Research Article HPLC-PAD-atmospheric pressure chemical ionization-MS metabolite profiling of cytotoxic carotenoids from the echinoderm Marthasterias glacialis (spiny sea-star) An HPLC-PAD-atmospheric pressure chemical ionization-MS metabolite profiling analysis was conducted on the marine echinoderm Marthasterias glacialis (spiny sea-star). Bio-guided purification of the methanolic extract led to the isolation of several carotenoids, namely zeaxanthin, astaxanthin and lutein. These compounds were characterized using both UV–Vis characteristics and MS spectra interpretation. No previous works addressed the MS analysis of carotenoids present in this organism. The purified carotenoid fraction displayed a strong cell proliferation inhibition against rat basophilic leukemia RBL-2H3 (IC 25 5 268 mg/mL) cancer cell line. Against healthy V79 (rat lung fibroblasts (IC 25 5 411 mg/mL)) cell line, however, toxicity was lower, as it is desired for anti-cancer mole- cules. This study suggests that M. glacialis may constitute a good source of bioactive compounds that can be used as lead compounds for the pharmaceutical industry. Keywords: Carotenoids / Cytotoxic / HPLC-PAD-atmospheric pressure chemical ionization-MS / Marthasterias glacialis DOI 10.1002/jssc.201000197 1 Introduction Carotenoids are a class of hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls). Their basic structure reflects their biosynthetic pathway and consists of eight isoprenoid units. A series of conjugated double bonds constitute the characteristic chromophore. Carote- noids are quite widespread in nature and marine organisms are no exception. In fact, the higher degree of diversity seems to be in the aquatic environment [1]. These compounds can play an important role in human health by acting as antioxidants, protecting cells and tissues from the damaging effects of reactive species. Lutein and zeaxanthin and xanthophylls found in corn and in leafy greens, such as kale and spinach, are believed to function as protective antioxidants in the macular region of the human retina, to protect against cataract formation, coronary heart diseases and stroke. Astaxanthin, a xanthophyll found in several marine organisms, is another naturally occurring compound with potent antioxidant properties. Other health benefits of carotenoids that may be related to their anti- oxidative potential include enhancement of immune system function and inhibition of the development of certain types of cancers [2]. The most well-known physiological role of carotenoids is provitamin A activity, although not all compounds exhibit this property [3]. Marthasterias glacialis (spiny sea-star) is an asteroid echinoderm, a group that has demonstrated to play one of the most influential roles in benthic ecosystems on a variety of scales [4]. During the period 1940–1950s, this species received great attention, mainly due to its asterosaponins [5]. At the same time, carotenoids in this organism were also studied by means of the analytical techniques common at the time, namely absorption spectrum, comparison of Rf values by TLC and partition coefficient [6]. To our knowl- edge, no previous study provided the LC-MS analysis of the carotenoid fraction of M. glacialis and its biological potential. Due to the heat-lability of carotenoids, GC-MS is not suitable for their analysis, the most convenient method being HPLC with UV–Vis detection or, if possible, MS detection. Xanthophylls and carotenes form both molecular ions and protonated molecules during positive-ion atmo- spheric pressure chemical ionization (APCI). APCI is an ideal method of ionization for low to medium polar compounds, which also include carotenoids and related compounds. Since their molecular mass does not exceed 2000 u, even in the case of glycosides or esters, this method is suitable for their analysis, as has been recently reviewed by Rezenka et al. [7]. Federico Ferreres 1 David M. Pereira 2Ã Angel Gil-Izquierdo 1 Patrı´cia Valenta ˜o 2 Joa ˜ o Botelho 2 Teresa Mouga 3 Paula B. Andrade 2 1 Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), Murcia, Spain 2 REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, Porto, Portugal 3 GIRM – Marine Resources Research Group, School of Tourism and Maritime Technology, Polytechnic Institute of Leiria, Santua ´ rio Na. Sra. Dos Reme ´ dios, Peniche, Portugal Received March 23, 2010 Revised May 10, 2010 Accepted May 11, 2010 Abbreviations: APCI, atmospheric pressure chemical ionization; FBS, fetal bovine serum; RBL, rat basophilic leukemia; SRB, sulphorhodamine B Ã Additional correspondence: Dr. David M. Pereira E-mail: david.ffup@gmail.com Correspondence: Professor Paula B. Andrade, REQUIMTE/ Department of Pharmacognosy, Faculty of Pharmacy, Porto University, R. Anı´bal Cunha 164, 4050-047 Porto, Portugal E-mail: pandrade@ff.up.pt Fax: 1351-222003977 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2010, 33, 2250–2257 2250