High-performance liquid chromatographic and field desorption mass spectrometric measurement of picomole amounts of endogenous neuropeptides in biologic tissue.

A unique combination of chromatographic separation and mass spectrometric techniques has been developed for a novel method for measurement of picomole amounts of endogenous oligopeptides in biologic tissue. High-performance liquid chromatography is utilized for rapid high-resolution separation of peptides. A new buffer system using dilute triethylamine-formic acid is utilized. The buffer system possesses excellent UV transparent properties enabling femtomole sensitivity for measurement of standard solutions of somatostatin. Use of porous polystyrenedivinylbenzene copolymer and octadecylsilyl columns facilitate retention of a peptide fraction from biologic extracts. Advantage was taken of field desorption mass spectrometric methods to eliminate chemical derivatization of peptides and to produce protonated molecular ions which retain total molecular information of the peptide. Use of appropriate internal standards and selected ion monitoring methods enabled nanogram sensitivity and, more importantly, optimized structural specificity of the compound being quantified. Results are compatible with radioimmunoassay data. Data obtained with field desorption mass spectrometry provide, for the first time, measurement of intact, chemically underivatized oligopeptides extracted form biologic matrices and significantly, provide and analytic method to calibrate radioimmunoassay data. This novel combination of methods is being applied to measurements of peptide (leu-enkephalin, met-enkephalin, somatostatin, etc.) in canine brain regions and dental pulp tissue.