On-line preconcentration techniques in determination of melatonin and its precursors/metabolites using micellar electrokinetic chromatography

Determination of melatonin (MT) (N-acetyl-5-methoxytryptamine) and related indole compounds using standard capillary electrophoresis (CE) system with UV detection was investigated. Satisfactory separations of six analytes i.e. l-tryptophan (l-TRP), 5-methoxyindoleacetic acid (5-MIAA), 6-hydroxymelatonin (6-HMT), MT, serotonin (SER) and 5-methoxytryptamine (5-MTRA) were performed employing micellar electrokinetic chromatography (MEKC). The optimal background electrolytes (BGE) used for separations were 20 mM tetraborate buffer (pH 9.2) and 20 mM phosphate buffer (pH 3.3) when employing techniques with normal and reverse migration of micelles, respectively. Fifty millimolar sodium dodecyl sulfate (SDS) was employed as the pseudostationary phase and voltage of ±20 kV was used throughout the investigation. On-line preconcentration techniques, stacking and sweeping, were applied in order to overcome high detection limits that are a serious drawback of CE with UV detection. A comparison of used techniques, concerning enhancement factors and limits of detection (LOD), is presented. Obtained results show that the use of stacking with reverse migrating micelles (SRMM) as one of preconcentration techniques allows obtaining the lowest estimated LODs for MT at the level of 30 ng/mL with injection time of 99 s at 0.5 psi. Estimated LODs for other analytes in these conditions were, 21, 26 and 100 ng/mL for l-TRP, 5-MIAA and 6-HMT, respectively. Signals of 5-MTRA and SER obtainable only with 10 s injection allowed reaching estimated LODs of 62.5 and 130 ng/mL, respectively. Analysis of spiked, diluted human serum was carried out as a preliminary application illustration of developed procedure.