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

摘要

Simple and reliable protocols are described for an extensive analysis of metabolites in extracts from different biological sources. Teh separation was performed by high performance ionic-exchange chromatography (HPIC) at alkaline pH using two types of chromatography columns and two detection methods. Organic acids and inorganic anions were separated on an ionPac AS11 column suing a 0.5 to 35 mM Na0H gradient. Detection limits in the range of milligrams per liter were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor. Twelve phosphorylated compounds belonging to the glycolytic and the pentose phosphate pathways could be resolved on a CarboPac PA1 column using a Na0H/Na-acetate gradient. Quantification was achieved by pulsed amperometry with detection limits in the micromolar range. Cell extracts obtained by extraction in boling buffered ethanol described previously could be directly injected onto PHIC columns for the separation of metabolites because the extraciton procedure affected neither the retention time nor the stability of most of themetabolites, and yielded very clean chromatograms. These improved protocols were applied for a dynamicd analysis of intracellular metabolites in Saccharomyces cerevisiae in response to a glucose pulse.