Cleanup and analysis of sugar phosphates in biological extracts by using solid-phase extraction and anion-exchange chromatography with pulsed amperometric detection.
A cleanup method based on anion-exchange solid-phase extraction (SPE) was developed to render biological extracts suitable for the analysis of hexose phosphates with a modified anion-exchange chromatography method and pulsed amperometric detection. The method was applied to cell extracts of Saccharomyces cerevisiae obtained by using cold methanol as quenching agent and chloroform as extraction solvent. It was shown that pretreatment of the cell extract with SPE markedly improved the quality of the liquid chromatography analysis with recoveries of the sugar phosphates close to 100%. Furthermore, the method allowed for sample enrichment and the original extraction procedure could be simplified by implementing SPE early in the extraction protocol.
Department of Biotechnology, Technical University of Denmark, Building 223, Lyngby DK-2800, Denmark.
H P
HP
Smits
A
A
Cohen
T
T
Buttler
J
J
Nielsen
L
L
Olsson
D002458:Cell Fractionation; D002623:Chemistry Techniques, Analytical; D002725:Chloroform; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004563:Electrochemistry; D006600:Hexosephosphates / Q000032:analysis* / Q000302:isolation & purification; D000432:Methanol; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012997:Solvents
D006600:Hexosephosphates; D012997:Solvents; D002725:Chloroform; D000432:Methanol