Simultaneous analysis of neuroendocrine tumor markers by HPLC-electrochemical detection

A validated, high pressure liquid chromatographic (HPLC) method for simultaneous quantitation of urinary catecholic acids 4-hydroxy-3-methoxymandelic acid (HMMA) (vanylmandelic acid) (VMA), 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) was developed. Sample preparation involved liquid–liquid extraction of acidified urine, containing iso-HMMA (IS) as internal standard, with ether, evaporation of the organic extract, followed by reconstitution of the residue in phosphate buffer at pH 3.3. After reversed-phase HPLC at 35 °C and separation on a Licrospher 125 mm × 4 mm C18 column (5 μm particle size) with phosphate buffer (pH 3.5)–acetone (950:50, v/v) as eluent, quantitation is achieved by electrochemical detection using coulometric detection at a potential of +350 mV. The method was successfully applied to routine diagnosis of neuroblastoma, carcinoid syndrome and pheochromocytoma.