Armindo Melo

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.

Effect of Beer/Red Wine Marinades on the Formation of Heterocyclic Aromatic Amines in Pan-Fried Beef

The effect of beer or red wine marinades on the reduction of heterocyclic aromatic amines (HAs) formation in pan-fried beef was compared. The cooking experiments were performed under well-controlled temperature and time conditions. The samples were analyzed for HAs contents using solid-phase extraction and high-performance liquid chromatography-diode array detection/fluorescence detection. Unmarinated samples cooked in similar conditions provided reference HAs levels. Marinating with beer or with red wine resulted in decreased levels of HAs. The amount of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline reduced significantly, respectively, around 88 and 40% after 6 h of marinating with beer or with wine. High variations were observed for reductions of AalphaC, ranging between 7 and 77%. Only beer marinade significantly reduced the levels of 4,8-DiMeIQx at 1, 2, and 4 h of marinating. Multivariate statistical treatment of results indicated that beer can be more efficient on the reduction of some HAs formation. In addition, results from descriptive sensory analysis of unmarinated and 2 h marinated beef samples, tested for by two trained sensory panels, pointed to beer marinade as the most adequate for maintaining the usual overall appearance and quality of the pan-fried steaks.

Monitoring pesticide residues in greenhouse tomato by combining acetonitrile-based extraction with dispersive liquid-liquid microextraction followed by gas-chromatography-mass spectrometry.

A multiclass and multiresidue method for pesticide analysis in tomato was validated. Extraction and pre-concentration of the pesticide residues from acetonitrile extracts was performed by using dispersive liquid-liquid microextraction (DLLME) technique, followed by gas chromatography-mass detection. DLLME was performed using carbon tetrachloride as extractive solvent and acetonitrile extract as dispersive solvent, in order to increase enrichment factor of the extraction procedure. Validation parameters indicated the suitability of the method for routine analyses of thirty pesticides in a large number of samples. In general, pesticide recoveries ranged between 70% and 110% and repeatability ranged between 1% and 20%. The proposed method was applied to the monitoring of pesticides in tomatoes grown during winter in greenhouses. Among the compounds considered in this work, cyprodinil was found in tomato at concentrations of 0.33mg/kg, other pesticides like azoxystrobin, fenhexanid, tolyfluanid, λ-cyhalothrin and trifloxystrobin were also detected, but, not quantified.

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.

Quantification of furanic compounds in coated deep-fried products simulating normal preparation and consumption: optimisation of HS-SPME analytical conditions by response surface methodology.

The validation of a method for the simultaneous quantification of furanic compounds in coated deep-fried samples processed and handled as usually consumed is presented. The deep-fried food was grinded using a device that simulates the mastication, and immediately analysed by headspace solid phase microextraction coupled to gas chromatography-mass spectrometry. Parameters affecting the efficiency of HS-SPME procedure were selected by response surface methodology, using a 2(3) full-factorial central composite design. Optimal conditions were achieved using 2g of sample, 3g of NaCl and 40min of absorption time at 37°C. Consistency between predicted and experimented values was observed and quality parameters of the method were established. As a result, furan, 2-furfural, furfuryl alcohol and 2-pentylfuran were, for the first time, simultaneously detected and quantified (5.59, 0.27, 10.48 and 1.77μgg(-1) sample, respectively) in coated deep-fried fish, contributing to a better understanding of the amounts of these compounds in food.

Sensitive quantitation of polyamines in plant foods by ultrasound-assisted benzoylation and dispersive liquid-liquid microextraction with the aid of experimental designs.

A new method involving ultrasound-assisted benzoylation and dispersive liquid-liquid microextraction was optimized with the aid of chemometrics for the extraction, cleanup, and determination of polyamines in plant foods. Putrescine, cadaverine, spermidine, and spermine were derivatized with 3,5-dinitrobenzoyl chloride and extracted by dispersive liquid-liquid microextraction using acetonitrile and carbon tetrachloride as dispersive and extraction solvents, respectively. Two-level full factorial design and central composite design were applied to select the most appropriate derivatization and extraction conditions. The developed method was linear in the 0.5-10.0 mg/L range, with a R(2) ≥ 0.9989. Intra- and interday precisions ranged from 0.8 to 6.9% and from 3.0 to 10.3%, respectively, and the limit of detection ranged between 0.018 and 0.042 μg/g of fresh weight. This method was applied to the analyses of six different types of plant foods, presenting recoveries between 81.7 and 114.2%. The method is inexpensive, versatile, simple, and sensitive.

EXTRACTION, DETECTION AND QUANTIFICATION OF HETEROCYCLIC AROMATIC AMINES IN PORTUGUESE MEAT DISHES BY HPLC/DIODE ARRAY

Abstract The present paper describes the results obtained in the analyses of heterocyclic aromatic amines (HAs) in bovine meat dishes prepared by three different coking methods; usually ingredients such as, salt, garlic, wine, olive oil, onion, and tomato were added. Control meat samples (cooked without ingredients) were also prepared. Analyses were performed by HPLC/diode array and detection of the eluted HAs was performed at 263 nm. The calibration graphs for fourteen amines injected into the column were linear up to approximately 2.0 ng/μL and the detection limits (signal‐to‐noise ratio 3:1) ranged from 0.06 to 0.4 ng injected. Nine HAs, namely 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), 2‐amino‐3,4,8‐trimethylimidazo[4,5‐f]quinoxaline (4,8‐DiMeIQx), 2‐amino‐1‐methyl‐6‐phenylimidazol[4,5]pyridine (PhIP), 3‐amino‐1,4‐dimethyl‐5H‐Pyrido[4,3‐b]indole (Trp‐P‐1), 3‐amino‐1‐methyl‐5H‐Pyrido[4,3‐b]indole (Trp‐P‐2), 2‐amino‐9H‐Pyrido [2,3‐b]indole (AαC) 2‐amino‐3‐methyl‐9H‐Pyrido [2,3‐b]indole (MeAαC), 2‐amino‐6‐methyldipyrido[1,2‐a:3′,2′‐d]imidazole (Glu‐P‐1) were encountered in meat samples. A paired Students t‐test showed no significant differences on HAs levels (p > 0.05) of control meat samples (cooked without ingredients) and meat samples cooked with ingredients usually in the Portuguese diet and rich in antioxidants. Changes in cooking habits are needed to make possible the use of ingredients not only as flavoring but also as protective agents against HAs formation during domestic cooking.

Identification and quantification of anthocyanins in fruits from Neomitranthes obscura (DC.) N. Silveira an endemic specie from Brazil by comparison of chromatographic methodologies.

Neomitranthes obscura (DC.) N. Silveira is a Brazilian fruit belonging to the Myrtaceae family that contains anthocyanins in the peel and was studied for the first time in this work. Delphinidin-3-O-galactoside, delphinidin-3-O-glucoside, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-galactoside, peonidin-3-O-glucoside, cyanidin-3-O-xyloside were separated and identified by LC/DAD/MS and by co-elution with standards. Reliable quantification of anthocyanins in the mature fruits was performed by HPLC/DAD using weighted linear regression model from 0.05 to 50mg of cyaniding-3-O-glucoside L(-1) because it gave better fit quality than least squares linear regression. Good precision and accuracy were obtained. The total anthocyanin content of mature fruits was 263.6 ± 8.2 mg of cyanidin-3-O-glucoside equivalents 100 g(-1) fresh weight, which was in the same range found in literature for anthocyanin rich fruits.

Application of a fast and cost-effective in situ derivatization method prior to gas chromatography with mass spectrometry to monitor endocrine disruptors in water matrices.

This work deals with the optimization of a rapid, cost-effective, and eco-friendly gas chromatography with mass spectrometry method for the simultaneous determination of four endocrine disruptor compounds in water matrices: estrone, 17β-estradiol, 17α-ethinylestradiol, and bisphenol A, that are currently considered to be of main concern in the field of water policy and that could became candidates for future regulations. The method involves simultaneous derivatization and extraction of compounds by dispersive liquid-liquid microextraction followed by gas chromatography with mass spectrometry analysis. Derivatization and extraction parameters were optimized with the aid of experimental design approach. An excellent linear response was achieved for all analytes (r(2) ≥ 0.999). Limits of detection and quantification are 0.003-0.005 and 0.0094-0.0164 μg/L, respectively. Intraday precision ranged between 1.1 and 12.6%, whereas interday precision ranged between 0.5 and 14.7%. For accuracy, bias values varied between -15.0 and 13.7%. Recoveries at three concentration levels ranged from 86.4 to 118.2%. The proposed method can be applied to the routine analysis of groundwater, river, sea, tap, and mineral water samples with excellent sensitivity, precision, and accuracy.

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.