An updated gradient PLE-SPE×HPLC-PDA system for the extraction, concentration, fractionation, and analysis of valuable compounds from cocoa bean shells.

Cocoa (Theobroma cacao L.) beans are extensively employed in the food industry. However, their shells (CBS), a by-product representing about 20 % of bean weight, contain valuable bioactive compounds such as phenolic compounds and methylxanthines. This study developed a PLE-SPE × HPLC-PDA system to extract, concentrate, fractionate, and analyze these metabolites from CBS. The optimized in-line PLE-SPE conditions (PoraPak Rxn RP Bulk adsorbent, 45-min gradient from pure H2O to pure EtOH) produced a first fraction enriched with theobromine (75.05 ± 9.22 %), a second fraction enriched with caffeine (80.66 ± 1.95 %), and a third fraction enriched with phenolics (69.21 ± 7.46 %). Additionally, the SPE adsorbent was evaluated over at least 10 reuses, with performance monitored in real-time using the updated PLE-SPE × HPLC-PDA system. Computational predictions using COSMO-RS described the metabolites × solvents × adsorbent interactions. Finally, this methodology reduces processing steps, time, and materials while producing valuable fractions for food, cosmetic, and pharmaceutical industries.