Determination of theobromine and caffeine in fermented and unfermented Amazonian cocoa (Theobromacacao L.) beans using square wave voltammetry after chromatographic separation

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The concentrations of theobromine (TB) and caffeine (CF) together with other substances such as phenolic compounds, contribute to the perception of the bitter taste characteristic of fermented and unfermented Amazonian cocoa samples and their edible derivatives, such as chocolates. Thus, an analytical method based on square wave voltammetry (SWV) with a boron doped diamond electrode (BDD) was proposed to determine them. The supporting electrolyte, pH buffer solutions and parameters of the SWV technique were studied, and after determining the optimal conditions, analytical curves were constructed and the limits of detection and quantification calculated as 0.027 μmol L−1 and 0.093 μmol L−1 for CF and 0.025 and 0.085 μmol L−1 for TB. Intra-day and inter-day repeatability tests were carried out and the relative standard deviation values were below 2.51% and 6.50%, respectively. Interference studies were carried out and no potential interferents were found, except for high levels of TB in the presence of low CF concentrations, and vice-versa. To overcome this drawback, a preparative chromatographic procedure was used and the isolated aliquots of TB and CF quantified using SWV and high performance liquid chromatography (HPLC) with photodiode array detectors (PAD) as the reference method. Due to the low concentration of CF in the Amazonian cocoa sample, a miniaturized electrochemical cell produced in a 3D printer was developed. Based on the results, the relative errors were lower than 10% and the critical values for the F and t tests were lower than those tabulated at the 95% confidence level, with n = 3. The recoveries ranged from 80% to 115%. According to the results one can determine the TB and CF contents with acceptable precision and accuracy in fermented and unfermented Amazonian cocoa samples with a reduced amount of residues, representing good analytical tools for the quality control of fermented cocoa and chocolate.