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.
Kai
M
M
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Ishida
J
J
Ohkura
Y
Y
D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D004743:Enkephalin, Leucine / Q000032:analysis*; D007651:Keto Acids / Q000032:analysis; D008297:Male; D051379:Mice; D010655:Phenylenediamines; D051381:Rats; D011919:Rats, Inbred Strains; D012794:Sialic Acids / Q000032:analysis; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000032:analysis
D007651:Keto Acids; D010655:Phenylenediamines; D012794:Sialic Acids; C047826:1,2-diamino-4,5-dimethoxybenzene; D014443:Tyrosine; D004743:Enkephalin, Leucine