Screening of Pharmaceuticals and Personal Care Products in Effluents and Surface Waters with LCMS Q-TOF

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1 Screening of Pharmaceuticals and Personal Care Products in Effluents and Surface Waters with LCMS Q-TOF Anthony Gravell National Laboratory Service Environment Agency

2 Agenda Pharmaceuticals in the environment - Why look for them? Analytical Challenges for LCMS Screening Compounds found in effluents and surface waters Confirmation using targeted MS/MS Summary

3 Pharmaceuticals Why look for them? Widespread detection in environmental samples Hormones have significant EDC effects Anti-biotic resistance Pseudo-persistent European Union s Water Framework Directive New proposal to add pharmaceuticals to Annex X

4 Pharmaceuticals Why look for them? Exposure of pharmaceuticals to aquatic life most likely from STW discharges Environmental risks due are an important issue for the EA regulator Chemical Investigation programme recently undertaken by UK water companies

5

6 National Geographic, April Pharmaceuticals in Fish

7 Analytical Challenges for LCMS Screening Detect and identify isobaric compounds Isolate compounds from matrix components Must Haves High Resolution Searching for unknowns Large number of compounds High mass accuracy Wide dynamic range

8 Resolution FWHM Resolution 100% Apex R m m 50% FWHM 400 Quadrupole MS R Time of Flight MS R m/z Mass Assignment

9 Resolution FWHM Resolution Desisopropyl-Atrazine C 5 H 8 ClN 5 Theoretical m/z: Experimental m/z: Mass Error: ppm Resolution: 25,207 Allidochlor C 8 H 12 ClNO Theoretical m/z: Experimental m/z: Mass Error: ppm Resolution: 25,556 Sample 0.3pg A:0.1pg B Blank No Chemical Interference

10 Measuring mass accuracy Mass error = (exact mass) (accurate mass) Mass error in parts per million(ppm) = mass error x 10 6 exact mass

11 Importance of resolution and mass accuracy Labetalol C19H24N2O3 Extracted Ion Chromatogram 400ppm

12 Importance of resolution and mass accuracy Significant improvement in selectivity Extracted Ion Chromatogram 10ppm Extracted Ion Chromatogram 400ppm

13 Sample collection & extraction Primary and final effluent POCIS SPE disks Disk extraction equipment

14 LC parameters 1290 Infinity LC system Column: Waters Atlantis T3, 2.1 x 150mm, C Flow: 0.4 ml/min Injection Volume: 5µL Solvent A: 2mM Ammonium Acetate, Water, 0.05% formic acid Solvent B: 2mM Ammonium Acetate, Methanol, 0.05% formic acid Gradient: Time (min) %B Equilibration time 10mins

15 MS parameters Source parameters : ESI Jetsream source Drying Gas Temp: 300 C Drying Gas Flow: 10l/min Nebuliser: 50 p.s.i. Sheath Gas Temp: 350 C Sheath Gas Flow: 12 l/min VCcap: 3000V Fragmentor: 175V Nozzle voltage: 1000V Aquisition parameters: ESI Positive: 2 & 4GHz modes Scan Range: m/z Scan Rate: 3Hz Internal Mass Calibration Refererence ions: m/z, m/z

16 Identification and scoring MS Score based on Monoisotopic mass (Mass Match) Isotope distribution (Abundance Match) Theoretical isotope distribution and spacing generated from the molecular formulae contained in the database. Score is assigned for each of the three criteria and combined into a final score for each target compound in sample Isotope spacing (Spacing Match)

17 What did we find? Compounds found in effluents and surface waters

18 Personal Care Product Cetirizine Antihistamine Extracted Ion Chromatogram Match factor > 98 <1ppm difference against database for M+H + <0.1mDa difference against database Theoretical v. actual Isotope abundance and spacing

19 Prescribed Pharmaceutical Lamotrigine Anti-convulsant Extracted Ion Chromatogram Match factor > 98 <1ppm difference against database for M+H + <0.1mDa difference against database Theoretical v. actual Isotope abundance and spacing

20 Personal Care Product Orlistat Weight loss aid sold under trade name alli Extracted Ion Chromatogram Log Kow: 8.19 Probably associated with suspended particulates in effluent Match factor > 98 <1ppm difference against database for M+H + <0.1mDa difference against database Theoretical v. actual Isotope abundance and spacing

21 PPCPs found in final effluent Tgt Name Cpd 1: Sotalol Cpd 2: Atenolol Cpd 3: Ranitidine Cpd 4: Trimethoprim Cpd 5: Amisulpride Cpd 6: Lidocaine Cpd 7: Metoclopramide Cpd 9: Tramadol Cpd 8: Tramadol Cpd 10: Lamotrigine Cpd 11: Mirtazapine Cpd 12: Celiprolol Cpd 13: Quinine Cpd 14: Labetalol Cpd 15: Venlaxafine Cpd 16: Bisoprolol Cpd 17: Propranolol Cpd 18: Citalopram Cpd 19: Flecainide Cpd 20: Mebeverine Tgt Score RT Diff Mass Error (ppm) Tgt Formula C12 H20 N2 O3 S C14 H22 N2 O3 C13 H22 N4 O3 S C14 H18 N4 O3 C17 H27 N3 O4 S C14 H22 N2 O C14 H22 Cl N3 O2 C16 H25 N O2 C16 H25 N O2 C9 H7 Cl2 N5 C17 H19 N3 C20 H33 N3 O4 C20 H24 N2 O2 C19 H24 N2 O3 C17 H27 N O2 C18 H31 N O4 C16 H21 N O2 C20 H21 F N2 O C17 H20 F6 N2 O3 C25 H35 N O5 Beta blocker Anti-depressant

22 PPCPs found in final effluent (cont.) Tgt Name Cpd 21: Diltiazem-Cis Cpd 22: Carbamazepine Cpd 23: Fexofenadine Cpd 24: Amitriptyline Cpd 25: Erythromycin Cpd 26: Losartan Cpd 27: Cetirizine Cpd 28: Naproxen Cpd 29: Bezafibrate Cpd 30: Clarithromycin Cpd 32: Valsartan Cpd 31: Dipyridamote Cpd 33: Irbesartan Cpd 34: Ketoconazole Cpd 35: Atorvastatin Cpd 36: Telmisartan Cpd 37: Clopidogrel Cpd 38: Dioctyl sulfosuccinate Cpd 39: Orlistat Tgt Score RT Diff Mass Error (ppm) Tgt Formula C22 H26 N2 O4 S C15 H12 N2 O C32 H39 N O4 C20 H23 N C37 H67 N O13 C22 H23 Cl N6 O C21 H25 Cl N2 O3 C14 H14 O3 C19 H20 Cl N O4 C38 H69 N O13 C24 H29 N5 O3 C24 H40 N8 O4 C25 H28 N6 O C26 H28 Cl2 N4 O4 C33 H35 F N2 O5 C33 H30 N4 O2 C16 H16 Cl N O2 S C20 H38 O7 S C29 H53 N O5 Calcium channel blocker angiotensin-ii receptor antagonist

23 PPCPs found in final effluent (cont.) Tgt Name Cpd 21: Diltiazem-Cis Cpd 22: Carbamazepine Cpd 23: Fexofenadine Cpd 24: Amitriptyline Cpd 25: Erythromycin Cpd 26: Losartan Cpd 27: Cetirizine Cpd 28: Naproxen Cpd 29: Bezafibrate Cpd 30: Clarithromycin Cpd 32: Valsartan Cpd 31: Dipyridamote Cpd 33: Irbesartan Cpd 34: Ketoconazole Cpd 35: Atorvastatin Cpd 36: Telmisartan Cpd 37: Clopidogrel Cpd 38: Dioctyl sulfosuccinate Cpd 39: Orlistat Tgt Score RT Diff Mass Error (ppm) Tgt Formula C22 H26 N2 O4 S C15 H12 N2 O C32 H39 N O4 C20 H23 N C37 H67 N O13 C22 H23 Cl N6 O C21 H25 Cl N2 O3 C14 H14 O3 C19 H20 Cl N O4 C38 H69 N O13 C24 H29 N5 O3 C24 H40 N8 O4 C25 H28 N6 O C26 H28 Cl2 N4 O4 C33 H35 F N2 O5 C33 H30 N4 O2 C16 H16 Cl N O2 S C20 H38 O7 S C29 H53 N O5 Antibiotic Treatment of hyperlipidaemia & cholesterol reducing

24 PPCPs found in surface water plus a lot more!

25 Confirmation by MS/MS - Standard Ofloxacin Propanolol Fluoxetine Oxytetracycline Erythromycin

26 Confirmation by MS/MS - Sample TIC Propranolol EICs Ofloxacin Oxytetracycline Erythromycin (not present) Fluoxetine

27 Confirmation of Fluoxetine by MS/MS Precursor ion A Mass error = 0.8ppm A

28 Confirmation of Propranolol by MS/MS Average mass error = -2.67ppm

29 Summary Significant number of pharmaceuticals identified Various therapeutic classes found in all matrices Mass accuracy is a valuable tool for target identification High resolution ensures good mass accuracy with real samples How much resolution is required depends on matrix

30 Summary (cont.) Use of MS/MS with accurate mass provides unequivocal compound identification Screening method shows potential for determining efficiency of various WWTW treatment options POCIS provides higher sampling rates and lower LODs

31 Further work Use passive samplers to detect low concentrations of EDC s Look at metabolites of pharmaceuticals Chirality and different effects / toxicity Link to High Resolution GC/MS

32 Acknowledgements Prof. Graham Mills University of Portsmouth Agilent Technologies Dwr Cymru / Welsh Water

33 Any questions?