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

Abstract

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 C(18) cartridge. Subsequent injection on a C(18) 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.