Analysis of urinary neurotransmitters by capillary electrophoresis: sensitivity enhancement using field-amplified...

Capillary electrophoresis (CE) has been investigated for the analysis of some neurotransmitters, dopamine (DA), 3-methoxytyramine (3-MT) and serotonin (5-hydroxytryptamine, 5-HT) at nanomolar concentrations in urine. Field-amplified sample injection (FASI) has been used to improve the sensitivity through the online pre-concentration samples. The cationic analytes were stacked at the capillary inlet between a zone of low conductivity - sample and pre-injection plug - and a zone of high conductivity - running buffer. Several FASI parameters have been optimized (ionic strength of the running buffer, concentration of the sample protonation agent, composition of the sample solvent and nature of the pre-injection plug). Best results were obtained using H(3)PO(4)-LiOH (pH 4, ionic strength of 80 mmol L(-1)) as running buffer, 100 μmol L(-1) of H(3)PO(4) in methanol-water 90/10 (v/v) as sample solvent and 100 μmol L(-1) of H(3)PO(4) in water for the pre-injection plug. In these conditions, the linearity was verified in the 50-300 nmol L(-1) concentration range for DA, 3-MT and 5-HT with a determination coefficient (r(2)) higher than 0.99. The limits of quantification (10 nmol L(-1) for DA and 3-MT, 5.9 nmol L(-1) for 5-HT) were 500 times lower than those obtained with hydrodynamic injection. However, if this method is applied to the analysis of neurotransmitters in urine, the presence of salts in the matrix greatly reduces the sensitivity of the FASI/CE-UV method.Therefore, a solid phase extraction (SPE) on a dedicated imprinted polymer (MIP) was developed to extract specific neurotransmitters, catecholamines, metanephrines and indolamines, from urine. Matrix salts were thus discarded after sample extraction on AFFINIMIP™ Catecholamine & Metanephrine (100mg) cartridge. Therefore, lower limits of quantification were determined in artificial urine (46 nmol L(-1) for DA, 11 nmol L(-1) for 3-MT and 6 nmol L(-1) for 5-HT).The application of this protocol MIP-SPE/FASI-CE-UV analysis of neurotransmitters in human urine gave rise to electropherograms with a very good base line and signal to noise ratios above 15.