Desenvolvimento e procedimentos de validação de uma metodologia analítica por GC/MS/MS para a determinação de antidepressivos em sangue total

A depressão é uma das doenças de foro psiquiátrico que mais prevalece na nossa sociedade, subsistindo evidências epidemiológicas que indicam um aumento substancial da sua incidência nos últimos anos. Esta evidência é consubstanciada pelo aumento significativo do consumo de antidepressivos em Portugal. Este cenário pressupõe a necessidade de uma metodologia que permita analisar, com rigor e numa perspectiva de rotina, os antidepressivos que podem ser encontrados em amostras de sangue. No contexto do Serviço de Toxicologia Forense do Instituto Nacional de Medicina Legal, Delegação do Norte, torna-se necessário o desenvolvimento de uma metodologia analítica para a determinação simultânea de 15 antidepressivos em sangue total e a sua validação relativamente a vários parâmetros analíticos. Os antidepressivos considerados foram Amitriptilina, Citalopram, Clomipramina, N-Desmetilclomipramina, Dotiepina, Fluoxetina, Imipramina, Maprotilina, Mianserina, Mirtazapina, Nortriptilina, Paroxetina, Sertralina, Trimipramina e Venlafaxina. A técnica utilizada para este efeito foi o GC/MS/MS, aplicando um procedimento extractivo prévio apropriado, baseado em procedimentos convencionais de extracção em fase sólida. A escolha desta técnica teve por base a possibilidade de identificar inequivocamente os compostos presentes na amostra, independentemente da complexidade da matriz, e de originar metodologias com uma sensibilidade elevada e com limites de detecção muito baixos. Os parâmetros analíticos considerados para validação da metodologia estabelecida foram selectividade/especificidade e capacidade de identificação; limites de detecção e de quantificação; linearidade e gama de trabalho; eficiência de extracção; arrastamento; exactidão (precisão, veracidade e incerteza de medição) e robustez. Com excepção da exactidão, um parâmetro que carece ainda de estudos complementares, todos os parâmetros estudados foram validados de acordo com os requisitos internos do Serviço. De uma forma geral, os resultados obtidos com o método desenvolvido revelaram-se selectivos e apresentaram respostas analíticas tanto para concentrações de antidepressivos em níveis terapêuticos como para níveis letais destas drogas. Os procedimentos extractivos revelaram-se eficazes e não foram verificados fenómenos de arrastamento em concentrações mais elevadas. O método foi ainda considerado robusto.Depression is one of the most prevalent psychiatric disorders in our society. Solid epidemiological evidence suggests a substantial incidence of the disease in recent years. This is supported by a significant increase in the consumption of antidepressant drugs, creating the need of suitable methods for their accurate analysis in blood samples, carried out in a routine fashion. Focusing the needs of the Serviço de Toxicologia Forense do Instituto Nacional de Medicina Legal, Delegação do Norte, it is important to develop an analytical methodology for the simultaneous determination of 15 antidepressant drugs in whole blood and to validate it with regard to several analytical parameters. The selected antidepressants are Amitriptyline, Citalopram, Clomipramine, N-Desmethylclomipramine, Dothiepin, Fluoxetine, Imipramine, Maprotiline, Mianserine, Mirtazapine, Nortriptyline, Paroxetine, Sertraline, Trimipramine e Venlafaxine. GC/MS/MS was selected for this purpose, after carrying out suitable pre-treating solid-phase extraction procedures. This selection was grounded on the fact that the identification of every compound is ensured and the resulting method offers high sensitivity with low limits of detection. The analytical parameters required for validation included selectivity/specificity and identification capability; limits of detection and quantification; linearity and working concentration range; extraction efficiency; carryover; accuracy (precision, trueness and uncertainty); and robustness. Excluding accuracy that still needs additional testing, all parameters were validated according to the requisites of the Serviço de Toxicologia Forense do Instituto Nacional de Medicina Legal, Delegação do Norte. In general, the obtained results offered good selectivity and analytical responses for the typical concentration ranges of therapeutic and lethal levels. The extraction procedures were effective and no carryover was observed for the highest concentrations. The overall method was found robust

The potential of molecular imprinting as a biosensing devices for monitoring the CEA cancer biomarker

6th Graduate Student Symposium on Molecular Imprinting6th Graduate Student Symposium on Molecular Imprinting, in Medway School of Pharmacy, Kent, UK, August 27-28, 2015Colorectal cancer is the third most common type of cancer and the major cause of the death throughout the world. Widely known, carcinoembryonic antigen (CEA) is an important tumour marker responsible for clinical diagnosis of 95% of all colon tumors1. The discovery of novel non-invasive biomarkers, as CEA, and its fast determination at low cost is presently required, to enable its use over wide screening programs and applications in point-of-care context, and, thus, its monitoring quite early. As a novel approach, this work proposes a novel support with molecular imprinted polymer (MIP) for CEA cancer biomarker based on carbon ink matrix linked by sol-gel chemistry on top of conductive glass covered by fluorine-doped tin oxide (FTO glass). In brief, the electrical biosensor was tailored on top of a disposable conductive glass electrode, following a bottom-up approach. The several stages of this process included the chemical modification of a homemade carbon ink layer and the assembly of a MIP or non-imprinted polymer (NIP) layer. The analytical performance of the obtained devices was followed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Chemical modifications of the surface were characterized using Fourier Transform Infrared (FTIR), and Raman spectroscopy with confocal microscopy. Overall, the MIP/FTO glass-based device displayed linear responses to CEA in EIS assays from 2.5×10-3 µg.mL-1 to 1.25 µg.mL-1 in PBS buffer, with detection limits of 2.5×10-3 µg.mL-1. Successful detection of CEA was, also, achieved in spiked samples of fetal bovine serum. In conclusion, the devices developed are a promising tool for the monitoring of CEA in a point-of-care applications, due to its detection capability below the normal physiological levels expected for this cancer biomarker, simplicity of manufacture, low-cost and good sensitivity and selectivity

Glass-based biosensing device for monitoring CA15-3 cancer biomarker

We thank funding to the European Research Council, through the Starting Grant 3P’s (GA311086, given to MGFS) and to Norte 2020 - Programa Operacional Regional do Norte, through the project CANCER (NORTE-01-0145-FEDER-000029). The authors also thank due to CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM/50011/2013).info:eu-repo/semantics/publishedVersio

Artificial antibodies based potentiometric sensors for monitoring diabetic ketoacidosis

Diabetic ketoacidosis is a pathological condition characterized for the complex disordered metabolic state (as hyperglycemia, metabolic acidosis, dehydration or ketosis), caused by the total failing of insulin production for beta cells in the islets of Langerhans (type 1 diabetes) or abnormalities in the peripheral insulin action and insulin secretion (type 2 diabetes). This pathogenesis can be also associated with the increase of counter-regulation hormones, leading to an increase in the hepatic glucose synthesis and a decrease in the peripheral tissues, resulting in hyperglycemia and hyperosmolarity. The effect of lipolysis increase leads to an increase in the production of free fatty acids, which are oxidized in the hepatic microsomal system and converted to acetyl-CoA. When acetyl-CoA production exceeds hepatic utilization capacity, this substance acts as a substrate for the production of ketone bodies (β-hydroxybutyrate (BHB), acetoacetate and acetone), causing ketonemia and metabolic acidosis.info:eu-repo/semantics/publishedVersio

A disposable glass-based immunosensor for monitoring the cancer biomarker CEA in urine

NanoPT 2014 International Conference, in Portugal, on February 12-14. Poster presentation based on topic Nanobio/NanomedicineImmunosensing is a fast and cost-effective method in clinical diagnosis, relying on antibody-antigen interaction for the quantitative detection a specific antigen. Different types of antigen species have been screened in this context, including tumor markers, assessed by enzyme-linked immunosorbent, chemiluminescence and electrochemical methods1. Tumor markers are biomolecules in tumor cells or body fluids that may be associated with cancer diseases. Carcinoembryonic antigen (CEA) is among such markers, being employed in clinical diagnosis of over 80% colorectal cancers, with relatively little expression in normal mucosa2,3. Fast determination of CEA at low cost is presently required, to enable its use over wide screening programs and application in point-of-care context. This work presents a new simple immunoassay method for CEA detection in urine using a disposable glass-based immunosensor coupled to electrochemical detection. Conductive glass covered by fluorine doped tin oxide (FTO) was used as conductive support and modified with anti-CEA by means of bottom-up approach. All stages involved in the biochemical modification were followed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The analytical performance of the device was fully characterized by EIS, proving sensitive readings of CEA from 2.5x10-3 to 0.10 ng/mL. The immunosensor was applied to real urine analysis from healthy individual spiked with the antigen. Overall, the combination of the sensory material with electrode design has lead to a promising tool for point-of-care applications, when applied to field monitoring of CEA in urine samples

Sol-gel biomimetic material designed to target CEA cancer biomarker

1st ASPIC International CongressPoster J3, presented at 1st ASPIC International Congress, in Fundação Calouste Gulbenkian, Lisbon, Portugal, November 25-26, 2014.Introduction Carcinoembryonic antigen (CEA) is an important tumor marker responsible for clinical diagnosis of over 95% of all colon tumors, 50% of breast tumors, as well as tumors of the lung cancer or ovarian carcinoma [1]. The detection of CEA levels in biological samples plays an important role in the pre-diagnosis evaluation and in the follow-up examination during therapy stage [2]. The most common tool for the analysis of CEA in hospitals and clinical laboratories relies on ELISA-based procedures using antibodies as capturing probe. The overall principal offers the selectivity and sensitivity out coming from the use of antibodies, but it could be further improved by assembling the biosensors over a receptor platform and establishing a label-free measure by electrical impedance spectroscopy (EIS). Thus, the present work proposes the development of an immunosensor for CEA. Materials and Methods Electrochemical signals were measured in a Methrom Autolab potentiostat/galvanostat (Autolab PGSTAT302N) interfaced to a computer and controlled by NOVA 1.9 software. The chemical modification of the surface of the conductive glass was characterized by Raman spectroscopy with confocal microscopy (Thermo Scientific). The immunosensor was assembled by modifying conductive glass (with ITO) with an amino silane compound (APTES), activating the antibody via carbodiimide chemistry (EDAC/NHS) and binding the antibody to the amine surface over the ITO glass. The performance of the imunosensor was evaluated by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SVW). Results and discussion The immunosensor made with an optimized composition displayed linear behavior against CEA concentration by EIS and SWV techniques. The corresponding linear ranges were 0.502-1.5 and 0.252-1.5ng/mL, with detection limits of 0.417 and 0.043 ng/mL, respectively. Overall, the obtained device may be potential method to apply for screening CEA in point-of-care due to the simplicity of fabrication, short time response, low cost and good sensitivity when compared to other analytical techniques, such as ELISA assays

Coupling gold nanoparticles to Dye-Sensitized Solar Cells for an increased efficiency

New approaches for coupling Au NPs to the photoanode of dye sensitized solar cells (DSSCs) were proposed herein, aiming to improve the typical energy conversion efficiency of these cells. For this purpose, colloidal Au NPs with different particles sizes, ∼5 nm and ∼22 nm, were chemically synthesized and attached (i) directly to titanium dioxide (TiO2) or (ii) to TiO2 surface modified with siliceous shells enriched in dithiocarbamate moieties (SiO2/SiDTC). Photoanodes composed by films of TiO2 anatase, TiO2@Au NPs (∼5 nm and ∼22 nm), or TiO2 functionalized with SiO2/SiDTC, loaded with colloidal Au NPs, were made. DSSCs were set-up in a typical sandwich configuration, using the photoanode, a Pt counter electrode, and an iodide electrolyte solution (I−/I3−). In general, a relevant contribution in the plasmonic DSSC performance was evidenced by using Au NPs of ∼22 nm loaded in different amounts 23.9 wt%, 31.0 wt% and 44.0 wt%. Photoanodes composed by 23.9% of Au yielded an increment of 14.40% in photocurrent and of 11.21% in the overall power conversion efficiency (PCE), when compared to the conventional one. In turn, the new strategy used in the chemical modification of the conventional photoanodes with dithiocarbamate groups showed also a significant improvement of the DSSC parameters.publishe

Desenvolvimento de biossensores para diagnóstico precoce de biomarcadores do cancro e aplicabilidade em células fotovoltaícas

Cancer diseases are exponentially growing in Portugal throughout the years, reflecting the same scenario as in Europe. Several efforts have been made to minimize cancer incidence, including adopting a healthier behaviour (as vaccination programs and lifestyle adjustments) and early diagnosis of the disease. Currently, the screening of the disease can be performed by monitoring tumour markers (CEA, CA15-3, CA125, CA19-9, PSA, among others) that circulate in the body fluids, as blood and urine. Thus, the development of new analytical tools for detecting these biomarkers in a clinical context is also of growing interest. This PhD thesis aimed to develop low-cost sensing platforms based on newly biomaterials to screen cancer biomarkers in biological samples towards on-site application. For the purpose, different analyte targets were presented herein, including CRT, which is a potential biomarker in ovarian cancer, and CEA, a tumor marker that despite being an indicator of other types of cancer such as breast, pancreas and lung, offers high specificity in colorectal cancer. Distinct approaches were developed for this purpose, based on the design of plastic antibodies by means of molecular imprinting technology, and on antibody-antigen recognition though the development of immunosensors for the detection of colorectal cancer biomarker. The sensing platforms built on a solid conductive glass support (composed by fluorine-doped tin oxide film (FTO)) allowed understanding the interaction between cancer biomarker and the sensing material through electrical signals. In order to allow the portability of these devices, a new approach was proposed in this project, establishing the integration of a sensing platform in a photovoltaic cell, namely in a DSSC, once these cells are capable of generating electrical energy from solar energy. In addition, efforts were made to improve the overall performance of these photovoltaic cells, modifying the DSSCs configuration by introducing metallic nanoparticles, such as gold nanoparticles, in the composition of photoanodes, or even, by the chemical functionalization of the semiconductor material, followed by the metallic nanoparticles binding. In general, the emerging biosensing materials and platforms out coming from this project may contribute for the development of new non-invasive or minimally invasive and self-sustained devices suitable for the early diagnosis of cancer biomarkers in a clinical application.O cancro é uma doença que tem vindo aumentar de forma exponencial em Portugal ao longo dos anos, à semelhança do que acontece no resto da Europa. Deste modo, diversos esforços têm vindo a ser feitos no sentido de mitigar a sua incidência, entre os quais a adesão a comportamentos saudáveis (programas de vacinação e modificação do estilo de vida) e ao diagnóstico precoce da doença. Atualmente, o rastreio da doença é concretizado através de marcadores tumorais (CEA, CA15-3, CA125, CA19-9, PSA, entres outros), não-invasivos, que circulam no organismo, em fluídos como sangue e urina. Neste sentido, esta tese de doutoramento teve o intuito de desenvolver plataformas sensoras de baixo custo, baseadas em novos biomateriais sensores que facultem o rastreio dos biomarcadores do cancro em amostras biológicas, de forma a serem aplicados num contexto clínico. Para este efeito foram consideradas como moléculas alvo a carnitina (CRT), um potencial biomarcador para o diagnóstico do cancro do ovário, e o antigénio carcinoembrionário (CEA), um marcador tumoral que, apesar de ser indicador de outros tipos de cancro como da mama, do pâncreas e do pulmão, apresenta uma elevada especificidade para o cancro do colorretal. Para a conceção destes biossensores foram concretizadas duas abordagens distintas que se basearam no desenvolvimento de anticorpos plásticos, recorrendo à síntese de polímeros de impressão molecular, e no reconhecimento anticorpo-antigénio, através da construção de imunossensores para a deteção do biomarcador do cancro do colorretal. As plataformas sensoras construídas sobre um suporte sólido de vidro condutor (composto por um filme de óxido de estanho dopado com flúor) permitiram avaliar a interação entre o biomarcador e o material sensor através de sinais de natureza elétrica. De modo a possibilitar a portabilidade destes dipositivos para a deteção de marcadores tumorais, neste projeto foi proposta uma nova abordagem que estabeleceu a integração de uma plataforma sensora numa célula fotovoltaica, nomeadamente, numa célula solar sensibilizada por corante (Dye-Sensitized Solar Cell, DSSC), uma vez que estas são, por si só, células capazes de gerar energia eléctrica por conversão da energia solar. Também foram realizados esforços no sentido de melhorar o desempenho geral destas células fotovoltaicas, alterando a sua configuração, através da implementação de nanopartículas metálicas, como as nanopartículas de ouro, na constituição do fotoânodo, ou até mesmo através da funcionalização química do material semicondutor que compõe estes fotoânodos e, posterior, ligação a estas nanopartículas metálicas. De uma forma geral, os dispositivos sensores desenvolvidos poderão ser uma ferramenta promissora, de baixo custo e portátil, para um diagnóstico precoce, ou para o acompanhamento e a monitorização terapêutica do cancro, de modo não invasivo ou minimamente invasivo, para aplicação num contexto clínico.Programa Doutoral em Nanociências e Nanotecnologi

Antidepressants detection and quantification in whole blood samples by GC–MS/MS, for forensic purposes

Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC–MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/ specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and subtherapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16 min) for typical concentration ranges, both for therapeutic and lethal levels.info:eu-repo/semantics/acceptedVersio

Novel optical PVC probes for on-site detection/determination of fluoroquinolones in a solid/liquid interface: Application to the determination of Norfloxacin in aquaculture water

A novel optical disposable probe for screening fluoroquinolones in fish farming waters is presented, having Norfloxacin (NFX) as target compound. The colorimetric reaction takes place in the solid/liquid interface consisting of a plasticized PVC layer carrying the colorimetric reagent and the sample solution. NFX solutions dropped on top of this solid-sensory surface provided a colour change from light yellow to dark orange. Several metals were tested as colorimetric reagents and Fe(III) was selected. The main parameters affecting the obtained colour were assessed and optimised in both liquid and solid phases. The corresponding studies were conducted by visible spectrophotometry and digital image acquisition. The three coordinates of the HSL model system of the collected image (Hue, Saturation and Lightness) were obtained by simple image management (enabled in any computer). The analytical response of the optimised solid-state optical probe against concentration was tested for several mathematical transformations of the colour coordinates. Linear behaviour was observed for logarithm NFX concentration against Hue+Lightness. Under this condition, the sensor exhibited a limit of detection below 50 μM (corresponding to about 16 mg/mL). Visual inspection also enabled semi-quantitative information. The selectivity was ensured against drugs from other chemical groups than fluoroquinolones. Finally, similar procedure was used to prepare an array of sensors for NFX, consisting on different metal species. Cu(II), Mn(II) and aluminon were selected for this purpose. The sensor array was used to detect NFX in aquaculture water, without any prior sample manipulation