Selective and Sensitive Determination of Pheomelanin in Biological Samples Using MEKC with Laser-Induced Fluorescence Detection Based on Intramolecular Excimer-Forming Fluorescence Derivatization

Abstract

A highly selective micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence (LIF) detection for the sensitive determination of pheomelanin in diverse biological materials was originally described. The derivatization reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), allowed for the selective detection of the two aminohydroxyphenylalanines (AHPs) markers for pheomelanin monitored at 500 nm. Multiple labeling of two AHPs with PSE allowed the formation of intramolecular excimers that emit at longer wavelengths (500 nm) than the mono-labeled analytes (360–420 nm) based on intramolecular excimer-forming fluorescence derivatization. Optimal separation of the labeled polyamines was achieved using a separation buffer consisting of 20 mM phosphate pH 7.4, 30 mM cholate, and 30% methanol. Using these conditions, the two AHPs were separated within 12 min, and the relative standard deviations (RSDs) were less than 1.5 and 1.6% (intra-run), 3.8 and 4.6% (inter-run, for a 6-day period) for the migration times and peak areas (n = 10), respectively. This method was successfully applied to the monitoring of pheomelanin in diverse biological samples with the spiked recoveries in the range of 94–101%. At a signal-to-noise ratio of 3, the detection limit for AHPs in the real samples was 31 pM for 3-AHP and 35 pM for 4-AHP, respectively, which are superior to those previously reported in the literature using fluorescence detection.