J. Sep. Sci. 2009, 32, 2827 – 2834 J. C. Pires Penteado et al. 2827 JosØ Carlo Pires Penteado 1 Marilda Rigobello-Masini 1 Cleber Wanderlei Liria 2 M. TerÞsa Machini Miranda 2 Jorge Cesar Masini 1 1 Departamento de Química Fundamental, Instituto de Química, Universidade de S¼o Paulo, S¼o Paulo, SP, Brazil 2 Departamento de Bioquímica, Instituto de Química, Universidade de S¼o Paulo, S¼o Paulo, SP, Brazil Original Paper Fluorimetric determination of intra- and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode This paper describes the development and application of an RP HPLC method using aC 18 monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mer- captoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution A1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 lmol/L) to 12% for Ile (0.10 lmol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facil- itates the analysis by shortening the separation time and saving solvents and instru- mentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns). Keywords: Amino acids / HPLC / Microalgae / Monolithic columns / Received: December 18, 2008; revised: February 12, 2009; accepted: February 15, 2009 DOI 10.1002/jssc.200800741 1 Introduction Amino acids (AAs) play a key role in plant and animal metabolisms. The types and total concentrations of such biomolecules in intra- and extracellular environments are modulated by the photosynthetic process and nitro- gen assimilation by living organisms [1]. AAs in marine ecosystems are responsible for 13 – 30% of the dissolved nitrogen [2]. Thus, these carbon – nitrogen compounds can be used as markers to characterize distinct physio- logical conditions of the phytoplankton and help the understanding of oceanic food webs. Some AAs such as serine (Ser), glycine (Gly), glutamine (Gln), and glutamic acid (Glu) can be used as markers for the photorespira- tory process [3], which occurs as phytoplanktonic cells are exposed to factors that limit the carbon assimilation by the rubisco enzyme. Ornithine (Orn) and citrulline (Ctr) are nonproteic AAs used by the cell as carriers of amino groups between Glu and Gln, and as carriers of amino and carboxyl groups released during the synthesis of Ser from Gly in the photorespiratory process. There- fore, they act as carriers of amino groups between cell compartments [4]. Other AAs, such as alanine (Ala) and arginine (Arg), can indicate the nutritional status of the cell, regarding to nitrogen assimilation and storage. Some authors use the AAs composition as the chemical print that differentiates among the several phytoplank- tonic species [5]. Determination of dissolved free amino acid (DFAA) can be made by precolumn derivatization with the o-phthal- dialdehyde reagent followed by RP HPLC using stationary phase consisted of octadecylsilane (C 18 ) particles and fluo- rescence detection [5, 6]. Depending on the chromato- graphic conditions, baseline separation requires chroma- tographic runs that may last about 50 min or more [7 – 9]. Correspondence: Dr. Marilda Rigobello-Masini, Departamento de Química Fundamental, Instituto de Química, Universidade de S¼o Paulo, C.P. 26077, 05513-970, S¼o Paulo, SP, Brazil E-mail: rmmarilda@gmail.com Fax: +55-11-3815-5579 Abbreviations: AA, amino acid; Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Ctr, citrulline; Cys, cysteine; DFAA, dissolved free amino acid; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; OPA-2MCE, o-phthaldialdehyde 2- mercaptoethanol; Orn, ornithine; Phe, phenylalanine; Ser, ser- ine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com