Determination of Cocoa Flavanols and Procyanidins (by Degree of Polymerization DP1-7) in Cocoa-Based Products by Hydrophilic Interaction Chromatography Coupled With Fluorescence Detection: Collaborative Study

J AOAC Int. 2022 Jun 29;105(4):1060-1068. doi: 10.1093/jaoacint/qsac007.


Background: Cocoa flavanols and procyanidins (CF) are flavonoids whose consumption is associated with health benefits, resulting in increasing attention from consumers, industry, researchers, and regulators. Methods that can provide appropriate characterization and quantification of the distinct mixture found in cocoa-based products thus offer important scientific and commercial value.

Objective: This study validated the precision of AOAC Official Method of AnalysisSM2020.05, which measures CF with a degree of polymerization DP1-7.

Method: Method precision (repeatability and reproducibility) was evaluated for seven cocoa matrixes in blind duplicates with total CF content from 1.0 to 500 mg/g. Ten of the 12 laboratories from multiple sectors invited to implement the method returned data for statistical analysis. Precision was evaluated per AOAC INTERNATIONAL guidelines for collaborative studies using RSDr and RSDR as indicators of method repeatability and reproducibility.

Results: RSDr ranged from 1.6 to 4.8%, and RSDR ranged from 5.8 to 22.4%, demonstrating excellent within-laboratory repeatability and good method precision across different laboratories. RSDR values were below 10% with the exception of chocolate, potentially due to very low CF content and sampling inhomogeneity.

Conclusions: These data demonstrate that acceptable method repeatability and reproducibility is achieved when measuring cocoa flavanols and procyanidins using AOAC Method 2020.05 and support the advancement of the AOAC Official Method of Analysis status to Final Action for evaluated matrixes.

Highlights: This collaborative study evaluated the repeatability and reproducibility of AOAC Official Method of Analysis 2020.05.

MeSH terms

  • Cacao* / chemistry
  • Chocolate* / analysis
  • Chromatography, High Pressure Liquid / methods
  • Hydrophobic and Hydrophilic Interactions
  • Polymerization
  • Polyphenols / analysis
  • Proanthocyanidins* / analysis
  • Reproducibility of Results


  • Polyphenols
  • Proanthocyanidins