Empfindliche Bestimmung von Lysinoalanin und 2,3-Diaminopropionsäure durch fluorimetrische Detektion mit ortho-Phthaldialdehyd

A highly sensitive fluorometric method for simultaneous determination of lysinoalanine (LAL) and dl-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).