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

A. Jámbor, I. Molnár-Perl

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21 Citations (Scopus)

Abstract

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 3.0 mM FMOC concentration, at pH 9; derivatization time = 20 min), 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 2.5 pmol, except for cystine, ornithine (5 pmol) and for the total of tyrosines (N-FMOC-tyrosine and N,O-FMOC-tyrosine 10 pmol).

Original languageEnglish
Pages (from-to)6218-6223
Number of pages6
JournalJournal of Chromatography A
Volume1216
Issue number34
DOIs
Publication statusPublished - Aug 21 2009

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High performance liquid chromatography
Chlorides
High Pressure Liquid Chromatography
Plasmas
Amino Acids
Tyrosine
Plasma (human)
Derivatives
Ornithine
Cystine
Photodiodes
Histidine
Tryptophan
9-fluorenylmethoxycarbonyl
Fluorescence

Keywords

  • 9-Fluorenylmethyloxycarbonyl chloride
  • Amino acids
  • Deproteinization
  • Derivatization
  • High performance liquid chromatography
  • HPLC-UV(DAD)/fluorescence detection
  • Plasma

ASJC Scopus subject areas

  • Analytical Chemistry
  • Organic Chemistry
  • Biochemistry

Cite this

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abstract = "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 3.0 mM FMOC concentration, at pH 9; derivatization time = 20 min), 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 2.5 pmol, except for cystine, ornithine (5 pmol) and for the total of tyrosines (N-FMOC-tyrosine and N,O-FMOC-tyrosine 10 pmol).",
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AB - 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 3.0 mM FMOC concentration, at pH 9; derivatization time = 20 min), 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 2.5 pmol, except for cystine, ornithine (5 pmol) and for the total of tyrosines (N-FMOC-tyrosine and N,O-FMOC-tyrosine 10 pmol).

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