269 Annexe III. Publications / Publicaciones ...... 272 Annexe IV. Participation of conference papers (oral communications and posters) / Participación en ponencias (comunicaciones orales y pósters)...... 274 Annexe V. Curriculum Vitae ...... 276

Dedication / Dedicación I

Dedication / Dedicación

This thesis is dedicated to:

The sake of Allah, my Creator and my Master,

My great teacher and messenger, Mohammed (May Allah bless and grant him), who taught us the purpose of life,

My homeland Palestine, the warmest womb;

The great martyrs and prisoners, the symbol of sacrifice;

My great parents, who never stop giving of themselves in countless ways,

My beloved brothers; who stands by me when things look bleak,

To all my family, the symbol of love and giving,

My friends who encourage and support me,

All the people in my life who touch my heart,

I dedicate this research.

Khaled Ali Murtada Ciudad Real, 2019

Acknowledgements / Agradecimientos II

Acknowledgements / Agradecimientos

Firstly, I would like to express my sincere gratitude to my advisors Prof. Ángel Ríos and Dr. Mohammed Zougagh for the continuous support of my Ph.D. study and related research, for their patience, motivation, and immense knowledge. Their guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisors and mentors for my Ph.D. study.

I want to express my deep gratitude to Virginia Moreno and her family. This thank you is long overdue. There are so many things I want to thank you for, and I’m sure I’m going to still be missing some by the end of this letter. Thank you for being you. You’re very own unique and incredible definition of beauty, and I know I’m one of many who see it.

I would like to thank my friends Carlos Adelantado and Fernando de Andrés, I’ll never forget your willingness to help me, to encourage me, to believe in my ability to achieve my dreams.

My colleagues at the laboratory, have all extended their support in a very special way, and I gained a lot from them, through their personal and scholarly interactions, their suggestions at various points of my research program.

"The Power of Love"

When I think of my family, I think of Love. I love all of you very much!

Last but not the least, I would like to thank my family for supporting me spiritually throughout writing this thesis and my life in general. My parents for love and inspiration throughout my life, my brothers for the excellent example their sets, for daring to follow their dreams.

Thanks for all your encouragement!

Khaled Ali Murtada Ciudad Real, 2019

Abstract / Resumen III

Abstract / Resumen

n this doctoral Thesis, new analytical methodologies based on nanosized and non- nanosized materials are presented. One of the major objectives is the potential gain I in sensitivity and selectivity that can be obtained when employing the proposed techniques.

In the first part of this Thesis, a new methodology based on magnetic multi-walled carbon nanotube poly(styrene-co-divinylbenzene) composite is presented. This nanocomposite was used as a sorbent material for extracting several types of antidepressants in human urine and their separation by capillary electrophoresis. The same magnetic sorbent was used for the extraction of different types of catecholamines in urine and at the dark ventral patch hair from Iberian male red deer(s) and their subsequent determination by liquid chromatography-mass spectrometry. On the other hand, a simple method based on poly(styrene-co-divinylbenzene)-coated glass blood spot is presented. This methodology is based on the use of lab manufactured poly(styrene-co-divinylbenzene)-coated glass blood spot for the extraction of seven types of antidepressants and their subsequent separation/detection by capillary liquid chromatography-mass spectrometry.

In the second part of this Thesis, different methods for improving the sensitivity of electrochemical detection of certain analutes have been developed. One methodology for the analysis of vanillin in food samples is presented, using aluminium doped titanium oxide nanoparticles. The prepared nanomaterials were used to modify screen-printed carbon electrodes. Another method is based on the modification of screen-printed carbon electrode with aluminium doped copper selenide nanoparticles for the electrochemical sensing of tyrosine in pharmaceutical samples. Finally, a novel approach for the synthesis of copper selenide-graphene oxide and its reduction by supercritical fluid carbon dioxide is presented. The resulting material was used for the modification the glassy carbon electrodes and to carry out the electrochemical detection of eugenol in various samples (clave, cinnamon and toothpaste).

Abstract / Resumen IV

n esta tesis doctoral, se presentan nuevas metodologías analíticas basadas en materiales nanométricos y no nanométricos. Uno de los objetivos principales es E la potencial ganancia en sensibilidad y selectividad que se puede obtener al emplear las técnicas propuestas. En la primera parte de esta tesis, se presenta una nueva metodología basada en un compuesto de poli(estireno-co-divinilbenceno) de nanotubos de carbono magnéticos de pared múltiple. Este compuesto se usó como un material sorbente para extraer varios tipos de antidepresivos en orina humana y su separación mediante electroforesis capilar. El mismo sorbente magnético se usó para la extracción de cinco tipos de catecolaminas en la orina y el parche ventral oscuro del venado rojo macho Ibérico y la separación mediante cromatografía líquida-espectrometría de masas. Por otro lado, se presenta un método simple basado en la mancha de vidrio recubierta con poli(estireno-co-divinilbenceno). Esta metodología se basa en el uso de manchas de sangre de vidrio recubierto de poli(estireno-co-divinilbenceno) fabricadas en el laboratorio para la extracción de siete tipos de antidepresivos y su posterior separación/detección mediante cromatografía líquida capilar-espectrometría de masas.

En la segunda parte de esta Tesis, se han desarrollado diferentes métodos para mejorar la sensibilidad del análisis electroquímico. Se presenta un método para el análisis de vainillina a partir de muestras de alimentos, utilizando nanopartículas de óxido de titanio dopadas con aluminio. Los nanomateriales preparados se utilizaron para modificar electrodos de carbono serigrafiados. Otro modelo se basa en la modificación electrodos de carbono serigrafiado con nanopartículas de seleniuro de cobre dopados con aluminio para la detección electroquímica de tirosina en muestras farmacéuticas. Finalmente, se presenta un nuevo enfoque para la síntesis de seleniuro de cobre-óxido de grafeno y su reducción mediante el empleo de dióxido de carbono supercrítico. El material decorado se usó para modificar el electrodo de carbono vítreo y la detección electroquímica de eugenol en varias muestras (clavo, canela y pasta dental).

Abbreviations / Acrónimos V

Abbreviations / Acrónimos

ACN Acetonitrile / Acetonitrilo AFM Atomic force microscopy / Microscópica de fuerza atómica AgNPs Silver nanoparticles / Nanopartículas de plata AIBN Azobisisobutyronitrile / Azobisisobutironitrilo Al-NPs Aluminium nanoparticles / Nanopartículas de aluminio AuNPs Gold nanoparticles / Nanopartículas de oro CE Capillary electrophoresis / Electroforesis capilar CLC Capillary liquid chromatography / Cromatografía líquida capilar CNTs Carbon nanotubes / Nanotubos de carbono CV Cyclic voltammetry / Voltametría cíclica CVD Chemical vapor deposition / Deposición de vapor química DA Dopamine / Dopamina DBS Dried blood spots / Manchas de sangre secas DHMA 3,4-dihydroxymandelic acid / Ácido 3,4-dihidroximandélico DHPG 3,4-dihydroxyphenyl glycol / 3,4-dihidroxifenilglicol DLS Dynamic light scattering / Dispersión dinámica de luz DPV Differential pulse voltammetry / Voltametría de pulsos diferenciados DVB Divinylbenzene / divinilbenceno ECD Electrochemical detection / Detección electroquímica EP Epinephrine / Epinefrina FT-IR Fourier transform infrared / Infrarrojo con transformada de Fourier GC Gas chromatography / Cromatografía de gases GCE Glassy carbon electrode / Electrodos de carbono serigrafiados GO Graphene oxide / Óxido de grafeno HFBA Heptafluorobutyric acid / Ácido heptafluorobutírico

Abbreviations / Acrónimos VI

HPLC High performance liquid chromatography /Cromatografía líquida de alta resolución ICP-MS Inductively coupled plasma-mass spectrometry / Espectrometría de masas por inducción de plasma LC Liquid chromatography / Cromatografía de líquidos LLE Liquid-liquid extraction / Extracción líquido-líquido LOD Limit of detection / Límite de detección LOQ Limit of quantification / Límite de cuantificación LSV Linear sweep voltammetry / Voltametría de barrido lineal MeOH Methanol / Metanol MIPs Molecularly imprinted polymers / Polímeros de impresión molecular MMWCNTs Magnetic multi-walled carbon nanotubes / Nanotubos magnéticos de carbono de pared múltiple MNPs Magnetic nanoparticles / Nanopartículas magnéticas MO-NPs Metal oxide nanoparticles / Nanopartículas de óxidos metálicos MS Mass spectrometry / Espectrometría de masas MSPE Magnetic solid phase extraction / Extracción en fase sólida magnética MWCNTs Multi-walled carbon nanotubes / Nanotubos de carbono de pared múltiple NE Norepinephrine / Norepinefrina NMs Nanomaterials / Nanomateriales NPs Nanoparticles / Nanopartículas Poly(STY-DVB) Poly(styrene-co-divinylbenzene) / Poli(estireno-co- divinilbenceno) PVD Physical vapor deposition / Deposición física de vapor QDs Quantum dots / Puntos cuánticos RAM Restricted access material / Material de acceso restringido rGO Reduced graphene oxide / Óxido de grafeno reducido RSD Relative standard deviation / Desviación estándar

Abbreviations / Acrónimos VII

relativa

sc-CO2 Supercritical carbon dioxide / Dióxido de carbono supercrítico SCFs Supercritical fluids / Fluidos supercríticos SEM Scanning electron microscopy / Microscopía de barrido electrónico SERS Surface enhanced Raman scattering / Dispersión Raman amplificada por superficie SFE Supercritical fluid extraction / Extracción fluidos supercríticos SPCE Screen printed carbon electrode / Electrodos de carbono serigrafiados electrodo SPE Solid phase extraction / Extracción en fase sólida µ-SPE Micro-solid phase extraction / Micro-extracción en fase sólida STY Styrene / Estireno SWCNTs Single-walled carbon nanotubes / Nanotubos de carbono de pared simple SWNHs Single-walled nanohorns / Nanocuernos de pared simple TEM Transmission electron microscopy / Microscopía de transmisión electrónica UV-Vis Ultraviolet-visible / Ultravioleta visible XRD X-ray diffraction / Difracción de rayos X

Aim / Objetivo

he development of new (nano)materials have promoted a new framework in Science and Technology. Analytical Chemistry has experienced, just like other T areas of science, a major change because of the needs and opportunities of Analytical Nanoscience and Nanotechnology. The exponential growth of the use of nanoparticles in the industry in recent years demands of the development of new analytical methods in the nanometric scale for the characterization and analysis of foods, as well as for biological monitoring and pharmaceutical studies. Furthermore, the possibility of using nanoparticles with unique properties allows the development of new strategies of analysis or the improvement of existing ones, for the analytical control of compounds with alimentary, biological and pharmaceutical interest.

The main aim of this Doctoral Thesis is to make new contributions to the role of (nano)materials in Analytical Chemistry. This general objective is addressed in the following three specific objectives:

I. To synthesize, characterize and use novel hybrid nanoparticles to preconcentrate, determine and quantify offering new opportunities for improving and developing of methods of analysis with analytical applications of interest. II. To develop new electrochemical methodologies based on novel nanomaterials for the improvement of food and pharmaceutical measurement processes. III. Development of new simple, inexpensive and environmentally friendly methods, based on non-nanosized materials, for the preconcentration, determination and quantification of interesting analytes.

Finally, it is important to remark that this Thesis has been developed under the research line of our group named "Simplification, automatization and miniaturization of analytical processes", which takes place at the University of Castilla-La Mancha (Department of Analytical Chemistry and Food Technology, from the Faculty of Sciences and Technologies, and the Regional Institute for Applied Scientific Research, IRICA).

Aim / Objetivo

l desarrollo de nuevos (nano)materiales ha impulsado un nuevo marco la Ciencia y la Tecnología. La Química Analítica ha experimentado, al igual que E otras áreas de la ciencia, un cambio importante debido a las necesidades y oportunidades de la Nanociencia Analítica y la Nanotecnología. Por un lado, el crecimiento exponencial del uso de nanopartículas en la industria que se está produciendo en los últimos años, exige el desarrollo de métodos analíticos en la escala nanométrica para la caracterización y análisis de alimentos, monitoreo biológico y estudios farmacéuticos. Por otro lado, la posibilidad de utilizar nanopartículas con propiedades únicas permite el desarrollo de nuevas estrategias de análisis o la mejora de las existentes, para el control analítico de compuestos de interés alimentario, biológico y farmacéutico.

El objetivo principal de esta Tesis Doctoral es desarrollar nuevas contribuciones al papel de los (nano)materiales en Química Analítica. Este objetivo general se aborda en los siguientes tres objetivos específicos:

I. Sintetizar, caracterizar y utilizar nuevas nanopartículas híbridas para la preconcentración, determinación y cuantificación ofreciendo nuevas oportunidades para mejorar y desarrollar métodos de análisis con aplicaciones analíticas de interés. II. Desarrollar nuevas metodologías electroquímicas basadas en nuevos nanomateriales para mejorar los procesos de medición de alimentos y productos farmacéuticos. III. Desarrollo de un nuevo método simple, económico y respetuoso con el medio ambiente basado en materiales no nanométricos para la pre-concentración, determinación y cuantificación de interés.

Por último, cabe destacar que esta Tesis se encuadra en la línea de investigación del grupo de investigación "Simplificación, automatización y miniaturización de procesos analíticos", que se desarrolla en la Universidad de Castilla-La Mancha (Departamento de Química Analítica y Tecnologías; y el Instituto Regional de Investigación Científica Aplicada, IRICA).

Introduction

1. INTRODUCTION

“Challenges are what make life interesting and overcoming them is what makes life meaningful.”

Joshua J. Marine

his introduction covers three main parts. The first one is related to the whole context of the Analytical Chemistry and Analytical Science including a brief and T general view of the analytical process. The second one deals with the view of new (nano)materials as samples and analytical tools for sample treatment and separation system. The last part covered in the introduction are directly connected with use of (nano)materials as tools for detection.

Introduction

1.1. Analytical Chemistry and Analytical Science

Analytical Chemistry is a branch of chemistry that deals with the separation, identification and quantification of chemical compounds. Chemical analyses can be qualitative, (i.e. identifying the chemical components in a sample), or quantitative, by determining the amount of a certain component in the sample. This description is misleading. After all, almost all chemists routinely make qualitative or quantitative measurements. The argument has been made that analytical chemistry is not a separate branch of chemistry, but simply the application of chemical knowledge.

The principle aims and objectives of Analytical Chemistry can be readily inferred from Figure 1.1.1. Thus, Analytical Chemistry has two main aims (an intrinsic one and an extrinsic one). The intrinsic aim is the achievement of metrological quality, i.e. ensuring full consistency between an analytical results delivered and the actual value of the measured parameters; in metrological terms, this translates into producing highly traceable results subject to very little uncertainty. The extrinsic aim is solving the analytical problems derived from (bio)chemical information needs posed by a variety of “clients” (e.g. private companies, research centers) or, in the other words, providing client satisfaction. As can be seen from Figure 1.1.1, realizing this aim entails achieving metrological quality. Broadly speaking, the principle objective of Analytical Chemistry is to obtain as much (bio)chemical information and of as high a quality as possible from objects and systems by using as little material, time, and human resources as possible, and with minimal costs and risks (see Figure 1.1.1).

Introduction

Figure 1.1.1. Global aims and objectives of Analytical Chemistry.

Analytical Chemistry presents a traditional and important tool in food, pharmacy industries, and bioanalytical science. In modern food chemistry, analytical methods are applied to study food constituents, additives and contaminants and their interactions and reactions during processing and storage. As well as, pharmacy is one of the subjects where Analytical Chemistry is studied vastly. It is used for purposes like checking the quality of inorganic compounds, organic compounds, drugs and other useful chemicals. Moreover, bioanalytical chemistry is the bridge between Biology and analytical process, bioanalytical chemistry relies on the identification and characterization of particles and compounds, particularly those involved with biochemical routes and related to the different health states. Through the study of modern techniques and chemical and biochemical methods and instrumental techniques for analysis and detection of biomolecules and biologically active molecules.

Analytical science is the science that seeks improving means of measuring the chemical composition, structure and morphology of natural and man-made substances (solids, fluids and gases), or entities (cells and complex materials) combined with the interpretation of the data obtained. In more general terms, its core is the art of measurement and interpretation. It can take us from a state where the chemical

Introduction

composition or properties of a sample are unknown, to a state in which we understand these parameters and we are able to use this information to our advantage.

Analytical science's impact is not only remarkable in fundamental research, but also in many industrial applications. Without analytical science modern life sciences would be impossible and innovation in chemicals – whether it be polymers, coatings or white biotechnology – would come to a standstill. Analytical science is also vital for the maintenance of public health and safety: quality control relies on analytical science to function, as does the safeguarding of food safety. Furthermore, analytical measurements are not merely confined to professional laboratories, but are also performed by non-specialists, such as the diabetic who monitors her blood sugar level. In short, analytical science is everywhere where measurement of the composition and/or of the (chemical) properties of substances or materials is required.

On the basis of the role plays by analytical science in chemical analysis? It has been contributed by the involvement and the integration of different type of (nano)materials and new systems at different steps of analytical processes. The exploitation of new materials properties in the whole steps of analytical process contribute to the development of innovative strategies or improvement of the conventional ones, in order to perform the analytical characteristics. The involved nanosized and non-nanosized materials can be used at different steps of analytical process.

The measurement process in Analytical Chemistry should be understood as a set of operations that separates the sample from the results obtained, expressed and interpreted according to the approach of the analytical problem to be solved. The analytical process involves several steps, as represented in Figure 1.1.2, namely: i) sample treatment (including sampling), ii) instrumental system, including chromatographic or electrophoretic separation systems such as liquid chromatography (LC), gas chromatography (GC) and capillary electrophoretic (CE) techniques, iii) detection and iv) data handling and treatment to offer the results as required. The role of new materials differs according to the different steps of the analytical process in which they are involved.

Introduction

Nowadays, every step of the analytical process has been subjected to miniaturization. However, they have not been miniaturized to the same extent. For instance, sample’s collection and preservation is the step less subjected to the benefits of miniaturization, even though some autonomous and remote sensing analytical microsystems have been reported [1]. Conversely, data acquisition and processing have achieved an excellent degree of miniaturization. Furthermore, it is generally accepted that the downscaling of sample preparation approaches has been developed after certain efforts to miniaturize both separation and detection systems [1].

Figure 1.1.2. The analytical process of chemical analysis.

It is worth mentioning that full miniaturization of analytical systems has also been addressed in the literature [2]. Size reduction is not the only reason towards miniaturization. In fact, the miniaturization of the different steps of the analytical process involves several additional benefits, as can be seen in Figure 1.1.3. The main benefits that can be obtained by downsizing the different steps of the analytical process are: reduction of sample amount, decreased consumption of chemicals and solvents, reduction of associated wastes, improved sensitivity, rapidity, portability and power consumption.

Introduction

Figure 1.1.3. Potential benefits derived from the miniaturization of the different steps of the analytical process.

As can be noticed, several of the above mentioned advantages are related to each other. It is important to note, however, that the introduction of miniaturized alternatives to conventionally performed steps of the analytical process have created novel challenges that need to be addressed. Therefore, the miniaturization of certain analytical processes may be not just useless but counterproductive. Besides, the use of highly reduced amounts of sample can seriously affect the necessary representativeness of samples subjected to analysis. Additionally, the fabrication of miniaturized detectors can also yield reduced resolution when compared with the corresponding full-sized counterpart [3].

The reduced volume of sample required for the use of miniaturized analytical separation techniques can also give rise to reduced sensitivity, since the miniaturization of the chromatographic column involves the reduction of the detector volume. For instance, the use of miniaturized LC for the analysis of easily available samples can give rise to problems of sensitivity that could be easily circumvented with conventional LC [4]. In addition, the development of novel analytical methods involving miniaturized sample preparation approaches introduces novel concerns derived from the limitations of the corresponding sample pre-treatment techniques.

Physicists, chemists and engineers are the scientists who are most directly involved, but their convergence with other areas such as information technology and communication,

Introduction

biotechnology and materials science, in a first approach, and medicine, pharmacy, agro- food and various types of industries such as textiles or energetic, in another, it must be observed. This fact allows to share and connect knowledge about methods and techniques from different fields in this area of science. Due to this reason, the advances made in the tools that now allow atoms and molecules to be examined and tested with great precision, have allowed the expansion and development of new (nano)materials.

It must be emphasized that Analytical Chemistry plays a key role in several fields, as the information it provides is of great interest for scientific decision making. Without exception, the various synthesized (nano)materials have found application in this field with remarkable achievements, which have largely promoted a multidisciplinary research that relates our subject to other sciences.

Analytical Chemistry cannot be left out of (nano)materials with words belonging to the analytical discipline such as “analysis” or “characterization” and others such as “use” or “employment” summarize the two key facets of the relationship between Analytical Chemistry and (nano)materials, namely, (i) Consideration of (nano)materials and (nano)structured materials as target analytes, which require analytical methods to detect and/or quantify (nano)materials in different types of samples, as well as a correct characterization for the incorporation of these (nano)materials in industrial applications. (ii) Consideration of (nano)materials and (nano)structured materials as analytical tools for innovation and improvement of (bio)chemical measurement processes. Taking all this into consideration, the relationship between (nano)materials and Analytical Chemistry is defined, as can be seen in Figure 1.1.4.

Introduction

Figure 1.1.4. The role of (nano)materials in Analytical Science.

1.2. New (nano)materials as analytical tools in non-chromatographic separation techniques: treatment/separation of analytes in food, pharmaceutical and bioanalytical samples

Use of new (nano)materials in analytical processes is the most extensively explored area of Analytical Chemistry. The objective is to exploit the excellent properties of (nano)materials to improve well-established analytical methods or to develop others for new analytes or matrices. In addition to the typical advantages of (nano)materials, their use should lead to improved selectivity, sensitivity, rapidity, miniaturizability or portability of the analytical system. As separation techniques progresses, new (nano)materials with properties that can be unequivocally ascribed to their (nano)metric dimensions are bound to emerge as powerful analytical chemical tools.

New (nano)materials can be incorporated or used in analytical methods either as such or chemically bonded. In the latter case, (nano)materials can be chemically bonded to a surface or functionalized with other organic or inorganic compounds in order to increase their solubility. Chemically unmodified (nano)materials can be used as raw randomized materials or as self-assembled raw materials. The explored (nano)materials property can

Introduction

be electrical, optical, thermal, magnetic or chemical. Frequently, however, two or more properties are explored at once.

The availability of reproducible methods for the synthesis of different types of (nano)materials and suitable techniques for their characterization has allowed their wide application to improve the features of separation techniques: treatment/separation methods, mainly their selectivity and sensitivity. Although the use of (nano)materials in the main three steps of analytical process (sample treatment, sample separation and detection) has been widely studied, considering individually each of these steps, this section is focused to assess the real benefits that (nano)materials have provided in separation techniques: treatment/separation.

1.2.1. Types of new (nano)materials used in separation by extraction

Extraction processes can be enhanced by means of efficient selection of the experimental conditions. SPE and solvent extraction are the most commonly used sample preparation techniques in analytical laboratories. In fact, many official and standardized analytical methodologies involve their application for the extraction, preconcentration and sample clean-up prior to determination of target analytes.

SPE and solvent extraction involve the partitioning of target analytes between the sample solution and a solid (adsorbent) phase or immiscible organic solvent, respectively. Both SPE and solvent extraction are exhaustive extraction techniques, so quantitative transfer of target analytes from the sample to the extractant phase is achieved under optimal conditions. However, the consumption of large amounts of organic solvents and subsequent generation of waste, and significant time consumption are among the inherent drawbacks associated with these classical sample preparation approaches. These limitations led to the introduction of modern sample preparation techniques that share the common benefits of miniaturization and solvent-free operation.

The incorporation of new (nano)materials in the sample treatment step, for preconcentration of the analytes and sample clean-up to removal of some toxic and hazardous pollutants from polluted water, soil, and biological, which allows

Introduction

simplification of the method and the possibility of treating small amounts of sample. Sample preparation has also profited from the use of (nano)materials, albeit a lesser extent than detection. A general view of using (nano)materials in sample preparation is depicted in Figure 1.2.1.1.

Different (nano)materials have been synthesized using several methods such as co- precipitation method, water-in-oil microemulsions, hydrothermal and solvothermal synthesis techniques. There are many (nano)materials available with very different properties, have been used in sample treatment.

Figure 1.2.1.1. Use of (nano)materials in sample preparation.

Recently, different materials including, nanomaterials and other materials have been purposed in sample treatment process for the preconcentration of many analytes, as well as for the elimination of interferences (clean-up).

 Nanomaterials

The role of NMs as analytical tools shows the combinations of several situations in Analytical Nanoscience. These situations are namely i) nanometer size, ii) physicochemical properties of NMs, and iii) the two mentioned facets. Accordingly, these cases are described below [5]:

Introduction

i. The first is that nanometric analytical systems refer to devices based on the nanoscale. In this is a step forward to miniaturization without exploiting the unique properties of NMs. ii. In the case of nanotechnology analytical systems, the use of their unique properties allows the development and the improvement of analytical processes, resulting to the analysis of new compounds in different matrices. iii. The last case consists on use of analytical nanosystems. This is considered the best situation because the both previous aspects are exploited: the nanometer size and the control by physicochemical laws of nanoscience. Furthermore, it is based on the combination of "nanometric analytical systems" (exploitation of nanosize) and "nanotechnological analytical systems" (exploitation of the unique properties of NMs).

How to reach the nanoscale?

The nanoparticles (NPs) are of different shape, size and structure. It be spherical, cylindrical, tubular, conical, hollow core, spiral, flat, etc. or irregular and differ from 1 nm to 100 nm in size. The surface can be a uniform or irregular with surface variations. Some nanoparticles are crystalline or amorphous with single or multi crystal solids either loose or agglomerated [6].

Numerous synthesis methods are either being developed or improved to enhance the properties and reduce the production costs. Some methods are modified to achieve process specific NPs to increase their optical, mechanical, physical and chemical properties [7], such as sol-gel [8], spinning [9], chemical vapor deposition (CVD) [10], physical vapor deposition (PVD) [11], plasma processes [12], self-assembly [13], high energy ball milling [14], gas condensation [15], severe plastic deformation [16], pyrolysis [17] and Biosynthesis [18].

Two approaches can be used to raise nanosized, namely, (i) “top-down” strategies, based on methodologies which achieve nanosized materials from macromaterials (NPs are directly generated from bulk materials via the generation of isolated atoms usually involving physical methods such as milling or attrition, repeated quenching and photolithography [19]) and (ii) “bottom-up” strategies, based on the creation of

Introduction

complex nanostructures from atomic or molecular functional elements. They comprise molecular components as starting materials linked with chemical reactions, nucleation, and growth processes to promote the formation of clusters. Figure 1.2.1.2 shows a scheme of the different strategies (“top-down” and “bottom-up”) used to achieve the nanoscale.

Figure 1.2.1.2. Scheme of the two approaches employed in the fabrication of nanomaterials: “top-down” and “bottom-up.”

Nanomaterials can be classified according to their participation and role in the sample treatment step as follows [20]: i) sorbent agents [21], as direct interaction between the analyte and the nanoparticle occurs; ii) inert support, such as silica nanoparticles functionalized with a complexation agent; iii) NMs with magnetic properties, which can either directly adsorb the analyte or can be functionalized with organic groups, so the use of a magnetic field can simplify the analytical procedure; iv) NMs acting as ionizations agent for the direct analysis of samples by ion secondary mass spectrometry. Carbon NMs have focused attention thanks to their singular π-π electron configuration, as well as metal oxides by virtue of their high surface area. In this sense, carbon nanotubes (CNTs) have been widely used as sorbents for SPE [22,23]. Packed nanotubes tend to aggregate to some extent, their inclusion as a Nanoscience application requires that aggregation be avoided and interactions between analytes and isolated

Introduction

nanoparticles favored [24]. Conical carbon NMs, such as single-walled carbon nanohorns (SWNHs) [25] or carbon nanocones/disks [26] have been also used for solid phase microextraction (SPME).

On the other hand, the use of magnetic NPs has been extensively used as SPE in many contaminants scavenging mechanisms. For example, magnetic nanoparticle adsorbents based on maghemite core and a silica mesoporous layer [27] were used for achieving simultaneous removal of polycyclic aromatic hydrocarbons, (PAHs) and metal contaminants in contaminated wastewaters. Moreover, functionalized magnetic NPs have been used in sample preparation matrices [28–30]. Magnetic molecularly imprinted polymer (MMIP) sorbent material based on dopamine hydrochloride as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linking agent and Fe3O4 magnetite as magnetic component was used for the extraction and determination of catecholamines from urine samples [31]. In addition to SPE sample treatment, NPs have been used in other sample treatments process such as membrane filtration, for example with membranes composed or modified by CNTs.

Generally, the effectiveness of NMs has been demonstrated based on the physicochemical characteristics of the NMs, which are used as analytical tools in sample preparation. Their special characteristics have been extensively exploited in different steps of analytical process (extraction/clean-up and preconcentration), proposing new strategies, facilitating and improving existing ones. Besides, the synergistic effect due to the combination of complementary properties (e.g., silica NPs with a magnetic core or CNTs combined with magnetite, etc.), makes possible to develop strategies characterized by materials with more stability, rapidity, selectivity and efficiency.

NMs can be classified as origin (natural and artificial), chemical nature (inorganic, organic, and mixed) and homogeneity (single or hybrid composition).The most used NMs in sample preparation procedures were organized into three types: metallic, silica and carbon-based nanomaterials.

Introduction

I. Metallic NPs Metallic NPs have fascinated scientist for over a century and are now heavily utilized in biomedical sciences and engineering. They are a focus of interest because of their huge potential in nanotechnology. Today these materials can be synthesized and modified with various chemical functional groups which allow them to be conjugated with antibodies, ligands, and drugs of interest and thus opening a wide range of potential applications in biotechnology, magnetic separation, and preconcentration of target analytes, targeted drug delivery, and vehicles for gene and drug delivery and more importantly diagnostic imaging [32].

Gold nanoparticles (AuNPs) are widely used in biotechnology and biomedical field because of their large surface area, and high electron conductivity [33]. Whereas silver nanoparticles (AgNPs) has many applications due to the large degree of commercialization. Silver is an attractive material for its distinctive properties, such as good conductivity, chemical stability, catalytic activity, and antimicrobial activity [34]. The strong affinity that exists between AuNPs and PAHs has been used for extraction and preconcentration of these compounds from drinking water [35]. On The other hand, poly(ethylene glycol methacrylate phosphate (PEGMP) was proposed for the preparation of AgNPs embedded polymer [36].

Metal oxide nanoparticles (MO-NPs) play a very important role in many areas of chemistry, physics and materials science, such as Al2O3, SiO2, MgO, ZrO2, CeO2, TiO2,

ZnO, SnO and iron oxides (FeOx). MO-NPs have unique properties (e.g., large specific surface area, high adsorption capacity and easy modification). Their adsorption strongly depends on morphology, crystal structure, defects, surface area and hydroxyl coverage. These unique properties make MO-NPs a good candidate for use as sorbents.

II. Silica NMs Silica is a very appealing material for analytical applications because it is relatively inexpensive, chemically inert and thermally stable. The silanol groups on the surfaces of silica NPs can be functionalized easily by treatment with the appropriate silane component [37]. Silica-coated magnetic NPs present the advantages of silica NPs with

Introduction

regard to surface reactivity and magnetic core, which contribute to the rapid separation of the sorbent carrying the target analytes from matrix solution.

III. Carbon-based NMs Carbon NMs, such as, graphene, CNTs, crystalline diamond and fullerenes, all display exceptional several properties which has resulted in their widespread application. Due to their high surface area, this NMs are employed as support, through each of them has unique structure, properties and applications. Figure 1.2.1.3 shows the main types of carbon-based NMs.

Figure 1.2.1.3. Main types of carbon-based nanomaterials.

The characteristic structures and electronic properties of CNTs allow them to interact strongly with organic molecules, via non-covalent forces (e.g., hydrophobic interactions, hydrogen bonding, π-π stacking, electrostatic and van der Waals forces). These interactions and hollow and layered nanosized structures make them a good candidate for use as sorbents [38–40].

 Other materials

Other materials for robust, efficient and highly selective sample preparation techniques is presented and critically discussed. In the sample preparation area, the main goal of this technology is obtain more selective materials higher recovery rates and less matrix effect.

The sol-gel technique has been widely used in different fields (e.g., separation techniques, chemical catalysis, biotechnology and drug delivery), mainly aiming to obtain materials [41,42]. In Analytical Chemistry applications, the sol-gel process is usually employed in the synthesis of materials as sorbents for sample preparation techniques and chromatography stationary phases. The main advantage of the sol-gel process nowadays is obtaining inorganic and organic-inorganic hybrid polymers by

Introduction

employing mild reaction conditions. Another feature is the possibility to generate thermally- and chemically-stable materials with controlled morphology, surface properties and pore structures, by carefully modifying the synthesis conditions [43,44].

The most used materials (non-nanomaterials) in sample preparation procedures were discussed in this section. Silica-based materials (SiO2) are the most used sorbents in separation techniques. However, other inorganic oxides (titania and zirconia) and mixed inorganic polymers (silica-titania, silica-zirconia) have also been used to improve the chemical and thermal stability of these materials [45,46].

Hybrid materials are an interesting strategy to alter sorbents polarity and selectivity. They are synthesized by the reaction of inorganic and organic precursors (building blocks). Due to the large number of organic and inorganic precursors, a considerable variety of hybrid sorbents with different characteristics maybe designed.

Alkoxysilanes are the silica precursors most used in sol-gel processes due to the low presence of metals incorporated into the polymer network. However, other silica precursors have also been investigated. Nazario et al. [47] synthesized C18 sorbent using silicate solution as precursor. Liang et al. [48] synthesized a hybrid membrane (poly(vinylidene fluoride/silica)) able to separate proteins (bovine serum albumin and bovinehemoglobin) using electrostatic interactions. Kumar et al. [49] prepared polytetrahydrofuran coating on cellulose membrane by sol-gel process for extraction and clean-up of 17α-ethynylestradiol, β-estradiol and bisphenol A in urine samples. The fiber SPME coatings commercially available (polydimethylsiloxane, divinylbenzene, carboxen, polyethylene glycol and polyacrylate), as well as their combinations, are able to extract analytes withdistinct polarities [50]. Mirnaghi et al. [51] prepared a C18 coating for automated 96-blade SPME. Rahim et al. [52] prepared a hybrid polymer methyltrimethoxysilane-cyanopropyltriethoxysilane as stir bar sorptive extraction (SBSE) coating for extraction of non-steroidal anti-inflammatory drugs in urine samples.

Mao et al. [53] developed a hybrid SBSE coating (sulfonated polystyrene/TiO2) for determination of seleno-amino acids and seleno-oligopeptides in biological samples.

Poly(styrene-co-divinylbenzene) (poly(STY-DVB)) resins are finding increasing use is sample treatment because of their efficiency, ruggedness and wide pH stability. Various

Introduction

hydrophilic functional groups were chemically attached to the benzene rings of porous, cross-linked polystyrene resins [54] as stationary phase for high-performance liquid chromatography (HPLC). Furthermore it was found that the efficiency of porous resins used in SPE varies with the porosity, those with the largest surface area giving the greatest retention [55]. Sun and Fritz [56] inserted an acetyl- or hydroxyl methyl group into a porous poly(STY-DVB) Amberchrome 161 resin that provides more hydrophilic surface and is easily wetted water alone.

In order to allow the use of large sample volume membranes derivatized with an acetyl group can be used, which shows excellent hydrophilicity and a lesser dependence on wetting prior to extraction. These membranes also yielded higher recoveries compared to the unmodified analogue. This is attributed to an increase in surface polarity allowing the aqueous sample to make better contact with the resin surface. Fritz et al. [57] also investigated membranes impregnated with poly(STY-DVB) or acetyl-poly(STY-DVB) resin beads for SPE of ppm (w/w) concentrations of phenols from aqueous samples.

In the last years, there has been a growing interest in the application of ionic liquids (ILs) on sample preparation techniques in environmental, food, clinical, and related fields [58–60]. The advantage of ILs over other extraction phases may be related to their molecular structure. These materials consist of organic cations derived from Lewis bases and polyatomic anions containing different inorganic or organic structure [61]. ILs are termed “designable solvents” due to the possibility of different combinations between cation and anion, their miscibility with water and organic solvents, and tunable viscosities [62]. The main application of ILs in analytical chemistry is related to the separation techniques, including their utilization as stationary phases for HPLC and GC, coatings for SPME fibers as extractor solvent in LLE, modifying electrodes and sorbents for SPE employing different supports (e.g. silica, MWCNTs, graphene, and so on) [61,63].

The high selectivity of molecular recognition of antigen-antibody, drug-receptor and enzyme-substrate have encouraged researchers to develop materials that act as synthetic receptors for the compounds of interest. The molecular imprinting technology exhibit high selectivity for a target molecule/ion or similar analytes [64]. MIPs have

Introduction

demonstrated applications in several fields, including sample preparation, as reviewed by Haginaka [65].

Polymeric monolithic materials consist on rigid polymers with continuous porous channels that have been used in various applications. Monolithic media acquired importance in chromatographic separation and sample preparation techniques due to its versatility in selectivity modification, fast mass transferring, good peak capacity and mild synthetic conditions. Further, the good permeability and low backpressure are important features of monolithic sorbents to avoid clogging in biological fluids applications. These materials have been prepared in different formats based on organic, inorganic or hybrid precursors for several sample preparation methods [66].

The reduction of the matrix interferences in biological samples with a minimal sample preparation procedure is of current interest to improve the overall analytical performance. Restricted access material (RAM) sorbents has the feature of dual surface configuration acting simultaneously [67,68].

Magnetic materials represent an important alternative that emerged recently for sample preparation. In principle they can be prepared starting from any material discussed previously in this text as well as their derivatives (e.g. carbon derived material, MIPs, RAM, ILs and compounds derived from sol-gel process). They are commonly used to overcome practical problems that can arise from the use of nanomaterials in sample preparation techniques such as frits obstruction, increase in pressure and desirable sorption characteristics.

The magnetic material is dispersed into the solution containing the target analytes and are thoroughly mixed; this process increase the contact between the sorbent and the analytes and can provide better recovery rates without longtime consumption. The iron oxides (Fe3O4 and ɣ-Fe2O3) are the most frequently materials used to provide magnetic characteristics due to their favorable properties including: easiness preparation, easiness of surface modification, easiness of operation, good recoverability and, excellent diffuseness in aqueous solution [69].

Introduction

1.2.2. Types of extraction techniques involving (nano)materials

Sample treatment is crucial in Analytical Chemistry and especially for the complex matrices encountered in food, pharmaceutical and biological samples. Most samples are not ready for direct introduction into instruments. For example, in the analysis of pesticides in fish liver, it is not possible to analyze the liver directly [70]. The pesticides have to be extracted into a solution, which can be analyzed by an instrument. Currently, analytical science tends to exploit and improve the chemical properties of (nano)materials, especially the capacity of adsorption for their use as a reversible solvent in extraction and preconcentration processes. On first impression, sample treatment together with sample collection and preservation, may seem the most routinary aspects of an analytical protocol. However, it is critical that analysts realize that the measurement of an analyte from a sample is as good as the sample preparation is. If an aliquant taken for analysis does not represent the original sample accurately, the results of this analysis are questionable. Sample preparation has profited from the use of (nano)materials because of their properties, incorporating new materials in to the sample treatment step, to simplify it.

Various sample preparation methods have been reviewed, especially for analysis of several types of sample matrices, including conventional solvent extraction, liquid-liquid extraction (LLE), solid-phase extraction (SPE), Soxhlet extraction, pressurized solvent extraction (PSE), supercritical fluid extraction (SFE), microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). A variety of extractant phases, for example polymeric sorbents and adsorbents, organic solvents, ionic liquids, water or carbon dioxide can be used depending on the sample preparation technique [1].

 Solid phase extraction

Solid phase extraction (SPE) has played a crucial role in sample preparation. In the past, traditional solid phase sorbents, such as silica NPs, C8, C18, poly(STY-DVB), methacrylate- divinylbenzene resins, macroporous poly(N-vinylpyrrolidone-divinylbenzene) polymers and some others, usually used mixed-mode ion-exchange sorbents, including mixed- mode/cationic-exchange (MCX), mixed-mode/anion-exchange (MAX) and weak anion- exchange (WAX), which have already been reviewed in detail by Alberti et al. [71].

Introduction

The use of (nano)materials as sorbents in conventional SPE has been widely revised [72], discussing in general their advantages (large specific surface area, easy functionalization, high adsorption capacity and pre-concentration factors, and reusability) and limitations (high pressure in the SPE column, low selectivity, formation of aggregates and potential leakage from the column). For instance, MWCNTs are widely used as sorbent materials in SPE due to their extremely large surface area and strong sorption properties. These properties are ascribed to electrostatic interactions, such as dipole-dipole, hydrogen bonds, π-π stacking, dispersion forces, dative bonds and hydrophobic effect. However, a limitation of these NMs is the formation of aggregates that diminishes the surface/area ratio. Also, the small particle size of the CNTs results in compaction of the sorbent when relatively high flow rates are used, which forces to decrease the flow rate, lengthening significatively the preconcentration time when high sample volumes are used. A possible solution to this problem is the use of a MWCNTs/poly(vinyl alcohol) cryogel composite, which has been described for the separation of PAHs from water samples [73].

A modification of conventional SPE is dispersive solid phase extraction (d-SPE), in which the extraction is carried out in the bulk solution instead of the column. This technique has found a wide development with the use of (nano)materials, which are usually coated with a sorbent and dispersed into the sample solution, interacting with the analytes. After extraction, the sorbent containing the target analytes is isolated by centrifugation or collected by filtration, and the analytes are eluted or desorbed with a suitable desorption solvent. This approach overcomes the limitations of conventional SPE, such as the time-consuming process of loading large-volume of sample and the high pressure and the blockage probability of the SPE cartridge in analyzing samples containing particulate matter [74,75].

The availability of magnetic nanoparticles (MNPs) has allowed the development of magnetic solid-phase extraction (MSPE), which is a type of d-SPE that involves the use of a magnetic nanosorbents uniformly dispersed into the sample solution. The analytes are adsorbed onto the nanosorbent under stirring, which is recovered from the solution using an external magnet. After the elution of the analytes from the nanosorbent, the external magnet is used again to separate the nanosorbent from the solution. This

Introduction

approach allows rapid extractions with high efficacies, owing to the large surface area- to-volume ratio of the magnetic nanosorbent, which facilitates mass transfer between the MNPs and sample solution, shortens the extraction time and avoids the centrifugation or filtration step required in conventional d-SPE.

Through the introduction of advanced analytical techniques and improved throughput, a wide variety of biological samples have been tested for analysis. Despite the studies carried out with biological samples by using different sample preparation. SPE and dried blood spots (DBS) persist as the two most common. In recent decades, SPE has played a crucial role in sample preparation, replacing the classic LLE, in environmental, food and biological analyses [76,77]. On the other hand, the scope of DBS testing utilizing mass spectrometric methods, has broadly expanded. Clinicians and researchers have become very enthusiastic about the potential applications of dried blood spot based mass spectrometric applications.

Dried blood spot (DBS) is a spot of blood is placed on filter paper and allowed to air dry. A circular punch (about 3 mm) is removed, eluted with solvent and analyzed for metabolic markers. The idea of collecting blood on a paper card and subsequently using the DBS for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge [78].

Advantages of DBS sampling include minimal volume requirements (approximately 30 – 100 µL per spot), ease of sample attainment by finger or heel stick with minimal training required, easy of transport and sample stability. Once dried, the sample is stable for months to years at ambient temperature or under refrigeration [79].

These superior traits have made DBS a common sampling method for gene screening and long-term genetic sample bio-banking. Within the last few years, DBS sampling has been used for clinical and pre-clinical pharmacokinetic studies, taking advantage of smaller sampling needs and simplified sample collection and handling. DBS sampling has also been used for disease surveillance in developing countries [80].

Introduction

Several methods have been developed for the DBS analysis. Chromatography combined off-line or online with (tandem) MS has been the major tool for DBS analysis for decades [81–84]. Direct MS allows automated handling of DBS without any treatment prior to MS analysis [85]. Moreover, the disadvantages of DBS sampling, hematocrit effect, uncertainty about long term stability and sample inhomogeneity. Applications of DBS sampling and analysis will continue to expand and close the circle from health monitoring and rapid diagnosis through drug development to personalized point of care therapy.

Blood samples dried on filter paper or substrate can be a successful option for population screening in remote areas, provided pre-analytical variations arising due to the method of blood spot preparation and storage are well controlled [86].

 Micro-solid phase extraction

Another technique in which different (nano)materials have shown their usefulness is micro-solid phase extraction (μ-SPE), which involves the use of a syringe-like device fitted with a needle that contains a fiber or a wire coated with a thin layer of a sorbent [87,88]. The extraction is performed by either immersing the fiber in the liquid sample or extracting the analytes from the headspace above the sample. After the extraction step, the fiber or the wire is withdrawn from the sample and the analytes are thermally desorbed when GC is used, or eluted with a suitable solvent in LC. Chemical, thermal and mechanical stability of the fiber and the reproducibility are critical factors in μ-SPE

[88]. A polypropylene hollow fiber containing a sol-gel-derived Fe3O4/SiO2/TiO2 core- double shell nanocomposite has been described as sorbent in μ-SPE coupled with LC for the determination of non-steroidal anti-inflammatory drugs in human hair [89].

 Liquid-liquid extraction

Liquid-liquid extraction (LLE) is a separation process consisting of the transfer of a solute from one solvent to another, the two solvents being immiscible or partially miscible with each other. Frequently, one of the solvents is water or an aqueous mixture and the other is a non-polar organic liquid. The most common method of LLE is performed using a separatory funnel. LLE is an important separation technique for environmental, clinical, and industrial laboratories.

Introduction

Solvent extraction is an old, established process and together with distillation constitute the two most important industrial separation procedures. The first commercially- successful LLE operation was developed for the petroleum industry in 1909 when Edeleanu’s process was employed for the removal of aromatic hydrocarbons from kerosene, using liquid sulfur dioxide as solvent. Since then many other processes have been developed by the petroleum, chemical, metallurgical, nuclear, pharmaceutical and food processing industries.

 Supercritical fluid extraction

Supercritical fluid extraction (SFE) is a separation process where the substances are dissolved in a fluid, which is able to modify its dissolving power under specific conditions, above their critical temperature and pressure. SFE is an innovative and environmentally friendly extraction process that uses a supercritical fluid as an alternative to commonly used organic solvents.

The SFE technology has advanced tremendously since its inception and is a method of choice in many food processing industries. Over the last two decades, SFE has been well received as a clean and environmentally friendly “green” processing technique and in some cases, an alternative to organic solvent-based extraction of natural products. The most recent advances of SFE applications in food science, natural products, by-product recovery, pharmaceutical and environmental sciences have been published in extensive reviews [90–92].

Throughout this Doctoral Thesis, NMs and other materials have been used in non- chromatographic separation techniques for the sample treatment/separation step as a MSPE sorbents for the analysis of antidepressants in human urine samples, and for determination of catecholamines and related compounds in deer urine and hair samples, as well as, for the selective extraction of antidepressants in human blood samples by using DBS. All of this will be explained in deep in Section 3.1.

Introduction

1.3. New nanomaterials as tools for analytical detection of residues in food, pharmaceutical and bioanalytical samples

Detection is the analytical step in which NPs have been more widely used by virtue of their ability to replace conventional materials as well as the advantages of electrochemical biosensors. Detection systems consist of two functional components: recognition elements for binding with target analytes and a transduction process to signal the binding event. The efficiency of these two components are critically related to the outcome of the detection process in terms of the response time, signal-to-noise (S/N) characteristics, sensitivity, and selectivity of the system. Thus, the challenges in development of novel detection systems have been concerned with improving the recognition process as well as designing new signal transduction mechanisms. NMs provide novel systems for the pursuit of new recognition and transduction processes, as well as increasing the signal-to-noise ratio through miniaturization of the system components [93].

The use of new NMs for the improvement of detection is another aspect in Analytical Chemistry, which has been recognized, in the last decades, a great techniques progression. NMs can be involved in the development of electrodes or to modify electrodes able to detect food, pharmaceutical and bioanalytical substances. So, the designed electrodes can be modified by antibodies and/or other substances, according to the type analytes, to successfully achieve the analytical requirements. Thus, NMs can be used as surface modifiers of transducers, or as optical or electro-active components to improve sensitivity, to accelerate times of response, to allow multi-detection or provide more stable responses. In this sense, biomolecules are stabilized by the NPs, which increase active surfaces and facilitate electron transfer. In addition, the development of optical nanosensors has been fostered by the exceptional optical properties of metallic nanoparticles derived from their Plasmon resonance, as well as photo fluorescent properties in the case of semiconducting NPs (quantum dots), carbon dots (CDs) or graphene quantum dots. Table 1.3.1 summaries the roles that NMs play and their involvements at sample detection step. A general view of using NMs for sample detection is depicted in Figure 1.3.1.

Introduction

Figure 1.3.1. Use of nanomaterials for sample detection.

Table 1.3.1. Contributions of the NMs at analytical detection step.

Technique Advantage Disadvantage NMs improvement SERS - Wide range of - Weak vibrational - Obtaining SERS of applications spectroscopic method some molecules - Difficulty in obtaining - SERS signal repeatable signal enhancements and enhancements improvement for some molecules Absorbance - Simplicity of use - Limited to molecules - Obtaining absorbance - Non destructive without a chromophore of some molecules - Rapid - Signal enhancements and improvement for some molecules Fluorescence - Simplicity of use - Limited to molecules - Obtaining fluorescence - Non destructive without a fluorophore of some molecules - Sensitive - Signal enhancements - Rapid and improvement for some molecules

 Types of sensors

Sensors have various applications in different fields (food, pharmaceutical, bioanalytical and other). The ability to sense for chemicals and biological agents that are present in the different samples is a concern to several agencies.

Due to its high surface area, chemical reactivity, catalytic efficiency and excellent adsorption capacity, NPs are excellent candidates for use in sensors fabrication [94]. Thus, it is possible to stabilize the molecules due to the transfer of electrons. In addition, the possibility of binding specific ligands on its surface increases the biocompatibility,

Introduction

selectivity and sensitivity of the sensor. On the other hand, non-nanomaterials can be used in sensor material and fabrication. Sensors can be classified depending upon structure (electrochemical and optical) and applications (chemical, deployable, electrometers and biosensors). Next, various types of sensors including electrochemical sensors and optical sensors are described.

. Electrochemical sensors Electrochemical sensors are devices that give information about the composition of a system in real time by coupling a chemically selective layer (the recognition element) to an electrochemical transducer. In this way, the chemical energy of the selective interaction between the chemical species and the sensor is transduced into an analytically useful signal.

Different families of electrochemical sensors can be recognized depending on the electrical magnitude used for transduction of the recognition event: potentiometric (change of membrane potential); conductometric (change of conductance); impedimetric (change of impedance); and voltammetric or amperometric (change of current for an electrochemical reaction with the applied voltage in the first case, or with time at a fixed applied potential in the latter) [95].

The first step when a chemical sensor is used as an analytical tool is the selective recognition of the species of interest for a given application (the analyte) [96]. In the real world, the concept of “interference-free samples” does not exist. Selectivity should therefore be the most important feature under study during the development of a new sensor. Although there is no apparent relation with other analytical characteristics, it is known that when there is a high concentration of an interfering compound, the linear range is reduced, and the limits of detection are higher. Therefore, highly selective recognition elements are required for the development of sensors.

Other features of interest at sensor design involve short response times, as well as a high mechanical strength and chemical resistance to changes of operating media. Nevertheless, many sensors have been developed under critical conditions, when the high selectivity of biological receptors such as antibodies or enzymes compensate for the weak mechanical and chemical strength of the device.

Introduction

The use of NMs for the development of electrochemical sensors has attracted growing interest in recent years. When compared to bulk materials, NMs have been found to possess enhanced physical and chemical characteristics including, but not limited to, thermal, optical, magnetic, plasmonic, and catalytic properties [97–103]. These enhanced features have led to the incorporation of NMs into electrochemical sensing systems as a means of improving current sensor technologies. Individual electrodes with nanoscale dimensions which offer fundamental improvements over micro- and macro- electrodes [104] are also being developed at a growing rate.

There are electrochemical sensors based on NMs, such as, CNTs and modified graphene with NPs that have been used for various purposes [105–107], AuNPs, AgNPs and MIPs [108]. CNTs and AuNPs are the types of NPs most widely used for developing electrochemical (bio)sensors [109]. Combining AuNPs and CNTs was found to enhance some electro-catalytic properties of electrodes [110]. However, metal NPs, specifically NPs of metal oxides, are currently the most exploited in this field.

On the other hand, electrochemical sensors can be modified by organic electroconductive polymers (i.e. poly(3,4-ethylendioxythiophene) [111], polyvinyl chloride, polyethylene, polymethacrylate, polyurethane and polypyrrole [112]). Khan and Wernet [113] fabricated a novel electrode by the use of a flexible conductive polymer film of polypyrrole doped with polyanions and a microporous layer of platinum black. Metals and carbon are commonly used to prepare solid electrode systems and supporting substrates. Metals such as platinum, gold, silver and stainless steel have long been used for electrochemical electrodes due to their excellent electrical and mechanical properties.

. Optical sensors Optical sensors are powerful analytic tools capable of providing analyte information remotely. Typically, optical sensors for chemical or biological molecules are composed of molecular recognition elements and signal transducers [114]. The molecular recognition elements interact with the target analytes under study and provide information about their presence, concentration, and other physical properties of interest. When target molecules enter the system, the sensors produce detectable

Introduction

changes in their signals – which are then transduced into easily measured and quantified optical signals.

Over the past few decades, NMs have increasingly been considered as to be attractive materials that has revolutionized the sensors sector. Optical sensors consist of structured sensor materials which show a distinct reaction to electromagnetic excitation at optical frequencies. Two major working principles can be distinguished. On the one hand, NMs can influence direct light-matter interactions such as, absorption or scattering events by locally increasing or decreasing the respective interaction cross section. On the other hand, the optical properties of the sensor nanomaterial itself can change due to conditions or processes occurring in the vicinity of the sensor material. The change of the optical properties can then be interrogated in a contact-free fashion from the macroworld. These changes may consist of surface resonance Plasmon alterations, such as: modification of its wavelength or intensity (localized surface Plasmon resonance spectroscopy, LSPR) [115]; visible changes in color as a result of the alteration of the state of aggregation of (nano)materials [116]; Raman inelastic dispersion increase (SERS) [117]; or increase in temperature due to non-radiative energy decay [118].

Optical measurement includes absorption, fluorescence, phosphorescence, Raman, dispersion, refraction, interference spectroscopy etc. The properties measured include amplitude, energy, polarization, decay time and/or decay phase [119,120].

Likewise, fluorescent NPs, such as QDs [121] and AuNPs and AgNPs, can alter their native fluorescence in the presence of the analyte (quenching or fluorescent inactivation), which allows the detection of compounds of different nature [122]. The SWCNTs are fluorophores in the near infrared zone (NIR) although their practical application as sensors is quite limited since their fluorescent properties vary depending on their stability [123]. Additionally, various metals have been examined for utilization in optical sensors [124]. Luminescent optical fiber oxygen sensors have been fabricated by platinum tetrakis pentrafluorophenyporphine [125,126]. Three different polyelectrolytes acting as the polymeric matrices embedding the sensing material have been studied, such as poly(diallyldimethylammoniumchloride), polyethyleneimine and

Introduction

poly(allylamine hydrochloride) have been used for the entrapment of the luminescent material [127].

In general, the applications presented above demonstrates the important roles that NMs play in the detection step, and it may be concluded that NMs can be involved in any level of analysis process and for many purposes, while adapting the tools to the corresponding objectives by the modification of NMs and their characterization taking into account the possibility of using chromatography and capillary electrophoresis for this goal.

In this Doctoral Thesis, NMs have been employed in the detection step for the determination of vanillin in food samples, the determination of eugenol in different matrices and the detection of tyrosine in pharmaceutical samples. All of this will be explained in deep in Section 3.2.

Introduction

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detection of intracellular O2•-using Au NPs/cytochrome c as SERS nanosensors, Anal. Chem. 85 (2013) 9549–9555. [118] T. Bora, D. Zoepfl, J. Dutta, Importance of Plasmonic Heating on Visible Light Driven Photocatalysis of Gold Nanoparticle Decorated Zinc Oxide Nanorods, Sci. Rep. 6 (2016) 1–10. [119] Y.P. Kim, W.L. Daniel, Z. Xia, H. Xie, C.A. Mirkin, J. Rao, Bioluminescent nanosensors for protease detection based upon gold nanoparticle-luciferase conjugates, Chem. Commun. 46 (2010) 76–78. [120] M. Fehr, W.B. Frommer, S. Lalonde, Visualization of maltose uptake in living yeast cells by fluorescent nanosensors, Proc. Natl. Acad. Sci. 99 (2002) 9846–9851. [121] Z. Yue, F. Lisdat, W.J. Parak, S.G. Hickey, L. Tu, N. Sabir, D. Dorfs, N.C. Bigall, Quantum-dot-based photoelectrochemical sensors for chemical and biological detection, ACS Appl. Mater. Interfaces. 5 (2013) 2800–2814. [122] F. Mustafa, R.Y.A. Hassan, S. Andreescu, Multifunctional nanotechnology-enabled sensors for rapid capture and detection of pathogens, Sensors (Switzerland). 17 (2017). [123] R. Chavan, U. Desai, P. Mhatre, R. Chinchole, A review : Carbon nanotubes, Int. J. Pharm. Sci. Rev. Res. 13 (2012) 124–134. [124] J.W. Sadowski, J. Lekkala, plasmons excited in non-noble metals *, 6 (1991) 439– 444. [125] C. Elosua, N. De Acha, M. Hernaez, I.R. Matias, F.J. Arregui, Sensors and Actuators B : Chemical Layer-by-Layer assembly of a water – insoluble platinum complex for optical fiber oxygen sensors, Sensors Actuators B. Chem. 207 (2015) 683–689. [126] C. Elosua, N. De Acha, I.R. Matias, F.J. Arregui, Luminescent Optical Fiber Oxygen Sensor following Layer-by- Layer method, Procedia Eng. 87 (2014) 987–990. [127] N. De Acha, C. Elosúa, D. Martínez, M. Hernáez, I.R. Matías, F.J. Arregui, Sensors and Actuators B : Chemical Comparative study of polymeric matrices embedding oxygen-sensitive fluorophores by means of Layer-by-Layer nanosassembly, Sensors Actuators B. Chem. 239 (2017) 1124–1133.

Materials, Equipment and Methods

2. MATERIALS, EQUIPMENT AND METHODS

“See the invisible, believe the incredible, achieve the impossible.”

Joel Brown

n the present Doctoral Thesis different analytical tools have been used, which are detailed below, such as reagents, standard solutions, and the preparation of the I samples. Also, the instrumentation, equipment and other materials used, describing in more detail those that have been most important in the experimental work.

Materials, Equipment and Methods

2.1. Standards, reagents and samples

Standards and reagents

All the standards, reagents and solvents used throughout the experimental works of this Thesis were of analytical purity or higher. The compounds used in this memory are listed below grouped by families (Table 2.1.1)

The working solutions were daily prepared into the proper solvent from the stock standard solutions. The standard solutions were always kept preserved from light at a temperature of 4°C.

The buffer solutions were prepared in deionized Milli-Q water and the pH of the solution was adjusted by using 0.1 M NaOH or 0.1 M HCl solutions.

The solvents used in the experimental work are listed below:

 Ultrapure water (purification system Milli-Q, Millipore, Molshem, France);  Methanol, HPLC purity grade, Sigma-Aldrich;  Ethanol, HPLC purity grade, Panreac;  Acetonitrile, HPLC purity grade, Sigma-Aldrich;  Acetone, HPLC purity grade, Panreac.

These solvents were used for different purposes: (i) purification and solubilization of the synthesized materials, (ii) as extractants, (iii) to prepare standard solutions of the analytes, (iv) as a reaction medium, and (v) as background electrolyte (BGE) used in the electrophoretic system.

Materials, Equipment and Methods

Table 2.1.1. Compounds used in the experimental work.

Compound Purity (%) Physical state (25°C) Supplier

For the synthesis of Al-TiO2-NPs Aluminium acetylacetonate 99 Solid Sigma-Aldrich Lithium aluminium hydride 95 Solid Sigma-Aldrich Mesitylene 97 Liquid Sigma-Aldrich Titanium (IV) oxide ≥ 99 Solid Sigma-Aldrich Nitric acid 69 Liquid LabKem For the synthesis of Poly(STY-DVB) Styrene ≥ 99 Liquid Sigma-Aldrich Divinylbenzene 80 Liquid Sigma-Aldrich 2,2'-Azobis(2-methylpropionitrile) ≥ 98 Solid Sigma-Aldrich Acetonitrile HPLC grade Liquid Sigma-Aldrich For the synthesis of Poly(STY-DVB)-MMWCNTs Styrene ≥ 99 Liquid Sigma-Aldrich Divinylbenzene 80 Liquid Sigma-Aldrich 2,2'-Azobis(2-methylpropionitrile) ≥ 98 Solid Sigma-Aldrich MWCNTs 95 Solid NanoLab Sodium acetate ≥ 99 Solid Sigma-Aldrich Ethylene glycol 99 Liquid Panreac For the synthesis of CuSe@rGO Graphite 99.99 Solid Sigma-Aldrich Sodium nitrate ≥ 99 Solid Sigma-Aldrich Sulfuric acid ≥ 98 Liquid LabKem Potassium permanganate ≥ 99 Solid Sigma-Aldrich Hydrogen peroxide 30 Liquid Panreac Selenium powder 99.95 Solid Sigma-Aldrich Sodium sulfite ≥ 98 Solid Sigma-Aldrich Copper(II) sulfate pentahydrate 99 Solid Fluka Trisodium citrate dihydrate ≥ 99 Solid Sigma-Aldrich For the synthesis of Al-CuSe-NPs Aluminium acetylacetonate 99 Solid Sigma-Aldrich Lithium aluminium hydride 95 Solid Sigma-Aldrich Mesitylene 97 Liquid Sigma-Aldrich Selenium powder 99.95 Solid Sigma-Aldrich Sodium sulfite ≥ 98 Solid Sigma-Aldrich Copper(II) sulfate pentahydrate 99 Solid Fluka Sigma-Aldrich Trisodium citrate dihydrate ≥ 99 Solid

Materials, Equipment and Methods

Table 2.1.1. (continued) Acids used Hydrochloric acid 37 Liquid Panreac Hydrogen peroxide 30 Liquid Panreac Nitric acid 69 Liquid Labkem Phosphoric acid 85 Liquid Panreac Sulfuric acid 98 Solid Labkem Formic acid 95-97% Liquid Sigma-Aldrich Heptafluorobutyric acid ≥ 99 Liquid Sigma-Aldrich For the preparation of buffer solutions Ammonium acetate ≥ 98 Solid Sigma-Aldrich Nitric acid 99 Solid Sigma-Aldrich Di-sodium hydrogen phosphate 98 Solid Panreac Hydrochloric acid 37 Liquid Panreac Phosphoric acid 85 Liquid Panreac Sulfuric acid 99 Solid Sigma-Aldrich Sodium hydroxide 98 Solid Sigma-Aldrich Acetic acid ≥ 99.7 Liquid Sigma-Aldrich Boric acid ≥ 99 Soilid Sigma-Aldrich Potassium chloride 99 Soilid Sigma-Aldrich Sodium chloride ≥ 99.5 Soilid Sigma-Aldrich Ammonium phosphate 99.999 Soilid Sigma-Aldrich Potassium hydroxide 90 Soilid Sigma-Aldrich Sodium acetate ≥ 99 Soilid Sigma-Aldrich Used as analytes Vanillin ≥ 98 Soilid Fluka Fluoxetine hydrochloride ≥ 98 Soilid Sigma-Aldrich Venlafaxine hydrochloride ≥ 98 Soilid Sigma-Aldrich Citalopram hydrobromide ≥ 98 Soilid Sigma-Aldrich Sertraline hydrochloride ≥ 98 Soilid Sigma-Aldrich Agomelatine ≥ 98 Soilid Sigma-Aldrich Bupropion hydrochloride ≥ 98 Soilid Sigma-Aldrich Mirtazapine ≥ 98 Soilid Sigma-Aldrich Paroxetine hydrochloride ≥ 98 Soilid Sigma-Aldrich Trazodone hydrochloride ≥ 99 Soilid Sigma-Aldrich Eugenol 99 Liquid Fluka DL-3,4-Dihydroxymandelic acid 95 Soilid Sigma-Aldrich DL-3,4-Dihydroxyphenyl glycol 95 Soilid Sigma-Aldrich Norepinephrine ≥ 97 Soilid Sigma-Aldrich Epinephrine ≥ 95 Soilid Sigma-Aldrich

Materials, Equipment and Methods

Table 2.1.1. (continued) Dopamine 98 Soilid Sigma-Aldrich Tyrosine ≥ 98 Soilid Sigma-Aldrich Used as interferences Vanillic acid ≥ 97 Soilid Fluka Vanillyl alcohol ≥ 99 Soilid Sigma-Aldrich p-hydroxybenzaldehyde 98 Soilid Sigma-Aldrich p-hydroxybenzoic acid ≥ 99 Soilid Fluka Ascorbic acid 99.7 Soilid Panreac Citric acid 99.5 Soilid Panreac Vitamin B2 ≥ 98 Soilid Sigma-Aldrich Sodium bicarbonate 99.5-100.5 Soilid Sigma-Aldrich Glutamic acid 99 Soilid Sigma-Aldrich Glycine ≥ 99 Soilid Sigma-Aldrich Glucose ≥ 99.5 Soilid Sigma-Aldrich Fructose ≥ 99 Soilid Panreac Potassium sodium tartrate 99-102 Soilid Panreac Tyrosine ≥ 98 Soilid Sigma-Aldrich Sorbic acid ≥ 99 Soilid Sigma-Aldrich

Samples

The samples analyzed in the experimental work were:

Vanillin extract samples (from local markets).

Human urine samples were supplied by volunteers.

Human blood samples were supplied by volunteers.

Clove samples (from local market).

Cinnamon samples (from local market).

Toothpaste (from local market).

Deer urine and hair samples.

Pharmaceutial formulation samples.

Materials, Equipment and Methods

The different samples were prepared as follows:

Vanillin extract samples filtered through a sintered filter, and diluted directly in phosphate solution.

Human blank urine samples were supplied by healthy volunteers, pooled and kept at −20°C until their analysis as blank or quality control samples by spiking with known concentrations of the antidepressants.

Human blood samples were obtained from several healthy volunteers pooled and kept at 4°C until analysis. Blank blood samples utilized for calibration (consisting of at least 5 different concentrations covering a sufficient range for each case) were spiked by diluting appropriately the working standard solution of each analyte.

Clove or cinnamon powder sample: A 2.5 g of clove or cinnamon powder sample was placed into a 50 mL Erlenmeyer flask and 5 mL of ethanol was added. The mixture was shaken vigorously for 30 min and transferred to a centrifuge tube, and centrifuged at 3500 rpm for 10 min. After a settling time of 5 min, the supernatant was transferred into a 50 mL volumetric flask. Then, the solution was diluted with ethanol. Suitable dilutions were made before measurements to obtain solutions in Britton-Robinson buffer (0.1 M, pH 2).

Toothpaste sample: Approximately 0.5 g of the toothpaste sample was weighed into a 50 mL beaker containing 15 mL of water. The mixture was boiled for 5 min to allow total dissolution of the suspension. Suitable dilutions were made before measurements to obtain solutions in Britton-Robinson buffer (0.1 M, pH 2).

We obtained urine and hair samples of wild Iberian male red deer Cervus elaphus hispanicus that were harvested in hunts in Spain. Within 1 h after deer death, 10 mL of urine was directly extracted from the urinary bladder using a syringe, and 250 μL of 1 M HCl was immediately added. Deer urine samples were first kept at 4 °C, and then stored at −80 °C within 4 h of extraction. Then, the urine samples were diluted with water (1:100 v/v).

The deer hair samples were stored at -80°C. Then, 10 strands of hair were collected and cut a 2-cm piece of the middle part of each one with scissors and rule. Starting materials:

Materials, Equipment and Methods

10 pieces, 10 strands of hair 2 cm long each. Chop every single piece in 4 smaller pieces 0.5 cm long each to introduce the bundle of hair pieces in a 1.5 mL Eppendorf tube and allow an easier immersion in the extraction buffer. Add 0.2 mL of a 20 mM sodium acetate-acetic acid buffer solution (pH 5) with 0.08% in Triton X-100. Close the tubes, vortex vigorously for a few seconds and allow slow orbital shaking at room temperature for 1h. Centrifuge, the supernatant was collected and injected into a HPLC-MS system.

Pharmaceutial formulation samples. Three tablets were crushed in a mortar and they were homogenized. Then, a suitable amount was dissolved in deionized water; the solution was sonicated for 20 min and filtered. Suitable dilutions were made before measurements to obtain solutions in H3PO4 (0.1 M, pH 6) with L-tyrosine concentration within the calibration range.

2.2. Equipment

The following instruments and apparatus were used to carry out the works reported in this Thesis are listed and described below.

Spectrophotometer UV-Vis

Spectrophotometer UVI-Vis model Uvi Light & UVIKON XS reference 0M8307, SECOMAN equipment with a LabPower V3 50 software for the data acquisition and using 10 mm quartz cuvettes.

Figure 2.2.1. Illustration of the spectrophotometer UVI-Vis “Uvi Light & UVIKON XS”.

Materials, Equipment and Methods

Capillary electrophoresis

Capillary electrophoresis model Agilent G1600AX (Palo Alto, CA, USA) equipped with a UV/Vis diode array detector was used. The equipment is connected to a computer for the control and data processing which were performed using the Agilent ChemStation software.

Figure 2.2.2. Illustration of the capillary electrophoresis “Agilent G1600AX”.

Liquid chromatography

The equipment used for the liquid chromatography was a Chromatography System Agilent 1200. This system consists of a degasser, a liquid chromatographic pump, an autosampler, a temperature-controlled column compartment and a diode array detector. The detector is coupled to a system of data acquisition and processing system (Agilent ChemStation HPLC). Figure 2.2.3 shows the different modules of the system described.

Materials, Equipment and Methods

Figure 2.2.3. System scheme of Agilent 1200 HPLC.

Mass spectrometry

An Agilent 6110 Series LC/MSD system (equipped with a quadrupole analyzer) via an electrospray atmospheric pressure ionization interface was also used.

Figure 2.2.4. Illustration of Agilent 6110 Series LC/MSD instrument.

Electrochemical system

The equipment used in the electrochemical experiments was performed with a CHI812D electrochemical analyzer from CH Instruments (Austin, Texas, USA) controlled with a

Materials, Equipment and Methods

computer for the control and data processing which were performed using the Electrochemical Analyzer software.

Figure 2.2.5. Illustration of CH Instruments.

Another electrochemical equipment was used in the electrochemical experiments: Wuhan Corrtest Instruments Corp., Ltd. (Hubei, China) controlled with a computer for the control and data processing which were performed using the Electrochemical Analyzer software.

Figure 2.2.6. Illustration of Wuhan Corrtest Instruments.

Electrodes and work cell

To perform the electrochemical studies of the developed sensors in this Thesis, a conventional three-electrodes system has been used, consisting of:

 Working electrode: Glassy carbon electrode of 3.0 mm diameter (CH instruments), modified with hybrid material.

 Reference electrode: Ag/AgCl/KClsat (CH instruments).  Auxiliary electrode: Platinium wire.  Electrochemical glass cell (10 mL).

Materials, Equipment and Methods

Figure 2.2.7. Conventional system of three-electrodes (working, reference and auxiliary) used in electrochemical measurements.

Screen-printed carbon electrodes (SPCEs) system (DRP-C110, Dropsens) strip’s general dimensions: 3.4 x 1.0 x 0.05 cm. Reference electrode and electric contacts made of silver.

Cable connector between SPEs and potentiostat (2 WE, BICAC, Dropsens). Using SPEs in conjunction with home made electrochemical flow cell, by dipping the SPEs in solution.

Figure 2.2.8. illustration of the screen-printed electrode and its cable connector.

Supercritical fluid system

Supercritical carbon dioxide (sc-CO2) extraction was carried out at JASCO System (Tokyo,

Japan) comprised of CO2 tube, high pressure CO2 delivery pump (PU-1580-CO2, JASCO,

Materials, Equipment and Methods

Japan), CO2 flow-meter (JASCO, Japan), temperature controller from Hewlett Packard 5890, Series II gas chromatograph (Bothell, WA, USA) and Automated Back Pressure Regulator (BP-1580-81, JASCO, Japan).

Figure 2.2.9. Analytical lab-scale supercritical fluid system.

Portable Raman spectrometer

Raman measurements were carried out by a portable Raman spectrometer model B&W TEK, known as i-Raman BWS415, with a wavelength of 785 nm and a maximum laser output power at 300 mW at the sample. This instrument consists of a CCD detector (12 μm x 12 μm pixel size, 2048 pixels). The Raman spectra were measured using a lens with a magnification of 100x and a detection range between 100-3000 cm-1. The acquisition and processing of data, as well as the control of the instrument, were carried out with the BWSpec4TM software.

Furthermore, the BAC151B is a Raman microscope that is compatible with B&W Tek Raman systems. It is designed to offer the highest level of flexibility in facilitating Raman sampling for a variety of applications. The BAC151B can be configured to fulfill the exact requirements of your application. The unique dual laser wavelength port provides the flexibility for one system to be coupled with two different laser wavelengths. The integrated camera allows for precision Raman sampling through camera monitoring of the laser beam and imaging details. When coupled with B&W Tek’s portable Raman spectrometers, it provides the advantages of a Raman microscope at a fraction of the

Materials, Equipment and Methods

cost of most research instruments. The laser beam is focused on the surface of the sample using the 20x / 0.4 Zeiss and 100x / 1.25 in oil immersion, from Nikon.

Figure 2.2.10. Illustration of the portable Raman spectrometer model B&W TEK, known as i- Raman BWS415 and BAC151B microscope.

Dynamic light scattering (DLS)

The dynamic light scattering instrument applied by Zetasizer Nano (model ZEN3600) from Malvern (Worcestershire, UK) measures the particle size distribution using laser dynamic light scattering and analysis of the Brownian motions of particles in the media. DLS is controlled with a computer for the control and data processing.

Figure 2.2.11. Illustration of Zetasizer Nano instrument model ZEN3600.

Materials, Equipment and Methods

Fourier transform infrared (FT-IR)

For infrared spectrum measurement a Fourier transform infrared (FT-IR) spectroscope, model FT/IR 4200 (Jasco, Japan) was utilized.

Figure 2.2.12. Illustration of FT-IR spectroscope instrument model FT/IR 4200.

X-Ray Diffraction (XRD)

X-Ray Diffraction (XRD) measurements were made using a Philips X´Pert MPD, X-Ray diffractometer with CuKα radiation and graphite monochromador. The angular 2 theta diffraction range was between 3 and 100°. The data were collected with an angular step of 0.02° at 1.5 s per step.

Electron microscopy (TEM and SEM)

For the characterization of the NMs, the following electronic microscopy equipment were used:

 Transmission electron microscopy (TEM) Jeol JEM 2011 operating at 200 kV and equipped with an Orius Digital Camera (2×2 MPi) from Gatan. The samples were prepared by deposition of a drop of the synthesized material suspension onto a lacey carbon/format-coated copper grid. The digital analysis of the HRTEM micrographs was done using Digital Micrograph TM 1.80.70 for GMS 1.8.0 Gatan. Also, energy dispersive X-ray (EDX) spectroscopy was performed.  Scanning electron microscopy (SEM) ZEISS GeminiSEM 500 (Germany) operating at acceleration voltage 0.02-30 kV, probe current 3 pA - 20 nA, store resolution up to 32k × 24k pixels, and magnification from 50 up to 2x106. Equipped with

Materials, Equipment and Methods

several detectors: in-lens secondary electron detector, in-lens energy selected backscatter detector (EsB), annular STEM detector (aSTEM 4) and EBSD detector (electron backscatter diffraction) investigation of crystalline orientation. The samples were prepared onto a lacey carbon by deposition of the synthesized material.

Other instruments, apparatus and materials

 Experimental configuration and materials used for synthesis of aluminium

nanoparticles (Al-NPs) and sodium selenosulfate (Na2SeSO3) (Figure 2.2.11), which is based on the following elements: three necks round flask, temperature control system with a Pt 100 temperature sensor heating mantle with magnetic agitation connected to temperature controller and reflux system. The magnetic stirrer AGIMAN with adaptable heating mantle is connected to an electronic controller ELECTEMP (JP Selecta, Barcelona, Spain) with a Pt 100 temperature sensor allowing the temperature regulation up to 350 °C by an electronic energy regulator synchronized with flashing lamp when the heater is working. Also, it allows adjusting the stirring speed to 1600 rpm using an electronic speed controller.

Figure 2.2.13. Mounting used in the synthesis of Al-NPs and Na2SeSO3.

Materials, Equipment and Methods

 High speed Ultracentrifuge controlled by microprocessor and temperature regulation (CENTROFRIGER-BL-II model 7001669, JP Selecta, Barcelona, Spain).  Analytical Balance Gram Precision (Mettler, model AE240).  Microcentrifuge Biosan Microspin 12 (LabNet Biotecnica S.L., Spain)  pH-meter model Crison Basic 20 combined with a glass electrode (Allela, Barcelona, Spain).  Ultrasonic bath 50 W, 60 Hz (J. P. Selecta, Barcelona, Spain).  Digital magnetic stirrer with heating and ceramic coated plate, LBX H03D series.  Vortex stirrer LBX V05 series, with speed control.  UV lamp 230 V (wavelength of 254/365 nm, model E2107, Consort nv, Turnhout, Belgium).

 Si/SiO2 wafers from Pure Wafer (San José, California, USA).  Hamilton 250 microsyringe (Analysis Vínicos, Spain).  Eppendorf vials.  Centrifuge tubes.  Syringes.  Needles.  Nylon syringe filters with pore size of 0.45 μm (Millipore, Madrid, Spain).  Quartz cuvettes with four transparent faces.  Glass and polypropylene vials.  Chromatographic paper (Whatman grade 1 CHR, Whatman, Maldstone, Kent, UK).  Micropipettes.  Laboratory Glassware, class A.  Fused-silica capillary 75 μm i.d., 365 μm outside (Polymicro Technologies, Phoenix, Arizone, USA).

2.3. Methods

The most relevant methodologies and procedures used in the experimental work of this Thesis are described in this section.

2.3.1. Synthesis, decoration and functionalization of nanoparticles

. Synthesis of magnetic multiwalled carbon nanotubes (MMWCNTs)

MMWCNTs were prepared by in situ high temperature combination of the magnetic precursor [iron(III)] and MWCNTs, according to the previously described procedure [2],

Materials, Equipment and Methods

with slight modifications. This synthesis involves the mixture of 140 mg of FeCl3.6H2O and 40 mg of MWCNTs, which was suspended in 7.5 mL of ethylene glycol in a glass vial. Then, 0.36 g of sodium acetate was added, and the solution was stirred, sonicated for 10 min and then left at room temperature for 60 min. Afterward, the vial was heated at 200°C for 48 hr to complete the reaction. After cooling down to room temperature, the synthetic product was washed with 10 mL of distillated water for five times. The MMWCNTs were then separated by applying a magnet, and the nanoparticles obtained were dried at 80°C and stored.

Figure 2.3.1.1. Scheme of synthesis and preparation of MMWCNTs.

. Synthesis of poly(styrene-co-divinylbenzene)

The polymerization mixture consisted of 4.5 mL of styrene (STY, 9.79 mM) and divinylbenzene (DVB, 23.69 mM), 0.36 g of 2,2'-Azobis(2-methylpropionitrile) (AIBN) and 25 mL of ACN which were added into an Erlenmeyer flask, sonicated for 5 min, and then purged for 10 min with N2 gas and closed. Then, the mixture was stirred in a water bath at 70°C for 24 h to complete the polymerization process and the resultant particles were repeatedly rinsed with MeOH. The final product was then filtered and dried under low pressure [3].

Materials, Equipment and Methods

Figure 2.3.1.2. Scheme of synthesis of poly(styrene-co-divinylbenzene).

. Functionalization and decoration of MMWCNTs

MMWCNTs were functionalized with poly(STY-DVB) by mixing (9.79 mmol) STY, (23.69 mmol) DVB, 0.15 g of MMWCNTs, 25 mL of ACN and 0.36 g of AIBN at 70°C for 24 h [3,

4]. The mixture was sonicated for 5 min, purged with N2 gas for 10 min and then closed. The resulting product was then collected, washed several times with water together with mechanical agitation and dried in a vacuum desiccator for 24 h.

Figure 2.3.1.3. Scheme of functionalization and decoration of MMWCNTs with poly(styrene-co- divinylbenzene).

. Synthesis of aluminium doped TiO2 nanoparticles (Al-TiO2-NPs)

The synthesis of hybrid NPs was carried out, according to the following method as Figure 2.3.1.4 illustrates. Firstly, Al-NPs were prepared according the described procedure [1].

The aluminium acetylacetonate (Al(acac)3, 10 mmol) was added to a three-neck round bottom which was already contained mesitylene solution with magnetic bar. Then lithium aluminium hydride (LiAlH4, 30 mmol) was added to the mixture. The reaction was purged with N2 gas during reflux with stirring for 72 hours at 165°C. After cooling to 25°C, a gray-colored precipitate was formed which was crushed and kept to dry under

Materials, Equipment and Methods

low pressure for 5 hours. Wash the isolated solid product well with 25 ml portions of cold MeOH three times, to avoid any high temperatures (exothermic reaction) between solvent and Al-NPs. The unreacted materials were washed three times with MeOH. The resulting product was filtered and dried at 25°C under low pressure. The preparation of

Al-TiO2-NPs started by mixing of TiO2 (0.5 g) previously digested in nitric acid (0.1 M, 25 mL) during 3 hours and Al-NPs (0.5 g) previously prepared. The final product was filtered and dried at 25°C under low pressure, obtaining an Al-TiO2-NPs as light-gray powder.

Figure 2.3.1.4. Scheme of synthesis of Al-TiO2-NPs.

. Synthesis of reduced graphene oxide docurated copper selenide nanoparticles (CuSe@rGO)

For the synthesis of reduced graphene oxide (rGO) with copper selenide nanoparticles (CuSe-NPs) in supercritical fluids (SCFs), it was necessary several steps presented in

Figure 2.3.1.5. Firstly, Na2SeSO3 was prepared by mixing 10 g of selenium powder with 100 g of anhydrous sodium sulfite in 500 mL of distilled water. Then, this reaction was stirring for 8-10 h at 80°C, obtaining a fresh Na2SeSO3 solution (Figure 2.2.13).

On the other hand, the synthesis of graphene oxide (GO) was prepared according to a modified Hummers method [5]. This synthesis started by mixing 0.5 g of graphite, 0.5 g of sodium sulphite and 23 mL of concentrated H2SO4 into 100 mL erlenmeyer flask in ice bath maintaining temperature below 20°C. This mixture was stirring for 4h. Then, 3.0 g of KMnO4 were added slowly to the reaction mixture and stirring the mixture for 1h. After this hour, the ice was removed, and the reaction was stirring another hour more

Materials, Equipment and Methods

and heating the mixture at 35°C. After this step, 46 mL of deionized water were added, and the reaction was stirring and heating at 95°C for 2h without allowing the mixture to boil. Subsequently, the heater was turned off and allowed it to cool in room temperature. Then, 100 mL of deionized water were added and stirred the mixture for

1h. The next step was the addition of 10 mL of 30 % H2O2 for 1 h with constant stirring. The GO was formed and finally, the product was washed with deionized water several times until the pH became neutral, indicating the final product doesn’t contain residual salts and acids.

The next step was the preparation of copper selenide-graphene oxide (CuSe-GO), which consists of an aqueous solution by mixing 2 mL of CuSO4.5H2O, 2 mL of sodium citrate,

2 mL of Na2SeSO3 solution and 0.2 g of synthesized GO. This solution was mixing with constant stirring for 10 min and sonicated for 10 min.

The last step was the reduction of copper selenide graphene oxide (CuSe@rGO) under supercritical conditions. The synthesis was carry through using sc-CO2 as a reaction medium adapted from [6]. For each reaction, 6 mL of CuSe-GO was taken in a 10 mL stainless steel column. Then, sc-CO2 was charged into the column. The synthesis was carried out at 200°C and 20 MPa and the reaction was maintained for 1 h. A depressurization time of the system was about 5 min and it was used to bring back the system until atmospheric pressure.

Figure 2.3.1.5. Schematic synthesis of copper selenide-reduced graphene oxide in supercritical fluids medium.

Materials, Equipment and Methods

. Synthesis of aluminium doped copper selenide nanoparticles (Al-CuSe-NPs)

Firstly, Al-NPs were prepared according the previous procedure (section 2.3.1). The synthesis of Al-CuSe-NPs consists of an aqueous solution of 1.0 mL of CuSO4.5H2O (0.5

M), 1.0 mL of trisodium citrate (0.1 M), 1.0 mL of Na2SeSO3 (0.25 M) solution and 0.25 g Al-NPs, after sonication for 15 min, the mixture was mixed with constant stirring for 2 h. The final product was filtered and dried at 25°C under low pressure, obtaining an Al- CuSe-NPs as powder.

Figure 2.3.1.6. Scheme of synthesis of Al-CuSe-NPs.

2.3.2. General Procedures used in the extraction/separation of analytes.

. Preconcentration of antidepressants by magnetic solid-phase extraction

Magnetic solid-phase extraction (MSPE) is a new procedure for the preconcentration of target analytes from large volumes based on the use of magnetic or magnetizable adsorbents. The procedure followed for the preconcentration of antidepressants by MSPE is illustrates in Figure 2.3.2.1.

Materials, Equipment and Methods

Figure 2.3.2.1. Schematic representation of steps required for the MSPE procedure.

At first, 500 mg of the poly(STY-DVB)-MMWCNT composite was put into a conical flask, conditioned with (2 × 3) mL of MeOH and then with deionized water. The supernatant was then separated with a magnet, discarded, and 50 mL of standard solutions of the analytes or of spiked urine samples, previously adjusted to pH 5.3, were added into the conical flask. The mixture was stirred at room temperature for 5 min to create a homogenous dispersion and the magnetic polymer containing the adsorbed antidepressants was rapidly removed from the solution applying a strong external magnetic field and the supernatant was discarded. The magnetic polymer was then washed with 10 mL of Milli-Q water, and the antidepressants were eluted by rinsing with 1.0 mL of MeOH containing 2.0% of acetic acid. The resultant extract was injected into the CE system.

. Preconcentration of catecholamines by magnetic solid-phase extraction

Firstly, 500 mg of the magnetic composite was put into a conical flask, conditioned with 2×3 mL of MeOH and then with deionized water. The supernatant was separated with a magnet, discarded, and 50 mL of the standard solutions of the analytes or of urine samples (pH 5.2), were added into the conical flask. For the extraction of the analytes from deer hair samples, 2 mL of standard solution of the analytes or of extracted deer hair samples, were added into 10 mL beaker. The mixture was then stirred at room

Materials, Equipment and Methods

temperature for 5 min to create a homogenous dispersion and the magnetic polymer containing the adsorbed catecholamines was rapidly removed from the solution applying a strong external magnetic field. The supernatant was discarded, and the catecholamines finally eluted by rinsing with 1.0 mL of MeOH containing 0.01% of HFBA, and the eluent dried under a stream of nitrogen gas at room temperature. The residue was finally dissolved in 250 μL of initial mobile phase and an aliquot of 1.0 μL was injected into the LC-MS system.

. Extraction of antidepressants by poly(styrene-co-divinylbenzene)-coated glass blood spot

Firstly, glass sheets were cut into 10×10mm squares, and the surface of each square was first activated by dipping in 1.0M NaOH solution for 2–3 h. Then, 30 mg of poly(styrene- co-divinylbenzene) powder were suspended in 5 mL of tetrahydrofuran (THF) containing 10 mg of PVC powder dissolved. A small amount (10 μL) of this mixture was dropped onto the center of the square glassy substrate, and the THF was then evaporated at room temperature to obtain a polymer coating on the square glassy substrate. After that, 10 μL of either each standard or a blood spiked sample was spotted onto each square glassy substrate containing the polymer and then the mixture was dried. The glass sheet was cut with hand-held diamond glass cutter to obtain 10×10mm squares glassy containing blood. After that, the antidepressants were eluted with 1.0 mL of 2.0% acetic acid in MeOH, and the eluent dried under a stream of nitrogen gas at room temperature. The residue was finally dissolved in 200 μL of mobile phase (5mM ammonium acetate, pH 3.0) and an aliquot of 5.0 μL was injected into the capillary liquid chromatography-mass spectrometry (CLC-MS).

Materials, Equipment and Methods

Figure 2.3.2.2. Scheme of the general procedure for the synthesis and extraction of the selected antidepressants by PS-DVB-coated glass blood spot.

. Preparation of modified screen-printed electrodes

Prior to modified screen-printed carbon electrodes (SPCEs), synthezised Al-TiO2-NPs or Al-CuSe-NPs were dispersed in water (0.5% Nafion, v:v) by ultrasonication, obtaining individual concentrations of 1 mg mL−1. Nafion was used for the SPCE modification, because it is a compound usually employed as a cation conduction membrane and electron barrier to prevent usual interfering agents in analytical determinations . After that, 2 µL of the dispersed Al-TiO2-NPs or 6 µL of the dispersed Al-CuSe-NPs was added onto the surface of the electrode and, after 15 min under infrared light, the electrode was ready to use.

Figure 2.3.2.3. Scheme of preparation the modified SPCEs by Al-TiO2-NPs and Al-CuSe-NPs.

Materials, Equipment and Methods

. Preparation of modified glassy carbon electrode (GCE)

The GCE was polished by using 0.3 and 0.05 μm α-alumina slurry on a polishing cloth until a mirror like appearance was observed. Later, as cleaned GCE was washed thoroughly with distilled water [7]. CuSe@rGO materials were dispersed in water (0.5% Nafion, v:v) by ultrasound to obtaining individual concentrations of 1.0 mg mL-1. Films formed from nafion-solubilized nanoparticles are more uniform than those casted by organic solvent [8]. Each modiﬁed electrode was prepared by casting 6.0 µL of the dispersed CuSe@rGO materials onto the surface of the electrode. After drying under infrared light for 10 min, it was rinsed with water and ready to use.

Figure 2.3.2.4. Scheme of preparation the modified GCE by CuSe@rGO.

Materials, Equipment and Methods

References

[1] S. Reddy, G. Krishnamurthi, Chemical synthesis of aluminum nanoparticles, J. Nanopart. Res. 15 (2013) 1715-1725. [2] V. Moreno, M. Zougagh, A. Ríos, Hybrid nanoparticles based on magnetic multiwalled carbon nanotube-nanoC18SiO2 composites for solid phase extraction of mycotoxins prior to their determination by LC-MS, Microchim. Acta 183 (2016) 871-880. [3] S.E. Shim, S. Yang, M. Jin, Y.H. Chang, S. Choe, Effect of the polymerization parameters on the morphology and spherical particle size of poly(styrene-co- divinylbenzene) prepared by precipitation polymerization, Colloid Polym. Sci. 283 (2004) 41-48. [4] M.J. Lerma-Garcia, M. Zougagh, A. Ríos, Magnetic molecular imprint-based extraction of sulfonylurea herbicides and their determination by capillary liquid chromatography, Microchim. Acta 180 (2013) 363-370. [5] W. S. Hummers Jr, R.E. Offeman, Preparation of graphitic oxide, J. Am. Chem. Soc. 80 (1958) 1339-1339. [6] V. Moreno, E.J. Llorent-Martínez, M. Zougagh, A. Ríos, Decoration of multi-walled carbon nanotubes with metal nanoparticles in supercritical carbon dioxide medium as a novel approach for the modification of screen-printed electrodes, Talanta 161 (2016) 775-779. [7] R.N. Hegde, B.E. Kumara Swamy, B.S. Sherigara, S.T. Nandibewoor, Electro-oxidation of atenolol at a glassy carbon electrode, Int. J. Electrochem. Sci. 3 (2008) 302-314.

[8] K. Murtada, S. Jodeh, M. Zougagh, A. Ríos, Development of an Aluminium Doped TiO2 Nanoparticles-modified Screen Printed Carbon Electrode for Electrochemical Sensing of Vanillin in Food Samples, Electroanalysis 30 (2018) 696-974.

Results and Discussion

3. RESULTS AND DISCUSSION

“Optimism is the one quality more associated with success and happiness than any other.”

Brian Tracy

his section includes all the results obtained throughout the experimental work carried out in this Thesis, with the corresponding discussion. Two different parts T are distinguished: (i) the use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation; and (ii) the utilization of nanoparticles in electrochemical sensors for food and pharmaceutical samples monitoring.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

3.1. Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

anomaterials are important scientific tools being vastly explored in various fields such as alimentary, pharmaceutical and bioanalytical. Their high Nsurface area and chemical reactivity, together with their catalytic efficiency, excellent adsorption capacity and optical properities, make the NMs as an excellent candidate to be used in the whole analytical process. The ability to bind specific ligands on their surface increases the selectivety and sensitivity of analytical methods. NMs can be used as a platform able to their modification in order to improve the sensitivity, rapidity of time-response and providing more stable signal.

On the other hand, sample preparation is an essential step of the analytical process, which must be carried out with great caution, since it may cause errors (e.g. loss of and contamination of analytes), significantly affecting the final result. This step includes operations related to sampling, preservation of samples, storage and isolation and/or enrichment of analytes. This is particularly significant for biological samples, which are complex matrices and they usually contain ultratraces levels of analytes. Therefore, it is usually necessary, not only to isolate the analytes from the sample, but also to enrich them before their detection and quantification. In this way, the analytes isolated from the matrix eliminate the impact of the substances that interfere in the sample.

Solid-phase extraction (SPE) is a sample preparation technique routinely used in analytical laboratories for the extraction of analytes from a complex matrix. This sample preparation technique enables the extraction, cleanup and concentration of analytes prior to their quantification. SPE prevents most problems encountered with liquid-liquid extraction and improves quantitative recovery yields. This technique is rapid (most extraction in less than 30 min), easy to perform and can be automated. In addition, low amount of solvents is handled and this technique is fully adapted for the pre-treatment of complex matrices such as urine, blood, food samples, water etc.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

It is commonly known that the sorbent plays a crucial role in the SPE technique, which is directly related to the analytical sensitivity, precision, and selectivity parameters. Analytical applications can use nanosized and/or non-nanosized as solid sorbents in SPE techniques as well as the formation of hybrid materials. For this purpose, there are several ways to use these materials; packed in cartridge, dispersed or as coatings to extract and preconcentrate analytes from complex matrices.

This section aims to investigate the use of (nano)materials and their analytical applicability through different extraction techniques focused on the determination of several analytes of bioanalytical interest. This aspect is depicted in this chapter by the developed:

 The first work is a review which provides a general overview of the strategies for extraction of antidepressants from biological species using nanomaterials. This work is presented with aiming to highlight the currently developed nanomaterials used as nano-sorbents for various biological specimens.  The second work is based on the development of a straightforward decoration method based on magnetic multi-walled carbon nanotube-poly(styrene-co- divinylbenzene) composites to analyse antidepressants in human urine samples by magnatic solid phase extraction prior to their determination by capillary electrophoresis.  The third work is focused on the synthesis of a synthetic sorbent material with magnetic susceptibility, decoration of magnetic multi-walled carbon nanotube- poly(styrene-co-divinylbenzene) for the selective extraction of catecholamines and some related coumpounds in urine and dark ventral patch hair of Iberian male red deers prior to their determination by liquid chromatography-mass spectrometry.  The fourth work consists of a new and simple method based on poly(styrene-co- divinylbenzene)-coated glass blood spot, that was developed and successfully checked for the determination and monitoring of seven types of antidepressants in just a humun blood drop, by capillary liquid chromatography-mass spectrometry.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

3.1.1. Strategies for Antidepressants Extraction from Biological Species Using Nanotechnology: A Review

In preparation to be Submitted

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Abstract

Accurate and precise monitoring of antidepressants drugs represents a crucial step for the adequate and personalized treatment of several psychological disorders such as depression, which nowadays represent a social, economic and health major concern. Electrochemical, chemical, and biological methods have been traditionally developed for extraction and detection of antidepressants, even though their several limitations such as the requirement of post-treatment, limited efficiency, and elevate costs. Nanotechnology, instead, has observed tremendous growth in the last two decades, especially regarding their many biological applications, such as antibacterial or biosensors, as well as in many different applications related to medicine. More recently, nanotechnology has emerged as an excellent alternative for extraction of antidepressants over conventional techniques, as nanomaterials can be efficiently used as sorbents due to their small size and their high specific surface area which enhances their high reactivity. This review focuses on the use of different nanomaterials for antidepressants extraction from biological species and discusses not only the advantages but also the major limitations of using such nanomaterials. Potential alternatives to overcome these drawbacks are discussed as well.

Keywords: Antidepressants extraction, biological specimens, nanotechnology, nanoparticles, nanocomposites.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

GRAPHICAL ABSTRACT

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

1. Introduction

It is estimated by The World Health Organization (WHO) that major depressive disorder currently affects more than 350 million people globally, a currently increasing prevalence due to the population growth and ageing. This disease, therefore, represents the leading cause of disability worldwide and, consequently, possess a remarkable impact not only on each patient’s health, but also has become a social concern as of their consequences in terms of costs (i.e. health systems direct costs, suicide-related and workplace costs) [1–3]. Even though non-pharmacological (psychological) treatments are available, a trend of the increased use, or even the misuse of pharmacological treatments with the different types of antidepressant medication (Table 3.1.1.1) is detected. The antidepressants usually exert their function by inhibiting the reuptake of neurotransmitters (e.g. serotonin, dopamine, noradrenaline or norepinephrine) through selective receptors thereby increasing the concentration of specific neurotransmitter around the nerves in the brain. Nevertheless, the role of neurotransmitters at improving mood and emotion is not fully understood [4–6].

Nevertheless, these agents should be prescribed based on the best available evidence, as far as it is widely recognized that each patient under antidepressants treatment should be individually monitored due to the wide interindividual and intraindividual variability observed in their clinical response, and the potential occurrence of side effects frequently detected. The confirmed correlation between plasma concentrations of these drugs and tissue concentrations should therefore be confirmed through this monitoring to obtain evidences on the drug concentration at effector sites in tissue compartments of interest.

Consequently, an accurate therapeutic monitoring of these treatments is required [7– 9], not only to ensure the patients therapy compliance, but also to individualize and optimize the dosage regimens, as well as to optimize resources and to improve the overall mental health care as well [1,10–13]. However, the adoption of such monitoring as a routine point of care is quite slow of psychiatrists, even though the awareness on the significance of inter- and intra-patient variability in pharmacokinetics may affect response to antidepressants therapy is currently growing to optimize antidepressant

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

treatment response. TDM can be therefore used to increase the safety of antidepressant pharmacotherapy with certain agents (e.g., tricyclic antidepressants) by avoiding toxic levels of medication, enhancing therapeutic response, and speeding the antidepressant response by improving the dose adjustment and also helping clinicians to confirm adherence to medication [14].

Thus, confirming the levels of the antidepressants prescribed within their corresponding therapeutic range has become essential for treating these diseases, and thus the concentration of these drugs needs to be determined in different biological species (i.e. blood, serum, urine, saliva and/or human milk) , as confirmed in previous studies (e.g. for the tricyclic antidepressant, 100-300 µg L-1 distinct ranges of therapeutically optimal plasma concentration are required) [15,16].

With regard to the quantitative analysis of the selected drugs, several separation and detection techniques have been applied: spectroscopy [17], capillary electrophoresis (CE) [18], capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS) [19], thin-layer chromatography (TLC) [20], gas chromatography-mass spectrometry coupling (GC-MS) [21], liquid chromatography with UV diode array detection (LC-DAD) [22], fluorescence [23], chemiluminescence [24] or electrochemical detection (ECD) [25], gas chromatography using flame ionization detection (GC-FID) [26] and liquid chromatography coupled with-mass spectrometry (LC-MS) [27]. Most of these techniques allow to separate different components from complex mixtures like biological matrices. Therefore, an efficient drug monitoring approach usually requires of different techniques for the biospecimen treatment, such as liquid-liquid microextraction [28,29], solid phase extraction [30,31], magnetic solid phase extraction [32], stir bar sorptive extraction [33], pressurized liquid extraction [34] or microwave-assisted extraction [35].

However, several limitations are also encountered. For instance, the aforementioned extraction techniques require the correct selection of a robust sorbent to be successfully applied under variable conditions. Additionally, the efficiency of extraction, the reusability and disposal of the sorbents with antidepressants must be estimated, representing some of the major issues to consider. Also, performing such techniques is

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

not exempt of difficulties and higher costs. Alternatively, nanotechnology possess certain features which makes this discipline attractive to overcome the drawbacks found when using the conventional extraction techniques, although the correct selection of the nanomaterials with an adequate conformation or structure must be necessary considered.

Nanotechnology is conducted at the nanoscale objects, usually ranging from 1 to 100 nanometer (nm), so the prefix nano represents a key to open many doors [36], as confirmed by their numerous applications in medicine, electronics, food, cosmetic industries, space, as well as in solar cell and water treatment processes [37]. More indeed, nanomaterials have excellent thermal, mechanical, optical, structural, and morphological properties which enable them to be applied in so many different applications [38,39]. The distinct advantages offered by nanoparticles in drug extraction can be ascribed to their physical stability and the possibility of modifying the formulating nanomaterials to achieve controlled and selective retention and release of the analytes. Consequently, this ability of sustained release offers an opportunity for product life cycle management by developing formulations of drugs that are going off patent.

Consequently, a new perspective on the application of nanomaterials to the extraction of drugs and drug delivery is being considered and new approaches are currently being developed [40,41]. In this sense, antidepressants are drugs with a remarkable frequency of prescription worldwide and, considering the importance of monitoring these pharmacological treatments, require of new extraction approaches, which are currently being developed and applied. In this review, the recent strategies for the extraction of antidepressants from biological samples using different types of nanomaterials and their modifications with different capabilities are reviewed and discussed.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Table 3.1.1.1. Classification of the main types of antidepressants. Class of antidepressants Examples Selective serotonin reuptake inhibitors Citalopram, fluoxetine Serotonin-norepinephrine reuptake inhibitors Duloxetine, venlafaxine Tricyclic antidepressants Desipramine, imipramine Monoamine oxidase inhibitors Phenelzine, tranylcypromine Reversible inhibitors of monoamine oxidase A Moclobemide, toloxatone Tetracyclic antidepressants Mirtazapine, setiptiline Noradrenergic and specific serotonergic antidepressants Aptazapine, mianserin

2. Mechanisms of antidepressants extraction

Adsorption represents the most common mechanism for antidepressants extraction. More precisely, the antidepressant molecule is attached on the surface of the sorbent either by chemisorption through chemical, usually covalent, bonds to stick the adsorbate to the sorbent, or by physisorption through Van der Waals interactions between the substrate and adsorbate. In the latter case, an electrostatic interaction occurs among antidepressants and sorbent. Due to π-π and hydrogen bonding interactions between the antidepressants and the sorbent, the extraction recoveries of antidepressants can be further improved, wherein the retention behavior is mainly due to π-π interactions and the lack of additional hydrophobic interactions between the sorbent and antidepressants [42]. Nanomaterials with excellent adsorption properties possess two main properties: their innate specific surface area and the ability for external functionalization [43]. High surface area, adsorption activity, the superficial location of atoms, lack of internal diffusion resistance, and a high surface binding energy are additional factors that determine the adsorption capability of the nanomaterial [44,45].

Another common mechanism involved at the extraction of antidepressants is absorption. This mechanism involves the antidepressant molecules crossing the surface and entering the volume of the sorbent material. Again, there can be either chemical and/or physical absorption. The binding properties are key parameters for understanding these sorption mechanisms, so the sorption isotherm has been studied to describe the mutual behavior between the sorbent and the solutes. More indeed,

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

four different isotherm models including Langmuir [46], Freundlich [46], Dubinin– Radushkevich [47] and Temkin [46] are used to evaluate these interactions between the sorbent and the solutes.

The Freundlich isotherm, presented by Freundlich in 1906 [48], was the first isotherm model proposed for sorption processes. This model is applied for the non-ideal sorption on heterogeneous surfaces, as well as for multilayer sorption. The linear form of the Freundlich equation that can be fitted, as defined

1 log q = logK + logC , f n e where q represents the metal’s uptake per weight unit of sorbent (mg g-1), whereas Ce corresponds to the residual concentration at equilibrium of the metal ion in solution (mg

-1 -1 L ), Kf is the sorption capacity (mg g ), and 1/n the sorption intensity [49].

The Langmuir isotherm describes the adsorption of an analyte or adsorbate onto the surface of the adsorbent assuming of monolayer coverage equivalent in active sites and on a homogeneous surface. Its basic features are represented by the dimensionless constant called the equilibrium parameter (RL), defined as

1 RL = , 1+KLC0

-1 where KL is the Langmuir constant related to the energy of adsorption (mL mg ) and C0 is the initial concentration of the adsorbate (mg mL-1) [50].

On the other hand, the Dubinin-Radushkevich isotherm model explains an empirical adsorption model which is generally applied to express the mechanism of adsorption with Gaussian energy distribution onto heterogeneous surfaces [51]. The Dubinin- Radushkevich isotherm is expressed as follows [52]:

2 ln qe = ln qm − βE 1 ϵ = RT ln(1 + ) Ce 1 E = √2B

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

where ϵ is Polanyi potential, β is Dubinin-Radushkevich constant, R the gas constant (8.314 J mol−1 K−1), T is the absolute temperature, and E the mean adsorption energy.

At last, the Temkin isotherm assumes that the fall in the heat of sorption is linear rather than logarithmic, as implied in the Freundlich equation. The adsorption experiment data are thus analyzed by the Temkin isotherm model in the linearized form,

qe = B ln Ce + B ln A, where B is equal to RT/b, being b the Temkin constant related to heat of sorption (J mol- 1); additionally, A is the equilibrium binding constant corresponding to the maximum binding energy (L g-1), R is the gas constant (8.314 J mol-1 K-1), and T the absolute temperature (K) [53].

At present, the most popular kinetic modes are the pseudo-first order, the pseudo- second order and Elovich [54]. The pseudo-first order or the Lagergren kinetic mode, is probably the earliest known one describing the rate of sorption in the liquid-phase systems [55]. Moreover, it has been one of the most widely used kinetic equations until now. The pseudo-second order kinetics is usually associated with the situation when the rate of direct adsorption/desorption process (seen as a kind of chemical reaction) controls the overall sorption kinetics [55]. The Elovich kinetic, this is due to neglecting the rate of simultaneously occurring desorption. Thus, in practice, the applicability of the Elovich equation is restricted to the initial times of sorption process, when the system is relatively far from equilibrium [55].

Therefore, the adsorption and absorption capability of a nanomaterial is found and described through different isotherm and kinetic models, and this adsorption efficiency as well as the sorption/desorption kinetics can vary with each nanomaterial utilized for the extraction of antidepressants (Table 3.1.1.2). Many currently existing nanomaterials have been applied for the extraction of antidepressants from biological sources by utilizing different extraction mechanisms which are being further analyzed and their current applications discussed in the following section. The nanomaterials that have been used for extraction of antidepressants are nanoparticles, nanotubes, nanofibers, nanoshells, nanocomposites, nanorods and polymer-based nanosorbents (Figure

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

3.1.1.1). The adsorption efficiency may vary with each nanomaterial. Figure 3.1.1.2 shows the sorption mechanism of antidepressants through different nanomaterials.

Table 3.1.1.2. Isotherm and kinetic models of different nano-sorbents used for antidepressants extraction form biological species. Adsorbent Isotherm model Kinetic model Ref. MIPs based on magnetic chitosan/GO Langmuir, Freundlich, Pseudo-first-order, [124] Temkin, and Dubinin– pseudo-second-order, Radushkevich Elovich and intra particle kinetic The surface grafting of poly[β- Langmuir and – [78] CD/allylamine-co-N-isopropylacrylamide] Freundlich onto modified Fe3O4 MNPs by 3- mercaptopropyltrimethoxysilane RAMIP-BSA Langmuir and – [135] Freundlich Imz ionic liquid- modified Fe3O4@SiO2 NPs Freundlich – [67]

PNCBCA grafted to Fe3O4 MNPs Freundlich [77]

MIPs coated Fe3O4 MNPs Langmuir and – [136] Freundlich MIPs: Molecularly imprinting polymers, GO: Graphene oxide, MNPs: Magnetic nanoparticles, Imz: Imidazolium, RAMIP-BSA: Restricted access molecularly imprinted polymer-bovine serum albumin, NPs: Nanoparticles and PNCBCA: Poly[N-isopropylacrylamide-co-1-(N,N-bis-carboxymethyl)amino-3- allylglycerol].

Figure 3.1.1.1. Nanomaterials used in antidepressants extraction.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Figure 3.1.1.2. Antidepressants extraction mechanism using different nanomaterials.

3. Nanomaterials for antidepressants extraction

Up to now, the nanomaterials used to extract antidepressants comprise nanoparticles, nanotubes, nanofibers, nanoshells, nanocomposites, nanorods and polymer-based nanosorbents, most of which are currently used for antidepressants extraction (Figure 3.1.1.3). The main parameters involved in these extraction process such as the type of surface modification, the extraction mechanism applied, the biological specimens analyzed, as well as the sample volume and extraction time required, the instrumentation utilized, and the extraction recovery achieved are also included and discussed (Table 3.1.1.3).

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Figure 3.1.1.3. Different nanomaterials currently used for antidepressants extraction.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Table 3.1.1.3. Efficiency of the extraction of antidepressants from biological specimens by using different nanomaterials.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Table 3.1.1.3. (continued)

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Table 3.1.1.3. (continued)

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Table 3.1.1.3. (continued)

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

3.1. Nanoparticles

Nanoparticles (ranging from 1 to 100 nm) can be either metallic, semiconductor, or of polymeric nature, and have found numerous fields of application such as medicine, food science and technology, cosmetic industries, or water treatment [56]. Moreover, these materials can be used as adsorbents for an efficient extraction of many drugs in different biological species [57]. Due to their small size, large surface area and excellent catalytic activity, nanoparticles emerged as an alternative to conventional treatments for extraction of antidepressants from biological species, which often result in tedious, expensive and slightly complex procedures. Thus, metallic nanoparticles (MNPs) can be considered as really useful materials in several areas of analytical chemistry as these MNPs possess excellent surface area, high adsorption capacity, are susceptible of being modified at relative low temperature and have good conducting properties as well [58].

Currently, different nanoparticles such as Fe3O4 [59], TiO2 [60], Al2O3 [61], and others previously modified with functional coatings have already been succesfully applied as sorbents.

Among them, Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) have attracted interest worldwide due to their excellent catalytic activities strong magnetism and large surface area [59,62]. In fact, Fe3O4 MNPs are becoming important as their role as sorbents remarkably contributes to save time and costs by simplifying the extraction process through their simple isolation from the sample matrix just by applying an external magnetic field. Many synthetic processes have been developed to fabricate these nanomaterials and a coating step (e.g. the use of silica used to allow the introduction of other functional groups) is usually added after their synthesis not only to improve the stability but also to avoid the formation of agglomerates of these NPs. Indeed, a wide variety of coatings can be used to improve selectivity [63,64]. Therefore, these MNPs, including Fe3O4 ones, can be considered as excellent sorbents for the extraction of drugs as demonstrated by different studies here reported [65].

The Fe3O4 MNPs directly synthesized from its source are widely used for antidepressants extraction [65–71]. First, Markovich et al. synthesized Fe3O4 MNPs by reacting aqueous ammonia with an aqueous solution containing FeCl3 and FeCl2 at a molar ratio of 2:1

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

[72]. Since then, different approaches are considered for their synthesis, from physical methods (i.e. size reduction process down to the nanometric range, or the condensation of precursors from either a liquid or gaseous phase) [73], to wet chemical approaches such as oxidation, electrochemical, reactions carried out in constrained environments, supercritical fluid process, hydrothermal, sol-gel reactions, polyol methods, flow injection syntheses, and aerosol/vapor methods.. Furthermore, methods using microorganisms, such as the MNPs formed by bio-mineralization have been performed [73].

MNPs synthesis generally requires of remarkable costs; however, when synthesized from natural sources such as microorganisms (i.e. bacteria or fungi) [74,75] the process becomes significantly more economic, as is the case of the synthesis of magnetic/non- magnetic nanocellulose from argan press cake plant [76]. When Fe3O4 MNPs synthesized from a natural source is compared with MNPs created by co-precipitation.

Bare Fe3O4 MNPs have been coated with different materials (e.g. organic compounds, polymers, surfactants, and others) for the simultaneous extraction of antidepressants from biological species (Table 3.2.3). For instance, when the extraction of antidepressants with pyrrole-coated Fe3O4 MNPs was evaluated, a pH-dependent slight variation of their extraction efficiency was detected. Nevertheless, good extraction recoveries, ranging from 85.2 to 118.7% for citalopram, fluoxetine and sertraline were found in human urine and plasma [65].

Fe3O4 MNPs has also been ensembled with other nanoparticles to increase the antidepressants extraction ability. In one such case, Fe3O4 MNPs had been used with zirconium dioxide (ZrO2) NPs for the simultaneous extraction of amitriptyline and nortriptyline from human plasma [32]. More specifically, a hydrophobic layer of N- cetylpyridinium surfactant was adsorbed onto the surface of the Fe3O4 MNPs@ZrO2 composites driven by both electrostatic attraction and strong hydrophobic interactions, thus enhancing the extraction of these basic compounds (from 89 to 105%), as this outer surface provides different mechanisms (i.e. hydrophobic or chain–chain interactions with the hydrocarbon chains of the surfactant, electrostatic interaction or hydrogen bonding with the polar groups) for the effective retention of these analytes.

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

Simultaneously, the ZrO2 and N-cetylpyridinium surfactant enhanced the endurance of

Fe3O4 MNPs, thus allowing their reuse for repetitive extractions [32].

While bare Fe3O4 MNPs, or Fe3O4 MNPs in conjunction with other NPs showed good results in adequate antidepressants extraction, these nanomaterials still tend to aggregate in aqueous solution as of their ease of oxidation under ambient conditions, causing a reduction in the extraction capacity of Fe3O4 MNPs. However, this issue can be overcome by immobilization of these NMs on supports, by synthesis of bimetallic nanoparticles, or by using an appropriate capping material to increase their stability as well as to enhance their properties [32].

Another strategy implies the synthesis of amino–functionalized Fe3O4 MNPs. For instance, novel Fe3O4 MNPs@SiO2-NH2 have been synthesized for the extraction of the antidepressant clomipramine from human plasma samples by ultrasound-assisted dispersive magnetic solid phase extraction [66]. The interaction between this nanosorbent and clomipramine is highly influenced by pH values. Thus, at pH= 9.0, and with 37 mg of Fe3O4 MNPs, a maximum extraction recovery of 90.6% was obtained mainly due to the sorption by hydrogen bonding of the drug onto the Fe3O4 MNPs@SiO2-

NH2 sorbent.

In another study, Fe3O4 MNPs were synthesized by graft co-polymerization of the thermosensitive agent N-isopropylacrylamide and the functional monomer 1-(N,N-bis- carboxymethyl)amino-3-allylglycerol onto Fe3O4 MNPs surface modified with 3- mercaptopropyltrimethoxysilane. These so-modified Fe3O4 MNPs showed effectiveness for the extraction of the inhibitor of noradrenaline or dopamine uptake fluvoxamine, ranging from 85.5 to 96.5% either from urine, plasma and pharmaceutical samples [77]. At neutral pH, the best sorption in about five minutes shaking occurred, while lower extraction of fluvoxamine at lower pH was observed, probably by an enhanced protonation of the -NH2 group of the drug and the subsequent increase of its ionic form and/or its consequent higher water solubility. When pH increased, the drug becomes into its neutral form and, therefore, lower water solubility occurred and higher extraction values were favored. Additionally, the rate of fluvoxamine sorption was increased at temperatures below the lower critical solution temperature of the poly(N-

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

isopropylacrylamide), a consequence of the increase in driving force with decreasing temperature. The mechanism proposed indicated that this rapid adsorption behavior of fluvoxamine on these grafted Fe3O4 MNPs could be fitted by the Freundlich isotherm model, and attributed to the accessibility of the bonding site in polymers on the CPG- MNP [77].

An additional study used a sorbent synthesized by the polymer-grafted Fe3O4 MNP by the free-radical graft copolymerization of β-CD/allylamine and N-isopropylacrylamide and modified with 3-mercaptopropyltrimethoxysilane for the extraction of venlafaxine from urine, plasma and pharmaceutical samples. In this case, pH 5 was selected as optimal for the effective drug sorption, also considering the re-dissolving of Fe3O4 MNPs at alkaline pH. The higher extraction recovery of venlafaxine at a lower pH may be due to the weakening of electrostatic force of interaction between the oppositely-charged adsorbate and adsorbent leading to a reduction in sorption capacity. Overall, novel polymer grafted Fe3O4 MNPs were successfully synthesized and reported as an efficient and suitable sorbent for venlafaxine extraction with recovery values ranging from 17.6% for plasma to 103.3% for urine sample [78].

3.2. Nanotubes

Nanotubes are one dimensional nanostructures with hollow cylindrical tubes which are widely used in pharmacy and medicine as of their variable chemical, physical, electrical, and structural properties [79,80]. More indeed, because of their high surface area and ability for π-π interactions, their relatively low price, wider accessibility, and easy functionalization [79] these nanomaterials are used as adsorbents for extraction of different drugs, including antidepressants [80]. These particular properties, together with their higher length to act as large platforms for the interactions with the analytes make CNTs one of the most used sorbents in the recent years [81–84]. However, the high hydrophobicity surface of CNTs requires of its proper modification for an efficient extraction of antidepressants.

In one such case, CNTs together with ionic liquids have been utilized to extract and to improve the determination of nine antidepressants in urine samples by HPLC-UV [85]. More specifically, multiwall carbon nanotubes (MWCNTs) were used SPE sorbent for the

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

extraction and pre-concentration of imipramine, desipramine, amitryptiline, nortryptiline, clomipramine, trimipramine, trazodone, fluoxetine, and mianserine as MWCNTs enable these analytes to remain on their surface bonded by π–π interactions or Van der Waals forces. Then, the matrix interferences could be removed just with water, without adding any organic solvent. In addition, the use of an ionic liquid as an additive for silanol suppression is proposed to improve the chromatographic behavior of these antidepressants, avoiding band tailing of chromatographic peaks. This study showed extraction recoveries from 72.4 to 97%, whereas LODs were determined in the 12.3–90.1 ng mL-1 range [85].

Magnetic adsorption techniques are also employed to simplify and improve the extraction of antidepressants from biological species [86,87]. Thus, magnetic multiwalled carbon nanotubes (MMWCNTs) can be easily synthesized through chemical deposition of Fe3O4 on the CNTs surface [88] for their application as a cleaner alternative for antidepressants extraction [89] due to the magnetic nature of these modified nanotubes, which enables them to be easily separated from the matrix and/or from the analytes, thus enhancing their properties as sorbents.

In this sense, CNTs were modified with magnetic nanoparticles by the chemical co- precipitation of FeCl2 and FeCl3 in an alkaline solution, and the ionic liquid 1,4 diazabicyclo[2.2.2]octane (DABCO) was then covalently attached to their surface. The resulting material is shown to be a selective sorbent for the isolation of the antidepressants citalopram, sertraline, fluvoxamine and fluoxetine from human plasma samples with ultrasound-assisted magnetic solid phase extraction and their determination by HPLC-UV [90]. The ionic liquid DABCO is an ionic liquid framework and cage-like compound, with a great potential in sample preparation due to its high hydrophobicity and thermostability. Furthermore, it is an economic, eco-friendly, and non-toxic organic material. However, this ionic liquid is not usually recovered and, therefore, it would be present in solutions, thus representing a remarkable issue, which can be overcome by immobilizing it on a magnetic surface like magnetic CNTs, to obtain a heterogeneous, recoverable and reusable derivative of DABCO, as well as to separate the sorbent from the sample matrix. Thus, the antidepressants analyzed were extracted

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

from plasma samples by hydrophobic and π-cation interactions with recovery values higher than 91% [90].

Besides that, the high mechanical strength, large surface area, high aspect ratios and their excellent electrical properties [91,92] make CNTs ideal to be used as electrodes [93–95]. For instance, The MWCNT-modified glassy carbon electrode (MWCNT/GCE) was constructed and the electrochemical behavior of trazodone was investigated [95]. The GCE electrode was coated by casting 15 µL of MWCNTs suspension and air-dried. The electroactive areas of the MWCNT/GCE and the bare GCE were analysed by cyclic voltammetry (CV) using 1.0 mM of K3Fe(CN)6, and the MWCNT/GCE showed much better performance than bare GCE. The analytical performance of MWCNT/GCE has been evaluated for detection of trazodone in urine samples. Under optimized conditions, the working concentration range and LOD are 0.2–10 µM and 24 nM, respectively, for trazodone. Furthermore, the recovery determined was in the range of 99.15–103.2% for the determination of trazodone in urine samples [95].

In another study, MWCNTs functionalized with the glycine (Gly) amino acid were synthesized and held in the pore of a hollow fiber by sol–gel technique [96] to extract and to determine venlafaxine (VEN) and o-desmethylvenlafaxine (ODV) from biological matrices. Important microextraction parameters including pH of donor phase, donor phase volume, stirring rate, the extraction time, and the optimal desorption conditions such as the type and volume of solvents and desorption time were thoroughly investigated and optimized [96].

3.3. Nanofibers

Nanofibers usually possess diameters lower than 100 nm and have been used in various medical applications [97]. They are environmentally safe due to their extreme length and can be easily incorporated onto different media or support. They also have high surface-to-volume ratio, adjustable functionality, and large porosity. As a consequence, they have been used in particulates filtration, airborne nanoscale particles, and other applications [98].

In this case, polymer nanofibers have been utilized to prepare electrospun polystyrene nanofibers for direct extraction of the antidepressant and anxiolytic trazodone from

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

human plasma [99]. Electrospinning was used to produce a polymer this nanoscale fibrous structure, resulting in nanofibers with high aspect ratio and, consequently, larger specific surface. The fiber diameter, fiber packing amount, eluted solvent, pH and ionic strength were additionally optimized for the extraction process. The target compound was then monitored by HPLC-UV, and acceptable extraction recoveries of 58.3-75.2% and relative recoveries of 94.6-105.5% were obtained, showing the effectiveness of extraction method proposed. Furthermore, the use of this nanomaterial provides a number of advantages in simplifying sample preparation and thus saving costs and time of analysis with acceptable reliability, selectivity, and sensitivity [99].

Liquid-liquid extraction, although being highly reproducible and showing high sample capacity, it is a time-consuming and labor-intensive procedure. As an alternative to decrease the solvent consumption, a miniaturized format called liquid phase microextraction (LPME) was developed [100]. More specifically, 8.8 cm length hollow fiber-based liquid phase microextraction (HF-LPME) was successfully applied for the extraction and preconcentration of amitriptyline, imipramine and sertraline in urinary and plasma samples [101]. The adsorption experiments were carried out using an accurel Q3/2 polypropylene hollow fiber membrane (Wuppertal, Germany) with a 0.2 µm pore size, 600 µm internal diameter and 200 µm wall thickness and target analytes were then monitored by HPLC-UV. The study achieved extraction recoveries of 65-68% and enrichment factors up to 300. Overall, the study exhibited good performance of the HF-LPME technique for the extraction of antidepressant drugs from biological samples [101].

3.4. Nanoshells

Nanoshells consisted of spherical particles composed by a dielectric core (i.e. silica or ferric oxides, alumina, titanium) and covered by a thin metallic shell (e.g. gold, silver, copper, etc.) with remarkable tendency towards the core to form this adsorbed layer of surfactant molecule [102].

For antidepressants extraction, the core-shell nanoparticles of the type Fe3O4

MNPs@ZrO2 modified at their surface by N-cetylpyridinium have been used for the quantitative extraction by MSPE of amitriptyline and nortriptyline from plasma samples

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

[32]. The study showed 89 to 105 % extraction relative recoveries and detection limits of 0.04 and 0.08 ng mL-1 for amitriptyline and nortriptyline, respectively, due to the enhancement of in adsorption mechanism through hydrophobic or chain-chain interactions, electrostatic interaction or hydrogen bonding with the polar groups, which are more selective and efficient than Fe3O4@ZrO2 or bare Fe3O4 NPs. Overall, this MSPE method exhibited selective and efficient preconcentration of these amine containing groups tricyclic antidepressants, even more considering their stability over a 3-12 pH range and that just 5 mg of sorbent is required to achieve these good recoveries [32].

3.5. Nanocomposites

Nanocomposites have been widely used in medical applications as well as for the extraction of drugs [39,103]. Nanocomposites consist of a combination of nanomaterials such as polymer [104,105], graphene based [106,107], and magnetic polymer [108,109] based nanomaterials, which enhance its overall adsorbing capability.

In this regard, a sensitive MSPE method based on Fe3O4 MNPs–MgSiO3 magnetic nanocomposites was developed for extraction of venlafaxine, escitalopram, paroxetine, sertraline and fluoxetine in serum and urine samples and their subsequent determination at trace levels using LC-UV [110]. In this case, Fe3O4 NPs are applied in MSPE due to considerable paramagnetism, a high magnetic saturation, and their synthesis. However, these bare NPs are prone to aggregation and oxidation, apart from not being selective toward complex matrices. To overcome these limitations, this inorganic composite magnetic nanoparticle was prepared to make a selective and appropriate sorbent, also considering that inorganic composite magnetic NPs are easier to prepare and safer. Thus, the Fe3O4–MgSiO3 nanocomposites were synthesized by in situ chemical co-precipitation of Fe2+ and Fe3+, at a 1:2 molar ratio 1:2, in an alkaline solution in the presence of MgSiO3. This optimized approach required just 12.5 mg of adsorbent at 7.4 pH and using only 1.3 mL of desorption solvent. The recoveries ranged between 72 and 115 %, with RSD lower than 4.75% [110].

Likewise, the conducting polymer polythionine (PTh) was coated by chemical oxidative polymerization on the surface of graphene oxide (GO)/Fe3O4 NPs to generate a sorbent for the extraction of duloxetine from human plasma [111]. First, the GO synthesized was

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

dispersed in water by applying ultrasonic radiation and, then, FeCl3.6H2O and FeCl2.4H2O were added dropwise at room temperature and pH value of 10 to form the GO/Fe3O4 MNPs. At basic conditions (9.0 pH), the designed MSPE could be reused at least 9 times with an extraction recovery of 87% at its last use. The composition of this sorbent significantly could control the selectivity and efficiency of the extraction process. Indeed, the hydrophilicity of GO along with its large surface area enhances the extraction ability and notable π-electron interaction with hydrocarbon ring structures. Moreover, the large surface area, considerable π–π interactions and their excellent chemical, mechanical and thermal stabilities make PTh an excellent additive for the sorbent in the extraction of such compounds. The high surface area of the support facilitated the dispersion of NPs and prevented their agglomeration [111].

In another study, bovine serum albumin (BSA) conjugated with Fe3O4@AuNPs was applied as novel stationary phase at microchip electrophoresis (MCE) for efficient enantioseparation process. First, Fe3O4@AuNPs were synthesized by sonochemical synthesis strategy, and the resulting Fe3O4@AuNPs are endowed with the excellent properties of the two independent components, such as the high load ability of Au shell and the magnetic nature of Fe3O4, which also favors the further immobilization of biomolecules and easy retrieval and separation of the sample from dispersion. Then,

BSA was immobilized on the surface of Fe3O4@AuNPs through the interaction between

Au coating and the amine groups of BSA to form the Fe3O4@AuNPs-BSA conjugates, which are subsequently organized by an external magnetic field in the PDMS microchannel. Then, the electrochromatographic enantioselectivity and reproducibility of this so-constructed MCE device were applied to the chiral separation of the amino acids tryptophan and threonine, as well as of the enantiomers of ofloxacin. The results obtained prove the good performance in terms of repeatability and efficiency for the enantiomers separation of this approach [112].

3.6. Nanorods

Nanorods are nanoscale objects (10-9 m) within a 1-100 nm size range. They can be synthesized by direct chemical synthesis from different metals such as gold [113], silver [114], or from semiconducting materials [115].

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

In one such case, arrays of SnO2 nanorods were fabricated to be applied at a solid phase microextraction (SPME) fibre method for the extraction of some polar selective serotonin reuptake inhibitors drugs, citalopram and fluoxetine, in human urine and plasma [116]. The extraction time, pH, the ion strength or salt percentage, and desorption time of analytes from the fibre were optimized by using a Box–Behnken design and the response surface equations were developed. The optimal experimental conditions for the plasma analysis thus obtained included a 30% w/v salt percentage, 6.5 µL from a 1 M solution NaOH, 10 min required for the extraction and 30 min for desorption of the drugs, whereas 100 µL from a 1 M NaOH solution, 18 min for extraction and 23 min as optimum desorption time (23 min) were applied for drugs extraction from urine samples [116]. Good reproducibility (RSD < 10%) as well as acceptable recoveries (79–94%) were found for the method proposed [116].

3.7. Polymer-based nanosorbents

Polymer-based nanosorbents such as those based on organic [117,118], inorganic and hybrid polymers [119,120], as well as on molecularly imprinted polymers (MIPs) [121,122] have been successfully applied in for dugs extraction with medical purposes [123].

Thus, a selective and sensitive MIP based on magnetic chitosan/GO method was developed for the spectrophotometric analysis of fluoxetine in water, pharmaceutical formulations and urine samples [124]. For this purpose, a sorbent of MIP was synthesized using magnetic chitosan/GO as supporting material to provide multi- imprinting sites, large surface area, and ease of separation of magnetic nanocomposites. The synthesized polymer was thoroughly characterized, and its capability as a selective sorbent in MSPE for fluoxetine was investigated by optimizing all the parameters affecting the extraction and determination of fluoxetine. This method was successfully applied to the separation, preconcentration and determination of fluoxetine in the aforementioned matrices, with satisfactory recoveries (95.7–104 %) [124].

Another selective method was developed for determination and extraction of amitriptyline from water and plasma samples using nano-sized molecularly imprinted polymer (MIP) with ultrasound-assisted extraction (UAE). The nano-sized amitriptyline

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

imprinted polymer particles were synthesized using suspension polymerization in silicon oil. All the parameters affecting the extraction and determination of amitriptyline were investigated and optimized such as, pH, sample volume, nature and volume of concentrative solvent, temperature, and ultrasound time. The relative recovery values ranged from 82.4 to 92.3% [125].

4. Future perspectives

Most of the studies here discussed were performed at a laboratory, and no green synthetic materials were utilized. Even considering the reusability of many nanomaterials and the low reagents consumption associated to their application, these alternative green compounds and methods should be considered as future approaches to be developed. In this sense, supercritical carbon dioxide (sc-CO2) used to prepare nanomaterials may be further developed and encouraged [126,127]. More indeed, naturally occurring nanomaterials such as the clay-based halloysite nanotubes could also be used for antidepressants extraction from biological species [128–130]. Consequently, these nanomaterials represent an eco-friendly, economic, and remarkably efficient, thus a highly suitable alternative, also considering their potential tunable surface chemistry [131–133] for biological applications and, more specifically, as potential excellent adsorbents for antidepressants.

Additionally, new kind of nanomaterials applicable at wide pH ranges could also represent a remarkably interesting alternative to applied for antidepressants extraction from biological samples, even considering the different pH values usually existent in the different biological matrices to be monitored. On the other hand, all of these methods here resumed and discussed are applied as sorbents for solid phase extraction approaches, while alternative extraction strategies such as liquid-liquid microextraction or, more interestingly, supercritical fluid extraction are not considered when antidepressants are to be extracted from biological samples for their subsequent determination.

With regard to supercritical fluid extraction (SFE), supercritical CO2 was proven to be a green alternative to organic extraction liquids, as it is no flammable and it does not leave environmentally hazardous waste. Moreover, greater potential effectiveness, mild

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

extraction conditions and short analysis times of SFE is derived from the rapid diffusion (gas-like) of the analytes in the fluid and the liquid-like solvation capacity of the supercritical fluids, thus enhancing its preconcentration effect, as well as its quantitativeness, expeditiousness, simplicity and selectivity [134]. All of these advantages could even be reinforced if nanomaterials were added to this extraction approach as of potential improvement of selectivity, rapidness and reduction of time and costs could be obtained.

Nevertheless, some difficulties must be overcome when SFE is to be used as of the challenging extraction of polar analytes, the rate of extraction efficiency obtained when spiked and natural samples are analyzed, and the clean-up process usually required before the analytes’ measurement.

5. Conclusion

Antidepressants drugs are widely used for the treatment of psychological disorders such as depression and therefore their monitoring in biological species is important considering that they need to be accurately monitored. Antidepressants extraction does not occur through common processes for biological species. Conventional techniques used for antidepressants extraction include adsorption, and other biological and chemical techniques. However, these techniques have several limitations. Nanotechnology is a suitable alternative to extract the antidepressants over the conventional techniques, as it is a cleaner and energy efficient method.

Nanomaterials such as nanoparticles, nanotubes, nanofibers, nanoshells, nanocomposites, nanorods and polymer-based nanosorbents have been used for antidepressants extraction. Many advantages associated with these materials are high surface area, easy synthetic routes, high catalytic activity, and good optical, electrical and mechanical properties. The nanomaterials have either been used as sorbents due to their large surface areas. The nanoparticles were used without any modification or immobilized on a support. Efficient antidepressants extraction rates have been achieved using these nanomaterials at differing modification, extraction type, biological species, linear range, LOD, instrumentation and extraction recovery. The concentration of antidepressants extracted have been determined through different techniques such as,

Use of nanosized and non-nanosized co-polymer for bio-analytical sample preparation

spectrophotometry, and chromatography techniques. Antidepressants extraction recoveries as high as 100% have been achieved using different nanomaterials. However, these nanomaterials have several limitations such as toxicity and particle agglomeration. The nanomaterials may also become unstable at varying conditions. Use of novel and eco-friendly materials such as nanotubes and its composites for antidepressants extraction may lead to an improvement in antidepressants extraction efficiency. With development of further techniques that overcome the limitations of nanomaterials may become the silver bullet needed for efficient antidepressants extraction from biological species.

Acknowledgments

The Spanish Ministry of Economy and Competitiveness (MINECO) and JJCC Castilla-La Mancha are gratefully acknowledged for funding this work with Grants CTQ2016-78793- P and JCCM SBPLY/17/180501/000262, respectively.

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3.1.2. Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites and separation by capillary electrophoresis

Electrophoresis 39 (2018) 1808 – 1815

DOI: 10.1002/elps.201700496

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Abstract

Poly(styrene-co-divinylbenzene)-coatedmagnetic multiwalled carbon nanotube composite synthesized by in-situ high temperature combination and precipitation polymerization of styrene-co-divinylbenzene has been employed as a magnetic sorbent for the solid phase extraction of antidepressants in human urine samples. Fluoxetine, venlafaxine, citalopram and sertraline were, afterwards, separated and determined by capillary electrophoresiswith diode array detection. The presence of magnetic multiwalled carbon nanotubes in native poly(styrene-co-divinylbenzene) not only simplified sample treatment but also enhanced the adsorption efficiencies, obtaining extraction recoveries higher than 89.5% for all analytes. Moreover, this composite can be re-used at least ten times without loss of efficiency and limits of detection ranging from 0.014 to 0.041 µg/mL were calculated. Additionally, precision values ranging from 0.08 to 7.50% and from 0.21 to 3.05% were obtained for the responses and for the migration times of the analytes, respectively.

Keywords: Antidepressants / Capillary electrophoresis / Magnetic multiwalled carbon nanotube / Magnetic solid phase extraction / Poly(styrene-co-divinylbenzene).

Abbreviations: CNTs, carbon nanotubes; DVB, divinylbenzene; ECD, electrochemical detection; FID, flame ionization detection; MMWCNT, magnetic multiwalled carbon nanotube; MNPs, magnetic nanoparticles; MWCNT, multiwalled carbon nanotube; NPs, nanoparticles; poly(STY-DVB), poly(styrene-co-divinylbenzene); SSRIs, selective serotonin reuptake inhibitors; STY, styrene; TEM, transmission electron microscopy.

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GRAPHICAL ABSTRACT

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1. Introduction

Mood disorders have a one-time prevalence of 4.4% worldwide and, according to the World Health Organization, major depressive disorder will be the second most common disability by 2030 [1,2]. Therefore, an increase in antidepressants consumption has occurred and treatments with antidepressants need to be accurately monitored. Among these drugs, the selective serotonin reuptake inhibitors (SSRIs) are the most significant class of antidepressants marketed in recent years, even though considering the occurrence of adverse effects associated to their consumption [3,4]. Among the SSRIs, fluoxetine, citalopram, and sertraline are some of the most prescribed active principles [5]. Furthermore, newer atypical antidepressants including target other neurotransmitters either alone or in addition to serotonin, such as venlafaxine, are currently administered and commonly prescribed worldwide [6]. Nevertheless, a precise clinical monitoring to ensure patient’s adherence to therapy and/or to individualize the dosage regimens is highly recommended to avoid therapeutic ineffectiveness or the occurrence of adverse effects. In this sense, urine specimens are preferred since collection is non-invasive, and the specimen can provide a longer detection window, as drug concentrations are usually higher than in serum.

Different techniques to determine these antidepressants in urine have been reported, including liquid or gas chromatography coupled to different detection techniques [6- 12]. Capillary electrophoresis is also applied for this purpose [13-16] as of its high efficiency, low reagents and sample consumption, thus representing a low-cost alternative. Sample cleaning procedures [7,17-19] are usually required to remove the interfering compounds within the biological matrix and to pre-concentrate the analytes. Among them, liquid-liquid extraction is time consuming, labor intensive, and it usually requires remarkable amounts of solvents which are potentially toxic. Likewise, traditional solid phase extraction (SPE) sorbents are universally employed for sample treatment as of their high separation capacity and rapid extraction dynamics, though poor analyte recoveries, insufficient cleanup, or irreproducibility can be detected. By contrast, the polymeric sorbent developed by polymerization of styrene and divinylbenzene (e.g. the manufactured cartridge Strata-X®, from Phenomenex®) has been designed to eliminate these common bottlenecks associated to the use of

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traditional SPE sorbents. Thus, the copolymerization of styrene and divinylbenzene by using different polymerization techniques has been extensively studied [20]. This commercial cartridge containing a styrene-co-divinylbenzene polymer represents a good sorbent for antidepressants [21,22] as it strongly retains neutral, acidic, or basic compounds under aggressive, high organic washing conditions based on three mechanisms of retention: π-π bonding, hydrogen bonding (dipole-dipole interactions), and hydrophobic interactions. Carbon nanotubes (CNTs) [23] and, particularly, multiwalled carbon nanotubes (MWCNTs) [24], have a great potential as SPE sorbent for antidepressants, because of their hydrophobicity, large specific surface area, and excellent chemical stability [25]. These materials are usually applied in a cartridge or column mode, which often results in tedious packing procedures, high backpressure and low flow rates. To overcome these disadvantages, the incorporation of CNTs and poly(styrene-co-divinylbenzene) in magnetic solid phase extraction is an excellent alternative. The experimental procedure for their application is simple [26,27] as the phases can be separated by just applying an external magnetic field, in addition to the potential. recycling of these magnetic nanoparticles (NPs) for further extractions.

Different magnetic MWCNTs composites have been previously prepared [27], while the in-situ magnetic combination at high temperature of the precursor [iron(III)] and MWCNTs in ethylene glycol holds much promise for large-scale synthesis. Although a high temperature is required for this synthesis for an efficient Fe3O4 decoration of MWCNTs with NPs, the reaction is relatively selective and sensitive. In addition, MWCNTs do not require any modification, and the size and density of the magnetic nanoparticles (MNPs) coverage can be easily adjusted through the precursor MWCNT/magnetite ratio, the temperature, and the time of synthesis. Poly(styrene-co- divinylbenzene)-coated magnetic composite could be prepared by mechanical and chemical methods, including high-speed-stirred mixer or a high-shear mill [28,29]. Considering these precedents, and the absence of previous research on the application of the highly poly(STY-DVB)-MWCNT-MNP composites for the extraction of antidepressants in urine, the advantages of the solvothermal approach are exploited in this work. This procedure is used for decorating poly(styrene-co-divinylbenzene) and MWCNTs with MNPs to prepare magnetic hybrid nanoparticles to develop an improved

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extraction approach of these antidepressants from human urine. Then, capillary electrophoresis-DAD technique is used to develop a methodology for their further separation and quantification.

2. Materials and methods 2.1. Materials, reagents and samples

Fluoxetine hydrochloride, venlafaxine hydrochloride, citalopram hydrobromide and sertraline hydrochloride, as well as styrene (STY) (≥99%), divinylbenzene (DVB) (80%), sodium hydroxide, acetic acid (≥99.7%), Iron (III) chloride hexahydrate, sodium acetate and ethylene glycol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium tetraborate anhydrous (Borax) and 2,2'-Azobis(2-methylpropionitrile) (AIBN) were purchased from Fluka Chemie (Buchs, UK). Methanol and hydrochloric acid were obtained from Panreac (Barcelona, Spain), while acetonitrile (HPLC grade) was purchased from Fisher Scientific (Geel, Belgium). MWCNTs of 30±15 nm diameter (95% purity) were obtained from NanoLab Inc. (Waltham, MA, USA).

The stock solutions of all analytes were prepared at 5.0 mg/mL in methanol and stored in darkness at -20 °C. Working standard solutions were prepared at 0.1 mg/mL by appropriate dilution in methanol and stored at -20 °C as well. Standard calibration samples were prepared by diluting appropriately the working standard solution of each analyte.

Human blank urine samples were supplied by healthy volunteers, pooled and kept at - 20 °C until their analysis as blank or quality control samples by spiking with known concentrations of the antidepressants. Samples for transmission electron microscopy (TEM) were prepared by deposition of a drop of the synthesized material suspension onto a lacey carbon/format-coated copper grid.

2.2. Apparatus

The oven used for preparation of MMWCNT was from Hewlett Packard 5890, Series II gas chromatograph (Bothell, WA, USA). Ultrapure water was obtained from a Milli-Q water system from Millipore (Merck KGaA, Darmstadt, Germany), and for infrared

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spectrum measurement a Fourier transform infrared (FT-IR) spectroscope, model FT/IR 4200 (Jasco, Japan) was utilized.

Raman measurements were performed with a portable Raman Spectrometer system (B&W TEK Inc., DE, USA) at a wavelength of 785 nm and a maximum laser output power at system’s excitation port of 348 mW and 285 mW in the probe. The output laser power in the probe was set to the total percentage without any damage. For measurements, the laser beam was focused to the sample through a 100/1.25 objective. Raman signals were acquired by a CCD array detector cooled at 10 °C with an acquisition time ranging from 5 to 65.5 ms.

TEM images were obtained with a Jeol JEM 2011 microscope operating at 200 kV and equipped with an Orius Digital Camera (2×2 MegaPixel) from Gatan (Pleasanton, CA, USA), while the zeta-potential and particles’ size distribution were measured by dynamic light scattering using a Zetasizer Nano instrument (model ZEN3600) from Malvern (Worcestershire, UK).

2.3. Preparation of magnetic multiwalled carbon nanotubes

MMWCNT was prepared by in situ high temperature combination of the magnetic precursor [iron(III)] and MWCNTs, according to the previously described procedure [28], with slight modifications and without octadecyltrichlorosilane. This synthesis involves the mixture of 140 mg of FeCl3.6H2O and 40 mg of MWCNTs, which was suspended in 7.5 mL of ethylene glycol in a glass vial. Then, 0.36 g of sodium acetate was added, and the solution was stirred, sonicated for 10 minutes and then left at room temperature for 60 minutes. Afterwards, the vial was heated at 200 °C for 48 h to complete the reaction. After cooling down to room temperature, the synthetic product was washed with 10 mL of distillated water for 5 times. The MMWCNTs were then separated by applying a magnet, and the nanoparticles obtained were dried at 80 °C and stored. This procedure was optimized to obtain nanoparticles as reduced as possible to increase the surface area and, therefore, the retention of the analytes onto the composite.

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2.4. Preparation of poly(styrene-co-divinylbenzene) and poly(styrene-co- divinylbenzene)-coated magnetic multiwalled carbon nanotube composite

Three non-magnetic (P1, P2 and P3), and three magnetic polymers (MP1, MP2 and MP3) were synthesized by preparing diverse polymerization mixtures (Table 3.1.2.S1) by mixing divinylbenzene (DVB), styrene (STY), magnetic multiwalled carbon nanotubes, ACN and AIBN at 70°C for 24 h [30,31]. The mixture was sonicated for 5 min, purged with N2 gas for 10 min and then closed. The resulting product was then collected, washed several times with water together with mechanical agitation and dried in a vacuum desiccator for 24 h. Last, 500 mg of non-magnetic polymers were packed in the cartridge, whereas magnetic polymers were used without packaging before extraction.

2.5. Extraction by poly(styrene-co-divinylbenzene)-coated magnetic multiwalled carbon nanotubes composite (poly(STY-DVB)-MMWCNT)

500 mg of the MP3 was put into a conical flask, conditioned with (2x3) mL of methanol and then with deionized water. The supernatant was then separated with a magnet, discarded, and 50 mL of standard solutions of the analytes or of spiked urine samples, previously adjusted to pH 5.3, were added into the conical flask. The mixture was stirred at room temperature for 5 min to create a homogenous dispersion and the magnetic polymer containing the adsorbed antidepressants was rapidly removed from the solution applying a strong external magnetic ﬁeld and the supernatant was discarded. The magnetic polymer was then washed with 10 mL of Milli-Q water, and the antidepressants were eluted by rinsing with 1.0 mL of methanol containing 2.0 % of acetic acid. The resultant extract was injected into the CE system.

2.6. Separation of antidepressants and operating conditions

Antidepressants were separated and detected in a CE instrument (Model G1600AX) from Agilent (Palo Alto, CA, USA) provided with a diode array detector (DAD). The carrier electrolyte, sodium tetraborate anhydrous (50 mM, pH 9.3) with 20% of methanol was used as running buffer. CE voltage and temperature were set at 20 kV and 20 ºC, respectively, and detection was performed at 200±10 nm (450±10 nm as reference). Moreover, hydrodynamic injection mode (50 mbar) was applied for 5s, and a fused-silica capillary (Beckman, Fullerton, CA, USA) of 50 cm (27 cm effective length) and 75 µm of

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internal diameter was used. Capillary was daily conditioned with freshly prepared 0.1M NaOH (10 min) followed by deionized water (10 min) and fresh running electrolyte (10 min), and applying 2.0 bar in the external pressure pump. Capillary was rinsed with running electrolyte for 10 min after each complete run.

3. Results and discussion

First, electrophoretic method was optimized to separate and quantify fluoxetine, venlafaxine, citalopram and sertraline. Then, the extraction procedure utilizing poly(STY- DVB)-MMWCNT was designed and optimized to extract the antidepressants from urine samples.

3.1. Optimization of separation and detection of antidepressants

The influence of key parameters affecting the sensitivity and separation efficiency of the studied antidepressants was analyzed utilizing 1.0 µg/mL standard solutions. According to previous works, the borate buffer (pH= 9.3) was chosen for an adequate separation of the antidepressants [32–34]. Under these conditions, and considering that the pKa values of the analytes range from 8.7 to 9.5 [35], they are weakly ionized and, consequently, will have similar electrophoretic mobility [29]. Thus, pH has a minor effect in their separation. The effect of borate buffer concentration in the background electrolyte was additionally evaluated at different concentration levels (10–80 mM). The resolution slightly increased when buffer concentration raised from 10 to 50 mM, whereas no further improvement but higher current were observed over 50 mM. To improve resolution, an increase of borate-analyte interactions was explored by using organic modifiers such as methanol, isopropanol or acetonitrile, as an increasing concentration of organic modifiers in the background electrolyte is correlated with increased borate-analyte interactions. Hence, longer migration times clearly departed from that of the electroosmotic flow could be observed, and the best results were obtained with 20% methanol. Next, a 10–30 kV interval of applied voltage to improve peaks’ resolution was studied. In all cases, migration times decreased when voltage was increased. Thus, a voltage of 20 kV was selected as a compromise between the migration time and the separation efficiency, whereas no reproducibility was observed at voltages

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higher than 20 kV due to the Joule effect and capillary heating. Moreover, good separation of antidepressants was obtained at a capillary temperature of 20°C.

On the other hand, hydrodynamic injection was selected since more reproducible results were obtained when compared to electrokinetic injection, and the injection time (5–20 s) and pressure were also analyzed. Sensitivity increased when higher times of injection were applied, though venlafaxine and citalopram did not separate when times longer than 10 s were selected. Consequently, 10 s was selected as an optimal time of injection. Regarding injection pressure, values ranging from 30 to 50 mbar were assayed, and a pressure of 50 mbar was selected as of the best sensitivity observed for all the analytes.

3.2. Characterization of MMWCNTs, poly(STY-DVB) and poly(STY-DVB)-MMWCNT sorbents

The nanoparticles showed a super operational stability and retained excellent adsorption. Furthermore, magnetic properties did not decay even after a 10-cycle run for the adsorption and desorption of antidepressants. The synthetized material was characterized by FT-IR analysis, Raman spectroscopy and TEM.

Regarding FT-IR spectra (Figure 3.1.2.S1), the characteristic peaks of aromatic (C=C) bonding and alkyl (C-H) stretches observed at 1700 and 2800 cm-1 were originated from the poly(STY-DVB), while the peaks at 3085 cm-1 correspond to the aromatic (C=C) stretching, as well as the broad band at 3400 cm-1 is indicative of the presence of hydroxyl (-OH) groups on nanoparticles’ surface. Likewise, the existence of the characteristic peaks of MMWCNT-poly(STY-DVB) agreed with those observed in poly(STY-DVB) and MMWCNT, thus confirming the successful incorporation of MMWCNT into the poly(STY-DVB). For further confirmation of the presence of MMWCNTs in MMWCNT-poly(STY-DVB) polymer, Raman spectroscopy was utilized (Figure 3.1.2.S1). The characteristic peaks of D and G bands at 1351 and 1588 cm-1, respectively, for MWCNT were evident in the prepared MMWCNT-poly(STY-DVB) polymer.

To confirm whether the MMWCNT, poly(STY-DVB) and MMWCNT-poly(STY-DVB) were effectively produced, these materials were characterized by TEM (Figure 3.1.2.S2).

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Some differences can be observed, as the micrographs for the MMWCNTs reveal the formation of nano-spheres and the attachment of the magnetic nanoparticles onto the surface of the nanotubes (Figure 3.1.2.S2 A), whereas the formation of spherical particles of poly(STY-DVB) can also be observed (Figure 3.1.2.S2 B). Finally, the micrograph of MMWCNT-poly(STY-DVB) composite material showed the aggregate formed by poly(STY-DVB) and MMWCNT (Figure 3.1.2.S2 C). The detailed view at 200 nm scale (Figure 3.1.2.S2 D) clearly revealed the attachment of the MNPs onto the MWCNTs and poly(STY-DVB).

The size distribution and zeta potential of MMWCNT-poly(STY-DVB) composite was characterized by dynamic light scattering (DLS). The microspheres are clearly observed (Figure 3.1.2.S2) with uniform sizes (Figure 3.1.2.S3). It is expected that the microspheres were obtained with controlled zeta potentials by AIBN as an initiator: - 38.2 mV (Figure 3.1.2.S3). The value of zeta potential should be ascribed to the charge of free radicals from the AIBN in emulsion polymerization, such as [(CH3)2˙C(CN)]2.

3.3. Absorption experiments of antidepressants by MMWCNTs, poly(STY-DVB) and poly(STY-DVB)-MMWCNT

The capacity of MMWCNT, P1, P2, P3, MP1, MP2 and MP3 sorbents to extract antidepressants was evaluated in 0.1M HCl. Briefly, 500 mg of each mixture were mixed with 10 mL of the solution of antidepressants at a concentration of 1.0 µg/Ml in 0.1M HCl. The solution was incubated for 24 h at room temperature, and then the supernatant was separated and analyzed by CE. The rate of absorption of the antidepressants onto the MMWCNT and polymers was then calculated. Extraction recoveries ranging from 66.6 to 70.3% were obtained on the performance of the native poly(STY-DVB), thus confirming a remarkable adsorption efficiency when using polymer P3 (Figure 3.1.2.1). This phenomenon is explained by strong hydrophobic, π–π, and hydrogen bonding interactions between the analytes and the polymer. When MMWCNTs were used for the synthesis of the polymer P3, the recoveries increased from 66.6% to 106% for fluoxetine, from 63.3% to 100% for venlafaxine, from 66.4% to 89.5% for citalopram, and from 70.3% to 102% for sertraline (Figure 3.1.2.1). These improvements are a consequence of the adsorption of the analytes, which are aromatic amines, onto the

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surface of the MMWCNTs nanocomposite. It is recognized the presence of carboxyl and hydroxyl groups onto the surface of the oxidized MMWCNTs, making the surface charges more negative than those of pristine MMWCNTs, which made several adsorption mechanisms, such as hydrogen bond, electrostatic and hydrophilic/hydrophobic interactions. It is also known that the aromatic amines adsorb onto the MMWCNTs surface through π–π interactions (aromatic ring) or by interaction throughout the amino group (Nitrogen lone pair :N, or the hydrogen of the NH2 group). In addition, the aromatic amine is also adsorbed through a hydrogen bonding interaction with the carboxyl groups onto the MMWCNTs. Therefore, the magnetic polymer (MP3) was selected for the next experiments.

Figure 3.1.2.1. Evaluation of the sorption capacity of MMWCNT, P1, P2, P3, MP1, MP2 and MP3 sorbents.

3.4. Optimization of the extraction conditions

Different parameters affecting the performance of the extraction (i.e. pH, adsorption time, elution solvent, sample volume and mass of sorbent) were optimized analyzing 1.0 µg/mL standard solution of the antidepressants.

The influence of pH was studied in the 1–12 range, with a maximum of adsorption of the analytes onto the poly(STY-DVB)-MMWCNT at pH 5.3. Then, the effect of the adsorption

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time was assayed between 1 and 30 min, but just 10 min was enough to achieve maximum recoveries.

Additionally, the optimization of desorption conditions using different organic solvents (methanol, ethanol and acetone) containing 2.0 % acetic acid was carried out using 500 mg of sorbent and 50 mL of a 1.0 μg/mL standard solution of the analytes at pH 5.3. The three acidic solvents eluted the antidepressants, though higher desorption was found for methanol, and it was therefore selected for subsequent experiments. Additionally, the minimum volume of solvent needed for an efficient elution of the adsorbed antidepressants was optimized. After testing a 1–5 mL range, the best results were obtained when 1.0 mL was used. Thus, methanol containing 2.0 % of acetic acid (2×0.5 mL) under agitation for 5 min was utilized for the desorption of the analytes. Under these conditions, the whole extraction procedure can be accomplished within 15 min.

Finally, the amount of poly(STY-DVB)-MMWCNT sorbent for the quantitative extraction of the antidepressants was optimized. For this purpose, different amounts from 100 mg to 1000 mg were assayed to extract antidepressants from a 50-mL mixture standard solution (1.0 μg/mL at pH 5.3). Recoveries improved with an increase of poly(STY-DVB)- MMWCNT up to 500 mg, whereas no further improvement was observed with more than 500 mg. After each extraction, sorbent was reconditioned by rinsing with methanol, and it was also confirmed that the sorbent can be re-used at least five times with the same extraction efficiency.

3.5. Determination of antidepressants in urine samples

The selected antidepressants were adequately separated and quantified in less than 6 min (Figure 3.1.2.2). The method was evaluated considering a hydrodynamic sample injection of 10 s (Table 3.1.2.1). External calibration curves using peak areas were constructed by injecting by triplicate the standard solutions previously preconcentrated using the poly(STY-DVB)-MMWCNT.

The limits of detection for each analyte, defined as the concentration of analyte giving a signal equivalent to the blank signal plus three times its SD, are also presented in Table 3.1.2.1. Since the blank signal is practically the same for all analytes, intercept values and their corresponding SDs of the calibration equations were used to calculate these

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values [36]. The LOD values obtained ranged from 0.014 to 0.041 µg/mL, which are better than the values previously obtained using capillary electrophoresis after a solid- phase extraction step [37]. The limit of quantification (LOQ), defined as the concentration of analyte giving a signal equivalent to the blank signal plus ten times its standard deviation [36], were calculated in the 0.026–0.077 µg/mL range.

Table 3.1.2.1. Calibration data and validation parameters obtained for the developed methodology and the commercial sorbent (StrataTM–X) method. LOD LOQ . 2 Analyte LR (µg/mL) Y= (A±SA) X + (B±SB) R Sy/x (µg/mL) (µg/mL)

Poly(STY-DVB)-MMWCNT

Fluoxetine 0.1-1.0 Y= (189±8)·X – (2.25±1.45) 0.9934 2.6 0.041 0.077

Venlafaxine 0.1-1.0 Y= (37.4±0.6)·X + (0.53±0.12) 0.9989 0.2 0.016 0.031

Citalopram 0.1-1.0 Y= (104±4)·X + (2.50±0.74) 0.9944 1.6 0.037 0.071

Sertraline 0.1-1.0 Y= (450±6)·X – (1.02±1.18) 0.9992 2.1 0.014 0.026

Commercial sorbent (StrataTM–X, from Phenomenex)

Fluoxetine 0.1-1.0 Y= (152.7±6.7)·X – (0.19±1.50) 0.9924 2.8 0.055 0.098

Venlafaxine 0.1-1.0 Y= (30.5±1.1)·X + (0.99±0.26) 0.9944 0.5 0.047 0.085

Citalopram 0.1-1.0 Y= (98.9±4.7)·X + (3.01±0.9) 0.9909 1.6 0.050 0.094

Sertraline 0.1-1.0 Y= (429.9±8.8)·X - (0.2±1.7) 0.9983 3.1 0.0213 0.040

A: slope, SA: standard deviation of slope; B: intercept, LOD: limit of detection; LOQ: limit of quantification; LR: linear range; R: regression coefficient; SB: standard deviation of intercept; Sy/x: standard deviation of residuals (n=11).

The applicability of the proposed method was then tested on human urine samples. First, 50 mL of blank urine samples at a pH 5.3 were filtered and directly injected into the CE system. Unstable baseline and no separation of the analytes were obtained due to the presence of interferences (Figure 3.1.2.2). However, when these samples were extracted as described in Section 2.5, a stable baseline and few interferences were obtained (Figure 3.1.2.2). As expected, no antidepressant peak was detected in these samples.

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Additionally, acceptable within-run and between-run precision values were calculated for the proposed methodology (Table 3.1.2.2). Consequently, these samples were spiked with different quantities of the selected antidepressants and then the analytes were determined by the proposed CE method (Figure 3.1.2.2). The accuracy results obtained for each analyte after spiking samples at different concentration levels of antidepressants are shown in Table 3.1.2.3. The error values, estimated from the measured values versus the added amounts, ranged between 7.8 and -13.3% (Table 3.1.2.3). Not only better accuracy, but also improved values of LOD and LOQ, as well as better regression coefficients, precision values and recoveries were obtained with this new sorbent when compared with a Strata-XTM commercial sorbent (Table 3.1.2.3). The method proposed in this work possess an adequate dynamic range, low LODs and LOQs like previous procedures including these drugs, and low sample consumption as well. Nevertheless, the linear range determined for the proposed method is adequate for the quantification of fluoxetine and citalopram in urine samples because the prescription doses of these antidepressants are 20–60 mg/day so the urine levels excreted are usually at µg/mL levels [38].

Figure 3.1.2.2. Electropherograms of a 1.0 µg/mL mixture of standard solutions of the antidepressants (A); blank urine sample without waterwash step (B); washed by water (C) and of urine spiked with 0.20 µg/mL of each antidepressant (D). Conditions: capillary (50 cm length × 75 µm i.d.); capillary temperature 20°C; hydrodynamic injection of 10 s; applied voltage 20 kV; buffer 50 mM borax with 20% methanol at pH 9.30; detection at 200 nm.

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Table 3.1.2.2 Precision values calculated for the proposed methodology. Analyses were performed by triplicate in all cases. Concentration Analyte Within-run-precision Between-run-precision (µg/mL) Migration Migration Response Response time time Fluoxetine 0.10 3.43 2.21 3.91 0.41 0.40 2.79 2.75 0.75 0.34 0.80 3.11 3.05 0.55 0.45 Venlafaxine 0.10 4.92 2.01 0.89 0.36 0.40 5.13 1.98 2.17 0.65 0.80 4.73 2.52 4.23 0.88 Citalopram 0.10 5.87 2.15 1.66 0.44 0.40 4.51 2.81 3.23 0.72 0.80 7.37 1.86 0.38 0.95 Sertraline 0.10 7.50 2.27 1.66 0.68 0.40 3.45 1.73 1.26 0.59 0.80 6.77 2.09 0.08 0.21 Precision data expressed as relative standard deviation (%)

Table 3.1.2.3. Accuracy results obtained for the analysis of human urine samples spiked with different concentrations of the antidepressants, extracted either by poly(STY-DVB)-MMWCNT or with a Strata-XTM commercial sorbent Added Founda) Foundb) Analyte Errora) (%) Errorb) (%) (µg/mL) (µg/mL) (µg/mL) Fluoxetine 0.100 0.102 0.072 2.0 ± 3.1 -28.1 ± 3.9 0.200 0.211 0.135 5.5 ± 4.5 -32.3 ± 4.8 0.500 0.464 0.367 -7.2 ± 2.4 -26.7 ± 3.2 1.000 0.980 0.699 -2.0 ± 3.2 -30.1 ± 1.4 Venlafaxine 0.100 0.094 0.071 -6.0 ± 1.8 -29.5 ± 4.1 0.200 0.203 0.132 1.5 ± 1.1 -33.9 ± 1.9 0.500 0.539 0.363 7.8 ± 2.6 -27.5 ± 2.1 1.000 1.028 0.606 2.8 ± 4.5 -39.4 ± 6.0 Citalopram 0.100 0.099 0.072 1.0 ± 2.0 -27.8 ± 2.1 0.200 0.178 0.151 -11.0 ± 2.0 -24.7 ± 3.8 0.500 0.487 0.384 -2.6 ± 1.1 -23.3 ± 1.8 1.000 0.965 0.735 -3.5 ± 4.3 -26.5 ± 5.7 Sertraline 0.100 0.093 0.069 -7.0 ± 3.1 -30.8 ± 2.7 0.200 0.204 0.149 2.0 ± 4.1 -25.5 ± 1.1

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Table 3.1.2.3 (continued) 0.500 0.445 0.388 -11.0 ± 1.6 -22.4 ± 2.5 1.000 0.867 0.712 -13.3 ± 0.7 -28.8 ± 3.4 Error data are expressed as mean ± standard deviation (SD); n = 3. a) current method. b) commercial sorbent (StrataTM – X, from Phenomenex).

Moreover, the solvent consumption is minimal, representing a cost effective alternative if compared with other techniques such as HPLC-DAD or MEKC-DAD. Likewise, the time of analysis (i.e. less than 6 min) is shorter than most of the previously reported methods using DAD to determine these analytes (Table 3.1.2.S2). Furthermore, when the volume of biological samples is limited, the relatively large injection volume requirement in HPLC makes it inferior to CE, and using CE is preferable in therapeutic drug monitoring and toxicological analyses when compared to chromatographic methods such as HPLC, as CE not only requires less amount of biological samples, but its operating cost is lower.

The poly(STY-DVB)-MWCNT-MNP composite used for the selective extraction of the analytes represents a remarkable alternative to liquid-liquid extraction (LLE) procedures, which are usually more time consuming and cannot be automated; hence, they should be better replaced by other extraction techniques such as the MMWCNT composite indicated in this work.

4. Concluding remarks poly(STY-DVB)-MMWCNT was successfully prepared and applied for the extraction of four antidepressants from human urine and their subsequent analysis by CE-DAD. This sample treatment presents several advantages when compared with previously developed traditional LLE [39] and/or SPE [21, 22]. MMWCNT application represents an important trend in solid phase extraction techniques because the application of magnetic separation technology simplifies sample treatment, the sorbent does not need to be packed into the cartridge, the separation phase can be carried out easily by applying an external magnetic field, and the MMWCNT showed great stability and selectivity. In addition, remarkable precision, sensitivity and recovery values were obtained. However, this sample treatment, as well as the separation and detection are

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carried out off-line, and in some pharmacological treatments, the urine samples can contain other drugs that can interfere in the separation of these analytes by CE.

Consequently, the next step could be the development of an on-line approach for the application of MMWCNT and its coupling with more selective and sensitive detectors (e.g., MS or MS/MS), thus expanding the applicability of the method to other clinical and biological samples and to other drugs which could be taken simultaneously by patients suffering from depressive disorders.

The support given through an “INCRECYT” research contract to M. Zougagh is also acknowledged. The Spanish Ministry of Economy and Competitiveness is gratefully acknowledged for funding this work with grant CTQ2016-78793-P.

The authors have no other relevant affiliations or financial involvement with any organization or entity with financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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[38] Unceta, N., Gómez-Caballero, A., Sánchez, A.,Millán, S., Sampedro, M. C., Goicolea, M. A., Sallés, J., Barrio, R. J., J. Pharm. Biomed. Anal. 2008, 46, 763–770.

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SUPPLEMENTARY INFORMATION

Table 3.1.2.S1. Composition of the polymerization mixtures assayed for the methodology developed. Polymer STY (mmol) DVB (mmol) AIBN (g) MMWCNT (g) ACN (mL) P1 29.36 7.90 0.36 --- 25 P2 19.57 15.80 0.36 --- 25 P3 9.79 23.69 0.36 --- 25 MP1 29.36 7.90 0.36 0.15 25 MP2 19.57 15.80 0.36 0.15 25 MP3 9.79 23.69 0.36 0.15 25 ACN: acetonitrile; AIBN: 2,2’-Azobis(2-methylpropionitrile); DVB: divinylbenzene; MMWCNT: magnetic multiwalled carbon nanotube; MP: magnetic polymer; P: non-magnetic polymer; STY: styrene.

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Table 3.1.2.S2. Comparison of several analytical features obtained for the proposed methodology with previous methodologies reported for UV-Vis determination, in human urine, of the antidepressant(s) included in this work. Analyte(s) Instrumentation LOD (ng Analysis Accuracy Sample pre- Extraction Ref. mL-1) time (%) treatment recovery (min) (%) CIT CE-DAD 25 10 92.3 – 106 SPE NS [38] FLX HPLC-UV 90.1 60 NS SPE- 90.1 [39] MWCNTs FLX UHPLC-PDA 80 10 86.6 – 97.1 MEPS 98 – 98.8 [S1] FLX UHPLC-PDA 60 10 93.2 – 103 DSPE 99.6 – 109 [S2] SER HPLC-DAD NA 21 NA SPE ca. 90 [S3] SER CE-UV 1.53 12 90.6 – 97.7 DLLME NS [S4] VFX HPLC-DAD 0.03 7 93.2 – 109 SPME-Gly- 86.4 – 90.1 [S5] MWCNTs CIT, FLX HPLC-DAD 13, 10 8 90 – 110 SPME <80 [S6] CIT, FLX HPLC-UV 0.2, 0.5 13 91, 94 ATN-SPME NS [S7] CIT, SER HPLC-UV 1.3, 1.2 8 97 – 107 UA-DLLME- >92.9 [S8] SFODs FLX, VFX HPLC-DAD 400, 800 7 99.9 – 110 SPE >77 [S9] CIT, FLX, HPLC-UV 3 15 93.6 – 105 USAEMEI- 93.3, 49.5, [S10] VFX SFO 58.2 CIT, FLX, HPLC-UV-Vis 0.51, 20 85.9 – 104 LLE- 96.2, 102, [S11] SER 0.89, chloroform 103 0.30 CIT, FLX, MEKC-DAD 10–20 11 NS LLE-ethyl NS [29] SER, VFX ether CIT, FLX, CE-DAD 37, 41, 6 86.7 – 108 SPE- 89.5 – 106 This SER, VFX 14, 16 MMWCNTs work ca: circa; NA: not analyzed; NS: not specified. ATN-SPME: arrays of tin oxide nanorods-solid phase microextraction fiber; CIT: citalopram; DAD: diode array detection; DSPE: dispersive solid phase extraction; DLLME: dispersive liquid–liquid microextraction; FLX: fluoxetine; Gly: glycine; LLE: liquid-liquid extraction; LOD: limit of detection; MEKC: micellar electrokinetic chromatography; MEPS: microextraction with packed sorbent; MMWCNTs: magnetic multiwalled carbon nanotubes; MWCNTs: multi-walled carbon nanotubes; PDA: photodiode array; Ref.: reference(s); SER: sertraline; SPME: solid phase microextraction; UA-DLLME-SFODs: ultrasound-assisted dispersive liquid–liquid microextraction based on solidification of floating organic droplets; USAEMEI-SFO: Ultrasound-assisted emulsification microextraction based on the solidification of floating organic droplet; VFX: venlafaxine.

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Figure 3.1.2.S1. Fourier transform infrared (A) and Raman spectra (B) of MMWCNT, Poly(STY- DVB) and MMWCNT- Poly(STY-DVB) composite.

Figure 3.1.2.S2. Micrographs obtained by TEM of the magnetic nanoparticles attached onto the surface of the nanotubes (A), spherical poly(STY-DVB) particles (B), and MMWCNT-poly(STY-DVB) composite material (C) and (D).

Figure 3.1.2.S3. Particle size distribution (A) and zeta potential (B) of MMWCNT-poly(STY-DVB) composite material.

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Additional References

[S1] Flores J. R., Salcedo A. M. C., Llerena M. J. V., Fernández L. M., J. Chromatogr. A. 2008, 1185, 281–290.

[S2] Cruz-Vera, M., Lucena, R., Cárdenas, S., Valcárcel, M., Anal. Bioanal. Chem. 2008, 391, 1139–1145.

[S3] Alves, V., Gonçalves, J., Conceição, C., Teixeira, H. M., Câmara, J. S., J. Chromatogr. A. 2015, 1408, 30–40.

[S4] Alves, V., Conceição, C., Gonçalves, J., Teixeira, H. M., Câmara, J. S., J. Anal. Toxicol. 2017, 41, 45–53.

[S5] Klinke, H. B., Linnet, K., Scand. J. Clin. Lab. Invest. 2007, 67, 778–782.

[S6] Huang, S-W., Hsieh, M-M., Chang, S. Y., Talanta. 2012, 101, 460–464.

[S7] Ghorbani, M., Chamsaz, M., Rounaghi, G. H., Anal. Bioanal. Chem. 2016, 408, 4247– 4256.

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[S11] Ebrahimzadeh, H., Saharkhiz, Z., Tavassoli, M., Kamarei, F., Asgharinezhad, A. A., J. Sep. Sci. 2011, 34, 1275–1282.

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3.1.3. Magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites for the extraction and LC-MS determination of catecholamines and related compounds in red deer urine and hair

Submitted to Journal of Chromatography A

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Abstract

A novel analytical method for the extraction and subsequent determination of some catecholamines (dopamine, epinephrine and norepinephrine) and their metabolites 3,4- dihydroxyphenyl glycol and 3,4-dihydroxymandelic acid by LC-MS is here developed and validated for application to human and animal urine and hair samples. The method is based on the preliminary extraction of analytes by a magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composite. This is followed by a < 9 min chromatographic separation of the target compounds in an Onyx Monolithic C18 column using a mixture of 0.01% heptafluorobutyric acid in methanol and water at a flow rate of 500 µL min-1. Limits of detection ranged from 0.055 to 0.093 µg mL-1, and precision values for the response of analytes and retention times were > 90%. Accuracy values comprised the range 79.5–109.5% when the analytes were extracted from deer urine samples using the selected MMWCNT-poly(STY-DVB) sorbent. This method was applied to real red deer urine and hair samples, resulting in concentrations ranging from 0.05 to 0.5 mg mL-1 for norepinephrine and from 1.0 to 44.5 mg mL-1 for its metabolite 3,4- dihydroxyphenyl glycol. Analyses of red deer hair resulted in high amounts of 3,4- dihydroxyphenyl glycol (0.9–266.9 µg mL-1).

Keywords: catecholamines, deer urine, deer hair, magnetic composites, LC-MS.

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GRAPHICAL ABSTRACT

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1. Introduction

The catecholamines dopamine (DA), norepinephrine (NE) and epinephrine (EP) are monoamines with a catechol structure that are synthesized in the adrenal gland of mammals. These compounds function as neurotransmitters or hormones [1], thus playing a crucial role in several physiological processes, affecting behavior by their receptor-specific binding at cells. For instance, EP and NE are involved in the fight-or- flight response by inducing muscle cell contraction, thus affecting survival [2], as well as in the stress response [3]. Variations in the levels of these hormones are usually associated to pathological conditions and, therefore, influence the performance of several drug and pharmacological treatments. Additionally, catecholamines have many other physiological effects. For instance, blood glucose and lactate levels may increase with their circulating levels, particularly with EP infusion [4]. On the other hand, catecholamines have diverse effects on immune cells. For example, continuous infusion of physiological concentrations of NE and EP enhance chicken leukocyte migration and differentially affect the phytohemagglutinin response [5].

Consequently, the determination of catecholamines is essential for understanding certain physiological regulation pathways regulated by the sympathetic nervous system, including some specific animal and human behavioral disorders [6]. Moreover, the qualitative and quantitative analysis of these hormones and their metabolites does not only aid to improve some drug treatments, but also the diagnosis of different diseases, providing a unique information about disease development and treatment effects [7,8].

Catecholamines have been quantified in different biological matrices, from both human (plasma [9], urine [10], and saliva [11]) and animal origin (rat kidney [12], rat brain [13,14], mouse bone marrow [15], rat heart tissue [16] and dog kidney [17]). Indeed, variable levels of catecholamines have been determined in human plasma (1-800 pg mL- 1), cerebrospinal fluid (0-300 µg mL-1), urine (100-400 µg mL-1) and brain tissue (10-1000 µg g-1) [18].

Different techniques have been used for the determination of these endogenous molecules, including gas chromatography-mass spectrometry [19], capillary electrophoresis [20], fluorescence [21], chemiluminescence [22], electrochemical

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detection [23], gas chromatography-flame ionization detection [24] and liquid chromatography-tandem mass spectrometry [25]. High-performance liquid chromatography (HPLC) probably is the most widely used separative technique for the simultaneous determination of catecholamines and their metabolites [8,26], due to its reliability and accuracy as compared to other techniques.

However, most of the approaches previously described are quite laborious and time consuming, especially at the extraction of catecholamines from the original tissues. Furthermore, the monitoring of endogenous molecules usually requires sampling internal biological material such as blood, which may be difficult and troublesome for the organisms being sampled. This is particularly evident when samples must be obtained from wild animals. Consequently, different approaches for sample treatment have been proposed to remove interfering substances and/or enrich the target compounds [27]: liquid-liquid extraction or microextraction (LLE) [28], solid phase extraction (SPE) [29] or magnetic solid phase extraction (MSPE) [30]. However, some of them are still time consuming and complex (e.g. LLE) or achieve poor analyte recoveries, insufficient cleanup or irreproducibility, which may occur even including SPE [31]. In contrast, more recent polymeric sorbents synthesized by polymerizing styrene and divinylbenzene (e.g. Strata-X®, from Phenomenex®) overcome common bottlenecks associated to the use of traditional SPE sorbents. This commercial support strongly retains neutral, acidic, or basic compounds even under aggressive or high organic washing conditions. The improvement arises because this material possesses three retention mechanisms: π-π bonding, hydrogen bonding (dipole-dipole interactions) and hydrophobic interactions. Thus, the use of polymeric sorbents represent a good alternative for the analysis of catecholamines [32,33]. Likewise, multiwalled carbon nanotubes (MWCNTs) [34] have a great potential as SPE sorbent for catecholamines due to their hydrophobicity, a large specific surface area and an excellent chemical stability [35]. However, MWCNTs are usually applied in a cartridge or in the column format, which often results in tedious packing procedures, high backpressure issues and low flow rates. To overcome these drawbacks, incorporating MWCNTs and poly(styrene-co- divinylbenzene) in MSPE could represent an excellent alternative [36].

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Here we propose a new methodology for the simultaneous determination of the catecholamines DA, NE, EP, and their methoxylated derivatives DL-3,4- dihydroxymandelic acid (DHMA) and DL-3,4-dihydroxyphenyl glycol (DOPEG) based on a magnetic MWCNT poly(styrene-co-divinylbenzene) composite for analyte extraction prior to determination by liquid chromatography-mass spectrometry (LC-MS). We optimized and validated the method in deer urine and hair samples.

2. Experimental

2.1. Chemicals, material and samples

DL-3,4-dihydroxymandelic acid (DHMA), DL-3,4-dihydroxyphenyl glycol (DOPEG), norepinephrine, epinephrine, dopamine, heptafluorobutyric acid (HFBA), styrene (STY) (≥99%), divinylbenzene (DVB) (80%), iron (III) chloride hexahydrate, sodium acetate and ethylene glycol, for the synthesis of the composites, were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC-MS grade methanol (MeOH) and acetonitrile (ACN) were obtained from Fisher Scientific (Loughborough, Leics, UK), while acetic acid (≥ 99.7% purity) and Triton X-100 were also purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2’-Azobis(2-methylpropionitrile) (AIBN) was purchased from Fluka Chemie (Buchs, UK), and ultrapure water was obtained from a Milli-Q water instrument from Millipore (Merck KGaA, Darmstadt, Germany).

The stock solutions of the analytes were prepared at 1 mg mL-1 in 0.5% acetic acid and stored in absence of light at -20°C. Working standard solutions were prepared at 10 μg mL-1 by appropriate dilution in water and also stored at -20°C, until further use.

We obtained urine and hair samples of wild Iberian male red deer Cervus elaphus hispanicus that were harvested in hunts in Spain. Within 1 h after deer death, 10 mL of urine were directly extracted from the urinary bladder using a syringe, and 250 μL of 1 M HCl was immediately added. Deer urine samples were first kept at 4 °C, and then stored at −80 °C within 4 h of extraction. Then, the urine samples were diluted with water (1:100 v/v). On the other hand, hair samples from the dark ventral patch of male red deer were cut and stored at −80 °C until their analysis. More specifically, 10 strands of hair were collected and 2 cm length pieces from the middle part of each strand were cut using scissors and a rule, thus the starting material was composed by 10 strands of

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hair 2 cm long each. Then, every single piece was trimmed in 4 smaller pieces 0.5 cm length each to finally introduce the bundle of hair pieces in a 1.5 mL tube for an easier immersion into the extraction buffer. Then, 0.2 mL of 20 mM sodium acetate-acetic acid buffer solution (pH 5) with 0.08% in Triton X-10 were added, and the tubes were vortexed vigorously for a few seconds and then subject to slow orbital shaking at room temperature during 1 h. Finally, the mixture was centrifuged at 3000 rpm for 10 min, and the supernatant was collected and injected into the LC-MS system.

2.2. Instrumentation and apparatus

An HPLC system (Agilent series 1200, Waldbronn, Germany) and an Onyx Monolithic C18 (100 × 4.6 mm) column from Phenomenex® (Torrance, CA, USA) were utilized for the chromatographic separation of analytes. This system consists of a degasser, a liquid chromatographic pump, an autosampler, a temperature controlled column compartment and a diode array detector (Agilent, 1260 infinity model). Detection was carried out with an Agilent 6110 series MS detector (Waldbronn, Germany) equipped with an atmospheric pressure ionization source electrospray (API-ES).

A Biosan Microspin 12 microcentrifuge (LabNet Biotecnica S.L., Spain) and a Basic 20 pH- meter with a combined glass electrode (Crison Instruments S.A., Spain) were also used. Raman spectroscopy analyses were performed with a portable Raman Spectrometer system (B&W TEK Inc., DE, USA) using a 785 nm excitation laser source. The laser beam was focused on the samples through a 100/1.25 objective. For infrared spectroscopy analyses, we used an FT/IR 4200 Fourier transform infrared (FT-IR) spectrometer (Jasco, Japan). Transmission electron microscopy (TEM) images were obtained with a Jeol JEM 2011 microscope operating at 200 kV and equipped with an Orius Digital Camera (2 × 2 MegaPixel) from Gatan (Pleasanton, CA, USA), while the distribution of particle sizes and the zeta-potential were measured by dynamic light scattering (DLS) using a Zetasizer Nano instrument (model ZEN3600) from Malvern (Worcestershire, UK).

2.3. Preparation of magnetic multiwalled carbon nanotubes (MMWCNTs)

MMWCNTs were prepared by in situ high temperature combination of the magnetic precursor [iron(III)] and MWCNTs, according to a previously described procedure [37].

Briefly, this synthesis involves the mixture of 140 mg of FeCl3.6H2O and 40 mg of

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MWCNTs, which was then suspended in 7.5 mL of ethylene glycol in a glass vial. Later, 0.36 g of sodium acetate was added and the solution was stirred, sonicated for 10 min and left at room temperature for 60 min. After that, the vial was heated at 200°C for 48 h to complete the reaction and, after cooling down to room temperature, the product was washed 5 times with 10 mL of distilled water. MMWCNTs were then separated by applying a magnet and the obtained nanomaterial was dried at 80°C and stored at room temperature. This procedure allowed to obtain nanomaterials as reduced as possible to increase the surface area and the retention capacity of analytes onto the composite.

2.4. Preparation of magnetic multiwalled carbon nanotube poly(styrene-co- divinylbenzene) composite (MMWCNT-poly(STY-DVB))

MMWCNT-poly(STY-DVB) was synthesized by mixing 9.79 mmol of DVB, 23.69 mmol of STY, 0.36 g of AIBN, 0.15 g of MMWCNTs and 25 mL of ACN at 70°C for 24 h [36]. The mixture was sonicated for 5 min, purged with nitrogen gas for 10 min and then closed. The resulting product was collected, washed several times with water together with mechanical agitation and dried in a vacuum desiccator for 24 h.

2.5. Extraction with MMWCNT-poly(STY-DVB) composite

500 mg of the magnetic composite was put into a conical flask, conditioned first with 2×3 mL of MeOH and then with deionized water. The supernatant was separated with a magnet, discarded, and 50 mL of the standard solutions of the analytes or of urine samples (pH 5.2) were added into the conical flask. For the extraction of the analytes from deer hair samples, 2 mL of standard solution of the analytes or of extracted deer hair samples were added into 10 mL beaker. The mixture was then stirred at room temperature for 5 min to create a homogenous dispersion and the magnetic polymer containing the adsorbed catecholamines was rapidly removed from the solution applying a strong external magnetic field. The supernatant was discarded, the catecholamines were finally eluted by rinsing with 1.0 mL of MeOH containing 0.01% of HFBA, and the eluent was dried under a stream of nitrogen gas at room temperature. The residue was finally dissolved in 250 μL of initial mobile phase and an aliquot of 1.0 μL was injected into the LC-MS system.

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2.6. LC-MS analysis

MS detection of analytes was carried out in positive ion mode under the following conditions: 12 L min-1 of drying gas flow, drying gas temperature at 300°C, a nebulizer pressure of 35 psi and a capillary voltage of 2500 V. Single ion monitoring (SIM) was used to detect the target analytes, which were quantified by using the standard addition method. Previously, the analytes were qualitatively determined at full scan mode matching the retention time and mass spectra with those previously obtained with standards. The m/z ions used for identification were: 167 (DHMA), 153 (DOPEG), 152 (NE), 166 (EP) and 137 (DA).

Chromatographic analysis was carried out using a gradient formed by a 0.01% HFBA in water as solvent A, and 0.01% HFBA in MeOH as solvent B. The gradient was carried out at a flow-rate of 500 µL min-1, starting from 10% B during 0.5 min, then increasing up to 20% B until 3 min, 60% B from 3-3.5 min, 85% B from 3.5 to 7 min, 90% of B until 8 min and decreasing back to 10% of B for a final duration of 12 min. Injection volume was 1.0 μL, and the column was maintained at a temperature of 25°C. The column was re- equilibrated for 20 min after each run. All solvents were filtered through a 0.45 μm nylon membranes before their use.

2.7. Method validation

The method was validated using calibration standard solutions and quality control (QC) samples for the calibration curve of each analyte. The validation was conducted with respect to selectivity, limits of detection (LODs) and lower limit of quantification, linearity, recovery, accuracy, and precision. The stability was also evaluated. The method validation followed the recommendations of the European Medicines Agency Guideline on bioanalytical method validation [38].

The evaluation of stability was made with assays to determine the freeze/thaw and short-term stability. More specifically, QC samples (low, medium and high) were analyzed after three freeze/thaw cycles (n=6). For each freeze/thaw cycle, the samples were frozen at -20°C, then thawed (23°C) and kept at room temperature for 3 h. The concentrations of the QC samples were calculated based on the daily calibration curves. For short-term stability, the QC samples were extracted, placed in the autosampler at

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room temperature for 24 h, and then analyzed. In this case, the measured concentrations were compared to those of the same QC samples that had been analyzed immediately after processing. CVs within 15% were considered acceptable values.

3. Results and discussion

To our knowledge, this is the first application of our extraction approach and separation method by LC-MS to catecholamines from biological samples.

3.1. Optimization of experimental parameters for catecholamine determination

The optimization of conditions of desorption was carried out using MeOH containing 0.01% of different organic solvents (i.e. acetic acid, formic acid and HFBA), with the sorbent and 5 mL of a 1.0 µg mL-1 standard solution of the analytes at pH 5.2. The three acidic solvents eluted the catecholamines, though higher desorption was found when HFBA was used, and it was therefore selected for subsequent experiments. Additionally, the minimum volume of solvent needed for an efficient elution of the adsorbed catecholamines was optimized. After testing a 0.5–3.0 mL range, the best results were obtained when 1.0 mL was used. Consequently, MeOH containing 0.01% of HFBA (2×0.5 mL) under agitation during 5 min was utilized for an optimal desorption of the analytes. Under these conditions, the whole extraction procedure is accomplished within 15 min.

Regarding the optimization of sensitivity and selectivity of the chromatographic analysis, different organic acids (i.e. acetic acid, formic acid and HFBA) were alternatively added to MeOH. The highest sensitivity and selectivity values were obtained when 0.01% HFBA was used. Then, different proportions of HFBA, ranging from 0.01 to 0.1%, were tested, and the best sensitivity and selectivity were obtained when a 0.01% HFBA ratio was used. On the other hand, MeOH and ACN as organic solvents of the mobile phase were studied, and 0.01% HFBA in MeOH gave the best results in terms of sensitivity and selectivity. Likewise, to optimize the MS detection of each analyte, drying gas flow and temperature, the nebulizer pressure and capillary voltage values were analyzed. A drying gas flow of 12 L min-1, 300°C of drying gas temperature, a nebulizer pressure of 35 psi and a papillary voltage of 2500 V were selected as the optimal values according to the peak areas obtained.

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3.2. Characterization of MMWCNT-poly(STY-DVB) sorbent

The synthesis of MMWCNT-poly(STY-DVB) composite was further confirmed by Raman and IR spectroscopy (Figure 3.1.3.1A). The characteristic D and G bands at 1351 and 1588 cm−1 for MWCNTs were observed in the MMWCNT-poly(STY-DVB) polymer that was prepared. In the the FT-IR spectra, the characteristic bands of aromatic (C=C) bonding and alkyl (C-H) stretches originated from the poly(STY-DVB) were observed at 1700 and 2800 cm−1, respectively. A band at 3085 cm−1 corresponding to the aromatic (C=C) stretching and a broad band at 3400 cm−1 indicative of the presence of hydroxyl (- OH) groups on nanoparticles’ surface were also observed (Figure 3.1.3.1B). To confirm whether the MMWCNT-poly(STY-DVB) composite was effectively produced, the material was characterized by TEM (Figure 3.1.3.1C). The micrograph of MMWCNT- poly(STY-DVB) composite material revealed the attachment of the MNPs onto the MWCNTs and poly(STY-DVB). The size distribution and zeta potential of MMWCNT- poly(STYDVB) composite was characterized by DLS. The microspheres are clearly observed with uniform sizes (Figure 3.1.3.1D). It is expected that the microspheres were obtained with controlled zeta potentials by AIBN as an initiator: -38.2 mV (Figure 3.1.3.1E). The value of zeta potential should be ascribed to the charge of free radicals from the AIBN in emulsion polymerization, such as [(CH3)2˙C(CN)]2.

Figure 3.1.3.1. Characterization of the MMWCNT-poly(STY-DVB) composite by means of (A) FT- IR spectra, (B) Raman spectrum, (C) TEM micrograph, (D) Particle size distribution and (E) Zeta- potential.

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3.3. Validation of the methodology and application to real samples

Different solutions of catecholamines ranging from 0.25 to 2.0 µg mL-1 were extracted using MMWCNT-poly(STY-DVB) sorbent under the aforementioned optimized conditions. The analytes were eluted with 1.0 mL of 0.01% HFBA in MeOH and an aliquot of 1.0 µL was injected into the LC-MS. All the catecholamines were baseline separated and quantified in less than 9 min (Figure 3.1.3.2).

The analytical parameters of the method were additionally examined. The linear range, intercept and slope of the curve were calculated for each catecholamine (Table 3.1.3.1). The limits of detection, defined as the concentration of the analyte giving a signal equivalent to the blank signal plus three times its SD, are also presented (Table 3.1.3.1). Considering that the blank signal was practically the same for all analytes, intercept values and their corresponding SDs of the calibration equations were used to calculate detection limit values. The precision of the method, expressed as RSD, was obtained by injecting a 1.0 µg mL-1 standard solution ten times during a working session. In all cases, acceptable run-to-run precision and inter-day precision values (expressed as RSD) were obtained for both the retention time and the response of each analyte (Table 3.1.3.1).

Figure 3.1.3.2. LC-MS chromatograms of a blank human urine (yellow), 2 µg mL-1 standard solution of the selected catecholamines (blue) and 0.5 µg mL-1 spiked human urine sample extracted using MMWCNT-poly(STY-DVB) sorbent (green). Peak identification: DHMA (1), DOPEG (2), NE (3), EP (4) and DA (5).

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Table 3.1.3.1. Calibration data and precision data obtained for the developed methodology.

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The chromatograms in Figure 3.1.3.2 obtained for human urine samples, which were then spiked at different concentrations with the selected catecholamines and determined by the proposed method. A human urine sample spiked at 2.0 µg mL-1 with each catecholamine is also shown to exemplify these determinations (Figure 3.1.3.2). The proposed method was applied to analyze the selected catecholamines in human urine samples obtained from healthy volunteers, but no peaks of interest were detected in any case (Figure 3.1.3.2). To evaluate the applicability of the present method, these samples were then spiked with selected levels of the catecholamines and subsequently analyzed by LC-MS after their extraction using MMWCNT-poly(STY-DVB) sorbent (Figure 3.1.3.2).

Additionally, recovery values for deer urine were calculated within a range from 79.14% to 102.3% at the low concentration (0.25 μg mL−1), from 85.66% to 101.5% at the medium concentration (1.0 μg mL−1) and from 80.41% to 105.67% at the high concentration (2.0 μg mL−1). The precision values of recovery deer urine and hair were found to be acceptable, as all CV values were lower than 15% at all concentrations (Table 3.1.3.2). Stability for at least 24 h in the autosampler at room temperature was confirmed for the analytes in the extracted deer urine samples, as no deviation greater than ± 15% of the nominal concentration was calculated, and a CV lower than 15% was found. Likewise, the stability of the analytes in the extracted deer urine samples after three freeze (−20°C)/thaw (23°C) cycles was also confirmed (Table 3.1.3.2).

Table 3.1.3.2. Extraction recovery and stability results calculated for red deer urine analyzed with the proposed methodology.

On the other hand, the applicability of the proposed method for the determination of catecholamines was evaluated by analyzing different samples of urine and hair obtained

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from male red deer. Chromatograms in Figure 3.1.3.2 illustrate the adequate separation and detection of the selected catecholamines obtained from an extract of deer urine and hair samples. Table 3.1.3.3 shows the results of the accuracy assays obtained after the analysis of quality control samples. These experiments demonstrated that the values of accuracy of spiked samples extracted by MMWCNT-poly(STY-DVB) sorbent ranged in an acceptable level of accuracy, between 79.5 and 109.5%. Remarkably improved accuracy results were obtained when the extraction method proposed in this study was applied and, therefore, the overall strategy demonstrates a good applicability in practice, thus offering a promising potential for its application in clinical, biological and physiological studies. Interestingly, the precursors DA and EP were not found in any of the 33 samples collected. In contrast, unusually high concentrations of NE were found in the urine samples from all subjects (mean ± SE, 0.18 ± 0.02 mg mL-1, range 0.05–0.48 mg mL-1) as shown in Figure 3.1.3.3. In congruence with the known metabolism of catecholamines, the alcoholic derivative of norepinephrine (DOPEG) was detected in remarkably high concentrations in all samples (mean ± SE, 8.90 ± 1.42 mg mL-1, range 0.91–44.52 mg mL-1), as shown in Figure 3.1.3.3. The acidic derivative DHMA, however, was only detected in four samples at considerably lower concentrations than DOPEG (mean ± SE, 0.15 ± 0.05 mg mL-1, and range 0.06–0.26 mg mL-1).

Figure 3.1.3.3. LC-MS chromatograms of: (a) a 10 μg mL-1 standard solution of DA, NE, EP, DHMA and DOPEG (red), a 1:100 diluted urine sample from a wild Iberian male red deer (black), and one extracted wild Iberian male red deer urine sample using MMWCNT-poly(STY-DVB) sorbent (green); (b) extracts from deer hair of the dark ventral patch (black), and extract of red deer hair of the dark ventral patch using MMWCNT-poly(STY-DVB) sorbent (red). Additional peaks in (b) different from that of DHPG are of unknown origin. Peak identification: DHMA (1), DOPEG (2), NE (3), EP (4) and DA (5).

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Table 3.1.3.3. Accuracy results obtained in deer urine samples with different concentrations of catecholamines.

Analyte Added (µg mL-1) Found (µg mL-1) Accuracy (%)

DHMA 1.0 0.851 85.1 ± 3.93

2.0 1.696 84.8 ± 1.52

DOPEG 1.0 1.091 109 ± 5.77

2.0 1.645 82.3 ± 2.41

NE 1.0 0.799 79.9 ± 1.28

2.0 2.191 109 ± 4.62

EP 1.0 0.817 81.7 ± 2.74

2.0 1.753 87.7 ± 4.11

DA 1.0 0.801 80.1 ± 1.79

2.0 1.590 79.5 ± 3.48

Accuracy data are expressed as mean ± standard deviation (SD); n=3.DHMA: DL-3,4-Dihydroxymandel acid; DOPEG: DL-3,4-Dihydroxyphenyl glycol; NE: Norepinephrine; EP: Epinephrine and DA: Dopamine.

In congruence with the known metabolism of catecholamines (Figure 3.1.3.S1), the alcoholic derivative of norepinephrine (DOPEG) was the only analyte found in the red deer hair samples [2]. Indeed, remarkably high concentrations of DOPEG were found in red deer ventral darkened hair (mean ± SE, 58.64 ± 10.32 µg mL-1, range 0.93–266.93 mg mL-1). (Table 3.1.3.S1) shows some results obtained from the analyzed male red deer urine and dark ventral patch hair samples.

In sum, this method shows clear advantages in terms of time of analysis and precision as compared with existing alternative methods involving the determination of these catecholamines in biological samples [39].

4. Conclusions

A novel and simplified analytical method for the determination of catecholamines in deer urine and hair based on MMWCNT-poly(styrene-co-divinylbenzene) composites was developed and validated. This methodology is highly selective for catecholamines and the novel extraction approach is simple and reproducible. The proposed method may thus be successfully employed in clinical chemistry and also in the study of

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catecholamine-related diseases, physiological and biological issues. On the other hand, the sample treatment developed and the separation and detection steps are carried out off-line, thus further research regarding an on-line extraction approach together with more sensitive detectors such as MS/MS are warranted.

Acknowledgments

Iberian red deer samples were collected by Juan Carranza and Eva de la Peña (Universidad de Córdoba). I. Galván benefits from a Ramón y Cajal Fellowship (RYC-2012- 10237) from Spain’s Ministry of Science, Innovation and Universities. Financial support was obtained from project CTQ2016-78793-P from the Spanish Ministry of Economy and Competitiveness (MINECO) and from project SBPLY/17/180501/000262 from Junta de Comunidades de Castilla-La Mancha.

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[28] L. Konieczna, A. Roszkowska, M. Niedźwiecki, T. Baczek, Hydrophilic interaction chromatography combined with dispersive liquid-liquid microextraction as a preconcentration tool for the simultaneous determination of the panel of underivatized neurotransmitters in human urine samples, J. Chromatogr. A 1431 (2016) 111-121.

[29] D. Talwar, C. Williamson, A. McLaughlin, A. Gill, D.S.J. O’Reilly, Extraction and separation of urinary catecholamines as their diphenyl boronate complexes using C18 solid-phase extraction sorbent and high-performance liquid chromatography, J. Chromatogr. B 769 (2002) 341-349.

[30] M. Bouri, M.J. Lerma-García, R. Salghi, M. Zougagh, A. Ríos, Selective extraction and determination of catecholamines in urine samples by using a dopamine magnetic molecularly imprinted polymer and capillary electrophoresis, Talanta 99 (2012) 897-903.

[31] S.C. Moldoveanu, Solutions and Challenges in Sample Preparation for Chromatography, J. Chromatogr. Sci. 42 (2004) 1-14.

[32] M.M. Sack, B.R. Cooke, Extraction of Catecholamines and Metanephrines from Urine Using Strata X-CW Solid Phase Extraction Cartridges, AACB 51th Annual Scientific Conference (2013).

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[33] J. Layne, M. Brusius, Extraction and Analysis of Urinary Catecholamines by LC- MS/MS Using Strata®-X-CW Microelution Solid Phase Extraction and Luna Omega and Kinetex Polar C18 LC Columns, Phenomenex, Inc. (2017).

[34] J.B. Ma, H.W. Qiu, Q.H. Rui, Y.F. Liao, Y.M. Chen, J. Xu, P.P. Zhan, Y.G. Zhao, Fast determination of catecholamines in human plasma using carboxyl-functionalized magnetic-carbon nanotube molecularly imprinted polymer followed by liquid chromatography-tandem quadrupole mass spectrometry, J. Chromatogr. A 1429 (2016) 86-96.

[35] M. Cruz-Vera, R. Lucena, S. Cárdenas, M. Valcárcel, Combined use of carbon nanotubes and ionic liquid to improve the determination of antidepressants in urine samples by liquid chromatography, Anal. Bioanal. Chem. 391 (2008) 1139-1145.

[36] K. Murtada, F. de Andrés, A. Ríos, M. Zougagh, Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styrene-co- divinylbenzene) composites and separation by capillary electrophoresis, Electrophoresis 39 (2018) 1808-1815.

[37] C. Adelantado, K. Murtada, A. Ríos, M. Zougagh, Magnetic multi-walled carbon nanotube poly(styrene-co-divinylbenzene) for propranolol extraction and separation by capillary electrophoresis, Bioanalysis 10 (2018) 1193-1205.

[38] Committee for Medical Products for Human Use, Guideline on bioanalytical method validation Guideline on bioanalytical method validation, European Medicinal Products Agency, London, UK (2011).

[39] Y. Sakaguchi, H. Yoshida, T. Hayama, M. Itoyama, K. Todoroki, M. Yamaguchi, H. Nohta, Selective liquid-chromatographic determination of native fluorescent biogenic amines in human urine based on fluorous derivatization, J. Chromatogr. A 1218 (2011) 5581-5586.

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SUPPLEMENTARY INFORMATION

Figure 3.1.3.S1. Metabolic pathway of catecholamines from precursor dopamine [2].

Table 3.1.3.S1. Concentration of the catecholamines determined at the analysis of Iberian male red deer urine and dark ventral patch hair.

Found concentration (mg mL-1) Sample DHMA DOPEG NE EP DA Urine Hair Urine Hair Urine Hair Urine Hair Urine Hair S1 ND ND 12.810 0.023 0.079 ND ND ND ND ND S2 ND ND 44.520 0.071 0.182 ND ND ND ND ND S3 0.2564 ND 1.662 0.002 0.076 ND ND ND ND ND S4 ND ND 13.230 0.060 0.265 ND ND ND ND ND S5 ND ND 12.450 0.052 0.240 ND ND ND ND ND S6 ND ND 10.350 0.039 0.220 ND ND ND ND ND S7 ND ND 3.633 0.028 0.115 ND ND ND ND ND S8 ND ND 9.933 0.022 0.186 ND ND ND ND ND S9 ND ND 9.413 0.067 0.207 ND ND ND ND ND S10 ND ND 7.979 0.042 0.204 ND ND ND ND ND S11 ND ND 2.987 0.022 0.125 ND ND ND ND ND S12 0.059 ND 1.343 0.075 0.092 ND ND ND ND ND S13 ND ND 9.799 0.100 0.323 ND ND ND ND ND S14 ND ND 0.994 0.008 0.058 ND ND ND ND ND S15 ND ND 13.245 0.012 0.172 ND ND ND ND ND S16 0.227 ND 7.377 0.031 0.140 ND ND ND ND ND S17 ND ND 0.906 0.001 0.050 ND ND ND ND ND S18 ND ND 3.831 0.006 0.207 ND ND ND ND ND

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Table 3.1.3.S1. (continued) S19 ND ND 2.126 0.005 0.101 ND ND ND ND ND S20 ND ND 3.275 0.013 0.138 ND ND ND ND ND S21 0.067 ND 2.719 0.014 0.089 ND ND ND ND ND S22 ND ND 2.471 0.093 0.092 ND ND ND ND ND S23 ND ND 15.173 0.144 0.368 ND ND ND ND ND S24 ND ND 9.044 0.056 0.168 ND ND ND ND ND S25 ND ND 18.259 0.073 0.099 ND ND ND ND ND S26 ND ND 3.641 0.016 0.133 ND ND ND ND ND S27 ND ND 15.752 0.195 0.094 ND ND ND ND ND S28 ND ND 2.985 0.018 0.125 ND ND ND ND ND S29 ND ND 8.042 0.056 0.345 ND ND ND ND ND S30 ND ND 8.156 0.139 0.254 ND ND ND ND ND S31 ND ND 17.315 0.088 0.480 ND ND ND ND ND S32 ND ND 8.550 0.097 0.317 ND ND ND ND ND S33 ND ND 9.728 0.267 0.310 ND ND ND ND ND DHMA: DL-3,4-Dihydroxymandelic acid; DOPEG: DL-3,4-Dihydroxyphenyl glycol; NE: Norepinephrine; EP: Epinephrine; DA: Dopamine and ND: Not detected.

Up to our knowledge, precise and personalized dosage of antidepressants is usually required for each patient to attain obtain an efficient clinical response as well as to avoid the potential occurrence of serious side effects. Consequently, monitoring these compounds in patients under treatment is of remarkable interest.

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Over the last decade, DBS sampling technique has emerged as a promising substitute of traditional liquid biometrics. Furthermore, DBS sampling and the subsequent analysis is gaining interest in therapeutic drug monitoring of antidepressants as this sampling approach possesses certain advantages when compared with conventional venous sampling. Moreover, the low blood volume required facilitates sample shipment and storage as no special treatment is needed. On the other hand, some limitations of the use of DBS, preprossessing may increase lab costs, hematocrit effect and uncertenity about long-term stability.

In this sense, in this section a new and simple method based on poly(styrene-co- divinylbenzene)-coated glass blood spot was developed and successfully checked. This method was developed for the determination and monitoring of seven types of antidepressants in just a humun blood drop, prior to their determination by CLC-MS.

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3.1.4. A simple poly(styrene-co-divinylbenzene)- coated glass blood spot method for monitoring of seven antidepressants using capillary liquid chromatography-mass spectrometry

Talanta 188 (218) 772 – 778

DOI: 10.1016/j.talanta.2018.06.059

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Abstract

A simple, rapid, selective and sensitive monitoring method for the simultaneous determination of the widely-prescribed antidepressants agomelatine, bupropion, citalopram, fluoxetine, mirtazapine, paroxetine, trazodone in just a human blood drop is here developed and validated. This methodology is based on the use of lab manufactured poly(styrene-co-divinylbenzene)-coated glass (PS-DVB) blood spot for the extraction of the analytes and their subsequent separation and detection by capillary liquid chromatography-mass spectrometry (CLC-MS). Briefly, 10 mm-side squares were punched out from blood spots collected on glass substrate coated by 10 μg of the PS- DVB polymer and eluted with 1.0 mL of 2.0% acetic acid in methanol. The analytes were then separated and detected in less than 20 min by capillary CLC-MS using a Jupiter 4 μm Proteo 90 Å column and water: acetonitrile (20:80 v/v) and ammonium acetate (5 mM, pH 3.0) as mobile phase. Limit of detection (LOD) ranged from 0.018 to 0.038 μg mL−1, and remarkable precision values for the responses and retention times lower than 5.89% and 1.92% were calculated, respectively. Moreover, accuracy values ranging between 85% and 104% were obtained.

Keywords: Antidepressants, Drug monitoring, Blood spot, Capillary liquid chromatography-mass spectrometry, Poly(styrene-co-divinylbenzene)-coated glass

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GRAPHICAL ABSTRACT

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1. Introduction

Antidepressant drugs are widely prescribed to treat and relieve depression disorder symptoms, as well as to support the treatment of chronic pain, panic disorder, social phobia and/or narcolepsy, which affect millions of people worldwide, of whom only about half of those affected receive treatment. Furthermore, the expected clinical response is not always attained and/or undesired effects frequently occur [1,2]. The different existing types of antidepressants can be classified according to the brain chemicals regulated. Among these, fluoxetine, citalopram, paroxetine (i.e. selective serotonin reuptake inhibitors - SSRI), norepinephrine and dopamine reuptake inhibitors such as bupropion, the serotonin reuptake inhibitor and serotonin receptor (5-HT2) antagonist trazodone, the noradrenergic and specific serotonergic antidepressants mirtazapine and the melatonin agonist and a 5HT2c receptor antagonist agomelatine are widely prescribed for different mental disorders [3]. Nevertheless, precise and personalized dosage is usually required for each patient to attain obtain an efficient clinical response as well as to avoid the potential occurrence of serious side effects [4,5], either because of pharmacodynamics or pharmacokinetic (e.g. drug interactions, drug metabolism or transport) issues. Consequently, monitoring these compounds in patients under treatment is of remarkable interest. In clinical practice, conventional monitoring is usually based on venous sampling methods using different approaches for sample treatments, such as liquid-liquid microextraction (LLME) [6], solid phase extraction (SPE) [7], magnetic solid phase extraction (MSPE) [8], stir bar sorptive extraction (SBSE) [9], pressurized liquid extraction (PLE) [10] or microwave-assisted extraction (MAE) [11]. More specifically, SPE provides a remarkable efficient sample cleanup, recovery, and pre-concentration of the analytes for an accurate quantitative analysis. This technique is certainly based on high efficiency, and the low reagents and sample consumption, thus representing relatively rapid and low-cost candidates when compared to alternative approaches. Over the last decade, dried blood spot (DBS) sampling technique has emerged as a promising substitute of traditional liquid biometrics [12,13]. Furthermore, DBS sampling and the subsequent analysis is gaining interest in therapeutic drug monitoring of antidepressants as this sampling approach possesses certain advantages when compared with conventional venous sampling. First, blood

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spot sampling simply requires a heel or finger puncture which is less painful and more time efficient, especially for those more vulnerable subjects, such as children or elderly patients. Moreover, the low blood volume required facilitates sample shipment and storage as no special treatment is needed, thus reinforcing the stability of analytes, decreasing the biohazard associated to the transport and manipulation of the samples, thus allowing its in-situ application [3]. On the other hand, a rising number of DBS assays for different antidepressants have been reported using commercial blood sampling cards [14,15]. Among them, DBS assays for the analysis of tricyclic antidepressants and their active metabolites, as well as for the SSRIs fluoxetine, reboxetine, and paroxetine, or for the atypical antidepressant venlafaxine and O-desmethylvenlafaxine have been previously described [14,16,17]. Here we developed a lab-made blood spot approach to collect blood on styrene-co-divinylbenzene polymer (PS-DVB) coated in a glass substrate. This procedure is less invasive and more cost-effective in terms of sample collection, storage and shipment than venipuncture and/or commercial DBS approaches. Indeed, blood samples are collected from a small finger prick with an automatic lancet, enabling the patients to perform their self-sampling at home. Furthermore, no anticoagulant or plasma separation process is required, improving the stability of the analytes without using a refrigeration device. PS-DVB polymer was chosen because it represents a good adsorbent material for antidepressants. This sorbent can be obtained from a commercial cartridge (e.g. Strata-SCX® cartridges, from Phenomenex) which elevates the cost per analysis [18,19]. Alternatively, this polymer can be prepared in the laboratory, though requiring a tedious column packaging procedure, and with the potential occurrence of high backpressure and low flow rates phenomena. To overcome these drawbacks, the advantages of the solvothermal approach for decorating a novel extraction method by using

PS-DVB are considered. With regard to the quantitative analysis of these drugs, they have been quantified in different biological matrices using different techniques such as thin-layer chromatography (TLC) [20], gas chromatography–mass spectrometry (GC-MS) [21], liquid chromatography using UV diode array (LC-DAD) [22], fluorescence [23], chemiluminescence [24], electrochemical detection (ECD) [25], gas chromatography- flame ionization detection (GC-FID) [26] and liquid chromatography-mass spectrometry

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(LC-MS) [27]. Most of them currently require laborious and time consuming sample treatment steps to extract antidepressants from different body fluids (i.e. plasma [28], serum [29], blood [6], urine [30], saliva [22] and milk [27]). Although the determination of antidepressants in whole blood will represent the most realistic approach for monitoring these compounds, some drawbacks, such as the presence of potential interferences (e.g. trace minerals and other elements) must be overcome with an efficient extraction and their subsequent selective and sensitive detection [31]. Consequently, a new, rapid and selective extraction procedure using PS-DVB-coated glass blood spot for the extraction of the selected antidepressants from just one drop of blood is here proposed and optimized for their further separation and sensitive detection by a new capillary liquid chromatography-mass spectrometry (CLC-MS) method.

2. Experimental 2.1. Chemical, materials and samples

Agomelatine, bupropion hydrochloride, citalopram hydrobromide, fluoxetine hydrochloride, mirtazapine, paroxetine hydrochloride, trazodone hydrochloride, ammonium acetate, styrene (≥99% purity), divinylbenzene (80% purity), sodium hydroxide and acetic acid (≥99.7% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC-MS grade acetonitrile (ACN) and methanol were purchased from Fisher Scientific (Loughborough, Leics, UK), while 2,2’-Azobis(2-methylpropionitrile) (AIBN) and low molecular weight poly(vinylchloride) (PVC) were obtained from Fluka (MO, USA). Tetrahydrofuran (THF) and hydrochloric acid were supplied by Panreac (Barcelona, Spain), and 1.0 mm thickness glass sheets (30mm x 50 mm, cut corners) for the polymer deposition and blood pretreatment was purchased from Labbox (Madrid, Spain). The stock solutions of all analytes were prepared at 5.0 mg mL−1 in methanol and stored in absence of light at −20 °C, and working standard solutions were prepared at 100 μg mL- 1 by appropriate dilution in methanol and stored at −20 °C as well. Drug-free blood to prepare blank and quality control samples to validate the proposed methodology was obtained from several healthy volunteers pooled and kept at 4 °C until analysis. Blank blood samples utilized for calibration (consisting of at least 5 different concentrations

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covering a sufficient range for each case) were spiked by diluting appropriately the working standard solution of each analyte.

2.2. Instrumentation

A capillary LC pump (Agilent series 1200, Waldbronn, Germany) was used for the chromatographic system, and a Jupiter 4 μm Proteo 90 Å (250mm×0.5 mm) column from Phenomenex® (Torrance, CA, USA) was utilized for the chromatographic separation of the analytes. Detection was carried out with a UV–Vis diode array detector (Agilent, 1260 infinity model) equipped with a 2 μL flow cell coupled in series to an Agilent 6110 series MS detector (Waldbronn, Germany) equipped with an atmospheric pressure ionization source electrospray (API-ES). Raman measurements were performed with a Portable Raman Spectrometer system (B&W TEK Inc., DE, USA) at a wavelength of 785 nm and a maximum laser output power at system's excitation port of 348mW and 285mW in the probe. The output laser power in the probe was set to the total percentage without any damage. For measurements, the laser beam was focused to the sample through a 100/1.25 objective. Raman signals were acquired by a CCD array detector cooled at 10°C with an acquisition time ranging from 5.0 to 65.5 ms. Transmission electron microscopy (TEM) images were obtained with a Jeol JEM 2011 microscope operating at 200 kV and equipped with an Orius Digital Camera (2×2 MegaPixel) from Gatan (Pleasanton, CA, USA). The digital analysis of the high resolution TEM micrographs were taken using Digital Micrograph TM 1.80.70 for GMS 1.8.0 Gatan. The characterization of samples from the polymer by TEM analysis was performed by deposition of a drop of the synthesized material suspension onto a lacey carbon/format- coated copper grid. Ultrapure water was obtained from a Milli-Q water instrument from Millipore (Merck KGaA, Darmstadt, Germany), and for infrared spectrum measurement a FT/IR 4200 spectroscopy from Jasco (Japan) was utilized. The particle size distribution was measured by a Zetasizer Nano ZEN3600 from Malvern (Worcestershire, UK).

2.3. Synthesis of poly(styrene-co-divinylbenzene)

The polymerization mixture consisted of 4.5 mL of styrene (9.79 mM) and divinylbenzene (23.69 mM), 0.36 g of AIBN and 25 mL of acetonitrile which were added into an Erlenmeyer flask, sonicated for 5 min, and then purged for 10 min with N2 gas

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and closed. Then, the mixture was stirred in a water bath at 70 °C for 24 h to complete the polymerization process and the resultant particles were repeatedly rinsed with methanol. The final product was then filtered and dried under low pressure [32].

2.4. Preparation of poly(styrene-co-divinylbenzene)-coated glass blood spot

Blood samples were conditioned for the subsequent extraction of antidepressants based on the use of PS-DVB-coated glass blood spot (Figure 3.1.4.1). Previously, glass sheets were cut into 10×10mm squares, and the surface of each square was first activated by dipping in 1.0M NaOH solution for 2–3 h. Then, 30 mg of PS-DVB powder were suspended in 5 mL of tetrahydrofuran (THF) containing 10 mg of PVC powder dissolved. A small amount (10 μL) of this mixture was dropped onto the center of the square glassy substrate, and the THF was then evaporated at room temperature to obtain a polymer coating on the square glassy substrate. After that, 10 μL of either each standard or a blood spiked sample was spotted onto each square glassy substrate containing the polymer and then the mixture was dried. The glass sheet was cut with hand-held diamond glass cutter to obtain 10×10mm squares glassy containing blood. After that, the antidepressants were eluted with 1.0 mL of 2.0% acetic acid in methanol, and the eluent dried under a stream of nitrogen gas at room temperature. The residue was finally dissolved in 200 μL of mobile phase (5mM ammonium acetate, pH 3.0) and an aliquot of 5.0 μL was injected into the CLC-MS.

Figure 3.1.4.1. Scheme of the general procedure for the synthesis and extraction of the selected antidepressants by PS-DVB-coated glass blood spot.

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2.5. Capillary liquid chromatography-mass spectrometry analysis

DAD detector was set at a wavelength of 210 nm, and MS detection of analytes was carried out in positive ionization mode under the following conditions: 5.0 L min−1 of drying gas flow, drying gas temperature at 350 °C, a nebulizer pressure of 25 psi and a capillary voltage of 2000 V. Selected ion monitoring (SIM) was used to detect and quantify the target analytes using external calibration. Previously, the analytes were qualitatively determined at full scan mode and matching their retention time and mass spectra with standards. The maximum peaks in mass spectra ions were: 244.0 (agomelatine), 240.3 (bupropion), 325.1 (citalopram), 310.1 (fluoxetine), 266.1 (mirtazapine), 330.2 (paroxetine) and 372.1 (trazodone).

Chromatographic analyses were carried out using a gradient consisting of a binary mixture of solvents: ammonium acetate (5 mM, pH 3.0) as solvent A, and a water: acetonitrile (20:80, v/v) mixture as solvent B. The gradient was carried out at a flow-rate of 20 μL min−1, starting from 30% B and then increased up to 70% B over 30 min. Injection volume was 5 μL and the column was maintained at a temperature of 25 °C. Re- equilibration of the column was performed in 30 min after each run. All solvents were filtered through a 0.45 μm nylon membrane before their use.

2.6. Method validation

The method was validated using calibration standard solutions and quality control (QC) samples for each analyte's calibration curve. The validation was conducted with respect to selectivity, lower limit of quantification (LLOQ), linearity, recovery, accuracy, and precision. The matrix effects and stability were also evaluated. The method validation followed the recommendations of the European Medicines Agency Guideline on bio- analytical method validation [33].

2.6.1. Matrix effects

Blood was tested for the presence of potential interferences at the analysis of the antidepressants. For each analyte, the matrix factor (MF) was determined by calculating the ratio of the peak area in the blank blood spiked with analyte after extraction to the peak area in the absence of matrix (pure solution of the analyte). Six lots of blood

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samples after the extraction step were spiked with QC concentrations of calibrators. Non-matrix standard samples were prepared by direct addition of the same concentrations of calibrators to the mobile phase. This analysis was performed at a low (maximum of 3 times the LLOQ) and a high level of concentration, and the relative standard deviation (RSD) of the peak areas of each analyte was used to assess interlot matrix variability. Values lower or higher than 100% are a consequence of ion suppression or ion enhancement, respectively. The overall RSD calculated for the concentration should not be greater than 15%.

2.6.2. Stability

Stability was assayed in triplicate with QC samples at different concentrations. Firstly, for freeze/thaw stability testing, the QC samples were determined after three freeze (−20 °C) and thaw (23 °C) cycles and analyzed together with freshly prepared calibration samples. For short-term stability, the QC samples were extracted and placed in the autosampler at room temperature for 24 h, and then analyzed. The measured concentrations were compared to those of the same QC samples that had been analyzed immediately after processing. The percentage deviation in concentration compared to the mean of back calculated values for the standards at the appropriate concentrations from the first day was used as an indicator of stability. Stability was considered acceptable when the RSD was within 15%.

3. Results and discussion 3.1. Characterization of poly(styrene-co-divinylbenzene) sorbent

To confirm the synthesis of PS-DVB, the material produced was characterized by Fourier transform infrared (FT-IR), Raman spectroscopy, Transmission Electron Microscopy and Malvern Zetasizer for particle size distribution. The FT-IR spectrum of the PS-DVB showed the characteristic peaks of aromatic (C=C) bending and alkyl (C-H) stretches, which can be observed at 1700 cm−1 and 2800 cm−1, respectively, while the peak at 3085 cm−1 corresponds to the aromatic (C=C) stretching (Figure 3.1.4.2a). The PS-DVB layers deposited on the micro-channel surfaces were analyzed by in-situ Raman spectroscopy and this material was found to exhibit a vibrational spectrum identical to that of commercial PS-DVB (Figure 3.1.4.2b). Remarkable peaks, including 1001 cm−1 (aromatic

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ring breathe) and 1604 cm−1 (C=C) stretches, were additionally identified, and the results indicating the formation of microsphere PS-DVB particles were confirmed by TEM micrograph (Figure 3.1.4.2c). Furthermore, the particle size distribution of the PS-DVB microsphere with uniform size was confirmed (Figure 3.1.4.2d).

Figure 3.1.4.2. Characterization of PS-DVB by (a) FT-IR spectra, (b) Raman spectrum, (c) TEM micrograph and (d) particle size distribution. 3.2. Optimization of experimental parameters

The amount of polymer, pH and sample volume were analyzed, and their values were optimized to enhance the efficiency of the extraction process. Different amounts of PS- DVB coating the surface of glass substrate, ranging from 5 to 100 μg, were studied, and higher recovery values were obtained when 10 μg was used (Figure 3.1.4.S1). On the other hand, after analyzing a pH range from 2 to 14, neutral pH gave the best results in terms of peaks area and recovery yields (Figure 3.1.4.S2). Additionally, a range from 10 μL to 30 μL of sample was tested; 10 μL of sample gave the best recovery results, and was therefore selected (Figure 3.1.4.S3). For chromatographic analysis, different ratios of mobile phase (ACN: H2O v/v) were tested (50:50, 60:40, 70:30 and 80:20), and higher sensitivity and selectivity values were obtained when 80:20 (v/v) ratio was used. A range of concentration of ammonium acetate (3.0–10 mM) and pH (from 2.0 to 14) were additionally studied, and 5.0mM of ammonium acetate and pH=3.0 gave the best results in terms of selectivity and sensitivity. To optimize the MS detection of each analyte, the drying gas flow was investigated within the 1.0–13 L min−1 range and 5.0 L min−1 gave

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the best results in terms of the peaks area. The temperature of the drying gas was also studied in a range from 0 to 350 °C, and the best results were obtained when 350 °C was applied. Additionally, the nebulizer pressure, ranging from 5.0 to 60 psi, was investigated and the 25 psi was selected, and a capillary voltage of 2000 V was selected as optimal after an optimization process carried out from 500 V to 6000 V.

3.3. Validation of the methodology

Using the previously optimized conditions, 10 μL of the antidepressants standard solutions in the 0.1–1.0 μg mL−1 concentration range were spotted onto the square glass substrate previously coated by PS-DVB and then the mixture was dried. After that, the analytes were eluted with 1.0 mL of 2.0% acetic acid in methanol, and the eluent was dried under a stream of nitrogen gas at room temperature. The residue was finally dissolved in 200 μL of mobile phase (5mM ammonium acetate, pH 3.0) and an aliquot of 5 μL was injected into the LC–MS. All the antidepressants were separated and quantified in less than 20 min with good resolutions (Figure 3.1.4.3).

Figure 3.1.4.3. LC-MS chromatograms of 1.00 μg mL−1 standard solution of each antidepressant analyzed (a), 0.25 μg mL−1 spiked blood sample extracted using PS-DVB (b), 0.25 μg mL−1 spiked blood sample extracted without using PS-DVB (c), and blank blood sample (d). Peak identification; 1: mirtazapine, 2: bupropion, 3: trazodone, 4: citalopram, 5: paroxetine, 6: fluoxetine and 7: agomelatine. The analytical parameters of the method were examined, and the linear range, intercept and slope of the curve along with the regression coefficient for each antidepressant

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were calculated (Table 3.1.4.1). The theoretical limits of detection (LODs) and limits of quantification (LOQs), defined as the concentration of analyte giving a signal equivalent to the blank signal plus three and ten times their SD, respectively, are also presented in Table 3.1.4.1. The precision of this method is also indicated (Table 3.1.4.1), and relative standard deviations (RSD) lower than 6% were calculated in all cases (Table 3.1.4.2). Moreover, practical LODs, defined as the minimum level at which an analyte can be detected in a real matrix at acceptable levels of accuracy (> 80%) and precision (< 10%) were calculated in blood samples spiked with different levels of the selected antidepressants. Thus, LOD values lower than 0.08 μg mL−1 were found for all analytes (Table 3.1.4.3). This method shows clear advantages in terms of sensitivity with respect to other existing alternative methods involving the determination of these antidepressants in human blood samples [34,35]. Additionally, recovery values were calculated within a range from 88.8% to 110% at the low concentration (0.1 μg mL−1) and from 89.7% to 106% at the high concentration value of 1.0 μg mL−1 assayed (Table 3.1.4.4). Likewise, acceptable values within the 81.1–93.5% and 86.3–98.3% ranges, were found respectively for the same low and high concentration levels assayed for the matrix effects analyses. The matrix components only caused relatively minor effects on the ionization efficiency of the analytes under the proposed conditions, but the precision values for both recovery and matrix effects were found to be acceptable, as all the RSD values calculated were lower than 15% at all the concentrations studied (Table 3.1.4.4).

Stability for at least 24 h at room temperature was confirmed for all the analytes in the extracted samples as no deviation greater than ±15% of the nominal concentration was calculated, and a RSD lower than 15% was found (ranging from 5.53% to 12.7% for mirtazapine and citalopram, respectively). Likewise, stability of the analytes after three freeze (−20 °C)/thaw (24 °C) cycles, was also confirmed (Table 3.1.4.4). The proposed method was applied to analyze the seven selected antidepressants in capillary human blood spots samples obtained from healthy volunteers, and no peaks of interest were detected in any case (Figure 3.1.4.3). To evaluate the applicability of the present method, these samples were then spiked with selected levels of the antidepressants and subsequently analyzed by capillary LC-MS after their extraction using the lab

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manufactured PS-DVB-coated glass (Figure 3.1.4.3). Remarkably improved results were obtained when the extraction method proposed in this study was applied and, therefore, the overall strategy demonstrates a good applicability in practice, thus offering a promising potential for its application in clinical routine laboratory analysis.

Table 3.1.4.1. Calibration data and figures of merit obtained for the proposed method. LOD LOQ LR (µg 2 Analyte -1 Y = (A ± SA)X + (B ± SB) R Sy/x mL ) (µg mL-1) (µg mL-1) Mirtazapine 0.1-1.0 Y= (31432 ± 522)X – (106 ± 346) 0.999 402 0.038 0.100 Bupropion 0.1-1.0 Y= (38940 ± 544)X – (48.8 ± 361) 0.999 419 0.032 0.095 Trazodone 0.1-1.0 Y= (13877 ± 111)X + (39.3 ± 73.4) 0.999 85.4 0.018 0.053 Citalopram 0.1-1.0 Y= (34760 ± 299)X + (224 ± 198) 0.999 230 0.019 0.061 Paroxetine 0.1-1.0 Y= (6938 ± 54)X + (32.4 ± 36.1) 0.999 42.0 0.018 0.052 Fluoxetine 0.1-1.0 Y= (6623 ± 98.6)X + (62.7 ± 65.4) 0.999 76.0 0.034 0.099 Agomelatine 0.1-1.0 Y= (36504 ± 527)X + (80.2 ± 349) 0.999 406 0.033 0.096

A: slope, SA: standard deviation of slope; B: intercept, SB: standard deviation of intercept; R: regression coefficient; Sy/x: SD of residuals; n=11.

Table 3.1.4.2. Precision values calculated for the antidepressants to be analyzed by the proposed methodology. Run-to-run precision Inter-day precision Concentration Analyte -1 Retention (µg mL ) Responses Responses Retention time time Mirtazapine 0.20 2.45 1.03 2.62 1.74 0.40 2.28 0.75 4.75 1.32 0.80 2.85 1.07 2.98 1.42 Bupropion 0.20 1.74 1.14 2.16 1.92 0.40 2.57 1.62 3.48 1.85 0.80 2.32 0.69 3.27 0.94 Trazodone 0.20 3.68 1.05 3.46 1.93 0.40 2.88 0.40 3.60 1.55 0.80 2.37 0.99 4.30 1.11 Citalopram 0.20 3.60 0.54 3.66 0.76 0.40 2.03 0.71 5.22 0.73 0.80 2.16 0.48 2.36 0.63 Paroxetine 0.20 1.79 0.50 4.52 0.91 0.40 2.07 0.74 3.32 0.96 0.80 2.47 0.50 3.63 1.13 Fluoxetine 0.20 3.62 0.45 5.89 0.49 0.40 1.98 0.35 5.87 0.54 0.80 2.18 0.42 2.30 0.66 Agomelatine 0.20 1.34 0.13 4.01 0.26 0.40 2.25 0.35 4.05 0.80 0.80 2.37 0.88 4.31 0.99 Data expressed as relative standard deviation (%), (n=11).

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Table 3.1.4.3. Accuracy results calculated for blood samples spiked with different concentrations of the antidepressants analyzed. Practical limit of Analyte detection (μg Added (µg mL-1) Found (µg mL-1) Accuracy (%) mL−1) Mirtazapine 0.08 0.25 0.26 103±3.0 0.50 0.47 93.0±4.2 0.75 0.68 90.3±2.1 1.00 0.99 99.0±5.4 Bupropion 0.07 0.25 0.25 99.0±5.1 0.50 0.48 95.8±3.0 0.75 0.75 99.6±5.0 1.00 0.93 93.4±4.6 Trazodone 0.06 0.25 0.24 95.5±6.2 0.50 0.46 92.1±4.3 0.75 0.67 88.7±6.2 1.00 0.87 87.4±2.7 Citalopram 0.05 0.25 0.23 92.8±3.6 0.50 0.44 88.9±3.6 0.75 0.65 86.0±2.8 1.00 0.85 85.0±3.1 Paroxetine 0.05 0.25 0.22 89.4±4.4 0.50 0.52 104±1.1 0.75 0.72 96.2±2.6 1.00 0.92 91.8±2.3 Fluoxetine 0.08 0.25 0.26 103±3.0 0.50 0.50 100±5.0 0.75 0.69 92.5±2.3 1.00 0.89 89.1±1.3 Agomelatine 0.08 0.25 0.22 86.9±1.4 0.50 0.46 91.7±1.9 0.75 0.77 103±2.0 1.00 0.96 96.2±2.8 Data are expressed as mean ± standard deviation (SD); (n=3).

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Conclusion

The method developed and validated in this study represents a simple, economic and reproducible methodology which emphasizes the potential of using PS- DVB-coated glass blood spot together with LC-MS for the direct analysis of seven types of the most prescribed antidepressants in human blood samples. The assay performance is accurate and precise, and shows sufficient sensitivity, reliability and rapidness. In addition, the sampling technique here proposed presents advantages over traditional blood sampling in terms of samples’ storage temperature, their transport and the simplicity of sample preparation. Consequently, this work demonstrates the capability of DBS analysis to provide qualitative and quantitative information on antidepressants consumption using significantly smaller volumes (10 μL) of blood than those volumes conventionally required. On the other hand, the limitation of this method is that the sample treatment, separation and detection are carried out off-line. Thus, the on-line extraction combined with more sensitive detectors, such as MS/MS, could represent the following step, together with a future validation focused on the clinical application of the method to real blood samples analysis.

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Acknowledgments

The Spanish Ministry of Economy and Competitiveness (MINECO) is gratefully acknowledged for funding this work with Grants CTQ2016-78793-P. The support given through an “INCRECYT” research contract to M. Zougagh is also acknowledged.

References

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[8] F. Zare, M. Ghaedi, A. Daneshfar, Solid phase extraction of antidepressant drugs amitriptyline and nortriptyline from plasma samples using core-shell nanoparticles of the type Fe3O4@ZrO2@N-cetylpyridinium, and their subsequent determination by HPLC with UV detection, Microchim. Acta (2015) 1893–1902.

[9] L.P. Melo, A.M. Nogueira, F.M. Lanc, M.E.C. Queiroz, Polydimethylsiloxane/polypyrrole stir bar sorptive extraction and liquid chromatography (SBSE/LC-UV) analysis of antidepressants in plasma samples, Anal. Chem. Acta 633 (2009) 57–64.

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[12] A.J. Wilhelm, J.C.G. den Burger, E.L. Swart, Therapeutic drug monitoring by dried blood spot: progress to date and future directions, Clin. Pharmacokinet. 53 (2014) 961– 973.

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[14] E.J.J. Berm, J. Paardekooper, E. Brummel-Mulder, E. Hak, B. Wilffert, J.G. Maring, A simple dried blood spot method for therapeutic drug monitoring of the tricyclic antidepressants amitriptyline, nortriptyline, imipramine, clomipramine, and their active metabolites using LC-MS/MS, Talanta 134 (2015) 165–172.

[15] J. Weber, S. Oberfeld, A. Bonse, K. Telger, R. Lingg, G. Hempel, Validation of a dried blood spot method for therapeutic drug monitoring of citalopram, mirtazapine and

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risperidone and its active metabolite 9-hydroxyrisperidone using HPLC–MS, J. Pharm. Biomed. Anal. 140 (2017) 347–354.

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[19] C. Gagnon, Distribution of antidepressant residues in wastewater and biosolids following different treatment processes by municipal wastewater treatment plants in Canada, Water Res. 46 (2012) 5600–5612.

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[21] L. Truta, A.L. Castro, S. Tarelho, P. Costa, M.G.F. Sales, H.M. Teixeira, Antidepressants detection and quantification in whole blood samples by GC–MS/MS, for forensic purposes, J. Pharm. Biomed. Anal. 128 (2016) 496–503.

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human plasma by liquid chromatography with fluorescence detection, Anal. Chem. Acta 591 (2007) 141–147.

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[31] S.M. Young, L.K. Gryder, W.B. David, Y. Teng, S. Gerstenberger, D.C. Benyshek, Human placenta processed for encapsulation contains modest concentrations of 14 trace minerals and elements, Nutr. Res. 36 (2016) 872–878.

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SUPPLEMENTARY INFORMATION

Figure 3.1.4.S1. Optimization of the amount of PS-DVB coated on the surface of the glass substrate.

Figure 3.1.4.S2. pH optimization for the extraction of the selected antidepressants. 10 g of the PS-DVB was applied on the surface of the coated glass.

Figure 3.1.4.S3. Analysis of different amounts of blood sample utilized for the proposed methodology.

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3.2. Nanoparticles in electrochemical sensors for food and pharmaceutical samples monitoring

s mentioned in the previous section, (nano)materials can be used for purposes such as the treatment of samples or even for the instrumental separation of A analytes. In addition, they are widely used for detection in both food and pharmaceutical samples.

Detection is another step in the analytical process in which NMs offer many benefits due to their exceptional properties. It is the step in which NMs have been used more widely thanks to their ability to replace conventional materials, as well as the advantages of electrochemical biosensors.

The most commonly used NPs in the field of analytical chemistry are (i) SiO2NPs, (ii) carbon nanoparticles (mainly CNTs and graphene), (iii) organic polymer nanoparticles (e.g, MIPs), (iv) metallic nanoparticles (QDs, AgNPs [1] and AuNPs [2]) , (v) supramolecular aggregates such as nanomicelles or nanovesicles or (vi) transition metal oxides such as, copper (Cu) [3], titanum oxide nanoparticles (TiO2NPs), cadmium oxide nanoparticles (CdONPs) [4], and ruthenium (Ru) [5].

Graphene has received increasing attention due to its unique physicochemical properties such as high surface area, excellent conductivity, high mechanical strength, and ease of functionalization and mass production, which has extraordinary electronic transport properties and high electrocatalytic activities. Thus, it is regarded to be an ideal material for construction of sensitive sensors [6,7].

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This topic, contains three developed works, which is devoted to the use different materials such as Al-TiO2-NPs, Al-CuSe-NPs and CuSe@rGO in the detection process in order to detect analytes of interest.

 “Development of an Aluminium Doped TiO2 Nanoparticles-modified Screen Printed Carbon Electrode for Electrochemical Sensing of Vanillin in Food Samples.” In this work, the determination of vanillin based on aluminium doped

TiO2 nanoparticles-modified screen printed carbon electrode.  “A Sensitive Electrochemical Sensor Based on Aluminium Doped Copper Selenide Nanoparticles-modified Screen Printed Carbon Electrode for Determination of L- tyrosine in Pharmaceutical Samples.” In this work, the determination of L- tyrosine based on aluminium doped CuSe nanoparticles-modified screen printed carbon electrode.  “Decoration of graphene oxide with copper selenide in supercritical carbon dioxide medium as a novel approach for electrochemical sensing of eugenol in

various samples.” In this work, the determination of eugenol based on reduced graphene oxide decorated copper selenide nanoparticles. The synthesized composite material was used to modify the glassy carbon electrode.

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References

[1] W. Wang, Y. Xie, C. Xia, H. Du, F. Tian, Titanium dioxide nanotube arrays modified with a nanocomposite of silver nanoparticles and reduced graphene oxide for electrochemical sensing, Microchim. Acta 181 (2014) 1325–1331.

[2] M. G. Hosseini, M. Faraji, M. M. Momeni, S. Ershad, An innovative electrochemical approach for voltammetric determination of levodopa using gold nanoparticles doped on titanium dioxide nanotubes, Microchim. Acta 172 (2011) 103-108. [3] S. Zhang, H. Ma, L. Yan, W. Cao, T. Yan, Q. Wei, B. Du, Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors, Biosens. Bioelectron. 59 (2014) 335-341. [4] R. Jain, P. Pandey, Electrochemical Sensor Mediated by Synthesis of CdO Nanoparticles-Titanium Dioxide Composite Modified Glassy Carbon Electrode for Quantification of Zolmitriptan, J. Electrochem. Soc. 160 (2013) 687-692. [5] N. P. Shetti, D. S. Nayak, S. J. Malode, R. M. Kulkarni, An electrochemical sensor for clozapine at ruthenium doped TiO2 nanoparticles modified electrode, Sens. Actuators B 2017, 247, 858-867. [6] M. Zhang, C. Liao, C.H. Mak, P. You, C.L. Mak, F. Yan, Highly sensitive glucose sensors based on enzyme-modified whole-graphene solution-gated transistors, Sci. Rep. 5 (2015) 1-6. [7] B. He, Y. XH, Modifications of Au Nanoparticle-Functionalized Graphene for Sensitive Detection of Sulfanilamide, Sensors 18 (2018) 846-859.

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3.2.1. Development of an Aluminium Doped

TiO2 Nanoparticles-modified Screen Printed Carbon Electrode for Electrochemical Sensing of Vanillin in Food Samples

Electroanalysis 30 (2018) 969 – 974

DOI: 10.1002/elan.201800032

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Abstract

A new chemically modified electrode based on titanium dioxide nanoparticles (TiO2-NPs) has been developed. Aluminium was incorporated into the TiO2-NPs to prepare aluminium doped TiO2 nanoparticles (Al-TiO2-NPs). Aluminium doped TiO2 nanoparticles-modified screen printed carbon electrode (Al-TiO2-NPs/SPCE) was employed as easy, efficient and rapid sensor for electrochemical detection of vanillin in various types of food samples. Al-TiO2-NPs were characterized by energy dispersive X- ray (EDX), transmission electron microscopy (TEM), and X-ray diffraction (XRD) and analyses showing that the average particle sizes varied for the Al-NPs (7.63 nm) and Al-

TiO2-NPs (7.47 nm) with spherical crystal. Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were used to optimize the analytical procedure. A detection limit of vanillin was 0.02 mM, and the relative standard deviation (RSD) was 3.50%, obtained for a 5.0 mM concentration of vanillin. The electrochemical 192esityle of several compounds, such as vanillic acid, vanillic alcohol, p-hydroxybenzaldehyde and p- hydroxybenzoic, etc., generally present in natural vanilla samples, were also studied to check the interferences with respect to vanillin voltammetric signal. The applicability was demonstrated by analyzing food samples. The obtained results were compared with those provided by a previous method based on liquid chromatography for determination of vanillin.

Keywords: Aluminium doped TiO2 nanoparticles · screen-printed carbon electrode · electrochemical detection · vanillin · food samples

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GRAPHICAL ABSTRACT

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1. Introduction

Vanillin (4-hydroxy-3-methoxybenzaldehyde, C8H8O3) is one of the world’s 194esity extracts obtained primarily from Vanilla, a specie of tropical climbing. Although the production of vanillin every year more than 12,000 tons, the natural vanillin from Vanilla is less than 1%; the remainder is synthesized cheaper using biochemical and/ or chemical processes [1]. For the time being, several analytical methods have been used for determination and detection of vanillin in various types of food samples or vanilla extracts, including fluorescence [2], capillary electrophoresis (CE) [3], liquid chromatography [4], and GC-MS [5]. These have high cost and involve time-consuming sample pre-treatment processes. Because vanillin is an electro-active compound and it is possible to measure the quantity of vanillin in vanilla and in the final products by electrochemical detection (ECD) through the study of its oxidation. ECD is important method for quantitative determination of vanillin due to its simplicity, fast response, high sensitivity, and cheap instrumentation [6–11]. Various electrochemical methods, such as amperometry, square-wave voltammetry (SWV) or differential pulse voltammetry (DPV) for the detection and determination of vanillin in various types of food samples have been reported and discussed in literature [12, 13].

In recent years, many reports on screen-printed carbon electrodes (SPCEs) technology have been used to develop various electrochemical sensors that detect target molecules in various sectors, such as biomedical environmental and agri-food [14, 15]. SPCE is planar shape, thus can be used as droplet on sensor, using typically few microliters (µL) of the sample in miniaturized system. Moreover, it is low-cost and can be used as a disposable sensor with possibility used in large-scale implementation. The oxidation of vanillin at SPCE and others most unmodified electrode surfaces represents a serious problem which arises from the poor reproducibility with high over potential resulted from a fouling effect, which results in rather poor sensitivity and selectivity [16, 17]. To avoid these problems, the modification of electrochemical working electrode is an excellent alternative. In this sense, many researchers have attempted to diminish the over potentials by using various modified electrodes such as, graphene [6, 18], silver nanoparticles (Ag-NPs) [8], gold nanoparticles (Au-NPs) [9, 10], and multi-walled carbon nanotubes (MWCNTs) [19]. However, the performance of some electrodes was still

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ineffective enough. Thus it is necessary to develop new types of electrode devices by the preparation synthesis and preparation of new materials (modifiers). That regard, as modifiers for electrode surface, titanium dioxide nanoparticles (TiO2-NPs) had highly interest of many researchers and a many amount of research was applied in the previous decades [12, 13, 19]. The increase in the TiO2-NPs efficiency such as to ensnare the charge carrier is accomplished by doping with transition metals and transition metal oxides such as, copper (Cu) [20], cadmium oxide nanoparticles (CdONPs) [21], silver nanoparticles (Ag-NPs) [22], gold nanoparticles (Au-NPs) [23] and ruthenium (Ru) [24].

Here, the synthesis of aluminium doped TiO2 nanoparticles (Al-TiO2-NPs) is carried out, which were characterized by TEM and XRD. The sensitivity of the developed electrode is compared with SPCE, TiO2-NPs/SPCE, and Al-TiO2-NPs/SPCE for vanillin detection. The results show that a composite film of Al-TiO2-NPs/SPCE is more sensitive compared to

SPCE and TiO2-NPs/SPCE. Furthermore, the electrochemical 195esityle of vanillin at modified SPCE with Al-TiO2 (Al-TiO2-NPs/SPCE) was investigated. The performance of the modified electrode (Al-TiO2-NPs/SPCE) is also demonstrated for the determination and detection of vanillin in various types of food samples obtaining good selectivity, stability and high sensitivity.

2. Experimental 2.1. Reagents, Standards and Samples

All the starting materials were purchased with very highly purity. Aluminium acetylacetonate (Al(acac)3, 99%), lithium aluminium hydride (LiAlH4, 95%), 195esitylene (97%), titanium (IV) oxide (anatase, powder, 235 mesh), 4-hydroxybenzaldehyde (98%) and 4-hydroxy-3-methoxybenzyl alcohol (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nafion 117 solution (5% in a mixture of lower aliphatic alcohols and water), vanillin, vanillic acid, 4-hydroxybenzoic acid were purchased from Fluka Chemie (Buchs, UK). Ethanol and phosphoric acid were purchased from Panreac Quimica S.L.U. (Barcelona, Spain). Vanillin solution was prepared in ethanol and stored while tightly covered in the dark until use. Vanillin stock solutions were frequently diluted to working standard solutions with phosphate buffer at pH 6.3. The pure water was obtained by a

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Milli-Q system (Millipore). Ethanol and phosphoric acid were obtained from Panreac Quimica S.L.U. (Barcelona, Spain).

Vanillin extract samples (sample A and sample B) were purchased from different local markets (Ciudad Real, Spain). These extracts were filtered through a sintered filter, and diluted directly in phosphate solution.

2.2. Apparatus, Instruments and Chromatographic Conditions

Electrochemical detection was carried out on a CH Instruments Model 800D Series (Austin, Texas, USA) all the experiments were carried out using a screen-printed carbon electrodes (SPCEs) system (Dropsens DRP-C110) housed in the home made electrochemical flow cell. Transmission electron microscopy (TEM) micrographs were measured on a Jeol JEM 2011 operating at 200 kV and equipped with an Orius Digital Camera (2 x 2 Mpi). The digital analysis of the HRTEM micrographs was done using Digital Micrograph TM 1.80.70 for GMS 1.8.0 Gatan. The samples were prepared onto a lacey carbon/format-coated copper grid by deposition of a drop of the synthesized material suspension. XRD patterns were measured on Philips model X’Pert MPD diffractometer using a CuKα source (λ=1.5418A˚), programmable divergence slit, graphite mono chromator and proportional sealed xenon gas detector. The samples were made with a voltage of 40 KV, intensity 40 mA and an angular range of 20 to 70 degrees (2θ), a step of 0.02 degrees 2θ and a time per step of 1.50 sec.

Agilent 1200 liquid chromatography system was used as a chromatographic system. It was consisted from a LC pump, a vacuum degasser, a micro well-plate autosampler (5 µL injection loop), a thermostatted column compartment and a Diode-Array Detector (DAD). The data was processed using the PC computer with ChemStation Software. An appropriate reversed-phase C18 analytical column Luna 5 µm PFP (2) 100 A (150 x 4.6 mm) was used for the separation of the analytes present in vanillin extract samples. Elution was done quite under isocratic conditions, by using a mixture of acetonitrile/phosphate (20:80 v/v) as a mobile phase, 40 µL the injection volume was injected and flow-rate was set at 1.0 mL min-1. The detection wavelength was at 265 nm.

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2.3. Preparation of Aluminium Doped TiO2 Nanoparticles (Al-TiO2-NPs)

At first, Al-NPs were prepared according the previously described procedures [25], with inserting some changes. The aluminium acetylacetonate (Al(acac)3, 10 mmol) was added to a three-neck round bottom which was already contained 197esitylene solution with magnetic bar. Then lithium aluminium hydride (LiAlH4, 30 mmol) was added to the mixture. The reaction was purged with N2 gas during reflux with stirring for 72 hours at 165 °C. After cooling to 25 °C, a gray-colored precipitate was formed which was crushed and kept to dry under low pressure for 5 hours. Wash the isolated solid product well with 25 ml portions of cold methanol three times, to avoid any high temperatures (exothermic reaction) between solvent and Al-NPs. The unreacted materials were washed three times with methanol. The resulting product was filtered and dried at 25

°C under low pressure. The preparation of Al-TiO2-NPs started by mixing of TiO2 (0.5 g) previously digested in nitric acid (0.1 M, 25 mL) during 3 hours and Al-NPs (0.5 g) previously prepared. The final product was filtered and dried at 25 °C under low pressure, obtaining an Al-TiO2-NPs as light-gray powder.

2.4. Preparation of Modified Electrodes

Nanoparticles were ultrasonically dispersed in pure water (with 0.5% Nafion, v:v), The concentrations of 1 mg mL-1 were obtained individually. On the SPCE forming a layer of thin films from nafion-solubilized nanoparticles are more uniform distribution than those casted by organic solvents [26]. TiO2-NPs and Al-TiO2-NPs were used. Dropsens SPCEs (DRP-110), with carbon as a working electrode with a disk-shaped of 4 mm of diameter, were used to fabricate the electrode. So, 2 µL of the dispersed NPs was casted onto the surface of the SPCE. After drying the modified SPCE by using infrared light lamp for 15 min, rinsing with pure water. The electrode is ready for using.

2.5. Electrochemical Measurements of Vanillin

A 25 mL of H3PO4 electrolyte (0.1 M) with proper concentration of vanillin was transferred to a cell, then the SPCE was assembled on it. The potential range was 0.00– 1.20 V, and the scan rate was 50 mV s−1. Before each measurement, the electrode was cycled between 0.00–1.20 V in a blank solution (H3PO4, 0.1 M) until no vanillin peak

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current was detected. After each run, the sensor was rinsed in methanol/acetic acid (9:1, v:v) and ultrapure water for 5 min to remove vanillin for reuse.

3. Results and Discussion

3.1. Characterization of Aluminium Doped TiO2 Nanoparticles (Al-TiO2-NPs)

Energy-dispersive X-ray (EDX) elemental mapping and transmission electron microscopy (TEM) micrograph for the aluminium and aluminium doped titanium nanoparticles (Al-

TiO2-NPs) are shown in Figure 3.2.1.1. This figure shows that different materials have different surface morphologies. Figure 3.2.1.1 (a and c), shows the TEM and EDX micrographs of the aluminium particles without doping (this image was obtained before aluminium was mixed with TiO2-NPs.), and a higher magnification is also presented. A large number of precipitates were distributed homogeneously in the aluminium grains. The distribution of the precipitates for aluminium, which leads to increase in hardness and tensile properties. Figure 3.2.1.1 (b and d), shows the TEM and EDX micrographs of the fully mixed precursor. It shows two large particles in the lower region, which are Al-

NPs and TiO2-NPs, were distributed homogeneously, indicating that a large amount of

Al-NPs has been doped in the TiO2-NPs.

Figure 3.2.1.1. TEM and EDX micrographs of the two precursors: (a and c) Al-NPs and (b and d) Al-TiO2-NPs.

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Figure 3.2.1.2, shows XRD patterns measured for Al-NPs (Figure 3.2.1.2 a) and Al-TiO2- NPs (Figure 3.2.1.2 b). The XRD patterns show that the nanoparticles involved aluminium as a major component. The XRD patterns and all the positions of the peaks are attributed to face-centered cubic (fcc) crystal structure of aluminium [25], and anatase TiO2 [27], as shown in Figure 3.2.1.2. A closer look at the figure shows that the

Al-TiO2-NPs involved aluminium as a major component mixed with TiO2-NPs as minor components, as shown in Figure 3.2.1.2 b [28]. Calculations based on the Scherrer equation (D=Kλ/βcosɵ) [29], show that the average particle sizes varied for the Al-NPs

(7.63 nm) and Al-TiO2-NPs (7.47 nm). The XRD results confirm the TEM micrograph results discussed above.

Figure 3.2.1.2. XRD patterns measured for different NPs: (a) Al-NPs and (b) Al-TiO2-NPs.

3.2. Voltammetric Behaviour of Vanillin at the Al-TiO2-NPs/SPCE

The modified screen printed carbon electrodes (SPCEs) by aluminium doped TiO2 nanoparticles (Al-TiO2-NPs/SPCE) were ﬁrst characterized by cyclic voltammetry (CV) to test their behaviour for the oxidation of vanillin, as shown in Figure 3.2.1.3 d. In order to find the role of Al-NPs, the cyclic voltammograms of (b) SPCE, (c) TiO2-NPs/SPCE, and

(d) Al-TiO2-NPs/SPCE in the presence of 250 µM vanillin were recorded, as shown in

Figure 3.2.1.3. The electrochemical behaviours of the TiO2-NPs/SPCE and Al-TiO2-

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NPs/SPCE were studied by using the CV technique, as shown in Figure 3.2.1.3 (c and d).

All cyclic voltammograms reported here were obtained in the presence of H3PO4 electrolyte (0.1M and pH=6.3) with 250 µM vanillin and scan rate at 50 mV s-1. The cyclic voltammograms observed for the bare SPCE electrode (without TiO2-NPs and Al-TiO2-

NPs, Figure 3a). In contrast, the cyclic voltammograms observed for the TiO2-NPs/SPCE and Al-TiO2-NPs/SPCE modified electrode exhibited only an oxidation peak in the presence of vanillin, within the potential window between 0.00 and 1.20 V. The data suggest that the oxidation reaction on Al-TiO2-NPs/SPCE for vanillin is totally irreversible. The oxidation peak current at the SPCE electrode for vanillin (Figure 3.2.1.3 b), we therefore have a rather low and broad peak current. The oxidation current of vanillin

(0.58 V) on Al-TiO2-NPs/SPCE (Figure 3.2.1.3 d) was better than that on the bare SPCE.

Compared with the SPCE, TiO2-NPs/SPCE and Al-TiO2-NPs/SPCE, a significant enhancement in the anodic current (0.58 V) was achieved at the Al-TiO2-NPs/SPCE

(Figure 3.2.1.3 d), indicating that the high conductivity and high surface area of the TiO2- NPs/SPCE improve the catalytic activity and increase the effective electrode area toward the vanillin oxidation obviously indicating that the Al-TiO2-NPs/SPCE can be used to determine vanillin.

Figure 3.2.1.3. Cyclic voltammograms of different electrodes: (a) blank, (b) SPCE, (c) TiO2- NPs/SPCE, and (d) Al-TiO2-NPs/SPCE, at 250 mM vanillin solution in 0.1 M H3PO4.

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3.3. Optimization of Experimental Parameters

The electrochemical response of the modified Al-TiO2-NPs/SPCE toward the determination of vanillin were optimized by analyzing a standard solution (10 µM) of vanillin using linear sweep voltammetry (LSV) technique. The parameters aﬀecting the determination of vanillin, such as electrolyte, pH, Al-TiO2-NPs amount, scan rates, adsorption time, accumulation conditions and stability of the electrodes, were investigated. In order to find the optimal parameters, one parameter was varied and the other parameters were held ﬁxed at their reference values.

The influence of various supporting electrolytes were tested, such as NH4Ac, HCl, H3PO4,

HNO3, H2SO4 and NaOH. The results indicated that when H3PO4 solution (0.1 M) was used, the oxidation peak current was a higher sensitivity than other. For this study, 0.1

M H3PO4 solution was chosen to act as supporting electrolyte. On the other hand, the effect of 0.1 M H3PO4 supporting electrolyte pH on the electrochemical behaviour of the modified Al-TiO2-NPs/SPCE for the vanillin determination was studied. The variations of the oxidation peak potential as well as the peak current with respect to changes in the pH of the electrolyte in range (1.40 – 6.30), with an increase the pH, manifesting that protons have taken part in the electrode reaction processes. Figure 3.2.1.S1 shows, the relation between the oxidation peak potential and supporting electrolyte pH. The effect of modification amount of Al-TiO2-NPs on the SPCE surface for the determination of vanillin was also studied by using LSV, different modification volumes were tested: 2.0, 4.0 and 6.0 μg. The 2.0 μg gave the best result of oxidation peak current sensitivity than other counterparts. The effect of scan rate on the oxidation peak current of vanillin (10 µM) by using LSV was studied. Different scan rates were tested 20, 30, 50 and 70 mV s- 1. The 50 mV s-1 scan rate gave the best result of oxidation peak current sensitivity than other counterparts. In this study, 50 mV s-1 scan rate was used. The accumulation step of 10 µM vanillin after 120 second was performed under 0.0 V, at a fixed accumulation potential 0.0 V, the peak current increased progressively with accumulation time up to 120. Thereafter, the peak current increased much slightly as further increasing the accumulation time, also shown in Figure 3.2.1.S2. This phenomenon could be explained to the saturated adsorption of vanillin on the surface of the electrode. Then, the stability of the modified electrodes was examined under the best conditions. Five electrodes

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were made by the same procedure. Moreover, the stability of the modified electrode was also studied. When the modified electrode (Al-TiO2-NPs/SPCE) was studied with eight segments, the peak current kept 99.0 % of the original peak is also shown in Figure 3.2.1.S3.

3.4. Analytical Application

According to the previous optimum conditions, the analytical purposes parameters of the method were recorded by linear sweep voltammetry (LSV) technique, with (50 mV s-1) scan rate. The calibration graph showed a linear range for standard vanillin solutions of 0.07 to 20 µM. The LSV voltammograms obtained for different concentrations of vanillin. Table 3.2.1.1, shows the results of linear range, slope, intercept and the regression coefficient (R2) of the calibration curve for vanillin.

Table 3.2.1.1. Analytical parameters of electrochemical detection of vanillin by aluminium doped TiO2 nanoparticles-modified screen printed carbon electrode. Parameter Vanillin Linear dynamic range/ µM 0.07-20 Calibration graph: Intercept 0.2868 Slope 2.7213 Correlation coefficient 0.9977 Detection limit/ µM 0.02 Quantification limit/ µM 0.07 RSD (%) (n=10)a 3.5 a 5 µM vanillin.

The precision of the method for standard solutions (investigated after analyzing 10 series of 10 replicates) and the relative standard deviation (RSD) was calculated to be 3.5 % at the 5 µM concentration of vanillin. The theoretical limit of detection (LOD) was founded to be 0.02 µM are expressed as the analyte concentration giving a signal equivalent to the blank signal plus three times its standard deviation (3σ). The developed method provides clear and good advantages in terms of sensitivity with method previously-published reports for the electrochemical determination of vanillin [6,12,16,17,31] that involve the use of others vanillin sensors and electrochemical detection (see Table 3.2.1.2).

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Table 3.2.1.2. Comparison of different electrochemical electrodes for vanillin determination.

Electrode Electrochemical technique Detection limit (µM) Ref.

SPCE SWV 0.40 [17] GCE LSV and SWV 16 [16] PVC/ graphite electrode Amperometric 290 [12] Graphene/GCE DPV 0.056 [6] MIP-SWNTs-COOH/GCE DPV 0.2 [31] Al-TiO2-NPs/SPCE LSV 0.02 This work LSV: linear sweep voltammetry; SWV: square-wave voltammetry; DPV: differential pulse voltammetry; GCE: glass carbon electrode; and MIP-SWNTs-COOH: molecularly imprinted ionic liquid polymer−carboxyl single-walled carbon nanotubes composite.

The influence of some components in the determination of vanillin was studied for various common interfering substances in the samples to be analyzed, including vanillic acid, vanillic alcohol, p-hydroxybenzaldehyde and p-hydroxybenzoic acid. These substances were chosen because they are known to be present in natural vanilla. A compound was considered as interference if it caused an analytical variation of more than 5% when compared to the analytical signal obtained in the absence of the interfering compound. The results were showed that no interferences were appeared for the interference analyte (w/w) ratios investigated (Table 3.2.1.3).

Table 3.2.1.3. Interference study in the detection of vanillin by aluminum doped TiO2 nanoparticles-modified screen printed carbon electrode. Interferents Tolerated interferent analyte (w/w) ratioa vanillic acid > 5b vanillic alcohol > 10b p-hydroxybenzaldehyde and p-hydroxybenzoic acid > 50b a For 5 µM vanillin concentration b Maximum ratio tested

The applicability of the proposed methodology for the determination of vanillin in two vanillin extract samples (sample A and sample B) was investigated. These vanillin extract samples were purchased from local markets and prepared according the section 2.1. Sample A and sample B were found to contain vanillin. The results obtained are shown in Table 3.2.1.4. This was compared with a blind analysis of the vanillin extract samples using the modified HPLC-DAD method [30] described in the section 2.2. The results showed, both methods are in close agreement for supporting the validity of this method.

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Table 3.2.1.4. Analysis of vanilla extract samples by the proposed and HPLC-DAD [30] methods. Vanillin content (µM)

LSV method at Al-TiO2- HPLC-DAD [30] Recovery (%) NPs/SPCE electrode Vanilla extract A 12.7±1.3 11.9±0.6 93.7 Vanilla extract B 8.4±0.9 7.9±0.3 94.0

4. Conclusion

The development of an aluminium doped TiO2 nanoparticles have been described, and the prepared hybrid nanoparticles have been checked in SPCE, as new working electrode for detection of vanillin in extract vanilla samples with good analytical performance. The use of the developed working electrode (Al-TiO2-NPs/SPCE) allows implementing a simple, effective and rapid method for electrochemical detection of vanillin in extract vanilla samples. The Al-TiO2-NPs/SPCE electrode was found to increase the sensitivity higher than the commercial electrodes checked for vanillin determination, thus evaluating the effectiveness of the proposed approach for sensitivity improvement of the working electrodes in screen printed carbon electrodes. This modification also introduces higher stability and appropriated selectivity, and the simplicity of the method allows to be used as a rapid screening test for vanillin, very useful for routine analytical works. On the other hand, some limitations of the use of Al-TiO2-NPs/SPCE have short electrode lifetime, poor inter-electrode reproducibility and not being user-friendly.

Acknowledgements

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SUPPLEMENTARY INFORMATION

Figure 3.2.1.S1: The relationship between the oxidation peak potential and pH, for Al-TiO2- NPs/SPCE electrode in 0.1M H3PO4 buffer with different pH values: 1.41, 1.66, 2.11, 3.40, 4.92, 5.69, 6.0 and 6.31.

Figure 3.2.1.S2: The accumulation step of 10 µM vanillin in 0.1M H3PO4 solution. After 120 second was performed under 0.0 V, scan rate 50 mV s-1.

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Figure 3.2.1.S3: The stability of Al-TiO2-NPs/SPCE modified electrode with eight segments, at 250 µM vanillin solution in 0.1M H3PO4.

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3.2.2. A Sensitive Electrochemical Sensor Based on Aluminium Doped Copper Selenide Nanoparticles-modified Screen Printed Carbon Electrode for Determination of L-tyrosine in Pharmaceutical Samples

In preparation to be submitted

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Abstract

A simple method for the direct quantitative and determination of L-tyrosine was proposed in this work. Aluminium doped CuSe nanoparticles (Al-CuSe-NPs), were used to modify screen printed carbon electrode (SPCE) to study the electrochemical behaviors of L-tyrosine using cyclic voltammetry (CV) and linear sweep voltammetry (LSV) techniques. Al-CuSe-NPs were characterized by X-ray diffraction (XRD) and Scanning electron microscope (SEM). The results demonstrated that the Al-CuSe- NPs/SPCE exhibited high electrocatalytic activity and good analytical performance towards the oxidation of L-tyrosine. The linear range of L-tyrosine was 0.15–10 µM with correlation coefficient of 0.9974. The detection limit of the analyte was 0.04 µM. In addition, the Al-CuSe-NPs/SPCE displayed good reproducibility, high sensitivity and good selectivity towards the determination of the L-tyrosine, making it suitable for the determination of L-tyrosine in pharmaceutical samples.

Keywords: Aluminium doped CuSe nanoparticles, screen-printed carbon electrode, electrochemical sensor, L-tyrosine, pharmaceutical samples.

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GRAPHICAL ABSTRACT

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1. Introduction

Tyrosine (4-hydroxyphenylalanine, C9H11NO3) is one of the 20 standard amino acids that are used by cells to synthesize proteins. Tyrosine can also be found in dairy products, meats, fish, eggs, nuts, beans, oats, and wheat [1]. Determination of tyrosine is important in various fields of research, particularly in food and biotechnology and pharmaceutical industries [2]. Up to now, there have been many analytical methods dealing with the determination of tyrosine, including spectrophotometric [3], fluorescence [4], capillary electrophoresis (CE) [5], liquid chromatography-mass spectrometry (LC-MS) [6], gas chromatography-mass spectrometry (GC-MS) [7], high- performance liquid chromatography (HPLC) [4] and electrochemistry [8,9]. These methods have high cost and involve time-consuming sample pre-treatment processes. Because tyrosine is an electro-active compound and it is possible to measure the quantity of tyrosine in the pharmaceutical products by electrochemical detection (ECD) through the study of its oxidation. ECD is important method for quantitative determination of tyrosine due to its simplicity, fast response, high sensitivity, and cheap instrumentation [10–14]. Various electrochemical methods, such as amperometric [15], square-wave voltammetry (SWV) [16] and differential pulse voltammetry (DPV) [17] for the detection and determination of tyrosine in various samples.

In recent years, many reports on screen-printed carbon electrodes (SPCEs) technology have been used to develop various electrochemical sensors that detect target molecules in various sectors, such as biomedical, pharmaceutical, environmental and agri-food [18–20]. SPCE is planar shape, thus can be used as droplet on sensor, using typically few microliters of the sample in miniaturized system. Moreover, it is low-cost and can be used as a disposable sensor with possibility used in large-scale implementation. The oxidation of tyrosine at SPCE and others most unmodified electrode surfaces represents a serious problem which arises from the poor reproducibility with high over potential resulted from a fouling effect, which results in rather poor sensitivity and selectivity [21– 23]. To avoid these problems, the modification of electrochemical working electrode is an excellent alternative. In this sense, many researchers have attempted to diminish the over potentials by using various modified electrodes such as, multi-walled carbon nanotubes (MWCNTs) [24], silver nanoparticles (Ag-NPs) [25] and gold nanoparticles

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(Au-NPs) [26]. However, the performance of some electrodes was still ineffective enough. Thus it is necessary to develop new types of electrode devices by the preparation synthesis and preparation of new materials. That regard, as modifiers for electrode surface, copper selenide nanoparticles (CuSe-NPs) had highly interest of many researchers and a many amount of research was applied in the previous decades [27– 29]. The increase in the CuSe-NPs efficiency such as to ensnare the charge carrier is accomplished by doping with transition metals and transition metal oxides such as, tin (Sn) [30], gallium (Ga) and indium (In) [31].

Here, the synthesis of aluminium doped CuSe nanoparticles (Al-CuSe-NPs) is carried out, which were characterized by XRD and SEM. The sensitivity of the developed electrode is compared with SPCE, Al-NPs/SPCE, and Al-CuSe-NPs/SPCE for L-tyrosine detection. The results show that a composite film of Al-CuSe-NPs/SPCE is more sensitive compared to SPCE and Al-NPs/SPCE. Furthermore, the electrochemical behavior of L-tyrosine at Al- CuSe-NPs/SPCE was investigated. The performance of the modified electrode (Al-CuSe- NPs/SPCE) is also demonstrated for the determination and detection of L-tyrosine in pharmaceutical samples obtaining good selectivity, stability and high sensitivity.

2. Experimental 2.1. Chemicals and reagents

All the starting materials were purchased with very highly purity. L-tyrosine, aluminium acetylacetonate (Al(acac)3, 99%), lithium aluminium hydride (LiAlH4, 95%), mesitylene (97%), Se-powder, sodium sulfite were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nafion 117 solution (5% in a mixture of lower aliphatic alcohols and water), copper sulfate pentahydrate and trisodium citrate were purchased from Fluka Chemie (Buchs, UK). Phosphoric acid were purchased from Panreac Quimica S.L.U. (Barcelona, Spain). L- tyrosine solution was prepared in water and stored while tightly covered in the dark until use. L-tyrosine stock solutions were frequently diluted to working standard solutions with H3PO4 buffer at pH 6.0.

2.2. Apparatus and instruments

Electrochemical detection was carried out on a Wuhan Corrtest Instruments Corp., Ltd. (Hubei, China) all the experiments were carried out using screen-printed carbon

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electrodes (SPCEs) system (Dropsens DRP-C110) (Oviedo, Spain) housed in the home made electrochemical flow cell. X-ray Diffraction (XRD) patterns were measured on Philips model X’Pert MPD diffractometer using a CuKα source (l=1.5418Å), programmable divergence slit, graphite mono-chromator and proportional sealed xenon gas detector. The samples were made with a voltage of 40 KV, intensity 40 mA and an angular range of 20 to 80 degrees (2θ), a step of 0.02 degrees 2θ and a time per step of 1.50 sec.

Scanning electron microscope (SEM) micrographs were measured on a ZEISS GeminiSEM 500 (Germany) operating at acceleration voltage 0.02-30 kV, probe current 3 pA - 20 nA, store resolution up to 32k × 24k pixels, and magnification from 50 up to 2x106. Equipped with several detectors: in-lens secondary electron detector, in-lens energy selected backscatter detector (EsB), annular STEM detector (aSTEM 4) and EBSD detector (electron backscatter diffraction) investigation of crystalline orientation. The samples were prepared onto a lacey carbon by deposition of the synthesized material.

Ultrapure water was obtained from a Milli-Q water instrument from Millipore (Merck KGaA, Darmstadt, Germany). An ultrasound bath (Selecta, Barcelona, Spain) and a Basic 20 pH-meter with a combined glass electrode (Crison Instruments S.A., Barcelona, Spain) were used.

2.3. Synthesis of aluminium doped copper selenide nanoparticles (Al-CuSe-NPs)

At first, aluminium acetylacetonate (Al(acac)3, 10 mmol) was added to a three-neck round bottom which was already contained mesitylene solution with magnetic bar. Then lithium aluminium hydride (LiAlH4, 30 mmol) was added to the mixture. The reaction was purged with N2 gas during reflux with stirring for 72 hours at 165 °C. After cooling to 25 °C, a gray-colored precipitate was formed which was crushed and kept to dry under low pressure for 5 hours. Wash the isolated solid product well with 25 mL portions of cold methanol three times, to avoid any high temperatures (exothermic reaction) between solvent and Al-NPs. The unreacted materials were washed three times with methanol. The resulting product was filtered and dried at 25 °C under low pressure [32].

The sodium seleniosulfide (Na2SeSO3) solution was prepared by mixing 10 g selenium powder with 100 g anhydrous sodium sulfite in 500 ml of distilled water. After stirring

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for 8–10 h at 80 °C, fresh Na2SeSO3 solution was filtrated and stored while tightly covered for process [33]. The synthesis of Al-CuSe-NPs consists of an aqueous solution of 1.0 mL of CuSO4.5H2O (0.5 M), 1.0 mL of trisodium citrate (0.1 M), 1.0 mL of Na2SeSO3 (0.25 M) solution and 0.25 g Al-NPs, after sonication for 15 min, the mixture was mixed with constant stirring for 2 h. The final product was filtered and dried at 25 °C under low pressure, obtaining an Al-CuSe-NPs as powder.

2.4. Electrode preparation and modification

The prepared nanoparticles were ultrasonically dispersed in pure water (with 0.5% Nafion, v:v), The concentrations of 1.0 mg/mL were obtained individually. On the SPCE forming a layer of thin films from nafion-solubilized nanoparticles are more uniform distribution than those casted by organic solvents [32]. Al-NPs and Al-CuSe-NPs were used. Dropsens SPCEs (DRP-110), with carbon as a working electrode with a disk-shaped of 4 mm of diameter, were used to fabricate the electrode. So, 6.0 µL of the dispersed NPs was casted onto the surface of the SPCE. After drying the modified SPCE by using infrared light lamp for 15 min, rinsing with pure water. The electrode is ready for using.

2.5. Electrochemical measurements of L-tyrosine

A 25mL of H3PO4 electrolyte (0.1M, pH=6.0) with proper concentration of L-tyrosine was transferred to a cell, then the SPCE was assembled on it. The potential range was 0.0 – 1.2 V, and the scan rate was 50 mV/s. Before each measurement, the electrode was cycled between 0.0 – 1.2 V in a blank solution (H3PO4, 0.1M) until no L-tyrosine peak current was detected. After each run, the SPCE was immersed in ultrapure water for 3 min to remove L-tyrosine for reuse and to regenerate the electrode surface. All experiments were conducted at room temperature.

2.6. Real sample preparation

The commercial pharmaceutical sample was purchased at local pharmacy, Levotiroxina Sanofi (Sanofi-aventis, S.A., Barcelona, Spain). Three tablets were crushed in a mortar and they were homogenized. Then, a suitable amount was dissolved in deionized water; the solution was sonicated for 20 min and filtered. Suitable dilutions were made before measurements to obtain solutions in H3PO4 (0.1 M, pH 6) with L-tyrosine concentration within the calibration range.

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3. Results and discussion 3.1. Characterization of Al-CuSe-NPs

Figure 3.2.2.1, shows XRD patterns measured for Al-NPs (Figure 3.2.2.1 a) and Al-CuSe- NPs (Figure 3.2.2.1 b). The XRD patterns show that the nanoparticles involved aluminium as a major component mixed with CuSe-NPs as minor components. The XRD patterns and all the positions of the peaks are attributed to face-centered cubic (fcc) crystal structure of aluminium [32]. No diffraction peaks of other impurities are found in the XRD pattern, indicating that the product is pure Al-CuSe-NPs.

Figure 3.2.2.1. XRD patterns of (a) Al-NPs and (b) Al-CuSe-NPs.

The surface morphology of Al-CuSe-NPs composite was analysed by SEM and Energy- dispersive X-ray (EDX) elemental mapping. The micrographs with the results are showed in Figure 3.2.2.2. These images indicate the homogeneous distribution and the doping of the CuSe nanoparticles over the aluminum. The composite’s aluminum surfaces were clearly decorated with CuSe nanoparticles. The SEM confirms the formation of Al-CuSe- NPs composite according to the XRD results.

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Figure 3.2.2.2. a) SEM micrographs of Al-CuSe-NPs and b) elemental mapping image of Al-CuSe- NPs. 3.2. Electrochemical behavior of L-tyrosine

The electrochemical determination of L-tyrosine at the Al-CuSe-NPs/SPCE has been studied at the optimum condition using cyclic voltammetry (CV) technique. Figure

3.2.2.3 shows the cyclic voltammograms for 20 µM L-tyrosine with H3PO4 supporting electrolyte (0.1M, pH=6.0) at bare SPCE, Al-NPs/SPCE and Al-CuSe-NPs/SPCE, respectively. In contrast, the cyclic voltammograms observed for the Al-NPs/SPCE and Al-CuSe-NPs/SPCE modified electrode exhibited only an oxidation peak in the presence of L-tyrosine, within the potential window between 0.00 and 1.20 V. The data suggest that the oxidation reaction on Al-CuSe-NPs/SPCE for L-tyrosine is totally irreversible. The oxidation peak indicating that the Al-CuSe-NPs/SPCE can be used to determine L- tyrosine.

Figure 3.2.2.3. The cyclic voltammograms of 20 µM L-tyrosine in 0.1 M H3PO4 buffer at pH=6.0, 50 mV/s scan rate of blank (0.1M H3PO4), bare SPCE, Al-NPs/SPCE and Al-CuSe-NPs/SPCE. 3.3. Optimization of experimental parameters

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The electrochemical response of the modified Al-CuSe-NPs/SPCE toward the determination of L-tyrosine were optimized by analyzing a standard solution of L- tyrosine (20 µM) using linear sweep voltammetry (LSV) technique. The parameters affecting the determination of L-tyrosine, such as supporting electrolyte, supporting electrolyte concentration, pH, Al-CuSe-NPs amount, scan rates and stability, were investigated.

The influence of the supporting electrolyte was studied using 20 µM of L-tyrosine. The following supporting electrolytes were tested (0.1M each one): ammonium phosphate,

CH3COOH, H2SO4, H3PO4, HCl, HNO3, NaCl and KOH. The results indicated that when

H3PO4 solution was used, the oxidation peak current was a higher sensitivity than other

(Figure 3.2.2.S1). For this study, H3PO4 solution was chosen to act as supporting electrolyte. The concentration of H3PO4 was studied between 0.05 and 0.15 M. The results indicated that when 0.1M H3PO4 was used, the oxidation peak current was a higher sensitivity than other (Figure 3.2.2.S2). Hence, 0.1M was selected as the concentration of H3PO4 supporting electrolyte.

The influence of the pH value on the oxidation current of the L-tyrosine has also been studied at the optimum potentials as shown in (Figure 3.2.2.S3). With increasing pH from 2.0 to 8.0, the oxidation current of L-tyrosine increased from pH 2.0 to 8.0 and reached the maximum at pH 6.0, and then decreased with higher pH value. Therefore,

H3PO4 at pH 6.0, was chosen for analytical experiments.

The effect of the amount of the Al-CuSe-NPs on the SPCE surface for the determination of L-tyrosine was also studied, different modification volumes were tested: 2.0, 4.0, 6.0 and 8.0 µL. The 6.0 µL gave the best result of oxidation peak current sensitivity than other (Figure 3.2.2.S4). The effect of scan rate on the oxidation peak current of L- tyrosine (20 µM) by using LSV was studied. Different scan rates were tested 20, 40, 50, 60, 80, 100, 120, 140 and 160 mV/s.

The accumulation step of 20 µM L-tyrosine after 3600 second was performed under 0.0 V, at a fixed accumulation potential 0.0 V, the peak current increased progressively with accumulation time up to 3600. Thereafter, the peak current increased much slightly as further increasing the accumulation time, also shown in (Figure 3.2.2.S5). This

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phenomenon could be explained to the saturated adsorption of L-tyrosine on the surface of the electrode.

The stability of the modified electrode has been studied by investigating the amperometric responses of 20 µM L-tyrosine at Al-CuSe-NPs/SPCE. Five electrodes were made by the same procedure (Figure 3.2.2.S6). The results showed that the remained percentage of initial response for L-tyrosine at Al-CuSe-NPs/SPCE was 99%, which demonstrated a good stability of the modified electrode. The reproducibility of Al-CuSe- NPs/SPCE electrode was investigated by repetitive measurements (N=5) of 20 µM L- tyrosine, and the resulting relative standard deviation (RSD) was 3.07%. Therefore, the Al-CuSe-NPs/SPCE has the advantage of good reproducibility.

The influence of some components in the determination of L-tyrosine was studied for various common interfering substances in the samples to be analyzed, including arginine, lysine, cysteine and histidine (30 times content), glucose and ascorbic acid (100

+ + 2+ − − 2− 3− − times content), K , Na , Ca , Cl , NO3 , SO4 , PO4 and CH3COO (150 times content). A compound was considered as interference if it caused an analytical variation of more than 30% when compared to the analytical signal obtained in the absence of the interfering compound. The results were showed that no interferences were appeared for the interference analyte (w/w) ratios investigated (Table 3.2.2.1).

Table 3.2.2.1. Effect of the presence of potentially interfering compounds in the electrochemical response of L-tyrosine. Foreign species Tolerated interferent/ analyte (w/w) ratioa

Arginine, lysine, cysteine, histidine > 30b Glucose, ascorbic acid > 100b + + 2+ − − 2− 3− − b K , Na , Ca , Cl , NO3 , SO4 , PO4 and CH3COO > 150 a For 6.0 µM L-tyrosine concentration. b Maximum ratio tested.

3.4. Analytical applications

According to the previous optimum conditions, the analytical purposes parameters of the method were recorded by linear sweep voltammetry (LSV) technique, with (50 mV/s) scan rate. The calibration graph showed a linear range for standard L-tyrosine solutions of 0.15 to 10 µM. The LSV voltammograms obtained for different concentrations of L-tyrosine. Table 3.2.2.2, shows the results of linear range, slope, intercept and the regression coefficient of the calibration curve for L-tyrosine. The

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precision of the method for standard solutions (investigated after analyzing 11 series of 11 replicates) and the relative standard deviation (RSD %) was calculated to be 4.93% at the 7.0 µM concentration of L-tyrosine. The limit of detection (LOD) was found to be 0.04 µM.

Table 3.2.2.2. Analytical parameters obtained for the L-tyrosine determination.

Parameter L-tyrosine Linear dynamic range / µM 0.15 – 10 Regression equation y=53.10x + 3.31 Correlation coefficient (R2) 0.9974 Limit of detection / µM 0.04 Limit of quantification / µM 0.15 RSD (%) (n=11)a 4.93 y = ax + b; y: peak current (µA), x: tyrosine concentration (µM), a: slope and b: intercept. a 5.0 µM of L-tyrosine.

The developed method provides clear and good advantages in terms of sensitivity with method previously-published reports for the electrochemical determination of L- tyrosine (Table 3.2.2.3). The applicability of the proposed methodology for the determination of L-tyrosine in pharmaceutical sample was investigated. The pharmaceutical sample was found to contain L-tyrosine. The results obtained are shown in Table 3.2.2.4.

Table 3.2.2.3. Comparison of the proposed sensor for determination of L-tyrosine with other methods.

Electrode type Linear range (µM) LOD (µM) Remarks Ref. CNF-CPE 0.2 – 109 0.1 PBS (0.1M, pH=7) [34] MWCNT-GCE 0.9 – 350 0.35 Citric acid (2mM, pH=6.5) [35] Ag/Rutin-WGE 0.3 – 10 0.07 PBS (0.1M, pH=7) [25] Butyrylcholine-GCE 4 – 100 0.4 PBS (0.1M, pH=7) [1]

BDDE 100 – 700 1.0 Na2PO4/NaOH (0.1M, [36] pH=11.2) SWCNT-GCE 5–20; 27–260 0.09 Citric acid-sodium citrate [37] (0.1M, pH=4)

Nanostructures modified 3.6 – 240 0.12 H3PO4 (0.1M, pH=1) [38] gold electrode

Al-CuSe-NPs/SPCE 0.15 – 10 0.04 H3PO4 (0.1M, pH=6) This work CNF: carbon nanofiber, CPE: carbon paste electrode, MWCNT: multiwall carbon nanotube, GCE: glassy carbon electrode, Ag: silver nanoparticle, WGE: paraffin-impregnated graphite electrode, BDDE: boron- doped diamond electrode, SWCNT: single-walled carbon nanotube, LOD: Limit of detection and Ref: References.

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Table 3.2.2.4. Determination results and recovery study of L-tyrosine in pharmaceutical sample by the proposed method.

Sample L-tyrosine Addeda Found (n=3) Recovery (%) Levotiroxina Sanofib 150 148 ± 2.1 98.6 200 202 ± 5.7 101.0 250 244 ± 3.3 97.6 a Expressed in µg L-tyrosine per tablet. b 200 µg per tablet.

4. Conclusions

A new electrochemical sensor based on aluminium doped CuSe nanoparticles was developed for direct determination of L-tyrosine in pharmaceutical samples with good analytical performance. The developed electrochemical sensor showed high sensitivity and selectivity towards L-tyrosine with a detection limit of 0.04 µM. In particular, the developed electrochemical sensor (Al-CuSe-NPs/SPCE) offers the advantages of simplicity and efficiency in target detection from pharmaceutical sample. In addition, this detection method allow for further sensor developments.

Acknowledgments

References

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SUPPLEMENTARY INFORMATION

Figure 3.2.2.S1. Analytical signals obtained for different supporting electrolytes; 20 µM L- tyrosine; 0.1M electrolyte concentration and 50 mV/s scan rate.

Figure 3.2.2.S2. Analytical signals obtained for different concentrations of H3PO4 (0.05, 0.1 and 0.15 M); 20 µM L-tyrosine; and 50 mV/s scan rate.

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Figure 3.2.2.S3. Analytical signals obtained for different pH (2, 4, 6, 8 and 10) of 0.1M H3PO4; 20 µM L-tyrosine; and 50 mV/s scan rate.

Figure 3.2.2.S4. Analytical signals obtained for different amount of Al-CuSe-NPs materials (2.0,

4.0, 6.0 and 8.0 µL), 0.1M H3PO4; 20 µM L-tyrosine; and 50 mV/s scan rate.

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Figure 3.2.2.S5. The accumulation step of 20 µM L-tyrosine in 0.1M H3PO4 solution. After 60 min (3600 s) was performed under 0.0 V, scan rate 50 mV/s.

Figure 3.2.2.S6. The stability of Al-CuSe-NPs/SPCE modified electrode with eight segments, at 20

µM L-tyrosine solution in 0.1M H3PO4.

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3.2.3. Decoration of Graphene oxide with Copper Selenide in Supercritical Carbon Dioxide Medium as a Novel Approach for Electrochemical Sensing of Eugenol in Various Samples

Submitted to The Journal of Supercritical Fluids

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Abstract

A supercritical carbon dioxide medium (sc-CO2) was used for the decoration of reduced graphene oxide (rGO) with copper selenide (CuSe). Graphene oxide (GO), rGO and CuSe@rGO were characterized by Raman spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). The CuSe@rGO prepared composite materials were used to modify glassy carbon electrodes (GCE), improving their electrochemical properties and allowing to obtain a wide range of working electrodes based on graphene. Using CuSe@rGO/GCE as the working electrode, a linear dynamic range between 1–82 μg Kg−1 and limit of detection (LOD) of 0.4 μg Kg−1 were obtained. These parameters represented a minimum 2-fold increase in sensitivity compared to the use of GCE, CuSe/GCE, GO/GCE, or rGO/GCE. The present work is meaningful to expand decorated graphene composites to sensor fields and promote the development of eugenol sensors.

Keywords: Reduced graphene oxide, copper selenide, supercritical carbon dioxide, glassy carbon electrode, eugenol

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GRAPHICAL ABSTRACT

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1. Introduction

Design and synthesis of functional nanostructured materials using supercritical fluids (SCF) are a promising alternative due to their unusual properties such as low viscosity, high diffusivity, near-zero surface tension and tunable properties [1]. The consequence is that supercritical fluids have hybrids properties of those normally associated with gases and liquids and which are continuously adjustable from gas to liquid with small variations of pressure and temperature. Consequently, the density is closer to that of a liquid while the viscosities and diffusivities are similar to those of the gas. Moreover, surface tension disappears above the critical point of the fluid; this is of particular interest in surface and interface chemistries [2]. Different solvents can be used in SCFs

[3]. Supercritical carbon dioxide medium (sc-CO2) is widely used as nonflammable, non- toxic, an environmentally benign solvent with an easily accessible critical point. In the last decade, the decoration of carbon materials by using SCF has attracted much attention because of their properties. Graphene has been the most intensively explored carbon allotrope in material science because of its electrical, thermal and mechanical properties provide some application in electrochemistry and material science [4–6]. The decoration of graphene oxide (GO) and reduced graphene oxide (rGO) with another material by different methods, such as metal nanoparticles [7–9], metal-organic frameworks (MOFs) [10], and semiconductors [11]. This decoration provides an improvement in the optical, electrical and mechanical properties of this material leading to electrochemical application and surface-enhanced Raman scattering (SERS) spectroscopy [12,13]. Electrochemical detection (ECD) is a powerful tool for the detection of electro-active analytes in food, environmental assessment, and others. ECD has the advantages of high sensitivity, fast response, low cost and simple operation.

At present, the problem of food security has become the focus of threat to human survival. Eugenol (C10H12O2) is a flavour component, which is present in cloves and some other spices (cinnamon, nutmeg, basil, and bay leaf). It is also added to cosmetics, pharmaceutical products and toothpaste as flavour [14]. Eugenol is hepatotoxic, meaning it may cause damage to the liver [15]. Overdose is possible, causing a wide range of symptoms from blood in the patient's urine, to convulsions, diarrhoea, nausea, unconsciousness, dizziness, or rapid heartbeat. Thus, due to the harmful effects of

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eugenol, it was considered to develop a simple method for the determination of eugenol in various samples. One of the most promising techniques is the ECD that can answer to such technological challenge. Various electrochemical methods, such as amperometric, cyclic voltammetry (CV) [16], square-wave voltammetry (SWV) or differential pulse voltammetry (DPV) [17], were developed for the detection and determination of eugenol in various types of samples.

In this study, a new material with favourable characteristics for their use in electrochemical analysis, using an approach compatible with the green chemistry principles was developed. Hence, the decoration of rGO with CuSe was carried out in sc-

CO2. The synthesized composite material CuSe@rGO was used to modify the commercially available glassy carbon electrode (GCE). The modified electrode (CuSe@rGO/GCE) was used for the sensitive and selective voltammetric determination of eugenol in real samples (clove, cinnamon and toothpaste).

2. Materials and methods

2.1. Reagents, standards and samples

All the starting materials were purchased with very highly purity. Graphite, sodium nitrate, potassium permanganate, hydrogen peroxide, Se-powder, sodium sulfite, eugenol and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nafion 117 solution (5% in a mixture of lower aliphatic alcohols and water), copper sulfate pentahydrate, trisodium citrate and boric acid were purchased from Fluka Chemie (Buchs, UK). Ethanol, sulphuric acid and phosphoric acid were purchased from Panreac Quimica S.L.U. (Barcelona, Spain).

Working standard solutions were made by appropriate dilution of the eugenol stock standard solution with Britton-Robinson buffer at pH 2.0. Water was purified with a Milli-Q system (Millipore). Clove, cinnamon and toothpaste extract samples were purchased at different local markets (Ciudad Real, Spain).

2.2. Apparatus and instruments

Supercritical carbon dioxide (sc-CO2) extraction was carried out at JASCO System (Japan) comprised of CO2 tube, high pressure CO2 delivery pump (PU-1580-CO2, JASCO, Japan),

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temperature controller from Hewlett Packard 5890, Series II gas chromatograph (Bothell, WA, USA) and Automated Back Pressure Regulator (BP-1580-81, JASCO, Japan). Electrochemical detection was carried out on a CH Instruments Model 800D Series (Austin, Texas, USA). All measurements were carried out using a three-electrodes system consisted of a glassy carbon (GC) working electrode (diameter 3.0 mm), silver/silver chloride (Ag/AgCl/KClsat) reference electrode, and a platinum (Pt) wire counter electrode. All electrodes were provided by CH Instruments Inc. (Austin, TX, USA).

Raman measurements were performed with a Portable Raman Spectrometer system (B&W TEK Inc., DE, USA) at a wavelength of 785 nm and a maximum laser output power at system’s excitation port of 348 mW and 285 mW in the probe. The output laser power in the probe was set to the total percentage without any damage. For measurements, the laser beam was focused to the sample through a 100/1.25 objective. Raman signals were acquired by a CCD array detector cooled at 10 °C with an acquisition time ranging from 5 to 65.5 ms.

XRD patterns were measured on Philips model X´Pert MPD diffractometer using a CuKα source (λ=1.5418 Å), programmable divergence slit, graphite monochromator and proportional sealed xenon gas detector. The samples were made with a voltage of 40 KV, intensity 40 mA and an angular range of 5.0 to 80 degrees (2 Theta), a step of 0.02 degrees 2 Theta and a time per step of 1.50 sec.

Ultrapure water was obtained from a Milli-Q water instrument from Millipore (Merck KGaA, Darmstadt, Germany). An ultrasound bath (Selecta, Barcelona, Spain) and a Basic 20 pH meter with a combined glass electrode (Crison Instruments S.A., Barcelona, Spain)

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were used. Centrifugation was carried out using a Centrofriger BL-II model 7001669, J.P Selecta (Barcelona, Spain) centrifuge.

3.2. Synthesis of reduced copper selenide graphene oxide (CuSe@rGO)

First, Na2SeSO3 and graphene oxide were prepared. Na2SeSO3 solution was prepared by mixing 10 g of selenium powder with 100 g of anhydrous sodium sulfite in 500 mL of distilled water. Then, this reaction was stirring for 8-10 h at 80 °C, fresh Na2SeSO3 solution was filtrated and stored while tightly covered for process [18]. Graphene oxide was prepared according to a modified Hummers method [19]. Firstly, 0.5 g of graphite and 0.5 g of sodium sulphite, were dispersed in 23 mL of concentrated sulphuric acid into 100 mL Erlenmeyer flask in ice bath maintaining temperature below 20 °C. This mixture, was stirring for 4 hours. Then, 3.0 g of potassium permanganate were added slowly to the reaction mixture and stirring the mixture for 1 hour. After this hour, the ice was removed, and the reaction was stirring another hour more and heating the mixture at 35 °C. After this step, 46 mL of deionized water were added. This reaction was stirring and heating at 95 °C for 2 hours without allowing the mixture to boil. Subsequently, turn off the heater and allow it to cool in room temperature. Then, 100 mL of deionized water were added and stirred the mixture for 1 hour. Now, 10 mL of 30 % hydrogen peroxide were added for 1 hour with constant stirring. The GO was formed and finally, the product was repeatedly washed with deionized water several times until the pH became neutral, indicating the final product doesn’t contain residual salts and acids.

The preparation of CuSe@GO consists of an aqueous solution by mixing 2 mL of

-1 -1 CuSO4.5H2O (0.5 mol L ), 2 mL of trisodium citrate (0.1 mol L ), 2 mL of Na2SeSO3 (0.25 mol L-1) solution and 0.2 g of GO. This solution was mixing with constant stirring for 10 min and then, the mixture was sonicated for 10 min.

The reduction of CuSe@GO was carried out in 10 mL stainless steel column using the assembly shown in Figure 3.2.3.1. In the extraction step, CO2 was aspirated from a dip- tube cylinder at a constant flow rate of 5 mL min−1 (liquid) by means of a PELTIER pump and passed through the column. A homogenized mixture of 6 mL of mixture (CuSe@GO) was manually placed into a 10 mL stainless column accommodated in the extraction

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chamber. Intimate contact with the extracting supercritical fluid was allowed through the combination of static and dynamic extraction steps. Thus, once the target pressure (20 MPa) and temperature (200 °C) were reached, the mixture was processed in the static mode for 15 min. Dynamic extraction was then performed for 1 hour, after which

CO2 continuously flows through the mixture, containing Cu, Se and GO, in the extraction column and out the restrictor to the trapping vessel containing solvent. The interferences present in mixture were collected in trapping vessel, and Cu, Se and GO remained in the extraction column forming the composite. A depressurization time of the system was about 5 minutes. After cooling the column to room temperature, the synthetic product was washed with 1 mL of water and nanoparticles were recovered and dried at 60 °C under vacuum.

Figure 3.2.3.1. Experimental set-up for the reduction of CuSe@GO. V1 and V2 are pressure selection valves; V3 and V4 are pressure injection valves; and P1 is a CO2 high-pressure pump.

3.4. Treatment of real samples

A 2.5 g of clove or cinnamon powder sample was placed into a 50 mL Erlenmeyer flask and 5 mL of ethanol was added. The mixture was shaken vigorously for 30 min and transferred to a centrifuge tube, and centrifuged at 3500 rpm for 10 min. After a settling time of 5 min, the supernatant was transferred into a 50 mL volumetric flask. Then, the solution was diluted with ethanol. Approximately 0.5 g of the toothpaste sample was

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weighed into a 50 mL beaker containing 15 mL of water. The mixture was boiled for 5 min to allow total dissolution of the suspension. Suitable dilutions were made before measurements to obtain solutions in Britton-Robinson buffer (0.1 mol L-1, pH 2) with eugenol concentration within the calibration range.

3.5. Preparation of modified electrode

The GCE was polished by using 0.3 and 0.05 μm α-alumina slurry on a polishing cloth until a mirror like appearance was observed. Later, as cleaned GCE was washed thoroughly with distilled water [20]. CuSe@rGO materials were dispersed in water (0.5% Nafion, v:v) by ultrasound to obtaining individual concentrations of 1.0 mg mL-1. Nafion was used for the GCE modification, because it is a compound usually employed as a cation conduction membrane and electron barrier to prevent usual interfering agents in analytical determinations. Films formed from nafion-solubilized nanoparticles are more uniform than those casted by organic solvent [21]. Each modiﬁed electrode was prepared by casting 6.0 µL of the dispersed CuSe@rGO materials onto the surface of the electrode. After drying under infrared light for 10 min, it was rinsed with water and ready to use.

The electroactive surface areas of the GCE, GO/GCE and CuSe@rGO/GCE were

-3 -1 estimated by using cyclic voltammetry technique of 1.0x10 mol L of K3Fe(CN)6 in 0.1 mol L-1 KCl, Figure 3.2.3.2.

-3 -1 -1 Figure 3.2.3.2. The cyclic voltammograms of 1.0x10 mol L of K3Fe(CN)6 in 0.1 mol L KCl, 50 mV s-1 scan rate. GCE, GO/GCE and CuSe@rGO/GCE.

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Results and discussion

Characterization of GO, rGO and CuSe@rGO

To confirm the synthesis of CuSe@rGO, the material produced was characterized Raman spectroscopy, X-ray diffraction (XRD) and scanning electron microscope (SEM). Figure 3.2.3.3 A shows a Raman spectrum of the GO, rGO and CuSe@rGO. The usual features of carbon materials in Raman spectra were the G band at ~1590 cm-1, which was assigned to the in-plane vibration of sp2 hybridized carbon atoms, and a D band at ~1325 cm-1, which was associated with structural defects such as bond-angel disorder, bond length disorder and hybridization that can break the symmetry [22,23]. The appearance of the peak at ~260 cm-1 in the CuSe@rGO composites, indicates that the CuSe are well anchored on rGO. The intensity ratios of the D to G (ID/IG) band allows us to estimate the size of the in-plane crystallites in nano-meters. The ratio of ID/IG of CuSe@rGO composites (~1.58) is larger for rGO (~1.23) indicating that the anchoring of CuSe with

2 rGO affect the crystalline sp region. The Raman spectra of GO exhibits an ID/IG ratio (~1.18) suggesting that the degree of defects was lower than for rGO. The XRD measurement was employed to identify the chemical composition and phase of the as- prepared composites. As presented in Figure 3.2.3.3 B, shows XRD patterns measured for GO, rGO and CuSe@rGO. The featured diffraction peak of GO appears at ~11.38°. In the peak of rGO, the XRD peak of GO at ~11.38° disappeared, but a new broad peak appeared and shows an obvious to higher 2θ angles (~26.68°), which is closer to that graphite, suggesting that the reduction of GO to rGO resulted in structural change, especially the distance between layers and was well ordered with two dimensional sheets with removal of surface functional groups [24]. The strong diffraction peaks shown in the CuSe@rGO pattern can be matched well with the standard phase of CuSe [25]. Meanwhile, the similar diffraction peaks were also exhibited in CuSe@rGO composites. Moreover, no traces of other peaks are observed, which effectively confirm that the high purity of the CuSe@rGO was obtained.

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Figure 3.2.3.3. (A) Raman spectrum of GO, rGO and CuSe@rGO, (B) XRD patterns of GO, rGO and CuSe@rGO, (C and D) SEM micrographs of CuSe@rGO and (E) elemental mapping images of CuSe@rGO composite.

The surface morphology of CuSe@rGO composite was analysed by SEM. The micrographs with the results are showed in Figure 3.2.3.3. As can be seen, CuSe nanoparticles had spherical shapes with an average particles sizes of decorated CuSe nanoparticles were evaluated and the particle sizes ranges were recorded as 30-35 nm. These images indicate the homogeneous distribution and the doping of the nanoparticles over the sheets of reduced graphene. The composite’s graphene sheets’ surfaces were clearly decorated with CuSe nanoparticles. The SEM confirms the formation of CuSe@rGO composite according to the XRD results.

Optimization of supercritical fluid parameters for synthesis and decoration of CuSe@rGO

The reduction of CuSe@GO was carried out under supercritical fluids conditions as described above. The sc-CO2 conditions were optimized by characterizing the reduced material at end of each extraction by XRD and SEM. The reduction of CuSe@GO is considered improved when XRD patterns indicated that the peak of GO at ~11.38° disappeared and new peak appeared at ~26.68° for rGO. Moreover, the total reduction of CuSe@GO is considered improved when SEM micrographs and elemental mapping images indicated that all oxygen atoms are reduced. The variables optimized were the temperature, CO2 extraction pressure, extraction time and CuSe@GO amounts. When

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one parameter was changed, the other parameters were fixed at their optimal values.

The influence of the CO2 pressure and temperature was studied simultaneously. In this way, the temperature was tested between 120 and 250 °C, and the CO2 pressure ranged from 5 to 30 MPa. The best results were achieved by using 20 MPa at 200 °C. Next, the effect of the reaction time on reduction of CuSe-GO was examined between 1 and 4 hours. 1 hour was enough to achieve maximum reduction, so this time was selected for following experiments. On the other hand, the CuSe@GO amounts were optimized between 0.1 – 0.5 g, and the best conditions were obtained when using 0.2 g of CuSe@GO amount.

As explained before, and to confirm the formation of the CuSe@rGO, the synthesized material was characterized by XRD and SEM (Figure 3.2.3.3). Figure 3.2.3.3B shows the XRD patterns of GO. As can be seen in this figure, the diffraction peak at ~11.38° confirmed the presence of GO. Figure 3.2.3.3B shows the XRD patterns of rGO. As can be seen in this figure, the diffraction peak at ~26.68° confirmed the presence of rGO. Moreover, Figure 3.2.3.3B shows the XRD patterns of CuSe@rGO composite. As can be seen in this figure, the diffraction peak at ~26.68° confirmed rGO. Peaks at ~26.68°, ~28.36°, ~31.64°, ~44.64°, ~52.96°, ~65.12° and ~71.78° correspond to the CuSe nanoparticles. Figure 3.2.3.3C-D shows the SEM micrographs and elemental mapping image of the synthesized CuSe@rGO. It can be seen that, the CuSe nanoparticles decorated onto the surface of rGO sheet. On the other hand, Figure 3.2.3.3E shows the elemental mapping image shows that all oxygen atoms of GO were reduced.

Voltammetric behaviour of eugenol at CuSe@rGO/GCE

The modified electrode CuSe@rGO/GCE was ﬁrst characterized by cyclic voltammetry (CV) to test their behaviour for the oxidation of eugenol, as shown in Figure 3.2.3.4. In order to realize the role of CuSe@rGO, the cyclic voltammograms of GO/GCE, rGO/GCE, CuSe/GCE and CuSe@rGO/GCE in the presence of 4.0x10-5 mol L−1 eugenol were recorded, as shown in Figure 3.2.3.4. All CV curves were obtained in the presence of (0.1 mol L−1) Britton-Robinson buffer at pH=2.0 together with 4.0x10-5 mol L−1 eugenol at a constant scan rate of 50 mV s-1.

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The CV curves observed for the GCE electrode (in the absence of GO, rGO, CuSe and CuSe@rGO/GCE (Figure 3.2.3.4). In contrast, the CV curves observed for the GO/GCE, rGO/GCE, CuSe/GCE and CuSe@rGO/GCE modified electrode exhibited two oxidation peaks and reduction peak in the presence of eugenol, within the potential window from 0.1 to 1.0 V. It suggests that the oxidation reaction of eugenol is totally reversible. The oxidation current of eugenol (0.77 V) on CuSe@rGO/GCE was higher than that at the GO/GCE, rGO/GCE and CuSe/GCE (Figure 3.2.3.4). Compared with the GO/GCE, rGO/GCE, CuSe/GCE and CuSe@rGO/GCE, a significant enhancement in the anodic current (0.77 V) was achieved at the CuSe@rGO/GCE (Figure 3.2.3.4), indicating that the high surface area and high conductivity of the CuSe@rGO/GCE increase the effective electrode area and improve the catalytic activity toward the eugenol oxidation obviously indicating that the CuSe@rGO/GCE can be used to determine eugenol.

Figure 3.2.3.4. The cyclic voltammograms of 4.0x10-5 mol L-1 eugenol in 0.1 mol L-1 Britton- Robinson buffer at pH=2.0, 50 mV s-1 scan rate. GO/GCE, rGO/GCE, CuSe/GCE and CuSe@rGO/GCE.

Optimization of experimental parameters

The electrochemical response of the sensor toward the determination of eugenol were optimized by analysing a standard solution (4.0x10-5 mol L−1) of eugenol using linear sweep voltammetry (LSV) technique. The parameters aﬀecting the determination of eugenol, such as supporting electrolyte, concentration of supporting electrolyte, pH, CuSe@rGO amount, scan rate and stability of the electrodes, were investigated.

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Different supporting electrolytes were tested, including NH4Ac, CH3COOH, H2SO4, H3BO3,

−1 H3PO4, HCl, HNO3, KCl, NaCl, NaOH and Britton-Robinson (each 0.1 mol L ). It was found that when 0.1 mol L−1 Britton-Robinson solution was used; the oxidation peak current was a higher sensitivity. For further study, 0.1 mol L−1 Britton-Robinson solution was chosen as the supporting electrolyte, Figure 3.2.3.S1. The concentration of Britton- Robinson solution was studied between 0.01 and 0.15 mol L−1. It was observed that the analytical signal increased up to a 0.1 mol L−1 concentration and then slightly decreased. Hence, 0.1 mol L−1 Britton-Robinson was selected as the supporting electrolyte, Figure 3.2.3.S2. On the other hand, the effect of 0.1 mol L−1 Britton-Robinson solution pH on the electrochemical response of the sensor toward the determination of eugenol was studied. The variations of peak current as well as the oxidation peak potential with respect to the change of the electrolyte in the pH range from 2.0 to 8.0, gradually with increasing the pH, indicating that protons have taken part in the electrode reaction processes. The relationship between the oxidation peak potential and pH is also shown in Figure 3.2.3.S3. The effect of modification amount of CuSe@rGO on the electrochemical sensor for the determination of eugenol was also studied by using LSV, different modification volumes were tested: 2.0, 4.0, 6.0, 8.0 and 10.0 μL. The 6.0 μL gave the best result of oxidation peak current sensitivity than other counterparts, Figure 3.2.3.S4. The effect of scan rate value on the oxidation peak current of 4.0x10-5 mol L−1 of eugenol by using LSV was studied. Different scan rates were tested 10, 30, 50, 70, 90, 110, 130 and 150 mV s-1. When the scan rate increased, the peak potential slightly shifted to positive value, Figure 3.2.3.S5. To investigate the stability of the modified electrode, the CuSe@rGO/GCE was prepared separately six times with the use of the same GCE, and measurements were carried out by comparing the oxidation peak current of 2.0x10-8 mol L−1 eugenol. The RSD (%) of the measurements taken from the three separately prepared electrodes were 5.39 for eugenol, which indicated that the reproducibility of the modified electrode was satisfactory.

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Interference study

The influence of selected interfering compounds such as ascorbic acid, citric acid, vitamin B2, sodium bicarbonate, glutamic acid, glycine, glucose, fructose, potassium sodium tartrate, tyrosine, sorbic acid, some metal and acid radical ions such as K+, Na+,

2+ 2+ 2+ 2+ − − − 2− − 2− 2− 3− − Ca , Fe , Mg , Zn , F , Cl , I , SO3 , NO3 , SO4 , CO3 , PO4 and CH3COO was studied. This study was carried out with a solution of 3.284 µg g-1 of eugenol prepared in Britton- Robinson buffer (0.1 mol L-1, pH 2.0). A compound was considered to interfere if a variation of more than 50% was observed in the analytical signal. All the results are shown in Table 3.2.3.1. It can be observed that no interferences were observed at the tested interferent/analyte ratios.

Table 3.2.3.1. Effect of the presence of potentially interfering compounds in the electrochemical response of eugenol. Interferents Tolerated interferent/ analyte (w/w) ratioa Ascorbic acid, citric acid, vitamin B2, sodium > 50b bicarbonate, glutamic acid, glycine, glucose, fructose, potassium sodium tartrate, tyrosine, sorbic acid and F− (Toothpaste)

+ + 2+ 2+ 2+ 2+ − − 2− − b K , Na , Ca , Fe , Mg , Zn , Cl , I , SO3 , NO3 , > 100 2− 2− 3− − SO4 , CO3 , PO4 and CH3COO a For 3284 µg Kg-1 eugenol concentration. b Maximum ratio tested.

Analytical applications

Using the previously optimized conditions, the analytical parameters of the system were examined by linear sweep voltammetry (LSV), using a scan rate of 50 mV s-1. Linear sweep voltammograms obtained for different concentrations of eugenol under the optimum conditions are shown in Figure 3.2.3.S6. A calibration graph was obtained using standard solutions of eugenol over the range 1–82 μg Kg−1. The linear range, intercept and slope of the curve are given in Table 3.2.3.2 along with the regression coefficient for eugenol. The precision of the method for aqueous standards (evaluated as the relative standard deviation obtained after analyzing 11 series of 11 replicates) was 4.1 % at the 8.2 μg Kg-1 eugenol. The limit of detection, defined as the concentration of the analyte giving a signal equivalent to the blank signal plus three times its standard deviation (3σ), was 0.4 μg Kg-1. This method shows clear advantages in terms of

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sensitivity with respect to other existing alternatives methods [17, 26]. The analytical applications for CuSe@rGO/GCE were investigated for the determination of eugenol in different real samples such as clove, cinnamon and toothpaste. The analytical applications were examined by linear sweep voltammetry under the optimized conditions described earlier. The quantitative levels of the eugenol found in clove, cinnamon and toothpaste are shown in Table 3.2.3.3. Finally, the trueness of the proposed instrumental method and potential matrix effects were studied by speaking these sample extracts with a known concentration eugenol and analyze them. Recoveries between 88.50 and 94.86 % were obtained in all cases.

Table 3.2.3.2. Analytical parameters obtained for the eugenol determination.

Parameter Eugenol

Linear dynamic range / µg Kg-1 1 – 82 Calibration curve Intercept 0.427 Slope 1.15x10-5 Correlation coefficient (R2) 0.9903 Detection limit / µg Kg-1 0.4 Quantification limit / µg Kg-1 0.7 RSD (%) (n=11)a 4.14 a 8.2 µg Kg-1

Table 3.2.3.3. Applications and recovery study of eugenol in various sample by the proposed method.

Sample Content (µg Kg-1) Found (µg Kg-1) Recovery (%) Clove 8.0 7.0 88.70 ± 3.15 10 9.0 94.86 ± 2.71

Cinnamon 10 9.0 93.35 ± 4.57 20 19 92.68 ± 3.86

Toothpaste 35 31 89.13 ± 5.58 60 53 88.50 ± 3.32 Recovery (%) data are expressed as mean ± standard deviation (SD); (n=3).

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Conclusions

The copper selenide decorated-reduced graphene oxide (CuSe@rGO) has been carried out in sc-CO2, and the obtained hybrid materials have been tested in glassy carbon electrode (GCE), as novel working electrodes for determination of eugenol in clove, cinnamon and toothpaste. In comparison with the poor response at conventional GCE, the modified GCE presented higher sensitivity, therefore demonstrating the suitability of the proposed approach to improve the sensitivity of the working electrodes in GCE. The preparation conditions for the sensor, suitable operating conditions, calibration curve, detection limit and selectivity in eugenol detection were presented and discussed. Under the optimal conditions, the presented method can provide wider linear range and lower detection limit compared with other previous procedure, Table 3.2.3.4.

Table 3.2.3.4. Comparison of analytical parameters obtained from CuSe@rGO/GCE with different electrodes in literature for electrochemical detection of eugenol. Electrode EC technique Linear range (µg Kg-1) LOD (µg Kg-1) Ref. Cu@AuNPs/GCE CV 50 – 800 41 [16] Graphene/CPE DPV 16 – 2791 1 [17] PGE DPV 49 – 8210 14 [26] Cu2O-TiNTs/GCE CV 755 – 75532 213 [27] AuNPs/CPE DPV 821 – 41050 328 [28] CuSe@rGO/GCE LSV 1 – 82 0.4 This work EC: Electrochemical; CPE: Carbon paste electrode; GCE: Glassy carbon electrode; PGE: Pencil graphite electrode; DPV: Differential pulse voltammetry; CV: Cyclic voltammetry; LSV: Linear sweep voltammetry; AuNPs: Gold nanoparticles; TiNTs: TiO2 nanotubes and Ref.: References.

Acknowledgment

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SUPPLEMENTARY INFORMATION

Figure 3.2.3.S1. Analytical signals obtained for different supporting electrolytes; 4.0×10−5 mol L−1 eugenol; 0.1 mol L−1 electrolyte concentration and 50 mV s-1 scan rate.

Figure 3.2.3.S2. Analytical signals obtained for different concentrations of Britton-Robinson buffer (0.01, 0.05, 0.1 and 0.15 mol L−1); 4.0×10−5 mol L−1 eugenol; and 50 mV s-1 scan rate.

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Figure 3.2.3.S3. Analytical signals obtained for different pH (2.0, 4.0, 6.0 and 8.0) of 0.1 mol L−1 Britton-Robinson buffer; 4.0×10−5 mol L−1 eugenol; and 50 mV s-1 scan rate.

Figure 3.2.3.S4. Analytical signals obtained for different amount of CuSe@rGO materials (2.0, 4.0, 6.0, 8.0 and 10.0 µL), 0.1 mol L−1 Britton-Robinson buffer; 4.0×10−5 mol L−1 eugenol; and 50 mV s-1 scan rate.

Figure 3.2.3.S5. The cyclic voltammograms of 4.0 × 10-5 mol L-1 eugenol in 0.1 mol L-1 Britton- Robinson buffer (pH 2.0) at scan rates (10, 30, 50, 70, 90, 110, 130 and 150 mV s-1).

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Nanoparticles in electrochemical sensors for food and pharmaceutical samples monitoring

Figure 3.2.3.S6. Linear sweep voltammograms of eugenol in 0.1 mol L-1 Britton-Robinson buffer at pH=2.0, 50 mV s-1 scan rate; concentrations of 1, 5, 10, 13, 17, 49 and 82 µg Kg-1 eugenol.

252

Conclusions / Conclusiones

4. Conclusions / Conclusiones

ith this doctoral work, we have expanded the development of new analytical methodologies by using nanosized and non-nanosized materials. W Also, the use of new extraction media has been investigated. The main outcome of this research is the design, development and assessment of tools for facilitating the simplification and automatization of alternative methodologies that are more expeditious and economical than existing choices.

The main conclusions drawn from the results of the research work described in this Report are as follows:

 New electrophoretic and chromatographic methods have been developed by using novel hybrid materials for the quantitative analysis of various compound families in a different types of samples. Specifically:

- Development of a simple analytical method for determining four types of antidepressants in human urine with capillary electrophoresis by using magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) as sorbent for the solid-phase extraction of samples prior to their processing.

- An effective analytical method for determining of catecholamines in urine and dark ventral patch hair of Iberian male red deer by magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites and their separation/detection by liquid chromatography-mass spectrometry have been developed.

- A simple, rapid, selective and sensitive monitoring method for the simultaneous determination of the widely prescribed antidepressants in just a human blood drop is here developed and validated. This methodology is based on the use of lab manufactured poly(styrene- co-divinylbenzene)-coated glass blood spot for the extraction of the analytes, and their subsequent separation and detection by capillary liquid chromatography-mass spectrometry.

253

Conclusions / Conclusiones

 Effective electrochemical methodologies using nanomaterials for qualitative and quantitative analyses of one or more analytes in the same sample have been developed. Thus:

- A new sensor based on aluminium doped titanium oxide nanoparticles, for modification of screen-printed carbon electrode for electrochemical sensing of vanillin in food samples.

- A novel approach for synthesis and supercritical fluid reduction of copper selenide-graphene oxide has been developed. To modify glassy carbon electrodes for the electrochemical detection of eugenol in various samples.

- A new electrochemical sensor for electrochemical sensing of tyrosine in pharmaceutical samples based on aluminium doped copper selenide nanoparticles, with screen-printed carbon electrodes has been developed.

254

Conclusions / Conclusiones

on este trabajo de doctorado, hemos ampliado el desarrollo de nuevas metodologías analíticas mediante el uso de materiales de tamaño nanométrico C y no nanométrico. Además, se ha investigado el uso de nuevos medios de extracción. El principal resultado de esta investigación es el diseño, desarrollo y evaluación de herramientas para facilitar la simplificación y automatización de metodologías alternativas que son más rápidas y económicas que las opciones existentes.

Las principales conclusiones extraídas de los resultados del trabajo de investigación descrito en este Informe son las siguientes:

 Se han desarrollado nuevos métodos electroforéticos y cromatográficos utilizando nuevos materiales híbridos para el análisis cuantitativo de varias familias de compuestos en diferentes tipos de muestras. Específicamente, tenemos - Desarrollo de un método analítico simple para determinar cuatro tipos de antidepresivos en orina humana con electroforesis capilar mediante el uso de nanotubos de carbono magnéticos de paredes múltiples poli(estireno-co-divinilbenceno) como sorbente para la extracción en fase sólida de muestras antes de su procesamiento.

- Se ha desarrollado un método analítico eficaz para la determinación de catecolaminas en orina y parches ventrales oscuros del venado rojo macho ibérico mediante nanocubos de carbono magnéticos de pared múltiple poli(estireno-co-divinilbenceno) compuestos y su separación/detección mediante cromatografía líquida- espectrometría de masas.

- Se ha desarrollado y validado un método de monitoreo simple, rápido, selectivo y sensible para la determinación simultánea de los antidepresivos ampliamente prescritos en una gota de sangre humana. Esta metodología se basa en el uso de manchas de sangre de vidrio recubierto de poli (estireno-co-divinilbenceno) fabricadas en el laboratorio para la extracción de los analitos y su posterior separación

255

Conclusions / Conclusiones

y detección mediante cromatografía líquida capilar-espectrometría de masas.  Se han desarrollado varios métodos electroquímicos efectivos utilizando nanomateriales para el análisis cualitativo y cuantitativo de uno o más analitos en la misma muestra. Así: - Un nuevo sensor basado ha sido empleado en nanopartículas de óxido de titanio dopado con aluminio, para la modificación de electrodos de carbono serigrafiado para la detección electroquímica de vainillina en muestras de alimentos.

- Se ha llevado a cabo un enfoque novedoso para la síntesis y reducción mediante fluidos supercríticos del seleniuro de cobre-óxido de grafeno. Los materiales compuestos preparados se utilizaron para modificar electrodos de carbono serigrafiados para la detección electroquímica de eugenol en varias muestras.

- Se ha desarrollado un nuevo sensor electroquímico para la detección electroquímica de tirosina en muestras farmacéuticas basadas en nanopartículas de seleniuro de cobre dopado con aluminio, con electrodos de carbono serigrafiados.

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Scientific Self-assessment / Autoevaluación científica

5. Scientific Self-assessment / Autoevaluación científica

his section presents a critical overall analysis of the outcome of this doctoral work as regards the advantages of the new methodologies, and the potential T deficiencies and limitations of our research work. Note that this Doctoral Thesis has contributed with a total of 7 scientific articles (3 of them already published) as well as a variety of communications in national and international conferences. The most immediate contribution of this work is the development of new electrophoretic and chromatographic methods for determining antidepressants and catecholamines by using (nano)materials. The need for little sample treatment and the operational simplicity of the detectors constitute two remarkable advantages over existing choices. In addition, using carbon nanotubes as a solid phase extraction sorbent material allows samples to be treated in a single step. The sorbents nanomaterials developed for the extraction of different analytes have been applied off-line. Perhaps, a future progress in this aspect would be the development of extraction in magnetic solid phase on-line, because its advantages such as: shorter time of sample preparation, the risk of contamination is reduced, improvement in the precision and the operators do not are exposed to infectious materials. Although the resulting detection limits are not especially low, they are quite acceptable for analysing the sample types addressed in this work in accordance with existing regulations and the client’s needs. The inability to quantify minor compounds in the sample types studied here and in other matrices, might be overcome by using microextraction techniques affording high preconcentration factors and a high selectivity in order to circumvent this analytical shortcoming of universal detectors. For example, it would be interesting to compare the response of CE with LC to minor components with a view to assessing their performance at low analyte concentration levels, as well as use different detectors instead of diode array detector (DAD) and Mass spectrometry, such as tandem mass spectrometry (MS/MS).

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Scientific Self-assessment / Autoevaluación científica

We have also developed a simple analytical methodology applied to blood samples. In this work, we have improved selectivity, one of the basic properties of Analytical Chemistry, by using lab manufactured poly(styrene-co-divinylbenzene)-coated glass blood spot prior to introduction of the analytes into LC-MS. The developed method for the extraction of different analytes have been applied off-line. Consequently, the next step could be the development of an on-line approach for the application and its coupling with more selective and sensitive detectors (e.g. MS/MS). The analytical methodologies proposed to carry out a possible analysis method of antidepressants in human urine and blood, they did not apply to real samples, so it would be convenient to carry out studies of this kind of methodologies in real matrices. Also, we have successfully expanded the electrochemical scope by using NMs to enable the analysis of samples by electrochemistry. Electrochemical sensors have more advantage over the others because; the electrodes can sense the materials which are present within the system without damage it. Moreover, low cost and involve time saving sample pre-treatment processes. On the other hand, some electrodes have short electrode lifetime, poor inter-electrode reproducibility and not being user-friendly. Finally, copper selenide-reduced graphene oxide composites (CuSe@rGO) would be interesting to assess the usefulness of CuSe@rGO composites with samples of clinical or environmental interest with a view to the overall analysis of contaminants.

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Scientific Self-assessment / Autoevaluación científica

sta sección presenta un análisis general crítico del resultado de este trabajo de doctorado con respecto a las ventajas de las nuevas metodologías y las posibles E deficiencias y limitaciones de nuestro trabajo de investigación. Ha de tenerse en cuenta que esta Tesis Doctoral ha contribuido con un total de 7 artículos científicos (3 de ellos ya publicados), así como una variedad de comunicaciones en conferencias nacionales e internacionales. La contribución más inmediata de este trabajo es el desarrollo de nuevos métodos electroforéticos y cromatográficos para determinar antidepresivos y catecolaminas utilizando nanomateriales y no nanomateriales. La necesidad de un pequeño tratamiento de la muestra y la simplicidad operativa de los detectores constituyen dos ventajas notables sobre las opciones existentes. Además, el uso de nanotubos de carbono como material sorbente de extracción en fase sólida permite tratar muestras en un solo paso. Los nanomateriales sorbentes desarrollados para la extracción de diferentes analitos se han aplicado fuera de línea. Quizás, un progreso futuro en este aspecto sería el desarrollo de la extracción en fase sólida magnética en línea, debido a sus ventajas tales como: menor tiempo de preparación de la muestra, reducción del riesgo de contaminación, mejora de la precisión y los operadores no están expuestos a materiales infecciosos. Aunque los límites de detección resultantes no son especialmente bajos, son bastante aceptables para analizar los tipos de muestras abordados en este trabajo de acuerdo con las regulaciones existentes y las necesidades del cliente. La incapacidad para cuantificar compuestos menores en los tipos de muestras estudiados aquí y en otras matrices, podría superarse mediante el uso de técnicas de microextracción que ofrezcan altos factores de preconcentración y una alta selectividad para evitar este defecto analítico de detectores universales. Por ejemplo, sería interesante comparar la respuesta de CE con LC a componentes menores con el fin de evaluar su desempeño a niveles de concentración de analito bajos, así como usar detectores diferentes en lugar de detectores de red de diodos (DAD) y espectrometría de masas, como la espectrometría de masas en tándem (MS/MS).

259

Scientific Self-assessment / Autoevaluación científica

También hemos desarrollado una metodología analítica simple aplicada a muestras de sangre. En este trabajo, hemos mejorado la selectividad, una de las propiedades básicas de la química analítica, mediante el uso de manchas de sangre en vidrio recubierto de poli (estireno-co-divinilbenceno) fabricado en el laboratorio antes de la introducción de los analitos en el LC-MS. El método desarrollado para la extracción de diferentes analitos se ha aplicado fuera de línea. En consecuencia, el siguiente paso podría ser el desarrollo de un enfoque en línea para la aplicación y su acoplamiento con detectores más selectivos y sensibles (por ejemplo, MS/MS). Las metodologías analíticas propuestas para llevar a cabo un posible método de análisis de antidepresivos en orina y sangre humana no se aplicaron a muestras reales, por lo que sería conveniente realizar estudios de este tipo de metodologías en matrices reales.

Además, hemos ampliado con éxito el alcance electroquímico mediante el uso de NM para permitir el análisis de muestras mediante electroquímica. Los sensores electroquímicos tienen más ventajas sobre los demás porque; Los electrodos pueden detectar los materiales que están presentes en el sistema sin dañarlo. Además, los procesos de pretratamiento de muestras de bajo coste - implican un ahorro de tiempo. Por otro lado, algunos electrodos tienen una vida útil corta, poca reproducibilidad entre los electrodos y no son fáciles de usar. Finalmente, los compuestos de óxido de grafeno reducido con seleniuro de cobre (CuSe@rGO) sería interesante para evaluar la utilidad de los compuestos de CuSe@rGO con muestras de interés clínico o ambiental con vistas al análisis general de los contaminantes.

260

Annexes

Annexes / Anexos

Annexe I. Figure captions / Índice de figuras

Annexe II. Table captions / Índice de tablas

Annexe III. Publications / Publicaciones

Annexe IV. Participation of conference papers (oral communications and posters) / Participación en ponencias (comunicaciones orales y pósters).

Annexe V. Curriculum vitae

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Figure captions / Índice de figuras

Annexe I. Figure captions / Índice de figuras

Figure 1.1.1 Global aims and objectives of Analytical Chemistry. 5 Figure 1.1.2 The analytical process of chemical analysis. 7 Figure 1.1.3 Potential benefits derived from the miniaturization of the 8 different steps of the analytical process. Figure 1.1.4 The role of (nano)materials in Analytical Science. 10 Figure 1.2.1.1 Use of (nano)materials in sample preparation. 12 Figure 1.2.1.2 Scheme of the two approaches employed in the fabrication 14 of nanomaterials: “top-down” and “bottom-up.” Figure 1.2.1.3 Main types of carbon-based nanomaterials. 17 Figure 1.3.1 Use of nanomaterials for sample detection. 27 Figure 2.2.1 Illustration of the spectrophotometer UVI-Vis “Uvi Light & 48 UVIKON XS”. Figure 2.2.2 Illustration of the capillary electrophoresis “Agilent 49 G1600AX”. Figure 2.2.3 System scheme of Agilent 1200 HPLC. Figure 2.2.4 Illustration of Agilent 6110 Series LC/MSD instrument. 50 Figure 2.2.5 Illustration of CH Instruments. 51 Figure 2.2.6 Illustration of Wuhan Corrtest Instruments. 51 Figure 2.2.7 Conventional system of three-electrodes (working, 52 reference and auxiliary) used in electrochemical measurements. Figure 2.2.8 Illustration of the screen-printed electrode and its cable 52 connector. Figure 2.2.9 Analytical lab-scale supercritical fluid system. 53 Figure 2.2.10 Illustration of the portable Raman spectrometer model 54 B&W TEK, known as i-Raman BWS415 and BAC151B microscope. Figure 2.2.11 Illustration of Zetasizer Nano instrument model ZEN3600. 54

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Figure captions / Índice de figuras

Figure 2.2.12 Illustration of FT-IR spectroscope instrument model FT/IR 55 4200.

Figure 2.2.13 Mounting used in the synthesis of Al-NPs and Na2SeSO3. 56 Figure 2.3.1.1 Scheme of synthesis and preparation of MMWCNTs. 58 Figure 2.3.1.2 Scheme of synthesis of poly(styrene-co-divinylbenzene). 59 Figure 2.3.1.3 Scheme of functionalization and decoration of MMWCNTs 59 with poly(styrene-co-divinylbenzene).

Figure 2.3.1.4 Scheme of synthesis of Al-TiO2-NPs. 60 Figure 2.3.1.5 Schematic synthesis of copper selenide-reduced graphene 61 oxide in supercritical fluids medium. Figure 2.3.1.6 Scheme of synthesis of Al-CuSe-NPs. 62 Figure 2.3.2.1 Schematic representation of steps required for the MSPE 63 procedure. Figure 2.3.2.2 Scheme of the general procedure for the synthesis and 65 extraction of the selected antidepressants by PS-DVB- coated glass blood spot.

Figure 2.3.2.3 Scheme of preparation the modified SPCEs by Al-TiO2-NPs 65 and Al-CuSe-NPs. Figure 2.3.2.4 Scheme of preparation the modified GCE by CuSe@rGO. 66 Figure 3.1.1.1 Nanomaterials used in antidepressants extraction. 80

Figure 3.1.1.2 Antidepressants extraction mechanism using different 81 nanomaterials.

Figure 3.1.1.3 Different nanomaterials currently used for antidepressants 82 extraction.

Figure 3.1.2.1 Evaluation of the sorption capacity of MMWCNT, P1, P2, P3, 129 MP1, MP2 and MP3 sorbents.

Figure 3.1.2.2 Electropherograms of a 1.0 µg/mL mixture of standard 132 solutions of the antidepressants (A); blank urine sample without waterwash step (B); washed by water (C) and of

263

Figure captions / Índice de figuras

urine spiked with 0.20 µg/mL of each antidepressant (D). Conditions: capillary (50 cm length × 75 µm i.d.); capillary temperature 20°C; hydrodynamic injection of 10 s; applied voltage 20 kV; buffer 50 mM borax with 20% methanol at pH 9.30; detection at 200 nm.

Figure 3.1.2.S1 Fourier transform infrared (A) and Raman spectra (B) of 141 MMWCNT, Poly(STY-DVB) and MMWCNT- Poly(STY-DVB) composite.

Figure 3.1.2.S2 Micrographs obtained by TEM of the magnetic 141 nanoparticles attached onto the surface of the nanotubes (A), spherical poly(STY-DVB) particles (B), and MMWCNT- poly(STY-DVB) composite material (C) and (D).

Figure 3.1.2.S3 Particle size distribution (A) and zeta potential (B) of 141 MMWCNT-poly(STY-DVB) composite material.

Figure 3.1.3.1 Characterization of the MMWCNT-poly(STY-DVB) 153 composite by means of (A) FT-IR spectra, (B) Raman spectrum, (C) TEM micrograph, (D) Particle size distribution and (E) Zeta-potential.

Figure 3.1.3.2 LC-MS chromatograms of a blank human urine (yellow), 2 154 µg mL-1 standard solution of the selected catecholamines (blue) and 0.5 µg mL-1 spiked human urine sample extracted using MMWCNT-poly(STY-DVB) sorbent (green). Peak identification: DHMA (1), DOPEG (2), NE (3), EP (4) and DA (5).

Figure 3.1.3.3 LC-MS chromatograms of: (a) a 10 μg mL-1 standard solution 157 of DA, NE, EP, DHMA and DOPEG (red), a 1:100 diluted urine sample from a wild Iberian male red deer (black), and one extracted wild Iberian male red deer urine sample using MMWCNT-poly(STY-DVB) sorbent (green); (b) extracts from

264

Figure captions / Índice de figuras

deer hair of the dark ventral patch (black), and extract of red deer hair of the dark ventral patch using MMWCNT- poly(STY-DVB) sorbent (red). Additional peaks in (b) different from that of DHPG are of unknown origin. Peak identification: DHMA (1), DOPEG (2), NE (3), EP (4) and DA (5).

Figure 3.1.3.S1 Metabolic pathway of catecholamines from precursor 164 dopamine [2].

Figure 3.1.4.1 Scheme of the general procedure for the synthesis and 174 extraction of the selected antidepressants by PS-DVB- coated glass blood spot.

Figure 3.1.4.2 Characterization of PS-DVB by (a) FT-IR spectra, (b) Raman 177 spectrum, (c) TEM micrograph and (d) particle size distribution.

Figure 3.1.4.3 LC-MS chromatograms of 1.00 μg mL−1 standard solution of 178 each antidepressant analyzed (a), 0.25 μg mL−1 spiked blood sample extracted using PS-DVB (b), 0.25 μg mL−1 spiked blood sample extracted without using PS-DVB (c), and blank blood sample (d). Peak identification; 1: mirtazapine, 2: bupropion, 3: trazodone, 4: citalopram, 5: paroxetine, 6: fluoxetine and 7: agomelatine.

Figure 3.1.4.S1 Optimization of the amount of PS-DVB coated on the 188 surface of the glass substrate. Figure 3.1.4.S2 pH optimization for the extraction of the selected 188 antidepressants. 10 g of the PS-DVB was applied on the surface of the coated glass. Figure 3.1.4.S3 Analysis of different amounts of blood sample utilized for 188 the proposed methodology.

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Figure captions / Índice de figuras

Figure 3.2.1.1 TEM and EDX micrographs of the two precursors: (a and c) 199

Al-NPs and (b and d) Al-TiO2-NPs.

Figure 3.2.1.2 XRD patterns measured for different NPs: (a) Al-NPs and 200

(b) Al-TiO2-NPs.

Figure 3.2.1.3 Cyclic voltammograms of different electrodes: (a) blank, (b) 201

SPCE, (c) TiO2-NPs/SPCE, and (d) Al-TiO2-NPs/SPCE, at 250

mM vanillin solution in 0.1 M H3PO4.

Figure 3.2.1.S1 The relationship between the oxidation peak potential and 208

pH, for Al-TiO2-NPs/SPCE electrode in 0.1M H3PO4 buffer with different pH values: 1.41, 1.66, 2.11, 3.40, 4.92, 5.69, 6.0 and 6.31.

Figure 3.2.1.S2 The accumulation step of 10 µM vanillin in 0.1M H3PO4 208 solution. After 120 second was performed under 0.0 V, scan rate 50 mV s-1.

Figure 3.2.1.S3 The stability of Al-TiO2-NPs/SPCE modified electrode with 209

eight segments, at 250 µM vanillin solution in 0.1M H3PO4.

Figure 3.2.2.1 XRD patterns of (a) Al-NPs and (b) Al-CuSe-NPs. 217

Figure 3.2.2.2 a) SEM micrographs of Al-CuSe-NPs and b) elemental 218 mapping image of Al-CuSe-NPs.

Figure 3.2.2.3 The cyclic voltammograms of 20 µM L-tyrosine in 0.1 M 218

H3PO4 buffer at pH=6.0, 50 mV/s scan rate of blank (0.1M

H3PO4), bare SPCE, Al-NPs/SPCE and Al-CuSe-NPs/SPCE.

Figure 3.2.2.S1 Analytical signals obtained for different supporting 228 electrolytes; 20 µM L-tyrosine; 0.1M electrolyte concentration and 50 mV/s scan rate.

Figure 3.2.2.S2 Analytical signals obtained for different concentrations of 228

H3PO4 (0.05, 0.1 and 0.15 M); 20 µM L-tyrosine; and 50 mV/s scan rate.

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Figure captions / Índice de figuras

Figure 3.2.2.S3 Analytical signals obtained for different pH (2, 4, 6, 8 and 229

10) of 0.1M H3PO4; 20 µM L-tyrosine; and 50 mV/s scan rate.

Figure 3.2.2.S4 Analytical signals obtained for different amount of Al-CuSe- 229

NPs materials (2.0, 4.0, 6.0 and 8.0 µL), 0.1M H3PO4; 20 µM L-tyrosine; and 50 mV/s scan rate.

Figure 3.2.2.S5 The accumulation step of 20 µM L-tyrosine in 0.1M H3PO4 230 solution. After 60 min (3600 s) was performed under 0.0 V, scan rate 50 mV/s.

Figure 3.2.2.S6 The stability of Al-CuSe-NPs/SPCE modified electrode with 230

eight segments, at 20 µM L-tyrosine solution in 0.1M H3PO4.

-3 -1 Figure 3.2.3.2 The cyclic voltammograms of 1.0x10 mol L of K3Fe(CN)6 239 in 0.1 mol L-1 KCl, 50 mV s-1 scan rate. GCE, GO/GCE and CuSe@rGO/GCE. Figure 3.2.3.3 (A) Raman spectrum of GO, rGO and CuSe@rGO, (B) XRD 241 patterns of GO, rGO and CuSe@rGO, (C and D) SEM micrographs of CuSe@rGO and (E) elemental mapping images of CuSe@rGO composite.

Figure 3.2.3.4 The cyclic voltammograms of 4.0x10-5 mol L-1 eugenol in 0.1 243 mol L-1 Britton-Robinson buffer at pH=2.0, 50 mV s-1 scan rate. GO/GCE, rGO/GCE, CuSe/GCE and CuSe@rGO/GCE.

Figure 3.2.3.S1 Analytical signals obtained for different supporting 251 electrolytes; 4.0×10−5 mol L−1 eugenol; 0.1 mol L−1 electrolyte concentration and 50 mV s-1 scan rate. Figure 3.2.3.S2 Analytical signals obtained for different concentrations of 251 Britton-Robinson buffer (0.01, 0.05, 0.1 and 0.15 mol L−1); 4.0×10−5 mol L−1 eugenol; and 50 mV s-1 scan rate. Figure 3.2.3.S3 Analytical signals obtained for different pH (2.0, 4.0, 6.0 and 252 8.0) of 0.1 mol L−1 Britton-Robinson buffer; 4.0×10−5 mol L−1 eugenol; and 50 mV s-1 scan rate.

267

Figure captions / Índice de figuras

Figure 3.2.3.S4 Analytical signals obtained for different amount of 252 CuSe@rGO materials (2.0, 4.0, 6.0, 8.0 and 10.0 µL), 0.1 mol L−1 Britton-Robinson buffer; 4.0×10−5 mol L−1 eugenol; and 50 mV s-1 scan rate. Figure 3.2.3.S5 The cyclic voltammograms of 4.0 × 10-5 mol L-1 eugenol in 252 0.1 mol L-1 Britton-Robinson buffer (pH 2.0) at scan rates (10, 30, 50, 70, 90, 110, 130 and 150 mV s-1). Figure 3.2.3.S6 Linear sweep voltammograms of eugenol in 0.1 mol L-1 253 Britton-Robinson buffer at pH=2.0, 50 mV s-1 scan rate; concentrations of 1, 5, 10, 13, 17, 49 and 82 µg Kg-1 eugenol.

268

Table captions / Índice de tablas

Annexe II. Table captions / Índice de tablas

Table 1.3.1 Contributions of the NMs at analytical detection step. 27 Table 2.1.1 Compounds used in the experimental work. 44-46 Table 3.1.1.1 Classification of the main types of antidepressants. 77

Table 3.1.1.2 Isotherm and kinetic models of different nano-sorbents 80 used for antidepressants extraction form biological species.

Table 3.1.1.3 Efficiency of the extraction of antidepressants from 83-86 biological specimens by using different nanomaterials. Table 3.1.2.1 Calibration data and validation parameters obtained for the 131 developed methodology and the commercial sorbent (StrataTM–X) method.

Table 3.1.2.2 Precision values calculated for the proposed methodology. 133 Analyses were performed by triplicate in all cases.

Table 3.1.2.3 Accuracy results obtained for the analysis of human urine 133- samples spiked with different concentrations of the 134 antidepressants, extracted either by poly(STY-DVB)- MMWCNT or with a Strata-XTM commercial sorbent

Table 3.1.2.S1 Composition of the polymerization mixtures assayed for the 139 methodology developed.

Table 3.1.2.S2 Comparison of several analytical features obtained for the 140 proposed methodology with previous methodologies reported for UV-Vis determination, in human urine, of the antidepressant(s) included in this work.

Table 3.1.3.1 Calibration data and precision data obtained for the 155 developed methodology. Table 3.1.3.2 Extraction recovery and stability results calculated for red 156 deer urine analyzed with the proposed methodology.

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Table captions / Índice de tablas

Table 3.1.3.3 Accuracy results obtained in deer urine samples with 157 different concentrations of catecholamines. Table 3.1.3.S1 Concentration of the catecholamines determined at the 164- analysis of Iberian male red deer urine and dark ventral 165 patch hair. Table 3.1.4.1 Calibration data and figures of merit obtained for the 180 proposed method.

Table 3.1.4.2 Precision values calculated for the antidepressants to be 180 analyzed by the proposed methodology.

Table 3.1.4.3 Accuracy results calculated for blood samples spiked with 181 different concentrations of the antidepressants analyzed. Table 3.1.4.3 Matrix effects, extraction recovery and stability results 182 calculated for the proposed methodology. Table 3.2.1.1 Analytical parameters of electrochemical detection of 203

vanillin by aluminium doped TiO2 nanoparticles-modified screen printed carbon electrode.

Table 3.2.1.2 Comparison of different electrochemical electrodes for 204 vanillin determination.

Table 3.2.1.3 Interference study in the detection of vanillin by aluminum 204

doped TiO2 nanoparticles-modified screen printed carbon electrode.

Table 3.2.1.4 Analysis of vanilla extract samples by the proposed and 205 HPLC-DAD [30] methods.

Table 3.2.2.1 Effect of the presence of potentially interfering 220 compounds in the electrochemical response of L-tyrosine. Table 3.2.2.2 Analytical parameters obtained for the L-tyrosine 221 determination. Table 3.2.2.3 Comparison of the proposed sensor for determination of L- 221 tyrosine with other methods.

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Table captions / Índice de tablas

Table 3.2.2.4 Determination results and recovery study of L-tyrosine in 222 pharmaceutical sample by the proposed method. Table 3.2.3.1 Effect of the presence of potentially interfering compounds 245 in the electrochemical response of eugenol.

Table 3.2.3.2 Analytical parameters obtained for the eugenol 246 determination.

Table 3.2.3.3 Applications and recovery study of eugenol in various 246 sample by the proposed method.

Table 3.2.3.4 Comparison of analytical parameters obtained from 247 CuSe@rGO/GCE with different electrodes in literature for electrochemical detection of eugenol.

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Annexe III. Publications

Annexe III. Publications / Publicaciones

1. Development of an Aluminium Doped TiO2 Nanoparticles-modified Screen Printed Carbon Electrode for Electrochemical Sensing of Vanillin in Food Samples Khaled Murtada, Shehdeh Jodeh, Mohammed Zougagh, Ángel Ríos Electroanalysis 30 (2018) 969–974

2. Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites and separation by capillary electrophoresis Khaled Murtada, Fernando de Andrés, Ángel Ríos, Mohammed Zougagh Electrophoresis 39 (2018) 1808–1815

3. A simple poly(styrene-co-divinylbenzene)-coated glass blood spot method for monitoring of seven antidepressants using capillary liquid chromatography- mass spectrometry. Khaled Murtada, Fernando de Andrés, Ángel Ríos, Mohammed Zougagh Talanta 188 (2018) 772–778

4. Decoration of Graphene oxide with Copper Selenide in Supercritical Carbon Dioxide Medium as a Novel Approach for Electrochemical Sensing of Eugenol in Various Samples Khaled Murtada, Virginia Moreno, Ángel Ríos, Mohammed Zougagh Submitted to The Journal of Supercritical Fluids.

5. Magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites for the extraction and LC-MS determination of catecholamines and related compounds in red deer urine and hair Khaled Murtada, Fernando de Andrés, Ismael Galván, Mohammed Zougagh, Ángel Ríos Submitted to Journal of Chromatography A

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Annexe III. Publications

6. Strategies for Antidepressants Extraction from Biological Specimens Using Nanotechnology: A Review Khaled Murtada, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos Submitted to xxxxxx

7. A Sensitive Electrochemical Sensor Based on Aluminium Doped Copper Selenide Nanoparticles-modified Screen Printed Carbon Electrode for Determination of L-tyrosine in Pharmaceutical Samples Khaled Murtada, Mohammed Zougagh, Ángel Ríos Submitted to xxxxxx

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Annexe IV. Participation of conference papers (oral communications and posters)

Annexe IV. Participation of conference papers (oral communications and posters) / Participación en ponencias (comunicaciones orales y pósters).

Oral communication

A simple poly(styrene-co-divinylbenzene)-coated glass blood spot method for monitoring of seven antidepressants using capillary liquid chromatography-mass spectrometry

Khaled Murtada, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos. XII Young Science Symposium, Castilla-La Mancha University, Ciudad Real (Spain), June- 2018.

Fabrication of nano-aluminum/titanium dioxide modified screen printed carbon electrode for electrochemical detection of vanillin in food samples Khaled Murtada, Mohammed Zougagh, Ángel Ríos. VIII International Congress on Analytical Nanoscience and Nanotechnology (NyNA 2017), Barcelona (Spain), July-2017.

Fabrication of nanoalumin/titanium dioxide modified screen printed carbon electrode for electrochemical detection of vanillin in food samples Khaled Murtada, Mohammed Zougagh, Ángel Ríos. XI Young Science Symposium, Castilla-La Mancha University, Ciudad Real (Spain), June- 2017.

Poster communication

A Green Approach to the Synthesis of Reduced Graphene Oxide Decorated Copper Selenide Nanoparticles for Electrochemical Sensing of Eugenol in Food Samples Khaled Murtada, Virginia Moreno, Mohammed Zougagh, Ángel Ríos. VIII Jornadas Doctorales de la Universidad de Castilla-La Mancha, Cuenca (Spain), October-2018.

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Annexe IV. Participation of conference papers (oral communications and posters)

A Sensitive Electrochemical Sensor Based on Aluminium Doped CuSe Nanoparticles- modified Screen Printed Carbon Electrode for Determination of L-tyrosine in Pharmaceutical Samples Khaled Murtada, Mohammed Zougagh, Ángel Ríos. The 2nd International Symposium on Materials, Electrochemistry and Environment (CIMEE 2018), Lebanese University, Tripoli (Lebanon), October-2018.

Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styreneco-divinylbenzene) composites and separation by capillary electrophoresis Khaled Murtada, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos. 1er Congreso sobre Materiales Multifuncionales para Jóvenes, Universidad de Granada, Granada (Spain), September-2018.

Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites and separation by capillary electrophoresis Khaled Murtada, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos. VII Jornadas Doctorales de la Universidad de Castilla-La Mancha, Albacete (Spain), Novermber-2017.

Synthesis of novel poly(styrene-co-divinylbenzene) with magnetic nanoparticles for urinary determination of antidepressants by capillary electrophoresis Khaled Murtada, Fernando de Andrés, Mohammed Zougagh, Ángel Ríos. XXVII International Symposium on Pharmacutical and Biomedical Analysis (PBA 2017), San Pablo CEU University, Madrid (Spain), July-2017.

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Annexe V. Curriculum Vitae

Khaled Ali Murtada Licenciado en Química Burqin, Jenin (Palestine) [email protected]

Formación Académica

Licenciado en Química Nablus – Palestine An-Najah National University, 2012.

Master en Química Nablus – Palestine An-Najah National University, 2014.

Estancias en centros de I+D+i públicos o privados

Entidad de realización: Escuela Nacional de Ciencias Aplicadas (ENSA), Universidad de IBN ZOHR (Agadir, Marruecos). Duración: 01/07/2018 – 08/10/2018. Tareas contrastables: Determinación electroquímica del aminoácido tirosina en muestras farmacéuticas.

Participación en Proyectos de I+D+i financiados en convocatorias competitivas de Administraciones o entidades públicas y privadas

Proyecto: “Metodologías analíticas basadas u orientadas a los nanomateriales en los campos ambiental, alimentario y bioanalítico (CTQ2016-78793-P)”. Financiación: Ministerio de Economía y Competitividad. Entidad de realización: Universidad de Castilla-La Mancha, Ciudad Real, Castilla-La Mancha, España. Investigador principal: Ángel Ríos Castro.

Proyecto: “Nanometrología analítica aplicada al campo alimentario (SBPLY/17/180501/000262)”. Financiación: Consejería de Educación y Ciencia de la J.CC. Castilla – La Mancha. Entidad de realización: Universidad de Castilla-La Mancha, Ciudad Real, Castilla-La Mancha, España. Investigador principal: Ángel Ríos Castro. Duración: 2018-2021.

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Producción Científica Publicaciones

Título: “Copper selenide film electrodes prepared by combined electrochemical/chemical bath depositions with high photo-electrochemical conversion efficiency and stability”. Autores: Ahed Zyoud, Khaled Murtada, Hansang Kwon, Hyun-Jong Choi, Tae Woo Kim, Mohammed H.S. Helal, Maryam Faroun, Heba Bsharat, Dae hoon PARK, Hikmat Hilal. Revista: Solid State Sciences 75 (2018) 53–62.

Título: “Development of an Aluminium Doped TiO2 Nanoparticles-Modified Screen Printed Carbon Electrode for Electrochemical Sensing of Vanillin in Food Samples”. Autores: Khaled Murtada, Shehdeh Jodeh, Mohammed Zougagh and Angel Rios. Revista: Electroanalysis 30 (2018) 969–974.

Título: “Combined Electrochemical/Chemical Bath Deposited Metal Selenide Nano-film Electrodes with High Photo-electrochemical Characteristics”. Autores: Hikmat S. Hilal, Ahed Zyoud, Khaled Murtada, Nour Nayef, Mohammed H.S. Helal, Naser Qamhieh Naser Qamhieh, AbdulRaziq HajiMohideed. Revista: IEEE Xplore Digital (2018) 140–142.

Título: “Determination of antidepressants in human urine extracted by magnetic multiwalled carbon nanotube poly(styrene-co-divinylbenzene) composites and separation by capillary electrophoresis”. Autores: Khaled Murtada, Fernando de Andrés, Ángel Ríos, Mohammed Zougagh. Revista: Electrophoresis 39 (2018) 1808–1815.

Título: “A simple poly(styrene-co-divinylbenzene)-coated glass blood spot method for monitoring of seven antidepressants using capillary liquid chromatography-mass spectrometry”. Autores: Khaled Murtada, Fernando de Andrés, Ángel Ríos, Mohammed Zougagh. Revista: Talanta 188 (2018) 772–778.

Título: “Magnetic multi-walled carbon nanotube poly(styrene-co-divinylbenzene) for propranolol extraction and separation by capillary electrophoresis”. Autores: Carlos Adelantado, Khaled Murtada, Ángel Ríos, Mohammed Zougagh. Revista: Bioanalysis 10 (2018) 1193-1205.

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Título: “Unprecedented high catecholamine production causing hair pigmentation after urinary excretion in red deer”. Autores: Ismael Galván, Francisco Solano, Mohammed Zougagh, Fernando de Andrés, Khaled Murtada, Ángel Ríos, Eva de la Peña, and Juan Carranza. Revista: Cellular and Molecular Life Sciences 76 (2019) 397-404.

Trabajos presentados en congresos nacionales o internacionales

Título: “A Green Approach to the Synthesis of Reduced Graphene Oxide Decorated Copper Selenide Nanoparticles for Electrochemical Sensing of Eugenol in Food Samples”. Autores: Khaled Murtada, Virginia Moreno, Mohammed Zougagh and Ángel Ríos. Participación: Póster. Evento: VIII Jornadas Doctorales de la Universidad de Castilla-La Mancha, Cuenca, España, Octubre 2018.

Título: “A simple poly(styrene-co-divinylbenzene)-coated glass blood spot method for monitoring of seven antidepressants using capillary liquid chromatography-mass spectrometry”. Autores: Khaled Murtada, Fernando de Andrés, Mohammed Zougagh and Ángel Ríos. Participación: Comunicación oral. Evento: XII Simposio Ciencia Joven de la Universidad de Castilla-La Mancha, Ciudad Real, España, Junio 2018.

Título: “Determination of antidepressants in human urine extracted by magnetic multi-walled carbon nanotubes poly(styrene-co-divinylbenzene) composites and separation by capillary electrophoresis”. Autores: Khaled Murtada, Fernando de Andrés,, Mohammed Zougagh and Ángel Ríos. Participación: Póster. Evento: VII Jornadas Doctorales de la Universidad de Castilla-La Mancha, Albacete, España, Noviembre 2017.

Título: “Fabrication of nanoalumina/titanium-modified screen printed carbon electrode for electrochemical detection of vanillin in food samples”. Autores: Khaled Murtada, Mohammed Zougagh and Ángel Ríos. Participación: Comunicación oral. Evento: VIII International Congress on Analytical Nanoscience and Nanotechnology (NyNA), Barcelona, España, Julio 2017.

Título: “Fabrication of nanoalumina/titanium-modified screen printed carbon electrode for electrochemical detection of vanillin in food samples”. Autores: Khaled Murtada, Mohammed Zougagh and Ángel Ríos. Participación: Comunicación oral. Evento: XI Simposio Ciencia Joven de la Universidad de Castilla-La Mancha, Ciudad Real, España, Junio 2017.

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Formación especializada

Título: Formación teórica y práctica en el manejo de un espectrómetro de masas con plasma acoplado inductivamente “ICP-MS modelo Agilent 7900x acoplado a técnicas de separación de electroforesis capilar y HPLC”. Entidad organizadora: ISC Science Fecha: Abril 2018 Duración: 16 h.

Título: Formación teórica y práctica en el manejo de un espectrómetro de masas con plasma acoplado inductivamente “ICP-MS modelo Agilent 7900”. Entidad organizadora: ISC Science Fecha: Noviembre 2017 Duración: 16 h.

Título: Aprendizaje para el uso del equipo Zetasizer Nano ZS (potencial Z y tamaño). Entidad organizadora: Instrumentación Específica de Materiales (IESMAT). Fecha: Noviembre 2017 Duración: 7 h.

Título: “Técnicas de Espectroscopía y Aplicaciones en Nanotecnología”. Entidad organizadora: Sociedad de Espectroscopía Aplicada. Fecha: Septiembre 2017 Duración: 24 h.

Título: “Avances en la Agricultura Ecológica en el Área Mediterránea”. Entidad organizadora: Instituto Agronómico Mediterráneo de Bari (MIBA). Fecha: Septiembre – December 2014 Duración: 76 días.

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