in 1989 along the coast of emilia romagna…
DESCRIPTION
In 1989 along the coast of Emilia Romagna…. DSP TOXINS. Mytilus galloprovincialis. Mouse Bioassay for DSP Toxins. Intraperitoneal (IP) injection of lipophylic extract of shell-fish digestive glands into 18-20 g mouse - PowerPoint PPT PresentationTRANSCRIPT
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In 1989 along the coast of Emilia Romagna…
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Mytilus galloprovincialis
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Mouse Bioassay for DSP Toxins
• Intraperitoneal (IP) injection of lipophylic extract of shell-fish digestive glands into 18-20 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
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O
OO
OOH
OHO
O O
O
O
OH O
O
Pectenotoxins
O
OH
O
OH
OH
O
CH3
OO
O
OH
OH
O
O
Okadaic acid
DSP Toxins: Chemistry and Toxicology
Yessotoxins
Diarrhea, nausea, vomiting, tumor promoting activity
DL50=192g/Kg
Diarrhea, gastrointestinal distress, epatotoxicity
DL50=260g/Kg
O
O
O
O
O
O
O
O
O
O
NaO3SO
NaO3SO Me
Me
Me
Me
OH
H
H
H H H HH
Me H
H
H
H
HH H
H
H
OH
MeHO
DL50=100g/Kg
Cardiotoxicity, ?
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~200 Kg of mussels~200 Kg of mussels
hundreds g of extracthundreds g of extract
FatsFats SterolsSterols ContaminantsContaminants
Toxins (g)
extractionextraction
chromatografic separationchromatografic separation
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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).
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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
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Let’s
now bo
unce i
nto 19
95!!!
Let’s
now bo
unce i
nto 19
95!!!
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Protoceratium reticulatum
A much
bette
r spec
trum,
isn’t
it?
ppm (f1)1.02.03.04.05.06.0
0.0
5.0
10.0
15.0
O
O
O
O
O
O
O
O
O
O
NaO3SO
NaO3SO Me
Me
Me
Me
OH
H
H
H H H HH
Me H
H
H
H
HH H
H
H
OH
MeHO
YESSOTOXIN
P. Ciminiello, E. Fattorusso, M. Forino, S. Magno, R. Poletti, M. Satake, R. Viviani and T. Yasumoto, Toxicon, 35, 177 (1997)
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Known Yessotoxin analogues
Known Yessotoxin analogues
O
O
O
O
O
O
O
O
O
O
NaO3SO
NaO3SO
O
OH
HO
1995:1995: YessotoxinYessotoxin
O
O
O
O
O
O
O
O
O
O
NaO3SO
NaO3SO
O
R n1
2
2
( )n
1
OH
HO
OH
OH
R
19971997 :: YessotoxinYessotoxin homoYessotoxinhomoYessotoxin 45-hydroxyYessotoxin45-hydroxyYessotoxin45-hydroxy-homoYessotoxin45-hydroxy-homoYessotoxin
TOXIC AREA
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O
O
O
O
O
O
O
O
O
O
NaO3SO
NaO3SO Me
Me
Me
Me
30
34
313233
3536 37
Me
OSO3NaOH
OH
H
H
H
H H H HH
H
H
H
H
HH H
H
H
Adriatoxin (1997)
New Yessotoxin analogues
New Yessotoxin analogues
O
O
O
O
O
O
O
O
O
O
NaO3SO
NaO3SO Me
Me
Me
Me
Me
O
Me
40
( )n
41
COOH
OH
H
H
H H H HH
H
H
H
H
HH H
H
H
H
H
HO
n = 1 Carboxy YTX (1997)
n = 2 CarboxyhomoYTX (1998)
O
O
O
O
O
O
O
O
O
O
NaO3SO
NaO3SO
Me
O
O
n = 1 OxoYTX (2001)
n = 2 OxohomoYTX (1998)
P.Ciminiello, E.Fattorusso, M.Forino, R.Poletti and R.Viviani. Eur. J. Org. Chem. 291-294 (2000).P.Ciminiello, E.Fattorusso, M.Forino, R.Poletti and R.Viviani. Chem. Res. Toxicol. 13, 770-774 (2000).P.Ciminiello, E.Fattorusso, M.Forino e R.Poletti Chem. Res. Toxicol. 14, 596-599 (2001).
P.Ciminiello, E.Fattorusso, M.Forino, S. Magno, R.Poletti, R.Viviani. Tetrahedron Lett. 39, 8897 (1998).
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CYTOTOXIC DERIVATIVESFROM MUSSELS OF ADRIATIC SEA
CYTOTOXIC DERIVATIVESFROM MUSSELS OF ADRIATIC SEA
*IC50 16.5 g/ml WEHI 164 (murine fibrosarcoma) cell lines IC50 10.9 g/ml on J774 (murine monocyte/macrophage) cell lines*IC50 16.5 g/ml WEHI 164 (murine fibrosarcoma) cell lines IC50 10.9 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. 49-53 (2001).
HN
O
HNO
OH
O
CN
HN
O
HNO
OH
OHHN
O
HNO
OH
1*
1'2'
3'
4'5'
6'
7'2
3
4
5
61
1'''
1''2''
3''
4''
5''
6''
7'' 8''
2 3
1'''
2 6
2'
4'
7'
2''
4''
62
7'' 2''
4''
2'
4'
7'
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FURTHER OXAZININESFROM ADRIATIC MUSSELS
HN
O
HN
OH
O
O
CN
SR
R
HN
O
HN
OH
O
O
CN
HN
O
HN
OH
O
OCH2CH2CN
HN
O
O
NH
OH
HO
OCH2CH2CN
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
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CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA
0
5
10
15
20
J 774 WEHI 164 P388
CLOROSULFOLI PI DE DTX
IC50 (
g/m
l)
Cl
Cl Cl
Cl
Cl
OSO3H
Cl
P. Ciminiello, M. Di Rosa, E. Fattorusso, M. Forino, A. Ianaro, and R. Poletti. J. Org. Chem. 66(2), 578-582 (2001).
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CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEACHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA
OH
O Cl
Cl
OSO3Cl
Cl
OH
Cl
Cl
Cl
OH
Cl
OH
Cl Cl
Cl
OH
C
O
(CH2)14CH3
IC50 (g/ml)
RAW 274.7: 20 g/ml
WEHI 164: 13.5 g/ml
0
5
10
15
20
RAW274.7 WEHI 164
CLOROSULFOLI PI DE DTX
6.2 mg
P. Ciminiello, C. Dell’Aversano, E. P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno and Fattorusso, M. Forino, S. Magno and Poletti J. Am. Chem. SocPoletti J. Am. Chem. Soc. . 124124, 13114 , 13114 (2002) (2002)
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221414= 16384= 16384
A FURTHER CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEAA 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)
.
OH
O Cl
Cl
OSO3Cl
Cl
OH
Cl
Cl
Cl
OH
Cl Cl Cl
Cl
OH
C
O
(CH2)14CH3
0
5
10
15
20
J 774 WEHI 164
CHLOROSULFOLI PI D DTX
IC50 (g/ml)
J774: 20 g/ml
WEHI 164: 10.4 g/ml
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High variation in toxins composition
New Biotoxins
New Cytotoxins
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• Monitoring program• qualitative analysis • quantitative analysis
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ripartizione
n-e,hexaneMeOH 80%
ripartizione
MeOH 40%CH2Cl
2LiChroprep RP-
8
AcOEt H2O
ripartizione
MeOH60%
MeOH80%
MeOH
90%
MeOH100%
Toyopearl HW-40
Yasumoto testCytoxicity tests
MeOH50 ml
RP-18
H2O/MeOH3:7
Luna RP-18H2O/MeOH/
CH3CN4:4:2
OMOYTX
CARBOXY-OMOYTX
45OH-OMOYTX
Acetone extraction
Mytilus galloprovincialis hepatopancreas
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Analysis Methods: LC-MSAnalysis 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
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Components of a Tandem Mass Spectrometer
InletSystem
Sanple
IonizationSource
MassAnalyzer 1
CollisionCell
MassAnalyzer 2
IonDetector
DataAcquisition
EI, CI, FABLD, FD, ESI Sector, Quadrupole,
TOF, Ion Trap
High Vacuum (10-5 to 10-6 torr)
Syringe, CE, HPLC
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COMPONENTS OF TANDEM MASS COMPONENTS OF TANDEM MASS SPECTROMETERSPECTROMETER
Inlet systemInlet system
HPLCHPLC
SSOOUURRCCEE ESIESI
Ion trapIon trap
DATADATA
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Advantage: •High sensitivity
in Full Scan
Drawback • Non linearcalibration
curves
XIC m/z 1173.5B
XICXIC
A
B
C
D
TIC MS
TICTIC
SELECTIVITY
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FSMS YTX
[M-Na]-
[M-2Na+H]-
Temp= 150°CTemp= 150°CVoltage = -10VVoltage = -10VTube Lens off set = -60VTube Lens off set = -60VSpray Voltage = 4,5KVSpray Voltage = 4,5KV
Hypersil C-8 BDS
14.1g/ml 4,7g/ml 1,6g/ml 0,52g/ml 0,17g/ml 0,057g/ml
YTXYTX
DL68pgYTXYTX
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HPLC-ESI-ion trap-MS Method for Direct Detection of Yessotoxins
O
O
O
O
OO
OO
OO
NaO3SO
NaO3SO
Me
O
MeMe
Me
Me
40
( )n
R
R n Tossina
1
1
COOH
2
CarboxyYTX
CarboxyhomoYTX
2 HomoYTX
2 Nor oxo Homo YTX
Nor oxo YTX1
Me
Me
O
1 45 OH-YTX
2 45OH-homoYTX
Me OH
OH
H
H
H H H HH
H
H
H
H
HH HH
H
H
H
YTXHO
HO
HO
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 SpectrometerHypersil 3m C8 BDS, 50 x 2.0 mmGradient H2O/ACN 2mM AF/50mM FA
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XIC
LC-MS LC-MS Mytilus galloprovincialis Cesenatico coasts in July 2001
TIC MS
FSMS
9.23 min
XIC
XIC
FSMS
8.65
min
8.60 min
FSMS
45-OHYTX45-OHhomoYTX
CarboxyYTXCarboxyhomoYTX
1141.5
1157.5
1171.5
1047.5
New YTX
YTX
P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, and R. Poletti Chem. Res. Toxicol. 15, 979 (2002)
8.25 min
8.30
min
FSMS
1187.51173.5
XIC
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Protoceratium reticulatum as the producing organism of…Protoceratium reticulatum as the producing organism of…
MeOH (125 L x 3)
Ultrafree (6000 rpm, 10 min)
+
Extract
(500 L)
SPE
O
O
O
O
O
O
O
OO
O
O
NaO3SO
NaO3SO
Me
O
Me
Me
Me
Me
OH
H
H
H H H HH
H
H
H
H
HH H
H
H
H
H
40
NoroxoYTX
HomoYTX
O
O
O
O
O
O
O
O
O
O
NaO3SO Me
Me
Me
Me
OH
H
H
H H H HH
Me H
H
H
H
HH H
H
H
O
H
MeHO
NaO3SO
45-OHYTX
O
O
O
O
O
O
O
O
O
O
NaO3SO Me
Me
Me
Me
OH
H
H
H H H HH
Me H
H
H
H
HH H
H
H
O
H
MeHO
NaO3SO
OH
O
O
O
O
O
O
O
O
O
O
NaO3SO Me
Me
Me
Me
OH
H
H
H H H HH
Me H
H
H
H
HH H
H
H
O
COOH
H
MeHO
NaO3SO
OH
CarboxyYTX
O
O
O
O
O
O
O
O
O
O
NaO3SO Me
Me
Me
Me
Me
O
Me
NaO3SO
OH
OH
H
H
H H H HH
H
H
H
H
HH H
H
H
H
HO
YTX
LC-multiple tandem MS
LoadWashEluate
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Some YTX analoguesSome YTX analoguespresent in large quantities present in large quantities in contaminated shellfish – in contaminated shellfish –
45-hydroxyYTX, carboxyYTX, 45-hydroxyYTX, carboxyYTX, noroxoYTX -noroxoYTX -
could to derive from oxidative could to derive from oxidative modification modification
of the absorbedof the absorbed YTX within the YTX within the mollusk .mollusk .
Conclusions
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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 timeIn the Adriatic Sea
From 1995 on:Yessotoxins have become the main Adriatic toxinsWhile Okadaic acid has slowly
subsided
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Anomalous toxicity of Adriatic mussels cultivated in November 2004
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ALLOWANCE LEVELS SUGGESTED BY EU
TOXINTOXIN 100g 100g SHELLFISHSHELLFISH
OKADAIC ACIDOKADAIC ACID 16 16 gg
YESSOTOXINYESSOTOXIN 100 100 gg
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Mytilus galloprovincialis hepatopancreas
Acetone extraction
AcOEt H2O
MeOH 80%
n-hexane
CH2Cl2MeOH 40%
Yasumoto test
OKADAIC ACIDYESSOTOXINS
H2O CH2Cl2
BuOH H2O
YESSOTOXINS OKADAIC ACID
OLD PROCEDURE
NEWPROCEDURE
Yasumoto test
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In summer 2004CH2Cl2 phase derivingFrom Adriatic mussels
Was foundTOXIC!!!
In summer 2004CH2Cl2 phase derivingFrom Adriatic mussels
Was foundTOXIC!!!
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AcetoneAcetoneextractextract
Isolation of the new toxinsIsolation of the new toxins
HH22O O HW
-40
HW
-40
RP
-8R
P-8
MeOH/HMeOH/H22OO6:46:4
OD
SO
DS
MeOH/HMeOH/H22OO8:28:2
CHCH22ClCl22
.
HPLC HPLC
RP-18H2O/MeOH/CH3CN
4:4:2
NEW NEW TOXINTOXIN
AA
NEW NEW TOXINTOXIN
BB
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New Toxin- A:Carboxyhomo-1-desulfoYTX
5.0 4.0 3.0 2.0 1.0
H2-1H-4
H-5
O
O
O
O
O
OO
OO
OH
MeMe
O
MeO
Me
Me
HO
Me
HOOC
R'O
RO
1008
869
940
727
12
2a 3
40
44
H H H H HH
H
H
H
H
H
H H H H H
H
H
H
467A B C D E
O
O
O
O
O
O
O
O
O
O
O
OH
HO
HOOC
HO3SO
HO
1
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O
O
HO3SO
HO
H H
HH
H H
Toxin A
Spin system C-1/C-2a
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New Toxin- B :Carboxyhomo-4-desulfoYTX
5.0 4.0 3.0 2.0 1.0
H2-1H-4
H-5
O
O
O
O
O
OO
OO
OH
MeMe
O
MeO
Me
Me
HO
Me
HOOC
R'O
RO
1008
869
940
727
12
2a 3
40
44
H H H H HH
H
H
H
H
H
H H H H H
H
H
H
467A B C D E
O
O
O
O
O
O
O
O
O
O
O
OH
HO
HOOC
HO
HO3SO
4
0
100
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Toxin B: spin system C-4/C-8
O
HO
HO3SO
O
O
4
5
6
8
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Two new toxins in Adriatic mussels: Desulfohomoyessotoxins
O
O
O
O
O
O
O
O
O
O
O
OH
HO
HOOC
HO3SO
HO
O
O
O
O
O
O
O
O
O
O
O
OH
HO
HOOC
HO
HO3SO
P. Ciminiello, C. Dell'Aversano, E. P. Ciminiello, C. Dell'Aversano, E. Fattorusso, M. Forino, L. Grauso, S. Fattorusso, M. Forino, L. Grauso, S. Magno, R. Poletti, L. TartaglioneMagno, R. Poletti, L. Tartaglione Chem. Res. in Toxicol.,Chem. Res. in Toxicol., 20, 95 (2007)20, 95 (2007)
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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
H2O CH2Cl2
BuOH H2O
YESSOTOXINS OKADAIC ACID
NEWPROCEDURE
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In November 2003…
Alexandrium ostenfeldiimassively bloomed
in the Adriatic Sea
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spirolides
OO
HO OO
O
N
OH
OO
HO OO
O
N
OH
OO
HO OO
O
N
OH
OON
O O
OHO
OH
OO
HO OO
O
N
OH
OO
HO OO
O
N
HO
Spirolide A
Spirolide B
Spirolide C
Spirolide DSpirolide G
13-desmethyl Spirolide C
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Extraction of Alexandrium ostenfeldii culture
H2O:CH3CN
HPLC Rp-8
H2O:CH3CN
90% A to 100% B
Three majorSpirolides @ m/z:
678,5 692,5 694,5
MPLC
MPLC C18
C18
MeOH: H2O8 : 2
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Spirolide @ 678
ppm (f1)1.02.03.04.05.06.07.0
5.0
10.0
N
O
O
HO
OO
O
OH
14
36
5
6
37
7
10 38
1215
1819
22
24
41
2728
29
3132
4243
13,19-didesmethylspirolideC
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Spirolide @ 692
ppm (f1)1.02.03.04.05.06.07.0
0.0
5.0
10.0
15.0
N
O
O
HO
OO
O 14
36
5
6
37
7
10 38
1215
1819
22
24
41
2728
29
3132
42
43
OH
13-desmethylspirolideC
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Spirolide @ 694
ppm (f1)1.02.03.04.05.06.07.0
0.0
5.0
10.0
15.0
N
O
O
HO
OO
O
OH
OH
14
36
5
6
37
7
10 38
1215
1819
22
24
41
2728
29
3132
4243
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.
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ACN/H2O 8:2 +0,1% formic acid
FiltrateFiltrate ResidueResiduepartition
ACN/HACN/H22OO n-hexanen-hexane
partition
CHCH22ClCl22 ACN/H2O 2:1ACN/H2O 2:1
deaddeadlivelive
Cesenatico and TriesteCesenatico and Trieste
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312.5
312.3
294.9
266.1182.1
DOMOIC ACID ?DOMOIC ACID ?DOMOIC ACID ?DOMOIC ACID ?
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NH
COOH
COOH
COOH
Domoic Acid
Glutamico Acid Aspartic cido
ConfusionConfusion
VomitingVomiting
AmnesiaAmnesia
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Optimization of ionization parameters
DLDA=123ng/ml
r2 >0.999
SIM (Selected Ion Monitoring)
MSMS (Massa Massa)
MRM (Multiple Reaction Monitoring)
Column:Column:TSK-gel amide 80TSK-gel amide 80Mobil phase:Mobil phase:H2O/ACN +H2O/ACN + 2mM formiato/3.53 mM formic 2mM formiato/3.53 mM formic acidacid
Calibration
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m/z 312 > 175
Time (min)
0 5 10 15
m/z 312 > 193
m/z 312 > 220
m/z 312 > 248
m/z 312 > 266
m/z 312 > 294
DLDA=12 ng/ml
DLPSP= 5-30 nM
r2 >0.999(0.12-10 g/ml)
Good sensitivity and linearity
C. Dell’Aversano, P. Hess, M. A. Quilliam J. Chromatogr. A, 1081 (2), 190-201 (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, 2030-2038 (2005)
API-2000 triple quadrupoleAPI-2000 triple quadrupole mass spectrometer equippedmass spectrometer equipped with a turbo-ionspray sourcewith a turbo-ionspray source (Applera, Thornhill, ON, Canada)(Applera, Thornhill, ON, Canada)
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Quantitative AnalysisQuantitative Analysis
m/z 310/266m/z 310/266
m/z 310/248m/z 310/248
m/z 310/222m/z 310/222
MRM-MRM-
Cesenatico=2.5 µg/gCesenatico=2.5 µg/gTrieste=0.25 µg/g Trieste=0.25 µg/g Limite legislativo= 20µg/gLimite legislativo= 20µg/g