title: Determination of trehalose-6-phosphate in Arabidopsis thaliana seedlings by hydrophilic-interaction liquid chromatography-mass spectrometry.
abstract: 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.
authors.affiliation: Biomolecular Analysis, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. j.sastretorano@uu.nl
authors.forename: Javier
authors.initials: J
authors.lastname: Sastre Toraño
authors.forename: Thierry L
authors.initials: TL
authors.lastname: Delatte
authors.forename: Henriette
authors.initials: H
authors.lastname: Schluepmann
authors.forename: Sjef C M
authors.initials: SC
authors.lastname: Smeekens
authors.forename: Gerhardus J
authors.initials: GJ
authors.lastname: de Jong
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D036226:Seedlings / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013403:Sugar Phosphates / Q000032:analysis*; D014199:Trehalose / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D013403:Sugar Phosphates; C082722:trehalose-6-phosphate; D014199:Trehalose