jf2048788_si_001.pdf (326.74 kB)
Improved Isolation Procedure for Azaspiracids from Shellfish, Structural Elucidation of Azaspiracid-6, and Stability Studies
journal contribution
posted on 2016-02-21, 17:51 authored by Jane Kilcoyne, Adela Keogh, Ger Clancy, Patricia LeBlanc, Ian Burton, Michael
A. Quilliam, Philipp Hess, Christopher O. MilesAzaspiracids are a group of lipophilic polyether toxins
produced
by the small dinoflagellate Azadinium spinosum. They
may accumulate in shellfish and can result in illnesses when consumed
by humans. Research into analytical methods, chemistry, metabolism,
and toxicology of azaspiracids has been severely constrained by the
scarcity of high-purity azaspiracids. Consequently, since their discovery
in 1995, considerable efforts have been made to develop methods for
the isolation of azaspiracids in sufficient amounts and purities for
toxicological studies, in addition to the preparation of standard
reference materials. A seven-step procedure was improved for the isolation
of azaspiracids-1–3 (1, 2, and 3) increasing recoveries 2-fold as compared to previous methods
and leading to isolation of sufficiently purified azaspiracid-6 (6) for structural determination by NMR spectroscopy. The procedure,
which involved a series of partitioning and column chromatography
steps, was performed on 500 g of Mytilus edulis hepatopancreas
tissue containing ∼14 mg of 1. Overall yields
of 1 (52%), 2 (43%), 3 (43%),
and 6 (38%) were good, and purities were confirmed by
NMR spectroscopy. The structure of 6 was determined by
one- and two-dimensional NMR spectroscopy and mass spectrometry. The
stability of 6 relative to 1 was also assessed
in three solvents in a short-term study that demonstrated the greatest
stability in aqueous acetonitrile.