Toxin profile of Alexandrium ostenfeldii (Dinophyceae)

from the Northern Adriatic Sea revealed by liquid

chromatography–mass spectrometry

Patrizia Ciminiello a,*, Carmela Dell’Aversano a, Ernesto Fattorusso a,

Silvana Magno a, Luciana Tartaglione a, Monica Cangini b, Marinella Pompei b,

Franca Guerrini c, Laurita Boni c, Rossella Pistocchi c

a Dipartimento di Chimica delle Sostanze Naturali, Universita degli Studi di Napoli ‘Federico II’, via D. Montesano 49, 80131 Napoli, Italyb Centro Ricerche Marine, Viale Vespucci 2, 47042 Cesenatico(FC), Italy

c Scienze Ambientali, Universita di Bologna, Via Tombesi Dall’Ova 55, 48100 Ravenna, Italy

Available online 24 March 2006

Abstract

This paper reports on the first occurrence of fairly high numbers of Alexandrium ostenfeldii along the Emilia Romagna coasts

(Italy). Detailed liquid chromatography–mass spectrometry (LC–MS) analyses of the toxin profile were performed on a strain

of the organism collected in November 2003, isolated during the event and grown in culture. Selected ion monitoring (SIM) and

multiple reaction monitoring (MRM) experiments were carried out for detection of spirolides and paralytic shellfish poisoning

(PSP) toxins. They revealed that the Adriatic A. ostenfeldii produces mainly spirolide 13-desmethyl C at levels of 3.7 pg/cell but

not PSP toxins. Interestingly, low levels of some spirolide isomers that have not been reported so far in other strains of the

dinoflagellate were also detected. This represents the first report of spirolide-type toxins in the Adriatic Sea.

q 2006 Elsevier Ltd. All rights reserved.

Keywords: Alexandrium ostenfeldii; Spirolide 13-desmethyl C; Spirolides; LC–MS; Liquid chromatography–mass spectrometry; Adriatic Sea

1. Introduction

The spirolides are a group of marine toxins detected in

European and North American seas. First episodes of this

kind of toxicity were reported in early 1990s in bivalve

mollusks collected along the eastern shore of Nova Scotia,

Canada (Hu et al., 1995). Intraperitoneal injection (IP) of

lipophilic shellfish extracts in mice caused an unusual toxin

syndrome expressed as piloerection, hyperextension of the

back, arching of the tail, neuro-convulsions and rapid death

(3–20 min). Toxicity studies in mice gave an LD50 of 40 mg/kg and 1 mg/kg for IP and oral dosing, respectively (Richard

0041-0101/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2006.02.003

* Corresponding author. Tel.: C39 081 678507; fax: C39 081

678552.

E-mail address: ciminiel@unina.it (P. Ciminiello).

et al., 2000). Some evidence indicates that spirolides are

muscarinic acetylcholine receptor antagonists and weak

L-type transmembrane calcium channel activators in

mammalian systems (Hu et al., 1995). Human toxicity is

still unknown, even if gastric distress and tachycardia,

following consumption of contaminated shellfish, were

reported in the period when spirolides were detected in

Nova Scotian shellfish.

Structurally, spirolides are characterized by a spiro-

linked tricyclic ether ring system and an unusual seven-

membered spiro-linked cyclic iminium moiety. The number

of toxins belonging to this class has grown significantly over

the years to include several isomers and compounds with

slightly modified structures (Fig. 1) (Hu et al., 1995, 2001).

Six major spirolides are known, namely spirolide A, B,

C, D, E and F, in addition to certain desmethyl derivatives

Toxicon 47 (2006) 597–604

www.elsevier.com/locate/toxicon

Fig. 1. Chemical structures of major spirolides and related protonated ions (m/z) and Q1OQ3 transitions (m/zOm/z) monitored in selected ion

monitoring (SIM) and multiple reaction monitoring (MRM) experiments (positive ions), respectively.

P. Ciminiello et al. / Toxicon 47 (2006) 597–604598

of spirolides C (13-desMeC) and D (13-desMeD). Small

alteration in structure can result in large differences in

toxicity. In fact, the bioactive part structure of these

compounds has been identified as the cyclic imine.

Consequently, hydrolysis to a keto-amine functional

group, such as in spirolides E and F, results in non-toxic

metabolites in shellfish (Hu et al., 1996).

Recently, the causative organism producing spirolide

toxins was identified as the dinoflagellate Alexandrium

ostenfeldii (Paulsen) Balech and Tangen (1985) from Ship

Harbour, Nova Scotia (Cembella et al., 1999, 2000, 2001),

even if no cases of human poisoning have been definitively

linked to blooms of such organism. A. ostenfeldii has been

found widely distributed in north temperate waters, occurring

off the coasts of Denmark (Moestrup and Hansen, 1988),

Norway (Aasen et al., 2005), Scotland (John et al., 2003),

Atlantic Canada (Cembella et al., 2001) and New Zealand

(Mackenzie et al., 1996). Crude extracts from different strains

of A. ostenfeldii collected from different coastal regions have

shown important and in some cases dramatic differences in

toxicity and toxin profile. Particularly, in Nova Scotia,

A. ostenfeldii cells contain a high level of spirolides A, B, C,

D, 13-desMeCand 13-desMeD, but these compoundshavenot

been found in populations from New Zealand, where they

produce, instead, toxins involved in paralytic shellfish

poisoning (PSP) (Mackenzie et al., 1996). Even more

confusing is that certain populations found in Scandinavia

produce both spirolides and PSP toxins, but at very low levels

(Cembella et al., 2000). In Italy, the presence of A. ostenfeldii

has not been reported so far.

This paper reports on the first occurrence of fairly high

numbers of A. ostenfeldii along the Emilia Romagna coasts

(Italy). Detailed analysis of the toxin profile was performed

by liquid chromatography–mass spectrometry (LC–MS) on

a strain of this organism collected in November 2003,

isolated during the event and grown in culture.

2. Materials and methods

2.1. Chemicals

All organic solvents were of distilled-in-glass grade (Carlo

Erba, Milan, Italy). Water was distilled and passed through a

MilliQ water purification system (Millipore Ltd, Bedford,

MA, USA). Formic acid (95–97%, Laboratory grade) and

ammonium formate (AR grade) were purchased from Sigma

Aldrich, Steinheim, Germany. Spirolide 13-desMeC standard

solution and aCanadianA. ostenfeldiiplankton extract, used as

references, were kindly provided by Dr Michael Quilliam

(Institute for Marine Biosciences, National Research Council

of Canada, Halifax, NS, Canada).

P. Ciminiello et al. / Toxicon 47 (2006) 597–604 599

2.2. Sampling and cultures of A. ostenfeldii (Paulsen)

Balech et Tangen

The sampling area is located in the North-Western

Adriatic Sea along Emilia Romagna coasts (Italy) and, since

1978, it is subjected to careful monitoring for toxic

phytoplankton and algal toxins in farmed shellfish.

Qualitative and quantitative analysis of phytoplankton

was performed by the Utermohl method (Utermohl, 1931)

on water samples stored in dark glass bottles. A. ostenfeldii

(Paulsen) Balech and Tangen (1985) was isolated by the

capillary pipette method (Hoshaw and Rosowski, 1973)

from a natural sample of phytoplankton collected in the

coastal waters of Emilia Romagna, in the nearby of

Cesenatico (FC), in November 2003. After an initial growth

in microplates, the unialgal cultures were grown in sterile

Erlenmeyer flasks sealed with cotton plugs at 20 8C under a

16:8 h LD cycle (ca. 166 mmol/m2 per s from cool white

lamp); nutrients were added at the f/2 concentration

(Guillard and Ryther, 1962), and water salinity was adjusted

to 30 psu. For toxicity studies, A. ostenfeldii was cultured in

two Erlenmeyer flasks each containing 1.5 l of medium.

Cells were collected at stationary phase on the 30th day of

growth first by gravity filtration through 0.45 mm Millipore

filters and then by centrifugation at 3000g for 15 min at

10 8C. Both cell pellets and growth mediums were saved for

analyses. Cell counts were made every other day in settling

chambers by the Utermohl method (Utermohl, 1931).

2.3. Sample extraction and clean-up

Cultured cells (49!106 cells in 3 l culture) were

suspended in methanol/water (8:2, v/v) solution (9 ml) and

sonicated for 5 min in pulse mode, while cooling in ice bath.

The mixture was centrifuged at 5000 rpm for 10 min and the

pellet was washed twice each with 9 ml of methanol. The

supernatants were combined and the volume was adjusted to

27 ml with extracting solvent. A 1 ml aliquot of the crude

extract was analyzed directly by LC–MS (5 ml injected).A 6 ml aliquot of the extract was evaporated to dryness. The

residue was dissolved in 1 ml of acetonitrile–water (1:9,

v/v), and the solution was loaded on a Sep-Pak C-18 plus

cartridge (Waters Corporation, Milford, MA, USA) equili-

brated with the same solution. The column was sequentially

eluted with 10 ml of acetonitrile–water (1:9, 3:7, 1:1, v/v)

solutions and acetonitrile 100%. The toxins were eluted with

the acetonitrile–water (1:1, v/v) solution. The residue in the

eluate was evaporated, redissolved in 1 ml of methanol and

directly injected into the LC–MS system.

2.4. Liquid chromatography–mass spectrometry analyses

Mass spectral experiments were performed by using an

API-2000 triple quadrupole mass spectrometer equipped

with a turbo-ionspray source (Applera, Thornhill, ON,

Canada), coupled to an Agilent (Palo Alto, CA, USA) model

1100 LC which included a solvent reservoir, in-line

degasser, binary pump and refrigerated autosampler. LC–

MS analyses for spirolides (Quilliam et al., 2001) were

performed using a 3 mm Hypersil C8 BDS, 50!2.00 mm

column (Phenomenex, Torrance, CA, USA) at room

temperature. Eluent A was water and B was 95%

acetonitrile–water solution, both eluents containing 2 mM

ammonium formate and 50 mM formic acid. The flow rate

was 200 ml/min. Either a gradient elution, 10–100% B in

10 min followed by 100% B for 15 min, or an isocratic

elution with 35% B were used. Selected ion monitoring

(SIM) experiments were carried out in positive ion mode by

selecting the ions reported in Fig. 1 for known spirolides.

Dwell time was generally set at 100 ms and a maximum

number of 10 ions per experiment were monitored. Four

MRM experiments were carried out by selecting the

following groups of transitions (692O150, 692O164,

692O444, 692O458), (694O150, 694O164, 694O444,

694O458), (706O150, 706O164, 706O444, 706O458)

and (708O150, 708O164, 708O444, 708O458), respect-

ively. Dwell time of 250 ms for each transition was used.

In these studies, a temperature of 100 8C, an ionspray

voltage of 5400 V, a declustering potential of 90 V, a

focusing potential of 230 V, and an entrance potential of

12 V were used. A collision energy of 53 eV and a cell exit

potential of 13 V were used in MRM experiments. Spirolide

13-desMeC was quantitatively determined in the crude

extract by direct comparison to individual standard solutions

of spirolide 13-desMeC at similar concentrations injected in

the same experimental conditions.

HILIC–MS analyses for PSP toxins (Dell’Aversano

et al., 2005) were performed using a 250!2.00 mm column

packed with 5 mm Tosohaas TSK-GEL Amide-80 material

at room temperature. Eluent A was water and B was 95%

acetonitrile–water solution, both eluents containing 2 mM

ammonium formate and 3.6 mM formic acid (pH 3.5).

Isocratic elution with 65% B at a flow rate of 200 ml/min

was used. A sample injection volume of 5 ml was used.

Selected ion monitoring (SIM) and multiple reaction

monitoring (MRM) experiments were carried out in positive

ion mode. The following ions were monitored in SIM mode:

m/z 300 for saxitoxin (STX) and gonyautoxin 5 (GTX5),m/z

316 for neosaxitoxin (NEO), gonyautoxins 2/3 (GTX2/3)

and N-sulfogonyautoxins 1/2 (C1/2), m/z 380 for GTX5, m/z

396 for GTX2/3 and C1/2, m/z 412 and 332 for both

gonyautoxins 1/4 (GTX1/4) and N-sulfogonyautoxins 3/4

(C3/4). The following transitions (precursor ion)O(frag-

ment ion) were monitored in MRMmode: m/z 300O282 for

STX, m/z 316O298 for NEO, m/z 380O300 for GTX5, m/z

396O316 and 396O298 for both GTX2/3 and C1/2, m/z

412O332 and 412O314 for both GTX1/4 and C3/4. Dwell

time of 150 ms for each ion or transition was used. For these

studies, a temperature of 50 8C, an ionspray voltage of

4500 V, a declustering potential of 55 V, a focusing

potential of 270 V, an entrance potential of 5 V, a collision

energy of 25 eV and a cell exit potential of 7 V were used.

P. Ciminiello et al. / Toxicon 47 (2006) 597–604600

3. Results and discussion

In the period November 2003–February 2004, increasing

concentrations of A. ostenfeldii cells were detected along

Emilia Romagna coasts, in the Cesenatico area, where

previously we had sporadically observed only a few A.

ostenfeldii cells. The highest concentration was recorded at

the end of November 2003 (15,612 cells/l) and then

decreased.

The cells presented the following morphological features

(Fig. 2), fully consistent with the morphology reported for

the dinoflagellate A. ostenfeldii (Balech and Tangen, 1985):

the cell was large and nearly spherical, with thin thecal

plates and a characteristic large ventral pore on the first

apical plate (1 0); the cingulum and the sulcus were shallow.

Unialgal cultures of A. ostenfeldii, after an initial lag

phase of about a week, reached a maximum density of about

18,500 cells/ml in 25–30 days. A sample containing

49,000,000 cells was provided for evaluation of toxin

profile by LC–MS analyses, which were directed to

detection of toxins so far identified in A. ostenfeldii cultures,

namely PSP toxins and spirolides (Mackenzie et al., 1996;

Cembella et al., 1999).

Cultured cells were extracted by sonicating with

methanol/water (8:2, v/v) and the crude extract was directly

analyzed by hydrophilic interaction liquid chromatography–

mass spectrometry (HILIC–MS). The chromatographic

separation was carried out by using a TSK gel Amide 80

column and a mobile phase containing ammonium formate

and formic acid, as recently reported for rapid, sensitive and

selective determination of PSP toxins (Dell’Aversano et al.,

2005). The MS was equipped with a turboionspray interface

and operated in positive ion mode. Selected ion monitoring

(SIM) experiments were carried out in positive ion mode for

major PSP toxins (saxitoxin, neosaxitoxin, gonyautoxins 1–

5 and N-sulfogonyautoxins 1–4). No peak corresponding to

PSP toxins was detected in the chromatogram. In order to

definitively exclude the presence of saxitoxin and its

Fig. 2. Vegetative cells of A. ostenfeldii (400!) at the optical microscope:

ventral pore on the first apical plate. Scale barZ10 mm.

derivatives, an aliquot of cultured cells was extracted with

0.1 M acetic acid, as recommended for exhaustive extrac-

tion of PSP toxins from phytoplankton (Ravn et al., 1995).

The crude extract was analyzed by HILIC–MS in multiple

reaction monitoring (MRM) mode and showed again not to

contain any of the most common PSP toxins.

The methanol/water (8:2, v/v) crude extract of the

Adriatic A. ostenfeldii was analyzed by LC–MS for most of

the known spirolides. LC–MS is the most common

analytical technique used for detection of spirolides, by

allowing the monitoring of known spirolides as well as the

identification of some unknown derivatives through

interpretation of the fragmentation pattern shown in

MS/MS product ion spectra (Sleno et al., 2004a,b).

The chromatographic separation was carried out by

using a reversed phase Hypersil C8 BDS column and a

mobile phase containing ammonium formate and formic

acid, as suggested by Quilliam et al. (2001) for the analyses

of spirolides and various lipophilic toxins. SIM experiments

were carried out by selecting [MCH]C ions for the

spirolides contained in Fig. 1. In fact, preliminary full

scan MS1 experiments on a standard solution of spirolide

13-desmethyl C, the only spirolide solution commercially

available, had shown that formation of the only pseudo-

molecular ion [MCH]C at m/z 692.5 was favored (Fig. 3a)

under the used ionization process parameters.

SIM traces of the A. ostenfeldii crude extract showed the

presence of a dominant chromatographic peak eluting at

7.48 min for m/z 692.5. This peak could be assigned to

spirolide 13-desMeC, based on the comparison of its

retention time and MW with those of an authentic sample

of 13-desMeC. In order to gain further evidence for the

obtained result we also used, as reference sample,

A. ostenfeldii extract provided by the Institute for Marine

Biosciences (IMB), Canada, which contained spirolide 13-

desMeC in addition to spirolide C, C3, D, and 13-desMeD

(Maclean et al., 2003). Comparison between SIM results for

the two plankton samples provided further support for the

A, not stained cell; B, cell stained with Calcofluor white showing the

OO

N

O

OO

HOOH

m/z

408

Inte

nsity

, cps - H2O

- H2O

- H2O

- H2O- H2O

- H2O

(b)

692

100 200 300 400 500 600 7000

1e+5

2e+5

3e+5

4e+5

674

656

638

462

444

426

164

(a)

500 600 700 800 9000

2e+5

4e+5

6e+5

8e+5 [M+H]+

692.5

m/z

Inte

nsity

, cps

NH+

O

OO OH

fragment aO

O

O

OO OH

fragment b

NH+

Fig. 3. Full scan MS1 (a) and MS2 product ion (b) spectra of a standard solution of spirolide 13-desMeC. MS2 product ion spectrum of the [MCH]C ion atm/z 692 was obtained using a collision

energy of 53 eV.

P.Ciminiello

etal./Toxico

n47(2006)597–604

601

P. Ciminiello et al. / Toxicon 47 (2006) 597–604602

presence of spirolide 13-desMeC in the Adriatic strain and

allowed to rule out the presence of other spirolides produced

by the Canadian strain.

Final confirmation for the identity of spirolide 13-

desMeC in the Adriatic A. ostenfeldii sample was obtained

by multiple reaction monitoring. Such experiment was set

up based on the results of the full scan MS2 product ion

spectrum of a standard solution of spirolide 13-desMeC

that was obtained using the [MCH]C ion at m/z 692.5 as

precursor ion and a collision energy of 53 eV (Fig. 3b). It

contained three characteristic fragment ion clusters: (i)

ions at m/z 674, 656, and 638 due to loss of water

molecules from the pseudo-molecular ion; (ii) ions at m/z

462, 444, 426, 408 due to loss of the structural ‘fragment

a’ and associated water loss from the pseudo-molecular

ion; (iii) an abundant ion at m/z 164 which derives from

the ion at m/z 444 by loss of the structural ‘fragment b’

(Sleno et al., 2004a). The most abundant ions (m/z 674,

444 and 164) were selected as product ions of the ion at

m/z 692 in MRM transitions. MRM traces of the

A. ostenfeldii crude extract (Fig. 4) showed the presence

of peaks eluting at 7.48 min for all three diagnostic

transitions (m/z 692O674, 692O444, and 692O164); the

observed ion ratios were identical to those obtained by

MRM analysis of the spirolide 13-desMeC standard

solution.

The whole of the above experiments were fully

consistent with the presence of spirolide 13-desMeC in the

analyzed sample. Quantitation was carried out by compari-

son of the peak area for the most abundant transition at m/z

692O164 with that obtained by injecting a standard solution

of spirolide 13-desMeC at similar concentration. This

revealed a concentration value for spirolide 13-desMeC of

3.7 pg/cell.

0 2 4 6 8Time, mi

7,

Fig. 4. MRM analysis in positive ion mode of the Adriatic A. ostenfeldii sa

fragmentation pattern of spirolide 13-desMeC (see Fig. 3b). *Chromatogr

Under the same ionization and fragmentation process

parameters optimized for spirolide 13-desMeC, a number of

MRM experiments were carried out by selecting transitions

characteristic for the known spirolides A, B, C, 13-desMeC,

D and 13-desMeD. As a result, the presence of such

spirolides could be definitively excluded in the crude

extract.

It has to be noted that in most of the analyzed strains

of A. ostenfeldii, the 13-desMeC derivative represents

by far the major spirolide component, usually constituting

O90% of the total spirolide content (Sleno et al.,

2004a,b). Since such toxin appeared to be present in

our crude extract at a relatively low level, a doubt

aroused about the possibility that matrix suppression

effect could have hampered detection of minor spirolides.

Thus, a clean-up step by solid phase extraction (SPE)

was carried out.

SPE was accomplished by using a C18 stationary

phase eluted with water–acetonitrile solutions of decreas-

ing polarity. All SPE eluates were analyzed by LC–MS

in MRM mode according to the following procedure: the

protonated ions for spirolides A and 13-desMeC (m/z

692), B and 13-desMeD (m/z 694), C (m/z 706), and D

(m/z 708) were selected as precursor ions and combined

with the four possible fragment ions at m/z 150, 164,

444, and 458. Such transitions were monitored in four

MRM experiments using 250 ms as dwell time in order

to gain highest sensitivity. Such a kind of experiment

gave the opportunity to check for the presence of the

above known derivatives as well as to individuate some

unknown spirolide analogues which differ in substitution

at part structures giving rise to ‘fragment a’ and

‘fragment b’ (Fig. 3b).

10 12 14

m/z 692 > 674

m/z  692 > 444

m/z 692 > 164

n

48

mple was performed on a series of ion transitions consistent with the

aphic conditions were as in Section 2.

692 > 164

692 > 444

Time, min6 10 12

desMeC

692 > 150692 > 458

X1

694 >164 694 >444

desMeC

X2

X3

708 > 458

708 > 164

x 600

x 20

x 300

8

Fig. 5. MRM analyses in positive ion mode of the acetonitrile–water (1:1) SPE eluate of A. ostenfeldii. For the m/z 694O444 and 694O164

transitions, a peak due to 13C isotope of spirolide desMeC is present. Chromatographic conditions were as in Section 2.

P. Ciminiello et al. / Toxicon 47 (2006) 597–604 603

The results of MRM analyses of the acetonitrile–water

(1:1) SPE eluate are shown in Fig. 5. They suggested

the presence of three novel spirolide analogues (X1, X2

and X3) in theA. ostenfeldii extract. Particularly, two of them

(X2 and X3) had the same fragmentation pattern as spirolide

13-desMeD (m/z 694O164 and 694O444) and D (m/z 708O164 and 708O458), respectively, but remarkably

different retention times, namely Rt(X2)Z6.77 min versus

Rt(13-desMeD)Z8.84 min and Rt(X3)Z7.36 min versus

Rt(D)Z7.79 min. On the contrary, X1 had the same retention

time as spirolide 13-desMeC (7.50 min) but different

fragmentation (m/z 692O150 and 692O458), suggesting

that it was a new derivative possibly with R1ZH, R2ZCH3,

and an additional methyl in the ‘fragment b’ moiety of the

molecule.

Mass spectral behavior of X1, X2 and X3 was

clearly indicative of spirolide structures. Unfortunately,

the small amount of available material prevented us to

fully assign their structures by extensive spectral

analyses.

4. Conclusions

This report highlights, for the first time, the presence of

toxic dinoflagellate A. ostenfeldii in the Adriatic Sea. The

careful LC–MS analysis of a culture of this organism

revealed that the Adriatic strain of A. ostenfeldii produces

spirolides but not PSP toxins, which were instead produced

by strains from New Zealand and Scandinavia (Mackenzie

et al., 1996; Cembella et al., 2000). Interestingly, the

Adriatic A. ostenfeldii in addition to spirolide 13-desMeC

also produces some unknown spirolide isomers. This

represents the first report of spirolide-type toxins in the

Adriatic Sea. The variety of spirolide isomers detected can

be explained in light of a classical poliketide synthesis

through joint participation of acetate and propionate units

which could occur by different coupling sequences acetate/

propionate. The aims of our future research are (i) verifying

whether spirolides were retained and accumulated in

mussels cultivated in Adriatic Sea; (ii) large-scale culturing

of A. ostenfeldii in order to isolate sufficient amount of the

P. Ciminiello et al. / Toxicon 47 (2006) 597–604604

new derivatives for structure elucidation both by MS/MS

and NMR means.

Acknowledgements

The authors wish to thank Dr Michael Quilliam and

William Hardstaff (NRC/IMB, Halifax, NS, Canada) for

providing the standard of 13-desMeC and the plankton

extract containing spirolides. This work is a result of a

research supported by MURST PRIN, Rome, Italy. LC–MS

experiments were performed at ‘Centro di Servizi Inter-

dipartimentale di Analisi Strumentale’, Universita degli

Studi di Napoli ‘Federico II’. The assistance of the staff is

gratefully appreciated.

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