Antimycotics are a broad group of drugs, active against infections caused by fungi, recognized as emerging environmental contaminants. Their most common way of use is the topic application, although they can be also administered orally or intravenous. Therefore, excretion and showering result in the introduction of these compounds in municipal sewage water[1 2]. These pharmaceuticals are suspected of being endocrine disruptors, since they work inhibiting some enzymes, such as aromatases, that are responsible for sexual differentiation of vertebrates and the balance between androgenic and estrogenic hormones in mammals[3]. Therefore, analytical methodologies that permit the analysis of different kinds of water, as well as sewage sludge, and the quantification of these dangerous pharmaceuticals are necessary. In this work we present the optimization of an improved selectivity enrichment method for the analysis of antimycotic drug residues in water and sludge samples.

1. Levels and fate of antimycotic
pharmaceuticals at sewage
treatment plants
Jorge Casado
Department of Analytical Chemistry
J Casado, G Castro, I Rodríguez, M Ramil, R Cela

2. Antimycotic drugs
• Treatment of infections caused by fungi
• Inhibition of sexual differentiation enzymes
– Disturbance of endocrine system of aquatic organisms
• Topic and oral application
– Introduction in municipal sewage water
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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3. Goals
• Development of accurate and sensible analytical
methodology
• Appropriate sample preparation
• Occurrence of antimycotic in environmental
samples:
Surface water Wastewater Sewage sludge
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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4. Selected substances
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Etaconazole, ETZ
pKa: 2.94
Log Kow: 3.6
Miconazole, MCZ
pKa: 6.64
Log Kow: 6.1
Fluconazole, FCZ
pKa: 11.01, 2.64
Log Kow: 0.4
Ketoconazole, KTZ
pKa: 6.88
Log Kow: 4.3
Terbinafine, TRB
pKa: 7.1
Log Kow: 5.6
Clotrimazole, CTZ
pKa: 6.12
Log Kow: 4.1
Econazole, ECZ
pKa: 6.68
Log Kow: 5.5

5. Water samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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SPE
MCX
150 mg
J. Casado, I. Rodríguez, M. Ramil, R. Cela, J. Chromatogr. A 1339 (2014) 42-49
Sample
R.W.150 mL
T.W. 300 mL
River water: 500 mL
MeOH
washing fraction
MeOH (NH3)
analytical fraction
LC-QTOF HR MS/MS
WASTE

6. LC separation
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Time
min
H2O MeOH Flow
mL min-1
5 mM NH4OAc
0 95 5 0.2
3 95 5 0.2
5 7 93 0.2
20 0 100 0.2
21 0 100 0.2
22 95 5 0.2
30 95 5 0.2

7. Water samples: method performance
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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y = 0,0038x + 0,0023
R² = 0,9999
0
0,2
0,4
0,6
0,8
0 50 100 150 200
Area/IS
Area
ng mL-1
CTZ
y = 0,0018x - 0,0001
R² = 0,9998
0
0,1
0,2
0,3
0,4
0 50 100 150 200
Area/IS
Area
ng mL-1
MCZ
Compound MCX MCX no washing HLB
FCZ 58 ± 2 27 ± 1 25 ± 3
ETZ 97 ± 3 60 ± 2 52 ± 2
KTZ 93 ± 2 47 ± 2 38 ± 3
CTZ 99 ± 3 58 ± 2 54 ± 2
ECZ 101 ± 3 49 ± 4 39 ± 4
MCZ 101 ± 5 66 ± 2 60 ± 4
TRB 87 ± 7 75 ± 1 81 ± 6

8. Water samples: method performance
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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River water, 500 mL Treated wastewater, 300 mL Raw wastewater, 150 mL
200
ng L-1
50
ng L-1
LOQs
(ng L-1)
500
ng L-1
100
ng L-1
LOQs
(ng L-1)
500
ng L-1
100
ng L-1
LOQs
(ng L-1)
FCZ 104 ± 5 113 ± 18 2 63 ± 4 117 ± 12 3 86 ± 9 80 ± 10 7
ETZ 94 ± 2 103 ± 13 2 90 ± 2 101 ± 1 3 82 ± 5 76 ± 8 7
KTZ 90 ± 10 105 ± 18 20 80 ± 2 100 ± 8 30 73 ± 12 75 ± 7 66
CTZ 102 ± 4 95 ± 11 2 96 ± 2 101 ± 5 3 104 ± 2 88 ± 4 7
ECZ 100 ± 5 84 ± 6 2 92 ± 3 105 ± 1 3 90 ± 1 85 ± 5 7
MCZ 111 ± 6 85 ± 20 5 103 ± 4 96 ± 7 10 92 ± 5 81 ± 5 15
TRB 99 ± 3 94 ± 7 4 94 ± 2 101 ± 4 7 101 ± 3 97 ± 6 13
y = 0,0038x + 0,0023
R² = 0,9999
0
0,2
0,4
0,6
0,8
0 50 100 150 200
Area/IS
Area
ng mL-1
CTZ
y = 0,0018x - 0,0001
R² = 0,9998
0
0,1
0,2
0,3
0,4
0 50 100 150 200
Area/IS
Area
ng mL-1
MCZ

9. Analysis of samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Sample Concentration (ng L-1) ± SD
Code Date Type FCZ KTZ CTZ MCZ
1
05/07/13
R.W. 86 ± 5 72 ± 7 47 ± 6 30 ± 10
2 T.W. 72 ± 12 23 ± 7 5 ± 1 < LOQ
3
12/07/13
R.W. 83 ± 3 110 ± 10 11 ± 1 20 ± 2
4 T.W. 95 ± 9 15 ± 3 6 ± 1 < LOQ
5
04/11/13
R.W. 20 ± 4 102 ± 23 48 ± 10 43 ± 5
6 T.W. 40 ± 14 34 ± 17 5 ± 1 11 ± 1
7
12/11/13
R.W. 93 ± 17 191 ± 15 49 ± 7 55 ± 17
8 T.W. 37 ± 2 36 ± 7 11 ± 1 12 ± 1
9
21/11/13
R.W. 41 ± 10 55 ± 6 80 ± 3 30 ± 4
10 T.W. 70 ± 7 10 ± 3 9 ± 1 < LOQ
11 River 32 ± 4 4 ± 1 7 ± 1 5 ± 1
12
27/11/13
R.W. 59 ± 22 58 ± 11 74 ± 15 80 ± 20
13 T.W. 69 ± 9 17 ± 1 8 ± 1 < LOQ
14 River 25 ± 8 11 ± 1 9 ± 2 < LOQ

10. Analysis of samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Sample Concentration (ng L-1) ± SD
Code Date Type FCZ KTZ CTZ MCZ
1
05/07/13
R.W. 86 ± 5 72 ± 7 47 ± 6 30 ± 10
2 T.W. 72 ± 12 23 ± 7 5 ± 1 < LOQ
3
12/07/13
R.W. 83 ± 3 110 ± 10 11 ± 1 20 ± 2
4 T.W. 95 ± 9 15 ± 3 6 ± 1 < LOQ
5
04/11/13
R.W. 20 ± 4 102 ± 23 48 ± 10 43 ± 5
6 T.W. 40 ± 14 34 ± 17 5 ± 1 11 ± 1
7
12/11/13
R.W. 93 ± 17 191 ± 15 49 ± 7 55 ± 17
8 T.W. 37 ± 2 36 ± 7 11 ± 1 12 ± 1
9
21/11/13
R.W. 41 ± 10 55 ± 6 80 ± 3 30 ± 4
10 T.W. 70 ± 7 10 ± 3 9 ± 1 < LOQ
11 River 32 ± 4 4 ± 1 7 ± 1 5 ± 1
12
27/11/13
R.W. 59 ± 22 58 ± 11 74 ± 15 80 ± 20
13 T.W. 69 ± 9 17 ± 1 8 ± 1 < LOQ
14 River 25 ± 8 11 ± 1 9 ± 2 < LOQ

11. Analysis of samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Sample Concentration (ng L-1) ± SD
Code Date Type FCZ KTZ CTZ MCZ
1
05/07/13
R.W. 86 ± 5 72 ± 7 47 ± 6 30 ± 10
2 T.W. 72 ± 12 23 ± 7 5 ± 1 < LOQ
3
12/07/13
R.W. 83 ± 3 110 ± 10 11 ± 1 20 ± 2
4 T.W. 95 ± 9 15 ± 3 6 ± 1 < LOQ
5
04/11/13
R.W. 20 ± 4 102 ± 23 48 ± 10 43 ± 5
6 T.W. 40 ± 14 34 ± 17 5 ± 1 11 ± 1
7
12/11/13
R.W. 93 ± 17 191 ± 15 49 ± 7 55 ± 17
8 T.W. 37 ± 2 36 ± 7 11 ± 1 12 ± 1
9
21/11/13
R.W. 41 ± 10 55 ± 6 80 ± 3 30 ± 4
10 T.W. 70 ± 7 10 ± 3 9 ± 1 < LOQ
11 River 32 ± 4 4 ± 1 7 ± 1 5 ± 1
12
27/11/13
R.W. 59 ± 22 58 ± 11 74 ± 15 80 ± 20
13 T.W. 69 ± 9 17 ± 1 8 ± 1 < LOQ
14 River 25 ± 8 11 ± 1 9 ± 2 < LOQ

12. Screening for aditional antymicotics
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Compound CAS number
Empirical
formula
Bifonazole 60628-96-8 C22H18N2
Ciclopirox 29342-05-0 C12H17NO2
Climbazole 38083-17-9 C15H17ClN2O2
Eberconazole 128326-82-9 C18H14Cl2N2
Fenticonazole 72479-26-6 C24H20Cl2N2O5
Griseofulvin 126-07-8 C17H17ClO6
Itraconazole 84625-61-6 C35H38Cl2N8O4
Naftifine 65472-88-0 C21H21N
Omoconazole 74512-12-2 C20H17Cl3N2O2
Oxiconazole 64211-45-6 C18H13Cl4N3O
Sertaconazole 99592-32-2 C20H15Cl3N2OS
Tioconazole 65899-73-2 C16H13Cl3N2OS

13. Climbazole
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Climbazole, CBZ
pKa: 5.66
Log Kow: 3.5
3
x10
0
2
4
6
8
Counts vs. Acquisition Time (min)
2 4 6 8 10 12 14 16 18 20 22 24 26 28
C15H17ClN2O2
Score 92.50
[M+H]+ calculated 293.1051
Error -0.63 ppm
12.572
2
x10
0
0.4
0.8
1.2
1.6 293.1050
([C15 H17 Cl N2 O2]+H)+
296.2494
295.1045
Counts (%) vs. Mass-to-Charge (m/z)
292 293 294 295 296
([C15 H17 Cl N2 O2]+H)+
297
2
x10
0
0.2
0.4
0.6
0.8
1
1.2
69.0706
197.0705
57.0702
293.1108
225.0680
129.0068
109.0374 166.1055
Counts (%) vs. Mass-to-Charge (m/z)
60 80 100 120 140 160 180 200 220 240 260 280 300
C12H14ClO2
+
Calculated 225.0677
Error 1.3 ppm
- C3H4N2
- CO
C11H14ClO+
Calculated 197.0728
Error -11.7 ppm [M+H]+

14. Sludge samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
14 J. Casado, G. Castro, I. Rodríguez, M. Ramil, R. Cela, Anal. Bioanal. Chem. 407 (2015) 907-917
MSPD
PSA 1 g
Sample 0.5 g
Dispersant C18 2 g
SPE
SCX
500 mg
MSPD MeOH
extraction fraction
SPE MeOH (NH3)
analytical fraction
LC-QTOF HR MS/MS
WASTE
MeOH
1 2
1 2
1 2

15. Sludge samples: method performance
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Analyte
Global recovery
(%) ± SD LOQs
(ng g-1)
500
ng g-1
50
ng g-1
FCZ 118 ± 7 81 ± 7 5
CBZ 109 ± 3 98 ± 8 6
ETZ 110 ± 14 104 ± 7 5
KTZ 109 ± 13 78 ± 4 5
CTZ 106 ± 5 111 ± 3 5
ECZ 97 ± 7 74 ± 2 5
MCZ 86 ± 5 70 ± 5 8
TRB 103 ± 8 89 ± 7 8
y = 0,0205x + 0,0248
R² = 0,9999
0
5
10
15
20
25
0 200 400 600 800 1000 1200
Area/IS
Area
ng mL-1
CTZ
y = 0,0073x + 0,016
R² = 0,9998
0
2
4
6
8
0 200 400 600 800 1000 1200
Area/IS
Area
ng mL-1
CBZ

16. Analysis of samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Sample code
Sample concentration (ng g-1) ± SD
CBZ KTZ CTZ MCZ TRB Sum
1 14 ± 1 518 ± 37 498 ± 26 167 ± 10 < LOQ 1197
2 42 ± 9 87 ± 19 334 ± 15 66 ± 5 < LOQ 529
3 11 ± 4 169 ± 1 283 ± 42 87 ± 18 < LOQ 550
4 28 ± 2 123 ± 5 349 ± 23 122 ± 1 < LOQ 622
5 58 ± 6 39 ± 3 311 ± 19 78 ± 6 < LOQ 486
6 18 ± 1 336 ± 31 800 ± 54 304 ± 6 10 ± 1 1458
7 8 ± 1 87 ± 3 584 ± 22 204 ± 8 < LOQ 883
8 32 ± 2 205 ± 18 310 ± 6 171 ± 16 < LOQ 718
9 98 ± 6 109 ± 10 285 ± 18 75 ± 2 < LOQ 567
Average
(ng g-1)
34 185 417 141 - 779

17. Analysis of samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Sample code
Sample concentration (ng g-1) ± SD
CBZ KTZ CTZ MCZ TRB Sum
1 14 ± 1 518 ± 37 498 ± 26 167 ± 10 < LOQ 1197
2 42 ± 9 87 ± 19 334 ± 15 66 ± 5 < LOQ 529
3 11 ± 4 169 ± 1 283 ± 42 87 ± 18 < LOQ 550
4 28 ± 2 123 ± 5 349 ± 23 122 ± 1 < LOQ 622
5 58 ± 6 39 ± 3 311 ± 19 78 ± 6 < LOQ 486
6 18 ± 1 336 ± 31 800 ± 54 304 ± 6 10 ± 1 1458
7 8 ± 1 87 ± 3 584 ± 22 204 ± 8 < LOQ 883
8 32 ± 2 205 ± 18 310 ± 6 171 ± 16 < LOQ 718
9 98 ± 6 109 ± 10 285 ± 18 75 ± 2 < LOQ 567
Average
(ng g-1)
34 185 417 141 - 779

18. Applicability to other basic drugs extraction
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Compound CAS number Formula
Amiodarone 1951-25-3 C25H29I2NO3
Amitryptiline 50-48-6 C20H23N
Azelastine 58581-89-8 C22H24ClN3O
Bromocriptine 25614-03-3 C32H40BrN5O5
Carbamazepine 298-46-4 C15H12N2O
Chlomipramine 303-49-1 C19H23ClN2
Chlorpromazine 50-53-3 C17H19ClN2S
Chlorprothixene 113-59-7 C18H18ClNS
Cimetidine 51481-61-9 C10H16N6S
Ciprofloxacine 85721-33-1 C17H18FN3O3
Clemastine 15686-518 C21H26ClNO
Codeine 76-57-3 C18H21NO3
Cyproheptadine 129-03-3 C21H21N
Dihydroergotamine 511-12-6 C33H37N5O5
Diphenhydramine 88637-37-0 C17H21NO
Domperidone 57808-66-9 C22H24ClN5O2
Doxepine 1668-19-5 C19H21NO
Doxycycline 564-25-0 C22H24N2O8
Duloxetine 116539-59-4 C18H19NOS
Escitalopram 128196-01-0 C20H21FN2O
Fluoxetine 54910-89-3 C17H18F3NO
Compound CAS number Formula
Haloperidol 52-86-8 C21H23ClFNO2
Lamotrigine 84057-84-1 C9H7Cl2N5
Levomepromazine 60-99-1 C19H24N2OS
Lidocaine 137-58-6 C14H22N2O
Meclizine 569-65-3 C25H27ClN2
Ofloxacine 82419-36-1 C18H20FN3O4
Paroxetine 61869-08-7 C19H20FNO3
Perphenazine 58-39-9 C21H26ClN3OS
Pizotifen 15574-96-6 C19H21NS
Promethazine 60-87-7 C17H20N2S
Quinacrine 83-89-6 C23H30ClN3O
Roxithromycin 80214-83-1 C41H76N2O15
Sertraline 79617-96-2 C17H17Cl2N
Sulfamethoxazole 723-46-6 C10H11N3O3S
Sulfapyridine 144-83-2 C11H11N3O2S
Tamoxifen 10540-29-1 C26H29NO
Tetracycline 60-54-8 C22H24N2O8
Tramadol 27203-92-5 C16H25NO2
Verapamil 52-53-9 C27H38N2O4
Zolpidem 82626-48-0 C19H21N3O

19. U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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Amitriptyline
Sertraline
Amiodarone
[M+H]+
2
x10
0
0.2
0.4
0.6
0.8
1
1.2
277.0359
158.9758
129.0692 306.0807
91.0558
Counts (%) vs. Mass-to-Charge (m/z)
60 80 100 120 140 160 180 200 220 240 260 280 300 320
[M+H]+
Calculated 306.0811
Error 1.4 ppm
C16H15
35Cl37Cl+
Calculated 277.0389
Error 10.8 ppm
275.0386
C16H13Cl2
+
Calculated 275.0389
Error 1.1 ppm
- CH5N
C7H5Cl2
+
Calculated 158.9763
Error 3.1 ppm
C10H9
+
Calculated 129.0699
Error 5.4 ppm - C9H8
- C6H4Cl2
C7H7
+
Calculated 91.0542
Error 17.6 ppm
- C3H2
[M+H]+
1
x10
0
1
2
3
4
5
6
91.0546
105.0697
191.0840
233.1327
278.1901
Counts (%) vs. Mass-to-Charge (m/z)
60 80 100 120 140 160 180 200 220 240 260 280
[M+H]+
Calculated 278.1909
Error 0.8 ppm
C18H17
+
Calculated 233.1325
Error -0.9 ppm
C15H11
+
Calculated 191.0855
Error 7.8 ppm
C8H9
+
Calculated 105.0699
Error 1.9 ppm
C7H7
+
Calculated 91.0542
Error -4.4 ppm
- C2H7N
- C3H6
- C7H2
- CH2
2
x10
0
0.2
0.4
0.6
0.8
1
Counts (%) vs. Acquisition Time (min)
11 12 13 14 15 16 17 18 19 20 21 22
5
x10
0
0.2
0.4
0.6
0.8
1
1.2 646.0314 [C25 H29 I2 N O3+H]+
Counts vs. Mass-to-Charge (m/z)
642 644 646 648 650
4
x10
0
0.2
0.4
0.6
0.8
1
1.2
100.1124
201.0910
Counts vs. Mass-to-Charge (m/z)
50 100 150 200 250 300 350 400 450 500 550 600 650
646.0314
277.0855
58.0661
[M+H]+
Calculated 646.0310
Error+0.7 ppm
- C7H17I2N
C18H13O3
+
Calculated 277.0859
Error -1.4 ppm
C13H13O2
+
Calculated 201.0916
Error -2.8 ppm
C6H14N+
Calculated 100.1121
Error +3.8 ppm
- C5O
159.0430
C10H7O2
+
Calculated 159.0441
Error -6.8 ppm
- C8H6O
C20H23N
Score 99.66
[M+H]+ calculated 278.1909
Error 0.79 ppm
2
x10
0
0.2
0.4
0.6
0.8
1
Counts (%) vs. Acquisition Time (min)
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
4
x10
0
1
2
3
278.1901
([C20 H23 N]+H)+
279.1932
([C20 H23 N]+H)+
Counts vs. Mass-to-Charge (m/z)
278 279 280
C17H17Cl2N
Score 97.54
[M+H]+ calculated 306.0811
Error 1.30 ppm
2
x10
0
0.2
0.4
0.6
0.8
1
Counts (%) vs. Acquisition Time (min)
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
4
x10
0
0.5
1
1.5
306.0807
([C17 H17 Cl2 N]+H)+
Counts vs. Mass-to-Charge (m/z)
306 308 310
C25H29I2NO3N
Score 99.02
[M+H]+ calculated 646.0310
Error 0.60 ppm

20. Metabolites
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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N-desethylamiodarone
Norsertraline
2
x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Counts (%) vs. Acquisition Time (min)
7 8 9 10 11 12 13 14 15 16 17 18
C16H13Cl2
Score 96.24
[M+H]+ calculated 275.0389
Error -0.20 ppm
2
x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Counts (%) vs. Acquisition Time (min)
10 11 12 13 14 15 16 17 18
C23H25I2NO3
Score 99.82
[M+H]+ calculated 617.9997
Error 0.27 ppm
4
x10
0
1
2
275.0389
277.0365
([C16 H12 Cl2]+H)+
Counts vs. Mass-to-Charge (m/z)
274 276 278 280
4
x10
0
1
2
3
4
Counts vs. Mass-to-Charge (m/z)
619.0023
618
620.0045
620
617.9995
619 621
([C23 H25 I2 N O3]+H)+
3
x10
0.5
1
1.5
2
2.5
3
3.5
4
158.9756
243.1489
129.0696 275.0388
Counts vs. Mass-to-Charge (m/z)
60 80 100 120 140 160 180 200 220 240 260 280 300
[M+H]+
Calculated 275.0389
Error -0.2 ppm
C7H5Cl2
+
Calculated 158.9763
Error -4.4 ppm
C10H9
+
Calculated 129.0699
Error -2.3 ppm
- C6H4Cl2
- C9H8
3
x10
0
1
2
72.0815
546.9247
372.8214
201.0913
617.9999
Counts vs. Mass-to-Charge (m/z)
50 100 150 200 250 300 350 400 450 500 550 600 650
- C4H9N
C19H17I2O3
+
Calculated 546.9262
Error -2.7 ppm
[M+H]+
Calculated 617.9997
Error 0.3 ppm
C7H3I2O2
+
Calculated 372.8217
Error -0.9 ppm
C13H13O2
+
Calculated 201.0910
Error +1.5 ppm
- C12H14O
- C6H4I2O
700 750

21. Conclusions
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
21
• Aqueous samples:
– Improvement of selectivity with mixed-mode SPE with
sequential elution
– Reduction of ESI signal attenuation effects
• Solid samples:
– Improvement of selectivity with on-line ionic exchange SPE
clean-up
– Reduction of ESI signal attenuation effects
• Clotrimazole, Climbazole, Ketoconazole and Miconazole
are ubiquitous in environmental samples
• Poorly degradation of Fluconazole

22. Future research
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
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• Assessment of stability in batch degradation
experiments
• Searching for degradation methodologies to
eliminate fluconazol

23. jorge.casado@usc.es
u n i v e r s i d a d e d e s a n t i a g o d e c o m p o s t e l a
Project CTQ2012-33080

24. Levels and fate of antimycotic
pharmaceuticals at sewage
treatment plants
Jorge Casado
Department of Analytical Chemistry
J Casado, G Castro, I Rodríguez, M Ramil, R Cela

25. MS/MS conditions
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
25
Analyte
Retention
time
(min)
Precursor
ion (Da)
Collision
energy
(eV)
Quantification
ion (Da)
Other
product
ions (Da)
Fluconazole 10.7 307.1113 18 220.0681 238.0783
Etaconazole 12.7 328.0614 20 158.9763 204.9818
Ketoconazole 13.0 531.1560 48 82.0530 489.1455
Clotrimazole 13.5 277.0788 20 165.0699 242.1035
Clotrimazole-d5 13.6 282.1092 20 170.1018 247.1449
Econazole 14.1 383.0293 22 125.0153 69.0447
Miconazole 15.1 416.9904 24 158.9763 69.0447
Terbinafine 15.3 292.2060 14 141.0699 93.0699

26. Water samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
26
• Filtered samples
– Untreated wastewater: 150 mL
– Treated wastewater: 300 mL
– River water: 500 mL
• Solid Phase Extraction
– OASIS MCX 150 mg
– pH 3 + 5% (v/v) MeOH
– Drying step 30 min N2
– Washing step 2.5 mL MeOH, 0.1% (v/v) formic acid
– Elution 2 mL MeOH, 2% (v/v) NH3
• LC-HRMS determination
– C18 reversed phase
– ESI+ ionization
– QTOF MS/MS
J. Casado, I. Rodríguez, M. Ramil, R. Cela, J. Chromatogr. A 1339 (2014) 42-49

27. Waters SPE sorbents
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
27

28. Water sample 12
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
28
Fluconazole
CTZ
3
x10
0
0.2
0.4
0.6
0.8
1
+ESI EIC Product Ion Frag=150.0V CID@20.0 (277.0788[z=1] -> 165.0699) INF_3.d
Counts vs. Acquisition Time (min)
13 13.2 13.4 13.6 13.8 14 14.2
2
x10
0
0.4
0.8
1.2
+ESI Product Ion (13.515-13.756 min, 6 Scans) Frag=150.0V CID@20.0 (277.0788…
…
165.0689
242.1069
199.0318
57.0701
Counts (%) vs. Mass-to-Charge (m/z)
60 100 140 180 220 260
277.0784
Clotrimazole
1
x10
0
1
2
3
4
5
+ESI EIC Product Ion Frag=150.0V CID@18.0 (307.1113[z=1] -> 220.0681) INF_3.d
Counts vs. Acquisition Time (min)
10 10.2 10.4 10.6 10.8 11 11.2 11.4
2
x10
0
0.4
0.8
1.2
+ESI Product Ion (10.718-10.759 min, 3 Scans) Frag=150.0V CID@18.0 (307.1113
…..
220.0678
238.0775
169.0447
70.0406
141.0482 307.1114
200.0639
Counts (%) vs. Mass-to-Charge (m/z)
60 100 140 180 220 260 300
Ketoconazole Miconazole
3
x10
0
0.2
0.4
0.6
0.8
1
1.2
+ESI EIC Product Ion Frag=150.0V CID@24.0 (416.9904[z=1] -> 158.9763) INF_3.d
Counts vs. Acquisition Time (min)
14.6 14.8 15 15.2 15.4 15.6 15.8
14.4
2
x10
0
0.4
0.8
1.2
+ESI Product Ion (15.107-15.190 min, 4 Scans) Frag=150.0V CID@24.0 (416.9904…
158.9764
416.9904
227.0114
69.0447
Counts (%) vs. Mass-to-Charge (m/z)
60 100 140 180 220 260 300 340 380 420
2
x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
+ESI EIC Product Ion Frag=150.0V CID@48.0 (531.1560[z=1] -> 82.0530) INF_3.
Counts vs. Acquisition Time (min)
12.4 12.6 12.8 13 13.2 13.4 13.6
2
x10
0
0.2
0.4
0.6
0.8
1
1.2
+ESI Product Ion (12.848-13.124 min, 7 Scans) Frag=150.0V CID@48.0 (531.1560…
…
82.0513
110.0694
255.0029 489.1438
Counts (%) vs. Mass-to-Charge (m/z)
100 150 200 250 300 350 400 450 500
531.1579
219.1128

29. Sludge samples
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
29
• On-line Matrix Solid-Phase Dispersion/Solid Phase Extraction
– MSPD:
• Sample: 500 mg lyophilized sludge
• Dispersion sorbent: C18, 2 g, 5 minutes
• On-line clean-up: PSA, 1 g
• Elution: 10 mL MeOH
– SPE:
• Bond Elut SCX, 500 mg
• Drying step 5 min N2
• Elution 10 mL MeOH, 0.5% (v/v) NH3
• Concentration 5 mL MeOH
• LC-HRMS determination
– C18 reversed phase
– ESI+ ionization
– QTOF MS/MS
J. Casado, G. Castro, I. Rodríguez, M. Ramil, R. Cela, Anal. Bioanal. Chem. 407 (2015) 907-917

30. 3
x10
0
2
4
6
8
+ESI EIC Product Ion Frag=150.0V CID@24.0 (416.9904[z=1] -> 158.9763
…
Counts vs. Acquisition Time (min)
14.5 15 15.5 16 16.5 17 17.5
Counts vs. Mass-to-Charge (m/z)
3
x10
0
2.5
5
7.5
+ESI Product Ion (15.108-15.301 min, 8 Sc
…
158.9758
416.9897
69.0452
100 200 300 400
C7H5Cl2
+
Calc.158.9763
Error -3.1 ppm
Sludge sample 6
U n i v e r s i d a d e d e S a n t i a g o d e C o m p o s t e l a
30
4
x10
0
1
2
3
4
5
+ESI EIC Product Ion Frag=150.0V CID@20.0 (277.0788[z=1] -> 165.0699
…
Counts vs. Acquisition Time (min)
13 13.5 14 14.5 15 15.5 16
Counts vs. Mass-to-Charge (m/z)
4
x10
0
1
2
3
+ESI Product Ion (13.632-13.859 min, 7 Scans)
…
165.0693
242.1077
50 100 150 200 250
277.0775
C13H9
+
Calc.165.0699
Error -3.6 ppm
2
x10
0
1
2
3
4
5
+ESI EIC Product Ion Frag=150.0V CID@16.0 (293.1051[z=1] -> 197.0705
…
Counts vs. Acquisition Time (min)
12 12.5 13 13.5 14 14.5 15
2
x10
0
2
4
6
+ESI Product Ion (12.599-12.737 min, 5 Sc
…
197.0722
141.0084
Counts vs. Mass-to-Charge (m/z)
100 150 200 250 300
293.1047
69.0456
C11H14ClO+
Calc.197.0705
Error +8.6 ppm
3
x10
0
0.5
1
1.5
2
2.5
+ESI EIC Product Ion Frag=150.0V CID@37.0 (531.1560[z=1] -> 489.1455
…
Counts vs. Acquisition Time (min)
12.5 13 13.5 14 14.5 15 15.5
3
x10
0
1
2
3
+ESI Product Ion (12.999-13.240 min, 6 Sc
…
289.1068
Counts vs. Mass-to-Charge (m/z)
100 200 300 400 500
531.1546
489.1442
82.0530
C24H27Cl2N4O3
+
Calc. 489.1455
Error -2.7 ppm
Climbazole Clotrimazole Miconazole
Ketoconazole Terbinafine
3
x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
+ESI EIC Product Ion Frag=150.0V CID@14.0 (292.2060[z=1] -> 141.0699
…
Counts vs. Acquisition Time (min)
15.5 16 16.5 17 17.5 18 18.5
3
x10
0
0.5
1
+ESI Product Ion (15.971-16.201 min, 12 S
…
141.0699
93.0686
Counts vs. Mass-to-Charge (m/z)
100 150 200 250 300
50
C11H9
+
Calc 141.0699
Error +0.1 ppm
292.2077

31. Levels and fate of antimycotic
pharmaceuticals at sewage
treatment plants
Jorge Casado
Department of Analytical Chemistry
J Casado, G Castro, I Rodríguez, M Ramil, R Cela