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UHPLC-HRMS Analysis of Theobromine in Theobroma cacao and its Products

Katarina Mladenovic, Yuriko Root and Dilrukshi Ramanathan

Theobroma cacao seed is the major ingredient in all types of chocolate products and contains Theobromine. Theobromine is a bitter alkaloid beneficial in the treatment of hypertension, arteriosclerosis and angina pectoris. Of all chocolate brand samples, Sample J containing 70% Cacao had the highest amount of theobromine. Sample A containing 11% Cacao had the least amount of theobromine. The 100% Cacao Chocolate Bar had the highest concentration of theobromine in comparison to roasted cocoa having the lowest concentration of theobromine. Quantitative analysis of Theobromine was completed on the Thermo Scientific UHPLC and LTQ Orbit rap Discovery equipped with an ESI ion source. A three-minute gradient method with a flow rate of 300 μL/min was developed on the UHPLC-HRMS using HPLC-grade water and acetonitrile. Ethyl ether was used to remove cacao fats and water was used to isolate theobromine. To obtain the precision of the theobromine extraction process, the recovery analysis was 86%.