Copyright © 2010 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 878–886
Research Article
Received 4 August 2009, Revised 21 October 2009, Accepted 2 November 2009 Published online in Wiley InterScience: 19 January 2010
(www.interscience.wiley.com) DOI 10.1002/bmc.1380
New protocol to obtain spirolides from
Alexandrium ostenfeldii cultures with high
recovery and purity
Paz Otero
a
, Amparo Alfonso
a
, Carmen Alfonso
a
, Mercedes R. Vieytes
b
,
M. Carmen Louzao
a
, Ana M. Botana
c
and Luis M. Botana
a
*
ABSTRACT: The aim of this work was to develop a method to purify large amounts of spirolide toxins from cultures of
Alexandrium ostenfeldii. The dinoagellates grew in batches under controlled conditions of salinity, light and temperature.
Analysis of the cultures demonstrated the existence of neurotoxins associated with paralytic shellsh poisoning toxins and
two spirolides, 13-desmethyl spirolide C and 13,19-didesmethyl spirolide C. The protocol designed presents several stages of
extraction, separation between spirolides and paralytic shellsh poisoning toxins, and cleanup in solid-phase extraction.
Finally, the purication of spirolides was conducted by a preparative high-performance liquid chromatography system coupled
to a mass spectrometer detector. The purity and the amount of both toxins in each step was monitored by analytical liquid
chromatographic–mass spectrometry. Large amounts of 13-desMeC, 97% pure, and 13,19-didesMeC, 99% pure, were obtained.
A novel and ecient method to separate and purify spirolide toxins from large amounts of phytoplankton is provided. The
protocol proposed shows, for the rst time, a complete and detailed methodology to separate and purify spirolide toxins with
high purity, recovery, repeatability and stability. Copyright © 2010 John Wiley & Sons, Ltd.
Keywords: spirolides; purication, 13-desmethyl spirolide C; 13,19-didesmethyl spirolide C; LC-MS; analytical and preparative
methodology
* Correspondence to: L. M. Botana, Departamento de Farmacología, Facultad
de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
E-mail: luis.botana@usc.es
a
Departamento de Farmacología, Facultad de Veterinaria, Universidad
de Santiago de Compostela, 27002 Lugo, Spain
b
Departamento de Fisiología, Facultad de Veterinaria, Universidad de
Santiago de Compostela, 27002 Lugo, Spain
c
Departamento de Química Analítica, Facultad de Ciencias, Universidad
de Santiago de Compostela, 27002 Lugo, Spain
Abbreviations used: dcGTX, decarbamoylgonyautoxins; dcSTX,
decarbamoylsaxitoxin; GTX, gonyautoxins; NEO, neosaxitoxin; PSP,
paralytic shellsh poisoning; STX, saxitoxin; TFA, triuoroacetic acid.
Introduction
Spirolides are macrocyclic polyether compounds that belong to
the cyclic imine group. This group includes other lipophilic com-
pounds such as gymnodimine, pinnatoxins, prorocentrolide, pte-
riatoxins and espiro-prorocentrimine (MacKinnon et al., 2004,
2006a, b). Spirolides are the most common cyclical imine toxins
due to their widespread global distribution. They were rst iden-
tied in extracts of the digestive glands of mussels (Mytilus edulis)
and scallops (Placopecten magellanicus) from the Atlantic coast
of Nova Scotia, Canada, in 1991 (Meilert and Brimble, 2006). That
year, routine toxin monitoring of bivalve molluscs in this location
revealed a toxic response in mice after intraperitoneal injections
of shellsh extracts. The clinical manifestations included abdomi-
nal muscle spasms, piloerection, ataxia, tail whipping and, imme-
diately prior to death, neurological symptoms, comprising
convulsions and cramps. Chemical investigations demonstrated
that the toxicity was due to a new class of toxins which were
called spirolides.
Spirolide toxins have now been detected in shellsh from a
number of other locations around the world. In Europe, these
toxins were found in Spain (Villar Gonzalez et al., 2006), Italy
(Ciminiello et al., 2006), Scotland (John et al., 2003), Norway
(Aasen et al., 2005) and Denmark (MacKinnon et al., 2006b). The
dinoagellate identied as the producer of spirolides, in all these
coasts, was Alexandrium ostenfeldii. Many studies report this
organism to be the main origin of spirolides, although this dino-
agellate may produce toxins associated with paralytic shellsh
poisoning (PSP).
There have been no reports of toxic eects in humans due to
ingestion of spirolides. However, general symptoms such as tach-
ycardia and gastric distress were reported following contami-
nated shellsh consumption from Nova Scotia during the spring
and summer of 1991, when spirolides were detected in mollusc
from this region (Richard et al., 2001; Sleno and Volmer, 2005).
Although the mechanism of action of spirolides in cells are not
yet well understood, it has been suggested that the muscarinic
acetylcholine receptors might be implicated in its mode of action
(Gill et al., 2003).
Nuclear magnetic resonance (NMR) and mass spectrometry
(MS) methods were used to achieve the structure elucidation of
spirolides (Aasen et al., 2005; Ciminiello et al., 2006, 2007; Hu et al.,
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