3. Results and discussion
3.1. Development and finalisation of HPLC method
Many of the published methods for examining the metabolites
found in pomegranate have focused on determining ellagic and
gallic acids, or the punicalagins A and B, but not all four of the
metabolites in a single HPLC analysis. This divided approach has
largely resulted from the differences in the chromatographic properties
of these four compounds when applied to C-18 stationary
phases. Gallic and ellagic acids represent the extremes in terms
of retention and are relatively simple to separate if the oligomeric
hydrolyzable polyphenols that elute in between them are ignored.
In contrast, punicalagins A and B are part of the oligomeric hydrolyzable
polyphenols that elute in the between the extremes and
require elongated chromatographic runs focused on that region
to resolve. We selected a 2.6 lm Kinetex C-18 column with the
hope that the additional resolution afforded by a smaller particle
size would facilitate resolution of the target compounds, while
minimising sample run time.