New LC/MS Water Screening PCDL for Environmental Contaminants · New LC/MS Water Screening PCDL for Environmental Contaminants Dorothy Yang Senior Application Scientist Agilent Technologies

New LC/MS Water Screening PCDL for Environmental Contaminants

Dorothy Yang

Senior Application Scientist

Agilent Technologies

1

6/20/2016

Outline

• Water Screening PCDL – Solution Overview

• Water Pollutants and Regulations

• The Value of the PCDL

• Analysis of Waste Water Treatment Plant Effluent

• Summary

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PCDL = Personal Compound Database and LibraryAgilent Technologies

6/20/2016

Water Screening PCDLNew Agilent Workflow Solution

• Comprehensive database with > 1,400 compounds covers

compounds from all major water regulations

• Library with accurate mass spectra for > 1,000 compounds for seamless

integration into the All Ions workflow

• Retention times for > 260 compounds to help avoid false positives

• Database and library are curated for highest quality in industry

• Guidance for easy development of screening methods provided

• Combined w/ high resolution 6200 Series TOF & 6500 Series Q-TOF LC/MS

Systems and 1260/1290 Infinity LC Series


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E.g. Agilent 1290 Infinity II UHPLC

E.g. Agilent 6550 iFunnel LC/Q-TOF

Target and suspect screening for a wide range of water contaminants

with highly confident compound identification using accurate mass

PCDL = Personal Compound Database and Library 6/20/2016

Water Pollutants

• Clean water is a prerequisite for humans, wildlife and in

agriculture

• Anthropogenic activities have effects on water quality

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• Industrial chemicals

• Industrial waste

• Pesticides

• Fertilizers

• Manure

• Veterinary Drugs

• Pharmaceuticals &

Personal Care Products

(PPCPs)

• Metabolites

• Transformation

products

Large number of diverse contaminants fuel multiple water regulations

Focus on contaminants of emerging concern, e.g. PPCPs


6/20/2016

Pharmaceutical and Personal Care Products (PPCPs)

• Prescription and over the counter therapeutic drugs

• Veterinary drugs

• Cosmetics, fragrances, sunscreen products

• Diagnostic agents

• Nutraceuticals (fortified foods or dietary supplements)

Compounds

• Human activity

• Residues from pharmaceutical manufacturing

• Illicit drugs

• Residues from hospital waste water treatment

• Veterinary drug use, especially antibiotics and steroids

Sources

www.natalieschibell.com

5

Lake Geneva

Lake Constance

Waste water content in Swiss surface

waters Red = sewage water influence

PPCPs and their transformation products enter rivers via

effluents from Waste Water Treatment Plants

ehp.niehs.nih.gov


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Pharmaceutical and Personal Care Products (PPCPs)

• Sewage systems are not fully or specifically equipped for PPCP removal

• Removal varies based on the chemical and treatment facilities

• Potential appearance in drinking water, especially with Direct Potable Reuse initiatives

Concerns

• Threat to aquatic organisms and to humans not fully known

• Resistance to antibiotics

• Natural and synthetic steroid disruption of aquatic endocrine systems

• Potential for synergistic effects due to mix of chemical contaminants present

Risks

www.natalieschibell.com

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Drugging the Environment (The Scientist, Aug 1,2015)Humans have spiked ecosystems with a flood of active pharmaceuticals. The

drugs are feminizing male fish, confusing birds, and worrying scientists.


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EU WFD

EPA

521, 535, 539, 1694

1698, 1699

Draft CCL 4

PPCPs

Japan specific

regulations

JPL

JDWQS

Over 1,400 compounds in the PCDL

Accurate mass MS/MS spectra for ~1,000 compounds

Polar

MetabolitesPesticides

Drugs

Vet. Drugs

Water Screening PCDL ContentCovering All Major Regulations

Cyanotoxins


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China-specific lists and regulations

CN-antibiotics list

CN-CDC survey list

CN-NY-193, 235, 265, 560

CN-GB 2763-2014

CN-EPA screening list

EU WFD

Water Regulations in Europe Covered in PCDLWater Framework Directive

EU guideline 2000/60/EG: Legal framework to protect

and restore clean water across Europe and

ensure its long-term, sustainable use

• Innovative approach for integrated water management

based on river basins even across the borders of

several member states

• Specifies common standards for the chemical,

biological, and ecological quality of water bodies

• Requires surveillance monitoring including a list of

41 priority pollutants and a large number of other pollutants

which may be discharged in significant quantities

• Priority pollutants include pesticides, perfluorinated compounds

(indicator compound), Nonyl- and octylphenol, polybrominated

flame retardants…

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EPA

521, 535, 539, 1694

1698, 1699

Draft CCL 4

Water Regulations in the US Covered in PCDLFor Waste water, Surface water, Drinking water & Soil

EPA regulations 521, 535, 539, 1694, 1698, 1699 cover

pesticides, hormones, antibiotics and general PPCPs

• Although some are GC methods, many compounds are in our LC method or in EPA

LC method

- 521: Nitrosamines (GC/MS) - a growing demand to switch over to LC/MS method

- 535: Chloroacetanilide and other acetamide herbicide degradates (LC/MS)

- 539: Hormones (LC/MS)

- 1694: PPCPs (LC/MS)

- 1698: Steroids and Hormones (GC/MS)

- 1699: Pesticides (GC/MS)

Draft CCL4 - Draft Contaminant Candidate List 4

• The Contaminant Candidate List (CCL) is a list of contaminants that are currently

not subject to any proposed or promulgated national primary drinking water

regulations, but are known or anticipated to occur in public water systems


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CN-antibiotics list

• Top sold/used antibiotics in China

Chinese CDC

• CN-CDC survey list

Chinese Department of Agriculture

• CN-NY-193, 235, 265, 560

- Regulation of antibiotics residues in food in China is mainly based on the 235 Announcement by the Agriculture Department (NY-235), and some list of prohibited compounds like NY-193, NY-265, and NY-560

China GB Regulation 2763-2014

Chinese EPA

• CN-EPA screening list

• China regulation agency normally refers to US EPA 1694 and 539 list, however there are compounds specific to China. Screening list Chinese EPA/other related agencies are currently working on will potentially be incorporated into one HJ/GB regulation in the near future


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6/20/2016

China-specific lists and regulations

CN-antibiotics list

CN-CDC survey list

CN-NY-193, 235, 265, 560

CN-GB 2763-2014

CN-EPA screening list

Water Regulations in China Covered in PCDL

Water Regulations/Lists in Japan Covered in PCDL

Japanese Ministry of Health, Labor and Welfare regulations/lists

• Japanese positive list (JPL)

Positive List System for Agricultural Chemical Residues in Foods.

• Japan Drinking Water Quality Standard (JDWQS)

GC/MS Regulated Pesticides in Drinking Water


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Japan specific regulations

JPL

JDWQS

6/20/2016

THE VALUE OF THE PCDL

6/20/2016


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The New Water Screening PCDL Adds Value

Agilent’s new Water Screening database and accurate mass library

combined with LC/MS TOF and Q-TOF

• Allows environmental screening with highly confident compound

identification.

• Carefully curated and highest quality database includes all compounds

from major regulations.

• Supports All Ions workflow for screening without the need for

standards.

• Full-spectrum acquisition enables retrospective data mining for the

presence of new chemical contaminants.

• Easy addition of new compounds to the PCDL to allow you staying on

top of increasing screening requirements.


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6/20/2016

The Scientist, Aug 1,2015

Identification Approach vs Identification ConfidenceSchymanski et al., Anal. Bioanal. Chem. 2015

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Start here with

PCDL!!!


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Spectra Collected and Curated by Experts

Our collaborators who provide the PCDL data are leading

experts in their field and have the most comprehensive

collection of reference standards.

Compounds and spectral are extensively curated by Agilent


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Curation verifies these entries for correctness:

Database Library

• Compound Common Name

• Accurate Mass of Neutral Molecule

• IUPAC Name

• Molecular Formula,

• Molecular Structure

• Ion Type (Anion, Cation, or Neutral)

• CAS Number/PubChem link (if existing)

• ChemSpider ID and Hyperlink (if existing)

• Chemical class tags and user notes

• Each precursor and product ion peak corrected to

theoretical accurate mass

• Spectra acquired at 10, 20, and 40eV Collision

Energy

• Spectra measured in positive and/or negative ion

mode where applicable

• Spectra are filtered for signal intensity and

curated for:

• Spectrum noise

• Chemical impurities

• Incorrectly set instrumentation parameters

6/20/2016

Easy PCDL Management with PCDL Manager SW

Library

- Normalized MS/MS spectra

- Positive and negative polarity

- Different species and collision energies


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Database

- Names, formulas, monoisotopic

masses, structures, common identifiers

- Retention times from experimental data

- Fully searchable and integrated

- Additional compounds can be added

- Class tags allow easy generation of

personalized/subset libraries

6/20/2016

Add YOUR curated accurate MS/MS spectra to new or existing compound entries


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PCDL Scalable – Add New Curated Spectra

Measured Spectrum of Standard Compound

FBF results with annotated MSMS spectra Library Spectrum

Open data in

MH Qual

Open method

(LibrarySearching-

Default.m)

FBF wt extract

MSMS spectra

Check annotation

SEND to PCDL

Export curated spectra to

customized PCDL

Untargeted QTOF acquisition allows retrospective review of historic data with new

compounds in PCDL

Send to PCDL

Filter on:

Peaks shifted to theoretical m/z (annotated)

Minimum base peak abundance (>1000 cts)

Noise (S/N must >1% abundance)

mass measured

[annotated formula]

6/20/2016FBF = Find by Formula

Screening StrategiesHow the Water Screening PCDL fits into Agilent Software Workflows

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Target screeningE.g. 14 cyanotoxins from Water

PCDL

All Ions MS/MS

Acquisition

- Batch overview

- Quantitation

- Identification

(Accurate Mass, Isotopes,

Qualifiers (fragments), RTs

- Find-by-Formula

Subset PCDL

- Assign fragments as

Qualifiers (Qual)

- Generate Quant Method

All Ions MS/MS

Reference standard

Suspect screening

„What else is in my sample?“

(beyond the 14 cyanotoxins)

All Ions MS/MS

or Auto MS/MS

- Find-by-Formula

Comprehensive PCDL

- Identify compounds

- fragment co-elution PCDL

- library searching PCDL

- Suspect List

- Identification by fragment

co-elution, library searching

- Tentative Identification by

in-silico fragmentation (MSC)

Non-target screeningDiscovering unknowns

Auto MS/MS

- Molecular Feature Extraction

- Mass Profiler

(2-sample comparison)

- Mass Profiler Professional

(Multivariate statistics)

- Differential results

- Identification by MS/MS

library searching PCDL

- Tentative Identification by

in-silico fragmentation (MSC)

6/20/2016


MSC =Molecular Structure Correlator

ANALYSIS OF WASTE WATER TREATMENT PLANT EFFLUENT


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Agriculture

Wastewater Analysis - Sampling Overview

Four wastewater treatment plants (WWTP) in Europe

• Different treatment principles and sizes

- Simple: Only mechanical and biological treatment, phosphate elimination

- Conventional (thorough): Simple PLUS nitrification and denitrification

Effluent sample collected over 14 days, 9 x 14-day periods- Analyzed with LC/Q-TOF using Water Screening PCDL

- Different collection areas

• Peri-urban, Agricultural, Industrial

-> Goal is to look for information

about the collection area,

and the efficiency of

the treatment process

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WWTP 1: Simple

water treatment

WWTP 2: Conventional

(thorough) water treatment

6/20/2016

Experimental Conditions

Water sample

Sample storage (3-8 °C)

Filter with glass fiber syringe

filters (opt.)

Inject 100 µL into LC/MS

Adjust pH, add ISTDs (opt.)

Agilent 1290 Infinity II UHPLC

Agilent 6550 iFunnel Q-TOF

Separation column ZORBAX RRHDx SB-Aq

2.1 x 150 mm; 1.8 µm

Mobile phase A 0.1 mM NH4 acetate + 0.1% acetic acid

Mobile phase B 0.1% acetic acid in acetonitrile

MS conditions Positive and negative ESI with

Agilent Jet Stream

- All Ions MS/MS

- Target MS/MS

- Auto MS/MS

Calibration range 5 to 1000 ng/L


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6/20/2016

ZORBAX StableBond-Aq (SB-Aq) for Polar Analytes

• Proprietary short chain bonding

chemistry optimized for polar analytes

(or samples with a wide range of

polarities)

• Rapid Resolution High Definition for

compatibility with the full 1300bar

pressure range of the 1290 Infinity II

• Compatible with low pH and 100%

Aqueous conditions without degradation

For more info, see Agilent brochures 5990-

8794EN and

For the best column

connections, couple with

Agilent A-line fittings

for easy, reliable, and

reproducible connections

up to 1300 bar

6/20/2016


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Compound Confirmation by Library Matching (Auto MS/MS)


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MS1 target score 91.4 (out of 100) Library score 84.8 (out of 100)

Compound chromatogram and peak spectrum

obtained by the Find-by-Formula algorithm

Comparison of the measured spectrum with

the library reference spectrum

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Easy Data Mining and Unambiguous Identification Non-targeted acquisition of precursor and product ions with All Ions

PCDL loads inside MassHunter Qual SW –

just run samples and then press search

• Find-by-Formula database search

• Automatic extraction of major

fragment ions from library


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Coelution scoring of precursor

and fragment ions

Diazinon in WWTP effluent (AI-8)

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What else is in my sample?Identification of emerging contaminants by All Ions MS/MS

Conventional treatment facility with > 50,000 population equivalents

• Lamotrigine (anticonvulsant)

• Sitagliptin (antidiabetic drug)

• Fexofenadine (antihistamine)

• Cetirizine (antihistamine)

• Irbesartan (angiotensin receptor blocker)

• Valsartan (angiotensin receptor blocker)


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Mass deviation < 5 ppm for

precursor and fragments

Perfect coelution

Consistent ion ratios

6/20/2016

Quantitation of Emerging ContaminantsPositive and negative All Ions MS/MS


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6/20/2016

Diclofenac

Negative ionization

MRPL (WFD): 10 ng/L

MixedStd_15ppt [Diclofenac]

12 12.5 13

3x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

294.0094 Area=6252

250.0196 Area=3159

214.0429 Area=2061

MixedStd_10ppt [Quinoxyfen]

13.5 14 14.5 15

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

308.0040 Area=133310

196.9789 Area=19406

213.9821 Area=9910

15 ng/L

10 ng/L

Quinoxyfen

Positive ionization

EQS (WFD): 15 to 150 ng/L

Larger city

More effective treatment

Less agricultural influence

Smaller city

Less effective treatment

More agricultural influence

Waste Water Treatment Sample Analysis Summary

Contaminant screening in effluents from Waste Water Treatment Plants reveals information about the collection area and efficiency of the treatment process

• Pesticides and pesticide metabolites in smaller city in agricultural area, with seasonal effects

• Pharmaceuticals and personal care products in urban areas, with more effective conventional treatment in larger city

Application note 5991-6627EN


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Pesticide Azoxystrobin has a seasonal surge correlated to its

application in the fields (only from March to November)

Therapeutic drug Diclofenac is evenly distributed across the

sampling period and shows similar concentration between

smaller population area and larger area, reflecting different

treatment efficiency (simple vs. conventional).

March

2014 June

2014Samples taken every 2 weeks

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Application Note 5991-6627EN

6/20/2016

Agilent Restricted

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Creation of the PCDL and application data (as shown

in previous slides)

Retention time information added to 268 compounds,

thus adding in retention time to the identification score

and increasing identification confidence

• Retention times are included for 268 compounds in the

Water_AMRT_PCDL. This PCDL is available with the Water

Screening PCDL installer (only)

• Retention times can be specified as optional or required in

your MassHunter Qualitative Analysis workflow

• A document describing necessary chromatographic setup

allows you to replicate retention times

Summary


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• Targeted & suspect screening of a wide range of water contaminants with high confidence

• Curated accurate mass database with over 1,400 compounds

• Curated accurate mass MS/MS spectral library for ~1,000 compounds

• Compound class/regulation tags allow creation of customized PCDLs

• Easy modification of PCDL w/ PCDL manager SW

• All Ions screening workflow is valuable for semi-targeted analysis and structure confirmation

• Full-spectrum acquisition allows retrospective investigation of data for the presence of new contaminants

• Auto MS/MS is optimal for non-targeted screening and identification of suspects

Confident Water Screening within Your Reach

6/20/2016

Acknowledgements

With slides and significant input from…

Tarun Anumol

Jean-Daniel Berset, Water and Soil ProtectionLaboratory, Bern, CH

Thomas Glauner

Craig Marvin

Emma Rennie

Maryann Shen

Mike Vagner

Dorothy Yang

Dr. Christian Zwiener Lab, University of Tuebingen, Germany

Ralph Hindle, Vogon Laboratories, Alberta, Canada

Regional field teams


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6/20/2016

Water Screening PCDL Product Structure

G6882CA Water Screening PCDL

• Curated compound database with more than 1,400 compounds

• Curated accurate mass MS/MS library with spectra for more than 1,000

compounds

• Quick Start Guide

• Familiarization Guide with data analysis exercises, example data files and methods

• Application note with retention times

• PCDL Manager Software

The complete Agilent solution includes:

• 6200/6500 Series TOF/Q-TOF LC/MS systems, 1260/1290 Infinity II Series LC


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6/20/2016

Documents

New LC/MS Water Screening PCDL for Environmental Contaminants · New LC/MS Water Screening PCDL for Environmental Contaminants Dorothy Yang Senior Application Scientist Agilent Technologies