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New LC/MS Water Screening PCDL for Environmental Contaminants
Dorothy Yang
Senior Application Scientist
Agilent Technologies
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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
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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
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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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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
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THE VALUE OF THE PCDL
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Agilent Technologies
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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.
Agilent Technologies
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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
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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
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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)
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Agilent Technologies
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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Agilent Technologies
WWTP 1: Simple
water treatment
WWTP 2: Conventional
(thorough) water treatment
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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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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
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Quantitation of Emerging ContaminantsPositive and negative All Ions MS/MS
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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
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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
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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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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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