Amino acid analysis by high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride. Literature overview and further study

A. Jámbor, I. Molnár-Perl

Research output: Article

73 Citations (Scopus)

Abstract

A literature overview is given of HPLC methods currently in use to determine amino acids as their 9-fluorenylmethyloxycarbonyl (FMOC) derivatives. On the basis of the detailed literature overview an exhaustive derivatization study was performed with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection simultaneously, in order to clear up the controversial points of FMOC derivatization. Model investigations have been carried out as a function of the reaction time and reaction conditions, such as the molar concentration of the reagent, the molar ratios of the reactants, the pH and the solvent composition of the reaction medium. Special emphasis was put (i) on the evaluation of the blank values of the reagents, as a function of the composition and that of the pH of the reaction medium, (ii) on the unambiguous quantitation of all amino acids, including the less reactive aspartic and glutamic acids, as well as on the formation and transformation of histidine and tyrosine, existing partly, as single (N-FMOC-histidine, N-FMOC-tyrosine), partly as double labeled species (N,NH-FMOC-histidine, N,O-FMOC-tyrosine). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 0.5 mM FMOC concentration corresponding to the molar ratios of [FMOC]/[amino acids]T = 5.5/1 (note: the superscript 'T' means the total of amino acids), with acetonitrile containing reagents, at pH 9, derivatization time = 20 min), and characterized with the relative standard deviation percentages of their responses (≤3.98%, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 1 pmol, except for serine, glycine, valine (2.5 pmol), for cystine, N,NH-FMOC-histidine and for the total of N-FMOC-tyrosine and N,O-FMOC-tyrosine (5 pmol) and for tryptophan (10 pmol).

Original languageEnglish
Pages (from-to)3064-3077
Number of pages14
JournalJournal of Chromatography A
Volume1216
Issue number15
DOIs
Publication statusPublished - ápr. 10 2009

Fingerprint

High performance liquid chromatography
Tyrosine
High Pressure Liquid Chromatography
Histidine
Amino Acids
Derivatives
Glutamates
Cystine
Valine
9-fluorenylmethoxycarbonyl
Photodiodes
Chemical analysis
Tryptophan
Glycine
Serine
Fluorescence

ASJC Scopus subject areas

  • Analytical Chemistry
  • Organic Chemistry
  • Biochemistry

Cite this

@article{29fd61ba096d40bc9364bdd4f919b46a,
title = "Amino acid analysis by high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride. Literature overview and further study",
abstract = "A literature overview is given of HPLC methods currently in use to determine amino acids as their 9-fluorenylmethyloxycarbonyl (FMOC) derivatives. On the basis of the detailed literature overview an exhaustive derivatization study was performed with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection simultaneously, in order to clear up the controversial points of FMOC derivatization. Model investigations have been carried out as a function of the reaction time and reaction conditions, such as the molar concentration of the reagent, the molar ratios of the reactants, the pH and the solvent composition of the reaction medium. Special emphasis was put (i) on the evaluation of the blank values of the reagents, as a function of the composition and that of the pH of the reaction medium, (ii) on the unambiguous quantitation of all amino acids, including the less reactive aspartic and glutamic acids, as well as on the formation and transformation of histidine and tyrosine, existing partly, as single (N-FMOC-histidine, N-FMOC-tyrosine), partly as double labeled species (N,NH-FMOC-histidine, N,O-FMOC-tyrosine). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 0.5 mM FMOC concentration corresponding to the molar ratios of [FMOC]/[amino acids]T = 5.5/1 (note: the superscript 'T' means the total of amino acids), with acetonitrile containing reagents, at pH 9, derivatization time = 20 min), and characterized with the relative standard deviation percentages of their responses (≤3.98{\%}, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 1 pmol, except for serine, glycine, valine (2.5 pmol), for cystine, N,NH-FMOC-histidine and for the total of N-FMOC-tyrosine and N,O-FMOC-tyrosine (5 pmol) and for tryptophan (10 pmol).",
keywords = "9-Fluorenylmethyloxycarbonyl-chloride, Amino acids, Derivatization optimization, High-performance liquid chromatography, HPLC-UV(DAD)/fluorescence detection",
author = "A. J{\'a}mbor and I. Moln{\'a}r-Perl",
year = "2009",
month = "4",
day = "10",
doi = "10.1016/j.chroma.2009.01.068",
language = "English",
volume = "1216",
pages = "3064--3077",
journal = "Journal of Chromatography",
issn = "0021-9673",
publisher = "Elsevier",
number = "15",

}

TY - JOUR

T1 - Amino acid analysis by high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride. Literature overview and further study

AU - Jámbor, A.

AU - Molnár-Perl, I.

PY - 2009/4/10

Y1 - 2009/4/10

N2 - A literature overview is given of HPLC methods currently in use to determine amino acids as their 9-fluorenylmethyloxycarbonyl (FMOC) derivatives. On the basis of the detailed literature overview an exhaustive derivatization study was performed with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection simultaneously, in order to clear up the controversial points of FMOC derivatization. Model investigations have been carried out as a function of the reaction time and reaction conditions, such as the molar concentration of the reagent, the molar ratios of the reactants, the pH and the solvent composition of the reaction medium. Special emphasis was put (i) on the evaluation of the blank values of the reagents, as a function of the composition and that of the pH of the reaction medium, (ii) on the unambiguous quantitation of all amino acids, including the less reactive aspartic and glutamic acids, as well as on the formation and transformation of histidine and tyrosine, existing partly, as single (N-FMOC-histidine, N-FMOC-tyrosine), partly as double labeled species (N,NH-FMOC-histidine, N,O-FMOC-tyrosine). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 0.5 mM FMOC concentration corresponding to the molar ratios of [FMOC]/[amino acids]T = 5.5/1 (note: the superscript 'T' means the total of amino acids), with acetonitrile containing reagents, at pH 9, derivatization time = 20 min), and characterized with the relative standard deviation percentages of their responses (≤3.98%, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 1 pmol, except for serine, glycine, valine (2.5 pmol), for cystine, N,NH-FMOC-histidine and for the total of N-FMOC-tyrosine and N,O-FMOC-tyrosine (5 pmol) and for tryptophan (10 pmol).

AB - A literature overview is given of HPLC methods currently in use to determine amino acids as their 9-fluorenylmethyloxycarbonyl (FMOC) derivatives. On the basis of the detailed literature overview an exhaustive derivatization study was performed with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection simultaneously, in order to clear up the controversial points of FMOC derivatization. Model investigations have been carried out as a function of the reaction time and reaction conditions, such as the molar concentration of the reagent, the molar ratios of the reactants, the pH and the solvent composition of the reaction medium. Special emphasis was put (i) on the evaluation of the blank values of the reagents, as a function of the composition and that of the pH of the reaction medium, (ii) on the unambiguous quantitation of all amino acids, including the less reactive aspartic and glutamic acids, as well as on the formation and transformation of histidine and tyrosine, existing partly, as single (N-FMOC-histidine, N-FMOC-tyrosine), partly as double labeled species (N,NH-FMOC-histidine, N,O-FMOC-tyrosine). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 0.5 mM FMOC concentration corresponding to the molar ratios of [FMOC]/[amino acids]T = 5.5/1 (note: the superscript 'T' means the total of amino acids), with acetonitrile containing reagents, at pH 9, derivatization time = 20 min), and characterized with the relative standard deviation percentages of their responses (≤3.98%, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 1 pmol, except for serine, glycine, valine (2.5 pmol), for cystine, N,NH-FMOC-histidine and for the total of N-FMOC-tyrosine and N,O-FMOC-tyrosine (5 pmol) and for tryptophan (10 pmol).

KW - 9-Fluorenylmethyloxycarbonyl-chloride

KW - Amino acids

KW - Derivatization optimization

KW - High-performance liquid chromatography

KW - HPLC-UV(DAD)/fluorescence detection

UR - http://www.scopus.com/inward/record.url?scp=62249160671&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=62249160671&partnerID=8YFLogxK

U2 - 10.1016/j.chroma.2009.01.068

DO - 10.1016/j.chroma.2009.01.068

M3 - Article

C2 - 19215925

AN - SCOPUS:62249160671

VL - 1216

SP - 3064

EP - 3077

JO - Journal of Chromatography

JF - Journal of Chromatography

SN - 0021-9673

IS - 15

ER -