Oslo, 20 June 2017 Determination of fluoroquinolones in fish tissues, biological fluids and environmental waters

Transcription

1 Oslo, 20 June 2017 Determination of fluoroquinolones in fish tissues, biological fluids and environmental waters H. Ziarrusta, N. Val, H. Dominguez, L. Mijangos, A. Prieto, A. Usobiaga, N. Etxebarria, O. Zuloaga, M. Olivares.

2 Pharmaceuticals

3 Fluoroquinolones (FQs)

4 Fluoroquinolones: effects

5 Fluoroquinolones: method development Lack of analytical methods

6 Objectives 1 Optimization LC-MS/MS analysis of FQs 2 Optimization of the extraction: fish tissue/biofluids + environmental water 3 Application to real samples

7 Experimental part Solid-Phase Extraction (SPE) Focused Ultrasound Solid-Liquid Extraction (FUSLE) Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS)

8 Fluoroquinolones Pefloxacin (PEFLO) Norfloxacin (NORF) Enofloxacin (ENO) Danofloxacin (DANO) Ofloxacin (OFLO) and Levofloxacin (LEVO) Ciprofloxacin (CIPRO) Sparfloxacin (SPAR) Lomefloxacin (LOME) Enrofloxacin (ENRO)

9 Environmental samples Environmental waters Fish tissues and biofluids

10 Results and Discussion 1 Optimization LC-MS/MS analysis of FQs 2 Optimization of the extraction: fish tissue/biofluids + environmental water 3 Application to real samples

11 Optimization of LC-MS/MS

12 Fragmentor and collision energy Analyte SRM transitions NORF ENO PEFLO OFLO/LEVO CIPRO DANO LOME ENRO SPAR 320 (104 V) 302/231/282 (17/41/29 ev) 321 (104 V) 303/232/204 (17/37/45 ev) 334 (104 V) 316/290/233 (17/13/25 ev) 362 (104 V) 318/261/344 (17/25/17 ev) 332 (104 V) 314/231/288 (17/41/13 ev) 358 (104 V) 340/82/255 (21/45/41 ev) 352 (104 V) 265/308/334 (21/13/17 ev) 360 (104 V) 342/316/286 (17/17/37 ev) 393 (104 V) 349/292/375 (17/21/17 ev)

13 Results and Discussion 1 Optimization LC-MS/MS analysis of FQs 2 Optimization of the extraction: fish tissue/biofluids + environmental waters 3 Application to real samples

14 Optimization of the extraction 2.1 Fish tissues: liver and muscle 2.2 Fish biofluids: plasma and bile 2.3 Environmental waters: seawater, estuarine water and WWTP water

15 Optimization of the extraction 2.1 Fish tissues: liver and muscle 2.2 Fish biofluids: plasma and bile 2.3 Environmental waters: seawater, estuarine water and WWTP water

16 Normalized revovery Fish tissues: FUSLE Extraction solvent 120% ACN ACN:Milli-Q ACN:EDTA ACN:HOAc MeOH:HOAc 100% 80% 60% 40% 20% 0% NORF ENO PEFLO OFLO CIPRO DANO LOME ENRO SPAR

17 FUSLE recovery Fish tissues: FUSLE Extraction time 120% 100% 2.5 min 30 s 80% 60% 40% 20% 0%

18 FUSLE recovery FUSLE recovery Fish tissues: FUSLE Extraction time 120% 100% 2.5 min 30 s 80% 60% 40% 20% 0% 140% 120% 100% 80% 60% 40% 20% 0% -20% 30 s -E1 30 s -E2 E1 + E2 Optimum: 30 s x 2

19 Normalized recovery Solid-Phase Extraction (SPE) Sorbent phase 120% MAX (ph=12) CX (ph=4) CX (ph=6.5) HLB (ph=7) 100% 80% 60% 40% 20% 0% NORF ENO PEFLO OFLO CIPRO DANO LOME ENRO SPAR

20 Liver: dirty extracts Muscle Liver

21 Normalized recovery Liver: dirty extracts Liver Positive matrix effect 350% 300% 250% 200% 150% 100% 50% 0% NORF ENO PEFL OFLO CIPRO DANO LOME ENRO SPAR

22 Liver: extra step to remove lipids Clean-up strategies

23 Liver: extra step to remove lipids Clean-up strategies 1. Increase HLB phase

24 Liver: extra step to remove lipids Clean-up strategies 1. Increase HLB phase 2. Florisil-HLB

25 Normalized recovery Liver: extra step to remove lipids Clean-up strategies 1. Increase HLB phase 2. Florisil-HLB 3. Liquid-liquid extraction (LLE) prior to HLB 350% 300% 250% 200% HLB LLE+HLB 150% 100% 50% 0% NORF ENO PEFL OFLO CIPRO DANO LOME ENRO SPAR

26 Optimization of the extraction 2.1 Fish tissues: liver and muscle 2.2 Fish biofluids: plasma and bile 2.3 Environmental waters: seawater, estuarine water and WWTP water

27 Biofluids: SPE HLB: signal suppression

28 Biofluids: SPE HLB: signal suppression MIPs cartridges

29 Optimization of the extraction 2.1 Fish tissues: liver and muscle 2.2 Fish biofluids: plasma and bile 2.3 Environmental waters: seawater, estuarine water and WWTP water

30 RP-SPE (Oasis HLB) Seawater Estuarine and effluent: dirty extracts Signal suppression ( > 50 %)

31 RP-SPE (Oasis HLB) Seawater Estuarine and effluent: dirty extracts Signal suppression ( > 50 %) Solution: WAX + HLB Cleaner extracts and less signal suppression (< 33 %)

32 Method validation 75 ng/g 200 ng/g 75 ng/l

33 Method validation Linearity R 2 : Limits of detection LOD < 2 ug/ml LOQ < 5 ug/ml Precision RSD < 15 % Accuracy

34 Method validation Apparent recoveries: % Analyte Fish tissues Fish biofluids Environmental waters Liver Muscle Plasma Bile Seawater Estuarine Effluent NORF a ENO a PEFLO b OFLO b CIPRO a DANO b LOME b ENRO b SPAR a a Corrected with [ 2 H 8 ] CIPRO. b Corrected with [ 2 H 5 ] ENRO.

35 Method validation Apparent recoveries: % Analyte Fish tissues Fish biofluids Environmental waters Liver Muscle Plasma Bile Seawater Estuarine Effluent NORF a ENO a PEFLO b OFLO b CIPRO a DANO b LOME b ENRO b SPAR a a Corrected with [ 2 H 8 ] CIPRO. b Corrected with [ 2 H 5 ] ENRO.

36 Results and Discussion 1 Optimization LC-MS/MS analysis of FQs 2 Optimization of the extraction: fish tissue/biofluids + environmental water 3 Application to real samples

37 Real Samples from Biscay Coast

38 Real Samples from Biscay Coast

39 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 Seawater < LOQ Grey mullet liver 4 ng/g OFLO/LEVO

40 Conclusions

41 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

42 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

43 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

44 Tussen takk! Oslo, 20 June 2017

45 Oslo, 20 June 2017 Determination of fluoroquinolones in fish tissues, biological fluids and environmental waters H. Ziarrusta, N. Val, H. Dominguez, L. Mijangos, A. Prieto, A. Usobiaga, N. Etxebarria, O. Zuloaga, M. Olivares.