Title page for ETD etd-10222009-123647

Document Type Master's Dissertation
Author Du Rand, Esther Elizabeth
URN etd-10222009-123647
Document Title Identification of digallated and methylated catechins using UPLC/MS/MS and development of a rapid analysis method for theanine in tea (Camellia sinensis (L.) O. Kuntze) utilizing evaporative light scattering detection
Degree MSc
Department Biochemistry
Advisor Name Title
Prof Z Apostolides Supervisor
  • Camellia sinensis
  • green tea quality parameter
Date 2009-09-02
Availability unrestricted
Theanine is a free, non-protein amino acid found in Camellia sinensis (tea) leaves. It is the main amino acid component in tea and contributes 1 to 2% of the dry weight of tea leaves. The content of amino acids is a key green tea quality parameter. High grade teas contain high amounts of theanine. It is responsible for the sweet, brothy umami taste of green tea. To date there is no international standard method for the analysis of theanine in tea samples. Several methods with different advantages and disadvantages have been reported but most of them are unsuitable for routine quality control analysis in commercial tea samples.

In this study, a high performance liquid chromatography (HPLC) method with evaporative light scattering detection for the determination of theanine in tea samples has been developed. Chromatographic separation of theanine was achieved by reverse phase HPLC using a phenyl-hexyl column and isocratic water elution within 8 minutes. The method requires no sample derivatization and includes simple sample clean-up. Applying the same chromatographic conditions to a widely available C18 column, separation of theanine was achieved within 10 minutes. The successful application of the C18 column renders this method widely applicable. The method is linear over several orders of magnitude and the LOD are 11.53 and 10.83 μg/ml and the LOQ are 39.44 and 35.10 μg/ml for the phenyl-hexyl and C18 column, respectively. Simple but effective sample preparation and polyvinylpolypyrrolidone pre-treatment, along with the short analysis time facilitates high sample throughput (~40 samples can be prepared in an 8 hour day with overnight analysis by HPLCELSD). The method is selective, precise, accurate and practical for the quantification of theanine in tea extracts and was successfully used to determine theanine content in a variety of tea samples. The sensitivity and simplicity of this method renders it suitable for use in routine theanine analysis in quality control laboratories.

Green tea, a simple refreshing beverage, has been believed to have therapeutic uses for centuries. Scientists have recognized that the wide range of physiological functions of green tea is due to the presence of catechins. An increasing body of evidence is showing that methylated forms of catechins have potent inhibitory activities to allergies. In addition to the methylated catechins, another relative novel group of catechins exists, namely digallated catechins. The medicinal value of these compounds is not yet known, but it is reasonable to speculate that the antioxidant capacity of digallated catechins will be superior to that of epigallocatechin gallate due to the 3 additional hydroxyl groups provided by the second gallic acid moiety.

In this study reverse phase UPLC/ESI-MS/MS was effectively applied to investigate the presence of digallated and methylated catechins in crude tea extract. Low abundance catechins (-)-epigallocatechin-(O-3-methyl) gallate ([M + H]+, m/z 473) and (-)-epicatechin-3-(O-3-methyl) gallate ([M + H]+, m/z 457) were successfully detected at 31.9 and 37.5 minutes, respectively. The 2 digallated catechins investigated could not be detected, possibly due to being present at concentrations below the limit of detection. Compared to conventional HPLC/ESI-MS/MS, increased resolution and sensitivity, 80% less use of solvent and significantly reduced analysis times were observed.

2009 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Du Rand, EE 2009, Identification of digallated and methylated catechins using UPLC/MS/MS and development of a rapid analysis method for theanine in tea [Camellia sinensis (L.) O. Kuntze] utilizing evaporative light scattering detection, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-10222009-123647/ >


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