Method for the determination of catechin and epicatechin enantiomers in cocoa-based ingredients and products by high-performance liquid chromatography: First Action 2013.04.

Single-laboratory validation data previously published in the Journal of AOAC INTERNATIONAL 95(2), 500-507 (2012) was reviewed by the Stakeholder Panel on Strategic Food Analytical Methods Expert Review Panel (ERP) at the AOAC INTERNATIONAL Mid-Year Meeting held on March 12-14, 2013 in Rockville, MD. The ERP determined the data presented met the established standard method performance requirement and approved the method as AOAC Official First Action on March 14, 2013. Using high-performance liquid chromatography (HPLC), flavanol enantiomers, (+)- and (-)-epicatechin and (+)- and (-)-catechin, are eluted isocratically using ammonium acetate and methanol mobile phase. The mobile phase is applied to a modified beta-cyclodextrin chiral stationary phase and the flavanols detected by fluorescence. Using several cocoa-based matrices, recoveries for the four enantiomers ranged from 82.2-102.1% at a 50% spike level, and 80.4-101.1% at a 100% spike level. Precision was determined to be 1.46-3.22% for (-)-epicatechin, 3.66-6.90% for (+)-catechin, 1.69-6.89% for (-)-catechin. (+)-Epicatechin was not detected in any of the samples used for this work, so precision could not be determined for this molecule.

Method for the determination of catechin and epicatechin enantiomers in cocoa-based ingredients and products by high-performance liquid chromatography: single-laboratory validation.

A single-laboratory validation study was performed for an HPLC method to identify and quantify the flavanol enantiomers (+)- and (-)-epicatechin and (+)- and (-)-catechin in cocoa-based ingredients and products. These compounds were eluted isocratically with an ammonium acetate-methanol mobile phase applied to a modified beta-cyclodextrin chiral stationary phase and detected using fluorescence. Spike recovery experiments using appropriate matrix blanks, along with cocoa extract, cocoa powder, and dark chocolate, were used to evaluate accuracy, repeatability, specificity, LOD, LOQ, and linearity of the method as performed by a single analyst on multiple days. In all samples analyzed, (-)-epicatechin was the predominant flavanol and represented 68-91% of the total monomeric flavanols detected. For the cocoa-based products, within-day (intraday) precision for (-)-epicatechin was between 1.46-3.22%, for (+)-catechin between 3.66-6.90%, and for (-)-catechin between 1.69-6.89%; (+)-epicatechin was not detected in these samples. Recoveries for the three sample types investigated ranged from 82.2 to 102.1% at the 50% spiking level, 83.7 to 102.0% at the 100% spiking level, and 80.4 to 101.1% at the 200% spiking level. Based on performance results, this method may be suitable for routine laboratory use in analysis of cocoa-based ingredients and products.