Available online at www.sciencedirect.com Journal of Chromatography A, 1172 (2007) 170–178 Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry Carla Antonio a , Tony Larson b , Alison Gilday b , Ian Graham b , Ed Bergstr¨ om a , Jane Thomas-Oates a,∗ a Department of Chemistry, University of York, Heslington, York YO10 5DD, UK b Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, UK Received 14 August 2007; received in revised form 2 October 2007; accepted 4 October 2007 Available online 10 October 2007 Abstract This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0–100 M, selectivity, short analysis time, and limits of detection of 0.1 M for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5 M for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase (pgm) mutant over a 12-h light/12-h dark growth cycle. © 2007 Elsevier B.V. All rights reserved. Keywords: Glycolytic intermediates; Sugar phosphates; Porous graphitic carbon; HPLC–MS/MS; Arabidopsis thaliana; pgm1 Mutant 1. Introduction Glycolytic intermediates and sugar phosphates are central compounds in metabolism and signalling pathways. They are important intermediates of cellular energy metabolic pathways, such as glycolysis, the pentose phosphate pathway, and starch and sucrose synthesis in plants. However, due to their high polarity, low in vivo concentrations, rapid metabolic turnover, structural variety, instability and poor UV absorbance, these compounds are a particularly challenging subclass of the metabolome to analyse. ∗ Corresponding author. Tel.: +44 1904 43 4459; fax: +44 1904 43 2516. E-mail addresses: cipa500@york.ac.uk (C. Antonio), trl1@york.ac.uk (T. Larson), adg5@york.ac.uk (A. Gilday), iag1@york.ac.uk (I. Graham), etb2@york.ac.uk (E. Bergstr¨ om), jeto1@york.ac.uk (J. Thomas-Oates). Glycolytic intermediates and sugar phosphates in plants have been traditionally measured using highly sensitive techniques such as enzyme assays [1,2]. Such assays offer the advantage of being very specific, which at the same time is their primary dis- advantage; it is not possible to perform parallel determinations of different metabolites in the same sample. To overcome this, mass spectrometry (MS), combined with on-line separations, has been widely applied in metabolome analysis allowing the detection and identification of a wide range of metabolites in a single run. Moreover, the low limits of detection and the ability to perform multistage tandem MS experiments has made MS a popular choice. Glycolytic intermediates and sugar phosphates are non- volatile and unstable compounds, and to analyse this class of metabolite by GC–MS, derivatization is required. The main dis- advantage associated with derivatization protocols is the time and sample losses associated with the additional step in sample 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.10.011