Cyanobacterial Toxins by LC-MS/MS
1. Relevance of cyanotoxins in food chemistry 2. What are cyanotoxins? - Chemical classes
3. Qualitative detection of cyanotoxins by LC-MS/MS 4. Quantitative analysis
5. Summary
Relevance of cyanotoxins in food chemistry
www.allcura.de www.bodensee-therme-konstanz.de
1. potable water 2. dietary supplements -
algal preparations
Cyanotoxins – Classes
PSTs (paralytic shellfish toxins) sodium channel inhibitors
Anatoxins nicotinic acetylcholin receptor agonists Anatoxin-a(s) acetylcholin esterase inhibitor
Cylindrospermopsins inhibition of uridine monophosphate synthase complex - hepatotoxins Microcystins block of protein serine/threonine
phosphatases PP1 and PP2A Nodularins block of protein serine/threonine
phosphatases PP1 and PP2A
Toxin class mode of action
HN
N N
H H N
NH
2OH OH O
H
2N O
H
2N
Anabaena
circinalis
lemmermannii Aphanizomenon
flos-aquae Cylindrospermopsis
raciborskii Lyngbya
wollei Microcystis
aeruginosa
Microcystis aeruginosa
Aphanizomenon flos-aquae Anabaena circinalis
Paralytic Shellfish Toxins (PSTs)
Saxitoxin
N
N N
H H N
NH 2 OH
OH O
H 2 N R
1R 2 R 3 R
4Toxin R1 R2 R3 R4
STX H H H
NEO OH H H
GTX1 OH H OSO3-
GTX2 H H OSO3-
GTX3 H OSO3- H
GTX4 OH OSO3- H
B1= GTX5 H H H
B2= GTX6 OH H H
C3 OH H OSO3-
C1 H H OSO3-
C2 H OSO3- H
C4 OH OSO3- H
dc-STX H H H
dc-NEO OH H H
dc-GTX1 OH H OSO3-
dc-GTX2 H H OSO3-
dc-GTX3 H OSO3- H
dc-GTX4 OH OSO3- H
H (Decarbamoyl-)
CO-NH-SO3- (N-Sulfocarbamoyl-)
CO-NH2 (Carbamoyl-)
STX = Saxitoxin NEO = Neosaxitoxin GTX = Gonyautoxin
Paralytic Shellfish Toxins (PSTs)
Anatoxin-a
NH O
Anatoxin-a
NH O
Homoanatoxin-a
Anabaena
circinalis flos-aquae Aphanizomenon
flos-aquae Planktothrix
formosa
Aphanizomenon flos-aquae Anabaena circinalis
Anabaena flos-aquae
Anatoxin-a(s)
Anatoxin-a(s)
HN N
HN
O N
P O
HO O
Anabaena
flos-aquae lemmermannii
Anabaena lemmermannii
Anabaena flos-aquae
Cylindrospermopsins
Cylindrospermopsin
N NH HN NH
N
O
O OH
H H
H HO3SO
N NH HN NH
N
O
O OH
H H
H HO3SO
7-epi-Cylindrospermopsin
N NH HN NH
N
O
O
H H
H HO3SO
deoxy-Cylindrospermopsin
Aphanizomenon flos-aquae ovalisporum Cylindrospermopsis
raciborskii Umezakia
natans
Cylindrospermopis raciborskii
Umezakia natans
Microcystins
NH H N
H N
HN O NH N
HN
O
O O
O
O O
R2 COOH
R1 COOH
OCH3
(1) D-Ala
(2) L-X
(3) D-erythro-β-methylAsp (iso) (4) L-Z
(5) Adda
(6) D-Glu (iso)(7) N-methyldehydroAla (Mdha)
Anabaena
circinalis flos-aquae Anabaenopsis
milleri Aphanizomenon
ovalisporum Microcystis
aeruginosa botrys
viridis Planktothrix
agardhii mugeotii rubescens Nostoc spp.
Planktothrix agardhii
Microcystis aeruginosa Nostoc
NH H N
H N
HN O NH N
HN
O
O O
O
O O
R2 COOH
R1 COOH
OCH3
(1) D-Ala
(2) L-X
(3) D-erythro-β-methylAsp (iso) (4) L-Z
(5) Adda
(6) D-Glu (iso) (7) N-methyldehydroAla (Mdha)
AA 1 AA 2 AA 3 AA 4 AA 5 AA 6 AA 7
D-Ala L-Leu D-MeAsp L-Arg Adda D-Glu Mdha
D-Ser L-Ala D-Asp L-Aib ADMAdda D-MeGlu Dha
L-Glu L-Ala DMAdda OC2H3(CH3)OH-Glu Dhb
L-GluMe L-Glu (6Z)Adda L-Ala
L-Har L-GluMe L-MeSer
L-Hil L-Har L-Ser
L-Hph L-Hph Mdhb
L-Hty L-Hty MeLan
L-Met L-Leu
L-Met(O) L-Met
L-Phe L-Met(O)
L-ThTyr L-Phe
L-Trp L-Trp
L-Tyr L-Tyr
L-Val
Microcystins
Nodularins
NH H
N NH
O
O
O N
H
COOH
OCH3 N
O O COOH
NH
NH2 HN
Adda D-Glu (iso) N-Methyl-
2-amino- butenic acid
D-erythro-β-methyl-iso-Asp L-Arg
Nodularia
spumigena
Nodularia spumigena
Aim:
Survey method for the qualitative detection of cyanobacterial freshwater toxins
Prerequisites:
All toxins soluble in the same extraction solvent Characteristic fragment for each toxin group
Toxin group (not single compound!) separation
Qualitative detection of cyanotoxins
2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time, min
0,0 2,0e6 4,0e6 6,0e6 8,0e6 1,0e7 1,2e7 1,4e7 1,6e7 1,8e7 2,0e7 2,2e7 2,4e7 2,6e7 2,8e7 3,0e7 3,2e7 3,4e7
3,6e7 14,47
5,28
15,09 5,04 6,12
Hiller et al. (2007) J. Mass Spectrom. 42(9), 1238-1250
Period 1: PSP & Anatoxin-a(s) Period 2: Anatoxins & Cylindrospermopsins Period 3: Nodularins & Microcystins
Phenomenex Luna C18 150x3 mm, 3 µm, 100 Å A: 2mM NH4HCOO, 50 mM HCOOH
B: 2mM NH4HCOO, 50 mM HCOOH in 95% MeOH Gradient: initial: 100% A
1 min 50% A
5 min 50% A
15 min 10% A 20 min 10% A 21 min 100% A Flow rate: 400 µl/min
Temperature: 20°C
Nodularia spumigena, Baltic Sea
Qualitative detection of cyanotoxins
Period 1: PSP & Anatoxin-a(s)
N
N N
H H N
NH2
OH OH O
H2N R1
R2 R3 R4 13
1
3 4
5 6
7 9
10 12
Anatoxin-a(s)
HN N
HN
O N
P O
HO O
PSP-toxins
No characteristic fragment
Only one toxin known
API 4000 QTrap, positive, MRM:
IS: 5000 V
CAD: high level TEM.: 550 °C GS 1: 50 L h-1 GS 2: 70 L h-1 CUR: 25 L h-1
CE: 30 eV
DP: 40 eV
Mass transtions:
412
→332 / 412
→314 (GTX1, GTX4, C3, C4) 396
→316 / 396
→298 (GTX2/3, B2, C1, C2) 380
→300 / 380
→282 (B1)
369
→289 (dcGTX1/4) 353
→273 (dcGTX2/3)
316
→298 (NEO, GTX2/3, B2, C1, C2) 316
→220 (NEO)
300
→282 / 300
→204 (STX, B1) 273
→255 (dcNEO, dcGTX2, dcGTX3) 257
→239 (dcSTX )
253
→235 / 253
→159 (ANAS)
Period 2: Anatoxins & Cylindrospermopsins
NH O
Anatoxin-a
Cylindrospermopsin
N NH HN NH
N
O
O OH
H H
H HO3SO
+
characteristic fragment: m/z 91
N NH2
N H
H HO
+
characteristic fragment: m/z 194
API 4000 QTrap, positive precursor ion (m/z): 194.0 scan range (m/z): 350-450 amuCUR: 25
CAD: High
IS: 5200
TEM: 550
GS 1: 50
GS 2: 70
CE: 50
DP: 80
API 4000 QTrap, positive precursor ion (m/z): 91.0
scan range (m/z): 100-300 amu CUR: 25
CAD: High IS: 5200 TEM: 550 GS1: 50 GS2: 70 ihe: OFF
DP: 80
EP: 10
CE: 30
CXP: 12
Experiment 1
Experiment 2
Period 3: Microcystins & Nodularins
NH H N
H N
HN O NH N
HN
O
O O
O
O O
R2 COOH
R1 COOH
OCH3
characteristic fragment: m/z 135
OCH3
+
experiment 1 experiment 2 experiment 3 experiment 4
scan range (m/z) 400 - 575 400 - 575 900 - 1150 800 - 850
protonated fragment ions
[M+H]+/ [M+2H]2+ [M+2H]2+ [M+2H]2+ [M+H]+ [M+H]+
collision energy (eV) 17 35 60 90
declustering potential (V) 46 40 60 175
cyanobacterial toxins:
microcystins / nodularins microcystins microcystins microcystins nodularins number of Arg residues within
the microcystin peptide 1, exceptional 0 2 0
Microcystis aeruginosa
Microcystins – precursor mode
MC-LR
dmMC-LR
NH H N
H N
HN O NH N
HN
O
O O
O
O O
COOH COOH
OCH3
HN
H2N NH
OCH3
+
N H2N
O
O COOH
O NH N H2N
O
O COOH
NH2
N HN
O
O O
COOH
OCH3
[Mdha-Ala-Leu-MeAsp-Arg + H]
+ H
N
H N
HN O NH HN
O
O O
O
COOH
HN
H2N NH
NH H
N NH2
O
O O
COOH OCH3
HN
H2N NH
