Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate‐related metabolites from Arabidopsis thaliana leaf tissue

Abstract

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.

Mass spectrometry as a quantitative tool in plant metabolomics

“…The method was rapid, and allowed the separation of a complex mixture containing several reducing and non-reducing sugars, sugar alcohols and sugar phosphates in less than 15 min [83]. In addition, a better LOD was obtained for stachyose (66 pg), which represents a considerable improvement over the previously reported HILIC method [80] (table 1).…”

“…In addition, a better LOD was obtained for stachyose (66 pg), which represents a considerable improvement over the previously reported HILIC method [80] (table 1). In that same study, António and co-workers also demonstrated that HILIC-ESI-QIT-MS/MS and PGC-ESI-QIT-MS/MS gave comparable quantitative results (and LODs) for the analysis of the same set of metabolites, and concluded that HILIC and PGC are two excellent alternatives to typical RP methods for the analysis of highly polar metabolites [83] (table 1). However, PGC separations have been reported to require extensive method development and strong conditions for the elution of sugar phosphates [81] and sugar nucleotides [84], which makes HILIC a more suitable approach for the quantitative analysis of these metabolites in routine plant metabolomics studies [83,85,86] (table 1).…”

Application of hydrophilic interaction chromatography in phytochemical analysis

“…[30]. Antonio et al described the use of HILIC coupled to negative ion mode trap ESI-MS for the analysis of several polar plant metabolites: soluble neutral sugars, sugar alcohols and phosphorylated sugars, which generally are not retained on RP-LC columns [31]. Chromatographic separation was performed on a zwitterionic ZIC-HILIC stationary phase, using gradient elution.…”

A novel ionic-bonded cellulose stationary phase for saccharide separation

“…It was also obtained important position among the separation strategies applied for pharmaceutically active compounds [12][13][14]. Because of its good retention of polar compounds, high selectivity and compatibility with mass spectrometry (MS), HILIC has become the most appropriate method for separating carbohydrates by chromatography [10,15,16].…”

“…Hydrophilic interaction liquid chromatography (HILIC), described by Alpert [7], has gained intense interest in the last few years. HILIC has been used to separate polar compounds including sugars, amino acids, and small peptides [8][9][10][11]. It was also obtained important position among the separation strategies applied for pharmaceutically active compounds [12][13][14].…”