Screening Environmental Water for a diverse range of Pollutants with UPLC High Resolution LC/MS - Waters Environmental Analysis
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There is a need to screen for an ever increasing number of chemically diverse contaminants that maybe present in the environment. Typically these contaminants may only be present at very low (ppb or even ppt) concentrations and due to the the complexity of the sample matrices encountered this screening is an increasingly demanding analytical challenge.
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Screening Environmental Water for a diverse range of Pollutants with UPLC High Resolution LC/MS - Waters Environmental Analysis
- 1. ©2015 Waters Corporation 1 Screening environmental water for a diverse range of pollutants with UPLC-high resolution MS
- 2. ©2015 Waters Corporation 2 Content Sources of environmental pollution – the challenges? PPCPs in water – targeted screening Expanding the scope; migrating to accurate mass screening Efficient & intelligent data interrogation; UNIFI workflows – Qualitative screening – Unknown screening – binary compare – Unknown screening – Met ID Summary
- 3. ©2015 Waters Corporation 3 Environmental Screening the challenge? Requirement to screen for an ever increasing number of chemical diverse contaminants Pollutants maybe present at very low concentrations & exhibit various toxicological effects; (DWD Maximum allowable concentrations ppb – ppq) Occurrence of new or unexpected residues including metabolites, biotransformation and degradation products... Screen for the presence of known / unexpected & unknown chemicals simultaneously – Cost effective screening process required
- 4. ©2015 Waters Corporation 4 Transformation products Targeted analysis Perfluorinated compounds Environmental Pollutants Marine studies Unknown analysis Dioxins Persistent organic pollutants e.g. PAHs, BfRs, PCBs, PCDD,PCDF, pesticides… Endocrine disrupting compounds Water analysis
- 5. ©2015 Waters Corporation 5 Steroids Fluoroquinones Β-lactams Macrolides Sulfa’s Beta Blockers Anti-convulsant Decongestant Anti-histamine Anti-bacterial Anti-inflamatory Illicit drugs Anti-Helmintic Vaso-active PPCPs in environmental water structural diversity
- 6. ©2015 Waters Corporation 6 Steroids 6a-methylprednisolone Corticosterone Cortisone Desoximethasone Dexamethasone Hydrocortisone Triamcinolone Triamcinolone acetonide Digoxigenin Budesonide Flumethasone Beclomethasone dipropionate Fluoroquinones Difloxacin Ofloxacin Enrofloxacin Cinoxacin Sparfloxacin Fleroxacin Lomefloxacin Flumequin Nalidixic acid Antibiotics Dicloxacillin Penicillin G Cloxacillin Ceflaxin Rifaximin Trimethoprim Macrolides Erythromycin Josamycin Lincomycin Roxithromycin Tilmicosin Azithromycin Tiamulin Sulfonamides Sulfabenzamide Sulfacetamide Sulfadiazine Sulfadoxine Sulfaguanidine Sulfamerazine Sulfameter Sulfamethazine Sulfamethoxypyridazine Sulfapyridine Sulfamethizole Sulfamthoxazole Sulfadimethoxine Tolbutamide Beta Blockers Atenolol Carazolol Metoprolol Oxprenolol Salbutamol (albuterol) Tulobuterol Carbamazepine Cotinine Decongestant Cimbuterol Chlorpheniramine Cimetidine Promethazine Tripolidine Diphenhydramine Ranitidine Anti-bacterial Cocaine Codeine Dapsone Ipronidazole-hydroxy Pyrimethamine Terbinafine Ternidazole Miconazole Triclocarban Roxarsone Anti-Helmintic Diethylcarbamazine Levamisole (tetramisole) Oxfendazole Praziquantel Benzocaine Procaine Xylazine Bromhexine Vaso-active Buflomedil Diltiazem Acetaminophen Ticlopidine Metformin Anti-inflamatory Flunixin Ketoprofen Tolfenamic acid Naproxen Ibuprofen Warfarin Gemfibrozil PPCPs: Target list
- 7. ©2015 Waters Corporation 7 MAX 3 cc 150 mg MCX 3 cc 150 mg 1000 mL Final extract 1 mL Enrichment 1000:1 Fraction 1: Acidics Fraction 2: Neutrals & some Basics Fraction 3: Polar Basics Pool Fractions Single analysis PPCP: Sample Preparation Strategy Stage 1: Xevo TQD targeted Stage 2: Tof screening
- 8. ©2015 Waters Corporation 8 UPLC Conditions UPLC system: ACQUITY UPLC H-Class System Runtime: 8.0 min Column: ACQUITY UPLC HSS T3, 2.1 x 100 mm, 1.7 m Column temp: 60 C Mobile phase A: 10 mM ammonium formate in Water Mobile phase B: 10 mM ammonium formate in Acetonitrile Elution: 5 minute linear gradient from 5% (B) to 95% (B) Flow Rate: 0.45 mL/min Injection volume: 100 L MS Conditions MS System: XEVO TQD Ionization mode: ESI Negative/Positive switching Capillary voltage: 3.5 kV Cone voltage: Optimized for each PPCP Source temp: 140 C Desolvation temp: 550 C Desolvation gas: 1100 L/hr Cone gas: 50 L/hr PPCP: Targeted screening LC-MS/MS conditions
- 9. ©2015 Waters Corporation 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Example TIC chromatogram PPCPs in environmental water
- 10. ©2015 Waters Corporation 11 Detected Conc: 0.32ppt Time 2.40 2.60 2.80 3.00 3.20 3.40 % 0 100 2.40 2.60 2.80 3.00 3.20 3.40 % 0 100 28: MRM of 2 Channels ES+ 267.2 > 145.1 (atenolol)x50 28: MRM of 2 Channels ES+ 267.2 > 145.1 (atenolol)2.96 Detected Conc: > 0.1ppt Time 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 % 0 100 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 % 0 100 69: MRM of 2 Channels ES+ 411.4 > 391.2 (flumethasone)5.59 69: MRM of 2 Channels ES+ 411.4 > 391.2 (flumethasone)x10 PPCP: Incurred residues Flumethasone Surface water Flumethasone Well water Atenolol Well water 10 X 50 X Atenolol Surface water
- 11. ©2015 Waters Corporation 12 Can we use exact mass screening to expand our method for the detection of additional analytes? But what else is in the water….
- 12. ©2015 Waters Corporation 13 Screening approaches have evolved to meet sensitivity & specificity requirements – From sectors to single quads to tandem quads to Tof (HR-MS) Current Gold Standard for Screening: The Tandem Quad Why move from TQ s to HRMS Screening? QQQ HRMS 2 nominal mass transitions Accurate Mass Precursor 1 Ion Ratio Accurate Mass Fragments Targeted Acquisition Isotopic Pattern Scoring Limited by Duty Cycle Adduct Presence Ion Ratios CCS Historical Data Review via RADAR Historical LE/HE Data Review Unknown Screening – What else of interest is present in the sample. Established Data Review Perceived complexity of data review
- 13. ©2015 Waters Corporation 14 UNIFI Workflow Capability Acquire the data once; interrogate many times Targeted Screening - Qualitative Unknown Screening – Binary Compare Targeted Screening Quan-Qual Unknown Screening – Met ID
- 14. ©2015 Waters Corporation 15 Screening Platform Solution overview UNIFI toxicology library Library of over 1000 compounds with detection results Exact mass, retention time, fragment ions Pharmaceuticals, veterinary medicines, drugs of abuse & pesticides
- 15. ©2015 Waters Corporation 16 Comprehensive Scientific Library Screening experiments are dependent on the quality of the libraries Libraries already available: – Over 2000 entries of which around 700 compounds contain method related information (RT, m/z for precursor and fragment ions) Easy to input existing data – Directly import Excel spreadsheets Critical information that is used for ID process Name (chemical, common, marker residue definition) Chemical formula Structure Retention time Accurate mass (precursor and product ions) Fragment ion(s) Isotopic patterns Isotope intensity s
- 16. ©2015 Waters Corporation 17 Xevo G2-S QTof Acquisition of the complete MS Dataset (MSE ) Low energy Simultaneous acquisition Elevated energy CE ramp applied
- 17. ©2015 Waters Corporation 18 Xevo G2-S QTof Spectral alignment of the complete dataset
- 18. ©2015 Waters Corporation 19 Fragment ion & retention time – why? Without Rt or fragment ions- 735 detects Using fragment ions- 86% reduction in detects Rt with a 0.5 window still results in many detects Adding fragment ions to this window results in a manageable number of detects Too narrow a Rt window introduces false negatives 36 spiked PPCPs in water
- 19. ©2015 Waters Corporation 20 PPCP: HRMS Screening results Extracted well water. Any incurred residues should be present in each of these samples Non-extracted well water. Only incurred residues of very high concentrations would be apparent High grade water also subjected to SPE – ideally should be blank
- 20. ©2015 Waters Corporation 21 Target list exact mass screening; Qualitative workflow
- 21. ©2015 Waters Corporation 22 UNIFI Component plot showing all identifications in well water
- 22. ©2015 Waters Corporation 23 PPCP in water: Qualitative screening results = Imidacloprid
- 23. ©2015 Waters Corporation 24 PPCP in water: Qualitative screening results = Carbamazepine
- 24. ©2015 Waters Corporation 25 Non-targeted workflow: Binary Comparison Finding the “unexpected”
- 25. ©2015 Waters Corporation 26 Binary Comparison workflow Unexpected pollutants in spinach Region of interestRed = reference Blue = control Green = differences
- 26. ©2015 Waters Corporation 27 Component Plot Candidate accurate masses
- 27. ©2015 Waters Corporation 28 Chemical elucidation process
- 28. ©2015 Waters Corporation 29 Library Query
- 29. ©2015 Waters Corporation 30 Library matches
- 30. ©2015 Waters Corporation 31 Unknown Screening: Met ID Workflow Unknown Screening – Met ID
- 31. ©2015 Waters Corporation 32 Metabolite identification functionality Component summary table showing confident matches in a well water sample Target molecule mol. file List of possible transformations
- 32. ©2015 Waters Corporation 33 PPCP: HRMS Screening results Identification of carbazapine and carbamazine oxidation
- 33. ©2015 Waters Corporation 34 Summary Complex analytical challenge! – Requirement to screen for target list compounds; unexpected & unknowns simultaneously; quan & qual workflows – Potential presence of degradation products & metabolites – Requirement for very low detection limits in complex matrices Accurate mass screening provides significantly more information than conventional QqQ Screening with UNIFI means a single acquisition can be interrogated using a variety of workflows
- 34. ©2015 Waters Corporation 35 Acknowledgements Claude Mallet Lauren Mullin Jennifer Burgess Mark Wrona