New high-performance liquid chromatographic method for sensitive determination of pheomelanin in biological materials by precolumn fluorescence derivatization with naphthalene-2,3-dicarboxaldehyde.
Pheomelanin is an important type of melanin distributed in the skin, eye and hair in the mammal, which is of great social, clinical and cosmetic significance. In this study, a new HPLC method with fluorescence detection is described originally for the sensitive determination of pheomelanin in biological materials. The pheomelanin polymer is decomposed into two specific degradation products, 3-amino-4-hydroxyphenylalanine (3-AHP) and 4-amino-3- hydroxyphenylalanine (4-AHP) with hydriodic acid. Then the two AHP isomers are derivatized using a fluorescent probe naphthalene-2,3-dicarboxaldehyde in the presence of cyanide. The resulting highly stable 2-substituted 1-cyanobenz[f] isoindole derivatives were separated on a 5NH(2)-MS aminopropyl packed HPLC column with binary isocratic elution profile and detected fluorimetrically. The assay shows high sensitivity of 0.11nM (2.2fmol per injection, the lowest reported) at signal-to-noise ratio of 3 for each AHPs, good accuracy and precision (RSDs<3.1%), and linearity (range of 0.02-10microM, r>0.995). The results obtained by using fluorescence detection have been compared with other detection systems (electrochemical and UV). The sensitivity can increase from 100 to respect electrochemical detection and 30000 times respect UV detection. The method has been used for the quantitative determination of pheomelanin in various biological samples, including cell cultures from five types of melanoma cell lines of human and rat origin, hair samples of various colors, melanoma tissue and the urines from human melanoma patients and healthy subjects. This original application of HPLC-fluorescence detection represents a powerful tool for investigating pheomelanin synthesis in vitro and in vivo under physiological and pathophysiological conditions.
Yang
Qiao
Q
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Zhang
Xiao-Ling
XL
Ma
Ming
M
Huang
Ke-Jing
KJ
Zhang
Jun-Xiang
JX
Ni
Wu-Zhong
WZ
Fang
Cheng-Xiang
CX
Zheng
Cong-Yi
CY
D000818:Animals; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006197:Hair / Q000737:chemistry; D006801:Humans; D008543:Melanins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D008545:Melanoma / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D009281:Naphthalenes / Q000737:chemistry*; D051381:Rats; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D005456:Fluorescent Dyes; D008543:Melanins; D009281:Naphthalenes; C018362:pheomelanin; C019253:3-aminotyrosine; D014443:Tyrosine; C027717:2,3-naphthalenedicarboxaldehyde; C071227:4-amino-3-hydroxyphenylalanine