RP-LC of phenylthiocarbamyl amino acid adducts in plasma acetonitrile extracts: Use of multiple internal standards and variable wavelength UV detection

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.