Quantitative separation of antioxidant pigments in purple sea urchin shells using a reversed-phase high performance liquid chromatography

Sea urchin shells are generally discarded as food waste after removal of edible gonads, although they possess significant amount of polyhydroxylated naphtoquinone pigments having potent antioxidant property. In this study, separation and quantification of the pigment mixtures from purple sea urchin shells were performed using a C18 reversed-phase HPLC with isocratic elution of 50% mixture of solvent (A) formic acid: water (0.1: 100, v/v) and solvent (B) MeOH: acetonitrile (5: 9, v/v) at a flow rate of 0.5 mL/min. The pigment mixtures were separated into four major peaks in 30 min, which assumed to be spinochromes A, B and C and echinochrome A on the basis of UV–Vis absorption spectra obtained by a diode array detector. LC–MS analysis also supported these results by producing the predominant base peaks at m/z 221.1, 279.1, 265.1, and 263.1 corresponding to the pseudomolecular ions [M–H]− of the respective compounds. The HPLC method also showed good correlations (r2 > 0.98) between injected amounts of these pigments and peak areas recorded at 340 and 520 nm. These results indicated that the HPLC method developed in this study would be applicable for the separation and quantification of these major pigments in sea urchin shells.