Determination of theobromine, theophylline and caffeine in cocoa samples by a high-performance liquid chromatographic method with on-line sample cleanup in a switching-column system

A simple reversed-phase high-performance liquid chromatographic method was developed for the determination of theobromine, theophylline and caffeine in cocoa samples. In the sample cleanup step, the procedure involves an on-line solid-phase extraction of analytes from cocoa samples into a home-made dry-packed pre-column with ODS-C18 using a column-switching system. The separation was performed on a C18 Nova-Pak column (150 mm × 3.9 mm, 4 μm) using a mobile phase consisting of a solution of 20% of methanol in water under isocratic conditions, at a flow-rate of 1.4 ml/min. The validation method revealed quantitative recoveries (>95.0%) with a coefficients of variation <3.2% and it also provided a good precision for data validation. The overlap of sample cleanup, analysis and recondition of the precolumn increases the sample throughput to 8 samples/h. Furthermore, the proposed method was successfully applied to analysis of cocoa samples “Trinitario”, “Forastero” and “Criollo” grown in different seasons of the year and fermented for 3 and 7 days, respectively. The results showed a slight reduction in the theobromine and caffeine content according to the fermentation times. In the same way, the theobromine/caffeine ratio was assessed, with the purpose of establishing a correlation with the genotype of the studied samples.