Epimers of Azaspiracids: Isolation, Structural Elucidation, Relative LC-MS Response, and in Vitro Toxicity of 37-epi-Azaspiracid-1

Abstract

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.

Mediterranean Azadinium dexteroporum (Dinophyceae) produces six novel azaspiracids and azaspiracid-35: a structural study by a multi-platform mass spectrometry approach

“…Recent studies on the toxicity of some purified AZA analogues relative to AZA-1 showed that all analyzed AZAs were toxic and that AZA-2, AZA-3, and AZA-6 were more toxic than AZA-1 by 8.3, 4.5, and 7 times, respectively [10], 37-epi-AZA-1 was 5 times more toxic [11], AZA-33 and AZA-34 were 5 times less toxic and 5 times more toxic, respectively [17], and AZA-36 and AZA-37 were 6 and 3 times less toxic, respectively [15]. As major differences in toxicity can be related to slightly different structures of AZAs, the actual toxicity of the different compounds produced by the Mediterranean A. dexteroporum is worthy of consideration in future toxicological studies.…”

“…Over the last years, several analogues of AZA-1 have been discovered either in shellfish [7][8][9][10][11] or in dinoflagellates [6,[12][13][14][15][16][17] and structurally characterized by tandem mass spectrometry (MS/MS) and partly also by nuclear magnetic resonance (NMR) spectroscopy. Some AZAs (AZA-1 and AZA-2, among others) are actually produced by the dinoflagellates, whereas others (e.g., AZA-3 to AZA-19) seem to derive from biotransformation occurring in shellfish [9].…”

A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification

“…Other experiments, including LSE with matrix matched calibration, MSPD with standard addition, and both LSE and MSPD with dilution, were conducted to support the values used for certification (ESM). The neutral pH mobile phase provided resolution of AZA epimers [25] from the parent peak as established previously [16] ( F i g .1A). Molar responses of the 37-epi-AZAs [25] are approximately equivalent to the parent toxins under the LC-MS conditions used.…”

“…The neutral pH mobile phase provided resolution of AZA epimers [25] from the parent peak as established previously [16] ( F i g .1A). Molar responses of the 37-epi-AZAs [25] are approximately equivalent to the parent toxins under the LC-MS conditions used. Epimer peak areas corresponded to approximately 6.5-7.5 % of total peak areas for AZA1, -2, and -3.…”

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids

“…The occurrence of isomeric forms of the major AZA analogs was previously reported [35], and these have recently been characterized as C37 epimers [49]. The presence of these epimers in CRM-AZA-Mus is illustrated in Fig.…”