Pesticide Residue Analysis Webinar Series PART TWO: Workflow Guide for the Use of LCMS

Richard Fussell Vertical Marketing Manager, Food and Beverage Claudia Martins Applications Program Manager, Environmental and Food Safety

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Webinar Series Objectives

• To provide pesticide residue analysts with valuable information on development and optimization of method workflows for the analysis of pesticide residues in food.

• To understand the challenges, limitations and advantages of multi-residue workflows for pesticides residues in food

• To review the latest developments in: •  sample preparation •  targeted and screening techniques using GC-MS/MS and LC-MS/MS •  software advances to help with data interpretation and reporting.

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1. Sample Prep: March 24th 2. LC-MS Analysis: April 29th

3. GC-MS Analysis: June 17th 4. Data Processing/Analysis: July 15th

Typical Pesticides Workflow Register at www.chromatographyonline.com/LCGCwebseminars

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Thermo ScientificTM TSQ QuantivaTM and TSQ EnduraTM Next generation Triple Quadrupole MS/MS

LC-MS Timeline: Developments and Impact

Electrospray QQQ-MS

1995   2015  

APCI, Single quadrupole MS Karen A Barnes et al, Rapid Comunications in Mass Spectrometry (1995) 9, 1441-1445.

Christel L. Hetherton et al, Rapid Communications in Mass Spectrometry, 2004, 18, 2443-2450.

Quantification Limits

QuEChERS

2002  -­‐  2011  

Total # Analytical

Results (x1000)

Avg # pesticides analyzed per

sample

Total # residues detected (x10)

# of different residues detected

Thermo ScientificTM

Q ExactiveTM Focus Routine HRAM MS/MS

2013  

2000   2005   2010  

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Pesticide Residues Analysis Laboratory

Sample & Analyte

Variability

Number of Analytes

High Throughput

Cost Reduction

Screening Identification Quantitation

LC-MS &

GC-MS

Sensitivity

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Different Steps in Analytical Methodology

Sample Preparation

Chromatographic Separation Detection

Data Processing

& Reporting

Original QuEChERS Protocol*

Weight 10 g of sample

* Anastassiades et al. J AOAC Int., 86 (2) 412-431

Analysis – GC-MS and LC-MS

Shake Vigorously 1 min

Shake Vigorously 1 min

Shake 30s + Centrifuge

Shake vigorously 30s + Centrifuge

Add 10 mL Acetonitrile

Add ISTD - Solution

Add 4 g MgSO4 + 1 g NaCl

Take Aliquot and Mix with MgSO4 and PSA

Pros & Cons – Most-used Sample Prep Techniques

QuEChERS + Standard Method + Effective + Simple

- Contains manual steps - Difficulties when dealing with high fat content and carotinoides or chlorophyll rich samples

Dilute & Shoot (Liquid Samples)

+ Simple

- Dilution needed - Highly Sensitive and Robust methods needed - Regular maintenance of instrumentation needed

Turbulent Flow Chromatography

+ Automated

- Needs upgrade of standard configuration - Setup can be time-consuming

OFFLINE

ONLINE

9 9

How Does Thermo Scientific TurboFlow Technology Work?

•  Big particle size + high flow rate turbulent flow

•  Small molecules diffuse into pores faster than large molecules

•  Components of interest (analytes) are retained •  Matrix components are flushed through

•  A change in mobile phase composition elutes analytes of interest to the column (or

an analytical detector)

small molecules column pores analytes

analytes

10 10

Examples of the Use of Turbulent Flow Chromatography

Analytes Application Field Sample TFC column Loading Flow Rate

Injection Volume Detection Sensitivity

PFOS Environmental Analysis River Water 50 x 1.0 mm, 50µm C18 1 mL/min

1mL APPI-MS

5.35 ng/L (LOD)

Anti-Infectives Environmental Analysis Waste Water 50 x 1.0 mm, 50µm C18 XL 3 mL/min

1mL ESI-MS/MS

15-53 ng/L (LOD)

Enrofloxacin Ciprofloxacin

Food Analysis Edible Tissues

50 x 1.0 mm, 50µm C18 Cyclone

5 mL/min 20µL

ESI-MS/MS 25 µg/Kg (LOQ)

Quinolones Food Analysis Honey 50 x 0.5 mm, 60µm Cyclone 1.5 mL/min

160µL ESI-MS/MS

5 µg/Kg (MLOQ)

Veterinary drugs Food Analysis Milk

50 x 0.5 mm, 60µm Cyclone – Cyclone P (connected in

tandem)

1.5 mL/min 50µL

ESI-MS/MS 0.1-5.2 µg/L

O.Núñez et al. J Chrom A 2012, 1228, 298-323.

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Vortex & C

entrifugation

0.5 g sample

+ 990 µl MeOH

+ IS

Squeeze Adjust pH 7

Application of Turbulent Flow Chromatography TFC

-ES

I-MS

/MS

analysis

12 12

Prior to Injection Time Savings - Comparison

Online Clean Up Method

Centrifugation/Filtration

~0.5 g sample in 1 mL solvent

(shake briefly)

TFC-ESI-MS/MS Analysis

~ 12 min/sample (+ 13 min online clean-up-LC analysis)

Manual Method (QuEChERS)

d-SPE clean-up (vortex)

Supernatant recuperation

Liquid-Liquid Extraction (Vortex)

Centrifugation

~ 10 g sample in 10 mL solvent (shake briefly)

Centrifugation, supernatant recuperation

(filtration)

~ 30 min/sample ( + ~10 min LC analysis)

LC-ESI-MS/MS Analysis

Extraction of solid samples

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Configuration of the System

Laszlo Hollosi et al. Chromatographia, 2012, 75:1377-1393.

0

0

50

100

Polar Pesticides in Food Extracts

Acephate in acetonitrile

Acephate in acetonitrile-H2O

Polar pesticides – dilution of QuEChERS extract necessary before injection to achieve good peak shape

Alternatives: •  Reduce injection volume •  Increase tubing diameter

between injector and column

Time (min)

% H2O 5mM Ammonium Formate 0.1% FA

% MeOH 5 mM Ammonium Formate 0.1% FA

0.0 98 2

0.5 98 2

2.0 60 40

20.0 5 95

22.0 5 95

22.1 98 2

25.0 98 2

Thermo Scientific™ Accucore™ aQ (100 x 2.1 mm, 2.6 µm)

Chromatographic Separation of 440 Pesticides

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Time (min)

0

50

100 0.64

Rel

ativ

e A

bund

ance

Glyphosate

MeOH:Water Gradient Elution

C18  column  

0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 Time (min)

0

50

100 0.80

Rel

ativ

e A

bund

ance

Glyphosate

ACN:Water Gradient Elution

HILIC  column  

Mix-­‐mode:  HILIC  +  Ion-­‐Exchange    ACN:0.1%HCOOH 600 µL/min

AMPA

Glyphosate

0 1 2 3 4 5 6 7 8 9 10 11 12 Time (min)

Rel

ativ

e A

bund

ance

2.75

6.25

Chromatography is Important….

0

50

100

0

50

100

17 17

Impact on Maintenance

After cleaning

After 32 direct injections

After 120 direct injections with TLX

18 18

Matrix Effects

Laszlo Hollosi et al. Chromatographia, 2012, 75:1377-1393.

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•  Recoveries close to 100% most of compounds in range 80-110%

•  Small initial sample size <1 g homogenised sample

• Multiresidual assay 48 pesticides of different classes - extendable

•  Minimal Matrix Effects

•  High Sensitivity all (except chlormequate) fullfills MRL criteria –below 10 ug/kg

•  Good Intermediate Precision below 25%

• High speed total run time 13 min/sample (excl. extraction time)

Method Validation Results

Laszlo Hollosi et al. Chromatographia, 2012, 75:1377-1393.

20 20

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

The Ideal Mass Analyzer?

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What Workflow Should I Follow?

SAMPLE

Target Analysis Non-target Analysis

Identification

Screening

Profiling Fingerprinting

Differential Analysis

Database & Spectrum Libraries

Identification Quantitation

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The Industry’s Leading Portfolio of MS Solutions

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

Transform Your Science

Ion Traps Triple Quads

Tribrid Orbitrap Exactives

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Thermo Scientific TSQ Quantiva and Thermo Scientific TSQ Endura

Thermo Scientific™ TSQ Endura™ Triple Quadrupole Mass Spectrometer

Thermo Scientific™ TSQ Quantiva™ Triple Quadrupole Mass Spectrometer

TSQ Quantiva TSQ Endura

Mass Range 10-1850 10-3400

Max SRM Number 30,000 SRMs 30,000 SRMs

SRM/Sec 500 SRMs/sec 500 SRMs/sec

Ion Optics Active Ion Management (AIM)

•  Ion Max NG source •  Electrodynamic

ion funnel •  ion beam guide with

neutral blocker •  6 mm HyperQuad

quadrupoles with asymmetric RF drive

S-LENS with Beam Blocker Technology

Quadrupole Design 4mm Quadrupoles with Asymetric RF

Reserpine Specification (RFQ)

100,000 : 1 S/N for 1 pg Reserpine

10,000 : 1 S/N for 1 pg Reserpine

Extreme Quantitative Value •  Designed for non-stop operation.

•  For scientists who need to run routine samples day in and day out.

Extreme Quantitative Performance

•  Designed for the most challenging assays.

•  For scientists needing to stay at the forefront of analytical technology.

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What’s the Alternative to Optimization by Infusion?

Select compounds from Thermo Scientific™ TraceFinderTM 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

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Typical Setup of a LC-QqQ-MS/MS Method

1. Compound Database 2. Master method

4. Data Review & Report 3. Acquisition

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Typical Triple Quad MS Technology in Action

Q1 q2

Q3

408,4

408,2

256,2

256,3

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Why use Hyperbolic rods ? •  Forms Pure Quadrupolar Fields •  Reduces Fringing Field Effects •  Significantly Improves Resolution •  Improves Transmission •  Improves Peak Shapes

TSQ Quantum HyperQuads Technology

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The Impact of H-SRM

Q1

Q2

Q3

408.8

408.4

0.7 Da

256.3

0.4 Da

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Advantages of H-SRM in the Analysis of Pesticides O.Núñez et al. J Chrom A ., 2012, 1249, 164-180.

m/z  

•  Remove background interferences

•  Smooth baselines •  Improve % CV •  Increase signal-to-noise

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T-SRM: Pictorial Representation

0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

0 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Rel

ativ

e A

bund

ance

29.85 15.15 29.68

27.16 39.01 34.40

26.28 40.72 51.67 34.02 32.99

48.24 3.99 42.93 15.53 36.34 30.18

7.93 55.39 22.97 20.40 49.61 6.44 14.53 10.61 47.98 52.10 2.83

NL: 3.59E6 TIC MS

•  Instead of continually monitoring all transitions across the entire chromatographic window, SRMs can be monitored in distinct time windows (based on the retention time of the analyte).

•  Increases number of transitions that can be monitored. •  Increases dwell time for each transition monitored. •  Therefore improved %CV.

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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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The Use of QqQ for Pesticide Residue Analysis

•  Compatibility with simple sample preparation: QuEChERS, extract-n-shoot •  Compatibility with UHPLC •  High Sensitivity BUT… •  Targeted analysis only •  Limited screening capability of MS/MS (~ 200 compounds) •  Data evaluation is time consuming •  Multiple instruments to cover whole scope of method

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Where’s the Crossover Point?

Kaufmann et al: Anal. Chim. Acta., 2010, 673, 60-72.

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Skip the Matrix: Dummy Transitions Concentrations

“…the selectivity of LC-HRMS exceeds LC-MS/MS, if high resolution mass spectrometry (HRMS) data is

recorded with a resolution of 50,000 full width at half maximum (FWHM) and a corresponding mass

window…”

Kaufmann et al: Anal. Chim. Acta., 2010, 673, 60-72.

35 COMPANY CONFIDENTIAL

Q Exactive & Q Exactive Plus •  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

Q Exactive HF •  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

Exactive Plus (EMR) •  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

Q Exactive Focus •  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 What’s the Alternative to LC-QqQ-MS/MS?

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Typical LC-HRMS workflow

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

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Pesticides in Fruits and Vegetables by LC-HRMS

•  QuEChERS •  5 X Dilution

Sample Treatment

•  Accucore aQ (13min)

•  FS/ddMS2 (Inclusion List)

LC-HRMS

Data presented at EPRW 2014

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Is Mass Resolving Power important?

Data presented at EPRW 2014

39

Add Sample Complexity…

Data presented at EPRW 2014

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What Scan Mode is Right for Your Workflow?

PRM/T-MS2

Full Scan MS

Matrix

Leek extract spiked with 0.1 ppb imazalil

Pre

curs

or io

n @

R=7

0,00

0 P

rodu

ct io

n @

R=7

0,00

0

3.275 ppm Product Ion

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

41

SANCO/12571/2013

•  Method Validation & Quality Control Procedures for Pesticide Residues Analysis in Food & Feed

•  Implemented 1/1/2014 HRAM criteria ≥ 2 ions (pref. including quasi molecular ion)

< 5 ppm mass accuracy At least one fragment ion

Resolution typically >20000FWHM

42

  EU 2002/657/EC  

SANCO  12571/2013  

EU-RL-MB SOP  

Gerssen  (2010)  

Mol   (2012)  

Domènech  (2014)  

Kumar  (2014)  

Pitarch  (2007)  

Analytes   -   Pesticides   Lipophilic Toxins  

Lipophilic Toxins  

Pesticides   Lipophilic Toxins   Ronidazole  Nitroimidazoles  

Priority organic micropollutants  

Matrix   Food   Food and Feed  

Molluscs   Shellfish   Vegetables &Fruits   Mussels   Muscle   Water  

Technique   HRMS   HRMS   LC-MS/MS   LC-MS/MS   LC-HRMS/MS   LC-HRMS/MS   LC-HRMS/MS   GC-MS/MS  

Mass Accuracy   -   ˂ 5ppm   -   -   ˂ 5ppm   ˂ 5ppm   ˂ 5ppm   -  Mass Resolving Power (FWHM)  

-   ≥20,000   -   -   ≥20,000   ≥20,000   ≥70,000   -  

Retention Time (RT) Tolerance  

2.5%   2.5%   Not exceed 3%  

5%   1%   Mean ±3SD (not relative to time)  

±1%   Agreement - RT samples & standards  

Diagnostic Ions   ≥ 2   ≥ 2   1 precursor   1 precursor   ≥ 2   1   2   1 or 2 precursors  Fragment Ions   -  

 At least one   At least 2

precursor-product

transitions  

2 products   At least one   1   At least 1 >20,000FWHM  

At least 2 precursor-product

transitions  

Isotope Ions   -   -   -   -   M+1  M+2  

M+1   -   -  

Ion Ratio   Relative intensity (% at

base peak)  

Relative intensity (% at base peak)  

Must be  recorded  

As described 2002/657/EC  

Fragment Ion Ratio: Diagnostic/Fragment  

Isotope Ion Ratio:  Diagnostic/M+1 (M

+2)  

Fragment Ion Ratio: Diagnostic/

Fragment  Isotope Ion Ratio:  Diagnostic/M+1  

At least one   Ratio between quantitative and

confirmation transition  

Fragment-Isotope Ion Ratio Tolerance  

2 IPs for precursor ion  2.5 IPs for a

product  

As described 2002/657/EC  

-   As described 2002/657/EC  

Independent of relative intensity

between ions: ±50%  

As described 2002/657/EC  

-   As described 2002/657/EC  

Identification Criteria - Other Examples P. Lucci and C.P.B. Martins in Fast Liquid Chromatography – Mass Spectrometry Methods in Food and Environmental Analysis - World Scientific Publishing Company (March 2015)

43

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

Trends in pesticide residue analysis

•  IC-MS/MS for polar pesticides

•  Low flow separations (microflow) for highest sensitivity

•  Fast separation (< 15 min/run)

•  Matrix effects compensation by dilution (1:100-1000?)

•  The use of LC-HRMS as an alternative to LC-MS/MS

•  Multi-analyte screening (eg. pesticide & mycotoxin)

Conclusions

•  It is critical to adapt methodology for maximum performance •  Sample preparation, chromatography, configuration, method set-up, etc

•  Different technologies and configurations complement each other to

suit multiple needs

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Thermo Scientific Food and Environmental Communities: Resources •  View application notes, on-demand webinars, product information, and

many more resources on our Pesticides and Food Communities Libraries: www.thermoscientific.com/pesticides www.thermoscientific.com/foodandbeverage