Original research articleImproved HPLC method for rooibos phenolics targeting changes due to fermentation

- Quantitative HPLC-DAD method for aspalathin oxidation products developed.
- 40% aqueous acetonitrile optimal for extraction of phenolic compounds from rooibos.
- Dihydrochalcones substantially decrease due to fermentation.
- C-Glucosyl eriodictyol isomers substantially increase during fermentation.
- Slight decreases in flavonols following fermentation.

The phenolic composition of rooibos (Aspalathus linearis) herbal tea is inherently linked to its health-promoting properties. “Fermentation” (oxidation) develops the characteristic flavour and colour of the herbal tea product. Changes to the phenolic composition during fermentation include conversion of aspalathin, a dihydrochalcone unique to rooibos and the major bioactive compound, to eriodictyol-glucopyranoside isomers. These compounds have not been quantified in rooibos plant material to date, due to separation challenges. In this study an improved HPLC method using a core shell column was developed to provide effective separation of a phenolic precursor (enolic phenylpyruvic acid-2-O-glucopyranoside, PPAG) and 15 rooibos tea phenolics (aspalathin, four eriodictyol-glucopyranoside isomers and 11 other major phenolic compounds) in a reasonable time. Compounds were tentatively identified using authentic reference standards and/or tandem mass spectrometry. The HPLC method utilising diode-array detection for quantitation was validated. After evaluation of different extraction solvents, a 40% aqueous acetonitrile solution was deemed optimal for extraction of phenolic compounds from rooibos plant material. Green, semi-fermented and fermented plant material from 10 sub-divided rooibos bushes were extracted and analysed. Water extracts, representing food ingredient extracts, were also analysed. The largest decreases with fermentation were observed for the dihydrochalcones, with moderate to small decreases for flavonols. Eriodictyol-glucopyranoside isomer concentrations increased following fermentation.