Exploring the rosmarinic acid profile of dark chocolate fortified with freeze-dried lemon balm extract using conventional and non-conventional extraction techniques

Abstract

The present study evaluated the feasibility of the fortification of dark chocolate with a freeze-dried lemon balm extract rich in rosmarinic acid (RA). Given the food matrix used, it was necessary to develop an analytical and extraction method for RA determination in fortified chocolate. A simple and efficient reversed-phase high-performance liquid chromatography method (RP-HPLC) has been developed for the estimation of RA in dark chocolate. The validity of the method was demonstrated by evaluating the system suitability, linearity, precision, accuracy, the limit of detection, and limit of quantification. The extraction of RA from chocolate was optimized using maceration with shaking (MACS) and microwave-assisted extraction (MAE). The experimental conditions, namely solvent type and extraction time, were optimized using the one-factor-at-a-time methodology. Under the optimized conditions, the proposed method showed good recovery within 95% and acceptable reproducibility with intra- and inter-day relative standard deviations of <2.3%. The method was linear from 5 to 800 μg/mL with a determination coefficient of 1.000. Moreover, the functional chocolate showed a homogeneous active ingredient distribution and retained its RA amount since no significant difference was observed during storage. Overall, the present method was successfully applied to determine RA content in fortified chocolate.