Reversed-phase liquid chromatography of the opioid peptides. 3. Development of a microanalytical system for opioid peptides involving microbore liquid chromatography, post-column derivatization and laser-induced fluorescence detection.

A microanalytical system has been developed for the determination of peptides in small samples. Naphthalene-2,3-dicarboxaldehyde-beta-mercaptoethanol (NDA-BME) was used as the labelling reagent system as an alternative to NDA-cyanide (NDA-CN) because of the faster labelling when CN was replaced by a thiol. The fluorescence characteristics of the NDA-thiol adducts, N-substituted 1-alkylthiobenz[f]isoindoles (TBIs), were found to be different from the previously described cyanobenz[f]isoindole (CBIs) adducts formed by the reaction of primary amines with NDA-CN. The excitation maximum of the TBI adducts was at 460 nm, which was closer to the 457.9 nm argon-ion laser line, than the 440-nm maximum of the CBI adduct. The limit of detection for leucine enkephalin was 36 fmol (S/N = 3) and linearity was proven for greater than 2 orders of magnitude, from 45 fmol to 9 pmol for an injection volume of 60 nl. The detectability was limited by the high background noise produced by the post-column derivatization system. The utility of the system was demonstrated for the analysis of methionine enkephalin and its potential oxidation products, using leucine enkephalin as a suitable internal standard.