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 excimerforming 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.

Micellar electrokinetic chromatography: A review of methodological and instrumental innovations focusing on practical aspects

“…Bile salts provide a useful alternative to SDS for the separation of hydrophobic compounds by virtue of the hydrogen bond interactions between the analytes and the two hydroxyl groups in the salts. There have been few new applications of this type of surfactant in MEKC in the reviewed period; most relied on sodium cholate or sodium deoxycholate instead. As with SDS, separations were done in an alkaline medium (pH ca.…”

“…In general, the argon‐ion laser emitting at 488 nm is the excitation source most commonly used for this purpose, and so is a BGE consisting of SDS and borate buffer at alkaline pH, which provides efficient baseline separation for labeled analytes. Other labels such as fluorescamine , 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole , 4‐(1‐pyrene)butyric acid N ‐hydroxysuccinimide ester , and 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐ s ‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) have also been used in this context for the determination of biogenic amines in lake water , ammonia and aliphatic amines in environmental water , pheomelanin in biological samples , and modified and normal deoxyribonucleotides in DNA and ribonucleoside‐5′‐monophosphates for the analysis of RNA . As with previous labels, argon‐ion laser and SDS are the most widely used laser source and PSP, respectively, but the He‐CD laser and other surfactants such as sodium cholate have also been employed.…”

“…Several new PSPs of variable selectivity other than SDS—the most common choice by virtue of its applicability to a wide range of analytes—have been tested to improve resolution in this technique. The alternatives used to overcome selectivity limitations in SDS‐MEKC methods include bile salts ; cationic and nonionic surfactants ; surfactants structurally similar to SDS ; and mixed micelles . A description of the contributions relying on the use of polymeric surfactants is beyond the scope of this review.…”