SUPPLEMENTARY MATERIAL FOR
Arsenolipids in oil from blue whiting Micromesistius poutassou – evidence
for arsenic-containing esters
Mojtaba S. Taleshi1,2, Georg Raber1, John S. Edmonds1, Kenneth B. Jensen1, and Kevin A.
Francesconi1*
1
Institute of Chemistry-Analytical Chemistry, University of Graz, Universitaetsplatz 1, 8010
Graz, Austria
2
Department of Marine Chemistry, Faculty of Marine Science, University of Mazandaran,
Babolsar, Iran
*Corresponding author
kevin.francesconi@uni-graz.at
The following Supplementary material accompanies this paper:
Figure S1. Classification of arsenolipids.
Figure S2. Flow diagram for fractionation of arsenolipids in blue whiting oil.
Figure S3. HPLC/ESI-MS chromatogram of arsenolipids in the isopropanol extract of bluewhiting oil, post-silica column.
Figure S4. High resolution accurate mass spectrum for As-HC440 in blue-whiting oil.
Figure S5. High resolution accurate mass spectrum for As-HC442 from blue-whiting oil.
Figure S6. High resolution accurate mass spectrum for As-HC444 in blue-whiting oil.
Figure S7. High resolution accurate mass spectrum for As-HC542 in blue-whiting oil.
Figure S8. HPLC/ESI chromatograms of As-HC444).
Figure S9. High resolution mass spectrum for synthesized As-HC 444.
Figure S10. HPLC/ICPMS chromatogram of the less polar arsenolipids (iso-propanol
fraction) of the blue-whiting oil.
Figure S11. HPLC/ESI-MS chromatograms of thio analogues of arsenolipids
1
Arsenolipids
Polar
Less polar
Non-polar
(As fatty acids
+ smaller As hydrocarbons)
(As hydrocarbons
+ unknowns)
(Unknown structures)
40000
Arsenic-containing hydrocarbons
Arsenic-containing fatty acids
intensity
30000
Low-polarity arsenolipids (unknown)
20000
10000
0
0
10
20
30
40
50
60
70
80
90
Time (min)
Figure S1. Classification of arsenolipids.
On the basis of their extraction by solvent partitioning, and their retention on reversed-phase
HPLC, arsenolipids in fish oils can be categorized into three broad polarity groups: polar
(mainly arsenic-containing fatty acids), less-polar (mainly arsenic-containing hydrocarbons),
and non-polar (containing unknown arsenolipids). In this example of crude blue whiting oil,
HPLC conditions were: Atlantis dC18 (1501.0 mm, 5 µm) at 30 oC and a mobile phase
comprising a mixture of 10 mM NH4OAc pH 6.0 and ethanol at a flow rate of 100 µL min-1.
The chromatography was performed with linear gradient elution: 060 min for 35%95%
ethanol).
2
Blue-Whiting Oil
2.16 µg As/g
200.7 g, 434  2 µg As
Dissolved in hexane (500 mL)
Extracted with MeOHaq (300 mL)
Hexane (1) fraction
MeOH (1) fraction
199 g, 333  7 µg As
1.0 g, 103  6 µg As
MeOHaq (300 mL)
DEAE Sephadex
MeOH (2) fraction
Hexane (2) fraction
1.0 g, 45  2 µg As
195 g, 297  7 µg As
Basic/neutral fraction
Acidic fraction
145 mg, 67  3 µg As
170 mg, 14  0.3 µg As
EtOHaq (300 mL)
EtOH fraction
Hexane (3) fraction
1.2 g, 37  1 µg As
190 g, 260  4 µg As
Hexane (3) fraction
184 g, 252  3 µg As
Isopropanolaq (IPA) (2300 mL)
IPA fraction
Hexane (4) fraction
9.5 g, 130  2 µg As
173 g, 143  9 µg As
Figure S2. Flow diagram for fractionation of arsenolipids in blue whiting oil.
3
a) Measured in SIM mode at m/z 91
60000
E
Intensity
40000
20000
A
B C
D
F
0
0
5
10
15
20
25
30
35
40
45
50
Time (min)
+
b) Measured at [M+H]
150000
Arsenolipid 542
Arsenolipid 442
Intensity
100000
Arsenolipid 444
Arsenolipid440
50000
0
0
5
10
15
20
25
30
35
40
45
50
Time (min)
Figure S3. HPLC/ESI-MS chromatogram of arsenolipids in the isopropanol extract of
blue-whiting oil, post-silica column. a) Measured at m/z 91 with fragmentor voltage 400 V
and b) Performed in scan mode (m/z 100-1000) with fragmentor voltage 150 V and the
masses were extracted from scan mode. HPLC conditions: Atlantis dC18 (150 × 4.6 mm, 5
µm) at 30 oC and a mobile phase comprising a mixture of 92% ethanol-buffer (100 mM
NH4OAc, pH 5.0) (85+15, v/v) and 8% chloroform at a flow rate of 0.5 mL min-1 (isocratic
elution).
4
O
H3C
As
CH3
8
As-HC440
kbj09070808_108_Recal_triton381 #1
RT: 0,01
T: FTMS + c NSI Full ms [120,00-1000,00]
441,3075
100
AV: 1
NL: 1,05E6
95
Arsenolipid 440
90
85
80
75
70
65
60
55
50
45
40
35
30
443,3141
25
20
467,1022
15
10
455,3346
413,2664
416,3737
432,2383
457,3024
473,2973
5
487,3609
423,3236
495,3296
0
410
420
430
440
450
m/z
460
470
480
490
500
Figure S4. High resolution accurate mass spectrum for As-HC440 in blue-whiting oil.
Molecular formula C25H49AsO: calculated for [M+H]+ 441.3077; found 441.3075; Δm/m =
0.5 ppm.
5
O
H3C
As
9
CH3
As-HC442
kbj09070806_108_Recal #72-146 RT: 1,36-2,44
T: FTMS + c NSI Full ms [150,00-2000,00]
215,1260
100
AV: 75
NL: 1,98E7
443,3235
95
239,1623
90
Arsenolipid 442
381,2981
85
80
75
70
65
60
353,2667
55
50
393,2981
45
40
35
30
309,2042
25
20
301,1416
15
255,1572
10
273,1678
467,1026
5
325,2355
416,3740
487,3612
0
250
300
350
m/z
400
450
Figure S5. High resolution accurate mass spectrum for As-HC442 from blue-whiting
oil. Molecular formula C25H51AsO: calculated for [M+H]+ 443.3234; found 443.3235; Δm/m
< 0.3 ppm.
6
O
H3C
As
10
CH3
AS-HC444
kbj09070807_108_Recal_triton381 #1 RT: 0,01 AV: 1
T: FTMS + c NSI Full ms [120,00-1000,00]
445,3385
100
NL: 5,95E6
95
90
85
80
Arsenolipid 444
75
70
65
60
55
50
45
40
35
30
447,9147
25
20
462,1466
471,3542
15
10
5
418,3528
404,3734
482,3243
437,2663
487,2795
0
400
420
440
460
m/z
480
500
515,3921
520
Figure S6. High resolution accurate mass spectrum for As-HC444 in blue-whiting oil.
Molecular formula C25H53AsO: calculated for [M+H]+ 445.3390; found 445.3385; Δm/m =
1.1 ppm.
7
O
H3C
As
11
CH3
As-HC542
kbj09070805_108_Recal_triton381 #1 RT: 0,01 AV: 1
T: FTMS + c NSI Full ms [150,00-2000,00]
543,3545
100
NL: 5,91E6
95
Arsenolipid 542
90
85
80
75
70
65
60
55
50
45
40
544,3578
35
30
25
20
536,1658
15
556,4939
10
531,3544
5
537,1647
522,3558
555,4236
545,3611
559,3496
565,3364
550,4675
575,3446
0
520
530
540
550
m/z
560
570
Figure S7. High resolution accurate mass spectrum for As-HC542 in blue-whiting oil.
Molecular formula C33H55AsO: calculated for [M+H]+ 543.3547; found 543.3545; Δm/m =
0.4 ppm.
8
O
H3C
As
10
CH3
AS-HC444 (synthesised sample)
kbj_AsLipid_445_+1pmPPG_090929174233_Recal #204-219
T: FTMS + p NSI Full ms [100,00-1000,00]
445,3389
100
RT: 2,97-3,11
AV: 16
NL: 7,87E6
95
90
85
80
75
70
65
447,2929
60
55
50
45
40
35
30
25
20
448,2963
15
10
5
0
435,3457
435
441,3380
440
449,2986
450,3010
443,3234
445
450
455,3162
457,3018
455
m/z
Figure S8. High resolution mass spectrum for synthesized As-HC 444. Molecular
formula C25H53AsO: calculated for [M+H]+ 445.3390; found 445.3389; Δm/m < 0.3 ppm.
9
Figure S9. HPLC/ESI-MS chromatograms of As-HC444). Purified As-HC444 from the
isopropanol-fraction of blue-whiting oil (gray line) and the same sample spiked with
synthesized As-HC444 (solid line). HPLC conditions: Atlantis dC18 (150 × 1.0 mm, 5 µm) at
30 oC and a mixture of 85% of ethanol-buffer (100 mM NH4OAc, pH 5.0) (9+1) and 15%
chloroform (isocratic elution) at a flow rate of 100 µL min-1. Selected ion monitoring was
performed for [M+H]+ at m/z 445 with fragmentor voltage 150 V.
10
Figure S10. HPLC/ICPMS chromatogram of the less polar arsenolipids (iso-propanol fraction)
of the blue-whiting oil. a) Pre-silica column and b) post-silica column. The newly appeared
arsenolipids showed peaks between those of arsenic-containing fatty acids (e.g. As-FA362) and
arsenic-containing hydrocarbons (e.g. As-HC332). HPLC conditions, for both a) and b) were: Atlantis
dC18 (1501.0 mm, 5 µm) at 30 oC and a mobile phase comprising a mixture of 10 mM NH4OAc pH
6.0 and ethanol at a flow rate of 100 µL min-1. The chromatography was performed with linear
gradient elution: 060 min for 35%95% ethanol.
11
Figure S11. HPLC/ESI-MS chromatograms of thio analogues of arsenolipids. (a) polar
fraction of blue whiting oil post-silica column after elution with H2S/acetone; and (b) thioarsenolipid standards prepared by bubbling of H2S gas into standard solutions of oxo
arsenolipids As-FA362, As-FA436, As-FA390, and As-HC404. HPLC conditions: Atlantis
dC18 (150 × 1.0 mm, 5 µm) at 30 oC and a mobile phase comprising a mixture of 10 mM
NH4OAc (pH 6.0) and ethanol at a flow rate of 100 µL min-1. The chromatography was
performed with linear gradient elution: 060 min with 35%95% ethanol. Selected ion
monitoring was performed for [M+H]+ at m/z 379, 453, 407, 421 and 377 with fragmentor
voltage 150 V. The slight differences in retention times are attributed to small changes in
column performance between the HPLC runs.
12