The Analysis of Algal Toxins Using Various
Scan Modes in LC/MS/MS
Toxic Algal Blooms
Distribution of Paralytic Shellfish Poisoning events
2005
1970
N H HOOC
HOOC
2
3 4
5 7 6
8
1'
5'
6'
7' 8'
COOH
Causative Organisms
Diatoms
Pseudo-Nitzschia pungens
Amnesic Shellfish Poisoning
Domoic acid
Causative Organisms
Dinoflagellates
Lingulodinium polyedrum
O
O O
O O
O
O O
O O O
OH
O
O O
O O
O O
O O
O O O O O O
O O O O O
O
OH
OH H
H H
OH
OH OH
H H H
H OH
H HO
OH
H OH
H OH
HOH H OH H H H H H H OH H H H H
OSO3Na OH H H H H
OHH H H H H H
OH H
OH OH H H H H H H
H H H H H OH HO
HO
H H OH H HO OH
OH
HO OH
NaO3SO
H
HN
N N
H H N
NH2 OH OH O
H2N O
H2N
Paralytic Shellfish Poisoning
Saxitoxin
O O
O O
O O
O O
O
O O
O
H H H
H H H
H H
H HO
H H H
H H
H
O
Brevetoxin
Maitotoxin Yessotoxin
O
O O
O O
O O
O O
O O
-O3SO
Me H
H
Me
H
H Me
Me Me H H H H H
Me H H H H H
H H
H H
H HO
-O3SO
OH
13-desmethyl spirolide C
O O
N
O
O O OH
HO
2 3
31
13
Azaspiracid-1
NH
O O
O O O O O O
OH
O
OH HO H
H H
H
H
H
H A
B
C D
E
F H G I
1
10
13
14
21
28 26
32 37
40
Okadaic acid
H O
O O O H
O
O O
O
O H O
O H
O H O
Neurotoxic Shellfish Poisoning
Diarrhetic Shellfish Poisoning
Spirolide determination - MRM
Chromatography
Luna C18 150x3 mm, 3 µm, 100 Å A: 2mM NH4HCOO, 50 mM HCOOH
B: 2mM NH4HCOO, 50 mM HCOOH in 95% ACN Linear Gradient: 22%B Æ65%B (0-50 min) Flow rate: 200 µl/min
Temperature: 35°C
API 4000 QTrap, positive, MRM CUR: 20
CAD: High IS: 5500 TEM: 650 GS1: 40 GS2: 70 ihe: ON
DP: 121
EP: 10
CE: 57
CXP: 22
Yessotoxin detection – Neutral Loss
O
O
O
O
O
O O
O O
O O
-O3SO
Me H
H
Me
H
H Me
Me Me H
H H
H H
Me H H H H H
H H
H H
H HO
-O3SO
OH
yessotoxin
Miles et al. (2005) Harmful Algae 4 : 1075-1091
List of Yessotoxin Pseudo-molecular and Fragment Masses (abridgement)
The most abundant fragment of all
YTXs is the loss of SO
3from the
sulfate groups
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 T i m e , m i n
0 , 0 2 , 0 e 5 4 , 0 e 5 6 , 0 e 5 8 , 0 e 5 1 , 0 e 6 1 , 2 e 6 1 , 4 e 6 1 , 6 e 6 1 , 8 e 6 2 , 0 e 6 2 , 2 e 6 2 , 4 e 6 2 , 6 e 6 2 , 8 e 6
2 , 9 e 6 8.24 min8 , 2 4
Protoceratium reticulatum, North Sea Neutral loss scan (m/z 80)
Peak @ 8.24 min
Yessotoxin, MW = 1142.5
1 0 5 0 1 0 6 0 1 0 7 0 1 0 8 0 1 0 9 0 1 1 0 0 1 1 1 0 1 1 2 0 1 1 3 0 1 1 4 0 1 1 5 0 1 1 6 0 1 1 7 0 1 1 8 0 1 1 9 0 1 2 0 0
m / z , a m u 5 0 0 0 , 0 0
1 , 0 0 e 4 1 , 5 0 e 4 2 , 0 0 e 4 2 , 5 0 e 4 3 , 0 0 e 4 3 , 5 0 e 4 4 , 0 0 e 4 4 , 5 0 e 4 5 , 0 0 e 4 5 , 5 0 e 4 6 , 0 0 e 4 6 , 5 0 e 4 7 , 0 0 e 4 7 , 5 0 e 4 8 , 0 0 e 4 8 , 5 0 e 4 9 , 0 0 e 4 9 , 5 0 e 4 1 , 0 0 e 5 1 , 0 5 e 5 1 , 1 0 e 5
1 , 1 5 e 5 1 1 4 1 , 7
1 1 4 2 , 8
1 1 4 3 , 7
1 1 4 4 , 8
1 1 4 6 , 1 1 1 5 7 , 6 1 1 5 9 , 3
m/z 1141.5
Yessotoxin detection – Neutral Loss
API 4000 QTrap, negative, NL m/z 80:
CUR: 10.00 CAD: Medium TEM: 0.00 GS1: 10.00 GS2: 0.00 ihe: ON IS: -4500.00
DP -50.00
EP -10.00
CE -55.00
CXP -15.00
Chromatography
Hypersil BDS C8 50x2 mm, 3 µm, 120 Å A: 2mM NH4HCOO, 50 mM HCOOH B: 2mM NH4HCOO, 50 mM HCOOH
in 95% MeOH/ACN (1:2, v/v)
Linear Gradient: 40%B Æ100%B (6-15 min) Flow rate: 300 µl/min
Temperature: 20°C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 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
7,67
7,80
Protoceratium reticulatum, Benguela Current Neutral loss scan (m/z 80)
6.24 min
Yessotoxin detection – Neutral Loss
Hypersil BDS C8 50x2 mm, 3 µm, 120 Å A: 2mM NH4HCOO, 50 mM HCOOH B: 2mM NH4HCOO, 50 mM HCOOH
in 95% MeOH/ACN (1:2, v/v)
Linear Gradient: 40%B Æ100%B (6-15 min) Flow rate: 300 µl/min
Temperature: 20°C
1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 m/z, amu
500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000
1175,8 1048,7
1047,7
1176,8
1177,8
1161,3 1105,7
1049,6
1163,9 1178,9 1106,7 1119,6
1051,6
1158,4
1131,4 1164,9
Protoceratium reticulatum, Benguela Current
Neutral loss scan (m/z 80) Peak @ 6.24 min
Yessotoxin detection – Neutral Loss
API 4000 QTrap, negative, NL m/z 80:
CUR: 10.00 CAD: Medium TEM: 0.00 GS1: 10.00 GS2: 0.00 ihe: ON IS: -4500.00
DP -50.00
EP -10.00
CE -55.00
CXP -15.00
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 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 1 1 0 0 m / z , a m u
2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0 2 2 0 0 2 4 0 0 2 6 0 0 2 8 0 0 3 0 0 0 3 2 0 0 3 4 0 0 3 6 0 0 3 8 0 0 4 0 0 0 4 2 0 0
9 2 5 ,6
8 5 5 ,3
1 0 5 3 , 6
7 4 3 ,4
1 0 9 6 , 6 7 1 3 , 2
4 8 9 , 4 3 6 3 , 3
4 3 3 ,2 8 2 5 , 4 8 3 9 , 4
924.5
855.5
713.4 743.4
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 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0
m / z , a m u 5 0 0 0 ,0
1 ,0 e 4 1 ,5 e 4 2 ,0 e 4 2 ,5 e 4 3 ,0 e 4 3 ,5 e 4 4 ,0 e 4 4 ,5 e 4 5 ,0 e 4 5 ,5 e 4 6 ,0 e 4 6 ,5 e 4
8 5 5 ,5
9 2 4 , 5
8 5 8 , 1
1 0 6 2 , 5 7 1 3 ,4
9 7 7 , 4 4 8 9 , 1
7 9 9 , 1 7 4 3 , 4
4 7 5 , 1 8 9 5 , 4
4 1 9 , 1 8 3 9 , 5 8 6 7 , 5
3 6 5 , 1 6 3 1 , 1
6 8 7 , 5 6 5 7 ,4
4 8 1 , 4 5 2 3 ,8 8 2 9 , 4 9 0 8 , 7
3 4 9 ,1
924.5 855.5
743.4 713.4 Protoceratium reticulatum, North Sea MS
3(1141.6)(1061.5) yessotoxin
Protoceratium reticulatum, Benguela Current MS
3(1175.6)(1095.5) peak @ 6.24 min
Yessotoxin confirmationtion – MS 3
API 4000 QTrap, negative, MS3:
CUR: 10 CAD: Medium TEM: 0
GS1: 10 GS2: 0 ihe: ON IS: -4500
DP -50
AF2 150
CES 0
CE -55
Causative Organisms
Cyanobacteria
(Blue-green algae)
Anabaena flos-aquae
NH H N
H N
HN O NH N
HN
O
O O
O
O O
R2 COOH
R1 COOH
OCH3 NH
O
Anatoxin-a
Anatoxin-a(s)
HN N
HN
O N
P O
HO O
Cylindrospermopsin
N NH HN NH
N
O
O OH
H H
H HO3SO
Microcystin
Hepatotoxins
Neurotoxins
Cyanotoxin detection – Precursor Scan
Prerequisites:
All toxins soluble in the same extraction solvent Characteristic fragment for each toxin group Toxin group (not single compound!) separation Aim:
Survey method for the qualitative detection of cyanobacterial
freahwater toxins
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. J. Mass Spectrom. submitted
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
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 amu CUR: 25CAD: 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
0 5 10 15 20 25 30
Intensity (cps)
0 5e+7 1e+8
m/z (amu)
350 375 400 425 450
0 5e+4 1e+5
Time (min)
3 4 5 6 7 8 9 10
0 5e+7 1e+8
period 1 period 2 period 3
0,0 2,5e+6
prec. of m/z 194
416.5 CYN
doCYN Time (min)
Intensity (cps)
400.4
Intensity (cps)
x 25
[M+H]+
[M+H]+ a)
b)
c)
d)
5.55
5.40
Lyngbya wollei, Australia
MS parameters for detection of CYNs
Precursor Ion mode protonated ion:
[M+H]+
precursor ion (m/z):
194.0
scan range (m/z):
350-450 amu
IS: 5200 V CAD: high level Temp.: 550 °C Gas 1: 50 L h-1 Gas 2: 70 L h-1 CUR: 25 L h-1
CE: 50 eV
DP: 80 eV
0 5 10 15 20 25 30
Intensity (cps)
0 1e+7 2e+7
m/z (amu)
400 450 500 550
0 2e+5 4e+5
Time (min)
10 15 20 25 30
0 5e+6 1e+7
period 1 period 2 period 3
0 1e+5 2e+5
prec. of m/z 135
dmMC-LR Time (min)
Intensity (cps)
492.2
Intensity (cps)
524.3 [M+2H]2+
MC-YR [M+2H]2+
a)
b)
c)
d)
14.79
15.56
MS parameters for detection of MCs
Precursor Ion mode protonated ion:
[M+2H]2+
precursor ion (m/z):
135.0
scan range (m/z):
400-550 amu
IS: 5500 V CAD: high level Temp.: 550 °C Gas 1: 50 L h-1 Gas 2: 70 L h-1 CUR: 15 L h-1
CE: 35 eV
DP: 40 eV
