Ecological Chemistry: Allelochemical Effects of Protists

Marine Biotoxins:

Determination of Spirolide Profiles in Phytoplankton by LC/MS/MS

1. Introduction

2. Search for unknowns

3. Mass spectral characterization

4. Phenotypic profiling of Alexandrium ostenfeldii strains 5. Pitfalls

Ecological Chemistry: Allelochemical Effects of Protists

Allelochemistry:

Interaction of biologically active components eliciting specific responses in target organisms.

These highly specific allelochemical compounds are typically secondary

metabolites and should be distinguished from low molecular weight inorganic and organic nutrients and complex but poorly defined dissolved organic

matter (DOM) that may be utilized as growth substrates by protists.

There are both stimulatory and inhibitory functions to be exploited via production of allelochemicals by protists. Among the putative functions of allelochemicals, their use as agents of chemical defence is most often invoked.

1. Introduction

Lytic effect of Alexandrium, here the example of Oxyrrhis marina (heterotrophic dinoflagellate).

Black arrows: Alexandrium;

Red arrows: remains of Oxyrrhis

Alexandrium ostenfeldii Oxyrrhis marina

Ecological Chemistry: Allelochemical Effects of Protists

1. Introduction

Alexandrium ostenfeldii

?

Defense against predators and/or competitors Lysis of other

protists

?

Accumulation in marine food

webs,

poisoning of vertebrates and humans

Spirolides

O O

N

O

O O OH

HO 2

3

31

13

Organism Effect chemical interaction ecological function

Ecological Chemistry: Allelochemical Effects of Protists

1. Introduction

1991-1992

During routine monitoring of shellfish aquaculture sites in Nova Scotia, extracts of the digestive glands of blue mussels (Mytilus edulis) from Ship Harbour and sea scallops (Placopecten magellanicus) (Graves Shoal) elicited a unique toxic response in the DSP mouse bioassay…….

Coincident consumer complaints of mild illness after shellfish consumption

The Discovery of Novel „Fast Acting Toxins“

1. Introduction

“Fast Acting Toxicity”: Lipophilic extracts (DSP toxins) Symptomology:

not PSP/DSP(!) strong convulsions tail whirling

body arching

rapid death (min)

High I.P. Toxicity

1. Introduction

Ship Harbour

Nova Scotia, Canada

Transect Station TACCS Station

Mussel farm

Nova Scotia Shelburne

Ship Harbour Mahone

“Fast Acting Toxicity”: Occurrence

1. Introduction

Limfjorden, Denmark

8^o 1 0^o 1 2^o

5 5^o

5 6^o

5 7^o

8^o 1 0^o 1 2^o

5 5^o

5 6^o

5 7^o

Limfjorden

Denmark

Sweden

Germany

“Fast Acting Toxicity”: Occurrence

Later also found in

Norway, Scotland

1. Introduction

Alexandrium tamarense Alexandrium ostenfeldii

Scanning electron micrographs of vegetative cells

APC APC

1’ VP 1’

VP

1. Introduction

Confirmation of spirolides in cultured isolates from Nova Scotia

Produces PSP toxins, but no spirolides

Produces spirolides, but no PSP toxins

Alexandrium ostenfeldii Alexandrium tamarense

Culprit species!!

Time (min)

0 2 4 6 8 10

m/z (x1)

m/z (x10)

m/z 706.5 (x10) m/z 708.5 (x50)

C

D desMe-C

desMe-D C3

* D3 (A)

(B)

(D2) (C2)

1. Introduction

Cause of “Fast Acting Toxicity”

Novel compounds identified as

“spirolides”

• macrocyclic imines

• structural similarity to pinnatoxin

& gymnodimine

• pharmacologically active/inactive forms

O O

R1

N

O H

O

R2

O O

OH

R1 R2 MW

A H CH₃ Δ^2,3 691.5

desMeC CH₃ H Δ^2,3 691.5 B H CH₃ 693.5 desMeD CH₃ H 693.5

C CH₃ CH₃ Δ^2,3 705.5 D CH₃ CH₃ 707.5

E H CH₃ Δ^2,3 709.5 F H CH₃ 711.5 O O

R1 NH₂

O H

O

R2

O O

OH O

2 3

2 3

13 31

13 31

toxic

non-toxic

1. Introduction

2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 T i m e , m i n

0 , 0 2 0 0 0 , 0 4 0 0 0 , 0 6 0 0 0 , 0 8 0 0 0 , 0 1 , 0 e 4 1 , 2 e 4 1 , 4 e 4 1 , 6 e 4 1 , 8 e 4 2 , 0 e 4 2 , 2 e 4 2 , 4 e 4 2 , 6 e 4 2 , 7 e 4

2. Search for unknowns

13-desMe C Strd

MRM: m/z 692.5 > 164.1

2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8

T i m e , mi n 0 , 0 0

5 , 0 0 e 4 1 , 0 0 e 5 1 , 5 0 e 5 2 , 0 0 e 5 2 , 5 0 e 5 3 , 0 0 e 5 3 , 5 0 e 5 4 , 0 0 e 5 4 , 5 0 e 5 5 , 0 0 e 5 5 , 5 0 e 5 6 , 0 0 e 5 6 , 5 0 e 5 7 , 0 0 e 5 7 , 5 0 e 5 8 , 0 0 e 5 8 , 5 0 e 5 9 , 0 0 e 5 9 , 5 0 e 5 1 , 0 0 e 6 1 , 0 5 e 6

1 , 1 0 e 6 1 3 , 0 0

1 2 , 7 2

2 6 , 2 7

AOSH 2

MRM: m/z 692.5 > 164.1

Identical mass transitions – different retention times

13-desMe C Strd: EPI m/z 692.5

AOSH 2: EPI m/z 692.5

2. Search for unknowns

MS/MS spectra of m/z 692.5

2. Search for unknowns

M+H = 692.5

0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0

6 7 4 ,3

1 6 4 ,1

4 4 4 ,2

6 5 6 ,3

6 3 8 ,4

4 2 6 ,3 6 9 2 ,3

1 7 7 ,1

2 3 0 ,2

4 6 2 ,3 1 1 9 ,0 1 4 7 ,0

2 2 0 ,2 2 5 8 ,2 4 0 8 ,2

1 5 7 ,1 3 0 2 ,2 3 2 0 ,2 3 5 8 ,2 4 5 4 ,2 5 2 2 ,3 5 5 8 ,4 6 2 0 ,2 6 3 0 ,4

1 3 1 ,0 4 7 2 ,3

Characteristic spirolide

fragment

O O

N

O

O O

OH

HO

2 3

31

13

+ m/z = 444.3

N

O

O O

HO

31

13

+

m/z = 164.1

N

31

+

O O

R1

N

O H

O

R2

O O

OH

R1 R2 MW

A H CH₃ Δ^2,3 691.5 desMeC CH₃ H Δ^2,3 691.5 B H CH₃ 693.5 desMeD CH₃ H 693.5 C CH₃ CH₃ Δ^2,3 705.5 D CH₃ CH₃ 707.5

E H CH₃ Δ^2,3 709.5 F H CH₃ 711.5 O O

R1 NH₂

O H

O O

O

OH O

2 3

2 3

13 31

13 31

toxic

non-toxic

2. Search for unknowns

Cyclic imino moiety accounts for toxicity and forms a

characteristic spirolide fragment

2. Search for unknowns

AOSH 2: Precursor m/z 164.1

Using the characteristic fragment for the detection of

unknown spirolides

2. Search for unknowns

AOSH 2: Precursor m/z 164.1

00 520 540 560 580 600 6 20 6 40 66 0 68 0 700 720 740 760 780 800

65 0,7

6 40,6

64 1,7

65 2,7

632 ,6

622,7

7 24,6

64 3,6 654,7 6 92,9 7 12,2

11.7 min m/z 640.6 m/z 650.7

00 520 540 560 580 600 6 20 6 40 66 0 68 0 700 720 740 760 780 800

7 20,7

7 06,9

692 ,7 708 ,0 722 ,9

688,7

65 1,1

64 1,0

622 ,9 725,2 739 ,0 760,1

12.6 min m/z 706.6 m/z 720.7

692,6

694 ,6

13.0 min m/z 692.6

m/z 694.6

Spirolide masses

706,6

70 8,4

68 8,9

67 2,7

64 0,5 690,9 710 ,7 738,1

12.0 min m/z 706.6

3. Mass spectral characterization

AOSH 2: m/z = 640.5

3. Mass spectral characterization

AOSH 2: m/z = 650.5

3. Mass spectral characterization

AOSH 2: m/z = 692.5

AOSH 2: m/z = 694.5

3. Mass spectral characterization

3. Mass spectral characterization

AOSH 2: m/z = 706.5

3. Mass spectral characterization

AOSH 2: m/z = 720.5

10,5 11,0 11,5 12,0 12,5 13,0 13,5 14,0 14,5 15,0 Time, min

0,0 2,0e5 4,0e5 6,0e5 8,0e5 1,0e6 1,2e6 1,4e6 1,6e6 1,8e6 2,0e6 2,2e6 2,4e6

11,51

O O

N

O

O O OH

HO 2

3

31

13

13-desMe C

O O

N

O

O O OH

2 3

31

13

HO 19

13,19-didesMe C

678.5 > 164.1 692.5 > 164.1

4. Penotypic profiling of Alexandrium ostrenfeldii strains

CCMP 1773, Denmark

10,5 11,0 11,5 12,0 12,5 13,0 13,5 14,0 14,5 15,0 0,0

1,0e5 2,0e5 3,0e5 4,0e5 5,0e5 6,0e5 7,0e5 8,0e5 9,0e5 1,0e6 1,1e6 1,2e6 1,3e6 1,4e6 1,5e6 1,6e6 1,7e6 1,8e6 1,9e6 2,0e6 2,1e6

11,95

O O

N

O

O O OH

HO 2

3

31

13

13-desMe C

O O

N

O

O O OH

HO

2 3

31

13

Spirolide C

?

692.5 > 150.1 692.5 > 164.1 694.5 > 164.1 706.5 > 164.1

4. Penotypic profiling of Alexandrium ostrenfeldii strains

AOSH 1, Canada

12,38

10,5 11,0 11,5 12,0 12,5 13,0 13,5 14,0 14,5 15,0 Time, min

0,0 2,0e5 4,0e5 6,0e5 8,0e5 1,0e6 1,2e6 1,4e6 1,6e6 1,8e6 2,0e6 2,2e6 2,4e6 2,6e6 2,8e6 3,0e6 3,2e6 3,4e6 3,6e6 3,8e6 4,0e6 4,2e6 4,4e6

12,38

12,00

12,85

?

?

? ?

? ?

O O

N

O

O O

HO 2

3

32

13 HO

17

20

20-Me Spirolide G

O O

N

O

O O OH

HO

2 3

31

13

Spirolide C

?

604.5 > 356.3 640.5 > 164.1 650.5 > 164.1 692.5 > 150.1 692.5 > 164.1 694.5 > 164.1 706.5 > 164.1 708.5 > 164.1 720.5 > 164.1

4. Penotypic profiling of Alexandrium ostrenfeldii strains

AOSH 2, Canada

5. Pitfalls

Identical retention time and mass transition,

AOSH 2: MRM 706.5 > 164.1 AOSH 1: MRM 706.5 > 164.1

5. Pitfalls

AOSH 2: EPI m/z 706.5; 12.37 min AOSH 1: EPI m/z 706.5; 12.37 min

but different mass spectra

O O

R1

N

O H

O

R2

O O

OH

R1 R2 MW

A H CH₃ Δ^2,3 691.5 desMeC CH₃ H Δ^2,3 691.5 B H CH₃ 693.5 desMeD CH₃ H 693.5 C CH₃ CH₃ Δ^2,3 705.5 D CH₃ CH₃ 707.5

E H CH₃ Δ^2,3 709.5 F H CH₃ 711.5 O O

R1 NH₂

O H

O O

O

OH O

2 3

2 3

13 31

13 31

5. Pitfalls

Compound or isotopic peak?

5. Pitfalls

AOSH 1: MRM 694.5 > 164.1

AOSH 2: MRM 694.5 > 164.1

Compound or isotopic peak?

5. Pitfalls

AOSH 1: EPI m/z 694.5; 12.0 min

AOSH 2: EPI m/z 694.5; 12.9 min

Product ion spectra reveal isotopic pattern

Isotopic fragments of m/z ¹³C₂-692.5

Monosotopic fragments of m/z ¹²C-694.5

Conclusions:

Triple quadrupole tandem mass spectrometry is a powerful tool for the determination and quantitation of spirolides

Unknown toxic spirolides can be detected in the precursor ion mode of the characteristic cyclo imino fragments at m/z 150 and 164,

respectively

Structural information can be obtained by product ion spectra of parent ions Co-eluting compounds with identical mass transitions can be

differentiated by their product ion spectra

Product ion spectra can be used to differentiate between isotope statellites and monoisotopic peaks

Acknowledgements:

Allan Cembella, AWI Urban Tillmann, AWI

Shawna MacKinnon, IMB - NRC Corinne Garnett, IMB - NRC

Thank You!