Quantitation and Identification of Organotin Compounds in
Food, Water, and Textiles Using LC-MS/MS
Yun Yun Zou and André Schreiber
AB SCIEX Concord, Ontario (Canada)
Overview
Organotin compounds are chemicals composed of tin linked to
hydrocarbons, used in industrial materials and various biocides
and fungicides. As a result, organotin compounds can enter the
environment through a number of channels, and can often be
found in seawater, seafood, fruits and vegetables, and consumer
goods. Due to the toxicity of these compounds, there is a need
for analytical methods allowing accurate quantitation and
identification. Here we present an LC-MS/MS method to
measure tributyltin, fentin, cyhexatin, and fenbutatin oxide in
different matrices. Triphenyl phosphate was used as the internal
standard.
Spiked apple, potato, synthetic seawater, and textile samples
were prepared using a quick and easy acetonitrile extraction.
Organotin compounds were detected using an AB SCIEX
®
4000 QTRAP system with Electrospray Ionization (ESI) using
Multiple Reaction Monitoring (MRM). Detection limits were
determined to be well below regulated levels, enabling extra
dilution of the sample extract to minimize possible matrix effects.
Introduction
Organotin (organostannic) compounds are chemical compounds
comprised of tin with hydrocarbon substituents. Organotin
compounds are widely used as additives in plastic material,
wood preservatives, marine biocides, and agricultural pesticides.
Tri-substituted organotin compounds were previously widely
used as antifouling agents in paints on ships. However, such
paints were found to release organotin compounds into the
aquatic environment, where they can accumulate in sediments
and organisms or degrade to less substituted toxic compounds.
Studies have shown that trace amounts of organotin compounds
can have significant detrimental effects on aquatic organisms.
For instance, tributyltin (TBT), present in sea water at ng/L
levels, has been identified as an endocrine disruptor promoting
harmful effects on aquatic organisms. Therefore, the use of
organotin compounds in antifouling paints is prohibited or
1-3
restricted in many countries.
to pose a risk to human health, particularly for children.
Therefore, the use of tri-substituted and di-substituted organotin
compounds, including TBT, tributyltin (TPhT), dibutyltin (DBT),
4-5
and dioctyltin (DOT) in consumer products is restricted.
Finally, organotin compounds enter the human diet through
contaminated seafood and the use as agricultural pesticides.
International maximum residue limits (MRL) have been
established by Codex Alimentarius and the EU for many food
commodities, with some MRL as low 50 μg/kg.
Traditionally gas chromatography coupled to mass spectrometry
(GC-MS) was used for analysis of organotin compounds.
However, the analysis by GC requires time consuming
derivatization, because of poor compound volatility, and long
chromatographic run times. Liquid chromatography with tandem
mass spectrometry (LC-MS/MS) allows simplifying sample
preparation and shortening run times due to increased selectivity
and sensitivity and, thus, is evolving as a preferred technique for
the analysis of organotin compounds.
The use of organotin compounds in consumer products, such as
textiles, footwear, wall and floor coverings, etc., has been found
p1
Method Details
Sample Preparation
TBT chloride, fentin acetate, cyhexatin and fenbutatin oxide were
purchased from Sigma-Aldrich and spiked into four matrices
(apple, potato, synthetic seawater (drinking water with 35 g salt
per liter), and textile material). Triphenyl phosphate (TPP) was
used as the internal standard.
CH3
Hom ogenize and weigh 10 g
of apple and potato.
Shred and weigh 1 g
of textile m aterial.
Add internal standard
(50 μL of 10 μg/m L TPP).
Add internal standard
(50 μL of 10 μg/m L TPP).
Transfer 1 m L of water
sam ple into autosam pler vial.
Add 10 m L acetonitrile and
shake vigorously for 1 m inute.
Add 20 m L acetonitrile and
sonicate for 5 m inutes.
Add internal standard
(10 μL of 10 ng/m L TPP).
Centrifuge at 5 rpm for 5 m in.
Centrifuge at 5 rpm for 5 m in.
(Dilute water sam ple to
reduce possible m atrix
effects.)
Transfer 100 μL of the extract
into autosam pler vial and add
900 μL water.
Transfer 100 μL of the extract
into autosam pler vial and add
900 μL water.
Inject directly.
O
Sn
Cl
Sn
O
Sn
CH3
OH
H3C
Figure 2. Sample preparation protocols for the analysis of organotin
compounds in fruit and vegetable, textiles, and water
CH3
H3C
CH3
H3C
Sn
O
CH3
Sn
O
O
P
O
MS/MS Detection
O
®
3
3
Figure 1. Target organotin compounds: TBT chloride, fentin acetate,
cyhexatin, fenbutatin oxide, and internal standard triphenyl phosphate
(top left to bottom right)
The AB SCIEX 4000 QTRAP LC/MS/MS system with Turbo V™
source and ESI probe was used. All the analytes and internal
standard were detected in positive polarity using MRM for best
selectivity and sensitivity. Two MRM transitions were monitored
for each compound to allow quantitation and identification using
the characteristic MRM ratio. The Scheduled MRM™ algorithm
was activated for best data quality (Table 1).
Spiked samples were extracted using acetonitrile and diluted 10x
with LC grade water prior to LC-MS/MS analysis. The spiked
synthetic seawater was directly injected for detection of
organotin compounds. Note that additional dilution is possible
depending on required limits of detection to reduce possible
matrix effects (Figure 2).
The data was processed in MultiQuant™ software version 2.1.
UHPLC Separation
A Shimadzu UFLCXR system was used with a Phenomenex
Kinetex 2.6u C18 50x3mm column at 40ºC. A gradient of water
with 2% formic acid + 5 mM ammonium formate and methanol
with 2% formic acid + 5 mM ammonium formate at a flow rate of
800 μL/min resulted in a total run time of 12 minutes.
The injection volume was set to 20 μL for apple and potato
extracts and 50 μL for textile extracts and synthetic seawater.
Table 1. MRM transitions and retention times (RT) of targeted organotin
compounds
Organotin compound
Q1 (amu)
Q3 (amu)
RT (min)
TBT 1
291.0
123.0
3.8
TBT 2
291.0
235.1
3.8
Fentin 1
351.0
120.0
3.0
Fentin 2
351.0
197.0
3.0
Cyhexatin 1
369.0
205.0
5.3
Cyhexatin 2
369.0
287.1
5.3
Fenbutatin oxide 1
519.1
351.0
6.2
Fenbutatin oxide 2
517.1
349.0
6.2
TPP (internal standard)
326.9
152.1
4.4
p2
Table 2. Signal-to-noise (S/N) in different matrices
Chromatography conditions were important for successful
determination of organotin compounds by LC-MS/MS. Organotin
compounds are known for strong interaction with reversed phase
material resulting in peak broadening. A strong acidic mobile
phase was used to reduce this effect and to optimize peak
8
shape.
Two chromatographic interferences were observed for TBT in all
matrices. Thus, stable retention times and good separation was
important. A core-shell column (Phenomenex Kinetex) was used
for improved UHPLC performance while operating at reduced
column pressure (Figure 3).
Organotin
compound
Apple
(2 μg/kg)
Potato
(2 μg/kg)
Textile
(0.1 mg/kg)
Seawater
(50 ng/L)
TBT 1
105
71
93
53
Fentin 1
355
315
209
186
Cyhexatin 1
240
197
51
133
Fenbutatin oxide 1
339
377
66
176
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 4 (apple 2ngml 1/10) of 21010117-apple & p...
3.72
2200
Max. 2499.1 cps.
Cyhexatin
TBT
2400
Apple extract
5.27
3.83
Fenbutatin oxide
2000
6.13
1800
1600
Intensity, cps
Results and Discussion
Fentin
1400
3.96
2.93
1200
1000
800
600
400
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 4 (SW) of 20120118-salty water test.wiff (Tu...
Max. 4000.4 cps.
200
3.73
4000
0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time, min
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 10 (potato 2ngml 1/10) of 21010117-apple &...
Blank synthetic seawater
3500
3.84
1800
Two chromatographic interferences for TBT
are separated well from the target analyte
2500
3.97
2000
1600
3.73
TBT
5.27
7.0
7.5
8.0
Max. 1826.2 cps.
Fenbutatin oxide
6.14
Potato extract
Cyhexatin
3.97
1400
Fentin
1200
1500
Intensity, cps
Intensity, cps
3000
6.5
1000
2.95
1000
800
500
600
0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time, min
XIC of +MRM (12 pairs): 326.900/77.000 amu Expected RT: 4.4 ID: triphenyl phosphate 1 from Sample 4 (SW) of 20120118-salty water test.wiff (Tu...
6.5
7.0
8.0
400
Max. 1.6e5 cps.
200
7.5
4.41
1.6e5
0
3.0
3.5
4.0
4.5
5.0
Time, min
5.5
6.0
6.5
7.0
7.5
8.0
Figure 4. Apple (top) and potato (bottom) sample spiked at 2 μg/kg and
diluted 10x after extraction
1.2e5
Intensity, cps
2.5
Internal standard (TPP)
1.4e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 3 (textile 0.1mg/kg 1/10) of 20120118-textile...
0.0
2.5
3.0
3.5
4.0
4.5
5.0
Time, min
5.5
6.0
6.5
7.0
7.5
8.0
3.84
7548
Max. 7547.7 cps.
TBT
Textile extract
7000
6000
Fentin
5000
Intensity, cps
Figure 3. Blank synthetic seawater, two chromatographic interferences
for TBT are separated well from the target analyte (top) and internal
standard (bottom)
2.93
Cyhexatin
4000
5.27
3000
Fenbutatin oxide
6.13
2000
3.74
3.98
1000
The achieved Signal-to-noise (S/N) ratios are listed in Table 1.
S/N values were measured in MultiQuant™ software after
applying a 2x Gaussian smooth. S/N values were used to
estimate limits of quantitation (LOQ) for all analytes in each
matrix.
0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time, min
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 7 (SW 50ppt) of 20120118-salty water test....
3.73
1903
1800
6.5
7.0
7.5
8.0
Max. 1903.4 cps.
Synthetic seawater
TBT
1600
1400
3.97
1200
Intensity, cps
Apple, potato, textile, and synthetic seawater samples were
spiked at different concentrations, extracted, and analyzed using
the fast LC-MS/MS method. Example chromatograms are shown
in Figures 4 and 5.
Cyhexatin
Fenbutatin oxide
5.27
1000
800
Fentin
600
6.14
2.95
400
200
0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Time, min
5.5
6.0
6.5
7.0
7.5
8.0
Figure 5. Textile material spiked with 0.1 mg/kg and diluted 10x after
extraction (top) and synthetic seawater spiked at 50 ng/L and analyzed
by direct injection (50 μL)
p3
Table 3. Estimated limits of quantitation (LOQ) in different matrices
based on S/N of 10
Fentin
Organotin
compound
Area Ratio
Area Ratio
TBT
TBT
Fentin
Cyhexatin
Area Ratio
Area Ratio
Fenbutatin oxide
Figure 6. Calibration lines of organotin compounds in apple matrix (2 to
100 μg/kg)
Fentin
Textile
(μg/kg)
Seawater
(ng/L)
0.2
0.3
10
10
< 0.1
< 0.1
< 10
< 10
0.1
0.1
20
< 10
< 0.1
< 0.1
15
< 10
Fenbutatin oxide
Area Ratio
Cyhexatin
The linear dynamic range was evaluated from 2 to 100 μg/kg for
apple and potato, from 0.1 to 1 mg/kg for textiles, and from 50 to
2000 ng/L for seawater. Example calibration lines of all four
organotin compounds in apple and synthetic seawater are shown
in Figures 6 and 7.
Repeatability was found to be less than 15% coefficient of
variation (%CV) and accuracy between 85 and 115% for all
compounds at all concentrations (Table 4).
Area Ratio
TBT
Area Ratio
Potato
(μg/kg)
Fenbutatin oxide
Cyhexatin
Area Ratio
Apple
μg/kg
Figure 7. Calibration lines of organotin compounds in synthetic seawater
(50 to 2000 ng/L)
Table 4. Repeatability (%CV) and accuracy of organotin compounds at the lowest point of the calibration line
Apple (2 μg/kg)
Potato (2 μg/kg)
Textile (0.1 mg/kg)
Seawater (50 ng/L)
Organotin compound
%CV
Accuracy (%)
%CV
Accuracy (%)
%CV
Accuracy (%)
%CV
Accuracy (%)
TBT
10.0
97.0
13.9
86.4
7.3
95.6
6.3
113.1
Fentin
9.9
101.4
12.4
96.8
4.7
95.8
7.9
112.6
Cyhexatin
5.9
108.5
2.4
88.4
3.6
93.3
4.2
115.0
Fenbutatin oxide
11.4
104.4
11.8
99.5
13.2
97.3
3.6
107.4
p4
Compound identification was achieved using the
‘Multicomponent’ query in MultiQuant™ software. This query
automatically calculates and compares MRM ratios for
identification and highlights concentrations above a user
specified residue level. Examples of the result table and peak
review after running the query file are shown in Figures 8 and 9.
Summary
A quick, easy, and robust LC-MS/MS method for the
determination of different organotin compounds in food,
seawater, and textile materials was developed. The method
allows accurate and reproducible quantitation using the
selectivity and sensitivity provided by the AB SCIEX
®
4000 QTRAP system operated in MRM mode. Detection limits
well below regulated levels allow sample extract dilution to
minimize possible matrix effects. Confident compound
identification was achieved through the automatic calculation of
MRM ratios using the ‘Multicomponent’ query in MultiQuant™
software.
References
1
2
Figure 8. Automatic compound identification using the ‘Multicomponent’
query (example cyhexatin in potato)
3
4
5
6
7
8
K. Fent: ‘Ecotoxicology of organotin compounds’ Crit. Rev.
Toxicol. 26 (1996) 1-117
E. Gonzalez-Toledo et al.: ‘Detection techniques in
speciation analysis of organotin compounds by liquid
chromatography’ Trends Anal. Chem. 22 (2003) 26-33
Regulation (EC) ‘on the prohibition of organotin compounds
on ships’ No 782/2003
Commission Decision ‘restrictions on the marketing and use
of organostannic compounds’ 2009/425/EC
International Association for Research and Testing in the
Field of Textile Ecology: OEKO-TEX Standard 100, Edition 4
(2012)
http://www.codexalimentarius.org/standards/pesticidemrls/en/
Council Directive ‘maximum levels for pesticide residues’
96/32/EC
EU Reference Laboratory for Single Residue Methods:
‘Analysis of Organotin Compounds via QuEChERS and LCMS/MS – Brief Description’ www.crl-pesticides.eu
Figure 9. Automatic compound identification using the ‘Multicomponent’
query (example fentin in textile)
For Research Use Only. Not for use in diagnostic procedures.
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