Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.