Complete capillary electrophoretic separation of substance P and its metabolites at neutral pH using ionic run buffer additives

Substance P (SP), a bioactive peptide that is present in the extracellular fluid of the brain at picomolar concentrations, has been proposed to be a neurotransmitter and a neuromodulator in the central and peripheral nervous systems. This paper describes the development of analytical methodology for the separation of SP and its metabolites by capillary electrophoresis at neutral pH. Since SP is a cationic peptide, it tends to adsorb to silica surfaces. In this work, phytic acid was employed as a run buffer additive to eliminate the interaction of SP and its cationic N-terminus metabolites with ionized silanol groups. The separation of the C-terminus metabolites was then accomplished through the use of sulfobutyl ether beta-cyclodextrin, which imparted a negative charge to these otherwise neutral metabolites through complexation of the phenylalanine residue. The final separation buffer consisted of 150 mM boric acid, 15 mM phytic acid and 5 mM SBE(IV)beta-cyclodextrin at pH 7. Under these conditions, it was possible to separate SP from all eight of its reported metabolites.