Investigating Glyoxal and Methylglyoxal Formation and Bioaccessibility in Chocolate with HPLC


The effects of simulated digestion on the formation of α-dicarbonyl compounds (α-DCs) in chocolates were assessed in a recent study with high-performance liquid chromatography (HPLC).

A recent study, published in Food Research International, focused on evaluating the formation of α-dicarbonyl compounds (α-DCs) in chocolates under replicated digestive system conditions (1). The concentrations of glyoxal and methylglyoxal in chocolates before and after the process were concluded using through high-performance liquid chromatography (HPLC) analysis.

Generally, α-DCs are formed from carbohydrates via the heating and storage process, mainly through the Maillard reaction (MR; the nonenzymic browning reaction of reducing sugars with amines and typically involves amino acids, proteins, and peptides), caramelization, lipid-peroxidation, and enzymatic reaction (2-4). Among these toxic compounds are glyoxal (GO, an organic compound that has been considered as an effective alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy [5]) and methylglyoxal (MGO, a by-product of glucose metabolism, known to be involved in microvascular dysfunction and is associated with reduced cognitive function [6]).

The production process of chocolate includes a variety of processes, such as fermentation, drying, roasting, conching (a process of heating and aerating chocolate to both introduce oxygen and to remove unwanted volatiles [7]), and tempering. Each of these processes play a critical part in the development of the resulting chocolate’s characteristic taste and smell. GO and MGO can be easily formed in chocolates due to high temperatures resulting from processing, pH, storage environments, high fat and sugar content, low moisture levels, and food processing techniques, including grinding, roasting, through MR, sugar autooxidation, lipid peroxidation, or caramelization.

The study revealed that initial concentrations for GO within the post-digested chocolate samples tested ranged from 0.0 and 228.2 µg/100 g, whereas initial concentrations for MGO concentrations ranged from 0.0 and 555.1. There was a notable increase in both GO and MGO levels following digestion, reaching up to 1804 % and 859 %, respectively. These findings indicate that digestive system conditions facilitate the formation of advanced glycation end product (AGE) precursors (modifications of proteins or lipids that become nonenzymatically glycated and oxidized after contact with aldose sugars [8]). Furthermore, GO and MGO levels were found to be low in chocolate samples containing dark chocolate. Conversely, they were found to be high in samples containing hazelnuts, almonds, pistachio, and milk.

The authors said that supplementary analysis should be performed that focuses on α-DCs formation under actual digestive system conditions to uncover the effects of gut microbiota to the process.


1. Elif Ede-Cintesun, R.; Jale Çatak, J.; Esra Ateş, E.; Mustafa Yaman, M. Glyoxal and Methylglyoxal Formation in Chocolate and their Bioaccessibility. Food Res. Int. 2024, 114552. DOI: 10.1016/j.foodres.2024.114552

2. Zheng, J., Ou, J., Ou, S. “Alpha-Dicarbonyl Compounds.” in Chemical Hazards in Thermally-Processed Foods, Wang, S. Editor. Springer, 2019. DOI:10.1007/978-981-13-8118-8_2

3. Yan, S.; Wu, L.; Xue, X. α-Dicarbonyl Compounds in Food Products: Comprehensively Understanding Their Occurrence, Analysis, and Control. Compr. Rev. Food Sci. Food Saf.2023. DOI: 10.1111/1541-4337.13115

4. (accessed 2024-05-28)

5. Richter, K.N.; Revelo, N. H.;Seitz, K. J.; Helm, M. S.; Sarkar, D.; Saleeb, R. S et al. Glyoxal as an Alternative Fixative to Formaldehyde in Immunostaining and Super-Resolution Microscopy. EMBO J. 2018, 37(1),139-159. DOI: 10.15252/embj.2016957096.

6. Berends, E.; van Oostenbrugge, R. J.; Foulquier, S. et al. Methylglyoxal, a Highly Reactive Dicarbonyl Compound, as a Threat for Blood Brain Barrier integrity. Fluids Barriers CNS 2023, 20, 75 (2023).

7. Conching and Refining. Chocolate Alchemy website. (accessed 2024-05-28)

8. Goldin, A.; Beckman, J. A.; Schmidt, A. M.; Creager. M. A. Advanced Glycation End Products Sparking the Development of Diabetic Vascular Injury. Circulation 2006,114, 597–605. DOI: 10.1161/CIRCULATIONAHA.106.621854

Chocolate pieces on a white background. © Anatoly Repin -

Chocolate pieces on a white background. © Anatoly Repin -

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Toby Astill | Image Credit: © Thermo Fisher Scientific