Supplementary Information
Figure S1. (A–C): Three methanol blanks screened for spirolides (SPX-1 to SPX-I, see
Table 1 in the main article) in the full MS scan at 10 ppm mass error tolerance. Blanks
were included in a batch of sixty samples (shellfish and SPATT samples) every six
samples. (A) First methanol blank; (B) Fifth methanol blank; (C) Tenth methanol blank.
Carryover was discarded by checking the methanol blanks analyzed after the most
concentrated level of the calibration curves, which never showed signal for PnTX-G of
SPX-1 at their retention times. Peaks did not correlate with the expected relative retention
time of the target analytes, but there are no standards available for PnTXs and SPXs other
than PnTX-G and SPX-1.
RT: 0.00 - 12.00 SM: 11G
NL: 0
m/z= 692.4452-692.4590 F: FTMS
+ p ESI Full ms [100.00-1000.00]
MS MeOH_3
100
50
0
100
4.77
NL: 5.13E2
m/z= 694.4608-694.4746 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_3
50
0
100
NL: 0
m/z= 706.4606-706.4748 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_3
50
0
100
NL: 0
m/z= 708.4763-708.4905 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_3
50
0
100
NL: 0
m/z= 710.4566-710.4708 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_3
50
0
100
NL: 0
m/z= 712.4723-712.4865 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_3
50
0
100
NL: 0
m/z= 650.4350-650.4480 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_3
50
0
100
NL: 0
m/z= 652.4507-652.4637 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_3
50
0
0
2
4
6
Time (min)
8
(A)
10
12
Relative Abundance
Mar. Drugs 2014, 12
S2
Figure S1. Cont.
RT: 0.00 - 12.00 SM: 11G
8.20
100
NL: 8.19E3
m/z= 692.4452-692.4590 F: FTMS + p
ESI Full ms [100.00-1000.00] MS
MeOH_6_120728202404
50
0
100
7.12
NL: 4.52E2
m/z= 694.4608-694.4746 F: FTMS + c
ESI Full ms2 [email protected]
[50.00-800.00] MS
MeOH_6_120728202404
50
0
100
NL: 0
m/z= 706.4606-706.4748 F: FTMS + c
ESI Full ms2 [email protected]
[50.00-800.00] MS
MeOH_6_120728202404
50
0
100
NL: 0
m/z= 708.4763-708.4905 F: FTMS + c
ESI Full ms2 [email protected]
[50.00-800.00] MS
MeOH_6_120728202404
50
0
100
3.80
NL: 4.80E2
m/z= 710.4566-710.4708 F: FTMS + c
ESI Full ms2 [email protected]
[50.00-800.00] MS
MeOH_6_120728202404
50
0
100
NL: 0
m/z= 712.4723-712.4865 F: FTMS + c
ESI Full ms2 [email protected]
[50.00-800.00] MS
MeOH_6_120728202404
50
0
100
NL: 0
m/z= 650.4350-650.4480 F: FTMS + c
ESI Full ms2 [email protected]
[50.00-800.00] MS
MeOH_6_120728202404
50
0
100
NL: 0
m/z= 652.4507-652.4637 F: FTMS + c
ESI Full ms2 [email protected]
[50.00-800.00] MS
MeOH_6_120728202404
50
0
0
2
4
6
Time (min)
8
(B)
10
12
Mar. Drugs 2014, 12
S3
Figure S1. Cont.
RT: 0.00 - 12.01 SM: 11G
NL: 0
m/z= 692.4452-692.4590 F: FTMS
+ p ESI Full ms [100.00-1000.00]
MS MeOH_9
100
50
0
100
NL: 0
m/z= 694.4608-694.4746 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_9
50
0
100
NL: 0
m/z= 706.4606-706.4748 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_9
50
0
100
NL: 0
m/z= 708.4763-708.4905 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_9
50
0
100
4.26
NL: 4.83E2
m/z= 710.4566-710.4708 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_9
50
0
100
NL: 0
m/z= 712.4723-712.4865 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_9
50
0
100
NL: 0
m/z= 650.4350-650.4480 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_9
50
0
100
NL: 0
m/z= 652.4507-652.4637 F: FTMS
+ c ESI Full ms2 [email protected]
[50.00-800.00] MS MeOH_9
50
0
0
2
4
6
Time (min)
8
(C)
10
12
Mar. Drugs 2014, 12
S4
Figure S2. Isotopic pattern simulation for PnTXG (C42H64NO7) calculated by Xcalibur® 2.07.
C42H64NO7: C42 H64 N1 O7 pa Chrg 1
100
694.4677
95
90
85
80
75
70
Relative Abundance
65
60
55
50
45
695.4711
40
35
30
25
20
15
696.4744
10
5
697.4778 698.4811 699.4820 700.4854 701.4887 702.4896
0
695
696
697
m/z
694.4677
695.4711
696.4744
697.4778
696.4720
695.4740
697.4753
695.4648
696.4774
695.4719
696.4681
698.4811
698.4787
696.4753
697.4807
697.4715
697.4787
699.4820
699.4845
698.4841
698
699
m/z
Intensity
618847.0
281117.8
62330.1
8988.6
8902.1
4555.2
4043.9
2285.8
2069.3
1650.1
1038.3
947.9
896.6
749.6
458.8
230.2
166.2
129.3
77.9
66.2
700
701
Relative
100.00
45.43
10.07
1.45
1.44
0.74
0.65
0.37
0.33
0.27
0.17
0.15
0.14
0.12
0.07
0.04
0.03
0.02
0.01
0.01
702
703
Mar. Drugs 2014, 12
S5
Figure S3. Contour Plotof LC-MS precursor scan for m/z164.2 (CE 45 V) with a Triple Quadrupole QTRAP 3200®. Sample of mussel from
Alfacs Bay (May 2012) with 60 μg/kg free PnTXG. The characteristic pattern with diagonal lines is evident in the figure, which could be
consistent with an acylation of PnTXG with fatty acids; although PnTXG esters described in literature [1] were formed by longer chains with
m/z values between m/z 904 (C14:0) and m/z 1034. (C24:5). The low concentration of these compounds constrained their characterization by MS2.
Contour Plot of 130126_pre ion.wiff, Sample 1, (0.012 min to 11.998 min)
Max. 3.6e4 cps.
0.0% - 100.0%
No Data
1060
1040
1020
1000
980
960
940
920
m /z , a m u
900
880
860
840
820
800
780
760
740
720
700
680
660
640
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
Time, min
9.0
9.5
10.0
10.5
11.0
11.5
Mar. Drugs 2014, 12
S6
Table S1. Assigned mass errors (Δ in ppm) to the molecular formula and theoretical m/z,
and ion intensities on the precursor ion (A) and the first three isotopes (A + m) of PnTX-G
(n = 2 for 19 and 1.9 ng/mL; n = 1 for 190 and 0.2 ng/mL). Mass errors larger than 5 ppm
are in bold, also the intensities associated with the analysis that yielded these high mass
errors. When intensity were below 1E4, mass accuracy dropped below 5 ppm.
Concentration—PnTX-G (ng/mL)
190
19
1.9
Δ (ppm)
0.2
(m/z 694.4677)
−0.1
−0.4
−0.1
0.4
(m/z 695.4711)
−0.1
−0.5
−1.2
−20.6
(m/z 696.4744)
0.1
−0.1
−1.4
−22.7
(m/z 697.4778)
0.7
1.1
−50.5
15.2
Isotopes—PnTX-G (m/z)
A (Prec ion)
C42H64NO7
A+1
13
C42 CH64NO7
A+2
C4213C2H64NO7
A+3
13
C42 C3H64NO7
Intensity Counts
A (Prec ion)
C42H64NO7
A+1
13
C42 CH64NO7
A+2
13
C42 C2H64NO7
A+3
C4213C3H64NO7
(m/z 694.4677)
1.56 × 107
1.95 × 106
2.17 × 105
1.74 × 104
(m/z 695.4711)
7.24 ×·106
8.88 ×·105
1.01 ×·105
9.36 ×·103
(m/z 696.4744)
1.80 ×·106
2.18 ×·105
1.57 ×·104
8.10 ×·102
(m/z 697.4778)
3.13 ×·105
2.81 ×·104
6.74 ×·103
7.24 ×·102
Table S2. Differences in (%) in intensity ratios between the isotopes (A + m) and the
precursor ion (A) compared to the simulated isotopic pattern for PnTX-G (n = 2 for 19 and
1.9 ng/mL; n = 1 for 190 and 0.2 ng/mL). For low concentrations (1.9 and 0.2 ng/mL
PnTX-G) the difference between the simulated isotopic ratios and the measured isotopic
ratios is very evident (over 35%).
Isotopes–PnTXG (m/z)
A + 1/A (Prec ion)
A + 2/A (Prec ion)
A + 3/A (Prec ion)
Concentration—PnTX-G (ng/mL)
190
19
1.9
0.2
−2
0
−2
−19
−15
−11
38
54
−39
0
−175
−187
Mar. Drugs 2014, 12
S7
Table S3. Isotopic pattern analysis in sample MUS120523. Assigned mass errors (in ppm)
to the molecular formula and theoretical m/z, and ion intensities on the precursor ion (A)
and the first three isotopes (A + m) of PnTX-G. Differences in (%) in intensity ratios
between the isotopes (A + m) and the precursor ion (A) compared to the simulated isotopic
pattern for PnTX-G. Only A + 1 could be measured with appropriate mass accuracy, and
its relative intensity compared to the precursor ion (A) similar to the simulated relative
intensity berween A + 1 and A (12.5% difference). A + 2 could not be measured accurately
and A + 3 was not found.
Isotopes—PnTXG (m/z)
A (Prec ion)
C42H64NO7
A+1
C4213CH64NO7
A+2
13
C42 C2H64NO7
A+3
13
C42 C3H64NO7
m/z Found
Sample MUS120523
Intensity
Mass Error
Counts
(ppm)
% Diff. Int.
Ratios
(m/z 694.4677)
694.4669
1.63 ×·105
1.15
(m/z 695.4711)
695.4702
6.46 ×·104
1.29
12.5
(m/z 696.4744)
696.4883
2.11 ×·104
−19.96
−28.7
(m/z 697.4778)
Not found
Not found
Not found
Not found
Reference
1.
McCarron, P.; Rourke, W.; Hardstaff, W.; Pooley, B.; Quilliam, M. Identification of pinnatoxins
and discovery of their fatty acid ester metabolites in mussels (Mytilus edulis) from eastern
Canada. J. Agric. Food Chem. 2012, 60, 1437–1446.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).