Unique Toxin Profile of a Mediterranean Ostreopsis cf. ovata Strain:HR LC-MSn Characterization of Ovatoxin-f, a New Palytoxin CongenerPatrizia Ciminiello,† Carmela Dell’Aversano,*,† Emma Dello Iacovo,† Ernesto Fattorusso,†

Martino Forino,† Luciana Tartaglione,† Cecilia Battocchi,§ Rita Crinelli,‡ Elisa Carloni,‡ Mauro Magnani,‡

and Antonella Penna§

†Department of Chemistry of Natural Products, University of Napoli Federico II, via D. Montesano 49, Napoli 80131, Italy‡Section of Biochemistry and Molecular Biology and §Section of Environmental Biology, Department of Biomolecular Sciences,University of Urbino, via A. Saffi 2, Urbino (PU) 61029, Italy

*S Supporting Information

ABSTRACT: Currently, the benthic dinoflagellate Ostreopsis cf.ovata represents a serious concern to human health in the wholeMediterranean basin due to the production of palytoxincongeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b,-c, -d/-e), listed among the most potent marine toxins. Highresolution liquid chromatography−mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain ofOstreopsis cf. ovata collected at Portonovo (Italy) in 2008 isreported herein. Toxin profile was different from those previouslyreported for other O. cf. ovata, both qualitatively andquantitatively. For the first time, ovatoxin-a did not dominatethe toxin profile, and a new palytoxin congener, here namedovatoxin-f, was detected. Ovatoxin-f and its elemental formulapresent C2H4 more than ovatoxin-a. HR CID MSn experimentsallowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region notpreviously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile ofthe analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of theMediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, thepresence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.

■ INTRODUCTIONTropical and subtropical benthic dinoflagellates belonging togenus Ostreopsis have been detected in temperate waters such asthose of the Mediterranean Sea since the late ‘70s.1,2 However,only in the past decade have blooms of O. confronta (cf.) ovataand less frequently of O. cf. siamensis been occurring along theMediterranean coasts of Europe and North Africa and theAtlantic coasts of Portugal3−5 resulting in benthic biocenosissuffering and associated human health effects. The mostextensive sanitary events associated with Ostreopsis bloomsoccurred in Italy in 20016 and 2005,7 in Spain in 2004 and2006,8,9 in France in the period 2006−2009,10,11 and in Croatiain 2010 (Pfannkuchen M. et al., personal communication).During these events, up to 200 people exposed to marineaerosols during recreational or working activities requiredmedical attention due to symptoms such as fever, cough,dyspnea, sore throat, rhinorrhea, etc.12,13 High concentration ofOstreopsis spp. (in most cases O. cf. ovata) were recorded inseawater both using traditional and innovative molecular

methods,14 and symptoms in humans disappeared as bloomintensity decreased.12

To date, resolving the taxonomy of Ostreopsis species basedonly on morphology has been difficult due to themorphological variability of both field material and culturedspecimens.15 Further, none of the original isolates from tropicalareas from which species of Ostreopsis species were describedhas yet been sequenced for the genotype assignment. Giventhat for many dinoflagellate genera there is a discrepancybetween morphology-based taxonomy and genetic studies,15

the species names applied to strains of different genetic lineagesmust be treated with caution. It is therefore advisable to use thedesignation O. cf. ovata when referring to isolates from theMediterranean sea.Liquid chromatography−mass spectrometry (LC-MS) stud-

ies on O. cf. ovata demonstrated it to produce several congenersof palytoxin (Figure 1), one of the most potent nonprotein

Received: February 27, 2012Published: April 13, 2012

Article

pubs.acs.org/crt

© 2012 American Chemical Society 1243 dx.doi.org/10.1021/tx300085e | Chem. Res. Toxicol. 2012, 25, 1243−1252

marine toxins known so far16 as well as one of the mostcomplex nonpolymeric water-soluble molecules ever de-scribed;17,18 particularly, a putative palytoxin (pPLTX)19 andseveral palytoxin analogues named ovatoxins (OVTX-a, -b, -c,-d, and -e)20,21 were identified in the O. cf. ovata toxin profilebased on a comparison of their HR LC-MS data with those ofpalytoxin, namely, (i) retention times, (ii) molecular formuladeduced by a cross-check of singly, doubly, and triply chargedions of each compound, and (iii) elemental composition offragment ions resulting from the favored cleavage between C-8and C-9 of palytoxin-like molecules. A recent NMR studyelucidated the planar structure of ovatoxin-a (Figure 1) anddemonstrated it to be the 42-hydroxy-17,44,64-trideoxypalytoxin.22

Toxin profiles of a number of field samples and cultured O.cf. ovata strains we have analyzed so far (Table 1) were alldominated by OVTX-a, which accounted for up to 89% of thetotal toxin content, followed by OVTX-b, OVTX-d+e, OVTX-c, and pPLTX. Only in two O. cf. ovata strains were some

minor components of the toxin profiles completely lacking,namely, OVTX-b and -c22 and pPLTX,23 respectively, but stillOVTX-a was the major component of the toxin profile.The environmental conditions appear to be one of the main

factors determining the proliferation of Ostreopsis species.24−26

However, laboratory studies on the effect of environmentalparameters on the growth and toxicity of O. cf. ovata are ratherscarce. Recent investigations have demonstrated that therelative abundance of ovatoxin types in a North West AdriaticO. cf. ovata isolate are not affected by temperature, salinity, ornutrient conditions which are known, by contrast, to influenceOstreopsis spp. proliferation and toxin production.In the present study, we report on HR LC-MS analysis of a

North West Adriatic O. cf. ovata strain collected at Portonovo(Italy) during a bloom in 2008. This O. cf. ovata strain CBA2-122 revealed a toxin profile that was different both qualitativelyand quantitatively than those previously reported; for the firsttime, OVTX-a did not dominate the toxin profile and a new

Figure 1. Planar structure of palytoxin (PLTX) and ovatoxin-a (OVTX-a). On the basis of HR LC-MSn data, ovatoxin-f (OVTX-f) presents the samestructural features as ovatoxin-a in the region stretching from the A-side terminal to C-94 and from C-103 to the B-side terminal with an additionalC2H4 in the region C-95 to C-102. Cleavages emerging from HR CID MSn spectra of PLTX, OVTX-a, and OVTX-f are reported in the structure andin scheme below. Relevant ion assignments are reported in Tables 3 and 4.

Chemical Research in Toxicology Article

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palytoxin congener, ovatoxin-f, was detected; the HR LC-MSn

based characterization of ovatoxin-f is presented herein.

■ MATERIALS AND METHODSCultures of O. cf. ovata CBA2-122. The O. cf. ovata strain

CBA2-122 was originally isolated from a field sample collected atPortonovo on October 2008 during a bloom of O. cf. ovata. PortonovoBay is a rocky beach with stones and artificial barriers along the beach,located in the Marche Region (Ancona), in the central West Adriaticcoast of Italy. During the bloom of O. cf. ovata local newspapers andbroadcast news reported cases of airborne syndrome in a few peopleattending the beaches, such as swimmers and divers, but not infishermen nor in operators who sampled Ostreopsis. In addition, noofficial source of information about cases of irritation to eyes, cough,and fever were reported, and the information was obtained frompeople visiting the rocky beach. Cultures were grown in 1 L glassbottles containing 0.6 L sterilized f/4 medium27 with an initial cellamount of 3.0 × 104 cells. The temperature was set at 23 ± 1 °C. Lightwas provided by cool white fluorescent bulbs (photon flux of 100 μEm−2 s−1) on a 14−10 h light dark cycle. Culture samples were fixedwith Lugol’s iodine28 and counted using the Utermhol method.29 Atotal volume of 8 L of culture was harvested by centrifugation at 4,000rpm for 15 min at room temperature; a pellet containing 3.0 × 106

cells was stored at −80 °C until chemical analysis.Taxonomic Molecular Identification by a PCR Based Assay.

Genomic DNA was extracted from a 10 mL culture of O. cf. ovataCBA2-122 in logarithmic growth phase using DNeasy Plant Kit(Qiagen, Valencia, CA, USA), according to the manufacturer’sinstructions. PCR amplification using genus- and species-specificprimers was performed as described in Penna et al.30 and Battocchi etal.31

Extraction. The O. cf. ovata cell pellet (3.0 × 106 cells) wasextracted by adding 20 mL of methanol/water (1:1 v/v) and 0.2%acetic acid and sonicating for 30 min in pulse mode, while cooling inan ice bath. The mixture was centrifuged at 3000g for 30 min, thesupernatant was decanted, and the pellet was washed twice more with20 mL of methanol/water (1:1, v/v) and 0.2% acetic acid. The extractswere combined, and the volume was adjusted to 60 mL with extractingsolvent. A 250 μL aliquot of the crude extract was filtered through a0.45 μm filter and directly analyzed by high resolution liquidchromatography−mass spectrometry (HR LC-MS).

HR LC-MS and MSn Experiments. HR LC-MS and MSn analyseswere performed on a hybrid linear ion trap LTQ Orbitrap XL Fouriertransform mass spectrometer (FTMS) equipped with an ESI IONMAX source (Thermo-Fisher, San Jose, CA, USA) and coupled to anAgilent 1100 LC binary system (Palo Alto, CA, USA) including asolvent reservoir, online degasser, binary pump, and thermostatedautosampler. A 3 μm Gemini C18 column (150 × 2.00 mm;Phenomenex, Torrance, CA, USA) was eluted at 0.2 mL/min withwater (eluent A) and 95% acetonitrile/water (eluent B), bothcontaining 30 mM acetic acid (control conditions). A gradient elution(20−50% B over 20 min, 50−80% B over 10 min, 80−100% B in 1min, and held for 5 min) was used. Injection volume was 5 μL.

HR full MS experiments (positive ions) were acquired either in therange m/z 800−1400 or m/z 2000−3000 at resolution setting 30.000to 100.000. The following source settings were used: spray voltage = 4kV, capillary temperature = 290 °C, capillary voltage = 45 V, sheath gas= 35 and auxiliary gas = 1 (arbitrary units), and tube lens voltage = 165V (m/z 800−1400) or 250 V (m/z 2000−3000).

HR collision induced dissociation (CID) MS2 experiments wereacquired on [M + H + Ca]3+ ions of palytoxin and ovatoxins (Table 2)by using 25% collision energy (CE). HR CID MS3 and MS4

experiments were carried out on m/z 905.5 > 327.1 and m/z 905.5> 327.1 > 309.1 of ovatoxin-f at CE = 25% and 20%, respectively. A60000 resolving power, an activation Q of 0.250, and an activationtime of 30 ms were used in all cases.

Calculation of elemental formulas in full MS and CID MSn spectrawas performed by using the monoisotopic ion peak of each ion cluster.T

able

1.Percentage(%

)andCon

tent

onaPer

CellBasis(pg/Cell)of

PutativePalytoxin

(pPLT

X)andOvatoxins

(OVTX-a,-b,-c,-d+-e,and-f)Con

tained

inFieldand

CulturedO.cf.ovataStrainsAnalyzedSo

Fara

pPLT

XOVTX-a

OVTX-b

OVTX-c

OVTX-d+-e

OVTX-f

cultured/

field

samplingsite

(sea,region,

country),d

ate(code)

%pg/cell

%pg/cell

%pg/cell

%pg/cell

%pg/cell

%pg/

cell

cultured

Portonovo(Adriatic,M

arche,Italy),2

008(C

BA2-122)

0.3

0.1

238

176

2.4

0.8

6.7

250

17cultured

Num

ana(Adriatic,M

arche,Italy),2006(O

OAN0601)2

1,22,25,26

0.6

0.2

5418

279

62

124

ndnd

field

Ancona(Adriatic,M

arche,Italy),2

0093

7nd-3

nd-2

55−65

8−49

14−20

2−15

nd-6

nd-4

15−21

3−13

ndnd

field

Trieste(Adriatic,F

riuliVeneziaGiulia,Italy),2009

23nd

nd77−89

45−64

5−14

4−8

nd-1

nd-0.7

4−9

3−5

ndnd

field

Rovinji(Adriatic,C

roatia),2010

(M.P

fannkuchen,p

ersonalcommun.)

0.6−

0.9

0.2−

0.7

58−64

14−46

18−25

5−13

4−5

0.9−

210−12

2−8

ndnd

cultured

Num

ana(Adriatic,M

arche,Italy),2008(O

OAN0816)2

23

77nd

nd20

ndnd

cultured

Num

ana(Adriatic,M

arche,Italy),2007(O

OAN0709)2

2Bari(Adriatic,

Puglia,Italy),2008

(OOAB0801)2

2Latin

a(T

yrrhenian,

Lazio,

Italy),

2007

(OOTL0

707)

22

1−4

53−57

24−26

4−5

12−14

ndnd

and

=notdetected.

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A mass tolerance of 5 ppm was used, and the isotopic pattern of eachion cluster was considered.Extracted ion chromatograms (XIC) were obtained from the HR

full MS spectra by selecting the most abundant and the monoisotopicion peaks of the [M+2H−H2O]

2+ and [M+H+Ca]3+ ion clusters ofeach compound (Table 2). A mass tolerance of 5 ppm was used. Thechromatographic peaks were identified by comparing their retentiontimes and associated HR full MS and CID MS2 spectra to those ofOVTX-a, OVTX-b, OVTX-c, OVTX-d and OVTX-e, and putativePLTX contained in a reference O. cf. ovata extract (OOAN0601)previously characterized21 and analyzed under the same experimentalconditions. In quantitative analyses (triplicate injection), peak areaswere measured and interpolated within the calibration curve ofpalytoxin standard (Wako Chemicals GmbH, Neuss, Germany) at fivelevels of concentrations (50, 25, 12.5, 6.25, and 3.13 ng/mL). Linearityof the calibration curve was indicated by a correlation coefficient (R2)of 0.9980. Because of the lack of standards for ovatoxins, their molarresponses were assumed to be the same as that of palytoxin, on thebasis of structural similarities between palytoxin and ovatoxins. Theobtained results were corrected based on recovery percentage of theextraction procedure (98%).20 Measured limit of quantitation (LOQ)and limit of detection (LOD) for the palytoxin standard under the LC-MS conditions used were 3.13 and 1.9 ng/mL, respectively.

■ RESULTS

Molecular Analysis. PCR amplification based assay of theOstreopsis sp. CBA2-122 strain using genus- and species-specificprimers confirmed the O. cf. ovata genotype identification.Toxin Profile of O. cf. ovata CBA2-122. Crude extract of

the cultured O. cf. ovata strain CBA2-122 from Portonovo(Italy) was analyzed by HR LC-MS in positive full MS and CIDMSn modes versus both a palytoxin standard and a previouslycharacterized culture extract of Adriatic O. cf. ovata(OOAN0601), which was used as reference in containing apPLTX and all the ovatoxins known so far, namely, OVTX-a,-b, -c, d-, and -e.21

Figure 2 shows the total ion chromatogram (TIC) of theCBA2-122 extract and extracted ion chromatograms (XIC) ofthe principal components of the toxin profile obtained selectingthe [M+2H−H2O]

2+ and [M+H+Ca]3+ ions of each com-pound. Recently, we demonstrated that palytoxin and ovatoxinstend to form adducts with various divalent cations and, aboveall, with calcium, which results in triply charged [M+H+Ca]3+

ions dominating their MS spectra.32 Chromatographic separa-tion was not complete; however, the use of a high resolutionMS setting (60.000) and a mass tolerance of 5 ppm in ionassignment, as well as the further confirmation provided by HRCID MSn experiments, avoided ambiguity in the identificationof individual compounds.Toxins eluting under the minor peak of the TIC were

identified as pPLTX, OVTX-b, -c, d-, and -e by comparison oftheir full MS and CID MS2 spectra with those previouslyreported for the reference sample (OOAN0601).21 Analysis offull MS spectrum associated with the most abundant peak of

the TIC (at 11.30 min) highlighted the presence of two sets ofions of different relative intensity, each including singly-,doubly-, and triply charged ions of a palytoxin-like compound:the first set of ions could be assigned to ovatoxin-a (Figure 3a),while the second and more intense set of ions suggested thepresence in the extract of a new palytoxin congener elutingalmost at the same retention time as ovatoxin-a (11.32 min vs11.24 min, respectively); we named it ovatoxin-f (Figure 3b).Because of chromatographic overlapping of OVTX-a and -f, theMS spectrum of each compound unavoidably contains lessintense ions belonging to the other compound.As recently reviewed by Ciminiello et al,33 the high number

of multiply charged ions contained in electrospray (ESI) MSspectra of palytoxin-like compounds, including protonated ions,adducts with monovalent (Na+, K+) and divalent (Ca2+, Mg2+,etc.) cations,32 and ions due to multiple water losses, may leadto ambiguity in assigning true molecular weights to newanalogues. High resolution MS is a prerequisite, but, even using

Table 2. Elemental Formula, Retention Times, and Principal Ions (m/z) of HR Full MS Spectra of Putative Palytoxin (pPLTX)and Ovatoxins (OVTX-) Used in Quantitative Analyses

pPLTX OVTX-a OVTX-b OVTX-c OVTX-d/-e OVTX-f

formula C129H223N3O54 C129H223N3O52 C131H227N3O53 C131H227N3O54 C129H223N3O53 C131H227N3O52

Rt (min) 10.56 11.24 10.98 10.65 10.89 11.32[M+2H−H2O]

2+ most intense 1331.7436 1315.7498 1337.7623 1345.7584 1323.7456 1329.7650monoisotopic 1331.2417 1315.2480 1337.2595 1345.2566 1323.2439 1329.2624

[M+H+Ca]3+ most intense 906.8167 896.1572 910.8318 916.1628 901.4884 905.4976monoisotopic 906.4851 895.8255 910.4976 915.8286 901.1533 905.1632

Figure 2. (a) Total ion chromatogram (TIC) of the cultured O. cf.ovata CBA2-122 and (b) extracted ion chromatograms (XIC) of theprincipal components of the toxin profile.

Chemical Research in Toxicology Article

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Figure 3. HR full MS spectra of ovatoxin-a (a) and ovatoxin-f (b) detected in the cultured O. cf. ovata CBA2-122. In each spectrum, triply, doubly,and singly charged ions appear in the mass range m/z 860−920, m/z 1290−1380, and m/z 2580−2700, respectively. Because of thechromatographic overlapping of OVTX-a and -f, the MS spectrum of each compound contains less intense ions (marked with an asterisk) belongingto the other compound.

Figure 4. HR CID MS2 spectrum of the [M+H+Ca]3+ ion of ovatoxin-f at m/z 905.5. The most abundant peak of each ion cluster is shown togetherwith cleavage numbers to which each fragment is relevant. Ion assignments are reported in Tables 3 and 4, while cleavages are shown in Figure 1.Mass expansions of selected regions of the spectrum are reported in Supporting Information.

Chemical Research in Toxicology Article

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Table

3.Assignm

entof

A-andB-SideFragments

Con

tained

inHRCID

MSn

Spectraof

Palytoxin,Ovatoxin-a,andOvatoxin-fto

RelevantCleavages

(Clv)a

palytoxin

ovatoxin-a

ovatoxin-f

Aside

Bside

Aside

Bside

Aside

Bside

Clv

m/z

(1+,2+

,+3)

form

ula,Δ

ppm

m/z

(1+,2+

,+3)

form

ula,Δppm

m/z

(1+,2+

,+3)

form

ula,Δ

ppm

m/z

(1+,2+

,+3)

form

ula,Δppm

m/z

(1+,2+

,+3)

form

ula,Δ

ppm

m/z

(1+,2+

,+3)

form

ula,Δppm

#2d

127.0867

(1+)

C6H

11ON

2,0.869

127.0866

(1+)

C6H

11ON

2,0.082

127.0867

(1+)

C6H

11ON

2,0.869

#3c,d

281.1861

(1+)

C15H

25O

3N2,0.465

281.1862

(1+)

C15H

25O

3N2,0.821

281.1860

(1+)

C15H

25O

3N2,0.110

#4b

327.1908

(1+)

(−1H

2O)

C16H

27O

5N2,−1.982

1187.1212(2+)

C113H

195O

48NCa,−1.664

327.1908

(1+)

(−1H

2O)

C16H

27O

5N2,−1.982

1171.1261(2+)

C113H

195O

46NCa,−1.845

327.1920

(1+)

(−1H

2O)

C16H

27O

5N2,1.686

1185.1437(2+)

C115H

199O

46NCa,−0.178

791.7502

(3+)

C113H

196O

48NCa,−1.274

781.0867

(3+)

C113H

196O

46NCa,−1.578

790.4315

(3+)

C115H

200O

46NCa,−0.252

#11b

446.2214

(2+)

C40H

72O

17N

2Ca,

−2.009

438.2239

(2+)

C40H

72O

16N

2Ca,

−2.143

438.2250

(2+)

C40H

72O

16N

2Ca,

0.367

#12b

544.2943

(2+)

C52H

92O

19N

2Ca,

−2.134

807.8892

(2+)

(−3H

2O)

C77H

125O

32NCa,−0.968

536.2967

(2+)

C52H

92O

18N

2Ca,

−2.431

799.8912

(2+)

(−3H

2O)

C77H

125O

31NCa,−1.656

536.2980

(2+)

C52H

92O

18N

2Ca,

−0.007

813.9080

(2+)

(−3H

2O)

C79H

129O

31NCa,−0.215

#13b

566.3074

(2+)

(−1H

2O)

C54H

96O

20N

2Ca,−2.064

803.8868

(2+)

(−1H

2O)

C75H

125O

33NCa,−0.795

566.3074

(2+)

(−1H

2O)

C54H

96O

20N

2Ca,−2.064

778.8858

(2+)

(−2H

2O)

C75H

123O

30NCa,−1.852

566.3085

(2+)

(−1H

2O)

C54H

96O

20N

2Ca,−0.121

792.9035

(2+)

(−2H

2O)

C77H

127O

30NCa,0.766

#14b

572.3074

(2+)

C55H

96O

20N

2Ca,

−2.042

797.8864

(2+)

C74H

125O

33NCa,

−1.303

ndnd

ndnd

#15b

596.3183

(2+)

C56H

100O

22N

2Ca,

−1.397

782.8815

(2+)

C73H

123O

32NCa,

−0.839

588.3204

(2+)

C56H

100O

21N

2Ca,

−2.169

774.8833

(2+)

C73H

123O

31NCa,

−1.807

588.3217

(2+)

C56H

100O

21N

2Ca,

−0.041

788.9003

(2+)

C75H

127O

31NCa,

−0.063

1526.8081(1+)

C73H

124O

32N,

−1.308

1510.8129(1+)

C73H

124O

31N,

−1.510

1538.8449(1+)

C75H

128O

31N,

−1.028

#16b

633.3363

(2+)

C59H

106O

24N

2Ca,

−1.931

745.8630

(2+)

C70H

117O

30NCa,

−1.029

625.3387

(2+)

C59H

106O

23N

2Ca,

−2.184

737.8650

(2+)

C70H

117O

29NCa,

−1.776

625.3401

(2+)

C59H

106O

23N

2Ca,

0.055

751.8821

(2+)

C72H

121O

29NCa,

0.186

1452.7717(1+)

C70H

118O

30N,

−1.113

1436.7765(1+)

C70H

118O

29N,

−1.324

1464.8087(1+)

C72H

122O

29N,

−0.684

#17b

647.3343

(2+)

C60H

106O

25N

2Ca,

−1.051

1406.7657(1+)

C69H

116O

28N,

−1.520

639.3361

(2+)

C60H

106O

24N

2Ca,

−2.226

1390.7711(1+)

C69H

116O

27N,

−1.311

639.3375

(2+)

C60H

106O

24N

2Ca,

−0.036

1418.8027(1+)

C71H

120O

27N,

−1.074

#18b

694.8289

(2+)

C65H

107O

28NCa,

−1.191

686.8319

(2+)

C65H

107O

27NCa,

−0.539

700.8485

(2+)

C67H

111O

27NCa,

0.827

#19b

948.4986

(2+)

C90H

156O

37N

2Ca,

−1.575

804.4369

(1+)

C39H

66O

16N,

−0.884

932.5036

(2+)

(−1H

2O)

C90H

156O

35N

2Ca,−1.693

804.4365

(1+)

C39H

66O

16N,

−1.381

932.5054

(2+)

(−1H

2O)

C90H

156O

35N

2Ca,0.237

832.4687

(1+)

C41H

70O

16N,

−0.254

#20e

744.4136

(1+)

C37H

62O

14N,

−3.871

ndnd

ndnd

#21b

1129.5956(2+)

(−3H

2O)

C107H

186O

45N

2Ca,−1.352

406.2218

(1+)

C22H

32O

6N,

−1.512

1113.6007(2+)

(−3H

2O)

C107H

186O

43N

2Ca,−1.359

406.2215

(1+)

C22H

32O

6N,

−2.250

1113.6016(2+)

(−3H

2O)

C107H

186O

43N

2Ca,−0.550

nd

#22b

1144.6023(2+)

C108H

188O

46N

2Ca,−0.096

1128.6057(2+)

C108H

188O

44N

2Ca,−1.591

1128.6052(2+)

C108H

188O

44N

2Ca,

−2.034

#23b

1174.6132(2+)

C110H

192O

48N

2Ca,−0.192

1158.6154(2+)

C110H

192O

46N

2Ca,−2.296

nd

#24b

1215.6328(2+)

(−1H

2O)

C115H

198O

49N

2Ca,−0.911

1199.6333(2+)

(−1H

2O)

C115H

198O

47N

2Ca,−4.745

nd

#25b

1222.6416(2+)

(−1H

2O)

C116H

200O

49N

2Ca,−0.108

1206.6488(2+)

(−1H

2O)

C116H

200O

47N

2Ca,1.643

1220.6630(2+)

(−1H

2O)

C118H

204O

47N

2Ca18.0,0

.436

#26b

1235.6482(2+)

(−1H

2O)

C118H

202O

49N

2Ca,−1.098

1219.6512(2+)

(−1H

2O)

C118H

202O

47N

2Ca,−2.822

1233.6681(2+)

(−1H

2O)

C120H

206O

47N

2Ca19.0,−

1.777

#27b

1236.6328(2+)

C117H

200O

50N

2Ca,−5.167

1220.6386(2+)

(−1H

2O)

C117H

200O

48N

2Ca,−4.649

1234.6559(2+)

(−1H

2O)

C119H

204O

48N

2Ca,−3.260

#28

1321.6819(2+)

C126H

214O

53N

2Ca,−3.357

ndnd

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a mass tolerance of 5 ppm, several plausible elemental formulasfor the monoisotopic ion peaks are possible due to the highvalues of the masses of palytoxin-like compounds associatedwith the high number of C, H, N, and O atoms contained intheir elemental formulas. This drawback can be overcomethrough a combined analysis of all the singly, doubly, and triplycharged ions contained in HR full MS spectra. Therefore,combining assignment of the most abundant singly, doubly, andtriply charged ions of full MS spectra of OVTX-f, namely, [M+H]+ at m/z 2676.5122 (monoisotopic at m/z 2675.5283,C131H228N3O52, Δ = −0.015 ppm), [M+H+K]2+ at m/z1357 . 7 4 78 (mono i s o t op i c a t m / z 1357 . 2 4 22 ,C131H228N3O52K, Δ = −2.615 ppm), [M+2H-1H2O]

2+ at m/z 1329.7640 (monoisotopic at m/z 1329.2624, C131H227N3O51,Δ = −0.095 ppm), [M+H+Ca]3+ at m/z 905.5003 (mono-isotopic at m/z 905.1632, C131H228N3O52Ca, Δ = −0.086ppm), and [M+H+Mg]3+ at m/z 900.1746 (monoisotopic atm/z 899.8364 C131H228N3O52Mg, Δ = −1.143 ppm), theelemental formula C131H227N3O52 was assigned to ovatoxin-f.Compared to OVTX-a (C129H223N3O52), the OVTX-felemental formula contains more C2H4 than that of OVTX-a.In order to individuate the region of the molecule where

structural differences between OVTX-a and -f occur, HR LC-MSn spectra of OVTX-f were acquired and analyzed in parallelto those of OVTX-a whose structure was recently determinedthrough NMR.22 In this regard, we recently demonstrated thatmuch structural information can be obtained from the positiveHR MSn spectra of palytoxin and its congeners, as they undergocharacteristic fragmentation at several sites of their backbone.32

Therefore, a comparative analysis of the fragmentation patternof OVTX-f versus that of OVTX-a was accomplished to gainstructural information on ovatoxin-f. In this comparative study,the [M+H+Ca]3+ ion at m/z 905.5 (ovatoxin-f) was used as theprecursor in MS2 experiments, and some of the resultingproduct ions were fragmented within MS3 and MS4 experi-ments.Figure 4 shows the HR CID MS2 spectrum of OVTX-f.

Tables 3 and 4 contain assignments of all the ions of HR CIDMSn spectra of ovatoxin-f to relevant cleavages shown in Figure1 in comparison to those previously reported for ovatoxin-a andpalytoxin, whose standard is commercially available.32 It has tobe noted that, under the used conditions, fragmentation occursat several sites of each palytoxin-like molecule generating foreach cleavage A-side fragments (containing 2 N atoms) and/orB-side fragments (containing 1 N atom) (Table 3). Each A-sideand B-side fragment can appear in MSn spectra as a singly and/or doubly charged ion and is followed by 1 to 7 ions due tosubsequent water losses (mass expansions are shown in FigureS1, Supporting Information); in some cases ions, correspondingto 1−3 water losses from the A- or the B-side fragments are theonly ones that can be detected; a very limited number of ionsare protonated fragments, whereas most of them are calcium-containing fragments. In addition, some internal fragments dueto a combination of two cleavages also occur, containingneither the A-side nor the B-side terminal of the molecule andthus having no N atom in their elemental formula (Table 4).With the only exception of the internal fragment due tocleavage #1 + #4 (Figure 1) which provides information on thepart of the structure ranging from C-8 to the A-side terminal ofthe molecule, all the other internal fragments originate from theinner portion of the molecule stretching from C-9 to C-48(Figure 1), namely, from the B-side fragment of cleavage #4after further fragmentation at cleavage sites #12, #13, #15, andT

able

3.continued

aElem

entalformulae

ofthemono-isotopicionpeaks(m

/z)arereported

with

ioncharge

state(1+,

2+,or3+

)anderrors(Δ

,ppm

).nd

=notdetected.bIons

intheHRCID

MS2

spectraof

the[M

+H+C

a]3+ionofpalytoxin(m

/z906.8),ovatoxin-a(m

/z896.2),and

ovatoxin-f(m

/z905.5),respectively.c IonsintheHRCID

MS3

spectraofpalytoxin(m

/z906.8>327.1),ovatoxin-a(m

/z896.2>327.1),

andovatoxin-f(m

/z905.5>327.1),respectively.dIons

intheHRCID

MS4

spectraofpalytoxin(m

/z906.8>327.1>309.1),ovatoxin-a(m

/z896.2>327.1>309.1),and

ovatoxin-f(m

/z905.5>327.1>

309.1),respectively.e Ionsin

theHRCID

MS2

spectraof

the[M

+2H−H

2O]2

+ionof

palytoxin(m

/z1331.7).

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#16 and from the A-side fragment of cleavage #12 after furtherfragmentation at cleavage sites #5, #7, #8, #9, and #10.Fragmentation pattern of ovatoxin-f closely paralleled that of

ovatoxin-a, suggesting that they share the same backbone;however, there were some key clues in the spectra thatindicated the region where structural differences between thetwo compounds occur. Such information could be obtained bycomparing elemental formulas of fragments of ovatoxin-a withthose of ovatoxin-f for each cleavage (Tables 3 and 4).In the part of the structure ranging from C-1 to C-94 (Figure

1), cleavages occurred at the same sites in both ovatoxin-a andovatoxin-f and generated A-side and internal fragments havingthe same elemental composition in both compounds (Tables 3and 4) as well as B-side fragments presenting C2H4 more inOVTX-f than the relevant fragments of OVTX-a. Thissuggested that ovatoxin-f and ovatoxin-a share the part of thestructure stretching from the A-side terminal to C-94, and thus,the structural difference between them is located in the regionranging from C-95 to the B-side terminal.A comparative analysis of fragmentation occurring in the part

of the structure close to the B-side terminal of the moleculesallowed us to restrict the region of structural difference betweenOVTX-a and OVTX-f to the region stretching from C-95 to C-102. In fact, the fragmentation pattern of the two molecules wasdifferent in this region: fragments due to cleavages #23 and #24in OVTX-a were lacking in OVTX-f, while the A-side fragmentdue to cleavage #25 (between C-102 and C-103), as well asthose due to cleavages #26, and #27, contained C2H4 more inOVTX-f than in OVTX-a.It has to be noted that within palytoxin analogues,22,34−36

including ovatoxin-a, the region close to the B-side terminal hasnever been described to have structural modifications. Inaddition, considering that, compared to palytoxin, ovatoxin-alacks the hydroxyl groups at C-17 and at C-64 and presents anadditional hydroxyl group at C-42, the whole of the obtainedresults suggest that the same structural features could bepresent in ovatoxin-f.For quantitative analysis, extracted ion chromatograms

(Figure 2b) were obtained from full MS spectrum of the

crude extract by selecting the most abundant bi- and trichargedions of ovatoxins (a−f) and palytoxin (Table 2). Because of thelack of pure standards of ovatoxins, their molar response wasassumed to be the same as palytoxin’s, and the calibration curveof palytoxin at four levels of concentration was used toextrapolate quantitative data. Total toxin content of the O. cf.ovata CBA2-122 strain was thus calculated to be 34 pg/cell.Ovatoxin-f resulted in being the major component of the toxinprofile (17 pg/cell, 50%) followed by ovatoxin-a (8 pg/cell,23%), ovatoxin-b (6 pg/cell, 17%), ovatoxin-c (0.8 pg/cell,2.4%), ovatoxin-d+e (2 pg/cell, 6.7%), and putative palytoxin(0.1 pg/cell, 0.3%). This is a unique toxin profile whencompared to the toxin profiles of most O. cf. ovata strains wehave analyzed so far (Table 1) because for the first time,ovatoxin-a does not represent the major component of thetoxin profile.The Ostreopsis isolate CBA2-122 was genetically identified as

O. cf. ovata, clade Atlantic/Mediterranean.3 Thus, the differentovatoxin profiles found among different isolates of O. cf. ovatacould be related to the different haplotypes within the Atlantic/Mediterranean clade based on the concatenated ITS regions,5.8S and LSU ribosomal genes. Studies are in progress in ourlaboratories to investigate this issue.Since the O. cf. ovata CBA2-122 extract was unique in

containing all the ovatoxins known so far, we believed itopportune to evaluate its bioactivity. The obtained results havebeen recently reported (Crinelli et al., unpublished) andshowed that the toxin extract was able to induce an increase inthe levels of mRNAs encoding inflammation-related proteins inprimary human macrophages via signaling through tran-scription factor-κB (NF−κB) and p38 MAPK (mitogenactivated protein kinases). This is the first report providingthe molecular basis of a direct correlation between ovatoxincellular activity and acute inflammatory reactions recorded inindividuals exposed to Ostreopsis species through inhalation orskin contact. In addition, the fact that the overall toxinscontained in the extract, although at a 10-fold higherconcentration, displayed an activity totally overlapping that ofthe palytoxin standard might suggest that chemically similar but

Table 4. Assignment of the Internal Fragments Contained in HR CID MSn Spectra of Palytoxin, Ovatoxin-a, and Ovatoxin-f toRelevant Cleavages (Clv)a

palytoxin ovatoxin-a ovatoxin-f

internal fragment internal fragment internal fragment

Clv m/z (1+,2+,3+) formula, Δ ppm m/z (1+,2+,3+) formula, Δ ppm m/z (1+,2+,3+) formula, Δ ppm

#1 + #4c,d 234.1127 (1+) C13H16O3N, 0.983 234.1123 (1+) C13H16O3N, −0.726 234.1126 (1+) C13H16O3N, 0.556#4 + #12b 372.1975 (2+) C36H64O13Ca, −1.593 364.2001 (2+) C36H64O12Ca, −1.471 364.2010 (2+) C36H64O12Ca, 1.000

743.3878 (1+) C36H63O13Ca, −1.492 727.3927 (1+) C36H63O12Ca, −1.780 727.3942 (1+) C36H63O12Ca, 0.282#4 + #13b 394.2105 (2+) C38H68O14Ca, −1.777 394.2104 (2+) C38H68O14Ca, −2.030 394.2113 (2+) C38H68O14Ca, 0.253#4 + #15b 424.2211 (2+) C40H72O16Ca, −1.568 416.2233 (2+) C40H72O15Ca, −2.421 416.2245 (2+) C40H72O15Ca, 0.462#4 + #16b 461.2389 (2+) C43H78O18Ca, −2.720 453.2417 (2+) C43H78O17Ca, −2.201 453.2426 (2+) C43H78O17Ca, −0.215#5 + #12b 657.3514 (1+) C32H57O11Ca, −1.111 641.3559 (1+) C32H57O10Ca, −2.051 641.3575 (1+) C32H57O10Ca, −2.051#6 + #12e 567.3189 (1+) C29H51O8Ca, −2.708 nd nd#7 + #12b 537.3075 (1+) C28H49O7Ca, −4.414 521.3137 (1+) C28H49O6Ca, −2.411 521.3151 (1+) C28H49O6Ca, 0.275#8 + #12b 507.2982 (1+) C27H47O6Ca, −2.182 507.2981 (1+) C27H47O6Ca, −2.379 507.2994 (1+) C27H47O6Ca, 0.184#9 + #12b 477.2878 (1+) C26H45O5Ca, −1.974 477.2868 (1+) C26H45O5Ca, −4.069 477.2888 (1+) C26H45O5Ca, 0.121#10 + #12b 447.2777 (1+) C25H43O4Ca, −1.068 447.2762 (1+) C25H43O4Ca, −4.421 447.2783 (1+) C25H43O4Ca, 0.274

aElemental formulas of the mono-isotopic ion peaks (m/z) are reported with ion charge state (1+, 2+, or 3+) and errors (Δ, ppm). nd = notdetected. bIons in the HR CID MS2 spectra of the [M+H+Ca]3+ ion of palytoxin (m/z 906.8), ovatoxin-a (m/z 896.2), and ovatoxin-f (m/z 905.5),respectively. cIons in the HR CID MS3 spectra of palytoxin (m/z 906.8 > 327.1),ovatoxin-a (m/z 896.2 > 327.1), and ovatoxin-f (m/z 905.5 >327.1), respectively. dIons in the HR CID MS4 spectra of palytoxin (m/z 906.8 > 327.1 > 309.1), ovatoxin-a (m/z 896.2 > 327.1 > 309.1), andovatoxin-f (m/z 905.5 > 327.1 > 309.1), respectively. eIons in the HR CID MS3 spectra of palytoxin (m/z 906.8 > 743.4).

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not identical compounds could exert their toxic effects via thesame signaling pathways. Thus, in light of these findings, itcould be hypothesized that ovatoxins a−f could indeed differnot in the mechanism of action but rather in their potency.Future experiments will be directed toward testing singleovatoxin types when it is possible to purify them in sufficientamounts to be tested in an in vitro cellular system.

■ CONCLUSIONS

A new palytoxin-like compound, ovatoxin-f, has been detectedin a NW Adriatic O. cf. ovata isolate. Its elemental compositioncompared to that of ovatoxin-a presents additional C2H4 andthe whole of the HR CID MSn data suggests that structuraldifferences between molecules reside in the region between C-95 and C-102.Ovatoxin-f represents the major component of the toxin

profile of the analyzed strain accounting for 50% of the totaltoxin content, while ovatoxin-a, the dominant toxin in most ofthe Mediterranean O. cf. ovata strains we have analyzed sofar,21−23,25,26,37 is the second major component of the toxinprofile (23%). Thus, the presence of ovatoxin-f should be takeninto account when monitoring programs for palytoxin-likecompounds in microalgal cells and/or seawater are carried out.The discovery of a new palytoxin congener in theMediterranean Ostreopsis strain is a major proof that we aredealing with an important emerging and evolving toxicologicalproblem with multiple consequences including the possibility ofMediterranean food chain contamination. Toxic effects exertedby single ovatoxin components should be evaluated both interms of the mechanism of action and potency in order toestablish a possible correlation between toxin profiles and riskassessment for human health.

■ ASSOCIATED CONTENT

*S Supporting InformationMonoisotopic ion peaks of all the fragment ions of the HR CIDMS2 spectra of ovatoxin-a and -f, mass expansions of the HRCID MS2 spectrum of ovatoxin-f. This material is available freeof charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Tel: +39-081-678502. Fax: +39-081-678552. E-mail:dellaver@unina.it.

FundingThis work was supported by PRIN 2009 and by ISPRA Grants.

NotesThe authors declare no competing financial interest.

■ ABBREVIATIONS

HR LC-MS, high resolution liquid chromatography−massspectrometry; CID, collision induced dissociation; PLTX,palytoxin; pPLTX, putative palytoxin; OVTX-a, ovatoxin-a;OVTX-b, ovatoxin-b; OVTX-c, ovatoxin-c; OVTX-d+e,ovatoxin-d+e; OVTX-f, ovatoxin-f; FTMS, Fourier transformmass spectrometry; ESI, electrospray ionization; XIC, extractedion chromatogram; LOQ, limit of quantitation; LOD, limit ofdetection; PCR, polymerase chain reaction; ITS, internaltranscribed spacer; LSU, large subunit; MAPK, mitogenactivated protein kinases; O. cf. ovata, Ostreopsis confronta ovata

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Chemical Research in Toxicology Article

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Chemical Research in Toxicology Article

dx.doi.org/10.1021/tx300085e | Chem. Res. Toxicol. 2012, 25, 1243−12521252