Rapid and highly automated determination of adenine and pyridine nucleotides in extracts of Saccharomyces cerevisiae using a micro robotic sample preparation-HPLC system.

An ion-pair reversed-phase chromatography method was adapted for the simultaneous separation and quantification of adenine and pyridine nucleotide concentrations in cell extracts of Saccharomyces cerevisiae. Microbial extracts including metabolites, macromolecular constituents and inorganic compounds were loaded onto a ODS pre-column in the presence of triethylamine phosphate (TEA-Pi) resulting in a selective binding of the nucleotides and removing of interfering compounds. After washing the enrichment cartridge with 30 mM TEA-Pi buffer, adenine and pyridine nucleotides were eluted with a gradient of Mg(II), the competing hetaeron. This combined cleaning and concentration step leads to remarkable improvement of the detection limit for all nucleotides of interest and column lifetimes. The clean up and separation procedures were performed automatically with a micro robotic-system and a control software package written in PASCAL. The paper reports about the application of the proposed method to separation of adenine and pyridine nucleotides in cells extracts of S. cerevisiae grown anaerobically in a continuous culture (D = 0.1 h-1). Rapidity of analysis, high sensitivity as well as reproducibility of the system and the accurate evaluation of the adenine and pyridine nucleotide concentrations make this method particularly useful for routine analysis.