Evaluation of capillary electrophoresis for the analysis of indigenous tea phenolics

Arries, William John (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: The endemic South African plant species, Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush) are consumed worldwide as popular herbal teas. In addition, recent trends in the consumption of these products as ready-to-drink commodities and the use of extracts as ingredients in food, beverage, cosmetic and nutraceutical products are partially ascribed to their potential health-promoting effects. Both rooibos and honeybush teas contain relatively high levels of rare anti-oxidative phenolic compounds. The health properties of these plants are largely associated with their content of phenolic compounds. The analysis of phenolics in these herbal teas is therefore important, not only in support of fundamental research, but also for quality control and marketing purposes. High performance liquid chromatography (HPLC) is the method of choice for the analysis of phenolic compounds in natural products, due to the proven performance of the technique in terms of robustness and sensitivity. However, HPLC also suffers from some drawbacks such as relatively high solvent consumption, long analyses and challenges to obtain sufficient resolution for highly complex samples such as these. Capillary electrophoresis (CE) is a potentially promising alternative technique for the separation of phenolics. CE offers cheap routine analyses, high speed and high efficiencies and an alternative separation mechanism compared to HPLC. The aim of this study was to evaluate CE as an alternative to HPLC for the analysis of rooibos and honeybush tea phenolics. Following extensive method optimisation, two capillary zone electrophoresis (CZE) methods which provided efficient separation of the principal rooibos (15) and honeybush (8) tea phenolics, were successfully developed for the first time. Experimental parameters evaluated and optimised include the background electrolyte (BGE) composition and pH, capillary dimensions, analysis temperature, applied voltage and injection volumes. A borate buffer was found to be essential due to the capability of this BGE to form anionic complexes with phenolics containing vicinal diols, thereby affecting the selectivity of the separation. The optimised BGE pHs for the rooibos and honeybush methods were 8.80 and 9.25, respectively, resulting in total analysis times (including conditioning of capillary) of 31 (honeybush) and 42 minutes (rooibos). Analysis times were slightly shorter than the routine HPLC methods. The optimised CZE methods were characterised by alternative selectivity compared to HPLC, and higher resolution of critical compound pairs was obtained. The methods were validated in terms of repeatability of electrophoretic mobilities, linearity of calibration curves and sensitivity (limits of detection (LODs) and limits of quantification (LOQs)). Sensitivity and repeatability of the CZE methods were however not comparable with HPLC. Finally, the developed methods were applied to the analysis of fermented and unfermented rooibos (Aspalathus linearis) and honeybush tea (C. subternata and C. maculata samples). Quantitative data obtained for 10 samples of fermented and unfermented rooibos and C. subternata and 9 of C. maculata each were compared to those obtained by routine HPLC methods, and were found to be statistically comparable for the majority of compounds, with a few exceptions. The two quantitative CZE methods demonstrated their utility for the routine quantitative analysis of phenolics in rooibos and honeybush teas, respectively, thereby confirming the potential of CE as an alternative to HPLC for the routine analysis of these samples.

AFRIKAANSE OPSOMMING: Die endemiese Suid-Afrikaanse plantspesies, Aspalathus linearis (rooibos) en die Cyclopia spesies (heuningbos) word wêreldwyd verbruik as gewilde kruietees. Verder word onlangse tendense ook gevolg met die verbruik van hierdie produkte as drink-gereed kommoditeite, asook die gebruik van ekstrakte daarvan as bestandele in voedsel, drank en kosmetiese produkte wat gedeeltelik toegeskryf kan word aan hul potensiële gesondheidsvoordele. Beide rooibos en heuningbos tee bevat relatief hoë vlakke van skaars anti-oksidante, sogenaamde fenoliese verbindings. Die gesondheidseienskappe van hierdie plante hou tot ’n groot mate verband met hul inhoud van fenoliese verbindings. Die analiese van fenoliese komponente in hierdie kruietees is dus belangrik, nie net ter ondersteuning van fundamentele navorsing nie, maar ook vir gehaltebeheer en bemarkings doeleindes. Hoë-druk vloeistof chromatografie (HDVC) is die mees algemene metode wat aangewend word vir die analise van fenoliese verbindings in natuurlike produkte as gevolg van die doeltreffendheid van die tegniek in terme van herhaalbaarheid en sensitiwiteit. Maar HDVC word egter ook gekenmerk deur ’n paar nadele soos relatief hoë oplosmiddel verbruik, lang analises en die uitdaging om voldoende resolusie vir baie komplekse monsters soos natuurlike produkte daar te stel. Kapillêre elektroforese (KE) is 'n belowende potensiële alternatiewe tegniek vir die skeiding van fenole. KE is goedkoop vir roetine analise, is vinnig en bied hoe effektiewiteit en ’n alternatiewe skeidingsmeganisme in vergelyking met HDVC. Die doel van hierdie studie was om KE te ondersoek as ’n alternatief vir HDVC vir die analiese van rooibos en heuningbos tee fenole. Na intensiewe metode optimisering is twee kapillêre zone elektroforese (KZE) metodes wat effektiewe skeiding van die hoof rooibos (15) en heuningbos (8) tee fenole verskaf het vir die eerste keer suksesvol ontwikkel. Veranderlikes wat eksperimenteel geëvalueerd en geoptimiseer is, sluit die samestelling en pH van agtergrond elektroliet (AE) in, kapillêre dimensies, analise temperatuur, aangewende hoogspanning en volume wat ingespuit word. Die gebruik van ’n boraat buffer was noodsaaklik, as gevolg van sy vermoë om anioniese komplekse te vorm met fenole met visinale diole en dit affekteer sodoende die selektiwiteit van die skeiding. Die optimale AE pH vir die rooibos en heuningbos metodes was 8.80 en 9.25 onderskeidelik, en die totale analise tyd (insluitend kondisionering van kapillêre) van 31 (heuningbos) en 42 minute (rooibos), onderskeidelik. Analise tyd was effens korter as die roetine HDVC metodes. Die optimale KZE metodes is gekenmerk deur alternatiewe selektiwiteit in vergelyking met HDVC, en hoër resolusie van kritiese pareverbindings is beweerkstellig. Die metodes is gevalideer in terme van herhaalbaarheid van elektroforetiese mobiliteite, lineariteit van kalibrasie kurwes en sensitiwiteit (limiete van deteksie (LVDs) en limiete van kwantifisering (LVKs)). Sensitiwiteit en herhaalbaarheid van die KZE metodes was nie vergelykbaar met die van HDVC nie. Ten slotte is die ontwikkelde metodes toegepas vir die analise van beide gefermenteerde en ongefermenteerde rooibos (Aspalathus linearis) en heuningbos tee (C.subternata en C. maculata) monsters. Kwantitatiewe data is verkry vir 10 monsters van gefermenteerde en ongefermenteerde rooibos en C. subternata en 9 van C. maculata. In vergelyking met data wat verkry is deur roetine HDVC was die resultate statisties vergelykbaar vir die meerderheid van komponente, met enekele uitsonderings. Die twee kwantitatiewe KZE metodes demonstreer hul nut vir die roetine kwantitatiewe analise van fenole in rooibos en heuningbos tee, onderskeidelik, en sodoende illustreer die potensiaal van KE as ’n alternatief vir HDVC vir roetine analise van hierdie monsters.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100265
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