Determination of Heavy Metals in Fish Tissues, Water and Sediment
Determination of fluoroquinolones in fish tissues, biological ......Analyte Fish tissues Fish...
Transcript of Determination of fluoroquinolones in fish tissues, biological ......Analyte Fish tissues Fish...
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H. Ziarrusta, N. Val, H. Dominguez, L. Mijangos, A. Prieto,
A. Usobiaga, N. Etxebarria, O. Zuloaga, M. Olivares.
Determination of fluoroquinolones
in fish tissues, biological fluids and
environmental waters
Oslo, 20 June 2017
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Pharmaceuticals
Introduction Objectives Experimental part Results Conclusions
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Fluoroquinolones (FQs)
Introduction Objectives Experimental part Results Conclusions
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Fluoroquinolones: effects
Introduction Objectives Experimental part Results Conclusions
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Introduction Objectives Experimental part Results Conclusions
Fluoroquinolones: method development
Lack of analytical methods
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Objectives
Introduction Objectives Experimental part Results Conclusions
Optimization LC-MS/MS analysis of FQs
Optimization of the extraction: fish tissue/biofluids + environmental water
Application to real samples
1
2
3
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Experimental part
Introduction Objectives Experimental part Results Conclusions
Focused Ultrasound
Solid-Liquid Extraction
(FUSLE)
Solid-Phase Extraction
(SPE)
Liquid Chromatography
tandem Mass Spectrometry
(LC-MS/MS)
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Fluoroquinolones
Norfloxacin (NORF)
Lomefloxacin (LOME)
Enofloxacin (ENO)
Ofloxacin (OFLO) and Levofloxacin (LEVO)
Ciprofloxacin (CIPRO)
Danofloxacin (DANO)
Sparfloxacin (SPAR)
Enrofloxacin (ENRO)
Pefloxacin (PEFLO)
Introduction Objectives Experimental part Results Conclusions
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Environmental samples
Introduction Objectives Experimental part Results Conclusions
Environmental waters Fish tissues and biofluids
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Results and Discussion
Optimization LC-MS/MS analysis of FQs
Optimization of the extraction: fish tissue/biofluids + environmental water
Application to real samples
1
2
3
Introduction Objectives Experimental part Results Conclusions
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Optimization of LC-MS/MS
Introduction Objectives Experimental part Results Conclusions
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Fragmentor and collision energy
Introduction Objectives Experimental part Results Conclusions
Analyte SRM transitions
NORF 320 (104 V) → 302/231/282 (17/41/29 eV)
ENO 321 (104 V) → 303/232/204 (17/37/45 eV)
PEFLO 334 (104 V) → 316/290/233 (17/13/25 eV)
OFLO/LEVO 362 (104 V) → 318/261/344 (17/25/17 eV)
CIPRO 332 (104 V) → 314/231/288 (17/41/13 eV)
DANO 358 (104 V) → 340/82/255 (21/45/41 eV)
LOME 352 (104 V) → 265/308/334 (21/13/17 eV)
ENRO 360 (104 V) → 342/316/286 (17/17/37 eV)
SPAR 393 (104 V) → 349/292/375 (17/21/17 eV)
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Results and Discussion
Optimization LC-MS/MS analysis of FQs
Optimization of the extraction: fish tissue/biofluids + environmental waters
Application to real samples
1
2
3
Introduction Objectives Experimental part Results Conclusions
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Optimization of the extraction
Fish tissues: liver and muscle
Fish biofluids: plasma and bile
Environmental waters: seawater, estuarine water and WWTP water
2.1
2.2
2.3
Introduction Objectives Experimental part Results Conclusions
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Optimization of the extraction
Fish tissues: liver and muscle
Fish biofluids: plasma and bile
Environmental waters: seawater, estuarine water and WWTP water
2.1
2.2
2.3
Introduction Objectives Experimental part Results Conclusions
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Fish tissues: FUSLE
• Extraction solvent
Introduction Objectives Experimental part Results Conclusions
0%
20%
40%
60%
80%
100%
120%
NORF ENO PEFLO OFLO CIPRO DANO LOME ENRO SPAR
No
rma
lize
d r
evo
ve
ry
ACN ACN:Milli-Q ACN:EDTA ACN:HOAc MeOH:HOAc
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Fish tissues: FUSLE
• Extraction time
Introduction Objectives Experimental part Results Conclusions
0%
20%
40%
60%
80%
100%
120%
FU
SL
E r
eco
very
2.5 min 30 s
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Fish tissues: FUSLE
• Extraction time
Introduction Objectives Experimental part Results Conclusions
0%
20%
40%
60%
80%
100%
120%
FU
SL
E r
eco
very
2.5 min 30 s
-20%
0%
20%
40%
60%
80%
100%
120%
140%
FU
SL
E r
eco
very
30 s -E1 30 s -E2 E1 + E2
Optimum: 30 s x 2
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Solid-Phase Extraction (SPE)
• Sorbent phase
Introduction Objectives Experimental part Results Conclusions
0%
20%
40%
60%
80%
100%
120%
NORF ENO PEFLO OFLO CIPRO DANO LOME ENRO SPAR
No
rma
lize
d r
ec
ove
ry
MAX (pH=12) CX (pH=4) CX (pH=6.5) HLB (pH=7)
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Liver: dirty extracts
Introduction Objectives Experimental part Results Conclusions
Muscle Liver
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Liver: dirty extracts
• Positive matrix effect
Introduction Objectives Experimental part Results Conclusions
0%
50%
100%
150%
200%
250%
300%
350%
NORF ENO PEFL OFLO CIPRO DANO LOME ENRO SPAR
No
rma
lize
d r
ec
ove
ry
Liver
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Liver: extra step to remove lipids
• Clean-up strategies
Introduction Objectives Experimental part Results Conclusions
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Liver: extra step to remove lipids
• Clean-up strategies
1. Increase HLB phase
Introduction Objectives Experimental part Results Conclusions
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Liver: extra step to remove lipids
• Clean-up strategies
1. Increase HLB phase
2. Florisil-HLB
Introduction Objectives Experimental part Results Conclusions
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Liver: extra step to remove lipids
• Clean-up strategies
1. Increase HLB phase
2. Florisil-HLB
3. Liquid-liquid extraction (LLE) prior to HLB
Introduction Objectives Experimental part Results Conclusions
0%
50%
100%
150%
200%
250%
300%
350%
NORF ENO PEFL OFLO CIPRO DANO LOME ENRO SPAR
No
rma
lize
d r
ec
ove
ry
HLB LLE+HLB
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Optimization of the extraction
Fish tissues: liver and muscle
Fish biofluids: plasma and bile
Environmental waters: seawater, estuarine water and WWTP water
2.1
2.2
2.3
Introduction Objectives Experimental part Results Conclusions
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Biofluids: SPE
• HLB: signal suppression
Introduction Objectives Experimental part Results Conclusions
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Biofluids: SPE
• HLB: signal suppression
• MIPs cartridges
Introduction Objectives Experimental part Results Conclusions
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Optimization of the extraction
Fish tissues: liver and muscle
Fish biofluids: plasma and bile
Environmental waters: seawater, estuarine water and WWTP water
2.1
2.2
2.3
Introduction Objectives Experimental part Results Conclusions
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RP-SPE (Oasis HLB)
• Seawater
• Estuarine and effluent: dirty extracts
• Signal suppression ( > 50 %)
Introduction Objectives Experimental part Results Conclusions
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RP-SPE (Oasis HLB)
• Seawater
• Estuarine and effluent: dirty extracts
• Signal suppression ( > 50 %)
• Solution:
WAX + HLB
Introduction Objectives Experimental part Results Conclusions
• Cleaner extracts and less
signal suppression (< 33 %)
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Method validation
Introduction Objectives Experimental part Results Conclusions
75 ng/g
200 ng/g
75 ng/L
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Method validation
• Linearity
• R2: 0.997-0.9997
• Limits of detection
• LOD < 2 ug/mL
• LOQ < 5 ug/mL
• Precision
• RSD < 15 %
• Accuracy
Introduction Objectives Experimental part Results Conclusions
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Analyte Fish tissues Fish biofluids Environmental waters
Liver Muscle Plasma Bile Seawater Estuarine Effluent
NORFa 107 96 93 96 90 84 93
ENOa 89 83 95 104 80 82 83
PEFLOb 103 89 91 97 101 109 109
OFLOb 96 94 110 105 105 93 125
CIPROa 92 92 96 97 87 85 109
DANOb 104 92 115 94 112 109 118
LOMEb 82 94 103 106 106 120 126
ENROb 90 94 100 101 101 97 99
SPARa 14 162 109 70 67 54 59
Method validation
Introduction Objectives Experimental part Results Conclusions
aCorrected with [2H8]–CIPRO. bCorrected with [2H5]–ENRO.
•Apparent recoveries: 80 - 126 %
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Analyte Fish tissues Fish biofluids Environmental waters
Liver Muscle Plasma Bile Seawater Estuarine Effluent
NORFa 107 96 93 96 90 84 93
ENOa 89 83 95 104 80 82 83
PEFLOb 103 89 91 97 101 109 109
OFLOb 96 94 110 105 105 93 125
CIPROa 92 92 96 97 87 85 109
DANOb 104 92 115 94 112 109 118
LOMEb 82 94 103 106 106 120 126
ENROb 90 94 100 101 101 97 99
SPARa 14 162 109 70 67 54 59
Method validation
Introduction Objectives Experimental part Results Conclusions
aCorrected with [2H8]–CIPRO. bCorrected with [2H5]–ENRO.
•Apparent recoveries: 80 - 126 %
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Results and Discussion
Optimization LC-MS/MS analysis of FQs
Optimization of the extraction: fish tissue/biofluids + environmental water
Application to real samples
1
2
3
Introduction Objectives Experimental part Results Conclusions
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Real Samples from Biscay Coast
Introduction Objectives Experimental part Results Conclusions
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Real Samples from Biscay Coast
Introduction Objectives Experimental part Results Conclusions
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Real Samples from Biscay Coast
• WWTP effluent • 143 ng/L NORF
• 160 ng/L CIPRO
• 234 ng/L OFLO/LEVO
• Estuarine water • 44 ng/L NORF
• 79 ng/L CIPRO
• 278 ng/L OFLO/LEVO
• Grey mullet liver • 4 ng/g OFLO/LEVO
Introduction Objectives Experimental part Results Conclusions
• Seawater < LOQ
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Conclusions
Introduction Objectives Experimental part Results Conclusions
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Conclusions
• Optimization of 10 FQs • Correction with labelled analogues
matrices with different clean-up strategies: Fish muscle and seawater: RP-SPE
Fish liver: LLE + RP-SPE
Fish bile and plasma: MIPs
Estuarine and effluent waters: WAX + RP-SPE
Real samples from the Biscay Coast: CIPRO, NORF and OFLO/LEVO
Water and fish liver samples
Introduction Objectives Experimental part Results Conclusions
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Conclusions
• Optimization of 10 FQs • Correction with labelled analogues
• 7 matrices with different clean-up strategies: • Fish muscle and seawater: RP-SPE
• Fish liver: LLE + RP-SPE
• Fish bile and plasma: MIPs
• Estuarine and effluent waters: WAX + RP-SPE
Real samples from the Biscay Coast: CIPRO, NORF and OFLO/LEVO
Water and fish liver samples
Introduction Objectives Experimental part Results Conclusions
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Conclusions
• Optimization of 10 FQs • Correction with labelled analogues
• 7 matrices with different clean-up strategies: • Fish muscle and seawater: RP-SPE
• Fish liver: LLE + RP-SPE
• Fish bile and plasma: MIPs
• Estuarine and effluent waters: WAX + RP-SPE
• Real samples from the Biscay Coast: • CIPRO, NORF and OFLO/LEVO
• Water and fish liver samples
Introduction Objectives Experimental part Results Conclusions
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Tussen takk!
Oslo, 20 June 2017
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H. Ziarrusta, N. Val, H. Dominguez, L. Mijangos, A. Prieto,
A. Usobiaga, N. Etxebarria, O. Zuloaga, M. Olivares.
Determination of fluoroquinolones
in fish tissues, biological fluids and
environmental waters
Oslo, 20 June 2017