High-performance liquid chromatography of neuropeptides using radially compressed polythene cartridges

This study was designed to assess practically the suitability of different C18 reversed-phase radially compressed polythene cartridges (Radial-Pak, Waters Assoc.) in two types of radial-compression systems, for the separation and analysis of various neuropeptides at both high (less than 5 micrograms) and low (greater than 100 pg) levels in biological extracts and to compare them with well established techniques using stainless-steel columns. A solvent system fully compatible with both radially compressed and steel columns is described. The completely volatile mobile phase (acetonitrile gradient containing trifluoroacetic acid) allows ultraviolet detection below 215 nm, gives good resolution and is readily compatible with the further radioimmunoassay and bioassay of collected fractions. The efficiency of radially compressed 5 and 10 microns "capped" and "non-capped" C18 silica supports and slurry-packed steel columns has been assessed by: (1) separation and recovery of a complex standard mixture of neuropeptides; (2) separation and subsequent identification of degradation products formed during the incubation of neurotensin with rat cortical synaptosomes; (3) analysis of alpha-melanotropin and corticotropin-(18-39) in tissue culture media containing varying amounts of foetal calf serum; and (4) characterization of corticotropin-like immunoreactivity in human cerebrospinal fluid. The Z-module fitted with the capped 10-microns irregular C18 silica cartridge gave better resolution than with the mu Bondapak steel column but the selective retention was similar. The back-pressures in the Z-module are much reduced (approximately 13 bar at 1 ml/min); therefore, flow-rates may be increased and analysis times greatly reduced. In order to obtain good resolution with the RCM-100 module which uses a non-capped stationary phase, a salt must be added (e.g. 15 mM sodium chloride) to the mobile phase to reduce polar interactions between the peptide and the free silanol groups on the stationary phase. This makes the solvent non-volatile and therefore less useful.