Evaluation of parameters that affect the 4-dimethylaminocinnamaldehyde assay for flavanols and proanthocyanidins

Proanthocyanidins are widely distributed in nature and represent the most abundant flavonoids consumed in the diet. Recent attention has been given to these compounds because of their health-promoting properties toward chronic diseases. Because of their large degrees of chemical variation and stereochemistry, isolation/quantification is difficult. The 4-dimethylaminocinnamaldehyde (DMAC) spectrophotometric assay has become increasingly popular as a rapid technique to quantify the amount of proanthocyanidins present in foods and beverages; however, there is no current industry standard. In this study, several parameters affecting the DMAC reaction with catechin were examined. Effects of acid nature (hydrochloric acid [HCl] and sulfuric acid [H(2)SO(4)]) and concentration (2 to 10 N), temperature (5 to 45 °C), reaction time (2 to 35 min), sample water content (1 to 100%), DMAC concentration (1 to 3%), and interference of 8 different substances were evaluated. A mixture of 2% DMAC in methanol (w/v) in 6 N H(2)SO(4) (50:50 v/v) showed the highest slope in the standard curve when allowed to react for 15 to 35 min prior to analysis. For samples containing high concentrations of oligomeric proanthocyanidins, a reaction time of 20 to 35 min is recommended. The reaction of catechin with DMAC at constant room temperature (21 to 25 °C), with a sample water content of less than 1% was found to increase reproducibility and better assess the amount of catechin. Sample water contents higher than 1% showed significant bleaching effects on the color produced, when catechin concentrations were high (lower levels showed no significant differences). None of the substances examined in this study interfered with the assay; however, other compounds present in various food matrices may have an effect on color development.