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.

Avaliação de métodos de controlo mecânicos e químicos da espécie invasora Hedychium gardnerianum Sheppard ex Ker Gawl. na ilha de São Miguel, Açores

Dissertação de Mestrado, Gestão e Conservação da Natureza, 6 de Abril de 2015, Universidade dos Açores.

Sulfadiazine-potentiometric sensors for flow and batch determinations of sulfadiazine in drugs and biological fluids

New PVC membrane electrodes for the determination of sulfadiazine (SDZ) are presented. The electrodes are fabricated with conventional and tubular configurations with a graphite-based electrical contact, and no internal reference solution. The selective membranes consist of bis(triphenylphosphoranilidene)ammonium·SDZ (electrode A), tetraoctylammonium bromide (electrode B), or iron(II)-phthalocyanine (FePC) (electrode C) electroactive materials dispersed in a PVC matrix of o-nitrophenyl octyl ether (o-NPOE) plasticizer. The sensors A, B, and C displayed linear responses over the concentration ranges 1.0*10-2 – 1.0*10–5, 1.0*10–2 – 7.5*10–6, and 3.2*10–2 – 7.0* 10–6 mol l–1 (detection limits of 1.09, 2.04 and 0.87 mg ml–1) with anionic slopes of –57.3 ± 0.1, –46.7 ± 0.5, and –65.1 ± 0.2 mV decade–1, respectively. No effect from pH was observed within 4.0 – 5.5, 4.8 – 10, and 4.5 – 8, respectively, and good selectivity was found. The sensors were applied to the analysis of pharmaceuticals and biological fluids in steady state and in flow conditions.

Biomimetic norfloxacin sensors made of molecularly-imprinted materials for potentiometric transduction

A biomimetic sensor for norfloxacin is presented that is based on host-guest interactions and potentiometric transduction. The artificial host was imprinted into polymers made from methacrylic acid and/or 2-vinyl pyridine. The resulting particles were entrapped in a plasticized poly(vinyl chloride) (PVC) matrix. The sensors exhibit near-Nernstian response in steady state evaluations, and detection limits range from 0.40 to 1.0 μgmL−1, respectively, and are independent of pH values at between 2 and 6, and 8 and 11, respectively. Good selectivity was observed over several potential interferents. In flowing media, the sensors exhibit fast response, a sensitivity of 68.2 mV per decade, a linear range from 79 μM to 2.5 mM, a detection limit of 20 μgmL−1, and a stable baseline. The sensors were successfully applied to field monitoring of norfloxacin in fish samples, biological samples, and pharmaceutical products

Determination of tartaric acid in wines by FIA with tubular tartrate-selective electrodes

A flow injection analysis (FIA) system comprising a tartrate- (TAT) selective electrode has been developed for determination of tartaric acid in wines. Several electrodes constructed for this purpose had a PVC membrane with a complex of quaternary ammonium and TAT as anion exchanger, a phenol derivative as additive, and a more or less polar mediator solvent. Characterization of the electrodes showed behavior was best for membranes with o-nitrophenyl octyl ether as solvent. On injection of 500 μL into a phosphate buffer carrier (pH = 3.1; ionic strength 10–2 mol/L) flowing at 3 mL/min, the slope was 58.06 ± 0.6 with a lower limit of linear range of 5.0 × 10–4 mol/L TAT and R2 = 0.9989. The interference of several species, e.g. chloride, bromide, iodide, nitrate, gallic acid, tannin, sucrose, glucose, fructose, acetate, and citrate, was evaluated in terms of potentiometric selectivity coefficients. The Hofmeister series was followed for inorganic species and the most interfering organic ion was citrate. When red and white wines were analyzed and the results compared with those from an independent method they were found to be accurate, with relative standard deviations below 5.0%.

Square-wave voltametric method for determination of molinate concentration in a biological process using a hanging mercury drop electrode

A square-wave voltammetric (SWV) method using a hanging mercury drop electrode (HMDE) has been developed for determination of the herbicide molinate in a biodegradation process. The method is based on controlled adsorptive accumulation of molinate for 10 s at a potential of -0.8 V versus AgCl/Ag. An anodic peak, due to oxidation of the adsorbed pesticide, was observed in the cyclic voltammogram at ca. -0.320 V versus AgCl/Ag; a very small cathodic peak was also detected. The SWV calibration plot was established to be linear in the range 5.0x10-6 to 9.0x10-6 mol L-1; this corresponded to a detection limit of 3.5x10-8 mol L-1. This electroanalytical method was used to monitor the decrease of molinate concentration in river waters along a biodegradation process using a bacterial mixed culture. The results achieved with this voltammetric method were compared with those obtained by use of a chromatographic method (HPLC–UV) and no significant statistical differences were observed.

Anodic adsorptive stripping voltammetric determination of atrazine in spiked soil samples with a gold microelectrode

Microwave-assisted solvent extraction was combined with anodic adsorptive stripping voltammetry at a gold microelectrode to extract and quantify the herbicide atrazine in spiked soil samples. A systematic study of the experimental parameters affecting the stripping response was carried out by square-wave voltammetry. The voltammetric procedure is based on controlled adsorptive accumulation of atrazine at the potential of 0.35V (versus Ag/AgCl) in the presence of Britton–Robinson buffer pH (2.0). The limit of detection obtained for a 30 sec collection time was 4.3x10-7 mol L-1. Recovery experiments, at the 1µgg-1 level of spiking, gave good results for the global procedure, and the values found were comparable to those obtained by HPLC.

Development of an FIA system with amperometric detection for determination of bentazone in estuarine waters

On the basis of its electrochemical behaviour a new flow-injection analysis (FIA) method with amperometric detection has been developed for quantification of the herbicide bentazone (BTZ) in estuarine waters. Standard solutions and samples (200 µL) were injected into a water carrier stream and both pH and ionic strength were automatically adjusted inside the manifold. Optimization of critical FIA conditions indicated that the best analytical results were obtained at an oxidation potential of 1.10 V, pH 4.5, and an overall flow-rate of 2.4 mL min–1. Analysis of real samples was performed by means of calibration curves over the concentration range 2.5x10–6 to 5.0x10–5 mol L–1, and results were compared with those obtained by use of an independent method (HPLC). The accuracy of the amperometric determinations was ascertained; errors relative to the comparison method were below 4% and sampling rates were approximately 100 samples h–1. The repeatability of the proposed method was calculated by assessing the relative standard deviation (%) of ten consecutive determinations of one sample; the value obtained was 2.1%.

Citrate selective electrodes for the flow injection analysis of soft drinks, beers and pharmaceutical products

Aiming the establishment of simple and accurate readings of citric acid (CA) in complex samples, citrate (CIT) selective electrodes with tubular configuration and polymeric membranes plus a quaternary ammonium ion exchanger were constructed. Several selective membranes were prepared for this purpose, having distinct mediator solvents (with quite different polarities) and, in some cases, p-tert-octylphenol (TOP) as additive. The latter was used regarding a possible increase in selectivity. The general working characteristics of all prepared electrodes were evaluated in a low dispersion flow injection analysis (FIA) manifold by injecting 500µl of citrate standard solutions into an ionic strength (IS) adjuster carrier (10−2 mol l−1) flowing at 3ml min−1. Good potentiometric response, with an average slope and a repeatability of 61.9mV per decade and ±0.8%, respectively, resulted from selective membranes comprising additive and bis(2-ethylhexyl)sebacate (bEHS) as mediator solvent. The same membranes conducted as well to the best selectivity characteristics, assessed by the separated solutions method and for several chemical species, such as chloride, nitrate, ascorbate, glucose, fructose and sucrose. Pharmaceutical preparations, soft drinks and beers were analyzed under conditions that enabled simultaneous pH and ionic strength adjustment (pH = 3.2; ionic strength = 10−2 mol l−1), and the attained results agreed well with the used reference method (relative error < 4%). The above experimental conditions promoted a significant increase in sensitivity of the potentiometric response, with a supra-Nernstian slope of 80.2mV per decade, and allowed the analysis of about 90 samples per hour, with a relative standard deviation <1.0%.

Determination of ametryn in soils via microwave-assisted solvent extraction coupled to anodic stripping voltammetry with a gold ultramicroelectrode

An extraction-anodic adsorptive stripping voltammetric procedure using microwave-assisted solvent extraction and a gold ultramicroelectrode was developed for determining the pesticide ametryn in soil samples. The method is based on the use of acetonitrile as extraction solvent and on controlled adsorptive accumulation of the herbicide at the potential of 0.50 V (vs. Ag/AgCl) in the presence of Britton-Robinson buffer (pH 3.3). Soil sample extracts were analysed directly after drying and redissolution with the supporting electrolyte but without other pre-treatment. The limit of detection obtained for a 10 s collection time was 0.021 µg g-1. Recovery experiments for the global procedure, at the 0.500 µg g-1 level, gave satisfactory mean and standard deviation results which were comparable to those obtained by HPLC with UV detection.

Electroanalytical study of the pesticide asulam

The electrochemical behaviour of the herbicide Asulam was studied by cyclic and square wave voltammetry. Asulam may be irreversibly oxidised at a glassy carbon electrode. Maximum currents were obtained at pH=1.9 in aqueous electrolyte solution. Based on the electrochemical behaviour of Asulam, two analytical methodologies were developed for its determination in water samples, using square wave voltammetry (SWV) and flow injection analysis (FIA) coupled with an amperometric detector. Limits of detection of 7.1x10-6 mol L-1 and 1.2x10-8 mol L-1 for SWV and FIA respectively, were achieved. Repeatability was calculated by assessing the relative standard deviation (%) for 10 consecutive determinations of one sample. The found values were 2.1% for SWV and 5.0% for FIA. Validation of the results provided by SWV and FIA methodologies was performed by comparison with results from an HPLC-DAD technique. Good relative deviations were found (<5%). Recovery trials were performed to assess the accuracy of the results and the obtained values were between 84% and 107% for both methods.

Construction and evaluation of cysteine selective electrodes for FIA analysis of pharmaceuticals

A flow injection analysis (FIA) system comprising a cysteine selective electrode as detection system was developed for determination of this amino acid in pharmaceuticals. Several electrodes were constructed for this purpose, having PVC membranes with different ionic exchangers and mediator solvents. Better working characteristics were attained with membranes comprising o-nitrophenyl octyl ether as mediator solvent and a tetraphenylborate based ionic-sensor. Injection of 500 µL standard solutions into an ionic strength adjuster carrier (3x10-3 M) of barium chloride flowing at 2.4mL min-1, showed linearity ranges from 5.0x10-5 to 5.0x10-3 M, with slopes of 76.4±0.6mV decade-1 and R2>0.9935. Slope decreased significantly under the requirement of a pH adjustment, selected at 4.5. Interference of several compounds (sodium, potassium, magnesium, barium, glucose, fructose, and sucrose) was estimated by potentiometric selectivity coefficients and considered negligible. Analysis of real samples were performed and considered accurate, with a relative error to an independent method of +2.7%.

Chromatographic techniques for the determination of free phenol in foundry resins

Phenol is a toxic compound present in a wide variety of foundry resins. Its quantification is important for the characterization of the resins as well as for the evaluation of free contaminants present in foundry wastes. Two chromatographic methods, liquid chromatography with ultraviolet detection (LC-UV) and gas chromatography with flame ionization detection (GC-FID), for the analysis of free phenol in several foundry resins, after a simple extraction procedure (30 min), were developed. Both chromatographic methods were suitable for the determination of phenol in the studied furanic and phenolic resins, showing good selectivity, accuracy (recovery 99–100%; relative deviations <5%), and precision (coefficients of variation <6%). The used ASTM reference method was only found to be useful in the analysis of phenolic resins, while the LC and GC methods were applicable for all the studied resins. The developed methods reduce the time of analysis from 3.5 hours to about 30 min and can readily be used in routine quality control laboratories.

Flow injection potentiometric determination of chlorpromazine

New chlorpromazine selective electrodes with a tubular arrangement and no internal reference solution are proposed. Selective membranes are of poly(vinyl chloride) (PVC) with the tetraphenylborate•chlorpromazine (TPB•CPZ) ion-exchanger dissolved in o-nitrophenyl octyl ether (oNPOE). Analytical features of the electrodes were evaluated on a single-channel flow assembly having 500 µl injection volumes and flow-rates of 4.5 ml min−1. For a carrier solution of 3.3×10−3Min sodium sulphate, Nernstian responsewas observed over the concentration range 1.0×10−5 to 1.0×10−2 M. Average slopes were about 59mVdecade−1 and squared correlation coefficients were >0.9984. Slight hiper-Nernstian behaviour was observed in buffer solutions of 4.4 pH; average slopes were of 62.06mVdecade−1. The electrode displayed a good selectivity for CPZ, with respect to, several foreign inorganic and organic species. The selective electrodes were successfully applied to the analysis of pure solutions and pharmaceutical preparations. Proposed method allows the analysis of 84 samples h−1, producing wastewaters of low toxicity. The proposed method offers the advantage of simplicity, accuracy, applicability to coloured and turbid samples, and automation feasibility.

Molecularly-imprinted materials for potentiometric transduction: application to the antibiotic enrofloxacin

Enrofloxacin (ENR) is an antimicrobial used both in humans and in food producing species. Its control is required in farmed species and their surroundings in order to reduce the prevalence of antibiotic resistant bacteria. Thus, a new biomimetic sensor enrofloxacin is presented. An artificial host was imprinted in specific polymers. These were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. The potentiometric sensors exhibited a near-Nernstian response. Slopes expressing mVΔlog([ENR]/M) varied within 48–63. The detection limits ranged from 0.28 to 1.01 µg mL 1. Sensors were independent from the pH of test solutions within 4–7. Good selectivity was observed toward potassium, calcium, barium, magnesium, glycine, ascorbic acid, creatinine, norfloxacin, ciprofloxacin, and tetracycline. In flowing media, the biomimetic sensors presented good reproducibility (RSD of ±0.7%), fast response, good sensitivity (47 mV/Dlog([ENR]/ M), wide linear range (1.0×10-5–1.0×10-3 M), low detection limit (0.9 µg mL-1), and a stable baseline for a 5×10-2 M acetate buffer (pH 4.7) carrier. The sensors were used to analyze fish samples. The method offered the advantages of simplicity, accuracy, and automation feasibility. The sensing membrane may contribute to the development of small devices allowing in vivo measurements of enrofloxacin or parent-drugs.