1 The world leader in serving science

Dipankar Ghosh, Ph.D Director, Environmental & Food Safety - LSMS San Jose, CA April 20, 2015

Thermo Scientific LCMS Solutions for Pesticide Analysis

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Routine Testing SOP Driven QQQ LC(GC)MS Methods

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What else could be present? Unknown screening

Emerging contaminants in environmental water

Food Authenticity and Fraud

Unknown contaminants in raw/processed food

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Analytical Methodology

Sample Preparation

Chromatographic Separation

Mass Spectrometry

Detection

Data Processing

& Reporting

5 Adapted from Alexander Makarov’s presentation 18th IMSC Bremen

The Ideal Mass Analzyer?

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HRAM

MS, MSn

App

lied

Mar

kets

Research

Markets

Non-targeted Analysis

Targeted Analysis

Quantitative Qualitative

• Biomarker Discovery • Proteomics • Metabolism

• Metabolomics • Proteomics • Bioanalysis

• Food Safety • Environmental • Clinical/Toxicology

• Metabolomics • PTM Analysis • Lipidomics

Thermo Scientific LSMS Portfolio

Ion Traps Triple Quads

Tribrid Orbitrap Exactives

The Industry’s Leading Portfolio of MS Solutions

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Thermo Scientific™ TSQ Quantiva™ MS

Extreme Quantitative Performance • Attogram sensitivity

• Unprecedented usability

• Exceptional robustness

QQQ : TSQ Endura & TSQ Quantiva LC-MS Systems

Thermo Scientific™ TSQ Endura™ MS

Extreme Quantitative Value • Best-in-class sensitivity

• Unprecedented usability

• Ultimate robustness

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Thermo Scientific™ Q Exactive™ MS & Q Exactive™ Plus MS • Orbitrap analyzer • Mass Range m/z 50 - 6000 • Mass Accuracy <1ppm • Max. Mass Resolution

>140,000 • Scan speed up to 12Hz MS and

MS/MS • Spectral Multiplexing • AQT & AABG (Q Exactive Plus

MS only) • Optional Intact Protein Mode

and Enhanced Resolution (280k) for Q Exactive Plus MS

Thermo Scientific™ Q Exactive™ HF MS • Ultra High Field Orbitrap analyzer • Mass Range m/z 50 - 6000 • Mass Accuracy <1ppm • Max. Mass Resolution >240,000 • Scan speed up to 18Hz MS and

MS/MS • Spectral Multiplexing • AQT & AABG • Optional Protein Mode

Thermo Scientific™ Exactive™ Plus (EMR) MS • Orbitrap analyzer • Mass Range m/z 50 - 6000 • Mass Range EMR: 300 - 20,000 • Mass Accuracy: <1ppm • Mass Resolution >140,000 • Scan Speed up to 12 Hz

Exactive Series Portfolio

Thermo Scientific™ Q Exactive™ Focus MS • Orbitrap analyzer • Mass Range m/z 50 - 2000 • Mass Accuracy <1ppm • Max. Mass Resolution >70,000 • Scan speed up to 12Hz MS and

12 Hz MS/MS • Polarity switching • Top 2 ddMS2 • No Multiplexing • Refined workflows that are

optimal for routine laboratories

VALUE

PER

FOR

MA

NC

E

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LC/MS Workflows for Environmental and Food Safety

Tools to develop methods for known and emerging contaminants

Raw Sample • Thermo Scientific™ Dionex™ ASE™

Accelerated Solvent Extractor

Dionex ASE Accelerated Solvent Extractor

EQuan MAX Plus System

• Thermo Scientific™ EQuan MAX Plus™ online sample prep LCMS

Customized Reporting

• Custom software to meet the needs of your workflow: Thermo Scientific™ TraceFinder™, Thermo Scientific™ SIEVE™, Thermo Scientific™ Compound Discoverer, and mzCloud software

TSQ Quantiva MS TSQ Endura MS

• TSQ Series mass spectrometers: triple stage quadrupole LC-MS/MS and GC-MS/MS for high sensitivity quantitation

• Thermo Scientific™ Exactive™ Plus and Thermo Scientific Q Exactive mass spectrometers: HRAM to identify, quantify and confirm

Orbitrap HR/AM

Orbitrap Fusion MS

UltiMate 3000 RSLC

• Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC systems

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Environmental and Food Safety Workflows

Quantitation Targeted Screening Non-Targeted and Unknown Screening

Hardware

Scan Modes

Software

Workflow

HRMS Series (QE)

HRMS Series (E+,QE)

Full Scan PRM TSIM

ddMS2 vDIA

Full Scan ddMS2

PRM, tSIM, vDIA Full Scan

ddMS2 vDIA

TraceFinder SW Compound

Discoverer SW SIEVE SW

SRM

m/zCloud

TSQ HRMS (E+,QE)

Thermo Scientific™ HRAM Spectral Library vDIA is not available in the U.S.

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QuEChERS

LC-MS/MS

Identification

Quantification

Reporting

Retention Time Product ions Ion ratios

SRM List

Typical LC-QqQ-MS/MS workflow

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Increasing Productivity: Alternative to Optimization by Infusion

Select compounds - TraceFinder Compound Database to create instrument method

Select Pre-configured TSQ method with LC and SRM conditions included - Load and Go!

Edit existing pre-configured method to create new

OR

OR

Built-in QQQ methods

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Toltrazuril is a triazinetrione derivative used in the prevention and treatment of coccidiosis of chicken, turkeys and pigs.

Metabolism → Two oxidation products, the sulfoxide and sulfone metabolites are obtained.

TOL has been included in the Annex I of Council Regulation (ECC) No 2377/90 and maximum residue levels (MRLs) on the animal tissues have been established (100-600 µg/kg)

O

N N

HN

SF3C

O O

OO

N N

HN

SF3C

O O

O

O

O

N N

HN

SF3C

O O

O

O

O

Toltrazuril (TOL) Toltrazuril Sulfoxide (TOLX) Toltrazuril Sulfone (TOLS)

H-SRM : The story of Toltrazuril and Frankfurt!

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Toltrazuril and metabolites – Frankfurt sausage

SRM 0.7 FWHM H-SRM 0.2 FWHM

0

100 0

100 0

100 0

100

Rela

tive

Abu

ndan

ce

1.6

1.6

1.0 2.0 3.0 4.0 Time (min)

0

100 0

100

0 100

0 100 1.8

1.8

1.8

1.8

0

100 0

100 0

100 0

100

1.6

1.6

0.0 1.0 2.0 3.0 4.0

Time (min)

0

100 0

100 0

100 0

100 1.8

1.8

1.8

1.8

m/z 371 371

m/z 371 271

m/z 388 288

m/z 388 248

m/z 356 256

m/z 356 124

m/z 359 256

m/z 359 124

m/z 371 371

m/z 371 271

m/z 388 288

m/z 388 248

m/z 356 256

m/z 356 124

m/z 359 256

m/z 359 124

A. Martínez-Villalba et al., Anal. and Bioanal. Chem, 2010 Aug; 397(7):2893-901

TOL-D3

TOLS

TOLX

TOL

2 µg/Kg

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QuEChERS

LC-HRMS

Identification

Quantification

Reporting

Retention Time Exact Mass (± 5 ppm) Product ions Ion ratios Isotopic Pattern Library Matching

Compound Inclusion List

Screening

Identification

Reporting

Generic Instrument Method

Typical LC-HRMS workflow

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Pesticides in Fruits and Vegetables by LC-HRMS

• QuEChERS Sample

Treatment

• Thermo Scientific™ Accucore™ aQ column(13min)

• FS/ddMS2 (Inclusion List)

LC-HRMS • Thermo Scientific™

TraceFinder™ software

Data Processing

Data presented at EPRW 2014

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Mass Resolving Power & Mass Accuracy – are they important?

The best a QTOF is going to get at low mass

Thermo Scientific™ Q Exactive Focus MS

Data presented at 10th European Pesticide Residue Workshop (EPRW 2014)

Do you want to see 94% or 97 % of pesticides?

Courtesy Prof Amadeo R Fernandez Albar

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YES - when you add sample complexity…

The best a QTOF is going to get at low mass

Q Exactive Focus MS

Data presented at 10th European Pesticide Residue Workshop (EPRW 2014)

Or worse !! Do you want to see 77% or 94 % of pesticides?

Courtesy Prof Amadeo R Fernandez Albar

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N. Cortés-Francisco, C. Flores, E. Moyano, J. Caixach. Anal. Bioanal. Chem. 2011 400(10):3595-606

Importance of Mass Accuracy on Unknown Screening

0

50

100

150

200

250

300

350

0.05 0.1 0.5 1 2 3 4 5 6 7 8 9 10

Mass Accuracy (ppm)

Num

ber o

f Pos

sibl

e C

andi

date

s

CHO CHON CHONS CHONSF

m/z 498.9302

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Which HRAM Scan Mode is Right for Your Workflow?

PRM/T-MS2

Full Scan MS

Matrix

Leek extract spiked with 0.1 ppb imazalil

Prec

urso

r ion

@ R

=70,

000

Prod

uct i

on @

R=7

0,00

0

3.275 ppm Product Ion

M.M.Gomez-Ramos et al. J Chrom A, 1287, 2013, 24-37

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Identification of Micropollutants and TPs by LC-HRMS

• Thermo Scientific™ Hypersil GOLD™ aQ column

• FS-ddMS2 (70K FWHM)

LC-HRMS

• TraceFinder SW (Targeted Screening)

Data Processing • Compound

Discoverer SW

Data Processing

• Sieve (Multivariate

Data Analysis PCA)

Data Processing

Data presented at SETAC and ASMS 2014

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Workflow for suspect and non-targeted analysis

Suspect Targeted Screening (TraceFinder SW

Search for Degradation Products (Compound Discoverer SW)

16:35 A Navigation through the Thermo Scientific™ Tracefinder™ Software Structure and Workflow Options; Frans Schoutsen, Thermo Fisher Scientific, Breda, Netherlands

Differential Analysis (Sieve SW)

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Q Exactive MS: HRAM Target Screening & Quantitation of Pesticides

GB National Food Standard method was created in China to specifically analyze 450 pesticides in fruits and vegetables using LC-MS/MS

To specifically focus on this issue we have created a turnkey (acquisition + processing) Thermo Scientific™ Q Exactive™ MS method that can Screen and Quantitate 500 pesticides

PARTNERS: FSANZ • Health Canada • IFIC Foundation • JIFSAN • NCFPD • USDA • IICA • FDA Advisory Council • CFIA • Food Communications Compass, Japan • CFSA • VAAS •

STEPRI

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500+ Pesticides Covering All Major Groups

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HRAM Library: Targeted Screening & Quantitation

Full MS / dd-MS2

Inclusion list activated: 500 pesticides

Quantitation &Confirmation: Fragment ion matching MS/MS library matching

Search by MS/MS spectrum

Q Exactive Focus MS

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R^2>0.998, Quan peak, Fragment ion matching and Spectra Library Scoring

Boscalid at 0.5ppb (Beet Extract)

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R^2>0.99, Quan Peak, fragment ion matching and Spectra Library Scoring

Trifloxystrobin at 0.1ppb (Beet Extract)

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Industry Leading HRAM MS/MS Spectral Library

Comprehensive HRAM Library and compound database created on Thermo Scientific™ Q Exactive™ MS at R 140,000

Searchable in TraceFinder software

Consists of: EFS (Pesticides, Mycotoxins, Veterinary Drugs, Environmental Contaminates, PFCs) Clin/Tox (Drugs of Abuse, Therapeutic Drugs, Poisons, Performance Enhancing Drugs) The new spectra library will include the following: 3 ramped CE @ 20, 30, 40 eV and 2

step collision energies @ 40 with 50% and 70 with 50% Contains RTs and RRTs using the same group of IS for both EFS + Clin/Tox

EFS Compounds: 1634 ++ EFS MS/MS spectra: 8900+ (5~6 spectra/compound)

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HRAM Ametryn Example - Different CEs Advantage

HCD 20 HCD 30 HCD 40

HCD 50 HCD Stepped 40 HCD Stepped 70

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Quantified and Confirmed

Chromatogram Fragment matching Spectra matching Calibration curve

Quantified and Confirmed with Built-in HRAM Spectra/Isotope/Fragments DB Imidacloprid at 0.64 ppb standard curve

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mzCloud

Novel highly curated MSn fragmentation library for next generation compound identification

TM

mzCloud™ is a unique approach to mass spectral fragmentation libraries Combining MSn, multiple fragmentation techniques, multiple energy levels, and a high level of curation Providing informative metadata https://mzcloud.org/

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mzCloud - Constantly Growing!

• 2,469 compounds with 78,813 spectra- 32 spectra per compound • Compounds currently available on-line and those in ‘que’

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FOOD: Status of Legislation in Different Regions

Japan

Well developed methods and standards Well developed infrastructure Food safety relatively under control

European Union

China

Developing methods Developing infrastructure Food safety control being developed.

USA

Developed methods and standards Prescribed methods by FDA and ISO, but no requirement to follow them Lack of testing resources

Well developed methods and standards Well developed infrastructure Food safety relatively under control No prescribed methods: must meet LOD’s, LOQs

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Overview of Identification Criteria LC-MS/MS

http://dx.doi.org/10.1016/j.aca.2015.03.007

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Environmental: Summary of Global Regulations & Enforcement

Country or Region

Regulation Enforcement

EU

European Commission sets directive, member states have 8 years to implement Directive 98;/83/EC - Drinking water Directive 86/278/EEC – metals Directive 2013/39/EU: SURFACE waters and BIOTA The new Directive 2008/50/EC of the European Parliament and of the Council of 21 May 2008 on ambient air quality

Labs can use any method, but must prove validation. Customer use ISO/DIN/AFNOR etc.

US

EPA Office of Ground Water and Drinking Water: Maximum Contaminant Limits (MCLs ) for drinking water under Safe Water Drinking Act (SWDA) EPA Office of Research and Development: Methods and MCL’s for compliance to the clean water act. EPA RCRA (formerly Office of Solid Waste): Solid Wastes

Labs must use approved methods; EPA , ASTM, Standard Methods EPA Regional Offices: 10 regions in the US. NELAC: Separate body which is responsible for compliance in some states. Customers are most concerned about enforcement bodies! Enforcement bodies often don’t know what the EPA has approved! Rely on you for information

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Standardization of Performance Criteria – yes ! Standardize global methods ? Standardize global technologies ? Sample prep Chromatography Detection – Mass Spectrometry analyzer Reporting

Your thoughts…

Moving Towards a Safer Way of Monitoring the Global Food & Environmental Chain…

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Thank You !

dipankar.ghosh@thermofisher.com