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

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 mu m 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 C18 column (2.7 mu m particle size, 50 x 4.6?mm i.d.), followed by a Thermo Hypersil Gold C18 column (1.9 mu m particle size, 50 x 2.1?mm i.d.) and the Thermo Hypersil BDS C18 column (2.4 mu m particle size, 100 x 2.1?mm i.d.). All columns required a mobile phase containing 1?m 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 5 mu m average particle size materials for clinical sample analysis.