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A hydrophilic-interaction chromatography (HILIC) method coupled to electrospray ionization mass spectrometry (ESI-MS) was developed for the determination of trehalose-6-phophate (Tre6P) in Arabidopsis thaliana seedlings. The method was optimized for MS detection and separation of Tre6P from its isomers, such as sucrose-6-phosphate, by testing eluent pH, type of organic solvent and alkalinizer, and gradient conditions. Tre6P could be resolved from matrix components within 28 min by using a water–acetonitrile gradient (0.2 ml/min) at pH 12 with piperidine as alkalinizer. The method was validated for concentrations between 25 and 4,000 nM Tre6P in A. thaliana seedling extracts. Seedlings were extracted with consecutive liquid-liquid and solid-phase extractions, and analyzed with HILIC-MS. Obtained accuracy (80–120 %) and precision (<24 %) demonstrated the suitability of HILIC-MS for determining Tre6P level variations in plants. The limit of detection (LOD) was 3.5 nM Tre6P in extracts corresponding to 4.1 pmol.g−1 fresh plant weight (FW). This is a considerable improvement with respect to anion-exchange chromatography (AEC)-MS (40 nM) and capillary electrophoresis-MS (80 nM). Furthermore, HILIC-MS analysis times were shorter than with AEC-MS (30 and 60 min, respectively). The applicability of the HILIC-MS method was demonstrated by the analysis of extracts from seedlings grown on medium containing 100 mM sorbitol or trehalose, resulting in mean Tre6P concentrations of 0.2 and 1.9 nmol.g−1 FW, respectively. Similar concentrations were found with AEC-MS. HILIC-MS was also evaluated at a high flow rate (2.0 ml/min). This high-speed method resolved the Suc6P and Tre6P peaks within 3 min yielding a detection limit of 1.3 nM Tre6P. Figure Extracted-ion chromatogram obtained during high-pressure hydrophillic-interaction liquid chromatography-mass spectrometry of a trehalose-grown Arabidopsis thaliana (photopgraph) seedling. Trehalose-6-phosphate (structural formula) is separated from its isomers sucrose-6-phosphate and lactose-1-phosphate
| Authors: | Javier Sastre Toraño, Thierry L. Delatte, Henriette Schluepmann, Sjef C. M. Smeekens, Gerhardus J. de Jong, Govert W. Somsen | |
| Journal: | Analytical and Bioanalytical Chemistry | |
| Year: | 2012 | |
| DOI: | 10.1007/s00216-012-5928-4 | |
| Publication date: | 26-03-2012 |
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