Tulip Carbohydrate Analysis by HPLC and Mass Spectrometry

YOUSEF GHOLIPOUR; HIROSHI NONAMI; ROSA ERRA-BALSELLS; ERRA BALSELLS, ROSA

Matsuyama, Japon. Septiembre 6-11

Congreso; The annual meeting of the Japanese Society of Agricultural, Biological and Environmental Engineers and Scientists-Matsuyama 2008; 2008

Japanese Society of Agricultural, Biological and Environmental Engineers and Scientists

Tulip Carbohydrate Analysis by HPLC and Mass Spectrometry   ??Yousef Gholipour (UGAS, Ehime University) Hiroshi Nonami (Ehime University) Rosa Erra-Balsells (University of Buenos Aires)   Keywords: MALDI; ESI; starch; fructan; degradation Abstract Carbohydrates play significant roles in agricultural yield and industry, and plant biology including whole carbon-energy budget, osmotic-water potential and whole-plant water relations. HPLC can provide precise detection-quantification of individual sugars. Mass spectrometry is a modern analytical technique that can be applied for accurate analysis of biological samples by molecular weight determination and also providing some structural information about compounds. In tulip, starch, fructans and sucrose are main carbohydrates stored in bulbs for supplying carbon and energy for flowering after rest period. Analysis of sugars in this plant, therefore, includes examination of insoluble starch and fructans, in addition to soluble fructans and sucrose.  Although there is possibility to analyze plant tissue directly by using MALDI MS without need to extraction-purification, here we discuss extracted carbohydrate analysis by HPLC and mass spectrometry. Separation and purification of interested metabolites from others eases analysis and inhibits probable chemical interaction between different metabolites during analysis. Freeze-drying of plant samples is the first step in the experiment to preserve metabolites in their original quantity and quality. The procedure continues by homogenizing of the sample in aqueous ethanol and centrifugation which results in the separation of soluble and insoluble contents of the sample. Water-ethanol insoluble phase which contains starch and insoluble fructans is boiled (to gelatinize) or dissolved in DMSO-HCl (to solubilize) the starch. Water-ethanol soluble phase is re-extracted by mixing vigorously in chloroform which removes hydrophobic compounds (e.g. lipids). Also more purification of neutral underivatized soluble sugars is carried out by passing through cation- and anion-adsorbing resins or commercial cartridges. Soluble sugars can be characterized in this stage by using both ESI and MALDI MS. However we shall follow some more steps in our analysis procedure to make possibility of accurate quantification of sugars by complete hydrolysis of starch, fructans and sucrose, followed by analysis of the products i.e. glucose and fructose by HPLC and/or mass spectrometry. Soluble and insoluble sugars are incubated with amyloglucosidase and fructofuranosidase which degrade glucose and fructose polymers, respectively. Then solutions are passed through resins. Analysis is carried out by HPLC, ESI  MS and MALDI MS. Information about different aspects of the method including experimental condition, procedure details, HPLC and mass spectrometry operation, and usefulness of different matrices for MALDI MS will be presented.