An easy-to-run method for routine analysis of eumelanin and pheomelanin in pigmented tissues Lucia Panzella 1,2 , Paola Manini 1 , Giuseppe Monfrecola 2 , Marco d’Ischia 1 and Alessandra Napolitano 1 * 1 Department of Organic Chemistry and Biochemistry, University of Naples ‘Federico II’, Naples, Italy 2 Department of Systematic Pathology, Dermatology Unit, University of Naples ‘Federico II’, Naples, Italy *Address correspondence to Alessandra Napolitano, e-mail: alesnapo@unina.it Summary A procedure for analysis of melanin-pigmented tissues based on alkaline hydrogen peroxide degra- dation coupled with high-performance liquid chro- matography (HPLC) ultraviolet determination of pyrrole-2,3,5-tricarboxylic acid (PTCA) for eumelanin and 6-(2-amino-2-carboxyethyl)-2-carb- oxy-4-hydroxybenzothiazole (BTCA) and 1,3-thiaz- ole-2,4,5-tricarboxylic acid for pheomelanin was recently developed. Despite advantages related to the degradation conditions and sample handling, a decrease of the reproducibility and resolution was observed after several chromatographic runs. We report herein an improved chromatographic meth- odology for simultaneous determination of PTCA and BTCA as representative markers of eumelanin and pheomelanin, respectively, based on the use of an octadecylsilane column with polar end-capping with 1% formic acid (pH 2.8)/methanol as the eluant. The method requires conventional HPLC equipments and gives very good peak shapes and resolution, without need of ion pair reagents or high salt concentrations in the mobile phase. The intra-assay precision of the analytical runs was satisfactory with CV values £4.0% (n ¼ 5) for the two markers which did not exceed 8% after 50 consecutive injections on the column over 1 week. The peak area ratios at 254 and 280 nm (A 280 /A 254 : PTCA ¼ 1.1, BTCA ¼ 0.6) proved a valuable param- eter for reliable identification of the structural markers even in the most complex degradation mixtures. The method can be applied to various eumelanin and pheomelanin pigmented tissues, including mammalian hair, skin and irides, and is amenable to be employed in population screening studies. Key words: high-performance liquid chromatography/ melanin structural markers/pyrrole-2,3,5-tricarboxylic acid/6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenz- othiazole/chemical degradation/pigmented tissues Received 18 October 2006, revised and accepted for publication 15 November 2006 Introduction Eumelanin and pheomelanin analysis by identification and quantification of specific structural markers derived by chemical degradation of pigmented tissues remains an active issue in studies of human pigmentation and associated pathological conditions (Hennessy et al., 2005; Ito, 1998; Naysmith et al., 2004; van Nieuwpoort et al., 2004; Pavel et al., 2004). This holds especially in the case of pheomelanin pigments, for which straight- forward analytical procedures such as spectrophotomet- ric determination of solubilized tissues (Ozeki et al., 1996) fail to provide safe identification of the pigments and a reliable estimate of their levels (Wakamatsu et al., 2006a). The discovery that some variant alleles of the gene encoding for the melanocortin-1-receptor (MC1R), the primary responsible for regulation of melanocyte pig- mentation (Garcı ´a-Borro ´ n et al., 2005; Kadekaro et al., 2003; Rees, 2003), are associated with the red hair color pheomelanic phenotype raised issues relating to possible relationships between the nature and content of cutaneous pigments and the type of MC1R poly- morphism. In several studies determination of the eumelanin/pheomelanin ratios in hair and melanocytes cultures from donors of different phenotypes was used to investigate the effects of MC1R genotype on human pigmentary status (Wakamatsu et al., 2006a; Naysmith et al., 2004). Sensitive methods for determination of the urinary levels of eumelanin and pheomelanin in body fluids were developed for the follow-up of melanoma, as an alternative to quantification of the colorless melanin-rela- ted metabolites (Wakamatsu et al., 2006b; Takasaki et al., 2003). Improved sensitivity in the determination of the eumelanin marker pyrrole-2,3,5-tricarboxylic acid (PTCA) in human skin biopsies was obtained by means of Liquid Chromatography/Mass Spectrometry/Mass Copyright ª 2006 The Authors Journal Compilation ª 2006 Blackwell Munksgaard doi: 10.1111/j.1600-0749.2006.00359.x 128 Pigment Cell Res. 20; 128–133