Sensitive determination of pheomelanin after 5-carboxyfluorescein succinimidyl ester precapillary derivatization and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection.
The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the determination of pheomelanin in biological materials has been investigated. 5-Carboxyfluorescein succinimidyl ester was chosen as the labeling reagent to precapillary derivatize the two marker aminohydroxyphenylalanine (AHP) isomers produced after reductive hydrolysis of pheomelanin with hydriodic acid (HI). Various parameters affecting derivatization and separation were systematically studied. Under optimal conditions, the analytes could be separated within 18 min, and the relative standard deviations (R.S.D.) of migration time and corrected peak areas were less than 5.5%. Compared with the conventional high-performance liquid chromatography (HPLC) method with electrochemical detection, the 100-fold improvements in sensitivity were achieved by applying LIF detection. As a preliminary application, this method has been successfully applied to the determination of pheomelanin in two human melanoma cell cultures, black hair, melanoma tissue and urine samples of human melanoma patients with the spiked recoveries in the range of 88-96%.
Zhang
Xiao-Ling
XL
College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310029, China.
Yang
Qiao
Q
Jiao
Bing-Hua
BH
Dai
Jun
J
Zhang
Jun-Xiang
JX
Ni
Wu-Zhong
WZ
D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D004952:Esters; D005452:Fluoresceins / Q000737:chemistry*; D007834:Lasers; D008543:Melanins / Q000737:chemistry*; D015394:Molecular Structure; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004952:Esters; D005452:Fluoresceins; D008543:Melanins; C018362:pheomelanin; C024098:6-carboxyfluorescein