Extraction of Purine Alkaloids from Cocoa Tissues and Determination by High-performance Liquid Chromatography

Protocols for the extraction and quantification by high-performance liquid chromatography of purine alkaloids from cocoa beans, Theobroma cacao plants and tissues cultivated in vitro have been developed. These allow the rapid determination of 7-methylxanthine, 3-dimethylxanthines and caffeine from complex mixtures and represent improvements over published methods. Alkaloids were extracted in boiling water and the extracts clarified by precipitation and filtration. Chromatography was carried out using C-18 reverse-phase columns with isocratic elution. The eluate was monitored routinely at 280 nm and compounds quantified using external standards. Estimations of purine alkaloid recoveries were better than 98% for caffeine, theobromine and theophylline. Reproducibility of replicate injections averaged 99% with coefficients of variation between replicate extractions typically less than 5% except for small samples (i.e., less than 0.2 g dry weight). Results for cocoa nibs and shells; roots, stem, green and red leaves of the cocoa plant; callus and suspension-cultured cells and growth media from one type of cocoa are reported.