-
p 1
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 restricted in many countries.1-3
The use of organotin compounds in consumer products, such as
textiles, footwear, wall and floor coverings, etc., has been
found
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), and
dioctyltin (DOT) in consumer products is restricted.4-5
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.
-
p 2
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.
Figure 1. Target organotin compounds: TBT chloride, fentin
acetate, cyhexatin, fenbutatin oxide, and internal standard
triphenyl phosphate (top left to bottom right)
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).
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.
Figure 2. Sample preparation protocols for the analysis of
organotin compounds in fruit and vegetable, textiles, and water
MS/MS Detection
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).
The data was processed in MultiQuant™ software version 2.1.
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
Sn OHSn O CH3
O
SnO
Sn
CH3CH3 CH3CH3
3 3
P
O
O
O
O
CH3
Sn
CH3
CH3
Cl
Hom ogenize and weigh 10 g of apple and potato.
Add internal standard(50 μL of 10 μg/m L TPP).
Add 10 m L acetonitrile and shake vigorously for 1 minute.
Centrifuge at 5 rpm for 5 m in.
Transfer 100 μL of the extract into autosampler vial and add
900 μL water.
Shred and weigh 1 gof textile m aterial.
Add internal standard(50 μL of 10 μg/m L TPP).
Add 20 mL acetonitrile and sonicate for 5 m inutes.
Centrifuge at 5 rpm for 5 min.
Transfer 100 μL of the extract into autosam pler vial and
add
900 μL water.
Add internal standard(10 μL of 10 ng/mL TPP).
(Dilute water sample to reduce possible matrix
effects.)
Inject directly.
Transfer 1 mL of water sample into autosampler vial.
-
p 3
Results and Discussion 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
shape.8
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).
Figure 3. Blank synthetic seawater, two chromatographic
interferences for TBT are separated well from the target analyte
(top) and internal standard (bottom)
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.
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.
Table 2. Signal-to-noise (S/N) in different matrices
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 (SW) of 20120118-salty water
test.wiff (Tu... Max. 4000.4 cps.
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
Inte
nsity
, cps
3.73
3.97
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... Max. 1.6e5 cps.
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
Inte
nsity
, cps
4.41
Blank synthetic seawater
Two chromatographic interferences for TBT are separated well
from the target analyte
Internal standard (TPP) Figure 4. Apple (top) and potato
(bottom) sample spiked at 2 μg/kg and diluted 10x after
extraction
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)
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... Max. 2499.1 cps.
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
Inte
nsity
, cps
3.72
3.83
3.96
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 &... Max. 1826.2 cps.
2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
Inte
nsity
, cps
3.84
3.733.97
Apple extract
Potato extract
2.93
6.13
5.27
2.95
6.145.27
Fentin
TBT Cyhexatin
Fenbutatin oxide
Fentin
TBTCyhexatin
Fenbutatin oxide
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... Max. 7547.7 cps.
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min
0
1000
2000
3000
4000
5000
6000
7000
7548
Inte
nsity
, cps
3.84
3.743.98
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.... Max. 1903.4 cps.
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min
0
200
400
600
800
1000
1200
1400
1600
18001903
Inte
nsity
, cps
3.73
3.97
Textile extract
Synthetic seawater
2.93
6.13
5.27
2.95
6.145.27
Fentin
TBT
Cyhexatin
Fenbutatin oxide
Fentin
TBT
CyhexatinFenbutatin oxide
-
p 4
Figure 6. Calibration lines of organotin compounds in apple
matrix (2 to 100 μg/kg)
Figure 7. Calibration lines of organotin compounds in synthetic
seawater (50 to 2000 ng/L)
Table 3. Estimated limits of quantitation (LOQ) in different
matrices based on S/N of 10
Organotin compound
Apple μg/kg
Potato (μg/kg)
Textile (μg/kg)
Seawater (ng/L)
TBT 0.2 0.3 10 10
Fentin < 0.1 < 0.1 < 10 < 10
Cyhexatin 0.1 0.1 20 < 10
Fenbutatin oxide < 0.1 < 0.1 15 < 10
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).
Are
a R
atio
Are
a R
atio
Are
a R
atio
Are
a R
atio
FentinTBT
Cyhexatin Fenbutatin oxideA
rea
Rat
io
Are
a R
atio
Are
a R
atio
Are
a R
atio
FentinTBT
Cyhexatin Fenbutatin oxide
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
-
For Research Use Only. Not for use in diagnostic procedures.
© 2012 AB SCIEX. The trademarks mentioned herein are the
property of AB Sciex Pte. Ltd. or their respective owners. AB
SCIEX™ is being used under license.
Publication number: 6690212-01 Headquarters International Sales
500 Old Connecticut Path, Framingham, MA 01701 USA For our office
locations please call the division Phone 508-383-7700 headquarters
or refer to our website at www.absciex.com
www.absciex.com/offices
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.
Figure 8. Automatic compound identification using the
‘Multicomponent’ query (example cyhexatin in potato)
Figure 9. Automatic compound identification using the
‘Multicomponent’ query (example fentin in textile)
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 K. Fent: ‘Ecotoxicology of organotin compounds’
Crit. Rev.
Toxicol. 26 (1996) 1-117 2 E. Gonzalez-Toledo et al.: ‘Detection
techniques in
speciation analysis of organotin compounds by liquid
chromatography’ Trends Anal. Chem. 22 (2003) 26-33
3 Regulation (EC) ‘on the prohibition of organotin compounds on
ships’ No 782/2003
4 Commission Decision ‘restrictions on the marketing and use of
organostannic compounds’ 2009/425/EC
5 International Association for Research and Testing in the
Field of Textile Ecology: OEKO-TEX Standard 100, Edition 4
(2012)
6
http://www.codexalimentarius.org/standards/pesticide-mrls/en/
7 Council Directive ‘maximum levels for pesticide residues’
96/32/EC
8 EU Reference Laboratory for Single Residue Methods: ‘Analysis
of Organotin Compounds via QuEChERS and LC-MS/MS – Brief
Description’ www.crl-pesticides.eu
http://www.absciex.comhttp://www.absciex.com/officeshttp://www.codexalimentarius.org/standards/pesticidehttp://www.crl-pesticides.eu