Determination of urinary biogenic amines' biomarker profile in neuroblastoma and pheochromocytoma patients by MEKC method with preceding dispersive liquid-liquid microextraction.

The unbalanced secretion of biogenic amines (BAs) is considered to be a relevant biochemical biomarker in the screening for neuroendocrine tumors, such as: neuroblastoma and pheochromocytoma. However, there is still a need to improve the bioanalytical procedures for BA determination in biological samples due to their instability (photo- and thermosensitivity, easy oxidation) and low concentration in the body fluids. In this study, the primary analytical challenge was to optimize the method of extraction of seven compounds from among BAs and their precursors from urine samples. Several methods based on liquid-liquid extraction (LLE) or solid phase extraction (SPE) techniques were tested. By optimization of the extraction and data analysis using chemometric tool, the dispersive liquid-liquid microextraction (DLLME) has been chosen due to its low solvents consumption, high efficiency of isolation, preconcentration and suitable clean-up of biological matrix. Further, α-cyclodextrin-modified micellar electrokinetic chromatography (MEKC) with ultraviolet detection (UV) has been applied for quantification of the analyzed biologically active compounds with limits of detection (LOD) and limits of quantification (LOQ) at 0.15 and 0.5μgmL, respectively. Finally, the optimized and validated DLLME-MEKC-UV method has been employed for the analysis of real urine samples, obtained from 6 children with neuroendocrine tumors and 6 healthy children. It was stated that concentrations of BA could serve to differentiate between the patients and healthy children. This pilot study indicates that the elaborated fast and sensitive DLLME-MEKC-UV method for determination of panel of biomarkers could be successfully applied in everyday clinical practice to help to confirm the clinical diagnosis of neuroendocrine tumors in children.