Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography Markus Ganzera a, * ,1 , Pamela Vrabl b,1 , Elisabeth Wo ¨rle a , Wolfgang Burgstaller b , Hermann Stuppner a a Institute of Pharmacy, Department of Pharmacognosy, University of Innsbruck, 6020 Innsbruck, Austria b Institute of Microbiology, University of Innsbruck, 6020 Innsbruck, Austria Received 29 August 2006 Available online 4 October 2006 Abstract 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 dinu- cleotide (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 per- formed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD 60.7 ng on-column), repeatable (r rel 6 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intra- day variation 69.4%, interday variation 66.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 °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. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Nucleotides; Penicillium; HPLC; Glucose limitation; Chemostat; Ethanol extraction Nucleotides play crucial roles in the regulation of the metabolic states of living cells. Free energy is stored via the displacement of phosphorylated adenine nucleotides (ATP, ADP, and AMP) far from their equilibrium concen- trations. Pyridine nucleotides (NAD and NADH) are cosubstrates in numerous redox reactions. Changes in envi- ronmental conditions can affect the total concentration of adenine and pyridine nucleotides and the distribution of nucleotide species [1–3]. These changes may influence the metabolism at several levels, for instance ATP-dependent membrane transport (e.g., the fungal plasma membrane H + -ATPase), ATP-dependent single-enzyme reactions (e.g., hexokinase), or—in form of the energy charge (EC) 2 —at the level of whole metabolic pathways (e.g., ATP-producing and ATP-consuming pathways). 0003-2697/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.09.012 * Corresponding author. Fax: +43 512 507 2939. E-mail address: markus.ganzera@uibk.ac.at (M. Ganzera). 1 These authors contributed equally to the work. 2 Abbreviations used: EC, energy charge; AMP, adenosine monophos- phate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinu- cleotide–reduced form; TEA, triethylamine; HPLC, high-performance liquid chromatography; CE, capillary electrophoresis; SPE, solid phase extraction; USP, United States Pharmacopoeia; LOD, limit of detection; LOQ, limit of quantitation; S/N ratio, signal to noise ratio; DAD, diode array detector; Hepes, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; PCA, perchloric acid; REC, recovery. www.elsevier.com/locate/yabio Analytical Biochemistry 359 (2006) 132–140 ANALYTICAL BIOCHEMISTRY