Quantitation of aspartate and glutamate in HPLC analysis of phenylthiocarbamyl amino acids.

In the quantitation of amino acids by precolumn derivatization with phenylisothiocyanate, the yields of N'-phenylthiocarbamyl (PTC)-aspartate and PTC-glutamate from protein hydrolysates are often suboptimal, particularly in analyses following rapid hydrolysis at 160 degrees C. In this paper we show that these losses are due to the presence of materials extracted from the glass container during hydrolysis. In the presence of these extracts, the repeated drying and neutralization steps which precede phenylthiocarbamylation result in samples not fully solubilized by the presently used derivatizing mixtures. Thus the coupling yields for the acidic residues are highly variable. A coupling buffer with the composition 35% H2O, 30% acetonitrile, 25% pyridine, and 10% triethylamine (v/v/v/v) is an efficient solvent for all amino acids in hydrolysates and permits consistent, quantitative derivatization of all amino acids, including aspartate and glutamate.