RP-HPLC Analysis of Polyphenols and Antioxidants in Dark Chocolate

Dark chocolates were characterized according to geographical origin, cocoa variety, and cocoa content in a recent study published in the Journal of Food Sciences (1), using the methylxanthine and polyphenolic composition and antioxidant activity as the data. The researchers were attempting to uncover sample patterns and identify possible markers of quality, variety, or origin to deal with authentication or fraud detection issues. Filtered extracts were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) with ultraviolet (UV) and spectrophotometric methods to determine individual phenolics and overall indexes of antioxidant and flavonoid content.

Dark chocolate’s popularity has increased considerably in recent years, both because of its taste and the health-related properties it offers, including antioxidant, anti-inflammatory, antimicrobial, anticancer, and cardioprotective activities (2-6). These benefits are primarily related to the high polyphenol content of dark chocolate due to its main ingredient being cocoa (7). The authentication and detection of possible adulterations can be done through a profiling approach based on various natural components as a source of information (8,9) including phenolic compounds (10,11) together with methylxanthines (12-14). Amino acids and biogenic amines have also been used to characterize cocoa or chocolate (15).

A set of 26 dark chocolates available in supermarkets and specialized shops was analyzed in this work. The cocoa used to produce the chocolates was from Africa (Ghana, Madagascar, Tanzania, Cameroun, or Ivory Coast), South America (Ecuador, Peru, or Venezuela), Central America (Mexico, Nicaragua, the Dominican Republic, Haiti, or Cuba), and Asia (Papua New Guinea and the Philippines). Criollo (and Nacional), Forastero, and Trinitario cocoa samples were available. Although the study design covered a small number of samples, the analyte concentrations depend on geographical and varietal factors, in addition to the content of cocoa. The research determined that chocolates made with South American cocoa were richer in bioactive compounds; similarly, Criollo cocoa also appears to contain higher levels of phenolics. Despite the multifactorial nature of the analyte composition, the authors said that their exploratory study can be the basis for addressing further cocoa or chocolate authentication studies. Furthermore, combining data from individual compounds and generic phenolic indices or antioxidant capacity for multivariate analysis may provide more comprehensive information on the sample behavior, both at the level of descriptors and to infer potential beneficial qualities for health (1).

Dark chocolate pieces crushed and cocoa beans, culinary background.
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References

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12. Nascimento, M. M.; Santos, H. M.; Coutinho, J. P.; Lobo, I. P.; da Silva Junior, A. L. S.; Santos, A. G.; de Jesus, R. M. Optimization of Chromatographic Separation and Classification of Artisanal and Fine Chocolate Based on its Bioactive Compound Content Through Multivariate Statistical Techniques. Microchem. J. 2020, 152, 104342. DOI: 10.1016/j.microc.2019.104342

13. Samaniego, I.; Espín, S.; Quiroz, J.; Ortiz, B.; Carrillo, W.; García-Viguera, C.; Mena, P. Effect of the Growing Area on the Methylxanthines and Flavan-3-ols Content in Cocoa Beans from Ecuador. J. Food Compos. Anal. 2020, 88, 103448. DOI: 10.1016/j.jfca.2020.103448

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