Analysis of Palmitoyl Apo-astaxanthinals, Apo-astaxanthinones, and their Epoxides by UHPLC-PDA-ESI-MS Yannick Weesepoel, †,§ Harry Gruppen, † Wouter de Bruijn, † and Jean-Paul Vincken* ,† † Laboratory of Food Chemistry, Wageningen University, P.O. Box 17, 6700 AA Wageningen, The Netherlands § FeyeCon Carbon Dioxide Technologies, Rijnkade 17a, 1382 GA Weesp, The Netherlands * S Supporting Information ABSTRACT: Food products enriched with fatty acid-esterified xanthophylls may result in deviating dietary apo-carotenoids. Therefore, free astaxanthin and its mono- and dipalmitate esters were subjected to two degradation processes in a methanolic model system: light-accelerated autoxidation and hypochlorous acid/hypochlorite (HOCl/OCl - ) bleaching. Reversed phase ultrahigh-performance liquid chromatography photodiode array with in-line electrospray ionization mass spectrometry (RP- UHPLC-PDA-ESI-MS) was used for assessment of degradation products. Apo-astaxanthinals and -astaxanthinones containing 3 (apo-9) to 10 (apo-8′) conjugated double bonds were found upon autoxidation for all three types of astaxanthin (except free apo-8′-astaxanthinal). Fragmentation of [M + H] + and [M + Na] + parent masses of apo-astaxanthins from dipalmitate astaxanthin indicated palmitate esterification. Astaxanthin monopalmitate degradation resulted in a mixture of free and palmitate apo-astaxanthins. HOCl/OCl - rapidly converted the astaxanthins into a mixture of epoxy-apo-9- and epoxy-apo-13- astaxanthinones. The palmitate ester bond was hardly affected by autoxidation, whereas for HOCl/OCl - the ester bond of the apo-astaxanthin palmitoyl esters was degraded. KEYWORDS: apo-astaxanthins, UHPLC-PDA-ESI-MS, fatty acid ester, xanthophyll, light-accelerated degradation, hypochlorite bleaching ■ INTRODUCTION For many years, the addition of arti ficially produced carotenoids to food products has been common practice in the food industry. Recent market demands toward natural substitutes have stimulated research on obtaining and utilizing carotenoids of natural origin. Algae, such as Haematococcus pluvialis, can be used for producing these natural carotenoids. 1 H. pluvialis produces the red carotenoid astaxanthin (3,3′- dihydroxy-β,β-carotene-4,4′-dione, Figure 1) in 2-3% (w/w) dry weight (dw) quantities. Different from its chemically synthesized astaxanthin analogue, H. pluvialis astaxanthin predominantly exists as mono- and diesterified fatty acid forms, 70-90 and 5-25%, respectively. 2,3 Carotenoids are regarded as powerful antioxidants, which degrade during processing and dietary uptake of foods. This process has been investigated especially for β,β-carotene. Various types of degradation reactions are associated with food processing and dietary uptake and evidently result in many degradation products. 4 In Figure 1, three reactions, generally known to occur upon carotenoid degradation, are putatively projected on astaxanthin (esters). Cleavage of conjugated double bonds (CDBs) results in a series of apo-astaxanthin (astaxanthins with a shortened carbon skeleton), aldehydes, and ketones (reactions 1, R1a and R1b, respectively). Depending on which CDB is disrupted, a pair of aldehydes (R1a) or a ketone and an aldehyde (R1b) is formed. Epoxidation of the 5-6 CDB of astaxanthin can result in 5,6-epoxy-astaxanthins (reaction 2, R2a). This process can also yield 5,8-furanoid-astaxanthins, which can be formed directly or via acidic rearrangement from the 5,6-epoxide form (R2b). Furthermore, the cleavage of the fatty acid ester might result in free astaxanthin or astaxanthin monoester (reaction 3, R3). Finally, the degradation products resulting from R1, R2, and R3 can undergo additional degradation via R1 or R2, until they end up as structures with little resemblance to the original carotenoid. 4-8 Also, cis- trans isomerization might be regarded as a degradation reaction, 4 but is not further considered here. A common carotenoid degradation reaction that results in R1 and R2 degradation products is oxidation. A relatively mild form of oxidation, occurring during product processing and storage, is autoxidation. This reaction can occur spontaneously and is accelerated by light sensitization, oxygen, and/or elevated temperatures. During autoxidation the CDBs of the carotenoid chromophore are reduced or cleaved, resulting in numerous products. Autoxidation is generally believed to proceed via carbon-peroxyl triplet radicals, which propagate the reaction via intramolecular homolytic substitution. 9 In contrast, a relatively aggressive oxidative agent is hypochlorous acid (HOCl). In the human body, HOCl can be formed upon inflammatory reactions by the enzyme myeloperoxidase in polymorphonuclear leukocytes. 10 There, HOCl is primarily produced to eliminate harmful bacteria or toxins, but it is also known to damage surrounding tissue. 11,12 Also, the dietary carotenoids present in the bloodstream 13,14 can be converted by HOCl into oxidation products that might be toxic. 15,16 HOCl occurs in equilibrium with its corresponding base OCl - , and together they are referred to as HOCl/OCl - or Received: July 23, 2014 Revised: September 24, 2014 Accepted: October 1, 2014 Published: October 3, 2014 Article pubs.acs.org/JAFC © 2014 American Chemical Society 10254 dx.doi.org/10.1021/jf503520q | J. Agric. Food Chem. 2014, 62, 10254-10263