Flavanol Research Studies

Funding provided in part by Mars, Incorporated

For more than 15 years, Mars, Incorporated has conducted and supported the vast majority of the research conducted on cocoa and cocoa flavanols reported in the peer-reviewed scientific literature. Mars Botanical will continue this global scientific leadership aimed at investigating the biomedical potential of cocoa and cocoa flavanols.

This leadership is represented by more than 100 published, peer-reviewed scientific articles and more than 30 patents resulting from work with institutions including Harvard University and the University of California, Davis.

In addition, Mars, Incorporated is actively researching topics focused on ecology, crop sustainability, ecological agro forestry, genetic characterization and breeding of cocoa plants and the analytics of flavanols in cocoa and other foods.

Several key research publications on flavanols are presented below.

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Analyses of procyanidins in foods using Diol phase HPLC
Liwei Gu¹, Aaron C. Hager¹, Rebecca J. Robbins², Ronald L. Prior¹
¹Arkansas Children’s Nutrition Center, 1212 Marshall Street, Little Rock, Arkansas, 72202; ²Analytical and Applied Sciences Group, Mars Snack Food US, 800 High Street, Hackettstown, New Jersey, 07840.

Separation of procyanidins using silica-based HPLC suffered from poor resolution for higher oligomers and low sensitivity due to the fluorescence quenching effects of methylene chloride in the mobile phase. Optimization of a published Diol-phase HPLC method resulted in higher resolution for pentamers to decamers at 29 min to 50 min and the elution of polymers with molecular weight higher than decamers at 66 min. By using excitation and emission wavelengths of 230 nm and 321 nm, the sensitivity was increased by 5 fold. Flavonoids other than proanthocyanidins did not generate signals at these wavelengths. Dimers and trimers in grape seed procyanidins eluted as a cluster of 5 peaks of isomers on normal phase HPLC, whereas they merged into a large peak with shoulders using the Diol-phase HPLC. Hexamers, heptamers, and octamers can be resolved on Diol-phase HPLC but not on normal-phase HPLC. Separation of procyanidins on Diol-phase HPLC was according the degree of polymerization and was not sensitive to isomeric differences for procyanidins of the same degree of polymerization. Proanthocyanidins in foods that were dominantly procyanidins, such as cocoa, sorghum, pear, and apple eluted as well separated oligomers and polymers using Diol-based HPLC.

Development of a robust, quantitative normal-phase HPLC method for flavan-3-ols and procyanidin oligomers in chocolate and cocoa-containing samples
REBECCA J. ROBBINS*, JADWIGA LEONCZAK, J. CHRISTOPHER JOHNSON, CATHERINE KWIK-URIBE, RONALD L. PRIOR§, AND LIWEI GU#
*Analytical and Applied Sciences, Mars, Incorporated, 800 High Street Hackettstown, NJ 07840
USDA, ARS Arkansas Children's Nutrition Center 1120 Marshall Street Little Rock, AR 72202
#Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205

A novel normal-phase HPLC method for the quantitative determination of flavan-3-ols and procyanidins in chocolate and cocoa-containing food products has been developed. Chromatographic separation based on degree of polymerization (DP = 1-10) was achieved on a diol stationary phase column (normal phase) with a binary gradient of acetonitrile (CH3CN:HOAc) and methanol-water (CH3OH:H2O:HOAc). Quantitation was achieved with fluorescent detection (FLD: ex 230 nm/em 321 nm) and the use of external standards. Under these chromatographic conditions, fluorescence quantum yields were significantly greater than observed in previous methods. To have oligomers (DP1 –10) within the detection range of the instrument in a single run, and behave linearly over a wide range of concentrations, an FLD photomultipler tube gain gradient was employed. Sample preparation investigations were conducted to eliminate common chromatographic interferences and assess extraction efficiencies. Method performance was examined on selected cocoa-containing foods (including NIST reference materials) with multiple analysts and laboratories. This method demonstrated accurate and reproducible quantitation of the flavanols and procyanidins in chocolate and cocoa-containing products.