Recovery of substance P and related C-terminal fragments on solid-phase extraction cartridges for subsequent high-performance liquid chromatographic separation and radioimmunoassay.

Abstract

The recoveries of substance P (SP) and five related peptides were evaluated on different types of solid-phase extraction sorbent. Best results were obtained by use of a C18 silica gel cartridge. Marked differences of extraction yields occurred for the different peptide fragments and, in general, recovery increased with increasing hydrophobicity of the peptide when reversed-phase materials like C18 and C8 cartridges were used. This observation is indicative of a sorption-desorption mechanism by prevailing solvophobic interactions. A similar trend was found when phenylpropyl silica gel (CPhenyl), generally known as a reversed-phase adsorbent of lower hydrophobicity, was used. It was concluded that a substantial participation of analyte-matrix pi-pi interactions has to be taken into account when extraction yields are compared with corresponding values obtained by use of a C8 cartridge. With CN silica gel cartridges, marked differences in extraction yields were obtained by use of acetonitrile or methanol as the organic modifier. As an attempt to explain this observation, conformational effects were assumed for the sorption-desorption behaviour of the peptides on the polar matrix.