Hydrophilic interaction liquid chromatographic analysis of aminohydroxyphenylalanines from melanin pigments

Malignant melanomas are more often seen in subjects with light colored skin who tan poorly than in persons who tan more rapidly. This has been attributed to the structure of their pigment, pheomelanin, which differs markedly from the eumelanin of persons with darker skin. To study the hydrolysis products of pheomelanin pigments a new method was developed for analysis of 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP). Pheomelanin samples were hydrolyzed and extracted with solid-phase extraction columns using strong cation-exchange (SCX) cartridges. Separation of 4-AHP and 3-AHP was achieved on a ZIC-HILIC column (150 mm × 2.1 mm I.D.) with a mobile phase consisting of acetonitrile:0.1 M ammonium acetate buffer, pH 4.5 (82:18, v/v). Detection was performed with an electrochemical detector at +400 mV. Run time was 30 min. The limits of detection were 73 pg and 51 pg for 4-AHP and 3-AHP respectively, using 2 μl injections. Good linearity was found within the range 0.05–5.0 μg/ml. Absolute recovery was 70% and relative recovery was 100%. The AHPs were stable for 1 year in the hydrolyzed samples, for 4 days in the eluates from solid-phase sorbents stored in the refrigerator, and for 2 days diluted with mobile phase and stored in the autosampler at 10 °C. The within-day imprecision was