Mass Spectrometry Based Investigation of Chlorogenic Acid Reactivity and Profile in Model Systems and Coffee Processing

Beneficial health and biological effects of coffee as well as its sensory properties are largely associated with chlorogenic acids (CGAs) since; coffee is the richest dietary source of CGAs and their derivatives. From green coffee beans to the beverage, chemical components of the green coffee undergo enormous transformations, which have been studied in great details in the past. Roasted coffee melanoidines are extensively contributed by the products formed by the most relevant secondary metabolite- chlorogenic acids. For every 1% of the dry matter of the total CGA content in the green coffee beans, 8-10% of the original CGAs are transformed or decomposed into respective derivatives of cinnamic acid and quinic acid. The non-volatile fraction of the roasted coffee remains relatively unravelled in the aspects of its chemistry and structural information. Coffee roasting, along with the other processes brings about considerable changes in the chlorogenic acid profile of green coffee through number of chemical processes. In roasting, chlorogenic acids evidently undergo various processes such as, acyl group migration, transesterification, thermal trans-cis isomerization, dehydration and epimerization. To understand the chemistry behind roasted coffee melanoidines, it is of utmost importance to study the changes occurring in CGAs and their derivatives through food processing. The isomeric transformations of the chlorogenic acids resulting due to the migration of hydroxycinnamoyl group from any of the four hydroxyl groups of quinic acid to another have been thoroughly investigated in this work by LC-MSn. In this thesis, the acyl migration phenomenon under the treatment of tetramethylammonium hydroxide (TMAH) hydrolysis, model roasting experiments and by brewing at pH 5 (water reflux, 5 h) of the seven commercially available mono- and di-caffeoylqunic acids was studied in detail. Intermolecular acyl migration (transesterification) was also studied by tetramethylammonium hydroxide (TMAH) hydrolysis and model roasting experiments in between 5-CQA and p-coumaric acid as well as in 5-CQA and ferulic acid. In this thesis, four diastereoisomers of quinic acid have been synthesized selectively, namely, epi-quinic acid, muco-quinic acid, cis-quinic acid and scyllo-quinic acid by applying appropriate hydroxyl group protection and deprotection strategies in order to study their behavior in LC-MSn along with commercially available (-)-quinic acid. We report for the first time that these diastereoisomers are distinguishable on the basis of their fragmentation behavior as well as their chromatographic elution order. In this study, we also observed that muco-quinic acid, scyllo-quinic acid and epi-quinic acid are present in hydrolyzed Guatemala roasted coffee sample as possible products of roasting. The synthetic work accomplished in present work will provide for the generation of the reference standards to identify remaining epimers of CGAs in roasted coffee. Considering the fact that relatively large amount of the degradation products of CGAs such as, quinic acid, caffeic acid along with the most prominent member of the CGAs profile in roasted coffee, 5-caffeoylquinic acid itself are present in the roasted coffee along with free small, non-volatile organic acids, we examined in details the further esterification phenomenon among themselves. To investigate transesterification in roasted coffee in details we designed a thorough analytical plan involving four experiments. A selection of small organic acids were heated in the presence of 5-CQA to check if simulated roasting conditions facilitate the formation of the transesterification, caffeic acid and quinic acid with the mixture of all the organic acids separately to check, which of the organic acid show greater affinity towards the formation of the condensed esters. With the experimental results in hand, we then identified transesterification products in different roasted coffee samples by LC-MSn, LC-TOF-MS and FT-ICR-MS. Data generated by different analytical techniques such as, NMR-, CD-, IR spectroscopy and LC-MS was used to differentiate the Arabica and Robusta green coffee extracts by principal component analysis (PCA) to determine, which spectroscopic technique allows the best discrimination of coffee varieties. A total of 38 green bean extracts were characterized using NMR-, CD-, and IR spectroscopy along with LC-MS and the data was further analyzed by PCA using different PCA processing parameters by unsupervised non-targeted approach. Distinction between different groups of samples, in particular, Arabica versus Robusta green coffee beans successfully achieved using IR- spectroscopy and LC-MS. Surprisingly, both CD- and NMR spectroscopy fail to achieve in this case, an adequate level of distinction. This is the first study that directly compares the value of various spectroscopic techniques if multivariant statistical techniques are employed to them.