The Plant Journal (2008) 55, 1047–1060
Metabolite profiling of Calvin cycle intermediates by HPLC-MS using mixed-mode stationary phases Jeffrey A. Cruz1, Caroline Emery1,2,3,†, Matthias Wu¨st3, David M. Kramer1 and B. Markus Lange1,2,* Institute of Biological Chemistry, 2 M.J. Murdock Metabolomics Laboratory, Washington State University, Pullman, P.O. Box 646340, Washington 99164-6340, USA, and 3 Insitute of Life Technologies, University of Applied Sciences of Western Switzerland Valais, Route du Rawyl 47, 1950 Sion, Switzerland
Received 19 February 2008; revised 7 April 2008; accepted 18 April 2008; published online 14 July 2008. * For correspondence (fax +1 509 335 7643; e-mail [email protected]
). † Present address: Swiss Laboratory for Doping Analyses, Chemin des Croisettes 22, 1066 Epalinges, Switzerland.
Summary A sensitive and robust mixed-mode high performance liquid chromatography–tandem mass spectrometry method was developed for the qualitative and quantitative determination of sugar phosphates, which are notoriously difficult to separate using reversed-phase materials. Sugar phosphates were separated on a Primesep SB column by gradient elution using aqueous ammonium formate and acetonitrile as mobile phases. Target analytes were identified by their precursor/product ions and retention times. Quantitative analysis was performed in negative ionization/multiple reaction monitoring mode with five different time segments. The method was validated by spiking authentic sugar phosphate standards into complex plant tissue extracts. Standard curves of neat authentic standards and spiked extracts were generated for concentrations in the low picomole to nanomole range, with correlation coefficients of R2 > 0.991, and the degree of ion suppression in the presence of a plant matrix was calculated for each analyte. Analyte recoveries, which were determined by including known quantities of authentic standards in the sugar phosphate extraction protocol, ranged from 40.0% to 57.4%. The analytical reproducibility was assessed by determining the coefficient of variance based on repeated extractions/measurements (