Analysis of complex tannins by multidimensional techniques

Venter, Pieter (2019-03)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Hydrolysable tannins are plant derived (poly)phenolics which for centuries have been used in tanning, the transformation of animal hides into leather. In addition to their industrial applications, epidemiological evidence suggests that the consumption of hydrolysable tannins can be linked to disease prevention and treatment, although their mode of action at a molecular level is still unknown. In order to investigate their biological and chemical properties, the determination of the molecular composition of hydrolysable tannins in plant material is a critical step. Liquid chromatography hyphenated to mass spectrometry (LC-MS) is the preferred analytical approach to obtain this information, although conventional methods often provide insufficient performance for highly complex mixtures of hydrolysable tannins. The goal of the work reported in this thesis was, therefore, to develop improved multidimensional methods for the analysis of hydrolysable tannins. In the first part of the study, a methodology for the comprehensive analysis of ellagitannins and gallotannins in chestnut and tara using reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) combined with ultraviolet (UV), ion mobility (IM) spectrometry and high-resolution mass spectrometry (HR-MS) detection is reported. Good chromatographic performance was achieved using both HILIC and RP-LC for chestnut tannins, with the complementary separation mechanisms proving useful for particular compound classes also pointing to the potential of the combination of HILIC and RP-LC in a comprehensive two-dimensional separation. In contrast, RP-LC provided much better separation of tara gallotannins than HILIC. Incorporation of IM into the LC-MS workflows offered several advantages for such non-targeted screening analyses, including improved mass spectral quality by filtering MS data according to IM arrival time, the availability of an additional identification criterion in the form of collisional cross section (CCS) values obtained from arrival times, and an improved MS duty cycle and, therefore improved sensitivity. Furthermore, additional isomeric species were resolved by IM and, in combination with UV spectral data, IM provided a simple methodology to differentiate between positional isomers. In the second part of the study, an on-line comprehensive two-dimensional HILIC×RPLC method was developed for the analysis of hydrolysable tannins by kinetic optimisation of the relevant experimental parameters. The use of a weak make-up flow to dilute the HILIC effluent and avoid second dimension injection band broadening resulted in excellent performance, with practical peak capacities in excess of 1000 determined for both samples. Despite on-column dilution and the short second dimension analysis times ultimately limiting the number of compounds detected in chestnut and tara, the group-type separations obtained show promise for the fast fingerprint analysis of hydrolysable tannins. Building on previous findings, the final part of the work evaluated the potential of IM spectrometry as a third dimension in a comprehensive three-dimensional HILIC×RPLC×IM configuration. Apart from the inherent advantages of IM alluded to above, the additional separation step offered by IM contributed to an increase in practical peak capacity by a factor of 13 when integrated into the LC×LC workflow. Certain instrumental constraints which limit the ultimate performance of LC×LC×IM-HR-MS are highlighted. Despite these, this approach shows significant promise for the high resolution separation of highly complex mixtures.

AFRIKAANSE OPSOMMING: Hidroliseerbare tanniene is plant gebaseerde (poli)fenole wat al eeue lank gebruik word in looiery, die omskakeling van diere velle in leer. Bykomend tot hul industriële toepassings dui epidemiologiese bewyse daarop dat die inname van hidroliseerbare tanniene gekoppel kan word aan die voorkoming en behandeling van siektes, alhoewel hul werkswyse op molekulêre vlak nog onbekend is. Ten einde hul biologiese en chemiese eienskappe te ondersoek, is die bepaling van die molekulêre samestelling van hidroliseerbare tanniene in plantmateriaal 'n kritieke stap. Vloeistofchromatografie gekoppel aan massa spektrometrie (LC-MS) is die analitiese benadering wat meestal gebruik word om hierdie inligting te verkry, hoewel konvensionele metodes dikwels onvoldoende resultate vir hoogs komplekse mengsels van hidroliseerbare tanniene lewer. Die doel van die werk wat in hierdie proefskrif gerapporteer is, was dus om verbeterde multidimensionele metodes vir die analise van hidroliseerbare tanniene te ontwikkel. In die eerste gedeelte van die studie is 'n metodologie vir die omvattende analise van ellagitanniene en gallotanniene in chestnut en tara deur die gebruik van omgekeerdefase vloeistofchromatografie (RP-LC) en hidrofiliese interaksie chromatografie (HILIC) gekombineer met ultraviolet (UV), ioon mobiliteit (IM) spektrometrie en hoë-resolusie massa spektrometrie (HR-MS) deteksie gerapporteer. Goeie chromatografiese skeiding is behaal met die gebruik van beide HILIC en RP-LC vir chestnut tanniene, met die komplementêre skeidingsmeganismes wat nuttig bewys is vir spesifieke komponent klasse, en dus ook dui op die potensiaal van die kombinasie van HILIC en RP-LC in 'n omvattende twee-dimensionele skeiding. In teenstelling hiermee lewer RP-LC ‘n baie beter skeiding van tara gallotanniene as HILIC. Die inkorporering van IM in die LC-MS werksvloei het verskeie voordele vir sulke nie-geteikende analise. Dit sluit in verbeterde massaspektrum kwaliteit deur MS-data volgens IM-aankomstyd te filtreer, die beskikbaarheid van 'n bykomende identifikasie parameter in die vorm van botsing-dwarssnit (CCS) waardes verkry vanaf aankoms tye, en 'n meer doeltreffende massa spektrometer wat lei tot beter sensitiwiteit. Verder, isomeriese spesies wat deur IM geskei kan word, in kombinasie met UV spektrale data, verskaf 'n eenvoudige metode om tussen posisionele isomere te onderskei. In die tweede deel van die studie is 'n aanlyn-omvattende twee-dimensionele HILIC×RP-LC metode vir die analise van hidroliseerbare tanniene deur kinetiese optimalisering van die relevante eksperimentele parameters ontwikkel. Verdunning van die eerste dimensie (1D) eluent met swak tweede dimensie (2D) mobiele fase om inspuitings-bandverbreding in die 2D te beperk het uitstekend skeiding gelewer, met praktiese piek kapasiteite van meer as 1000 wat vir beide monsters bepaal is. Ten spyte van in-kolom verdunning en kort tweede dimensie analise tye wat die aantal verbindings wat in chestnut en tara opgespoor word beperk, toon die groep-tipe skeidings potensiaal vir die vinnige vingerafdruk analise van hidroliseerbare tanniene. Gebaseer op vorige bevindinge, het die finale deel van die werk die potensiaal van IM-spektrometrie as 'n derde dimensie in 'n omvattende drie-dimensionele HILIC×RPLC×IM opset geëvalueer. Afgesien van die inherente voordele van IM wat hierbo genoem word, het die bykomende skeidingsstap wat deur IM gelewer word die praktiese piek kapasiteit met ‘n faktor van 13 verhoog wanneer dit in die LC×LC werksvloei geïntegreer is. Verskeie instrumentele limitasies wat die uiteindelike prestasie van LC×LC×IM-HR-MS beperk, word uitgelig. Ten spyte hiervan toon hierdie benadering betekenisvolle potensiaal vir die hoë resolusie skeiding van hoogs komplekse mengsels.

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