Fluorescence assay for small peptides and amino acids: High-performance liquid chromatographic determination of selected substrates using activated S-flunoxaprofen as a chiral derivatizing agent

Abstract

The applicability of the fluorescent S-flunoxaprofen, activated to the corresponding acyl chloride, as a chiral derivatizing agent for amino acids and small peptides was evaluated. The amino acid or peptide solution was evaporated to dryness. The carboxylic moieties were esterified with 3 M hydrochloric acid in 2-propanol. The solvent and hydrochloric acid were evaporated, and the residue was dissolved in aqueous sodium heptanesulphonate and extracted with benzene-butanol (85:15, v/v). The organic phase was evaporated and S-flunoxaprofen chloride reagent solution (in dichloromethan) added, together with 20 mg of anhydrous sodium carbonate. After 60 min at 40°C the solvent was evaporated and the residue reconstituted in mobile phase ( n-hexane-dichloromethane-ethanol). Resolution of the diastereomeric derivatives was accomplished on a Zorbax Sil high-performance liquid chromatography column. The eluate was monitored either fluorimetrically at 305/355 nm or by measuring the UV absorption at 305 nm. The procedure leads to highly fluorescent derivatives of amino acids or small peptides, which are resolvable on silica gel stationary phases.