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RP-LC of Phenylthiocarbamyl Amino Acid Adducts in Plasma Acetonitrile Extracts: Use of Multiple Internal Standards and Variable Wavelength UV Detection

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Amino Acid Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 828))

Abstract

The measurement of physiological amino acids in body fluids and circulating cells can be relevant in the search for biological correlates of neuropsychiatric, neurological, body weight, and pain diseases. Several techniques are available for the quantitative analysis of free amino acids, including UV detection after precolumn derivatization. These systems have low specificity due to possible interferences at the analytical wavelength. Another problem linked to these methods is variations potentially occurring during extraction, derivatization, and chromatography of amino acids in biological matrices. We present here a modified reversed-phase LC of phenylthiocarbamyl amino acids in plasma deproteinated by the organic solvent acetonitrile. Specificity was monitored by UV-photodiode array detection and accuracy was controlled by a plasma spiking procedure with three internal standards. A dual-wavelength spectrophotometry (254, 283 nm) was used to quantify coeluting ornithine and tryptophan adducts. The method is simple and economical and enables the measure of most plasma amino acids for clinical research, also during therapeutic drug monitoring. Dual UV-fluorimetric detection solutions can improve its sensitivity.

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Authors and Affiliations

  1. Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy

    Lionella Palego, Gino Giannaccini & Antonio Lucacchini

Authors
  1. Lionella Palego
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  2. Gino Giannaccini
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  3. Antonio Lucacchini
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Corresponding author

Correspondence to Antonio Lucacchini .

Editor information

Editors and Affiliations

  1. Ctr. Biologics Evaluation & Research, Div. Cellular & Gene Therapies, U.S. Food and Drug Administration, Rockville Pike 8800, Bethesda, 20892, Maryland, USA

    Michail A. Alterman

  2. Functional Genomics Center Zürich, Protein Analysis Group, Universität Zürich, Winterthurerstr. 190, Zürich, 8057, Switzerland

    Peter Hunziker

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Palego, L., Giannaccini, G., Lucacchini, A. (2012). RP-LC of Phenylthiocarbamyl Amino Acid Adducts in Plasma Acetonitrile Extracts: Use of Multiple Internal Standards and Variable Wavelength UV Detection. In: Alterman, M., Hunziker, P. (eds) Amino Acid Analysis. Methods in Molecular Biology, vol 828. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-445-2_17

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  • DOI: https://doi.org/10.1007/978-1-61779-445-2_17

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-444-5

  • Online ISBN: 978-1-61779-445-2

  • eBook Packages: Springer Protocols

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