Determination of 5-hydroxyindoleacetic acid using HPLC with electrochemical detection

The diploma thesis focuses on a determination of 5-hydroxyindoleacetic acid (5-HIAA) in human urine using a boron-doped diamond film electrode (BDDFE). 5-HIAA is the main final metabolite in the serotonin metabolic pathway and it is used as a biomarker for the diagnosis of carcinoid tumors. These tumors are mostly localized in gastrointestinal tract and lungs. The determination method was successfully optimized, the separation conditions were as follows: column LiChroCART® 250-4, Purospher® RP-18 (5µm), mobile phase flow rate 0,5 ml·min-1 , mobile phase was composed of methanol and acetate-phosphate (AF) buffer with pH 3, the pump was working in gradient mode with following gradient programme: 0 min: 10/90, 10 min: 80/20, 15 min 80/20 (MeOH/AF-buffer,v/v), the amount of analyte solution used was 20 µl. The detection parameters were also optimized for both UV detection (using 276 nm wavelength) and amperometric detection (BDDF electrode with applied voltage of 1,1 V and silver chloride electrode as reference electrode). Concentration dependence of 5-HIAA was measured within the concetration range of 1·10- 7 - 1·10-4 mol∙l-1 and the linearity in this range was proved. The limit of detection (LOD) and limit of quantification (LOQ) were established based on the signal/noise ratio from the lowest..