In 1989 along the coast of Emilia Romagna…

Mytilus galloprovincialis

Mouse Bioassay for DSP Toxins Intraperitoneal (IP) injection of lipophylic extract of shell- fish digestive glands into g mouse Measurement time to death; one mouse unit = amount that kills mouse in 24 hours Regulatory level: 0.05 MU per g of edible tissue (equivalent to 200 ng/g of okadaic acid) A validated method accepted by several countries Rapid and inexpensive Poor reproducibility and long assay time False positives mostly due to the presence of high levels of fatty acids No information on toxins profile

Pectenotoxins Okadaic acid DSP Toxins: Chemistry and Toxicology Yessotoxins Diarrhea, nausea, vomiting, tumor promoting activity DL50= 192  g/Kg Diarrhea, gastrointestinal distress, epatotoxicity DL50= 260  g/Kg DL50= 100  g/Kg Cardiotoxicity, ?

~200 Kg of mussels hundreds g of extract Fats Sterols Contaminants Toxins (  g) extraction chromatografic separation

NMR spectrum of okadaic acid… Can you spot it??? It was 1989 !!! E. Fattorusso, P. Ciminiello, V. Costantino, S. Magno, A. Mangoni, A.Milandri, R. Poletti, M. Pompei, R. Viviani, Mar. Poll. Bull., 24,234 (1992).

From 1989 on: Okadaic Acid has been the main Adriatic toxin Adriatic toxin profile: chronicle 1989: for the very first time okadaic acid was spotted in the Adriatic Sea

Let’s now bounce into 1995!!!

Protoceratium reticulatum A m u c h b e t t e r s p e c t r u m, i s n ’ t i t ? YESSOTOXIN P. Ciminiello, E. Fattorusso, M. Forino, S. Magno, R. Poletti, M. Satake, R. Viviani and T. Yasumoto, Toxicon, 35, 177 (1997)

Known Yessotoxin analogues Known Yessotoxin analogues 1995: Yessotoxin 1997 : Yessotoxin 1997 : Yessotoxin homoYessotoxin homoYessotoxin 45-hydroxyYessotoxin 45-hydroxyYessotoxin45-hydroxy-homoYessotoxin TOXIC AREA

Adriatoxin (1997) New Yessotoxin analogues n = 1 Carboxy YTX (1997) n = 2 CarboxyhomoYTX (1998) n = 1 OxoYTX (2001) n = 2 OxohomoYTX (1998) P.Ciminiello, E.Fattorusso, M.Forino, R.Poletti and R.Viviani. Eur. J. Org. Chem (2000). P.Ciminiello, E.Fattorusso, M.Forino, R.Poletti and R.Viviani. Chem. Res. Toxicol. 13, (2000). P.Ciminiello, E.Fattorusso, M.Forino e R.Poletti Chem. Res. Toxicol. 14, (2001). P.Ciminiello, E.Fattorusso, M.Forino, S. Magno, R.Poletti, R.Viviani. Tetrahedron Lett. 39, 8897 (1998).

CYTOTOXIC DERIVATIVES FROM MUSSELS OF ADRIATIC SEA CYTOTOXIC DERIVATIVES FROM MUSSELS OF ADRIATIC SEA *IC  g/ml WEHI 164 (murine fibrosarcoma) cell lines IC  g/ml on J774 (murine monocyte/macrophage) cell lines *IC  g/ml WEHI 164 (murine fibrosarcoma) cell lines IC  g/ml on J774 (murine monocyte/macrophage) cell lines P.Ciminiello, C. Dell’Aversano, E.Fattorusso, M. Forino, S. Magno, A. Ianaro, M. Di Rosa Eur.J.Org.Chem (2001).

FURTHER OXAZININES FROM ADRIATIC MUSSELS Ciminiello P., Dell’Aversano C., Fattorusso E., Forino M., Magno S., Santelia F.U., Couladouros E.A., Pitsinos E.N., Moutsos V.I., Tetrahedron, in corso di stampa

CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA IC 50 (  g/ml) P. Ciminiello, M. Di Rosa, E. Fattorusso, M. Forino, A. Ianaro, and R. Poletti. J. Org. Chem. 66(2), (2001).

CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA IC 50 (  g/ml) RAW 274.7: 20  g/ml WEHI 164: 13.5  g/ml 6.2 mg P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno and Poletti J. Am. Chem. Soc. 124, (2002)

2 14 = A FURTHER CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA 2.1 mg P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, M. Di Rosa, A. Ianaro, and R.Poletti, Tetrahedron, 60, 7093 (2004). P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, M. Di Rosa, A. Ianaro, and R.Poletti, Tetrahedron, 60, 7093 (2004). IC 50 (  g/ml) J774: 20  g/ml WEHI 164: 10.4  g/ml

 High variation in toxins composition  New Biotoxins  New Cytotoxins

Monitoring program qualitative analysis quantitative analysis

ripartizione n-e,hexaneMeOH 80% ripartizione MeOH 40% CH 2 Cl 2 LiChroprep RP-8 AcOEtH2OH2O ripartizione MeOH 60% MeOH 80% MeOH 90% MeOH 100% Toyopearl HW-40 Yasumoto test Cytoxicity tests MeOH 50 ml RP-18 H 2 O/MeOH 3:7 Luna RP-18 H 2 O/MeOH/CH 3 CN 4:4:2 OMOYTX CARBOXY-OMOYTX 45OH-OMOYTX Acetone extraction Mytilus galloprovincialis hepatopancreas

Analysis Methods: LC-MS Molecular weight & elemental composition (HRMS) Structural information (MS/MS) Wide range of analytes possible Universal AND selective High sensitivity Excellent quantitation Automated analysis Legally-acceptable confirmation Discovery of new toxins, analogs and metabolites Multiple toxin determinations Molecular weight & elemental composition (HRMS) Structural information (MS/MS) Wide range of analytes possible Universal AND selective High sensitivity Excellent quantitation Automated analysis Legally-acceptable confirmation Discovery of new toxins, analogs and metabolites Multiple toxin determinations

Components of a Tandem Mass Spectrometer Inlet System Sanple Ionization Source Mass Analyzer 1 Collision Cell Mass Analyzer 2 Ion Detector Data Acquisition EI, CI, FAB LD, FD, ESI Sector, Quadrupole, TOF, Ion Trap High Vacuum (10 -5 to torr) Syringe, CE, HPLC

COMPONENTS OF TANDEM MASS SPECTROMETER Inlet system HPLC SOURCE ESI Ion trap DATA

Advantage: High sensitivity in Full Scan Drawback Non linear calibration curves XIC m/z B XIC A B C D TIC MS TIC SELECTIVITY

FSMSYTX [M-Na] - [M-2Na+H] - Temp= 150°C Voltage = -10V Tube Lens off set = -60V Spray Voltage = 4,5KV Hypersil C-8 BDS 14.1  g/ml 4,7  g/ml 1,6  g/ml 0,52  g/ml 0,17  g/ml 0,057  g/ml YTX DL68pg YTX

HPLC-ESI-ion trap-MS Method for Direct Detection of Yessotoxins DL 70 pg P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, and R. Poletti J. Chromat. A, 968, 61 (2002). FINNIGAN LCQ Mass Spectrometer Hypersil 3  m C8 BDS, 50 x 2.0 mm Gradient H 2 O/ACN 2mM AF/50mM FA

XICLC-MS Mytilus galloprovincialis Cesenatico coasts in July 2001 TIC MS FSMS min XIC FSMS min min FSMS 45-OHYTX 45-OHhomoYTX CarboxyYTX CarboxyhomoYTX New YTX YTX P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, and R. Poletti Chem. Res. Toxicol. 15, 979 (2002) min min FSMS XIC

Protoceratium reticulatum as the producing organism of… MeOH (125  L x 3) Ultrafree (6000 rpm, 10 min) + Extract (500  L) SPE NoroxoYTX HomoYTX 45-OHYTX CarboxyYTX YTX LC-multiple tandem MS Load Wash Eluate

Some YTX analogues present in large quantities in contaminated shellfish – 45-hydroxyYTX, carboxyYTX, noroxoYTX - could to derive from oxidative modification could to derive from oxidative modification of the absorbed YTX within the mollusk. Conclusions

From 1989 on: Okadaic Acid has been the main Adriatic toxin Adriatic toxin profile: chronicle 1989: for the very first time okadaic acid was spotted in the Adriatic Sea At the beginning of the new millennium: Okadaic Acid had virtually disappeared In 1995: Yessotoxin was detected for the first time In the Adriatic Sea From 1995 on:Yessotoxins have become the main Adriatic toxins While Okadaic acid has slowly subsided

Anomalous toxicity of Adriatic mussels cultivated in November 2004

ALLOWANCE LEVELS SUGGESTED BY EUTOXIN 100g SHELLFISH OKADAIC ACID 16  g YESSOTOXIN 100  g

Mytilus galloprovincialis hepatopancreas Acetone extraction AcOEt H2OH2O MeOH 80% n-hexane CH 2 Cl 2 MeOH 40% Yasumoto test OKADAIC ACID YESSOTOXINS H2OH2O CH 2 Cl 2 BuOH H2OH2O YESSOTOXINSOKADAIC ACID OLD PROCEDURE NEW PROCEDURE Yasumoto test

In summer 2004 CH 2 Cl 2 phase deriving From Adriatic mussels Was found TOXIC!!! In summer 2004 CH 2 Cl 2 phase deriving From Adriatic mussels Was found TOXIC!!!

Acetoneextract Isolation of the new toxins H2OH2OH2OH2O HW-40 RP-8 MeOH/H 2 O 6:4 ODS 8:2 CH 2 Cl 2. HPLC RP-18 H 2 O/MeOH/CH 3 CN 4:4:2 NEWTOXINA NEW TOXIN B

New Toxin- A: Carboxyhomo-1-desulfoYTX H 2 -1 H-4 H-5 O O O O O O O O O OH Me Me O Me O Me Me HO Me HOOC R'O RO a HHHH H H H H H H H H HHHH H H H A BC D E 1

Toxin A Spin system C-1/C-2a

New Toxin- B : Carboxyhomo-4-desulfoYTX H 2 -1 H-4 H-5 O O O O O O O O O OH Me Me O Me O Me Me HO Me HOOC R'O RO a HHHH H H H H H H H H HHHH H H H A BC D E 4

Toxin B: spin system C-4/C-8

Two new toxins in Adriatic mussels: Desulfohomoyessotoxins P. Ciminiello, C. Dell'Aversano, E. Fattorusso, M. Forino, L. Grauso, S. Magno, R. Poletti, L. Tartaglione Magno, R. Poletti, L. Tartaglione Chem. Res. in Toxicol., 20, 95 (2007) Chem. Res. in Toxicol., 20, 95 (2007)

EU official protocol for extracting Marine Biotoxins needs to be updated What issues do CarboxyhomodesulfoYTXs Raise? CarboxyhomodesulfoYTX toxicology should be investigated Who is responsible for the lack of the sulfate ester in carboxyhomodesulfoYTXs? Mytilus galloprovincialis hepatopancreas Acetone extraction H2OH2O CH 2 Cl 2 BuOH H2OH2O YESSOTOXINSOKADAIC ACID NEW PROCEDURE

In November 2003… Alexandrium ostenfeldii massively bloomed in the Adriatic Sea

spirolides

Extraction of Alexandrium ostenfeldii culture H 2 O:CH 3 CN HPLC Rp-8 H 2 O:CH 3 CN 90% A to 100% B Three major m/z: 678,5 692,5 694,5 MPLC C18 MPLC C18 MeOH: H 2 O 8 : 2

,19-didesmethylspirolideC

desmethylspirolideC

694 P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, L. Grauso, S. G. Magno, R. Poletti, L. Tartaglione. Chem. Res. Toxicol. 20, 95-98, 2007.

ACN/H 2 O 8:2 +0,1% formic acid FiltrateResidue partition ACN/H 2 O n-hexane partition CH 2 Cl 2 ACN/H2O 2:1 dead live Cesenatico and Trieste Cesenatico and Trieste

DOMOIC ACID ?

Domoic Acid Glutamico AcidAspartic cido Confusion Vomiting Amnesia

Optimization of ionization parameters DL DA =123ng/ml r 2 >0.999 SIM (Selected Ion Monitoring) MSMS (Massa Massa) MRM (Multiple Reaction Monitoring) Column:TSK-gel amide 80 Mobil phase: H2O/ACN + 2mM formiato/3.53 mM formic acid Calibration

DL DA =12 ng/ml DL PSP = 5-30 nM r 2 >0.999 (  g/ml) Good sensitivity and linearity C. Dell’Aversano, P. Hess, M. A. Quilliam J. Chromatogr. A, 1081 (2), (2005) P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, G. S. Magno, L. Tartaglione, M. A. Quilliam, A. Tubaro, R. Poletti Rapid Commun. Mass Spectrom., 19, (2005) API-2000 triple quadrupole mass spectrometer equipped mass spectrometer equipped with a turbo-ionspray source with a turbo-ionspray source (Applera, Thornhill, ON, Canada) (Applera, Thornhill, ON, Canada)

Quantitative Analysis m/z 310/266 m/z 310/248 m/z 310/222 MRM- Cesenatico=2.5 µg/g Trieste=0.25 µg/g Limite legislativo= 20µg/g