1.2.2. HPLC of amino acids as phenylthiocarbamoyl derivatives

Abstract

Some of the general problems encountered in the analysis of amino acids derivatized with various isothiocyanates, determined both as phenylthiocarbamoyl (PTC) and as phenylthiohydantoin (PTH) derivatives, are presented. Based on our own experience and the literature associated with sample preparation, stability, optimum chromatographic and quantitation conditions are discussed. Twenty-seven PTC-AAs have been separated on seven columns, with the same gradient program, applying various temperatures and eluent flow rates, within 40 min, including equilibration time. Response values and spectral characteristics of the 27 AAs proved to be independent of the chromatographic conditions performed. Particular attention was paid to the issue of sensitive amino acids under hydrolysis (cystine/cysteine, methionine, tryptophan), and derivatization (cystine/cysteine) conditions as well. The special behavior of the PTC-cystine/cysteine has been examined by LC/API/MS measurements showing that, irrespective of the initial compound to be derivatized, the same stereoisomer pairs are formed at m/z = 255, PTC-cysteines (∼80 % of the total) and their oxidized version at m/z = 287 (∼20 % of the total). Whether there is an advantage of the microwave irradiation procedure is still not decided: however the hydrolysis time of lysozyme has been shortened considerably, but optimum hydrolysis conditions were not uniformly found for all essential amino acids.