Application of Probe Sonication for Defatting and Extraction of Theobromine and Caffeine from Cocoa-Based Samples

Methylxanthines are naturally occurring purine alkaloids widely present in cocoa-based products. Due to the relatively high fat content of these matrices and their complexity, appropriate sample preparation is an important step for their determination. This research examines the applicability of direct sonication with an ultrasonic probe as an alternative approach for defatting and extracting methylxanthines from cocoa-based samples. Three defatting procedures were compared: Soxhlet extraction, indirect ultrasound-assisted extraction using an ultrasonic bath, and direct ultrasound-assisted extraction using a probe. The percentage of extracted fat was 13.56 ± 0.52%, 8.94 ± 1.52%, and 18.46 ± 2.89%, respectively. A single 10 min probe sonication treatment with petroleum ether as solvent proved to be a promising and time-efficient alternative for defatting cocoa samples. For methylxanthine extraction, the method used by del Rosario Brunetto et al. was adapted by replacing 20 min water-bath extraction with 10 min direct probe sonication. The adapted procedure was applied to samples of cocoa powder, cocoa beans and 85% cocoa chocolate. Determined theobromine concentrations ranged from 15.61 ± 0.24 to 31.01 ± 0.91 mg/g, and caffeine concentrations from 1.64 ± 0.05 to 2.95 ± 0.07 mg/g. Theophylline concentrations in all samples were below the limit of quantification (LOQ), which is why the results were considered indicative of its presence rather than reliable quantitative values. The extraction yield of theobromine and caffeine from defatted cocoa powder was estimated by spike-recovery testing. Obtained mean recovery values were 112.31% for theobromine and 109.65% for caffeine. We assessed the greenness of the analytical procedure and sample preparation, including the defatting step, using AGREE and AGREEprep, with scores of 0.50 and 0.53, respectively. Assessment results indicate the possibility of further improving the greenness of the process. The results show that direct sonication with an ultrasound probe shortens defatting and methylxanthine extraction times and indicate the potential of this approach to simplify the preparation of complex cocoa-based samples.