Carina Pinho

Quantification of endocrine disruptors and pesticides in water by gas chromatography–tandem mass spectrometry. Method validation using weighted linear regression schemes

A multi-residue methodology based on a solid phase extraction followed by gas chromatography-tandem mass spectrometry was developed for trace analysis of 32 compounds in water matrices, including estrogens and several pesticides from different chemical families, some of them with endocrine disrupting properties. Matrix standard calibration solutions were prepared by adding known amounts of the analytes to a residue-free sample to compensate matrix-induced chromatographic response enhancement observed for certain pesticides. Validation was done mainly according to the International Conference on Harmonisation recommendations, as well as some European and American validation guidelines with specifications for pesticides analysis and/or GC-MS methodology. As the assumption of homoscedasticity was not met for analytical data, weighted least squares linear regression procedure was applied as a simple and effective way to counteract the greater influence of the greater concentrations on the fitted regression line, improving accuracy at the lower end of the calibration curve. The method was considered validated for 31 compounds after consistent evaluation of the key analytical parameters: specificity, linearity, limit of detection and quantification, range, precision, accuracy, extraction efficiency, stability and robustness.

Optimization of Conditions for Anthocyanin Hydrolysis from Red Wine Using Response Surface Methodology (RSM)

Optimization of conditions for anthocyanin hydrolysis from red wine was investigated using response surface methodology. The aglycon forms of the anthocyanins were quantified by high-performance liquid chromatography with diode array detection. The combined effects of three independent variables, HCl amount, heating temperature, and hydrolysis time, were studied using a 2(3) full-factorial central composite design. Anthocyanin hydrolysis yield depended mainly on the heating temperature and time of hydrolysis. HCl amount was the factor that least influenced the hydrolysis of anthocyanins. From experimental results, the maximum yield of anthocyanidins was reached with 9.8 mL of HCl (32% v/v), a heating temperature of 166.2 °C, and a hydrolysis time of 46.6 min. Five anthocyanidins, namely, delphinidin, cyanidin, petunidin, peonidin, and malvidin, were quantified in red wine. The reliability of the method was confirmed by recovery experiments, performed under optimal conditions. Recoveries indicated that anthocyanidins resisted the hydrolysis conditions.

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, 17β-estradiol and 17α-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.

Fatty acids derived from a food frequency questionnaire and measured in the erythrocyte membrane in relation to adiponectin and leptin concentrations

Background/objectives:Evidence on the association between fatty acids and adiponectin and leptin concentrations is scarce and inconsistent, which may in part be due to limitations of dietary reporting methods. We aimed to estimate the association of fatty acids, derived from a food frequency questionnaire (FFQ) and measured in the erythrocyte membrane, with adiponectin and leptin concentrations.Subjects/methods:We studied 330 non-institutionalized inhabitants of Porto (52.4% women; age range: 26–64 years) evaluated in 2010–2011, as part of the EPIPorto cohort study. Fatty acids were derived from a validated semiquantitative FFQ and measured in the erythrocyte membrane by gas chromatography. Serum concentrations of adiponectin and leptin were determined through radioimmunoassay. Regression coefficients (β) and 95% confidence intervals (95% CI) were obtained from linear regression models, after controlling for gender, age, education, leisure time physical activity and total body fat percentage (obtained from dual energy X-ray absorptiometry).Results:Fatty acids measured by FFQ showed no significant associations with both adipokines. Lauric and linoleic acids, measured in the erythrocyte membrane, were significantly and positively associated with adiponectin (β=0.292, 95% CI: 0.168–0.416; β=0.150, 95% CI: 0.020–0.280) and leptin (β=0.071, 95% CI: 0.003–0.138; β=0.071, 95% CI: 0.002–0.140), whereas total n-3, eicosapentaenoic and docosahexaenoic acids were significantly but negatively associated with adiponectin (β=−0.289, 95% CI: −0.420 to −0.159; β=−0.174, 95% CI −0.307 to −0.040; β=−0.253, 95% CI −0.383 to −0.124) and leptin (β=−0.151, 95% CI: −0.220 to −0.083; β=−0.080, 95% CI: −0.151 to −0.009; β=−0.146, 95% CI: −0.214 to −0.078). Positive significant associations of palmitic and trans-fatty acids with adiponectin were also observed.Conclusions:A positive association of lauric and linoleic acids and a negative association of total n-3 fatty acids with both adipokines were observed only with fatty acids measured in the erythrocyte membrane.

Response surface methodology for optimization of cyanamide analysis by in situ derivatization and dispersive liquid-liquid microextraction

Cyanamide is widely used for agricultural purposes; therefore, its residues can be found in water. A new method was developed for its quantification using in situ derivatization with 2,6‐dimethyl‐4‐quinolinecarboxylic acid N‐hydroxysuccinimide ester followed by dispersive liquid–liquid microextraction (DLLME) and high‐performance liquid chromatography/fluorescence analysis. Multivariate chemometric techniques were successfully used to obtain the optimum conditions for direct derivatization and DLLME extraction. Derivatization parameters and DLLME extraction conditions were optimized by a two‐step design, 2k factorial design for screening, and central composite design for optimization. Best derivatization conditions were addition of 600 μL of derivatizing reagent, a temperature of 4 ºC, and pH 8.5, whereas for optimum extraction 800 μL of solvent, 30% NaCl conc. w/v, and pH 3.8 were chosen.

Impact of Freezing on Flavonoids/Radical-Scavenging Activity of Two Onion Varieties

Pinho C., Soares M.T., Almeida I.F., Aguiar A.A.R.M., Mansilha C., Ferreira I.M.P.L.V.O. (2015): Impact of freezing on flavonoids/radical-scavenging activity of two onion varieties. Czech J. Food Sci., 33: 340–345. Flavonols, anthocyanins, and radical-scavenging activity of two Portuguese onion cultivars (Branca da Povoa, white; and Vermelha da Povoa, red) were evaluated simulating domestic freezing conditions (–18°C). Frozen portions of onions with different periods of domestic storage at ambient temperature presented increased flavonoid content when compared with the respective composition before freezing. No significant differences were observed on radicalscavenging activity. Domestic freezing of onion portions extended its shelf life. Thus, domestic freezing can be a good alternative to prevent the loss of unused fresh onions, preserving its antioxidant capacity, since frozen onions can be a useful natural antioxidant source.