Cristiano Augusto Ballus

Use of multivariate statistical techniques to optimize the simultaneous separation of 13 phenolic compounds from extra-virgin olive oil by capillary electrophoresis.

Characterization of phenolic compounds in olive oil has not been achieved as yet, owing to the complexities of their chemical structures and analytical matrix. The aim of this work is to optimize and validate a method for simultaneous separation and quantification of 13 phenolic compounds from extra-virgin olive oil: tyrosol, hydroxytyrosol, oleuropein glycoside, ferrulic acid, p-coumaric acid, cinnamic acid, p-hydroxybenzoic acid, gallic acid, caffeic acid, luteolin, apigenin, vanillic acid and 3,4-dihydroxybenzoic acid. A statistical central composite design, response surface analysis and the simultaneous optimization method of Derringer and Suich were used to separate all the peaks. These multivariate procedures were efficient in determining the optimal separation condition, using five peak-pair resolutions and runtime as responses. The optimized method employed a fused-silica capillary of 50 μm i.d.× 60 cm effective length with extended light path, 50 mmol L(-1) boric acid electrolyte, 10.2 pH, 25°C, injection of 50 mbar for 25s with application of reverse voltage (-30 kV for 5s) before setting the running voltage (+30 kV) with detection at 210 nm and a run time of 12 min. Peak resolutions are found to be very sensitive to pH values outside the 10.15-10.25 range but acceptable electropherograms can be obtained for a wide range of boric acid concentrations within this pH interval.

Profile of phenolic compounds of Brazilian virgin olive oils by rapid resolution liquid chromatography coupled to electrospray ionisation time-of-flight mass spectrometry (RRLC–ESI-TOF-MS)

In recent years, agronomical researchers began to cultivate several olive varieties in different regions of Brazil to produce virgin olive oil (VOO). Because there has been no reported data regarding the phenolic profile of the first Brazilian VOO, the aim of this work was to determine phenolic contents of these samples using rapid-resolution liquid chromatography coupled to electrospray ionisation time-of-flight mass spectrometry. 25 VOO samples from Arbequina, Koroneiki, Arbosana, Grappolo, Manzanilla, Coratina, Frantoio and MGS Mariense varieties from three different Brazilian states and two crops were analysed. It was possible to quantify 19 phenolic compounds belonging to different classes. The results indicated that Brazilian VOOs have high total phenolic content because the values were comparable with those from high-quality VOOs produced in other countries. VOOs from Coratina, Arbosana and Grappolo presented the highest total phenolic content. These data will be useful in the development and improvement of Brazilian VOO.

Doehlert design-desirability function multi-criteria optimal separation of 17 phenolic compounds from extra-virgin olive oil by capillary zone electrophoresis

In Brazil, the consumption of extra-virgin olive oil (EVOO) is increasing annually, but there are no experimental studies concerning the phenolic compound contents of commercial EVOO. The aim of this work was to optimise the separation of 17 phenolic compounds already detected in EVOO. A Doehlert matrix experimental design was used, evaluating the effects of pH and electrolyte concentration. Resolution, runtime and migration time relative standard deviation values were evaluated. Derringers desirability function was used to simultaneously optimise all 37 responses. The 17 peaks were separated in 19min using a fused-silica capillary (50μm internal diameter, 72cm of effective length) with an extended light path and 101.3mmolL(-1) of boric acid electrolyte (pH 9.15, 30kV). The method was validated and applied to 15 EVOO samples found in Brazilian supermarkets.

Chemometrics optimization of carbohydrate separations in six food matrices by micellar electrokinetic chromatography with anionic surfactant

Multivariate statistical design modeling and the Derringer-Suich desirability function analysis were applied to micellar electrokinetic chromatography (MEKC) results with anionic surfactant to separate carbohydrates (CHOs) in different food matrices. This strategy has been studied with success to analyze compounds of difficult separation, but has not been explored for carbohydrates. Six procedures for the analysis of different sets of CHOs present in six food matrices were developed. The effects of pH, electrolyte and surfactant concentrations on the separation of the compounds were investigated using a central composite design requiring 17 experiments. The simultaneous optimization of the responses for separation of six sets of CHOs was performed employing empirical models for prediction of optimal resolution conditions in six matrices, condensed milk, orange juices, rice bran, red wine, roasted and ground coffee and breakfast cereal samples. The results indicate good separation for the samples, with appropriate detectability and selectivity, short analysis time, low reagent cost and little waste generation, demonstrating that the proposed technique is a viable alternative for carbohydrate analysis in foods.

Comparison of capillary electrophoresis and high performance liquid chromatography methods for caffeine determination in decaffeinated coffee

Decaffeinated coffee accounts for 10 percent of coffee sales in the world; it is preferred by consumers that do not wish or are sensitive to caffeine effects. This article presents an analytical comparison of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) methods for residual caffeine quantification in decaffeinated coffee in terms of validation parameters, costs, analysis time, composition and treatment of the residues generated, and caffeine quantification in 20 commercial samples. Both methods showed suitable validation parameters. Caffeine content did not differ statistically in the two different methods of analysis. The main advantage of the high performance liquid chromatography (HPLC) method was the 42-fold lower detection limit. Nevertheless, the capillary electrophoresis (CE) detection limit was 115-fold lower than the allowable limit by the Brazilian law. The capillary electrophoresis (CE) analyses were 30% faster, the reagent costs were 76.5-fold, and the volume of the residues generated was 33-fold lower. Therefore, the capillary electrophoresis (CE) method proved to be a valuable analytical tool for this type of analysis.O comercio de cafe descafeinado constitui 10% das vendas mundiais de cafe, sendo preferido pelos consumidores que nao desejam ou sao sensiveis aos efeitos da cafeina. Este artigo apresenta uma comparacao analitica de metodos por eletroforese capilar (CE) e cromatografia liquida de alta eficiencia (HPLC) para a quantificacao de cafeina residual em cafe descafeinado, quanto aos parâmetros de validacao, custos, tempo de analise, composicao e tratamento dos residuos gerados, bem como quantificacao de cafeina em 20 amostras comerciais. Ambos os metodos apresentaram parâmetros de validacao adequados. O teor de cafeina nao diferiu estatisticamente pelos dois metodos. A vantagem do metodo por HPLC foi o limite de deteccao 42 vezes mais baixo. Nao obstante, o limite de deteccao da CE foi 115 vezes menor do que o permitido pela legislacao brasileira. A analise por CE foi 30% mais rapida, os custos com reagentes foram 76,5 vezes mais baixos e o volume de residuos gerados foi 33 vezes menor. Portanto, o metodo por CE mostrou-se uma valiosa ferramenta analitica para este tipo de analise.

A new extraction approach to correct the effect of apparent increase in the secoiridoid content after filtration of virgin olive oil.

In the current study, a new approach has been developed for correcting the effect that moisture reduction after virgin olive oil (VOO) filtration exerts on the apparent increase of the secoiridoid content by using an internal standard during extraction. Firstly, two main Spanish varieties (Picual and Hojiblanca) were submitted to industrial filtration of VOOs. Afterwards, the moisture content was determined in unfiltered and filtered VOOs, and liquid-liquid extraction of phenolic compounds was performed using different internal standards. The resulting extracts were analyzed by HPLC-ESI-TOF/MS, in order to gain maximum information concerning the phenolic profiles of the samples under study. The reduction effect of filtration on the moisture content, phenolic alcohols, and flavones was confirmed at the industrial scale. Oleuropein was chosen as internal standard and, for the first time, the apparent increase of secoiridoids in filtered VOO was corrected, using a correction coefficient (Cc) calculated from the variation of internal standard area in filtered and unfiltered VOO during extraction. This approach gave the real concentration of secoiridoids in filtered VOO, and clarified the effect of the filtration step on the phenolic fraction. This finding is of great importance for future studies that seek to quantify phenolic compounds in VOOs.

Optimization of Electrophoretic Separations of Thirteen Phenolic Compounds using Single Peak Responses and an Interactive Computer Technique

An interactive computer method is proposed for the electrophoretic separation of 13 phenolic compounds from extra-virgin olive oil using single peak response values. A central composite design was executed for optimization of the sodium tetraborate concentration, pH and applied voltage. Statistical models were determined for eight resolution responses and thirteen effective mobilities. Six of the resolution models had highly significant ANOVA lack of fit values, limiting their accuracies for use in Derringer´s desirability function search for optimal separation conditions. None of the 13 effective mobility models suffered from significant lack of fit. Since it is not possible to define effective mobility target values for the desirability function, an interactive computer program developed in our laboratories was applied to the single peak models. Mouse or cursor movements were executed to define experimental conditions in model simulations of the electropherogram. These simulations resulted in superior peak separations, especially for the apigenin and luteolin peaks, in 35 min, compared with those obtained in close to 50 min with the resolution models. Verification experiments performed 2 and 3 years later confirmed the robustness of the models.

Perfil de ácidos graxos e teor de colesterol de mortadela elaborada com óleos vegetais

The aim of this study was to evaluate the fatty acid profile and the cholesterol content of mortadella prepared with different vegetable oils. Nine treatments were made with four different vegetable oils (canola, linseed, olive and soy) using two substitution levels (50% and 100%). The fatty acids and the cholesterol content were separated and identified by gas chromatography. There was a prevalence of the oleic, linoleic and palmitic fatty acids. The saturated fatty acids were lower in the products with vegetable oils. The PUFA/SFA relation was lower in the control treatment while the higher value was observed in the 100% linseed oil treatment. The cholesterol levels found were similar between all the treatments, but a reduction of these levels were observed in the treatments, being superior only in treatment containing 100% of soybean oil. Thus, except the treatment with 100% of soybean oil, all the other treatments with vegetable oils showed a better nutritional quality in relation to the control treatment.

The influence of different combinations of probiotic bacteria and fermentation temperatures on the microbiological and physicochemical characteristics of fermented lactic beverages containing soybean hydrosoluble extract during refrigerated storage

Lactic beverages containing probiotics were prepared with whole UHT milk, whey of Mozzarella cheese, soybean hydrosoluble extract and sugar. Three formulations were studied, each one containing a different combination of probiotic/starter bacteria, fermented at two different temperatures (37 and 45 °C). The aim of this work was to verify the influence of these variables on the viability of probiotic microorganisms and on the physicochemical stability of lactic beverages during storage under refrigeration (21 days at 7 °C). The results indicated that the fermentation temperature had a significant effect on the viability of probiotic bacteria. Counts for Lactobacillus acidophilus were affected by storage time, resulting appropriate after 21 days only for the beverage fermented at 37 °C. Physicochemical parameters did not exhibit drastic variations - proving the stability of formulations during storage. Cells of Bifidobacterium spp. showed high survival ability, probably due to the presence of growth promoters from soybean and cheese whey. The fermentation temperature of 37 °C allowed counts above the minimum limit for all the studied microorganisms, being preferred to the temperature of 45 °C. The inclusion of soybean hydrosoluble extract, a prebiotics source, resulted in a symbiotic product with more benefits to the health of consumers.