High-performance liquid chromatographic determination of substance p-like arginine-containing peptide in rat brain by on-line post-column fluorescence derivatization with benzoin

Abstract

A high-performance liquid chromatographic method with fluorescence detection is described for the determination of substance P, one of the neuropeptides, in the hypothalamus tissue of rat brain. The detection is based on on-line post-column fluorescence derivatization selective for arginine-containing peptides. The endogenous substance P-like arginine-containing peptide extracted from the tissue and [D-Phe11]-neurotensin as an internal standard were separated from various interfering substances on a reversed-phase column (TSKgel ODS-120T) by gradient elution with acetonitrile-phosphate buffer (pH 2.3). The peptides in the eluate were then automatically converted into fluorescent derivatives for detection by reaction with benzoin. Arginine-containing fragments produced by the enzyme reaction of substance P in the chromatographic fraction with trypsin were also detected, for the identification of the endogenous substance P-like arginine-containing peptide. The method was sensitive enough to permit the quantitative determination of the peptide at a concentration as low as 580 fmol/mg of protein in the brain homogenate. The concentration values of the substance P-like arginine-containing peptide in the tissue were 9.45 +/- 1.50 pmol/mg of protein (six determinations).