High-performance liquid chromatographic determination of leucine-enkephalin-like peptide in rat brain by pre-column fluorescence derivatization involving formylation followed by reaction with 1,2-diamino-4,5-dimethoxybenzene.

Fluorimetrically reactive leucine-enkephalin, one of the opioid peptides in rat brain tissues such as the striatum, cortex and hypothalamus, was assayed by reversed-phase high-performance liquid chromatography with fluorescence detection based on pre-column derivatization of the tyrosyl residue. The tyrosine-containing peptides extracted from the tissue were first formylated with chloroform in an alkaline medium, and the resulting aldehydes were then converted into highly fluorescent derivatives by reaction with 1,2-diamino-4,5-dimethoxybenzene. The derivative of leucine-enkephalin-like peptide in tissue was separated from various other fluorescent compounds on a reversed-phase column (TSK gel ODS-120T) by isocratic elution and detected by fluorimetry. The concentrations of the leucine-enkephalin-like peptide in the tissues were 20-245 pmol/g. The method is sensitive enough to permit the quantitative determination of the endogenous peptide at concentrations as low as 5.6 pmol/g in brain tissues.