Enantiomeric fraction evaluation of pharmaceuticals in environmentalmatrices by liquid chromatography-tandem mass spectrometry

The interest for environmental fate assessment of chiral pharmaceuticals is increasing and enantioselective analytical methods are mandatory. This study presents an enantioselective analytical method for the quantification of seven pairs of enantiomers of pharmaceuticals and a pair of a metabolite. The selected chiral pharmaceuticals belong to three different therapeutic classes, namely selective serotonin reuptake inhibitors (venlafaxine, fluoxetine and its metabolite norfluoxetine), beta-blockers (alprenolol, bisoprolol, metoprolol, propranolol) and a beta2-adrenergic agonist (salbutamol). The analytical method was based on solid phase extraction followed by liquid chromatography tandem mass spectrometry with a triple quadrupole analyser. Briefly, Oasis® MCX cartridges were used to preconcentrate 250 mL of water samples and the reconstituted extracts were analysed with a Chirobiotic™ V under reversed mode. The effluent of a laboratory-scale aerobic granular sludge sequencing batch reactor (AGS-SBR) was used to validate the method. Linearity (r2 > 0.99), selectivity and sensitivity were achieved in the range of 20–400 ng L−1 for all enantiomers, except for norfluoxetine enantiomers which range covered 30–400 ng L−1. The method detection limits were between 0.65 and 11.5 ng L−1 and the method quantification limits were between 1.98 and 19.7 ng L−1. The identity of all enantiomers was confirmed using two MS/MS transitions and its ion ratios, according to European Commission Decision 2002/657/EC. This method was successfully applied to evaluate effluents of wastewater treatment plants (WWTP) in Portugal. Venlafaxine and fluoxetine were quantified as non-racemic mixtures (enantiomeric fraction ≠ 0.5). The enantioselective validated method was able to monitor chiral pharmaceuticals in WWTP effluents and has potential to assess the enantioselective biodegradation in bioreactors. Further application in environmental matrices as surface and estuarine waters can be exploited.

Implementação, validação e análise técnico-económico de alguns parâmetros analíticos no controlo de águas

Mestrado em Engenharia Química

Determination of total petroleum hydrocarbons in soil from different locations using infrared spectrophotometry and gas chromatography

Total petroleum hydrocarbons (TPH) are important environmental contaminants which are toxic to human and environmental receptors. Several analytical methods have been used to quantify TPH levels in contaminated soils, specifically through infrared spectrometry (IR) and gas chromatography (GC). Despite being two of the most used techniques, some issues remain that have been inadequately studied: a) applicability of both techniques to soils contaminated with two distinct types of fuel (petrol and diesel), b) influence of the soil natural organic matter content on the results achieved by various analytical methods, and c) evaluation of the performance of both techniques in analyses of soils with different levels of contamination (presumably non-contaminated and potentially contaminated). The main objectives of this work were to answer these questions and to provide more complete information about the potentials and limitations of GC and IR techniques. The results led us to the following conclusions: a) IR analysis of soils contaminated with petrol is not suitable due to volatilisation losses, b) there is a significant influence of organic matter in IR analysis, and c) both techniques demonstrated the capacity to accurately quantify TPH in soils, irrespective of their contamination levels.

Interlaboratory validation of an environmental monitoring method for trace analysis of endocrine disrupting compounds

Environmental pollution continues to be an emerging study field, as there are thousands of anthropogenic compounds mixed in the environment whose possible mechanisms of toxicity and physiological outcomes are of great concern. Developing methods to access and prioritize the screening of these compounds at trace levels in order to support regulatory efforts is, therefore, very important. A methodology based on solid phase extraction followed by derivatization and gas chromatography-mass spectrometry analysis was developed for the assessment of four endocrine disrupting compounds (EDCs) in water matrices: bisphenol A, estrone, 17b-estradiol and 17a-ethinylestradiol. The study was performed, simultaneously, by two different laboratories in order to evaluate the robustness of the method and to increase the quality control over its application in routine analysis. Validation was done according to the International Conference on Harmonisation recommendations and other international guidelines with specifications for the GC-MS methodology. Matrix-induced chromatographic response enhancement was avoided by using matrix-standard calibration solutions and heteroscedasticity has been overtaken by a weighted least squares linear regression model application. Consistent evaluation of key analytical parameters such as extraction efficiency, sensitivity, specificity, linearity, limits of detection and quantification, precision, accuracy and robustness was done in accordance with standards established for acceptance. Finally, the application of the optimized method in the assessment of the selected analytes in environmental samples suggested that it is an expedite methodology for routine analysis of EDC residues in water matrices.

Contaminação ambiental por pesticidas: análise metodológica

Este trabalho teve como objectivo pesquisar os pesticidas que são mais usados em Portugal, os métodos de análise mais frequentemente utilizados na determinação de pesticidas, a contaminação ambiental por pesticidas e como minimizar o impacto ambiental provocado pelo uso indiscriminado de pesticidas. Os pesticidas usados variam de acordo com as diferentes características das regiões de Portugal e os grupos de pesticidas mais usados são: organofosforados, derivados de anilina, derivados triazinicos, tiocarbamatos, derivados do ácido fenoxiacético, derivados de acetamida, benzotiadiazole, derivados de ureia e azol. A determinação destes grupos é usualmente feita usando métodos cromatográficos, uma vez que estes métodos permitem a separação de misturas complexas. A contaminação ambiental por pesticidas pode ser provocada por diversos factores, contudo esta contaminação pode ser minimizada se estes forem usados de forma racional e de um modo sustentável conforme o descrito na directiva 2009/128/CE de 21 de Outubro.

Distribuição do mercúrio nas águas termais e engarrafadas do Norte de Portugal

O aumento da utilização do mercúrio, Hg, tanto para fins industriais como na aplicação de compostos mercuriais na agricultura, originaram um aumento significativo da contaminação ambiental, especialmente das águas e dos alimentos. Foi aprofundado o estudo sobre o mercúrio, nomeadamente as suas principais fontes de emissão, a sua toxicidade, os principais efeitos nos seres humanos, algumas medidas preventivas, os limites máximos que a legislação permite, que no caso das águas analisadas é de 0,001mg/L, os métodos analíticos para a sua determinação e alguns métodos de amostragem, tanto para o mercúrio em águas como em sedimentos. Em Almadén (Espanha), existe uma mina que pode ser considerada como uma das maiores anomalias geoqímica de mercúrio na Terra. Com este trabalho pretendeu-se perceber a possível existência de mercúrio nas águas termais ao longo da zona de falha Penacova-Régua-Verin e avaliar a existência de uma relação entre os níveis de mercúrio e o seu contexto geológico. Neste trabalho, foi desenvolvido um método simples para a determinação dos níveis de mercúrio em águas, utilizando a espectrometria de absorção atómica de fonte contínua de alta resolução acoplado à técnica de vapor frio. O objectivo deste trabalho foi avaliar os níveis de mercúrio existente em algumas águas engarrafadas e termais. Os limites de detecção da técnica utilizada foram 0,0595 μg/L e os de quantificação foram 0,2100 μg/L. Depois de analisadas as amostras verificou-se que os níveis de mercúrio encontrados nas águas eram inferiores ao limite de quantificação da técnica e por isso, não foi possível extrapolar qualquer relação entre os níveis de mercúrio e as alterações do fundo geológico. Como o limite de quantificação é inferior ao limite máximo permitido por lei, pode então dizer-se que todas as águas se encontram abaixo do limite máximo permitido.

New biomimetic sensors for the determination of tetracycline in biological samples: batch and flow mode operations

New potentiometric membrane sensors with cylindrical configuration for tetracycline (TC) are described based on the use of a newly designed molecularly imprinted polymer (MIP) material consisting of 2-vinylpyridine as a functional monomer in a plasticized PVC membrane. The sensor exhibited significantly enhanced response towards TC over the concentration range 1.59 10 5–1.0 10 3 mol L 1 at pH 3–5 with a lower detection limit of 1.29 10 5 mol L 1. The response was near-Nernstian, with average slopes of 63.9 mV decade 1. The effect of lipophilic salts and various foreign common ions were tested and were found to be negligible. The possibility of applying the proposed sensor to TC determination in spiked biological fluid samples was demonstrated.

Flow injection electrochemical determination of apomorphine

Few analytical methods are currently available for determination of apomorphine, the active substance of a new oral formulation used in the treatment of erectile dysfunction. In this way a flow injection electrochemical method (FIA-EC) was developed for its quantification and applied to pharmaceutical dosage forms. Based in previous findings regarding the stability of apomorphine in borate buffer and after optimization of several analytical parameters a single channel flow injection manifold was set up that enables the determination of this drug over the concentration range of 3 to 16 µmol L-1 with a detection limit of 0.5 µmol L-1 at a sampling rateof 90 h-1. The simplicity and rapidity of the FIA-EC method used, its reproducibility and sensitivity make it suitable for quality control of pharmaceutical preparations of apomorphine intended for clinical use and research.

Electrochemical determination of dihydrocodeine in pharmaceuticals

Two analytical methods for the quality control of dihydrocodeine in commercial pharmaceutical formulations have been developed and compared with reference methods: a square wave voltammetric (SWV) method and a flow injection analysis system with electrochemical detection (FIA-EC). The electrochemical methods proposed were successfully applied to the determination of dihydrocodeine in pharmaceutical tablets and in oral solutions. These methods do not require any pretreatment of the samples, the formulation only being dissolved in a suitable electrolyte. Validation of the methods showed it to be precise, accurate and linear over the concentration range of analysis. The automatic procedure based on a flow injection analysis manifold allows a sampling rate of 115 determinations per hour.

Selective recognition in potentiometric transduction of amoxicillin by molecularly imprinted materials

The indiscriminate use of antibiotics in foodproducing animals has received increasing attention as a contributory factor in the international emergence of antibiotic- resistant bacteria (Woodward in Pesticide, veterinary and other residues in food, CRC Press, Boca Raton, 2004). Numerous analytical methods for quantifying antibacterial residues in edible animal products have been developed over years (Woodward in Pesticide, veterinary and other residues in food, CRC Press, Boca Raton, 2004; Botsoglou and Fletouris in Handbook of food analysis, residues and other food component analysis, Marcel Dekker, Ghent, 2004). Being Amoxicillin (AMOX) one of those critical veterinary drugs, efforts have been made to develop simple and expeditious methods for its control in food samples. In literature, only one AMOX-selective electrode has been reported so far. In that work, phosphotungstate:amoxycillinium ion exchanger was used as electroactive material (Shoukry et al. in Electroanalysis 6:914–917, 1994). Designing new materials based on molecularly imprinted polymers (MIPs) which are complementary to the size and charge of AMOX could lead to very selective interactions, thus enhancing the selectivity of the sensing unit. AMOXselective electrodes used imprinted polymers as electroactive materials having AMOX as target molecule to design a biomimetic imprinted cavity. Poly(vinyl chloride), sensors of methacrylic acid displayed Nernstian slopes (60.7 mV/decade) and low detection limits (2.9×10-5 mol/L). The potentiometric responses were not affected by pH within 4–5 and showed good selectivity. The electrodes were applied successfully to the analysis of real samples.

New sensing materials of molecularly-imprinted polymers for the selective recognition of Chlortetracycline

As a result of the stressful conditions in aquaculture facilities there is a high risk of bacterial infections among cultured fish. Chlortetracycline (CTC) is one of the antimicrobials used to solve this problem. It is a broad spectrum antibacterial active against a wide range of Gram-positive and Gram-negative bacteria. Numerous analytical methods for screening, identifying, and quantifying CTC in animal products have been developed over the years. An alternative and advantageous method should rely on expeditious and efficient procedures providing highly specific and sensitive measurements in food samples. Ion-selective electrodes (ISEs) could meet these criteria. The only ISE reported in literature for this purpose used traditional electro-active materials. A selectivity enhancement could however be achieved after improving the analyte recognition by molecularly imprinted polymers (MIPs). Several MIP particles were synthesized and used as electro-active materials. ISEs based in methacrylic acid monomers showed the best analytical performance according to slope (62.5 and 68.6 mV/decade) and detection limit (4.1×10−5 and 5.5×10−5 mol L−1). The electrodes displayed good selectivity. The ISEs are not affected by pH changes ranging from 2.5 to 13. The sensors were successfully applied to the analysis of serum, urine and fish samples.

Optimization of QuEChERS procedure coupled to GC-ECD for organochlorine pesticide determination in carrot samples

An optimised version of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for simultaneous determination of 14 organochlorine pesticides in carrots was developed using gas chromatography coupled with electron-capture detector (GC-ECD) and confirmation by gas chromatography tandem mass spectrometry (GC-MS/MS). A citrate-buffered version of QuEChERS was applied for the extraction of the organochlorine pesticides, and for the extract clean-up, primary secondary amine, octadecyl-bonded silica (C18), magnesium sulphate (MgSO4) and graphitized carbon black were used as sorbents. The GC-ECD determination of the target compounds was achieved in less than 20 min. The limits of detection were below the EUmaximum residue limits (MRLs) for carrots, 10–50 μg kg−1, while the limit of quantification did exceed 10 μg kg−1 for hexachlorobenzene (HCB). The introduction of a sonication step was shown to improve the recoveries. The overall average recoveries in carrots, at the four tested levels (60, 80, 100 and 140 μg kg−1), ranged from 66 to 111% with relative standard deviations in the range of 2– 15 % (n03) for all analytes, with the exception of HCB. The method has been applied to the analysis of 21 carrot samples from different Portuguese regions, and β-HCH was the pesticide most frequently found, with concentrations oscillating between less than the limit of quantification to 14.6 μg kg−1. Only one sample had a pesticide residue (β-HCH) above the MRL, 14.6 μg kg−1. This methodology combines the advantages of both QuEChERS and GC-ECD, producing a very rapid, sensitive and reliable procedure which can be applied in routine analytical laboratories.

Validation of a single-extraction procedure for sequential analysis of vitamin E, cholesterol, fatty acids, and total fat in seafood

Food lipid major components are usually analyzed by individual methodologies using diverse extractive procedures for each class. A simple and fast extractive procedure was devised for the sequential analysis of vitamin E, cholesterol, fatty acids, and total fat estimation in seafood, reducing analyses time and organic solvent consumption. Several liquid/liquid-based extractive methodologies using chlorinated and non-chlorinated organic solvents were tested. The extract obtained is used for vitamin E quantification (normal-phase HPLC with fluorescence detection), total cholesterol (normal-phase HPLC with UV detection), fatty acid profile, and total fat estimation (GC-FID), all accomplished in <40 min. The final methodology presents an adequate linearity range and sensitivity for tocopherol and cholesterol, with intra- and inter-day precisions (RSD) from 3 to 11 % for all the components. The developed methodology was applied to diverse seafood samples with positive outcomes, making it a very attractive technique for routine analyses in standard equipped laboratories in the food quality control field.

Auditoria à qualidade do ar interior de um edifício de serviços - RSECE QAI

O objectivo principal deste trabalho é a realização de uma auditoria, à qualidade do ar interior (QAI), a um edifício de serviços – COCIGA, SA, tendo como base o Regulamento dos Sistemas Energéticos de Climatização dos Edifícios (RSECE). A auditoria QAI implica a medição de vários parâmetros físicos, químicos, microbiológicos e também a inspecção aos componentes do sistema de climatização com a finalidade de averiguar o seu estado de limpeza e manutenção. Assim, foram seleccionados 3 espaços, para a realização de amostragens designados por Comercial - Produtos, AVAC e Mezaninne das oficinas, nos quais foi efectuada a medição de diversos parâmetros, de acordo com as imposições do RSECE, utilizando medidores portáteis ou recorrendo a métodos analíticos. Relativamente aos parâmetros físicos, registaram-se valores de temperatura, para os três espaços estudados, entre os 21 e os 24 ºC e valores médios de humidade relativa de cerca de 50 %. Outro parâmetro medido, e de grande importância para garantir o conforto dos ocupantes, foi a velocidade do ar nos postos de trabalho. De acordo com o RSECE este valor não deve ser superior a 0,2 m/s, o que se verificou em todos os pontos medidos. O último parâmetro físico medido foi a concentração de partículas (PM10) tendo-se obtido valores de cerca de 23 μg/m3ar, valor bastante inferior ao máximo permitido pelo RSECE (150 μg/m3ar). Também no que diz respeito aos parâmetros químicos, ou seja, CO2, CO, formaldeído e ozono, não se verificaram valores superiores aos regulamentares. No caso do CO2, o valor máximo encontrado, nestes três espaços, foi de 745 ppm na Mezaninne das Oficinas e para o CO, na zona AVAC com uma concentração de 0,73 ppm. A medição do formaldeído registou valores perto dos 45 μg/m3ar e o ozono apenas foi detectado, em concentração muito reduzida, na zona Comercial – Produtos. Por fim, as concentrações de bactérias e fungos, de acordo com o RSECE, não devem ultrapassar as 500 UFC/m3ar (parâmetros microbiológicos). Em qualquer dos espaços, os valores medidos foram inferiores ao máximo legal, não ultrapassando as 50 UFC/m3ar. Da avaliação do projecto AVAC, e através da medição dos caudais de insuflação/ extracção em cada zona, concluiu-se que os seus valores não estão de acordo com os valores do projecto inicial que poderá ser imputada a uma insuficiência no funcionamento do sistema detectada na altura das medições. No que diz respeito ao estado de limpeza do sistema AVAC, apenas foi possível inspeccionar as unidades de tratamento de ar, tendo-se constatado que se encontram em boas condições. Ou seja, do ponto de vista do RSECE, e referindo-nos apenas à vertente da Qualidade do Ar Interior, o edifício em causa, cumpre todos os limites impostos para as concentrações de poluentes mas, apresenta algumas deficiências no que respeita aos caudais de ar novo insuflados em cada espaço.

A New Approach in the Design of Air Stripping Columns for the Treatment of Groundwater Contaminated With Volatile Organic Compounds

Volatile organic compounds are a common source of groundwater contamination that can be easily removed by air stripping in columns with random packing and using a counter-current flow between the phases. This work proposes a new methodology for column design for any type of packing and contaminant which avoids the necessity of an arbitrary chosen diameter. It also avoids the employment of the usual graphical Eckert correlations for pressure drop. The hydraulic features are previously chosen as a project criterion. The design procedure was translated into a convenient algorithm in C++ language. A column was built in order to test the design, the theoretical steady-state and dynamic behaviour. The experiments were conducted using a solution of chloroform in distilled water. The results allowed for a correction in the theoretical global mass transfer coefficient previously estimated by the Onda correlations, which depend on several parameters that are not easy to control in experiments. For best describe the column behaviour in stationary and dynamic conditions, an original mathematical model was developed. It consists in a system of two partial non linear differential equations (distributed parameters). Nevertheless, when flows are steady, the system became linear, although there is not an evident solution in analytical terms. In steady state the resulting ODE can be solved by analytical methods, and in dynamic state the discretization of the PDE by finite differences allows for the overcoming of this difficulty. To estimate the contaminant concentrations in both phases in the column, a numerical algorithm was used. The high number of resulting algebraic equations and the impossibility of generating a recursive procedure did not allow the construction of a generalized programme. But an iterative procedure developed in an electronic worksheet allowed for the simulation. The solution is stable only for similar discretizations values. If different values for time/space discretization parameters are used, the solution easily becomes unstable. The system dynamic behaviour was simulated for the common liquid phase perturbations: step, impulse, rectangular pulse and sinusoidal. The final results do not configure strange or non-predictable behaviours.