In this work, we investigate the suitability of a commercial available and low cost polyethersufone (PES) sorbent for the microextraction of 1H-benzotriazole (BTri), and four polar derivatives (4 and 5-methyl-1H-benzotriazole, 4-TTri and 5-TTri; 5,6-dimethyl-1H benzotriazole, XTri; and 5-chloro-1H-benzotriazole, 5-ClBTri) from surface and wastewater samples. The performance of liquid chromatography (LC) combined with quadrupole time-of-flight mass spectrometry (QTOF-MS) for the selective determination of target compounds is also discussed. Parameters affecting the efficiency of the microextraction step, such as sample’s pH, ionic strength, stirring speed and extraction lapse of time, and the PES membrane desorption process have been thoroughly investigated. Analytes were extracted from 15 mL samples, containing a 30% of sodium chloride and adjusted at pH 4.5, using a tubular PES sorbent (5 cm length x 0.7 mm o.d., sorbent volume 42 μL). After methanol desorption and solvent exchange, benzotriazoles were determined by LC-MS, with chromatograms extracted using a mass window of 20 ppm, centered in their [M+H]+ ions. The identity of chromatographic peaks was confirmed with accurate ion product scan (MS/MS) spectra. The method provided limits of quantification (LOQs) between 0.005 and 0.1 ng mL-1, and relative recoveries from 81% to 124% (except for XTri in sewage samples, ca. 60%) with associated standard deviations between 2% and 9%. The efficiency of the PES sorbent for the extraction of these compounds has been compared with that attained by stir-bar sorptive extraction (SBSE), with polydimethylsiloxane (PDMS) covered stir bars. The PES polymer achieved significant higher responses (5- to 20-fold) for these polar pollutants. To the best of our knowledge, this research constitutes the first application of both techniques (microextraction using a PES sorbent and LC-QTOF-MS) for benzotriazoles determination in water samples. The method was used to provide data regarding the levels of target compounds in river and urban wastewater samples, including the individual quantification of 4-methyl and 5-methyl-benzotriazole isomers. Obtained results confirmed the ubiquity of benzotriazole, 4-methyl and 5-methyl-benzotriazole in urban wastewater and their incomplete removal at sewage treatment plants

1. DETERMINATION OF BENZOTRIAZOLES IN WATER
SAMPLES BY POLYETHERSULFONE SOLID-PHASE
MICROEXTRACTION AND LIQUID CHROMATOGRAPHY
QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETRY
Jorge Casado Agrelo
Young Scientists Session 1
J. Casado, I. Rodríguez, M. Ramil, R. Cela
Department of Analytical Chemistry, Institute of Food Analysis and Research, University of
Santiago de Compostela. R/ Constantino Candeira, s/n - 15782 - Santiago de Compostela,
Galicia, Spain

2. INTRODUCTION
2
Deuterated Benzotriazole (BTri-d4)
Internal Surrogate
Benzotriazole (BTri)
log Kow: 1.44
4-methylbenzotriazole (4-TTri)
log Kow: 1.71
5-methylbenzotriazole (5-TTri)
log Kow: 1.71
Dimethylbenzotriazole (XTri)
log Kow: 2.25
5-chlorobenzotriazole (5-ClBTri)
log Kow: 2.13
N
N
H
N
N
N
H
N
CH3
N
N
H
N
H3C
N
N
H
N
H3C
H3C
N
N
H
N
Cl
D.A. Cancilla, J.C. Baird, S.W. Geis, S.R. Corsi, Environ. Toxicol. Chem. 22 (2003)134
W. Vetter, J. Lorenz, Environ. Sci. Pollut. Res. 19 (2012), http://dx.doi.org/10.1007/s11356-12-1386-y
H. Tangtian, L. Bo, L. Wenhua, P.K.S. Shin, R.S.S. Wu, Ecotoxicol. Environ. Saf. 80(2012) 327
A.G. Asimakopoulos, L. Wang, N.S. Thomaidis, K. Kannan, Environ. Int, 59 (2013) 274
• Benzotriazoles Emerging Contaminants
• Risk
• Legislation
► Carcinogenic and Toxicity  LC50
► Anti-estrogen
► High mobility in water
► Slow biodegradation rates
► Recent detection in Urine samples
► Anti-freeze and De-icer
► Anti-corrosive
► Anti-microbial
► Lack of regulations

3. • Sample preparation technique
• Separation instrument
► LC-ESI-QTOF
► Orbitrap
► QqQ
► Polyethersulfone Solid-Phase Microextraction
► SPE
► DLLME
High Resolution Hybrid Mass Spectrometer
3
H. Wolschke, Z. Xie, A. Möller, R. Sturm, R. Ebinghaus, Water Res. 45 (2011) 6259
J.A. van Leerdam, A.C. Hogenboom, M.M.E. van der Kooi, P. de Voogt, Int. J. Mass Spectrom. 282 (2009) 99
M.T. Pena, X. Vecino-Bello, M.C. Casais, M.C. Mejuto, R. Cela, Anal. Bioanal. Chem.402 (2012) 1679
PDMS
PES
PP
Kv

4. AIMS
• Evaluate the suitability of PES sorbents for solid-phase microextraction of
benzotriazoles.
• Investigate the performance of LC-ESI-QTOF for their selective determination.
• Test the fate and the behaviour of benzotriazoles in WWTPs.
4

5. • LC-ESI-MS/MS optimized parameters
EXPERIMENTAL
Agilent 1200 LC
Chromatographic column Zorbax SB-Phenyl
Stationary phase Phenyl diisopropyl
Column size 100 mm x 2.1 mm (i.d.)
Particle size 3.5 μm
Oven temperature 30º C
Injector
Injection volume 10 μL
Mobile phase
Flow 0.2 mL min-1
Aqueous phase Ultrapure water (0.1 % formic acid)
Organic phase Acetonitrile (0.1 % formic acid)
5

6. Agilent 6520 QTOF MS; MS/MS
Electrospray Ionisation
Mode Positive
Needle voltage 3500 V
Drying gas temperature (N2) 330 ºC
Drying gas flow (N2) 10 L min-1
Nebulizing gas flow (N2) 30 psi
Acquisition
Mode Full Scan Spectra; Centroid
Ion reference solution 121.0509 Da; 922.0098 Da
Time-of-flight analyzer
Collision cell
Quadrupole
6

7. • LC-MS performance
► Repeatability: RSD < 5 % (25 ng mL-1)
► Mass error < 0.5 mDa
► LOQ: 0.03 – 9.1 ng mL-1
► Linearity: LOQ – 500 ng mL-1 (R2 > 0.9984)
• LC-MS/MS performance
► Repeatability: RSD < 14 % (25 ng mL-1)
► Mass error < 0.7 mDa
► LOQ: 7.7 – 26 ng mL-1
► Linearity: LOQ – 500 ng mL-1 (R2 > 0.9983)
BTri
y = 0.0402x – 0.055
R2
= 0.9998
0
5
10
15
20
25
0 100 200 300 400 500 600
Concentration (ng mL-1
)
Area
/
I.
S.
Area
BTri
y = 0.0553x + 0.0617
R2
= 0.9996
0
5
10
15
20
25
30
0 100 200 300 400 500 600
Concentration (ng mL-1
)
Area
/
I.
S.
Area
7

8. 0
BTri
0
0
0
0
5-ClBTri
0
Time ( min )
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Counts
4-OHBTri
5-CBTri
4-TTri 5-TTri
XTri
x102
x102
x103
x103
x10
2
x104
m/z 136.0505
m/z 164.0455
m/z 120.0556
m/z 134.0713
m/z 154.0167
m/z 148.0869
• MS
• MS/MS
10 ng mL-1
10 ng mL-1
8
0
0
XTri
0
4-TTri
0
5-CBTri
x102
0
4-OHBTri
0
Time (min)
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Counts
BTri
5-TTri
5-ClBTri
x102
x102
x101
x103
x103
136.0505 → 80.0495
164.0455 → 80.0495
120.0550 → 65.0386
134.0713 → 77.0386
154.0167 → 98.9998
148.0869 → 77.0386
Rs: 1.0
Rs: 1.0

9. • Optimization of microextraction
20 mL wine sample
1)
2)
3
)
Concentration to
100 µL Mobile Phase
MeOH
Desorption
Water Sample PES membrane
Stir
1) 2) 3) 4)
9

10. Preliminary experiments
pH of the sample: NaCl added to the sample:
Sample volume:
0%
20%
40%
60%
80%
100%
120%
BTri 4-TTri 5-TTri XTri 5-ClBTri
Normalized
response
pH 6 pH 2
0%
50%
100%
150%
200%
250%
BTri 4-TTri 5-TTri XTri 5-ClBTri
Normalized
response
0 g 2 g
0%
20%
40%
60%
80%
100%
120%
BTri 4-TTri 5-TTri XTri 5-ClBTri
Normalized
response
10 mL 50 mL
DISCUSSION
10

11. Design of experiments 23 + 4
► A: pH (2 – 6)
► B: NaCl concentration (5 – 30%, w/v)
► C: Stirring speed (400 – 800 rpm)
► pH: Significant for almost all the analytes (optimal 6), except for 5-ClBTri.
► NaCl concentration: Low significance (optimal 30%), except for Btri.
► Stirring speed: Not significant.
BTri
0 1 2 3 4 5
AC
B
C
BC
AB
A +
-
5-ClBTri
0 0.5 1 1.5 2 2.5 3
AC
BC
C
A
B
AB +
-
11

12. Most important parameter: pH of the sample
Desorption solvent
1.5 mL MeOH fractions:
pH 4
0%
20%
40%
60%
80%
100%
BTri 4-TTri 5-TTri XTri 5-ClBTri
1st Fraction 2nd Fraction 3rd Fraction
1.5 mL
MeOH
0%
20%
40%
60%
80%
100%
120%
BTri 4-TTri 5-TTri XTri 5-ClBTri
Normalized
response
Diethyl ether AcOEt MeOH
12

13. Extraction lapse of time
13
0
1
2
3
4
5
0 5 10 15 20 25
Time (h)
Peak
area
/
I.S.
area
5-TTri 5-ClBTri
6 hours

14. PES vs PDMS
0%
20%
40%
60%
80%
100%
120%
BTri 4-TTri 5-TTri XTri 5-ClBTri
Normalized
response
PES PDMS
PES: 42 μL PDMS (SBSE): 24 μL
14

15. ► Superficial water
► Untreated wastewater
► Treated wastewater Pseudo-external calibration
• Validation of the method
Precision: RSD < 7% (n = 5, 1 ng mL-1)
Global quantification limits (LOQ Extraction+LC-MS): 0.001 – 0.204 ng mL-1
Recoveries (relative to ultrapure water): 81 – 124% after I. S. correction
15
Different environmental water samples:

16. • Real samples analysis
Surface Water from Santiago de Compostela n = 3
# Place Date
Concentration (ng mL-1) ± standard deviation
BTri 4-TTri 5-TTri
1 River Sarela 01/03/2012 < LOQ < LOQ < LOQ
2 River Sar 27/03/2012 0.71 ± 0.04 0.46 ± 0.03 0.35 ± 0.04
3 River Sar 24/04/2012 < LOQ 0.18 ± 0.03 0.14 ± 0.01
4 River Sar 26/04/2012 < LOQ 0.38 ± 0.04 0.11 ± 0.01
Detection Frecuency (%) 100% 100% 100%
Maximum Concentration (ng mL-1) 0.71 0.46 0.35
16

17. # Place Date
Concentration (ng mL-1) ± standard deviation
BTri 4-TTri 5-TTri
5 Untreated WWTP 11/01/2012 2.31 ± 0.04 0.47 ± 0.01 0.49 ± 0.07
6 Treated WWTP 11/01/2012 0.90 ± 0.03 1.21 ± 0.01 1.52 ± 0.06
7 Untreated WWTP 27/03/2012 2.03 ± 0.02 0.36 ± 0.01 0.39 ± 0.03
8 Treated WWTP 27/03/2012 1.62 ± 0.05 1.22 ± 0.01 1.00 ± 0.05
9* Untreated WWTP 18/04/2012 5.93 ± 0.21 1.51 ± 0.01 1.44 ± 0.08
10 Treated WWTP 18/04/2012 1.32 ± 0.05 1.82 ± 0.09 1.63 ± 0.08
11* Untreated WWTP 24/04/2012 0.49 ± 0.03 0.54 ± 0.02 0.48 ± 0.02
12 Treated WWTP 24/04/2012 0.29 ± 0.02 0.62 ± 0.06 0.49 ± 0.04
13* Untreated WWTP 26/04/2012 1.58 ± 0.11 0.44 ± 0.01 0.37 ± 0.02
14 Treated WWTP 26/04/2012 1.57 ± 0.11 0.81 ± 0.01 0.58 ± 0.04
Detection Frecuency (%) 100% 100% 100%
Maximum Concentration (ng mL-1) 5.93 1.82 1.63
Urban Raw Water from Santiago de Compostela
17

18. • Relative Stability of each TTri isomer
Date
[4-TTri]/[5-TTri]
Untreated Treated
11/01/2012 0.52  0.06 0.45  0.03
27/03/2012 0.46  0.09 0.52  0.01
18/04/2012 0.45  0.02 0.50  0.02
24/04/2012 0.50  0.01 0.54  0.02
26/04/2012 0.51  0.01 0.59  0.04
Average 0.49  0.04 0.52  0.02
18
4-methylbenzotriazole (4-TTri)
N
N
H
N
CH3
5-methylbenzotriazole (5-TTri)
N
N
H
N
H3C

19. 4
x10
0
0.5
1
1.5
2
2.5
3
3.5
+ESI EIC(120.0556) Scan Frag=160.0V Residual_Salida_27mar12_2.d
Counts vs. Acquisition Time (min)
5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8
BTri
• Chromatogram samples 7 (solid line) and 8 (dotted line):
119
4
x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
+ESI EIC(134.0713) Scan Frag=160.0V Residual_Salida_27mar12_2.d
Counts vs. Acquisition Time (min)
11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18
4-TTri
5-TTri
2
x10
0
0.2
0.4
0.6
0.8
1
+ESI Product Ion (6.965-7.386 min, 7 scans) Frag=160.0V CID@30.0 (120.0556
65.0376
94.0382 120.0530
Counts (%) vs. Mass-to-Charge (m/z)
50 70 90 110 130 150 170 190
2
x10
0
0.2
0.4
0.6
0.8
1
+ESI Product Ion (14.032-14.724 min, 16 scans) Frag=160.0V CID@30.0 (120.0
…
77.0392
62.9298
134.0709
Counts (%) vs. Mass-to-Charge (m/z)
50 70 90 110 130 150 170 190
2
x10
0
0.2
0.4
0.6
0.8
1
+ESI Product Ion (14.861-15.507 min, 15 scans) Frag=160.0V CID@30.0 (120.
…
77.0392
62.9298
134.0708
Counts (%) vs. Mass-to-Charge (m/z)
50 70 90 110 130 150 170 190
2
x10
0
0.2
0.4
0.6
0.8
1
+ESI Product Ion (6.960-7.311 min, 6 scans) Frag=160.0V CID@30.0 (120.0556
65.0373
94.0378 120.0527
Counts (%) vs. Mass-to-Charge (m/z)
50 70 90 110 130 150 170 190
2
x10
0
0.2
0.4
0.6
0.8
1
+ESI Product Ion (14.122-14.814 min, 16 scans) Frag=160.0V CID@28.0 (134.0
…
77.0391
62.9298
134.0709
106.0651
Counts (%) vs. Mass-to-Charge (m/z)
50 70 90 110 130 150 170 190
2
x10
0
0.2
0.4
0.6
0.8
1
+ESI Product Ion (14.724-15.463 min, 17 scans) Frag=160.0V CID@28.0 (134.
…
77.0392
62.9298 134.0708
Counts (%) vs. Mass-to-Charge (m/z)
50 70 90 110 130 150 170 190
Standard solution

20. CONCLUSIONS & FUTURE
WORK
► Extraction by means of sorption on PES polymer  Low-cost: small sample volume,
small amounts of sorbents and solvents.
► PES polymer provides higher efficiency than PDMS for relatively polar compounds.
► QTOF instrument permits the unambiguous identification of the benzotriazoles by
accurate full scan MS/MS spectra.
► WWTPs release benzotriazoles to aquatic environment.
► Evaluation of the performance of SBSE bars covered with Polyethylene glycol
(PEG) for the extraction of benzotriazoles.
►Assessment of the benzotriazoles stability in batch degradation experiments.
20

21. DETERMINATION OF BENZOTRIAZOLES IN WATER
SAMPLES BY POLYETHERSULFONE SOLID-PHASE
MICROEXTRACTION AND LIQUID CHROMATOGRAPHY
QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETRY
Jorge Casado Agrelo
Young Scientists Session 1
J. Casado, I. Rodríguez, M. Ramil, R. Cela
Department of Analytical Chemistry, Institute of Food Analysis and Research, University of
Santiago de Compostela. R/ Constantino Candeira, s/n - 15782 - Santiago de Compostela,
Galicia, Spain

22. 0%
20%
40%
60%
80%
100%
120%
BTri 4-TTri 5-TTri XTri 5-ClBTri
Normalized
response Extract injection
Direct sample injection
CONCENTRATION FACTORS