Annibal Duarte Pereira Netto

Avaliação da contaminação humana por hidrocarbonetos policíclicos aromáticos (HPAs) e seus derivados nitrados (NHPAs): uma revisão metodológica

Polycyclic aromatic hydrocabons (PAHs) and their nitroderivatives (NPAHs) are ubiquitous in the environment and they are produced in several industrial and combustion processes. Some of these compounds are potent carcinogens/mutagens and their determination in biological samples is an important step for exposure control. A review of the analytical methodologies used for the determination of PAHs and their metabolites in biological samples is presented.

Optimization and application of methods of triacylglycerol evaluation for characterization of olive oil adulteration by soybean oil with HPLC-APCI-MS-MS.

Triacylglycerols (TAGs) are the main constituents of vegetable oils where they occur in complex mixtures with characteristic distributions. Mass spectrometry using an atmospheric pressure chemical ionization interface (APCI-MS) run in positive mode and an Ion Trap mass analyser were applied in the study of olive and soybean oils and their mixtures. Direct injections of soybean and olive oil solutions allowed the identification of ions derived from the main TAGs of both oils. This procedure showed to be a simple and powerful tool to evaluate mixtures or addition of soybean to olive oil. TAG separation was optimized by high performance liquid chromatography (HPLC) using an octadecylsilica LiChrospher column (250mm x 3mm; 5microm) and a gradient composed of acetonitrile and 2-propanol allowed the separation of the main TAGs of the studied oils. APCI vaporization temperature was optimized and best signals were obtained at 370 degrees C. Multiple reaction monitoring (MRM) employing the transition of the protonated TAG molecules ([M+H](+)) to the protonated diacylglycerol fragments ([M+H-R](+)) improved the selectivity of TAG detection and was used in quantitative studies. Different strategies were developed to evaluate oil composition following TAG analysis by MRM. The external standard calibration and standard additions methods were compared for triolein quantification but the former showed to be biased. Further quantitative studies were based on the estimates of soybean and olive oil proportions in mixtures by comparison of TAG areas found in mixtures of known and unknown composition of both oils. Good agreement with expected or labeled values was found for a commercial blend containing 15% (w/w) of olive oil in soybean oil and to a 1:1 mixture of both oils, showing the potential of this method in characterizing oil mixtures and estimating oil proportions. Olive oils of different origins were also evaluated by mass spectra data obtained after direct injections of oil solutions and principal component analysis (PCA). Argentinean olive oils were clustered in a different area of the principal components plot (PC2 xP C1) in comparison with European olive oils. The commercial blend containing 15% (w/w) of olive oil in soybean oil appeared in a completely different area of the graphic, showing the potential of this method to screen out for olive oil adulterations.

Multivariate optimization of a liquid-liquid extraction of the EPA-PAHs from natural contaminated waters prior to determination by liquid chromatography with fluorescence detection.

This paper reports the multivariate optimization of a liquid-liquid extraction procedure for the determination of 15 EPA-polycyclic aromatic hydrocarbons (PAHs) by high-performance liquid chromatography with fluorescence detection. A Doehlert design was used to find optimum conditions for the procedure through Response Surface Methodology. Three variables (total volume of hexane, number of extraction steps and duration of such steps) were elected as factors in the optimization study. A principal component analysis (PCA) was run with optimized data, resulting in four groups of PAHs, ordered according to their molecular weight. Final working conditions were established in order to achieve a more robust methodology in relation to all fifteen PAHs under study. Best results could be observed when 77 mL of hexane were divided in four consecutive extraction steps with 18 min each. These experimental conditions were applied in the analysis of a spiked river water sample, and the recoveries varied between 80.9 and 106%, with an average value of 97.1+/-6.8%. The application of the methodology to river water showed that the method has a good average precision for the studied PAHs.

Is it possible to screen for milk or whey protein adulteration with melamine, urea and ammonium sulphate, combining Kjeldahl and classical spectrophotometric methods?

The Kjeldahl method and four classic spectrophotometric methods (Biuret, Lowry, Bradford and Markwell) were applied to evaluate the protein content of samples of UHT whole milk deliberately adulterated with melamine, ammonium sulphate or urea, which can be used to defraud milk protein and whey contents. Compared with the Kjeldahl method, the response of the spectrophotometric methods was unaffected by the addition of the nitrogen compounds to milk or whey. The methods of Bradford and Markwell were most robust and did not exhibit interference subject to composition. However, the simultaneous interpretation of results obtained using these methods with those obtained using the Kjeldahl method indicated the addition of nitrogen-rich compounds to milk and/or whey. Therefore, this work suggests a combination of results of Kjeldahl and spectrophotometric methods should be used to screen for milk adulteration by these compounds.

Optimization and comparison of HPLC and RRLC conditions for the analysis of carbonyl-DNPH derivatives

Analytical conditions for the analysis of 15 carbonyl-DNPH derivatives were optimized and compared by high performance liquid chromatography (HPLC) and rapid resolution liquid chromatography (RRLC). Binary, ternary and quaternary mixtures of acetonitrile, isopropanol, methanol, tetrahydrofuran and water were evaluated under RRLC conditions employing a Zorbax Eclipse Plus C18 (50 mm x 4.6 mm x 1.8 microm) column and a Zorbax Eclipse Plus C18 (50 mm x 2.1 mm x 1.8 microm) column. The optimized conditions obtained employing the two RRLC columns were compared with those obtained using a Supelcosil C18 (250 mm x 4.6 mm x 5 microm; Supelco) that is designed for HPLC separation of DNPH derivatives. Chromatograms run with a Zorbax Eclipse Plus C18 (50 mm x 2.1 mm x 1.8 microm) column and a mobile phase composed of isopropanol, methanol, tetrahydrofuran and water led to the best separation conditions considering reduced analysis time (approximately 6 min per run), solvent consumption rate (approximately 2 mL per run) and resolution of propanone, acrolein and propionaldehyde hydrazones. Quantification limits and linear ranges were adequate for direct application of EPA TO-11 conditions in all sets of RRLC and HPLC conditions. The analytical method was applied in the determination of carbonyl compounds (CCs) in Niterói City, RJ, Brazil in samples that were collected during periods of 2h. Formaldehyde (8.22-9.78 ppbv) predominated in all periods followed by acetaldehyde (1.77-3.99ppbv) and propanone (1.89-3.26 ppbv). Heavy CCs such as butyraldehyde and benzaldehyde were also detected in most samples. Total CCs varied along the studied day. The obtained results showed that RRLC can be applied to CCs determination without any change in the conditions of sample preparation of the Method EPA TO-11.

Characterization of newfound natural luminescent properties of melamine, and development and validation of a method of high performance liquid chromatography with fluorescence detection for its determination in kitchen plastic ware

Experimental studies of the natural photoluminescence of melamine in aqueous solutions showed that its fluorescence intensity (at 250/365 nm) was appropriated for analytical purposes. The exploitation of such melamine property provided the basis of development of a new, simple, precise and accurate method based on high performance liquid chromatography with fluorescence detection (HPLC-Fluo) to determine melamine in kitchen plastic ware following aqueous extraction using a microwave oven. Optimization of analytical parameters such as solvent composition, pH and extraction conditions led to limits of detection and quantification of melamine of 0.0081 and 0.027 μg mL(-1), respectively, with a linear range up to 10 μg mL(-1). Sample extracts fortified with melamine at three concentration levels produced an average recovery of 98±6%, which was in agreement with the results achieved with a reference HPLC-UV method. Different samples of kitchen plastic ware analyzed by the developed and optimized method showed melamine concentrations in the aqueous extract up to 17 µg mL(-1), which corresponded to 86.0 mg kg(-1) in these utensils. The results obtained indicate that the use of kitchen plastic ware made of melamine can contaminate food with this compound after heating in a microwave oven.

Polycyclic aromatic hydrocarbons (PAHs) in street dust of Rio de Janeiro and Niterói, Brazil: Particle size distribution, sources and cancer risk assessment

Fifteen polycyclic aromatic hydrocarbons (PAHs) were analyzed in two fractions (<63μm and 63-850μm) of street dust samples collected in different urban areas of Rio de Janeiro and Niterói cities, Brazil. Individual PAH concentrations and total PAH concentrations (∑PAH) were evaluated considering geographic and particle size distributions, PAH source and cancer risk. In 53% of the samples, ∑PAH values were higher in the smallest particles than in the largest ones, whereas the inverse was true for 23.5% of the samples. In 23.5% of the samples, both classes of particles showed comparable ∑PAH. The highest ∑PAH concentrations were found in areas of limited air dispersion, resulting in PAH accumulation in street dust. The diagnostic ratios and principal component analysis (PCA) indicated a strong influence of pyrogenic sources on PAH concentration, mainly of vehicular emissions. The cancer risk levels for children and adults, for both particulate size fractions (<63μm and 63μm-850μm), were comparable for dermal contact and by ingestion, and ranged from 10-8 to 10-6, whereas in certain cases the cancer risk level for adults by dermal contact was 2 to 3 times larger than for children. The cancer risk levels via inhalation always ranged from 10-13 to 10-10, so this exposure pathway was almost 104 less severe than through ingestion and dermal contact, and thus negligible.

Polycyclic aromatic hydrocarbons in Tripuí River, Ouro Preto, MG, Brazil

This paper reports the determination of 15 EPA-polycyclic aromatic hydrocarbons (PAHs) and benzo[e]pyrene in water samples collected in Tripuí River, Ouro Preto City, MG, Brazil. Samples were collected between September 2006 (dry season) and November 2006 (wet season) in the neighborhood of an aluminum smelter. Detection limits and quantification limits were sufficiently low to accomplish PAH determination below the maximum concentration levels established by the Brazilian and USEPA legislations. Recoveries from water spiked samples were always larger than 89%. Fluoranthene, pyrene, phenanthrene, chrysene and benzo[b]fluoranthene predominated in the studied samples. The concentrations of PAHs upstream the aluminum smelter were systematically lower than those found downstream indicating a possible role of the smelter in the local pollution by PAHs. Principal component analysis and cluster analysis also showed remarkable differences of the characteristics of samples collected upstream and downstream the aluminum smelter and also of samples from wet and dry seasons.

Development and validation of a method for the determination of low-ppb levels of macrocyclic lactones in butter, using HPLC-fluorescence.

An analytical method was developed and validated for the simultaneous determination of four macrocyclic lactones (ML) (abamectin, doramectin, ivermectin and moxidectin) in butter, using liquid chromatography with fluorescence detection. The method employed heated liquid-liquid extraction and a mixture of acetonitrile, ethyl acetate and water, with preconcentration and derivatization, to produce stable fluorescent derivatives. The chromatographic run time was <12.5 min, with excellent separation. The method validation followed international guidelines and employed fortified butter samples. The figures of merit obtained, e.g. recovery (72.4-106.5%), repeatability (8.8%), within-laboratory reproducibility (15.7%) and limits of quantification (0.09-0.16 μg kg(-1)) were satisfactory for the desired application. The application of the method to real samples showed that ML residues were present in six of the ten samples evaluated. The method proved to be simple, easy and appropriate for simultaneous determination of ML residues in butter. To our knowledge, this is the first method described for the evaluation of ML in butter.

The validation of a new high throughput method for determination of chloramphenicol in milk using liquid–liquid extraction with low temperature partitioning (LLE-LTP) and isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS)

Chloramphenicol (CAP) is an antibiotic banned for treatment of food-producing animals. The minimum required performance limit (MRPL) of 0.3 μg kg−1 for analytical methods was set by the European Community for the detection of its residues in different matrices, including milk. A highly sensitive analytical method using isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and liquid–liquid extraction with low temperature partitioning (LLE-LTP) was developed and validated for CAP residues in milk. Selected validation parameters such as selectivity, working range and linearity, trueness (recoveries), precision (repeatability and intermediate precision), limit of detection (LOD) and limit of quantification (LOQ) were evaluated. The validation procedures were based on the new Eurachem Guide and the European Union Commission Decision 2002/657/EC. The matrix effect was evaluated by the ion suppression test and by comparison of the matrix-matched analytical curve and the solvent standard analytical curve. A linear working range between 0.1 and 5.0 μg kg−1 was observed. The homoscedasticity was demonstrated by the Cochran test, the coefficient of determination (R2) was higher than 0.99 and the residual plot was free of trends. The limits of detection and quantification were 0.015 μg kg−1 and 0.05 μg kg−1, respectively. Mean recoveries evaluated at three levels (0.3, 1.0 and 3.0 μg kg−1) ranged from 94% to 114% with RSDs lower than 6.7% (repeatability). For intermediate precision, different analytes were compared and the RSDs were lower than 7.3%. The method was accurate and reproducible, and was successfully applied to the evaluation of CAP residues in milk samples.