Analyzing Polyphenols and Antioxidants in Dark Chocolate with RP-HPLC

To determine sample patterns and identify possible markers of quality, variety, or origin to deal with authentication or fraud detection issues, researchers from the University of Barcelona’s Department of Chemical Engineering and Analytical Chemistry characterized dark chocolate samples according to geographical origin, cocoa variety, and cocoa content. Filtered extracts were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) and spectrophotometric methods to establish individual phenolics and overall indexes of antioxidant and flavonoid content. LCGC International spoke to Sonia Sentellas, corresponding author of the paper that resulted from her team’s work, about their findings.

Your research, as stated in the resulting paper (1), set out to uncover sample patterns and identify possible markers of quality, variety, or origin to deal with authentication or fraud detection issues regarding dark chocolate. What inspired you to pursue this research topic?

In our research group (2), we mainly follow two different research lines related to food analysis. In the first one, we work on recovering bioactive compounds from agri-food waste. The second one, more linked to the present study, is related to the characterization, classification, and authentication of foodstuffs. Indeed, we have been working for years in the characterization of different kinds of foods and beverages, such as honey, wine, tea, and paprika, and now, obviously, also chocolate.

Why dark chocolate?

Chocolate is a highly consumed product and very appreciated by consumers, but the origin of cocoa used for its production could be very different, both in terms of geographical origin as well as botanical origin. Depending on the origin, the aromatic properties and quality can differ substantially. For such a reason, we think it is important to establish reliable methodologies that allow us to characterize the final product, and to know which kind of cocoa has been used for its production.

Specifically, you set out to characterize polyphenolic profiles and antioxidant activity. Why did you choose these specific metabolites for characterization?

Polyphenols are secondary metabolites of plants, and their occurrence strongly depends on not only plant species and botanical varieties but also on environmental characteristics, including the type and components of soil, the weather, etc. Thus, we thought these features could be potential descriptors for chocolate characterization. Regarding the antioxidant capacity, it is a way of evaluating the overall activity of polyphenols without the need to determine them individually.

Your research team selected reversed-phase (RP) high performance liquid chromatography (HPLC) in their efforts. Why did you choose the methods and tools that you used?

In this case, we wanted to look for simple and fast methods to evaluate their potential for the characterization of chocolates. For that reason, we select HPLC in reverse mode, an instrumentation common in analysis laboratories and very versatile for the determination of alkaloids and polyphenols. We also used spectroscopic assays to determine total polyphenols content (TPC), total flavonoid content (TFC), and antioxidant power —using ferric reducing antioxidant power (FRAP) assay—. These complementary methods allow us to estimate the total content without the looking for specific compounds. In this way, not only those main components are considered but also all minor antioxidant compounds.

What are the key findings of your work?

In this preliminary work, we demonstrate that the alkaloid and polyphenol profiles of chocolates differ depending on the cocoa used for its production, specifically depending on the quality, botanical variety, or geographical origin. Overall, American cocoa is richer in bioactive compounds; likewise, Criollo cocoa also appears to contain higher levels of phenolics. Although a full differentiation could not be achieved, it has been demonstrated that combining data from individual compounds and generic phenolic indices or antioxidant capacity for multivariate analysis may provide comprehensive information on the sample behavior, both at the level of descriptors and to infer potential beneficial qualities for health.

Did any unexpected results change your approach?

We can consider this study as a proof of concept, meaning that, at this stage, we were able to demonstrate the potential of our hypothesis, i.e. that chocolates of different characteristics have differentiated polyphenol profiles as well as antioxidant capacity. Then, this is a starting point for studying deeply the role of polyphenols in the characterization, classification, and authentication of cocoa products.

Results from your analysis indicated that American samples are richer than African counterparts in alkaloids and phenolics, and you confirmed this earlier in the interview. Why do you think that this is the case?

Indeed, as indicated earlier, the occurrence of polyphenols and alkaloids may depend on many factors, including plant species, botanical varieties, and environmental characteristics (such as soil, weather, and water), among others. With this kind of study, a cause-effect relationship cannot be deduced; only characteristic features of one or another class can be defined.

How do you imagine your work will impact the discussion surrounding authentication and fraud detection?

With this and other work we have previously performed, we try to develop methodologies to differentiate foodstuff according to different features. Being able to differentiate between products of distinct qualities, products with a protected designation of origin (PDO), those of a specific variety, or geographical origin is of great interest to later apply these methods to detect food fraud. We apply several strategies, including target and non-target analysis, and explore different chemometric tools for analyzing the huge amount of data generated; all this know-how gives society the necessary skills to control fraudulent practices in the food industry. Overall, we hope that all this research helps agencies (or companies) to control the food trade, thus avoiding fraud.

What challenges did you encounter in your research?

Research does not always yield positive results. Indeed, before obtaining good results to be published, you have already performed multiple tests, and some of the experiments you perform fail or bring you to an unexpected point. This is research!! Thus, the work behind a paper is huge.

In this particular field, the characterization and authentication of foodstuffs is especially difficult to find enough authentic samples to build the models. For a reliable characterization, a representative number of samples of each studied class is needed, and not always is easy to find these authentic samples.

How do you stay focused and productive during those periods?

In our research group, alongside the academic staff, several students are beginning their research. In general, they are PhD or Master's students whose enthusiasm and dedication allow us to achieve interesting results and thus continue to advance towards our stated objectives.

What practical applications or future research could come from your work?

As previously stated, we expect that our research will be useful to society as we hope that the methods we develop can be generalized to avoid frauds that not only imply an undue economic benefit but can also cause health problems to people.

As for future research, each new result opens up more possibilities for us to develop increasingly simpler and faster methods aimed at facilitating their application in a more generalized way. Or, on the other hand, the knowledge we acquire allows us to gain experience, which allows us to face new challenges and try to solve more complex cases.

Can you imagine this research inspiring similar analysis on other food types? If so, what types?

Indeed, as I have commented previously, we apply similar methods to characterize and authenticate several foodstuffs. We have already dealt with some beverages (e.g., wine, tea, and coffee) and other products such as nuts, paprika, and honey.

Can you please summarize the feedback that you have received from others regarding this work?

This work has only recently been published; thus, we have not received specific comments on it. However, in general, our research dealing with the characterization, classification, and authentication of foods is extensively cited in other research publications.

What are your next steps regarding this research?

In the recently published research, we focused our efforts on characterizing dark chocolates by profiling alkaloids and main polyphenols with LC-UV and by determining the TPC, TFC, and antioxidant capacity. We have also been working on the profiling of biogenic amines and amino acids. The determination of these compounds is challenging, but we developed a methodology based on LC–MS/MS which allows us their accurate determination. Once identified and quantified, the concentrations of these compounds were used as descriptors of the different cocoa characteristics. Again, good results were obtained, which will be published in the following months. Additionally, more complex analytical techniques have been evaluated; in this way, LC–MS non-targeted analysis demonstrated to be adequate for differentiating fine aroma cocoa from bulk cocoa.

Apart from our interest in cocoa products, we want to extend our research to other products susceptible to being adulterated and that can be of interest to society.

References

1. Parada, T.; Pardo, P.; Saurina, J.; Sentellas, S. Characterization of Dark Chocolates Based on Polyphenolic Profiles and Antioxidant Activity. J. Food Sci. 2024. DOI: 10.1111/1750-3841.17451

2. Food and Art: Authentication & Sustainability Challenges. University of Barcelona website. https://www.ub.edu/FAAST (accessed 2024-11-19)

Sònia Sentellas has been an Associate Professor Serra Húnter at the Department of Analytical Chemistry of the University of Barcelona since 2020. Previously, and for almost 20 years, she developed her research career in the pharmaceutical company Almirall, S.A. centered on the study of the metabolism of drugs and bioanalysis. Her research interest is also focused on food analysis, including characterization, classification, and authentication of food products and fraud prevention, and the recovery of bioactive compounds from agrifood waste. She is member of the Research Institute in Food Nutrition and Food Safety at the University of Barcelona. Photo courtesy of Sentellas.