Optimization of high-performance liquid chromatography-radioimmunoassay protocols for the analyses of substance P and some of its metabolic fragments.

A reversed-phase high-performance liquid chromatographic procedure combined with radioimmunoassay (HPLC-RIA) was developed and optimized for the concomitant quantitation of substance P (SP) and some of its C- and N-terminal fragments in the extracts of the spinal cord of mice. A selective and efficient solid-phase extraction protocol was used for preparative purification of sample homogenates prior to analyses. The sensitivity of the HPLC assay was 18.75 ng for SP and some of its fragments of interest. Recoveries of peptides were calculated from spiked aqueous standards carried through the experimental protocol and ranged from 53 to 98%. The precision of the peptide recoveries from aqueous-based standards, expressed as coefficient of variation, ranged from 2 to 28%. The sensitivities for the RIA procedure using SP antiserum were 1.5, 3.4 and 4.6 fmol SP1-11, SP2-11 and SP5-11, respectively. The percentage cross-reactivity of SP1-11 antiserum with the C-terminal fragments was complete whereas the cross-reactivities of the N-terminal fragments were essentially zero. The molar limits of detectability of SP and some of its C-terminal fragments determined by HPLC alone were several orders of magnitude greater than those determined from the same spinal cord samples using RIA after HPLC fractionation.