Kinetic optimisation of the reversed phase liquid chromatographic separation of rooibos tea (Aspalathus linearis) phenolics on conventional high performance liquid chromatographic instrumentation

Rooibos tea, produced from the endemic South African shrub Aspalathus linearis, has various health-promoting benefits which are attributed to its phenolic composition. Generating reliable, quantitative data on these phenolic constituents is the first step towards documenting the protective effects associated with rooibos tea consumption. Reversed phase liquid chromatographic (RP-LC) methods currently employed in the quantitative analysis of rooibos are, however, hampered by limited resolution and/or excessive analysis times. In order to overcome these limitations, a systematic approach towards optimising the RP-LC separation of the 15 principal rooibos tea phenolics on a 1.8 μm phase using conventional HPLC instrumentation was adopted. Kinetic plots were used to obtain the optimal configuration for the separation of the target analytes within reasonable analysis times. Simultaneous optimisation of temperature and gradient conditions provided complete separation of these rooibos phenolics on a 1.8 μm C18 phase within 37 min. The optimised HPLC–DAD method was validated and successfully applied in the quantitative analysis of aqueous infusions of unfermented and fermented rooibos. Major phenolic constituents of fermented rooibos were found to be a phenylpropanoid phenylpyruvic acid glucoside (PPAG), the dihydrochalcone C-glycoside aspalathin, the flavones isoorientin and orientin, and a flavonol O-diglycoside tentatively identified as quercetin-3-O-robinobioside. Content values for PPAG, ferulic acid and quercetin-3-O-robinobioside in rooibos are reported here for the first time. Mass spectrometric (MS) and tandem MS detection were used to tentatively identify 13 additional phenolic compounds in rooibos infusions, including a new luteolin-6-C-pentoside-8-C-hexoside and a novel C-8-hexosyl derivative of aspalathin reported here for the first time.