©2015 Waters Corporation 1

Screening environmental water for a diverse

range of pollutants with UPLC-high

resolution MS

©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

©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

©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

©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

©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

©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

©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

©2015 Waters Corporation 9

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Example TIC chromatogram PPCPs in environmental water

©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

©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….

©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

©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

©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

©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

©2015 Waters Corporation 17

Xevo G2-S QTof

Acquisition of the complete MS Dataset (MSE )

Low energy

Simultaneous acquisition

Elevated energy

CE ramp applied

©2015 Waters Corporation 18

Xevo G2-S QTof

Spectral alignment of the complete dataset

©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

©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

©2015 Waters Corporation 21

Target list exact mass screening;

Qualitative workflow

©2015 Waters Corporation 22

UNIFI Component plot showing all identifications in well water

©2015 Waters Corporation 23

PPCP in water: Qualitative screening results = Imidacloprid

©2015 Waters Corporation 24

PPCP in water: Qualitative screening results = Carbamazepine

©2015 Waters Corporation 25

Non-targeted workflow: Binary Comparison

Finding the “unexpected”

©2015 Waters Corporation 26

Binary Comparison workflow Unexpected pollutants in spinach

Region of interest Red = reference Blue = control Green = differences

©2015 Waters Corporation 27

Component Plot Candidate accurate masses

©2015 Waters Corporation 28

Chemical elucidation process

©2015 Waters Corporation 29

Library Query

©2015 Waters Corporation 30

Library matches

©2015 Waters Corporation 31

Unknown Screening: Met ID Workflow

Unknown Screening – Met ID

©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

©2015 Waters Corporation 33

PPCP: HRMS Screening results

Identification of carbazapine and carbamazine oxidation

©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

©2015 Waters Corporation 35

Acknowledgements

Claude Mallet

Lauren Mullin

Jennifer Burgess

Mark Wrona