Antibiotic resistance in enterobacteriaceae isolated from Portuguese deli meats

This study aimed to identify the presence of β-lactam-resistant bacteria in different types of Portuguese deli meats. The numbers of ampicillin resistant bacteria varied from negative in 25 g to 1.0 × 108colony-forming units/g. Within 78 randomly selected β-lactam-resistant bacteria, 24 different resistant phenotypes were found and 35.9% were multidrug resistant (MDR). The majority (87.2%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the blaTEM gene was detected in 23 out of 67 isolates (34.3%) and 16 of them presented MDR phenotypes. Four Klebsiella oxytoca isolates (6%) harbored a gene for the CTX-M/OXY-type enzyme. The direct sequencing of their purified amplicons confirmed the presence of three types of blaOXYgenes (blaOXY-1, blaOXY-2 and blaOXY-5). These results suggest that without good hygienic practices, deli meats may act as a vehicle of transfer of β-lactam-resistant bacteria to the gastrointestinal tract of consumers.

The development and optimization of a modified single-drop microextraction method for organochlorine pesticides determination by gas chromatography-tandem mass spectrometry

We have developed a new method for single-drop microextraction (SDME) for the preconcentration of organochlorine pesticides (OCP) from complex matrices. It is based on the use of a silicone ring at the tip of the syringe. A 5 μL drop of n-hexane is applied to an aqueous extract containing the OCP and found to be adequate to preconcentrate the OCPs prior to analysis by GC in combination with tandem mass spectrometry. Fourteen OCP were determined using this technique in combination with programmable temperature vaporization. It is shown to have many advantages over traditional split/splitless injection. The effects of kind of organic solvent, exposure time, agitation and organic drop volume were optimized. Relative recoveries range from 59 to 117 %, with repeatabilities of <15 % (coefficient of variation) were achieved. The limits of detection range from 0.002 to 0.150 μg kg−1. The method was applied to the preconcentration of OCPs in fresh strawberry, strawberry jam, and soil.

Optimization of QuEChERS method for the analysis of organochlorine pesticides in soils with diverse organic matter

A QuEChERS method has been developed for the determination of 14 organochlorine pesticides in 14 soils from different Portuguese regions with wide range composition. The extracts were analysed by GC-ECD (where GC-ECD is gas chromatography-electron-capture detector) and confirmed by GC-MS/MS (where MS/MS is tandem mass spectrometry). The organic matter content is a key factor in the process efficiency. An optimization was carried out according to soils organic carbon level, divided in two groups: HS (organic carbon>2.3%) and LS (organic carbon<2.3%). Themethod was validated through linearity, recovery, precision and accuracy studies. The quantification was carried out using a matrixmatched calibration to minimize the existence of the matrix effect. Acceptable recoveries were obtained (70–120%) with a relative standard deviation of ≤16% for the three levels of contamination. The ranges of the limits of detection and of the limits of quantification in soils HS were from 3.42 to 23.77 μg kg−1 and from 11.41 to 79.23 μg kg−1, respectively. For LS soils, the limits of detection ranged from 6.11 to 14.78 μg kg−1 and the limits of quantification from 20.37 to 49.27 μg kg−1. In the 14 collected soil samples only one showed a residue of dieldrin (45.36 μg kg−1) above the limit of quantification. This methodology combines the advantages of QuEChERS, GC-ECD detection and GC-MS/MS confirmation producing a very rapid, sensitive and reliable procedure which can be applied in routine analytical laboratories.

QuEChERS: a new sample preparation approach for the determination of ibuprofen and its metabolites in soils

Ibuprofen is one of the most used active pharmaceutical ingredients worldwide. A new method for the analysis of ibuprofen and its metabolites, hydroxyibuprofen and carboxyibuprofen, in soils is presented. The extraction of these compounds from the soil matrices was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. The method involves a single extraction of the investigated compounds with purified water (acidified at pH 2.5 with hydrochloric acid), and a slow and continuous addition of the QuEChERS content, followed by the addition of acidified acetonitrile (1% acetic acid), prior to the determination by liquid chromatography coupled with fluorescence detection (LC–FLD). Validation studies were carried out using soil samples with a range of organic carbon contents. Recoveries of the fortified samples ranged from 79.5% to 101%. Relative standard deviations for all matrix–compound combinations did not exceed 3%. The method quantification limits were ≤22.4 μg kg−1 in all cases. The developed method was applied to the analysis of sixteen real samples.

Extraction of ochratoxin A in bread samples by the QuEChERS methodology

A QuEChERS method for the extraction of ochratoxin A (OTA) from bread samples was evaluated. A factorial design (23) was used to find the optimal QuEChERS parameters (extraction time, extraction solvent volume and sample mass). Extracts were analysed by LC with fluorescence detection. The optimal extraction conditions were: 5 g of sample, 15 mL of acetonitrile and 3 min of agitation. The extraction procedure was validated by systematic recovery experiments at three levels. The recoveries obtained ranged from 94.8% (at 1.0 μg kg -1) to 96.6% (at 3.0 μg kg -1). The limit of quantification of the method was 0.05 μg kg -1. The optimised procedure was applied to 20 samples of different bread types (‘‘Carcaça’’, ‘‘Broa de Milho’’, and ‘‘Broa de Avintes’’) highly consumed in Portugal. None of the samples exceeded the established European legal limit of 3 μg kg -1.

Monitorização de pesticidas em águas de Esposende

De forma a proteger o ambiente e a saúde humana, é imperativo evitar, prevenir ou reduzir as concentrações prejudiciais de poluentes nocivos na água subterrânea. A necessidade da obtenção de níveis de protecção da água subterrânea, encontra-se estabelecida em normas de qualidade e devem ser desenvolvidas metodologias que permitam a avaliação do estado químico da água subterrânea. Este trabalho experimental centrou-se no desenvolvimento de uma metodologia analítica de detecção e quantificação por cromatografia gasosa com detector de captura de electrões dos pesticidas atrazina e respectivos metabolitos (desetilatrazina e deisopropilatrazina), simazina, terbutilazina e o metabolito desetiterbutilazina, folpete, dimetoato, diazinão, malatião, cloropirifos e o azinfos-metilo em águas de poços. O estudo progressivo baseou-se na colheita de água a 20 poços agrícolas na zona de Esposende, área considerada pelo Ministério da Agricultura do Desenvolvimento Rural e Pescas como sendo uma zona vulnerável. O método utilizado para a validação da técnica cromatográfica baseou-se na norma ISO 8466-1:1990. Os parâmetros de validação considerados foram: especificidade/selectividade, capacidade de identificação, limites de detecção e quantificação, relação sinal/ruído, linearidade e curva de calibração, precisão (repetibilidade, precisão intermédia e reprodutibilidade), eficiência de extracção e arrastamento. O método demonstrou ser capaz de identificar e quantificar os analitos, sem interferência de outros compostos. Obteve-se um valor para os parâmetros da precisão inferior a 10%, enquanto os mais baixos limites de detecção e de quantificação foram, respectivamente, 0,014 e 0,047 μg L-1. Na preparação de amostras optou-se pelo método de extracção em fase sólida, tendo sido testadas cinco diferentes tipos de colunas extractivas; Lichrolut® EN/RP-18; Strata SDB-L e C18-E; Chromabond HR-P e HR-X, sendo que as colunas Lichrolut® EN/RP- 18 apresentaram melhores resultados para a globalidade dos pesticidas. Da análise efectuada aos 20 poços agrícolas verificou-se que apenas 3 não apresentavam qualquer vestígio dos pesticidas monitorizados, sendo que as restantes apresentavam valores entre 0,05 e 53,2 μg L-1, valores superiores aos impostos pela legislação em vigor (Decreto-Lei n.º 208/2008 de 28 de Outubro para água subterrânea e Decreto-Lei nº306/2007 referente a água para consumo). Verificou-se que os proprietários dos poços agrícolas, dos quais se procedeu à amostragem de água para análise não têm a consciência da falta de qualidade dessa água, nem dos malefícios que possam advir do seu consumo.

Monitorização de cobre em vinhos verdes

A presente dissertação descreve o desenvolvimento e a caracterização de sensores potenciométricos com base em polímeros de impressão molecular e de sensores ópticos com base em membranas de poli(cloreto de vinilo), PVC, para a determinação de cobre em vinhos verdes. Os sensores potenciométricos foram preparados a partir de diferentes solventes (metanol e clorofórmio), tendo o seu crescimento decorrido na presença ou ausência da molécula molde (cobre). Os sistemas sensores selectivos ao cobre continham partículas de polímeros com ou sem impressão molecular como material electroactivo, dispersas em solvente plastificante, PVC e, em alguns casos, aditivo aniónico. A avaliação dos vários sistemas baseou-se na comparação das características operacionais dos diversos eléctrodos onde foram aplicados. Estas características foram obtidas a partir de curvas de calibração, cujos declives e limites de detecção variaram entre -39,9 – 37,0 mV decada-1 e 4,2 – 29,1 μg mL-1, respectivamente. Os sensores não são independentes do pH uma vez que o complexo formado entre o cobre e a difenilcarbazida é favorecido por valores de pH próximos de 5. Assim, obtiveram-se melhores resultados usando água desionizada ou solução tampão de HEPES revelando-se um método rápido e relativamente eficaz nestas condições. Os sensores ópticos basearam-se na reacção colorimétrica entre o cobre e um complexante. Os reagentes complexantes escolhidos foram a neocuproína, a difenilcarbazida e o dietilditiocarbamato de sódio. Avaliou-se o efeito de vários parâmetros experimentais na resposta destes sensores, tais como o pH (avaliado para os valores 3,00 e 5,00), a concentração de cobre (que variou entre 0,06 e 317,7 mg L- 1) e as próprias características da membrana. Os melhores resultados foram obtidos a pH 3, numa gama de concentrações de 0,06 e 31,8 mg L-1 usando a difenilcarbazida como reagente complexante. A aplicação destes sensores a vinhos requer ainda estudos adicionais, especialmente no que diz respeito à necessidade de implementar algum procedimento de pré-tratamento de amostra.

Rapid determination of tartaric acid in wines

A flow-spectrophotometric method is proposed for the routine determination of tartaric acid in wines. The reaction between tartaric acid and vanadate in acetic media is carried out in flowing conditions and the subsequent colored complex is monitored at 475 nm. The stability of the complex and the corresponding formation constant are presented. The effect of wavelength and pH was evaluated by batch experiments. The selected conditions were transposed to a flowinjection analytical system. Optimization of several flow parameters such as reactor lengths, flow-rate and injection volume was carried out. Using optimized conditions, a linear behavior was observed up to 1000 µg mL-1 tartaric acid, with a molar extinction coefficient of 450 L mg-1 cm-1 and ± 1 % repeatability. Sample throughput was 25 samples per hour. The flow-spectrophotometric method was satisfactorily applied to the quantification of tartaric acid (TA) in wines from different sources. Its accuracy was confirmed by statistical comparison to the conventional Rebelein procedure and to a certified analytical method carried out in a routine laboratory.

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.

FIA potentiometric system based on periodate polymeric membrane sensors for the assessment of ascorbic acid in commercial drinks

Ascorbic acid is found in many food samples. Its clinical and technological importance demands an easyto- use, rapid, robust and inexpensive method of analysis. For this purpose, this work proposes a new flow procedure based on the oxidation of ascorbic acid by periodate. A new potentiometric periodate sensor was constructed to monitor this reaction. The selective membranes were of PVC with porphyrin-based sensing systems and a lipophilic cation as additive. The sensor displayed a near-Nernstian response for periodate over 1.0x10-2–6.0x10-6 M, with an anionic slope of 73.9 ± 0.9 mV decade-1. It was pH independent in acidic media and presented good selectivity features towards several inorganic anions. The flow set-up operated in double-channel, carrying a 5.0x10-4 M IO- 4 solution and a suitable buffer; these were mixed in a 50-cm reaction coil. The overall flow rate was 7 ml min-1 and the injection volume 70 µl. Under these conditions, a linear behaviour against concentration was observed for 17.7–194.0 µg ml-1, presenting slopes of 0.169 mV (mg/l)-1, a reproducibility of ±1.1 mV (n = 5), and a sampling rate of ~96 samples h-1. The proposed method was applied to the analysis of beverages and pharmaceuticals.

Contribution of hospital effluents to the load of pharmaceuticals in urban wastewaters: Identification of ecologically relevant pharmaceuticals

The impact of effluent wastewaters from four different hospitals: a university (1456 beds), a general (350 beds), a pediatric (110 beds) and a maternity hospital (96 beds), which are conveyed to the same wastewater treatment plant (WWTP), was evaluated in the receiving urban wastewaters. The occurrence of 78 pharmaceuticals belonging to several therapeutic classes was assessed in hospital effluents and WWTP wastewaters (influent and effluent) as well as the contribution of each hospital in WWTP influent in terms of pharmaceutical load. Results indicate that pharmaceuticals are widespread pollutants in both hospital and urban wastewaters. The contribution of hospitals to the input of pharmaceuticals in urban wastewaters widely varies, according to their dimension. The estimated total mass loadings were 306 g d− 1 for the university hospital, 155 g d− 1 for the general one, 14 g d− 1 for the pediatric hospital and 1.5 g d− 1 for the maternity hospital, showing that the biggest hospitals have a greater contribution to the total mass load of pharmaceuticals. Furthermore, analysis of individual contributions of each therapeutic group showed that NSAIDs, analgesics and antibiotics are among the groups with the highest inputs. Removal efficiency can go from over 90% for pharmaceuticals like acetaminophen and ibuprofen to not removal for β-blockers and salbutamol. Total mass load of pharmaceuticals into receiving surface waters was estimated between 5 and 14 g/d/1000 inhabitants. Finally, the environmental risk posed by pharmaceuticals detected in hospital and WWTP effluents was assessed by means of hazard quotients toward different trophic levels (algae, daphnids and fish). Several pharmaceuticals present in the different matrices were identified as potentially hazardous to aquatic organisms, showing that especial attention should be paid to antibiotics such as ciprofloxacin, ofloxacin, sulfamethoxazole, azithromycin and clarithromycin, since their hazard quotients in WWTP effluent revealed that they could pose an ecotoxicological risk to algae.

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.

Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata

The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmolL−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The shortterm assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μgL−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.

Optimization of HS-SPME analytical conditions using factorial design for trihalomethanes determination in swimming pool water samples

Trihalomethanes (THMs) are widely referred and studied as disinfection by-products (DBPs). The THMs that are most commonly detected are chloroform (TCM), bromodichloromethane (BDCM), chlorodibromomethane (CDBM), and bromoform (TBM). Several studies regarding the determination of THMs in swimming pool water and air samples have been published. This paper reviews the most recent work in this field, with a special focus on water and air sampling, sample preparation and analytical determination methods. An experimental study has been developed in order to optimize the headspace solid-phasemicroextraction (HS-SPME) conditions of TCM, BDCM, CDBM and TBM from water samples using a 23 factorial design. An extraction temperature of 45 °C, for 25min, and a desorption time of 5 min were found to be the best conditions. Analysis was performed by gas chromatography with an electron capture detector (GC-ECD). The method was successfully applied to a set of 27 swimming pool water samples collected in the Oporto area (Portugal). TCM was the only THM detected with levels between 4.5 and 406.5 μg L−1. Four of the samples exceeded the guideline value for total THMs in swimming pool water (100 μgL−1) indicated by the Portuguese Health Authority.

SPE-LC-FD Determination of Polycyclic Aromatic Hydrocarbon Monohydroxy Derivatives in Cephalopods

A new analytical methodology, based on liquid chromatography with fluorescence detection (LC-FD), after extraction, enzymatic hydrolysis, and solid-phase extraction (SPE) through Oasis HLB cartridges, was developed and validated for the simultaneous determination of three monohydroxy derivatives of polycyclic aromatic hydrocarbons (PAHs). The optimized analytical method is sensitive, accurate, and precise, with recoveries between 62 and 110% and limits of detection of 227, 9, and 45 ng/g for 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, respectively. Their levels were estimated in different cephalopod matrices (edible tissues and hemolymph). The methodology was applied to samples of the major cephalopod species consumed worldwide. Of the 18 samples analyzed, 39% were contaminated with 1-hydroxynaphthalene, which was the only PAH metabolite detected. Its concentration ranged from 786 to 1145 ng/g. This highly sensitive and specific method allows the identification and quantitation of PAH metabolites in forthcoming food safety and environmental monitoring programs.