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Quantitation of amino acids in plasma by high performance liquid chromatography: Simultaneous deproteinization and derivatization with 9-fluorenylmethyloxycarbonyl chloride

Jámbor, A., Molnár-Perl, I.
Journal of chromatography 2009 v.1216 no.34 pp. 6218-6223
acetonitrile, cystine, derivatization, fluorescence, free amino acids, high performance liquid chromatography, histidine, humans, hydrolysis, ornithine, pH, tryptophan, tyrosine
This paper, as a novelty to this field, presents the deproteinization and derivatization of plasma's free amino acids (PFAAs), simultaneously, in a single step, with the acetonitrile (ACN) containing 9-fluorenylmethyloxycarbonyl chloride (FMOC) reagent. Deproteinization and derivatization, were studied with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection, simultaneously. Model investigations have been carried out as a function of the FMOC concentration, reaction time and reaction conditions: with standard solutions, with human plasma samples in its initial condition and fortified with standard amino acids (excluding tryptophan because it co-elutes with the hydrolyzed FMOC). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 3.0mM FMOC concentration, at pH 9; derivatization time=20min), and characterized with the relative standard deviation percentages of their responses (<=4.4%, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 2.5pmol, except for cystine, ornithine (5pmol) and for the total of tyrosines (N-FMOC-tyrosine and N,O-FMOC-tyrosine 10pmol).