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Determination of eumelanin and pheomelanin in melanomas using solid-phase extraction and high performance liquid chromatography-diode array detection (HPLC-DAD) analysis.

Abstract

Determination of eumelanin and pheomelanin in melanomas that exhibit different pigmentation was carried using a solid-phase extraction (SPE) preparation method based on weak anion exchange chemistry. This extraction significantly enhanced the chromatographic profile obtained by reverse phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD). The SPE method was developed using aqueous standards of melanin markers: thiazole-2,4,5-tricarboxylic acid (TTCA), thiazole-4,5-dicarboxylic acid (TDCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) and non-pigmented cell lines spiked with those markers. An excellent average recovery, above 90%, was obtained for the four markers with a relative standard deviation below 7%. We have also optimized the stationary phase and the mobile phase (phosphate concentration and pH) to improve sensitivity and to reduce the analysis time. Elution of the four markers is achieved in 5 min and total analysis of biological samples is completed in 15 min. The quantification limits for TDCA, TTCA, PDCA and PTCA are 60, 50, 47 and 48 ng/mL respectively. Furthermore, DAD detection improves the marker identification in complex matrices through the analysis of UV spectra. We have successfully applied this method to melanoma tumors and cells. Murine B16BL6 tumor are highly pigmented with mostly eumelanin (98.1% of eumelanin) while human SK-MEL-3 tumor contain about 30% pheomelanin. B16BL6 and B16F10 are eumelanic cells lines and NHEM melanocytes contain about 24% of pheomelanin.

Authors+Show Affiliations

UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France.UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France.UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France.UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France.UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France.UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France.UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France.UMR 1240 INSERM UCA, 58 Rue Montalembert, 63005 Clermont-Ferrand Cedex, France. Electronic address: mercedes.quintana@uca.fr.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30897406

Citation

Rioux, Benjamin, et al. "Determination of Eumelanin and Pheomelanin in Melanomas Using Solid-phase Extraction and High Performance Liquid Chromatography-diode Array Detection (HPLC-DAD) Analysis." Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 1113, 2019, pp. 60-68.
Rioux B, Rouanet J, Akil H, et al. Determination of eumelanin and pheomelanin in melanomas using solid-phase extraction and high performance liquid chromatography-diode array detection (HPLC-DAD) analysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2019;1113:60-68.
Rioux, B., Rouanet, J., Akil, H., Besse, S., Debiton, E., Bouchon, B., Degoul, F., & Quintana, M. (2019). Determination of eumelanin and pheomelanin in melanomas using solid-phase extraction and high performance liquid chromatography-diode array detection (HPLC-DAD) analysis. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 1113, 60-68. https://doi.org/10.1016/j.jchromb.2019.03.010
Rioux B, et al. Determination of Eumelanin and Pheomelanin in Melanomas Using Solid-phase Extraction and High Performance Liquid Chromatography-diode Array Detection (HPLC-DAD) Analysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 15;1113:60-68. PubMed PMID: 30897406.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determination of eumelanin and pheomelanin in melanomas using solid-phase extraction and high performance liquid chromatography-diode array detection (HPLC-DAD) analysis. AU - Rioux,Benjamin, AU - Rouanet,Jacques, AU - Akil,Hussein, AU - Besse,Sophie, AU - Debiton,Eric, AU - Bouchon,Bernadette, AU - Degoul,Françoise, AU - Quintana,Mercedes, Y1 - 2019/03/11/ PY - 2018/12/06/received PY - 2019/03/05/revised PY - 2019/03/09/accepted PY - 2019/3/22/pubmed PY - 2019/5/29/medline PY - 2019/3/22/entrez KW - Eumelanin KW - High performance liquid chromatography–diode array detection (HPLC-DAD) KW - Melanoma tumors KW - Pheomelanin KW - Solid-phase extraction (SPE) SP - 60 EP - 68 JF - Journal of chromatography. B, Analytical technologies in the biomedical and life sciences JO - J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. VL - 1113 N2 - Determination of eumelanin and pheomelanin in melanomas that exhibit different pigmentation was carried using a solid-phase extraction (SPE) preparation method based on weak anion exchange chemistry. This extraction significantly enhanced the chromatographic profile obtained by reverse phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD). The SPE method was developed using aqueous standards of melanin markers: thiazole-2,4,5-tricarboxylic acid (TTCA), thiazole-4,5-dicarboxylic acid (TDCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) and non-pigmented cell lines spiked with those markers. An excellent average recovery, above 90%, was obtained for the four markers with a relative standard deviation below 7%. We have also optimized the stationary phase and the mobile phase (phosphate concentration and pH) to improve sensitivity and to reduce the analysis time. Elution of the four markers is achieved in 5 min and total analysis of biological samples is completed in 15 min. The quantification limits for TDCA, TTCA, PDCA and PTCA are 60, 50, 47 and 48 ng/mL respectively. Furthermore, DAD detection improves the marker identification in complex matrices through the analysis of UV spectra. We have successfully applied this method to melanoma tumors and cells. Murine B16BL6 tumor are highly pigmented with mostly eumelanin (98.1% of eumelanin) while human SK-MEL-3 tumor contain about 30% pheomelanin. B16BL6 and B16F10 are eumelanic cells lines and NHEM melanocytes contain about 24% of pheomelanin. SN - 1873-376X UR - https://www.unboundmedicine.com/medline/citation/30897406/Determination_of_eumelanin_and_pheomelanin_in_melanomas_using_solid_phase_extraction_and_high_performance_liquid_chromatography_diode_array_detection__HPLC_DAD__analysis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1570-0232(18)31821-X DB - PRIME DP - Unbound Medicine ER -