Improved High-Performance Liquid Chromatography Method to Determine Theobromine and Caffeine in Cocoa and Cocoa Products

<p align="justify">At present, the commonly used HPLC method for the analysis of caffeine and theobromine contents
in aqueous cocoa extracts employs direct application of the extracts on the column. This practice
gradually reduces the efficiency of the column and shortens its life. Also, this method gives inflated
values due to interfering substances and difficulty in achieving baseline resolution. In the improved
method, the interfering cocoa pigments are effectively removed by passing the aqueous extract
through a Sep-pak C₁₈ cartridge. Subsequent injection on a C₁₈ reverse-phase column employing
acetonitrile and water (20:80) as the mobile phase reduces the analysis time without affecting either
resolution of the peak or the accuracy of caffeine and theobromine determination or achieving
baseline resolution. Therefore, this method is ideally suited for rapid routine analysis of cocoa and
its products.</p>