Empfindliche Bestimmung von Lysinoalanin und 2,3-Diaminopropions�ure durch fluorimetrische Detektion mit ortho-Phthaldialdehyd

Abstract

A highly sensitive fluorometric method for simultaneous determination of lysinoalanine (LAL) anddl-2,3-diaminopropionic acid (DAP) was developed. After acid hydrolysis (6 N HCl/23 h/110 °C) and direct neutralization of the hydrolysate with sodium citrate/sodiumhydroxide — solution LAL, DAP, ornithine, amino sugars and normal basic amino acids were completely separated by means of an amino acid analyzer technique (0.2 M sodium citrate buffer pH 4.50/60 °C) described by the authors in another publication for LAL-ninhydrin detection. LAL and DAP were eluted after 45 min and 67 min resp.; the whole procedure inclusive regeneration and equilibration of the resin took about 90 min. Compared to the colorimetric ninhydrin method a considerable increase in sensitivity and accuracy was achieved with fluorometric detection using an o-phthalaldehyde-reagent (1 M potassium borate buffer pH 10.0; 3.73 mM o-phthaldialdehyde; 2.86 mM mercaptoethanol; 2 ml Brij 35 30% solution/l). Normally 4 pmole DAP (0.4 ng) and 7 pmole LAL (1.6 ng) could be detected in protein hydrolysates; 50 pmole amounts were recovered at 100 ± 3%. In complex foods the minimum detectable level for LAL and DAP was 1–5 ppm in the protein, in some protein isolates generally used for model experiments detection was possible also in the ppb-range. A very short reaction coil corresponding to a short reaction time was responsible for a more sensitive detection of LAL and DAP, whereas — in the interest of the described method — amino sugars, neutral and acidic amino acids reacted to a lesser extent; lysine and ornithine produced a nearly unchanged fluorescence. Up to now DAP had only been found in dietetic formulated protein concentrates containing granulated milk protein (0–50 ppm DAP in the protein) and in whipping protein products originating from milk protein (1,660–3,660 ppm in the protein) and from soy protein (10,270 ppm).