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Chocolate Sample Preparation for Chromatographic Determination of (-)-Epicatechin and Procyanidin B2

A number of studies have shown that natural antioxidants from plant sources can effectively inhibit oxidation in food and reduce the risk of age-dependent diseases. Polyphenols, abundant in fruits, vegetables, teas, medicinal plants, have received the greatest attention and have been studied extensively, since they are a kind of highly effective antioxidant and less toxic than synthetic antioxidants. Cocoa beans, chocolate, and cocoa powder are known to be rich sources of polyphenols. The concentrations of all polyphenols can vary tremendously between cocoa-containing foods, and this can vary depending on the source of the beans, the processing conditions, and how the chocolates are manufactured. Due to these facts, the ratio and type of polyphenols found in the beans are unlikely to be exactly the same as those found in the final product. The main polyphenols in chocolate are (-)-epicatechin (EC) and procyanidin B2 (pB2). The aim of this work was to extract EC and pB2 from chocolate samples with high efficiency and less matrix effect. The extraction of these phenolic compounds from plants and other solid samples has been traditionally performed using solvent extraction (Soxhlet) or steam distillation techniques. Traditional methods of extraction are labour-intensive, time consuming and require large volumes of solvents. In this work microwave-assisted extraction (MAE) procedure was developed for the fast extraction of EC and pB2 from chocolate. Several critical parameters of the MAE procedure (extraction time and temperature, volume of solvent) were studied for the optimization of the extraction protocol. The maximum yield of flavonoids with MAE was obtained by extraction with 80% methanol, 5 mL/25 mg of defatted chocolate powder, at 50 oC for 5 min. No degradation of the EC and pB2 was observed using the developed extraction protocol. Optimized microwave assisted extraction (MAE) was compared with ultrasonic solvent extraction and matrix solid-phase dispersion (MSPD). The optimal yield with MAE was close to that of ultrasonic solvent extraction (80% methanol 5 mL/25 mg defatted chocolate powder, at 30 oC for 25 min) and matrix solid-phase dispersion (C18 sorbent, methanol:water:0.05 M H2C2O4=70:29.5:0.5 as elution solvent, 5 mL/25 mg defatted chocolate powder). Extraction efficiency was determined by thin-layer chromatography.

sample preparation; chocolate; (-)-epicatechin; procyanidin B2; thin-layer chromatography

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