RESUMO
A simple, specific, high-throughput colorimetric method based on the reaction of 4-dimethylaminocinnamaldehyde (DMAC) with flavan-3-ols was developed to determine total procyanidins in selected cacao-based products. Extracts of defatted samples were dispensed into a 96-well plate and reacted with DMAC. The absorbance of the reaction products was measured at 640 nm and compared to commercially available procyanidin B2 as a standard. The use of the 96-well plates and a plate reader dramatically improved sample throughput. A standard protocol was established and used for further studies. The calibration was found to be linear from 1-100 ppm. The DMAC reagent reacted relatively specifically to (-)-epicatechin, (+)-catechin, epigallocatechin, gallocatechin, the gallates of catechin, epicatechin, gallocatechin, and epigallocatechin, oligomeric procyanidins of cocoa up to n=4, and A-type procyanidins. Little or no reaction occurred with cyanidins and representative compounds of phenolic acids, flavones, flavanones, flavonols, anthocyanidins, isoflavones, and stilbenes. Sample precision studies were carried out on 10 different test materials over several weeks, and yielded RSD values of 4.0 to 9.5%. The method was ring-tested in three laboratories using blinded test materials including cocoa beans, cocoa powder, chocolate liquor, dark chocolate, and milk chocolate. There was excellent agreement of the results between laboratories.