Profiling of phenolic glycosidic conjugates in leaves of Arabidopsis thaliana using LC/MS

Abstract

Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arabidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri-, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase HPLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated.

Profiling methods for the determination of phenolic compounds in foods and dietary supplements

“…Compromises in extraction and separation will affect both identification and quantification. Tables 1, 2, 3, 4 present 14 profiling methods for phenolic compounds that have been selected from the literature of the last 15 years [18][19][20][21][22][23][24][25][26][27][28][29][30][31]. This is not meant to be a comprehensive list.…”

“…In some cases, the methods were directed at a limited number of food materials, but could be readily modified for application to a greater breadth of materials than originally described [19,20,22,25,27]. In recent years, however, methods have appeared that were designed to cover a wide range of compounds in any type of food material [21,23,24,26,28,29] or as metabolite profiling methods [30,31]. For these methods, liquid chromatography (LC) is the method of choice for separation and diode array detection) and mass spectrometry (MS) detection are the detection methods of choice.…”

“…There are some exceptions, however. Methods are included that use multiple extractions [22], multiple chromatographic runs [28], and tandem MS (MS/MS) [30] or ion trap MS (IT/MS) [27,31] detection. Both of the latter methods are used in conjunction with collision-induced dissociation (CID).…”

Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin and sulfur deficiency

“…However, many of these secondary metabolites of Arabidopsis are as yet unidentified. Some metabolites were identified from a comprehensive analysis of mass fragmentation patterns and by comparison with published literature (Graham, 1998;Von Roepenack-Lahaye et al, 2004;Tohge et al, 2005;Kerhoas et al, 2006;Routaboul et al, 2006;Stobiecki et al, 2006;Besseau et al, 2007;Stracke et al, 2007). Additionally, compounds were identified by molecular weight determinations from protonated [M+H] + and deprotonated [MÀH] À molecular ions in the first order mass spectra and then MS/MS spectra where sufficient collision energy was applied to obtain maximum structural information.…”

“…GC-MS gives possibilities of analyzing different classes of compounds, including organic and amino acids, sugars, sugar alcohols, sterols, phosphorylated and lipophilic compounds (Wagner et al, 2003;Lisec et al, 2006). However, LC-MS is also a powerful technology to separate and analyze semi-polar secondary metabolites that can comprise large and unique groups of compounds in plants (Brown et al, 2003;von Roepenack-Lahaye et al, 2004;Stobiecki et al, 2006;de Vos et al, 2007). Therefore, the combination of GC-MS and LC-MS allows for a broad view of metabolic differences when comparing plants grown on different nutrient media.…”