Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection

Simple and reliable protocols are described for an extensive analysis of me tabolites in extracts from different biological sources. The separation was performed by high performance ionic-exchange chromatography (HPIC) at alka line pH using two types of chromatography columns and two detection methods . Organic acids and inorganic anions were separated on an ionPac AS11 colum n using a 0.5 to 35 mM NaOH gradient. Detection limits in the range of mill igrams per liter were achieved by use of a conductivity detector equipped w ith an anion self-regenerating suppressor. Twelve phosphorylated compounds belonging to the glycolytic and the pentose phosphate pathways could be res olved on a CarboPac PA1 column using a NaOH/Na-acetate gradient. Quantifica tion was achieved by pulsed amperometry, with detection limits in the micro molar range. Cell extracts obtained by extraction in boiling buffered ethan ol described previously could be directly injected onto HPIC columns for th e separation of metabolites because the extraction procedure affected neith er the retention time nor the stability of most of the metabolites, and yie lded very clean chromatograms. These improved protocols were applied for a dynamic analysis of intracellular metabolites in Saccharomyces cerevisiae i n response to a glucose pulse. (C) 2000 Elsevier Science Inc. All rights re served.