Isabel M. P. L. V. O. Ferreira

Quality evaluation of Portuguese honey

Abstract The present work was conducted to evaluate the quality of 25 brands of honey commercially available on the Portuguese market, in a total of 50 samples. The brands included unifloral and multifloral honeys, which were studied and botanically typified. Carbohydrate composition was determined by HPLC-RI to evaluate the contents of monosaccharides fructose and glucose; the disaccharides saccharose, maltose and trehalose and of the trisaccharide melizitose. 5-Hydroxymethyl-2-furfuraldehyde (HMF) was quantified by HPLC-UV and other physicochemical quality parameters were also carried out according to the European (Directive 74/409/EC) and Portuguese Regulations (Decreto-lei No. 131/85, 1985) in order to determine moisture, ash content, diastase activity, free acidity, free acidity and water-insoluble solids. All samples were organoleptically and microscopically examined. Only 13 brands were found to meet all major national and international specifications, the remaining 12 did not agree with one or more of the analysed parameters (HMF, diastase activity, or carbohydrate composition); such observations may reflect an inadequate manufacturing or storing process and a certain ageing of these honeys.

Detection and quantification of bovine, ovine and caprine milk percentages in protected denomination of origin cheeses by reversed-phase high-performance liquid chromatography of beta-lactoglobulins.

A method for detecting and quantifying bovine, ovine and caprine milk mixtures in milk and cheeses by means of reversed-phase high-performance liquid chromatography (RP-HPLC) of beta-lactoglobulins is described. Gradient elution was carried out with a flow rate of 0.5 ml/min and a temperature of 45 degrees C, using a mixture of two solvents: solvent A (0.1% TFA in water) and solvent B (0.09% TFA in 80% aqueous acetonitrile, v/v). The effluent was monitored at 215 nm. Under the conditions used different chromatographic patterns were obtained for bovine, ovine and caprine whey proteins. Each milk type presented different retention times for beta-lactoglobulin peaks. Binary mixtures of bovine and ovine or bovine and caprine raw milks containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of bovine milk, as well as ovine and caprine milk mixtures containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of ovine milk were used for cheese making. Cheeses were prepared and ripened, according to traditional methods. Milk mixtures, fresh and ripened cheeses were analyzed. A linear relationship was established between log 10 of beta-lactoglobulin peaks ratio (calculated as peak area values ratio) and log 10 of the relative percentage of bovine or ovine milk. The ratio between beta-lactoglobulin peaks was not affected by the degree of ripening. Thus, enabling the quantification of milk type percentage, with a detection limit of 2%. This technique allowed quantification of milk species within the concentration range of 5-95%. The method was successfully applied for authenticity evaluation and quantitative determination of ovine and caprine milk percentages of commercial protected denomination of origin (PDO) cheeses.

Quantification of residual nitrite and nitrate in ham by reverse-phase high performance liquid chromatography/diode array detector

Nitrite and nitrate are used as additives in ham industry to provide colour, taste and protect against clostridia. The classical colorimetric methods widely used to determine nitrite and nitrate are laborious, suffer from matrix interferences and involve the use of toxic cadmium. The use of chromatography is potentially attractive since it is more rapid, sensitive, selective and provides reliable and accurate results. A rapid and cost-effective RP-HPLC method with diode array detector was optimized and validated for quantification of nitrites and nitrates in ham. The chromatographic separation was achieved using a HyPurity C18, 5 microm chromatographic column and gradient elution with 0.01 M n-octylamine and 5mM tetrabutylammonium hydrogenosulphate to pH 6.5. The determinations were performed in the linear range of 0.0125-10.0mg/L for nitrite and 0.0300-12.5 g/L for nitrate. The detection limits were 0.019 and 0.050 mg/kg, respectively. The reliability of the method in terms of precision and accuracy was evaluated. Coefficients of variation lower than 2.89% and 5.47% were obtained for nitrite and nitrate, respectively (n=6). Recoveries of residual nitrite/nitrate ranged between 93.6% and 104.3%. Analysis of cooked and dried ham samples was performed, and the results obtained were in agreement with reference procedures.

Optimisation of extraction procedures for analysis of benzoic and sorbic acids in foodstuffs

Benzoic and sorbic acids are the most commonly used preservatives in foodstuffs. They are usually analysed by RP-HPLC. However, in view of the complexity and diversity of foodstuffs composition, appropriate sample preparation procedures are required for reliable extraction of these preservatives from the matrices. Specific extraction procedures for analysis of jams, table olives, spreadable fats, sauces, fruit juices and wines were optimised. Thus, different types of food matrices were chosen, including those with high sugar content, with high fat content and beverages (with and without alcohol). A significant set of validation data was performed through recovery and precision studies. Chromatographic separation was achieved using a C18 column (S10 ODS2) and acetate buffer 0.005 M (pH=4.4)—methanol (65:35) as mobile phase, 1.4 ml/min flow rate and UV detection at 235 nm. The concentration of preservatives in the samples was calculated by external standard method. Benzoic and sorbic acids in jams, jellies and table olives were efficiently extracted with methanol after ground homogenization. Fortified samples, at 4 different concentration levels of benzoic and sorbic acids, presented average recoveries (after discarding outliers) for each preservative greater than 91% with a coefficient of variation (CV) less than 2.6%. Sorbic acid was extracted from spreadable fats and emulsified sauces with n-hexane and was back-extracted to an aqueous phase with acetate buffer 0.005 M (pH=4.4). Recoveries were higher than 98% for two levels of concentration and CV lower than 2.9%. The preservatives extraction from fruit juices (orange, apple and pineapple) and wines required purification using a Sep-Pak C18 cartridge, and its elution with methanol. Average recoveries of benzoic and sorbic acids at two levels of concentration were greater than 94% with CV less than 4.0%. Eighty-seven commercial brands were analysed including table olives (29), jams (24), jellies (2) spreadable fats (25), sauces (3), fruit juices (10) and table wines (3). All samples conformed to the legal prescriptions.

Effect of temperature on evolution of free amino acid and biogenic amine contents during storage of Azeitão cheese

Abstract A study on the evolution of free amino acids and biogenic amines in Azeitao cheese during 4 weeks at different temperatures of storage (4 and 25°C) was performed. Free amino acids and biogenic amines were determined by RP-HPLC with visible detection, following extraction from the cheese and derivatization with dabsyl chloride. The method presented a linear relation between peak area and concentration from 2–200 mg/l. The detection limit value was less than 1.5 mg/l. The average repeatability was less than 4%. The major free amino acids were proline, valine, isoleucine and leucine and the major amines were tyramine, cadaverine and histamine. Room temperature (25°C) promoted a significant increase of the contents of valine, leucine, tyramine and putrescine, expressed as g/kg of dry matter. These two free amino acids and two biogenic amines may serve as indicators of temperatures changes in ripened cheese.

Separation and quantification of the major casein fractions by reverse-phase high-performance liquid chromatography and urea-polyacrylamide gel electrophoresis: Detection of milk adulterations

The separation and quantification of bovine kappa-, alpha- and beta-caseins by HPLC-UV using an RP column which contained polystyrene-divinilbenzene copolymer based packing was optimized and validated. Gradient elution was carried out at a flow-rate of 1 ml/min and a temperature of 46 degrees C, using a mixture of two solvents. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was acetonitrile-water-trifluoroacetic acid (95:5:0.1). The effluent was monitored by a UV detector at 280 nm. The determinations were performed in the linear range of 0.038-0.377 mg/ml for kappa-casein, 0.188-1.883 mg/ml for alpha-casein and 0.151-1.506 mg/ml for beta-casein. The detection limits were 0.006, 0.019 and 0.015 mg/ml for kappa-casein, alpha-casein and beta-casein, respectively. The validity of the method was verified. The recoveries ranged from 91 to 100% for bovine milk. The precision of the method was also evaluated, the RSD being less than 3.67%. The same HPLC procedure was used for the separation of caprine and ovine caseins. Different chromatographic profiles were obtained for bovine, ovine and caprine milks, although it was only possible to detect and quantify additions of 5% or more of bovine milk to caprine milk. With respect to detection of milk adulterations, electrophoresis using urea-polyacrylamide gel electrophoresis (PAGE) analysis was more sensitive. The evolution of casein proteolysis in cheeses made from bovine milk and cheeses made from ovine milk, during 30 days of ripening was followed by HPLC-UV and urea-PAGE methodologies. The results obtained by these techniques were similar.

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.

SIMULTANEOUS DETERMINATION OF BENZOIC AND SORBIC ACIDS IN QUINCE JAM BY HPLC

Abstract An isocratic HPLC technique is described for the determination of benzoic acid and sorbic acid in industrial quince jam. The preparation procedure was optimized. Precipitation of proteins and fat by the addition of methanol, followed by centrifugation and/or filtration provided an extract suitable for chromatographic analysis. The chromatographic separation was achieved with a C18 column and acetate buffer (pH=4.4) - methanol (65:35) as the mobile phase. The effluent was monitored at 235 nm. Effective separation and quantification was achieved in less than 7 min. Specificity of the method was checked against common food additives added to industrial quince jam, such as l -ascorbic acid and citric acid. Diode array detection was used for confirmation of the preservatives. Mean recoveries of 95–104% were obtained with a precision less than 2.6%, detection limits of 25 and 6.25 mg/kg were obtained for benzoic and sorbic acids, respectively. Results were in good agreement with the reference methods. The presence of benzoic and sorbic acids in quince jams available on the Portuguese market, was also determined. Eleven commercial brands of quince jam were analysed. All contained benzoic acid. The concentration ranged from 413.9±10.4 to 1501±4.2 mg of benzoic acid/kg of quince jam. Only two brands also contained sorbic acid. The concentrations were 515.0±7.0 and 908.3±5.3 mg of sorbic acid/kg of quince jam.

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.