Species-selective determination of cobalamin analogues by reversed-phase HPLC with ICP-MS detection

Abstract

The coupling of reversed-phase HPLC with ICP-MS was optimized for the species-selective determination of cobalt complexes with macrocyclic ligands (vitamin B ₁₂ and its analogues). The use of high efficiency microcolumns (33-50 mm) with porous and non-porous small diameter (1.5-2 µm) stationary phases was evaluated and compared with conventional (5 µm packing) HPLC. High concentrations of organic solvent (up to 50% of acetonitrile or methanol) could be introduced into the ICP using a Meinhard-type nebulizer and a cooled spray chamber. The choice of the column was dictated by the compromise among the analysis time, sensitivity and separation efficiency required. The best results in terms of resolution and sensitivity were obtained using a 15 cm×4.6 mm id C ₈ column. Absolute detection limits of 80-160 pg (4-8 ng ml ^–1 in the injected solution) were achieved. The method developed was applied to the determination of the active component in pharmaceutical cobalamin preparations and for monitoring its degradation.