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 s imultaneous separation and quantification of adenine and pyridine nucl eotide concentrations in cell extracts of Saccharomyces cerevisiae. Mi crobial extracts including metabolites, macromolecular constituents an d inorganic compounds were loaded onto a ODS pre-column in the presenc e of triethylamine phosphate (TEA-P-i) resulting in a selective bindin g of the nucleotides and removing of interfering compounds. After wash ing the enrichment cartridge with 30 mM TEA-P-i buffer, adenine and py ridine nucleotides were eluted with a gradient of Mg(II), the competin g hetaeron. This combined cleaning and concentration step leads to rem arkable improvement of the detection limit for all nucleotides of inte rest and column lifetimes. The clean up and separation procedures were performed automatically with a micro robotic-system and a control sof tware package written in PASCAL. The paper reports about the applicati on of the proposed method to separation of adenine and pyridine nucleo tides in cells extracts of S. cerevisiae grown anaerobically if a cont inuous 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. (C) 1998 Elsevier Science B .V. All rights reserved.