Analysis of phosphorylated carbohydrates by high-performance liquid chromatography electrospray ionization tandem mass spectrometry utilising a β-cyclodextrin bonded stationary phase

Chromatographic separation of phosphorylated carbohydrates was achieved on a beta-cyclodextrin (CD) bonded HPLC column. Applying acetonitrile-aqueous ammonium acetate gradient elution, the separation of sugar phosphates relied on the combination of anion-exchange and hydrophilic interaction properties of CD bonded phases in the presence of ammonium ions. Furthermore, this solvent system allowed on-line coupling to electrospray ionization mass spectrometry (ESI-MS) and direct structural characterisation of carbohydrate phosphates by tandem mass spectrometry (MS-MS). In addition to the analysis of various pentose and hexose phosphates, the HPLC-ESI-MS-MS method was successfully applied to demonstrate the enzymatic formation of D-1-deoxyxylulose 5-phosphate from pyruvate and glyceraldehyde phosphate catalysed by yeast transketolase. (C) 1998 Elsevier Science B.V.