An updated gradient PLE-SPE×HPLC-PDA system for the extraction, concentration, fractionation, and analysis of...

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.