The opioid peptides are biologically important products resulting from the biochemical processing of three precursor proteins: preproopiomelanocortin, preproenkephalin A and preproenkephalin B. The primary objective of this research is the development of direct chemical assay methodology which has the required selectivity and sensitivity to unambiguously quantify opioid peptides, especially, Leu-enkephanlin and Met-enkephalin, in biologically relevant tissues. This overall objective is being accomplished via the unique combination of fluorogenic derivatization, multidimensional HPLC and chemiluminescence detection. Specifically, the primary amine groups of the peptides are reacted with the novel NDA reagent (naphthalene-2,3- dialdehyde plus cyanide) to form stable and intensely fluorescent N-substituted 1-cyanobenz f isoindole (CBI) derivatives. Next, multidimensional chromatography is employed to achieve the desired selectivity. The CBI derivatives are initially fractionated on an HPLC column which retains the analytes by one mechanism (eg., reversed phase) and then switched automatically to a second column with a completely different mechanism (eg., ion-pair). Finally, the resolved analytes are detected via chemiluminescence of the CBI moiety, generated by an aryloxalate/H2O2 post-column reaction detector. The following studies will be carried out to optimize the approach: 1) the rate and yield of the NDA reaction will be studied using several peptides including those with multiple reaction sites, 2) the luminescent properties of the CBI peptides wil be characterized paying particular attention to the products of multiple derivatization, 3) the systematic characterization of the retention of CBI peptides on HPLC columns with different retention mechanisms will be carried out, 4) the characterization of the post-column chemiluminescent process will include: reactor design, flow-cell design, use of novel aryloxalate esters and reaction conditions. The results obtained will permit the unification of these various components into a system that will permit the direct chemical determination of opioid peptides with excellent selectivity and sensitivity (limits of detection of 1-10 femtomole on-column). The resulting methodology will be applicable to the quantification of endogenous or exogenously administered opioid peptides that could be easily transferred to other laboratories engaged in this area of peptide research without the addition of highly specialized equipment.
