Ultra high-performance liquid chromatography of porphyrins in clinical materials: Column and mobile phase selection and optimisation

摘要

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7μm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C 18 column (2.7μm particle size, 50×4.6mm i.d.), followed by a Thermo Hypersil Gold C 18 column (1.9μm particle size, 50×2.1mm i.d.) and the Thermo Hypersil BDS C 18 column (2.4μm particle size, 100×2.1mm i.d.). All columns required a mobile phase containing 1m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5μm average particle size materials for clinical sample analysis.