Analytical and Bioanalytical Chemistry

Electronic Supplementary Material

Determination of lewisite metabolite 2-chlorovinylarsonous acid in urine by

use of dispersive derivatization liquid-liquid microextraction followed by gas
chromatography-mass spectrometry

Mohammad Taghi Naseri, Mojtaba Shamsipur, Mehran Babri, Hamid Saeidian, Mansour Sarabadani,
Davood Ashrafi, NaserTaghizadeh

1
Fig. S1. EI-MS total ion chromatogram obtained from a solution of 500 µg/ml of 2-
chlorovinyldichloroarsine after Butanethiol derivatization. Lewisite purity was checked by GC-MS (as
Butanethiol derivative) and determined through measurement of chloride ion concentration after aqueous
hydrolysis (Volhard’s Method)

Abundance

T IC : 9 0 0 4 0 8 -L T . D
2400000 1 9 .6 3

2200000
L1-(BuS)2
2000000

1800000

1600000

1400000

1200000

1000000

800000

600000
L2-BuS
400000

200000
1 7 .2 6

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
T im e - - >

Determination of cysteine in human urine

A volume of 300 µL urine sample was diluted to 4.0 mL with water. The diluted urine sample,

was spiked with standard solution of Cys (300 µg L-1) and norleucine as an internal standard

(200 µg L-1). Then it was placed in a 10 mL capacity conical-bottom glass centrifuge tube and 80

µL of 12 M NaOH was added to obtain the desired pH. A mixture of acetonitrile (200 µL),

pyridine (200 µL ) and carbon tetrachloride (50 µL) was rapidly injected into the aqueous

solution by a 500 µL syringe (Hamilton, Reno, NV, USA). The tube was tightly capped and

shaken by hand vigorously for about 15 s to mix the phases which resulted in fine dispersed

droplets. An aliquot of 250 µL isobutyl chloroformate as derivatizing agent was added and

2
vigorously shaken for about 30 s. The liberated carbon dioxide was allowed to escape from the

tube during shaking. The resulting solution was left to stand for 1 min so that the cysteine, N.S-

Bis(isobutoxycarbonyl) isobutyl ester was formed. The mixture was then centrifuged at 5000

rpm for 3 min to separate the phases; the fine droplets of extraction solvent sedimented at the

bottom of the centrifuge tube. The aqueous (top) phase was removed using a glass Pasteur

pipettes, quantitatively, ensuring that no small water bubbles were left on the bottom layer.

Finally, one µL of the sediment phase was injected into GC-MS for analysis. An overlay

chromatogram (spike and without spike), CI and EI mass spectra of cysteine, N.S-

Bis(isobutoxycarbonyl) isobutyl ester are shown in Figure. S2.

3
Fig. S2. Overlay total ion chromatogram (TIC), EI and CI mass spectra of cysteine, N.S-
Bis(isobutoxycarbonyl) isobutyl ester

Abundance

T IC : 9 0 0 6 2 8 N C Y 3 .D
T IC : 9 0 0 6 2 8 N C Y 4 .D
2200000

2000000

1800000
Spike urine with Cys
1600000

1400000

1200000

1000000

800000
Non-spike urine
600000

400000

200000

1 9 .9 5 2 0 .0 0 2 0 .0 5 2 0 .1 0 2 0 .1 5 2 0 .2 0 2 0 .2 5 2 0 .3 0 2 0 .3 5 2 0 .4 0 2 0 .4 5 2 0 .5 0 2 0 .5 5
T im e - - >

57
100

O O

EI-MS spectrum S
O
50
O
NH O
220
41 O
102 120

[M-101]+
276
74
160 176 204
130
148
87 188 230 244 260
0
30 50 70 90 110 130 150 170 190 210 230 250 270 290 310 330 350 370 390
( n a se ri n ist) C y ste in e ‐D iiso b u to xy c a rb o n y l iso b u ty l e ste r

378
100
[M+H]+
CI-MS spectrum

O O

50
S
O

O
NH O
204
O
244 304
278 416
0
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420
( n a se ri n ist) C y ste in e ‐D iiso b u to xy c a rb o n y l iso b u ty l e ste r

4
Fig. S3. Negative and positive LC-ESI-MS chromatograms and mass spectra CVAOA and BCVAOA in
aqueous solution after oxidation of CVAA and BCVAA by hydrogen peroxide

LC-(-) ESI-MS
CVAOA

BCVAOA

Negative ESI [M+H]+ Positive ESI OH

- +
[M-H]- O As OH
Negative ESI
As O Cl OH
[2M-H-H2O]- [M+Na]+
Cl OH m/z 187

m/z 185
[2M-H]- [2M+H-H2O]+

- Cl
O [M+H]+
Cl
OH
[M-H]- As
As
O Cl [M+Na]+ + Cl
HO
m/z 229
m/z 231

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Fig. S4. a) Comparison of GC-MS peak area of CVAA-EDT vs. different dispersive solvents: acetonitrile
(AN), acetone (AC), methanol (MeOH) and ethanol (EtOH) using CHCl3 as extractive solvent, b) Effect
of MeOH volume on the average peak area of CVAA-EDT

6,E+07
5,E+07 a b
5,E+07
4,E+07
4,E+07 4,E+07
3,E+07
Peak Area

Peak Area
3,E+07 3,E+07
2,E+07
2,E+07 2,E+07
1,E+07
5,E+06 1,E+07
0,E+00
AC AN MeOH EtOH 0,E+00
0 250 500 750
Disperser Solvent
MeOH Volume (µL)

Fig. S5. GC-MS peak area of CVAA-EDT vs. pH

5,E+07
Peak Area

4,E+07

3,E+07

2,E+07

1,E+07

0,E+00
0 5 10 15
pH

6
Fig. S6. Overlay of TIC of standard CVAA in spiked water samples, derivatized with EDT in the
presence of cysteine (0, 5 and 10 µg L-1) as interference

A b u n d a n c e

T IC : 9 0 0 8 1 1 L 1 6 9 .D

2 1 0 0 0 0 0 10 ppm Cys T IC : 9 0 0 8 1 1 L 1 7 1 .D
T IC : 9 0 0 8 1 1 L 1 7 2 .D
2 0 0 0 0 0 0

5 ppm Cys
1 9 0 0 0 0 0

1 8 0 0 0 0 0

1 7 0 0 0 0 0

1 6 0 0 0 0 0

1 5 0 0 0 0 0
0 ppm Cys
1 4 0 0 0 0 0

1 3 0 0 0 0 0

1 2 0 0 0 0 0

1 1 0 0 0 0 0

1 0 0 0 0 0 0

9 0 0 0 0 0

8 0 0 0 0 0

7 0 0 0 0 0

6 0 0 0 0 0

5 0 0 0 0 0

4 0 0 0 0 0

3 0 0 0 0 0

2 0 0 0 0 0

1 0 0 0 0 0

0
1 3 .0 6 1 3 .0 8 1 3 .1 0 1 3 .1 2 1 3 .1 4 1 3 .1 6 1 3 .1 8 1 3 .2 0 1 3 .2 2 1 3 .2 4 1 3 .2 6
T im e - - >